David Feldman

All Publications

• The role of vitamin D in reducing cancer risk and progression NATURE REVIEWS CANCER Feldman, D., Krishnan, A. V., Swami, S., Giovannucci, E., Feldman, B. J. 2014; 14 (5): 342-357

  Abstract

  Vitamin D is not really a vitamin but the precursor to the potent steroid hormone calcitriol, which has widespread actions throughout the body. Calcitriol regulates numerous cellular pathways that could have a role in determining cancer risk and prognosis. Although epidemiological and early clinical trials are inconsistent, and randomized control trials in humans do not yet exist to conclusively support a beneficial role for vitamin D, accumulating results from preclinical and some clinical studies strongly suggest that vitamin D deficiency increases the risk of developing cancer and that avoiding deficiency and adding vitamin D supplements might be an economical and safe way to reduce cancer incidence and improve cancer prognosis and outcome.

  View details for DOI 10.1038/nrc3691

  View details for Web of Science ID 000335562000012

  View details for PubMedID 24705652

• Mutations in the vitamin D receptor and hereditary vitamin D-resistant rickets. BoneKEy reports Feldman, D., J Malloy, P. 2014; 3: 510-?

  Abstract

  Heterogeneous loss of function mutations in the vitamin D receptor (VDR) interfere with vitamin D signaling and cause hereditary vitamin D-resistant rickets (HVDRR). HVDRR is characterized by hypocalcemia, secondary hyperparathyroidism and severe early-onset rickets in infancy and is often associated with consanguinity. Affected children may also exhibit alopecia of the scalp and total body. The children usually fail to respond to treatment with calcitriol; in fact, their endogenous levels are often very elevated. Successful treatment requires reversal of hypocalcemia and secondary hyperparathyroidism and is usually accomplished by administration of high doses of calcium given either intravenously or sometimes orally to bypass the intestinal defect in VDR signaling.

  View details for DOI 10.1038/bonekey.2014.5

  View details for PubMedID 24818002

  View details for PubMedCentralID PMC4015455

• Mechanisms of the Anti-Cancer and Anti-Inflammatory Actions of Vitamin D ANNUAL REVIEW OF PHARMACOLOGY AND TOXICOLOGY, VOL 51, 2011 Krishnan, A. V., Feldman, D. 2011; 51: 311-336

  Abstract

  Calcitriol, the hormonally active form of vitamin D, is being evaluated in clinical trials as an anti-cancer agent. Calcitriol exerts multiple anti-proliferative, pro-apoptotic, and pro-differentiating actions on various malignant cells and retards tumor growth in animal models of cancer. Calcitriol also exhibits several anti-inflammatory effects including suppression of prostaglandin (PG) action, inhibition of p38 stress kinase signaling, and the subsequent production of pro-inflammatory cytokines and inhibition of NF-κB signaling. Calcitriol also decreases the expression of aromatase, the enzyme that catalyzes estrogen synthesis in breast cancer, both by a direct transcriptional repression and indirectly by reducing PGs, which are major stimulators of aromatase transcription. Other important effects include the suppression of tumor angiogenesis, invasion, and metastasis. These calcitriol actions provide a basis for its potential use in cancer therapy and chemoprevention. We summarize the status of trials involving calcitriol and its analogs, used alone or in combination with known anti-cancer agents.

  View details for DOI 10.1146/annurev-pharmtox-010510-100611

  View details for Web of Science ID 000287956600014

  View details for PubMedID 20936945

• The development of androgen-independent prostate cancer NATURE REVIEWS CANCER Feldman, B. J., Feldman, D. 2001; 1 (1): 34-45

  Abstract

  The normal prostate and early-stage prostate cancers depend on androgens for growth and survival, and androgen ablation therapy causes them to regress. Cancers that are not cured by surgery eventually become androgen independent, rendering anti-androgen therapy ineffective. But how does androgen independence arise? We predict that understanding the pathways that lead to the development of androgen-independent prostate cancer will pave the way to effective therapies for these, at present, untreatable cancers.

  View details for Web of Science ID 000180397000012

  View details for PubMedID 11900250

• Successful long-term treatment of refractory Cushing's disease with high-dose mifepristone (RU 486) JOURNAL OF CLINICAL ENDOCRINOLOGY & METABOLISM Chu, J. W., Matthias, D. F., Belanoff, J., Schatzberg, A., Hoffman, A. R., Feldman, D. 2001; 86 (8): 3568-3573

  Abstract

  An extremely ill patient, with Cushing's syndrome caused by an ACTH-secreting pituitary macroadenoma, experienced complications of end-stage cardiomyopathy, profound psychosis, and multiple metabolic disturbances. Initially treated unsuccessfully by a combination of conventional surgical, medical, and radiotherapeutic approaches, he responded dramatically to high-dose long-term mifepristone therapy (up to 25 mg/kg x d). Treatment efficacy was confirmed by the normalization of all biochemical glucocorticoid-sensitive measurements, as well as by the significant reversal of the patient's heart failure, the resolution of his psychotic depression, and the eventual unusual return of his adrenal axis to normal. His 18-month-long mifepristone treatment course was notable for development of severe hypokalemia that was attributed to excessive cortisol activation of the mineralocorticoid receptor, which responded to spironolactone administration. This case illustrates the efficacy of high-dose long-term treatment with mifepristone in refractory Cushing's syndrome. The case also demonstrates the potential need for concomitant mineralocorticoid receptor blockade in mifepristone-treated Cushing's disease, because cortisol levels may rise markedly, reflecting corticotroph disinhibition, to cause manifestations of mineralocorticoid excess.

  View details for Web of Science ID 000170430200016

  View details for PubMedID 11502780

• 1 alpha,25-dihydroxyvitamin D-3 down-regulates estrogen receptor abundance and suppresses estrogen actions in MCF-7 human breast cancer cells CLINICAL CANCER RESEARCH Swami, S., Krishnan, A. V., Feldman, D. 2000; 6 (8): 3371-3379

  Abstract

  1alpha,25-Dihydroxyvitamin D3 [1,25(OH)2D3], the active metabolite of vitamin D, is a potent inhibitor of breast cancer cell growth. Because the estrogen receptor (ER) plays a key role in breast cancer progression, we have studied the effects of 1,25(OH)2D3 on the regulation of ER in the estrogen-responsive MCF-7 human breast cancer cell line, which is known to predominantly express ERalpha. 1,25(OH)2D3 causes significant inhibition of MCF-7 cell growth, and it also decreases the growth-stimulatory effect of 17beta-estradiol (E2). Treatment of MCF-7 cells with 1,25(OH)2D3 reduces ER levels in a dose-dependent manner, as shown by ligand binding assays and Western blot analysis. The 1,25(OH)2D3 analogues EB-1089, KH-1060, Ro 27-0574, and Ro 23-7553 are more potent than 1,25(OH)2D3 in both their antiproliferative actions as well as ER down-regulation. There is a striking correlation (R2 = 0.98) between the growth-inhibitory actions of 1,25(OH)2D3 or analogues and their ability to down-regulate ER levels. Treatment with 1,25(OH)2D3 shows that the reduction in ER is accompanied by a significant decrease in the steady-state levels of ER mRNA. The decrease in ER mRNA is not abolished by the protein synthesis inhibitor cycloheximide. Inhibition of mRNA synthesis with actinomycin D reveals no significant differences between ER mRNA half-life in control and 1,25(OH)2D3-treated cells. Nuclear run-on experiments demonstrate significant decreases in ER gene transcription at the end of 17 h of treatment with 1,25(OH)2D3. These findings indicate that 1,25(OH)2D3 exerts a direct negative effect on ER gene transcription. Coincident with the decrease in ER levels there is an attenuation of E2-mediated bioresponses after 1,25(OH)2D3 treatment. Induction of progesterone receptor by E2 is suppressed by 1,25(OH)2D3, and the E2-mediated increase in breast cancer susceptibility gene (BRCA1) protein is reduced by 1,25(OH)2D3 treatment. Overall, these results suggest that the antiproliferative effects of 1,25(OH)2D3 and its analogues on MCF-7 cells could partially be mediated through their action to down-regulate ER levels and thereby attenuate estrogenic bioresponses, including breast cancer cell growth.

  View details for Web of Science ID 000088753300057

  View details for PubMedID 10955825

• Glucocorticoids can promote androgen-independent growth of prostate cancer cells through a mutated androgen receptor NATURE MEDICINE Zhao, X. Y., Malloy, P. J., Krishnan, A. V., Swami, S., Navone, N. M., Peehl, D. M., Feldman, D. 2000; 6 (6): 703-706

  Abstract

  The androgen receptor (AR) is involved in the development, growth and progression of prostate cancer (CaP). CaP often progresses from an androgen-dependent to an androgen-independent tumor, making androgen ablation therapy ineffective. However, the mechanisms for the development of androgen-independent CaP are unclear. More than 80% of clinically androgen-independent prostate tumors show high levels of AR expression. In some CaPs, AR levels are increased because of gene amplification and/or overexpression, whereas in others, the AR is mutated. Nonetheless, the involvement of the AR in the transition of CaP to androgen-independent growth and the subsequent failure of endocrine therapy are not fully understood. Here we show that in CaP cells from a patient who failed androgen ablation therapy, a doubly mutated AR functioned as a high-affinity cortisol/cortisone receptor (ARccr). Cortisol, the main circulating glucocorticoid, and its metabolite, cortisone, both equally stimulate the growth of these CaP cells and increase the secretion of prostate-specific antigen in the absence of androgens. The physiological concentrations of free cortisol and total cortisone in men greatly exceed the binding affinity of the ARccr and would activate the receptor, promoting CaP cell proliferation. Our data demonstrate a previously unknown mechanism for the androgen-independent growth of advanced CaP. Understanding this mechanism and recognizing the presence of glucocorticoid-responsive AR mutants are important for the development of new forms of therapy for the treatment of this subset of CaP.

  View details for Web of Science ID 000087438300044

  View details for PubMedID 10835690

• The vitamin D receptor and the syndrome of hereditary 1,25-dihydroxyvitamin D-resistant rickets ENDOCRINE REVIEWS Malloy, P. J., PIKE, J. W., Feldman, D. 1999; 20 (2): 156-188

  View details for Web of Science ID 000079668600003

  View details for PubMedID 10204116

• Treatment of early recurrent prostate cancer with 1,25-dihydroxyvitamin D3 (calcitriol) JOURNAL OF UROLOGY Gross, C., Stamey, T., Hancock, S., Feldman, D. 1998; 159 (6): 2035-2039

  Abstract

  Substantial experimental and epidemiological data indicate that 1,25-dihydroxyvitamin D3 (calcitriol) has potent antiproliferative effects on human prostate cancer cells. We performed an open label, nonrandomized pilot trial to determine whether calcitriol therapy is safe and efficacious for early recurrent prostate cancer. Our hypothesis was that calcitriol therapy slows the rate of rise of prostate specific antigen (PSA) compared with the pretreatment rate.After primary treatment with radiation or surgery recurrence was indicated by rising serum PSA levels documented on at least 3 occasions. Seven subjects completed 6 to 15 months of calcitriol therapy, starting with 0.5 microg. calcitriol daily and slowly increasing to a maximum dose of 2.5 microg. daily depending on individual calciuric and calcemic responses. Each subject served as his own control, comparing the rate of PSA rise before and after calcitriol treatment.As determined by multiple regression analysis, the rate of PSA rise during versus before calcitriol therapy significantly decreased in 6 of 7 patients, while in the remaining man a deceleration in the rate of PSA rise did not reach statistical significance. Overall the decreased rate of PSA rise was statistically significant (p = 0.02 Wilcoxon signed rank test). Dose dependent hypercalciuria limited the maximal calcitriol therapy given (range 1.5 to 2.5 microg. daily).This pilot study provides preliminary evidence that calcitriol effectively slows the rate of PSA rise in select cases, although dose dependent calciuric side effects limit its clinical usefulness. The development of calcitriol analogues with decreased calcemic side effects is promising, since such analogues may be even more effective for treating prostate cancer.

  View details for Web of Science ID 000073584400078

  View details for PubMedID 9598513

• ANTIPROLIFERATIVE EFFECTS OF 1,25-DIHYDROXYVITAMIN-D(3) ON PRIMARY CULTURES OF HUMAN PROSTATIC CELLS CANCER RESEARCH Peehl, D. M., Skowronski, R. J., Leung, G. K., Wong, S. T., Stamey, T. A., Feldman, D. 1994; 54 (3): 805-810

  Abstract

  Cultures of adult human prostatic epithelial and fibroblastic cells were established from normal, benign hyperplastic, and malignant tissues. Vitamin D receptors were detected by ligand binding of [3H]1,25-dihydroxyvitamin D3 [1,25(OH)2D3] in cytosolic extracts prepared from all types of cell cultures as well as from fresh prostatic tissues. Vitamin D receptor transcripts were demonstrated by Northern blot analysis. 1,25-(OH)2D3 inhibited the growth of epithelial cells with half-maximal inhibition at approximately 1 nM. The growth of fibroblasts was also inhibited by 1,25(OH)2D3 but to a lesser extent. This is consistent with the apparently lower level of vitamin D receptors in fibroblasts compared to epithelial cells determined by ligand binding and Northern analysis of RNA transcripts. The growth inhibition of epithelial cells by 1,25(OH)2D3 was irreversible even after a short 2-h exposure, but morphology and keratin expression were not appreciably altered by long-term exposure to the hormone. A physiological role for 1,25(OH)2D3 in the prostate is postulated, and the inhibitory effect of 1,25(OH)2D3 on cancer-derived prostate cells may provide a basis for new preventive or therapeutic strategies.

  View details for Web of Science ID A1994MU67700036

  View details for PubMedID 7508338

• BISPHENOL-A - AN ESTROGENIC SUBSTANCE IS RELEASED FROM POLYCARBONATE FLASKS DURING AUTOCLAVING ENDOCRINOLOGY Krishnan, A. V., Stathis, P., PERMUTH, S. F., Tokes, L., Feldman, D. 1993; 132 (6): 2279-2286

  Abstract

  In studies to determine whether Saccharomyces cerevisiae produced estrogens, the organism was grown in culture media prepared using distilled water autoclaved in polycarbonate flasks. The yeast-conditioned media showed the presence of a substance that competed with [3H]estradiol for binding to estrogen receptors (ER) from rat uterus. However, it soon became clear that the estrogenic substance in the conditioned media was not a product of the yeast grown in culture, but was leached out of the polycarbonate flasks during the autoclaving procedure. [3H]Estradiol displacement activity was monitored by ER RRA, and the active substance was purified from autoclaved medium using a series of HPLC steps. The final purified product was identified as bisphenol-A (BPA) by nuclear magnetic resonance spectroscopy and mass spectrometry. BPA could also be identified in distilled water autoclaved in polycarbonate flasks without the requirement of either the organism or the constituents of the culture medium. Authentic BPA was active in competitive RRAs, demonstrating an affinity approximately 1:2000 that of estradiol for ER. In functional assays, BPA (10-25 nM) induced progesterone receptors in cultured human mammary cancer cells (MCF-7) at a potency of approximately 1:5000 compared to that of estradiol. The BPA effect on PR induction was blocked by tamoxifen. In addition, BPA (25 nM) increased the rate of proliferation of MCF-7 cells assessed by [3H]thymidine incorporation. Thus, BPA exhibited estrogenic activity by both RRA and two functional bioresponse assays. Finally, MCF-7 cells grown in media prepared with water autoclaved in polycarbonate exhibited higher progesterone receptor levels than cells.grown in media prepared with water autoclaved in glass, suggesting an estrogenic effect of the water autoclaved in polycarbonate. Our findings raise the possibility that unsuspected estrogenic activity in the form of BPA may have an impact on experiments employing media autoclaved in polycarbonate flasks. It remains to be determined whether BPA derived from consumer products manufactured from polycarbonate could significantly contribute to the pool of estrogenic substances in the environment.

  View details for Web of Science ID A1993LE17000002

  View details for PubMedID 8504731

• VITAMIN-D AND PROSTATE-CANCER - 1,25-DIHYDROXYVITAMIN-D3 RECEPTORS AND ACTIONS IN HUMAN PROSTATE-CANCER CELL-LINES ENDOCRINOLOGY Skowronski, R. J., Peehl, D. M., Feldman, D. 1993; 132 (5): 1952-1960

  Abstract

  It has been suggested that vitamin D deficiency may promote prostate cancer, although the mechanism is not understood. In this study three human prostate carcinoma cell lines, LNCaP, DU-145, and PC-3, were examined both for the presence of specific 1,25 dihydroxyvitamin D3 [1,25(OH)2D3] receptors (VDRs) and also employed to study the effects of hormone on cell proliferation and differentiation. Ligand binding experiments demonstrated classical VDR in all three cell lines examined with an apparent dissociation constant of 7.5, 5.4, and 6.3 x 10(-11) M for LNCaP, DU-145, and PC-3 cells, respectively. Corresponding binding capacity for the three prostate carcinoma cell lines were 27, 31, and 78 fmol/mg protein, respectively. The presence of VDR in the three cell lines was also confirmed by immunocytochemistry. In addition, one major 4.6-kilobase messenger RNA transcript hybridizing with a specific human VDR complementary DNA probe was identified in all three cell lines. Interestingly, both DU-145 and PC-3 but not LNCaP cell lines exhibited 1,25(OH)2D3-stimulated induction of 24-hydroxylase messenger RNA employed as a marker of 1,25(OH)2D3 action. Physiological levels of 1,25(OH)2D3 dramatically inhibited proliferation of the LNCaP and PC-3 cell lines. However, in spite of the presence of high affinity VDR, proliferation of DU-145 cells was not inhibited by 1,25(OH)2D3 at the doses tested. Treatment with 1,25(OH)2D3 caused a dose-dependent stimulation of prostate-specific antigen secretion by LNCaP cells. In conclusion, these results demonstrate that these three human prostate carcinoma cell lines all possess specific VDR and that 1,25(OH)2D3 treatment can elicit both an antiproliferative and a differentiating action on these cancer cells. The findings lend support to the hypothesis that vitamin D might exert beneficial actions on prostate cancer risk.

  View details for Web of Science ID A1993KZ69900011

  View details for PubMedID 7682937

• THE MOLECULAR-BASIS OF HEREDITARY 1,25-DIHYDROXYVITAMIN-D3 RESISTANT RICKETS IN 7 RELATED FAMILIES JOURNAL OF CLINICAL INVESTIGATION Malloy, P. J., Hochberg, Z., Tiosano, D., PIKE, J. W., Hughes, M. R., Feldman, D. 1990; 86 (6): 2071-2079

  Abstract

  Hereditary 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] resistant rickets (HVDRR) is an autosomal recessive disease caused by target organ resistance to the action of 1,25(OH)2D3, the active form of the hormone. The defect in target cells is heterogenous and commonly appears to be a mutation in the gene encoding the vitamin D receptor (VDR). We have studied cultured skin fibroblasts and Epstein-Barr virus transformed lymphoblasts of seven family branches of an extended kindred having eight children affected with HVDRR. We have previously shown that cells from three affected children in this group contain an "ochre" nonsense mutation coding for a premature stop codon in exon 7 within the steroid-binding domain of the VDR gene. In the current studies, we found that cells from affected children failed to bind [3H]1,25(OH)2D3 and had undetectable levels of VDR as determined by immunoblots using an anti-VDR monoclonal antibody. Measurement of VDR mRNA by hybridization to a human VDR cDNA probe showed undetectable or decreased abundance of steady-state VDR mRNA. Parents, expected to be obligate heterozygotes, showed approximately half the normal levels of [3H]1,25(OH)2D3 binding, VDR protein, and mRNA. The mutation at nucleotide 970 (counting from the mRNA CAP site) results in the conversion of GTAC to GTAA, which eliminates an Rsa I restriction enzyme site and facilitates identification of the mutation. We found that polymerase chain reaction (PCR) amplification of exons 7 and 8 from family members and subsequent Rsa I digestion allows detection of the specific genotype of the individuals. When Rsa I digests of PCR-amplified DNA are subjected to polyacrylamide gel electrophoresis, children with HVDRR exhibit a homozygous banding pattern with loss of an Rsa I site. Parents exhibit a heterozygotic DNA pattern with detection of both normal and mutant alleles. In summary, our data show that the genetic abnormality is a point mutation within the steroid-binding domain of the VDR in all seven related families with HVDRR. Analysis of restriction fragment length polymorphism at the 970 locus of PCR-amplified DNA fragments can be used to diagnose this mutation in both affected children and parents carrying the disease.

  View details for Web of Science ID A1990EL76300040

  View details for PubMedID 2174914

  View details for PubMedCentralID PMC329846

• AN OCHRE MUTATION IN THE VITAMIN-D RECEPTOR GENE CAUSES HEREDITARY 1,25-DIHYDROXYVITAMIN-D3-RESISTANT RICKETS IN 3 FAMILIES PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ritchie, H. H., Hughes, M. R., Thompson, E. T., Malloy, P. J., Hochberg, Z., Feldman, D., PIKE, J. W., OMALLEY, B. W. 1989; 86 (24): 9783-9787

  Abstract

  Hereditary 1,25-dihydroxyvitamin D3-resistant rickets is a rare autosomal-recessive disease resulting from target-organ resistance to the action of the active hormonal form of vitamin D. Four affected children from three related families with the classical syndrome of hereditary 1,25-dihydroxyvitamin D3-resistant rickets and the absence of detectable binding to the vitamin D receptor (VDR) in cultured fibroblasts or lymphoblasts were examined for genetic abnormalities in the VDR gene. Genomic DNA from Epstein-Barr virus-transformed lymphoblasts of eight family members was isolated and amplified by polymerase chain reaction techniques. Amplified fragments containing the eight structural exons encoding the VDR protein were sequenced. The DNA from all affected children exhibited a single C----A base substitution within exon 7 at nucleotide 970 that resulted in the conversion of the normal codon for tyrosine (TAC) into a premature termination codon (TAA) at amino acid 292. This mutation causes a truncation of the VDR protein thereby deleting a large portion of the steroid hormone binding domain (amino acids 292-424). Although the affected children were all homozygotic for the mutation, the four parents tested all exhibited both wild-type and mutant alleles, indicating a heterozygous state. The functional consequences of this mutation were confirmed after expression of the recreated mutant VDR cDNA in mammalian cells. Recreated mutant receptor exhibited no specific 1,25-[3H]dihydroxyvitamin D3 binding and failed to activate a cotransfected VDR promoter-reporter gene construct. Thus these findings identify an ochre mutation in a human steroid hormone receptor in patients with hereditary 1,25-dihydroxyvitamin D3-resistant rickets.

  View details for Web of Science ID A1989CE97600031

  View details for PubMedID 2557627

  View details for PubMedCentralID PMC298586

• POINT MUTATIONS IN THE HUMAN VITAMIN-D RECEPTOR GENE ASSOCIATED WITH HYPOCALCEMIC RICKETS SCIENCE Hughes, M. R., Malloy, P. J., Kieback, D. G., Kesterson, R. A., PIKE, J. W., Feldman, D., OMALLEY, B. W. 1988; 242 (4886): 1702-1705

  Abstract

  Hypocalcemic vitamin D-resistant rickets is a human genetic disease resulting from target organ resistance to the action of 1,25-dihydroxyvitamin D3. Two families with affected children homozygous for this autosomal recessive disorder were studied for abnormalities in the intracellular vitamin D receptor (VDR) and its gene. Although the receptor displays normal binding of 1,25-dihydroxyvitamin D3 hormone, VDR from affected family members has a decreased affinity for DNA. Genomic DNA isolated from these families was subjected to oligonucleotide-primed DNA amplification, and each of the nine exons encoding the receptor protein was sequenced for a genetic mutation. In each family, a different single nucleotide mutation was found in the DNA binding domain of the protein; one family near the tip of the first zinc finger (Gly----Asp) and one at the tip of the second zinc finger (Arg----Gly). The mutant residues were created in vitro by oligonucleotide directed point mutagenesis of wild-type VDR complementary DNA and this cDNA was transfected into COS-1 cells. The produced protein is biochemically indistinguishable from the receptor isolated from patients.

  View details for Web of Science ID A1988R492900045

  View details for PubMedID 2849209

• INHIBITION OF ADRENAL STEROIDOGENESIS BY THE ANESTHETIC ETOMIDATE NEW ENGLAND JOURNAL OF MEDICINE Wagner, R. L., White, P. F., KAN, P. B., Rosenthal, M. H., Feldman, D. 1984; 310 (22): 1415-1421

  Abstract

  The use of the intravenous anesthetic etomidate for prolonged sedation has been associated with low levels of plasma cortisol and increased mortality. We measured the cortisol and aldosterone responses to ACTH stimulation in five patients receiving etomidate, and we also studied the direct effects of etomidate on enzymes in the rat steroidogenic pathway. One patient who was receiving a 20-hour infusion of etomidate (1.3 to 1.5 mg per kilogram of body weight per hour) had marked adrenocortical suppression that was still evident four days after etomidate was discontinued. Four surgical patients receiving etomidate during their operations were all found to have adrenal suppression four hours after the operation; mean (+/- S.D.) increases in cortisol and aldosterone after ACTH stimulation were only 1.8 +/- 0.5 micrograms per deciliter and 0.5 +/- 1.1 ng per deciliter, respectively. In rat adrenal cells, etomidate produced a concentration-dependent blockade of the two mitochondrial cytochrome P-450-dependent enzymes, cholesterol-side-chain cleavage enzyme, and 11 beta-hydroxylase, without evident inhibition of the microsomal enzymes in the glucocorticoid pathway. Physicians should be aware that etomidate inhibits adrenal steroidogenesis, and they should consider treating selected patients with corticosteroids if etomidate is used.

  View details for Web of Science ID A1984ST14400002

  View details for PubMedID 6325910

• 1,25-DIHYDROXYVITAMIN-D3 AND MALIGNANT-MELANOMA - THE PRESENCE OF RECEPTORS AND INHIBITION OF CELL-GROWTH IN CULTURE ENDOCRINOLOGY Colston, K., Colston, M. J., Feldman, D. 1981; 108 (3): 1083-1086

  Abstract

  In this study we demonstrate the presence of specific, high-affinity receptors for 1,25-dihydroxyvitamin D3 in malignant melanoma. Receptors are present both in cultured melanoma cells and in melanoma tumor tissue produced by inoculation of cells into athymic rats. The receptor sediments at 3.25 on sucrose density gradients, possesses a preferential affinity for 1,25-(OH)2D3 and has an apparent Kd of 0.18 nM by Scatchard analysis. We also demonstrate that human melanoma cells are responsive to 1,25-(OH)2D3 in vitro. Inclusion of 1,25-(OH)2D3 in the culture medium produced a marked increase in cell doubling time. This inhibitory effect of the hormone on melanoma cell proliferation was dose-related and represents the first demonstration of a 1,25-(OH)2D3 mediated action on tumor cells.

  View details for Web of Science ID A1981LD51700054

  View details for PubMedID 6257495

• DEMONSTRATION OF 1,25-DIHYDROXYVITAMIN-D3 RECEPTORS IN HUMAN-SKIN BIOPSIES JOURNAL OF CLINICAL ENDOCRINOLOGY & METABOLISM Feldman, D., Chen, T., Hirst, M., Colston, K., Karasek, M., Cone, C. 1980; 51 (6): 1463-1465

  Abstract

  In this study we report the demonstration of receptors for 1,25-(OH)2 vitamin D3 in fresh and cultured human skin. Cultured fibroblasts grown from infant foreskin exhibit a binding site which by Scatchard analysis had a Kd for [3H]1,25-(OH)2D3 of 0.2 nM and an Nmax of approximately 40 fmol/mg cytosol protein. On sucrose density gradients the receptor sediments at 3.2S. Receptors could also be identified in skin biopsies from adult patients when assayed either in fresh epidermis or cultured keratinocytes and dermal fibroblasts. The human receptors are similar to rodent receptors assessed in classical target organs such as intestine, bone and kidney. The findings that receptors can be measured in cultured human skin after several arterial passages indicates that skin biopsy may provide a means of assessing the 1,25-(OH)2D3 receptor status of patients.

  View details for Web of Science ID A1980KT18400046

  View details for PubMedID 6255007

• Vitamin D and malabsorptive gastrointestinal conditions: A bidirectional relationship? Reviews in endocrine & metabolic disorders Giustina, A., di Filippo, L., Allora, A., Bikle, D. D., Cavestro, G. M., Feldman, D., Latella, G., Minisola, S., Napoli, N., Trasciatti, S., Uygur, M., Bilezikian, J. P. 2023

  Abstract

  This paper is one of the outcomes of the 5th International Conference "Controversies in Vitamin D" held in Stresa, Italy from 15 to 18 September 2021 as part of a series of annual meetings which was started in 2017. The scope of these meetings is to discuss controversial issues about vitamin D. Publication of the outcomes of the meeting in international journals allows a wide sharing of the most recent data with the medical and academic community. Vitamin D and malabsorptive gastrointestinal conditions was one of the topics discussed at the meeting and focus of this paper. Participants to the meeting were invited to review available literature on selected issues related to vitamin D and gastrointestinal system and to present their topic to all participants with the aim to initiate a discussion on the main outcomes of which are reported in this document. The presentations were focused on the possible bidirectional relationship between vitamin D and gastrointestinal malabsorptive conditions such as celiac disease, inflammatory bowel diseases (IBDs) and bariatric surgery. In fact, on one hand the impact of these conditions on vitamin D status was examined and on the other hand the possible role of hypovitaminosis D on pathophysiology and clinical course of these conditions was also evaluated. All examined malabsorptive conditions severely impair vitamin D status. Since vitamin D has known positive effects on bone this in turn may contribute to negative skeletal outcomes including reduced bone mineral density, and increased risk of fracture which may be mitigated by vitamin D supplementation. Due to the immune and metabolic extra-skeletal effects there is the possibility that low levels of vitamin D may negatively impact on the underlying gastrointestinal conditions worsening its clinical course or counteracting the effect of treatment. Therefore, vitamin D status assessment and supplementation should be routinely considered in all patients affected by these conditions. This concept is strengthened by the existence of a possible bidirectional relationship through which poor vitamin D status may negatively impact on clinical course of underlying disease. Sufficient elements are available to estimate the desired threshold vitamin D level above which a favourable impact on the skeleton in these conditions may be obtained. On the other hand, ad hoc controlled clinical trials are needed to better define this threshold for obtaining a positive effect of vitamin D supplementation on occurrence and clinical course of malabsorptive gastrointestinal diseases.

  View details for DOI 10.1007/s11154-023-09792-7

  View details for PubMedID 36813995

• Controversies in Vitamin D: A Statement From the Third International Conference. JBMR plus Giustina, A., Bouillon, R., Binkley, N., Sempos, C., Adler, R. A., Bollerslev, J., Dawson-Hughes, B., Ebeling, P. R., Feldman, D., Heijboer, A., Jones, G., Kovacs, C. S., Lazaretti-Castro, M., Lips, P., Marcocci, C., Minisola, S., Napoli, N., Rizzoli, R., Scragg, R., White, J. H., Formenti, A. M., Bilezikian, J. P. 2020; 4 (12): e10417

  Abstract

  The Third International Conference on Controversies in Vitamin D was held in Gubbio, Italy, September 10-13, 2019. The conference was held as a follow-up to previous meetings held in 2017 and 2018 to address topics of controversy in vitamin D research. The specific topics were selected by the steering committee of the conference and based upon areas that remain controversial from the preceding conferences. Other topics were selected anew that reflect specific topics that have surfaced since the last international conference. Consensus was achieved after formal presentations and open discussions among experts. As will be detailed in this article, consensus was achieved with regard to the following: the importance and prevalence of nutritional rickets, amounts of vitamin D that are typically generated by sun exposure, worldwide prevalence of vitamin D deficiency, the importance of circulating concentrations of 25OHD as the best index of vitamin D stores, definitions and thresholds of vitamin D deficiency, and efficacy of vitamin D analogues in the treatment of psoriasis. Areas of uncertainly and controversy include the following: daily doses of vitamin D needed to maintain a normal level of 25OHD in the general population, recommendations for supplementation in patients with metabolic bone diseases, cutaneous production of vitamin D by UVB exposure, hepatic regulation of 25OHD metabolites, definition of vitamin D excess, vitamin D deficiency in acute illness, vitamin D requirements during reproduction, potential for a broad spectrum of cellular and organ activities under the influence of the vitamin D receptor, and potential links between vitamin D and major human diseases. With specific regard to the latter area, the proceedings of the conference led to recommendations for areas in need of further investigation through appropriately designed intervention trials. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.

  View details for DOI 10.1002/jbm4.10417

  View details for PubMedID 33354643

• Treatment of Hereditary Vitamin D-Resistant Rickets with Nocturnal Calcium Infusion: A 20-Year Follow-up Report Morrissey, C., Malloy, P. I., Feldman, D., Nayak, S. KARGER. 2020: 37–38

  View details for Web of Science ID 000544736900051

• Pre-diagnostic 25-hydroxyvitamin D concentrations in relation to tumor molecular alterations and risk of breast cancer recurrence. Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology Peng, C., Heng, Y. J., Lu, D., DuPre, N. C., Kensler, K. H., Glass, K., Zeleznik, O. A., Kraft, P., Feldman, D., Hankinson, S. E., Rexrode, K., Eliassen, A. H., Tamimi, R. M. 2020

  Abstract

  BACKGROUND: Although vitamin D inhibits breast tumor growth in experiments, the findings from population-based studies remain inconclusive. Our goals were to investigate the association between pre-diagnostic plasma 25-hydroxyvitamin D [25(OH)D] and breast cancer recurrence in the Nurses' Health Studies (NHS) and to explore the molecular underpinnings.METHODS: Plasma 25(OH)D was measured with a high-affinity-protein-binding-assay/a radioimmunoassay. We profiled transcriptome-wide-gene-expression in breast tumors using microarrays. Hazard ratios (HRs) of breast cancer recurrence were estimated from covariate-adjusted-Cox-regressions. We examined differential gene expression in association with 25(OH)D. We derived a gene expression score for 25(OH)D, and assessed associations between the score and cancer recurrence.RESULTS: Although 25(OH)D was not associated with breast cancer recurrence overall (HR=0.97; 95% confidence interval (CI): 0.88-1.08), the association varied by estrogen-receptor (ER) status (p-for-interaction=0.005). Importantly, among ER-positive stage I-to-III cancers, every 5ng/ml increase in 25(OH)D was associated with a 13% lower risk of recurrence (HR=0.87; 95%-CI: 0.76-0.99). A null association was observed for ER-negative cancers. Pathway analysis identified multiple gene-sets (proliferation, migration, and inflammation) that were significantly down-regulated in ER-positive tumors of women with high 25(OH)D (≥30ng/ml), compared to those with low levels. 25(OH)D score derived was marginally associated with reduced risk of recurrence in ER-positive diseases in NHS, however, no association was noted in the replication dataset.CONCLUSIONS: Our findings support an intriguing line of research to better understand the mechanisms underlying the role of vitamin D in breast tumor progression, particularly for the ER-positive subtype.IMPACT: Vitamin D may present a personal-level secondary-prevention strategy for breast cancer.

  View details for DOI 10.1158/1055-9965.EPI-19-1217

  View details for PubMedID 32238406

• PMID: 32180081. Consensus statement from 2nd International Conference on Controversies in Vitamin D. Rev Endocr Metab Disord. Mar;21(1):89-116. Giustina, A. 2020

  View details for DOI 10.1007/s11154-019-09532-w.

• 27-Hydroxycholesterol, an Endogenous SERM, and Risk of Fracture in Postmenopausal Women: A Nested Case-Cohort Study in the Women's Health Initiative JOURNAL OF BONE AND MINERAL RESEARCH Chang, P., Feldman, D., Stefanick, M. L., McDonnell, D. P., Thompson, B. M., McDonald, J. G., Lee, J. S. 2019; 34 (1): 59–66

  View details for DOI 10.1002/jbmr.3576

  View details for Web of Science ID 000456724900006

• 27-hydroxycholesterol, an endogenous SERM, and risk of fracture in postmenopausal women: A nested case-cohort study in the Women's Health Initiative. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research Chang, P., Feldman, D., Stefanick, M. L., McDonnell, D. P., Thompson, B. M., McDonald, J. G., Lee, J. S. 2018

  Abstract

  27-hydroxycholesterol (27HC) is a purported, novel endogenous SERM. In animal models, 27HC has an anti-estrogen effect in bone, and 17beta-estradiol mitigates this effect. 27HC in relation to fracture risk has not been investigated in humans. Depending on the level of bioavailable 17beta-estradiol (bioE2), 27HC may increase fracture risk in postmenopausal women and modify the fracture risk reduction from menopausal hormone therapy (MHT). To test these a priori hypotheses, we conducted a nested case-cohort study of 868 postmenopausal women within the Women's Health Initiative Hormone Therapy trials (WHI-HT). The WHI-HT tested conjugated equine estrogens versus placebo and separately conjugated equine estrogens plus progestin versus placebo. Fracture cases were 442 women who had an adjudicated incident hip or clinical vertebral fracture during the WHI-HT follow-up. The sub-cohort included 430 women randomly selected at WHI-HT baseline, 4 of whom had a subsequent fracture. Of 868 women, 266 cases and 219 non-cases were assigned to the placebo arms. Cox models estimated hazard ratios for incident fracture in relation to pre-randomization circulating levels of 27HC and 27HC/bioE2 molar ratio. Models adjusted for age, race/ethnicity, total cholesterol, bioE2, sex hormone-binding globulin, 25-hydroxyvitamin D, diabetes, osteoporosis, prior MHT use, BMI, falls history and prior fracture. In women assigned to placebo arms, those in the middle and the highest tertiles of 27HC/bioE2 had an up to 1.9-fold (95% confidence intervals: 1.25-2.99) greater risk of fracture than women in the lowest tertile. In women assigned to MHT arms, fracture risk increased with continuous 27HC/bioE2 levels but not with categorical levels. 27HC levels alone were not associated with fracture risk. 27HC and 27HC/bioE2 did not modify the fracture risk reduction from MHT. In postmenopausal women, circulating levels of 27HC relative to bioE2 may identify those at increased risk of fracture. This article is protected by copyright. All rights reserved.

  View details for PubMedID 30138538

• Association of 25-hydroxyvitamin D levels and cutaneous melanoma: A nested case-control study of the Women's Health Initiative Observation Study. Journal of the American Academy of Dermatology Kwon, G. P., Gamba, C. S., Stefanick, M. L., Swetter, S. M., Li, S., Shi, R. Z., Clarke, C. A., Feldman, D., Millen, A. E., Messina, C., Shikany, J. M., Manson, J. E., Chlebowski, R. T., Tang, J. Y. 2018; 79 (1): 145–47

  View details for PubMedID 29908819

• Effects of Vitamin D on Skeletal Muscle and Athletic Performance. The Journal of the American Academy of Orthopaedic Surgeons Abrams, G. D., Feldman, D., Safran, M. R. 2018; 26 (8): 278–85

  Abstract

  Vitamin D is known to be important for calcium homeostasis and bone metabolism. It also has important direct effects on skeletal muscle. Unlike authentic vitamins, which cannot be synthesized in the body, vitamin D is produced in the skin using sunlight. Through its nuclear receptor (ie, vitamin D receptor) located throughout the body, including skeletal muscle, vitamin D initiates genomic and nongenomic pathways regulating multiple actions, including myocyte proliferation and growth. In some studies, vitamin D supplementation has been shown to increase muscle strength, particularly in people who are vitamin D deficient. Higher serum levels of vitamin D are associated with reduced injury rates and improved sports performance. In a subset of the population, vitamin D appears to play a role in muscle strength, injury prevention, and sports performance.

  View details for PubMedID 29561306

• Vitamin D and obstructive sleep apnea: a systematic review and meta-analysis SLEEP MEDICINE Neighbors, C. P., Noller, M. W., Song, S. A., Zaghi, S., Neighbors, J., Feldman, D., Kushida, C. A., Camacho, M. 2018; 43: 100–108

  Abstract

  Several studies have reported an association between 25-hydroxyvitamin D (25(OH)D) levels and obstructive sleep apnea (OSA) patients. The objective of the current study was to perform a systematic review and meta-analysis of these studies and report the findings.Authors searched for studies (through January 1, 2017) reporting 25(OH)D serum levels in OSA patients. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement was followed.Fourteen studies with 4937 subjects met inclusion criteria. There were 1513 controls and 3424 OSA patients. The 25(OH)D serum levels for controls and mild OSA patients were 28.16 ± 9.39 ng/mL (95% CI 27.64, 28.68) and 27.41 ± 9.42 ng/mL (95% CI 26.87, 27.95), respectively. The 25(OH)D serum levels for controls and moderate OSA patients were 28.21 ± 9.38 ng/mL (95% CI 27.70, 28.72) and 25.48 ± 10.34 ng/mL (95% CI 24.68, 26.28), respectively. The 25(OH)D serum levels for controls and severe OSA patients were 28.32 ± 9.65 ng/mL (95% CI 27.80, 28.84) and 21.88 ± 10.24 ng/mL (95% CI 21.08, 22.68), respectively. Using random effects modeling, the 25(OH)D serum levels were decreased for patients with OSA when compared to control groups (mean differences were -2.7% for mild OSA, -10.1% for moderate OSA and -17.4% for severe OSA).There was a relative insufficiency in serum 25(OH)D levels among OSA patients compared to control patients, which was incrementally exacerbated with increasing severity of sleep apnea. It was unclear whether a low 25(OH)D was a risk factor for OSA or if OSA was a risk factor for 25(OH)D. It was also possible that the association between 25(OH)D and OSA was due to body mass index (BMI).

  View details for PubMedID 29482804

• Williams syndrome transcription factor (WSTF) acts as an activator of estrogen receptor signaling in breast cancer cells and the effect can be abrogated by 1α,25-dihydroxyvitamin D3. The Journal of steroid biochemistry and molecular biology Lundqvist, J. n., Kirkegaard, T. n., Laenkholm, A. V., Duun-Henriksen, A. K., Bak, M. n., Feldman, D. n., Lykkesfeldt, A. E. 2018; 177: 171–78

  Abstract

  A majority of estrogen receptor positive (ER+) breast cancers are growth stimulated by estrogens. The ability to inhibit the ER signaling pathway is therefore of critical importance in the current treatment of ER+ breast cancers. It has been reported that 1α,25-dihydroxyvitamin D3down-regulates the expression of the CYP19A1 gene, encoding the aromatase enzyme that catalyzes the synthesis of estradiol. Furthermore, 1α,25-dihydroxyvitamin D3has also been reported to down-regulate the expression of estrogen receptor α (ERα), the main mediator of ER signaling. This study reports a novel transcription factor critical to 1α,25-dihydroxyvitamin D3-mediated regulation of estrogenic signaling in MCF-7 breast cancer cells. We have investigated the molecular mechanisms for the 1α,25-dihydroxyvitamin D3-mediated down-regulation of CYP19A1 and ERα gene expression in human MCF-7 breast cancer cells and found that Williams syndrome transcription factor (WSTF) plays a key role by binding to the promoters of CYP19A1 and ERα. Although sometimes reported as an inhibitor of gene expression, we found that WSTF acts as an activator of the promoter activity of both CYP19A1 and ERα. Silencing of WSTF by siRNA transfection resulted in decreased aromatase-dependent cell growth as well as decreased ER signaling in the cells. When cells were treated with 1α,25-dihydroxyvitamin D3, WSTF was dissociated from the promoters and the promoter activities of CYP19A1 and ERα were decreased. We have measured the expression of WSTF in ER-positive tumor-samples from breast cancer patients and found that WSTF is expressed in the majority of the investigated samples and that the expression is higher in cancer tissue than in normal tissue. However, we were not able to show any significant association between the WSTF expression in the tumor and the disease free and overall survival in this patient group who have received adjuvant tamoxifen treatment, nor between the WSTF expression and the expression of ERα, progesterone receptor or HER2. The major conclusions of this study are that WSTF acts as an activator of ER signaling in MCF-7 breast cancer cells, that this action can be inhibited by 1α,25-dihydroxyvitamin D3, and that the expression of WSTF is higher in breast cancer tissue than in normal tissue. WSTF may by a new target for treatment of estrogen-dependent breast cancer cell growth.

  View details for PubMedID 28610873

• Vitamin D supplementation decreases serum 27-hydroxycholesterol in a pilot breast cancer trial. Breast cancer research and treatment Going, C. C., Alexandrova, L. n., Lau, K. n., Yeh, C. Y., Feldman, D. n., Pitteri, S. J. 2017

  Abstract

  27-hydroxycholesterol (27HC), an endogenous selective estrogen receptor modulator (SERM), drives the growth of estrogen receptor-positive (ER+) breast cancer. 1,25-dihydroxyvitamin D (1,25(OH)2D), the active metabolite of vitamin D, is known to inhibit expression of CYP27B1, which is very similar in structure and function to CYP27A1, the synthesizing enzyme of 27HC. Therefore, we hypothesized that 1,25(OH)2D may also inhibit expression of CYP27A1, thereby reducing 27HC concentrations in the blood and tissues that express CYP27A1, including breast cancer tissue.27HC, 25-hydroxyvitamin D (25OHD), and 1,25(OH)2D were measured in sera from 29 breast cancer patients before and after supplementation with low-dose (400 IU/day) or high-dose (10,000 IU/day) vitamin D in the interval between biopsy and surgery.A significant increase (p = 4.3E-5) in 25OHD and a decrease (p = 1.7E-1) in 27HC was observed in high-dose versus low-dose vitamin D subjects. Excluding two statistical outliers, 25OHD and 27HC levels were inversely correlated (p = 7.0E-3).Vitamin D supplementation can decrease circulating 27HC of breast cancer patients, likely by CYP27A1 inhibition. This suggests a new and additional modality by which vitamin D can inhibit ER+ breast cancer growth, though a larger study is needed for verification.

  View details for PubMedID 29116467

• Identification of tumor-autonomous and indirect effects of vitamin D action that inhibit breast cancer growth and tumor progression. The Journal of steroid biochemistry and molecular biology Aggarwal, A. n., Feldman, D. n., Feldman, B. J. 2017

  Abstract

  Several epidemiological studies have found that low vitamin D levels are associated with worse prognosis and poorer outcomes in patients with breast cancer (BCa), although some studies have failed to find this association. In addition, prior research has found that BCa patients with vitamin D deficiency have a more aggressive molecular phenotype and worse prognostic biomarkers. As vitamin D deficiency is common in patients diagnosed with BCa, elucidating the cause of the association between poor outcomes and vitamin D deficiency promises to have a significant impact on improving care for patients with BCa including enabling the development of novel therapeutic approaches. Here we review our recent findings in this area, including our data revealing that reduction of the expression of the vitamin D receptor (Vdr) within BCa cells accelerates primary tumor growth and enables the development of metastases, demonstrating a tumor autonomous effect of vitamin D signaling to suppress BCa metastases. We believe that these findings are likely relevant to humans as we discovered evidence that a mechanism of VDR regulation identified in our mouse models is conserved in human BCa. In particular, we identified a negative correlation between serum 25(OH)D concentration and the level of expression of the tumor progression factor ID1 in primary tumors from patients with breast cancer.

  View details for PubMedID 28710021

• Vitamin D mitigates the adverse effects of obesity on breast cancer in mice ENDOCRINE-RELATED CANCER Swami, S., Krishnan, A. V., Williams, J., Aggarwal, A., Albertelli, M. A., Horst, R. L., Feldman, B. J., Feldman, D. 2016; 23 (4): 251-264

  Abstract

  Obesity is an established risk factor for postmenopausal breast cancer (BCa), insulin resistance and vitamin D deficiency, and all contribute to increased synthesis of mammary estrogens, the drivers of estrogen receptor-positive (ER+) BCa growth. Since both dietary vitamin D and calcitriol treatments inhibit breast estrogen synthesis and signaling, we hypothesized that vitamin D would be especially beneficial in mitigating the adverse effects of obesity on ER+ BCa. To assess whether obesity exerted adverse effects on BCa growth and whether vitamin D compounds could reduce these unfavorable effects, we employed a diet-induced obesity (DIO) model in ovariectomized C57BL/6 mice. Breast tumor cells originally from syngeneic Mmtv-wnt1 transgenic mice were then implanted into the mammary fat pads of lean and obese mice. DIO accelerated the initiation and progression of the mammary tumors. Treatments with either calcitriol or dietary vitamin D reduced the adverse effects of obesity causing a delay in tumor appearance and inhibiting continued tumor growth. Beneficial actions of treatments with vitamin D or calcitriol on BCa and surrounding adipose tissue included: repressed Er, aromatase and Cox-2 expression, decreased tumor derived estrogen and PGE2 and reduced expression of leptin receptors and increased adiponectin receptors. We demonstrate that vitamin D treatments decreased insulin resistance, reduced leptin and increased adiponectin signaling and also regulated the LKB1/ AMPK pathway contributing to an overall decrease in local estrogen synthesis in the obese mice. We conclude that calcitriol and dietary vitamin D, acting by multiple interrelated pathways, mitigate obesity enhanced BCa growth in a postmenopausal setting.

  View details for DOI 10.1530/ERC-15-0557

  View details for Web of Science ID 000377691700007

  View details for PubMedID 26817629

• Androgen-glucocorticoid interactions in the era of novel prostate cancer therapy. Nature reviews. Urology Narayanan, S., Srinivas, S., Feldman, D. 2016; 13 (1): 47-60

  Abstract

  Great strides have been made in the treatment of castration-resistant prostate cancer (CRPC) with the development of new antiandrogens (enzalutamide) and more potent androgen synthesis inhibitors (abiraterone) that have both improved patient outcomes. These new drugs have also helped unravel the complex biology of androgen-androgen receptor driven prostate cancer and brought into prominence various mechanisms triggering the development of drug resistance and tumour cell survival despite use of androgen deprivation therapy (ADT). The complex role of glucocorticoids in the treatment, management and progression of patients with CRPC is integral to these advances. Historically, glucocorticoid treatment has resulted in both subjective and objective responses in patients with advanced-stage prostate cancer. With the use of these new therapeutic agents, however, unexpected glucocorticoid-related mechanisms that can cause iatrogenic stimulation of prostate cancer growth have emerged, which might contribute to drug resistance and disease progression despite optimal ADT. For example, the upregulation of glucocorticoid receptors (GRs) during enzalutamide therapy results in glucocorticoid-GR-mediated regulation of androgen target genes, leading to escape from enzalutamide blockade. Thus, understanding the biological role of glucocorticoids in patients with prostate cancer is of major importance in the era of new and evolving antiandrogen therapies.

  View details for DOI 10.1038/nrurol.2015.254

  View details for PubMedID 26643568

• Low Circulating 25-Hydroxyvitamin D Concentrations Are Associated with Defects in Insulin Action and Insulin Secretion in Persons with Prediabetes JOURNAL OF NUTRITION Abbasi, F., Blasey, C., Feldman, D., Caulfield, M. P., Hantash, F. M., Reaven, G. M. 2015; 145 (4): 714-719

  Abstract

  Individuals with prediabetes mellitus (PreDM) and low circulating 25-hydroxyvitamin D [25(OH)D] are at increased risk of type 2 diabetes mellitus (T2DM).We aimed to determine whether low 25(OH)D concentrations are associated with defects in insulin action and insulin secretion in persons with PreDM.In this cross-sectional study, we stratified 488 nondiabetic subjects as having PreDM or normal fasting glucose (NFG) and a 25(OH)D concentration ≤20 ng/mL (deficient) or >20 ng/mL (sufficient). We determined insulin resistance by steady state plasma glucose (SSPG) concentration and homeostasis model assessment of insulin resistance (HOMA-IR) and insulin secretion by homeostasis model assessment of β-cell function (HOMA-β). We compared insulin resistance and secretion measures in PreDM and NFG groups; 25(OH)D-deficient and 25(OH)D-sufficient groups; and PreDM-deficient, PreDM-sufficient, NFG-deficient, and NFG-sufficient subgroups, adjusting for age, sex, race, body mass index, multivitamin use, and season.In the PreDM group, mean SSPG concentration and HOMA-IR were higher and mean HOMA-β was lower than in the NFG group (P < 0.001 for all comparisons). In the 25(OH)D-deficient group, mean SSPG concentration was higher (P < 0.001), but neither mean HOMA-IR nor HOMA-β was significantly different from that in the 25(OH)D-sufficient group. In the PreDM-deficient subgroup, mean (95% CI) SSPG concentration was higher (P < 0.01) than in the PreDM-sufficient, NFG-deficient, and NFG-sufficient subgroups [192 (177-207) mg/dL vs. 166 (155-177) mg/dL, 148 (138-159) mg/dL, and 136 (127-144) mg/dL, respectively]. Despite greater insulin resistance, mean HOMA-β was not significantly higher in the PreDM-deficient subgroup than in the PreDM-sufficient, NFG-deficient, and NFG-sufficient subgroups [98 (85-112) vs. 91 (82-101), 123 (112-136), and 115 (106-124), respectively].Subjects with PreDM and low circulating 25(OH)D concentrations are the subgroup of nondiabetic individuals who are the most insulin resistant and have impaired β-cell function, attributes that put them at enhanced risk of T2DM.

  View details for DOI 10.3945/jn.114.209171

  View details for Web of Science ID 000352180500007

  View details for PubMedID 25740907

  View details for PubMedCentralID PMC4381771

• Relationship among 25-hydroxyvitamin d concentrations, insulin action, and cardiovascular disease risk in patients with essential hypertension. American journal of hypertension Abbasi, F., Feldman, D., Caulfield, M. P., Hantash, F. M., Reaven, G. M. 2015; 28 (2): 266-272

  Abstract

  Although low plasma 25-hydroxyvitamin D (25(OH)D) concentrations have been shown to predict risk of hypertension and associated cardiovascular disease (CVD), vitamin D repletion has not consistently lowered blood pressure or decreased CVD. One possibility for this discrepancy is the presence of considerable metabolic heterogeneity in patients with hypertension. To evaluate this possibility, we quantified relationships among insulin resistance, 25(OH)D concentration, and CVD risk factor profile in patients with essential hypertension.Measurements were made of 25(OH)D concentrations, multiple CVD risk factors, and insulin resistance by the steady-state plasma glucose concentration during the insulin suppression test in 140 otherwise healthy patients with essential hypertension.As a group, the patients were overweight/obese and insulin resistant and had low 25(OH)D concentrations. The more insulin resistant the patients were, the worse the CVD risk profile was. In addition, the most insulin-resistant quartile had significantly lower 25(OH)D concentrations than the most insulin-sensitive quartile (20.3±1.4 vs. 25.8±1.4ng/ml; P = 0.005). In the entire group, 25(OH)D concentration significantly correlated with magnitude of insulin resistance (steady-state plasma glucose concentration; r = -0.20; P = 0.02).There was considerable metabolic heterogeneity and substantial difference in magnitude of conventional CVD risk factors in patients with similar degrees of blood pressure elevation. The most insulin-resistant quartile of subjects had the lowest 25(OH)D concentration and the most adverse CVD risk profile, and they may be the subset of patients with essential hypertension most likely to benefit from vitamin D repletion.

  View details for DOI 10.1093/ajh/hpu136

  View details for PubMedID 25138785

• Inhibition of Mouse Breast Tumor-Initiating Cells by Calcitriol and Dietary Vitamin D. Mol Cancer Therapeutics Jeong, Y., Swami, S., Krishnan, A. V., Williams, J. D., Martin, S., Horst, R. L., Albertelli, M. A., Feldman, B. J., Feldman, D., Diehn, M. 2015; 14 (8): 1951-1961

  View details for DOI 10.1158/1535-7163

• Global clinical response in Cushing's syndrome patients treated with mifepristone. Clinical endocrinology Katznelson, L., Loriaux, D. L., Feldman, D., Braunstein, G. D., Schteingart, D. E., Gross, C. 2014; 80 (4): 562-569

  Abstract

  Mifepristone, a glucocorticoid receptor antagonist, improves clinical status in patients with Cushing's syndrome (CS). We examined the pattern, reliability and correlates of global clinical response (GCR) assessments during a 6-month clinical trial of mifepristone in CS.Post hoc analysis of secondary end-point data from a 24-week multicentre, open-label trial of mifepristone (300-1200 mg daily) in CS. Intraclass correlation coefficient (ICC) was used to examine rater concordance, and drivers of clinical improvement were determined by multivariate regression analysis.Forty-six adult patients with refractory CS along with diabetes mellitus type 2 or impaired glucose tolerance, and/or a diagnosis of hypertension.Global clinical assessment made by three independent reviewers using a three-point ordinal scale (+1 = improvement; 0 = no change; -1 = worsening) based on eight broad clinical categories including glucose control, lipids, blood pressure, body composition, clinical appearance, strength, psychiatric/cognitive symptoms and quality of life at Weeks 6, 10, 16, and 24.Positive GCR increased progressively over time with 88% of patients having improved at Week 24 (P < 0·001). The full concordance among reviewers occurred in 76·6% of evaluations resulting in an ICC of 0·652 (P < 0·001). Changes in body weight (P < 0·0001), diastolic blood pressure (P < 0·0001), two-hour postoral glucose challenge glucose concentration (P = 0·0003), and Cushingoid appearance (P = 0·022) were strong correlates of GCR.Mifepristone treatment for CS results in progressive clinical improvement. Overall agreement among clinical reviewers was substantial and determinants of positive GCR included change in weight, blood pressure, glucose levels and appearance.

  View details for DOI 10.1111/cen.12332

  View details for PubMedID 24102404

• Vitamin D receptor mutations in patients with hereditary 1,25-dihydroxyvitamin D-resistant rickets MOLECULAR GENETICS AND METABOLISM Malloy, P. J., Tasic, V., Taha, D., Tutunculer, F., Ying, G. S., Yin, L. K., Wang, J., Feldman, D. 2014; 111 (1): 33-40

  Abstract

  Hereditary vitamin D resistant rickets (HVDRR), also known as vitamin D-dependent rickets type II, is an autosomal recessive disorder characterized by the early onset of rickets with hypocalcemia, secondary hyperparathyroidism and hypophosphatemia and is caused by mutations in the vitamin D receptor (VDR) gene. The human gene encoding the VDR is located on chromosome 12 and comprises eight coding exons and seven introns.We analyzed the VDR gene of 5 previously unreported patients, two from Singapore and one each from Macedonia (former Yugoslav Republic), Saudi Arabia and Turkey. Each patient had clinical and radiographic features of rickets, hypocalcemia, and the 4 cases that had the measurement showed elevated serum concentrations of 1,25-dihydroxyvitamin D (1,25(OH)2D). Mutations were re-created in the WT VDR cDNA and examined for 1,25(OH)2D3-mediated transactivation in COS-7 monkey kidney cells.Direct sequencing identified four novel mutations and two previously described mutations in the VDR gene. The novel mutations included a missense mutation in exon 3 causing the amino acid change C60W; a missense mutation in exon 4 causing the amino acid change D144N; a missense mutation in exon 7 causing the amino acid change N276Y; and a 2bp deletion in exon 3 5'-splice site (IVS3∆+4-5) leading to a premature stop.These 4 unique mutations add to the previous 45 mutations identified in the VDR gene in patients with HVDRR.

  View details for DOI 10.1016/j.ymgme.2013.10.014

  View details for Web of Science ID 000330158100005

  View details for PubMedID 24246681

• Equivalent anticancer activities of dietary vitamin D and calcitriol in an animal model of breast cancer: Importance of mammary CYP27B1 for treatment and prevention JOURNAL OF STEROID BIOCHEMISTRY AND MOLECULAR BIOLOGY Krishnan, A. V., Swami, S., Feldman, D. 2013; 136: 289-295

  Abstract

  Calcitriol [1,25(OH)2D3], the hormonally active form of vitamin D exerts anti-proliferative, pro-apoptotic, anti-inflammatory effects and other anticancer actions in breast cancer (BCa) cell cultures and animal models of BCa. Our research is focused on investigating the potential beneficial effects of dietary vitamin D3 compared to calcitriol and the underlying mechanisms in BCa treatment and chemoprevention. We recently found that dietary vitamin D3 exhibits significant tumor inhibitory effects in xenograft models of BCa that are equivalent to those elicited by the administration of the active hormone calcitriol. At the easily achievable dose tested in our studies, dietary vitamin D3 exhibited substantial tumor inhibitory activity and, unlike calcitriol, did not cause hypercalcemia demonstrating its relative safety. We found elevations in circulating calcitriol as well as increased CYP27B1 expression in the tumor and the intestine in tumor-bearing mice ingesting a vitamin D3-supplemented diet. We hypothesize that the elevation in circulating 25(OH)D induced by dietary vitamin D3 supplements stimulates local synthesis of calcitriol in the mammary tumor microenvironment and the ensuing paracrine/autocrine actions play a major role in the anticancer activity of dietary vitamin D3. Our findings suggest that the endocrine activity of calcitriol derived from tumor and other extra-renal sources such as the intestine, probably also plays a role in mediating the anticancer effects of dietary vitamin D3. Thus it appears that multiple sites of 1α-hydroxylation contribute to the anticancer effects of dietary vitamin D3. Our data strongly suggest that dietary vitamin D will be useful in the chemoprevention and treatment of BCa since it is a safe, economical and easily available nutritional agent that is equivalent to calcitriol in exerting anticancer effects, at least in mouse models. Furthermore, adequate vitamin D nutrition and avoidance of vitamin D deficiency appear to be important in reducing BCa risk. These findings warrant clinical trials in BCa patients and in women at high risk for BCa to evaluate the benefits of dietary vitamin D3 supplementation. This article is part of a Special Issue entitled 'Vitamin D Workshop'.

  View details for DOI 10.1016/j.jsbmb.2012.08.005

  View details for Web of Science ID 000321405800058

  View details for PubMedID 22939886

  View details for PubMedCentralID PMC3554854

• Enteral calcium infusion used successfully as treatment for a patient with hereditary vitamin D resistant rickets (HVDRR) without alopecia: A novel mutation GENE Huang, K., Malloy, P., Feldman, D., Pitukcheewanont, P. 2013; 512 (2): 554-559

  Abstract

  We report a novel mutation in a case of hereditary vitamin D resistant rickets (HVDRR) without alopecia and successful management of this condition with the intravenous formulation of calcium chloride delivered via gastric tube.A 22 month old male (length -3.4 SDS; weight -2.1 SDS) presented with failure to thrive, short stature, severe hypocalcemia and gross motor delay. He did not have alopecia. Initial blood tests and history were thought possibly to suggest vitamin D deficiency rickets: calcium 5.1mg/dL, (8.8-10.8); phosphorus 4.1mg/dL, (4.5-5.5); alkaline phosphatase 1481 U/L (80-220); intact PTH 537.1 pg/mL (10-71). Subsequently, vitamin D studies returned that were consistent with HVDRR: 25-hydroxyvitamin D 34 ng/mL (20-100); 1,25-dihydroxyvitamin D 507 pg/mL. This diagnosis was confirmed by DNA sequencing. His subsequent clinical course was complicated by the fact that IV calcium was not a viable option for this patient, and his calcium levels could not be well controlled on oral calcium citrate or calcium glubionate therapy. Eventually, we were able to maintain calcium levels above 8 mg/dL using the intravenous preparation of calcium chloride administered via gastric tube. GENETIC STUDIES: A unique homozygous T to C base substitution was found in exon 6 in the vitamin D receptor (VDR) gene. This mutation causes leucine to be converted to proline at position 227 in helix 3 in the VDR ligand binding domain (LBD). The mutation rendered the VDR non-functional, leading to HVDRR, with absence of alopecia.HVDRR should be considered in a patient with profound hypocalcemia which is refractory to conventional therapy of vitamin D deficiency rickets even without evidence of alopecia. We report the first case of HVDRR with a novel mutation in the LBD that was successfully treated with enteral treatment using a calcium chloride infusion.

  View details for DOI 10.1016/j.gene.2012.09.078

  View details for Web of Science ID 000313768900060

  View details for PubMedID 23026218

• Transrepression of the estrogen receptor promoter by calcitriol in human breast cancer cells via two negative vitamin D response elements. Endocrine-related cancer Swami, S., Krishnan, A. V., Peng, L., Lundqvist, J., Feldman, D. 2013; 20 (4): 565-577

  Abstract

  Calcitriol (1,25-dihydroxyvitamin D3), the hormonally active metabolite of vitamin D, exerts its anti-proliferative activity in breast cancer (BCa) cells by multiple mechanisms including the downregulation of the expression of estrogen receptor α (ER). We analyzed an ∼3.5 kb ER promoter sequence and demonstrated the presence of two potential negative vitamin D response elements (nVDREs), a newly identified putative nVDRE upstream at -2488 to -2473 bp (distal nVDRE) and a previously published sequence (proximal nVDRE) at -94 to -70 bp proximal to the P1 start site. Transactivation analysis using ER promoter deletion constructs and heterologous promoter-reporter constructs revealed that both nVDREs functioned to mediate calcitriol transrepression. In the electrophoretic mobility shift assay, the vitamin D receptor (VDR) showed strong binding to both nVDREs in the presence of calcitriol, and the chromatin immunoprecipitation assay demonstrated the recruitment of the VDR to the distal nVDRE site. Mutations in the 5' hexameric DNA sequence of the distal nVDRE resulted in the loss of calcitriol-mediated transrepression and the inhibition of protein-DNA complex formation, demonstrating the importance of these nucleotides in VDR DNA binding and transrepression. A putative nuclear factor-Y (NFY) binding site, identified within the distal nVDRE, led to the findings that NFY bound to the distal nVDRE site interfered with the binding of the VDR at the site and reduced calcitriol-mediated transrepression. In conclusion, the ER promoter region contains two negative VDREs that act in concert to bind to the VDR and both nVDREs are required for the maximal inhibition of ER expression by calcitriol. The suppression of ER expression and estrogen-mediated signaling by calcitriol in BCa cells suggests that vitamin D may be useful in the treatment of ER+ BCa.

  View details for DOI 10.1530/ERC-12-0281

  View details for PubMedID 23744764

• Combination of calcitriol and dietary soy exhibits enhanced anticancer activity and increased hypercalcemic toxicity in a mouse xenograft model of prostate cancer PROSTATE Wang, J. Y., Swami, S., Krishnan, A. V., Feldman, D. 2012; 72 (15): 1628-1637

  Abstract

  The potential role of vitamin D and soy in prostate cancer (PCa) prevention/treatment has gained much attention in recent years. In this study, we evaluated the anticancer activity of calcitriol, the active form of vitamin D, dietary soy, and their combinations in a mouse model of PCa.Athymic male nude mice bearing PC-3 human PCa xenografts received diets containing 10 or 20 kcal% soy, calcitriol injections, or a combination of dietary soy and calcitriol. Changes in tumor growth, serum levels of 1,25(OH)(2)D and calcium, and regulation of tumor gene expression were examined.The combination treatments resulted in substantially greater inhibition of tumor growth than either agent alone. Soy diets alone caused a modest elevation in serum 1,25(OH)(2)D, whereas the calcitriol-soy combinations led to substantially elevated serum 1,25(OH)(2) D, hypercalcemia, and in some cases lethal toxicity. The combinations enhanced calcitriol activity in regulating target gene expression, including greater up-regulation of anti-proliferative (p21, IGFBP-3) and pro-apoptotic (Bax) genes, increased inhibition of anti-apoptotic (Bcl-2) and cell cycle promoting (cyclin D1) genes, and suppression of prostaglandin (PG) synthesis and signaling (COX-2, 15-PGDH, PG receptors). Increases in serum calcium were accompanied by elevated expression of intestinal calcium absorption genes (TRPV6, calbindin-9k).Soy increases the bioavailability of endogenous and administered calcitriol, thereby enhancing its anticancer effects and risk of hypercalcemia. Since both agents are easily available as dietary supplements, the increased potential for hypercalcemic toxicity becomes an important factor when considering the combined use of vitamin D and soy in PCa therapy.

  View details for DOI 10.1002/pros.22516

  View details for Web of Science ID 000309405800004

  View details for PubMedID 22457201

  View details for PubMedCentralID PMC3389566

• The potential therapeutic benefits of vitamin D in the treatment of estrogen receptor positive breast cancer STEROIDS Krishnan, A. V., Swami, S., Feldman, D. 2012; 77 (11): 1107-1112

  Abstract

  Calcitriol (1,25-dihydroxyvitamin D(3)), the hormonally active form of vitamin D, inhibits the growth of many malignant cells including breast cancer (BCa) cells. The mechanisms of calcitriol anticancer actions include cell cycle arrest, stimulation of apoptosis and inhibition of invasion, metastasis and angiogenesis. In addition we have discovered new pathways of calcitriol action that are especially relevant in inhibiting the growth of estrogen receptor positive (ER+) BCa cells. Calcitriol suppresses COX-2 expression and increases that of 15-PGDH thereby reducing the levels of inflammatory prostaglandins (PGs). Our in vitro and in vivo studies show that calcitriol decreases the expression of aromatase, the enzyme that catalyzes estrogen synthesis selectively in BCa cells and in the mammary adipose tissue surrounding BCa, by a direct repression of aromatase transcription via promoter II as well as an indirect effect due to the reduction in the levels of PGs, which are major stimulator of aromatase transcription through promoter II. Calcitriol down-regulates the expression of ERα and thereby attenuates estrogen signaling in BCa cells including the proliferative stimulus provided by estrogens. Thus the inhibition of estrogen synthesis and signaling by calcitriol and its anti-inflammatory actions will play an important role in inhibiting ER+BCa. We hypothesize that dietary vitamin D would exhibit similar anticancer activity due to the presence of the enzyme 25-hydroxyvitamin D-1α-hydroxylase (CYP27B1) in breast cells ensuring conversion of circulating 25-hydroxyvitamin D to calcitriol locally within the breast micro-environment where it can act in a paracrine manner to inhibit BCa growth. Cell culture and in vivo data in mice strongly suggest that calcitriol and dietary vitamin D would play a beneficial role in the prevention and/or treatment of ER+BCa in women.

  View details for DOI 10.1016/j.steroids.2012.06.005

  View details for Web of Science ID 000309021700011

  View details for PubMedID 22801352

  View details for PubMedCentralID PMC3429709

• Successful long-term treatment of Cushing disease with mifepristone (RU486). Endocrine practice Basina, M., Liu, H., Hoffman, A. R., Feldman, D. 2012; 18 (5): e114-20

  Abstract

  We describe a girl with Cushing disease for whom surgery and radiation treatments failed and the subsequent clinical course with mifepristone therapy.We present the patient's clinical, biochemical, and imaging findings.A 16-year-old girl presented with classic Cushing disease. After transsphenoidal surgery, Cyberknife radiosurgery, ketoconazole, and metyrapone did not control her disease, and she was prescribed mifepristone, which was titrated to a maximal dosage of 1200 mg daily with subsequent symptom improvement. Mifepristone (RU486) is a high-affinity, nonselective antagonist of the glucocorticoid receptor. There is limited literature on its use as an off-label medication to treat refractory Cushing disease. Over her 8-year treatment with mifepristone, her therapy was complicated by hypertension and hypokalemia requiring spironolactone and potassium chloride. She received a 2-month drug holiday every 4 to 6 months to allow for withdrawal menstrual bleeding with medroxyprogesterone acetate. Urinary cortisol, serum cortisol, and corticotropin levels remained elevated during mifepristone drug holidays. While on mifepristone, her signs and symptoms of Cushing disease resolved. Repeated magnetic resonance imaging demonstrated stable appearance of the residual pituitary mass. Bilateral adrenalectomy was performed, and mifepristone was discontinued after 95 months of medical therapy.We describe the longest duration of mifepristone therapy thus reported for the treatment of refractory Cushing disease. Mifepristone effectively controlled all signs and symptoms of hypercortisolism. Menstruating women who take the drug on a long-term basis should receive periodic drug holidays to allow for menses. The lack of reliable serum biomarkers to monitor the success of mifepristone therapy requires careful clinical judgment and may make its use difficult in Cushing disease.

  View details for DOI 10.4158/EP11391.CR

  View details for PubMedID 22441000

• Dietary Vitamin D-3 and 1,25-Dihydroxyvitamin D-3 (Calcitriol) Exhibit Equivalent Anticancer Activity in Mouse Xenograft Models of Breast and Prostate Cancer ENDOCRINOLOGY Swami, S., Krishnan, A. V., Wang, J. Y., Jensen, K., Horst, R., Albertelli, M. A., Feldman, D. 2012; 153 (6): 2576-2587

  Abstract

  1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3) or calcitriol], the hormonally active vitamin D metabolite, exhibits anticancer actions in models of breast cancer and prostate cancer. Because CYP27B1 (1α-hydroxylase), the enzyme catalyzing 1,25(OH)(2)D(3) formation in the kidney, is also expressed in extrarenal tissues, we hypothesize that dietary vitamin D(3) will be converted to 25(OH)D(3) in the body and then to 1,25(OH)(2)D(3) locally in the cancer microenvironment in which it will exert autocrine/paracrine anticancer actions. Immunocompromised mice bearing MCF-7 breast cancer xenografts showed significant tumor shrinkage (>50%) after ingestion of a vitamin D(3)-supplemented diet (5000 IU/kg) compared with a control diet (1000 IU/kg). Dietary vitamin D(3) inhibition of tumor growth was equivalent to administered calcitriol (0.025, 0.05, or 0.1 μg/mouse, three times a week). Both treatments equivalently inhibited PC-3 prostate cancer xenograft growth but to a lesser extent than the MCF-7 tumors. Calcitriol at 0.05 μg and 0.1 μg caused modest but statistically significant increases in serum calcium levels indicating that the dietary vitamin D(3) comparison was to a maximally safe calcitriol dose. Dietary vitamin D(3) did not increase serum calcium, demonstrating its safety at the concentration tested. The vitamin D(3) diet raised circulating 1,25 dihydroxyvitamin D levels and did not alter CYP27B1 mRNA in the kidney but increased it in the tumors, suggesting that extrarenal sources including the tumors contributed to the elevated circulating 1,25 dihydroxyvitamin D(3). Both calcitriol and dietary vitamin D(3) were equipotent in suppressing estrogen synthesis and signaling and other proinflammatory and growth signaling pathways. These preclinical data demonstrate the potential utility of dietary vitamin D(3) supplementation in cancer prevention and therapy.

  View details for DOI 10.1210/en.2011-1600

  View details for Web of Science ID 000304370700010

  View details for PubMedID 22454149

  View details for PubMedCentralID PMC3359605

• Relations between obesity, insulin resistance, and 25-hydroxyvitamin D AMERICAN JOURNAL OF CLINICAL NUTRITION Lamendola, C. A., Ariel, D., Feldman, D., Reaven, G. M. 2012; 95 (5): 1055-1059

  Abstract

  Although low circulating 25-hydroxyvitamin D [25(OH)D] concentrations have been associated with insulin resistance and obesity, the relations between these 3 variables have not been completely resolved.The objective was to compare circulating 25(OH)D concentrations in apparently healthy individuals who were matched for degree of obesity or insulin sensitivity.This was a case-control study in which 78 apparently healthy individuals were classified as being normal weight (NW) or obese (OB) on the basis of their BMI and as being insulin sensitive (IS) or insulin resistant (IR) on the basis of their steady state plasma glucose (SSPG) concentration during the insulin suppression test.Groups did not differ in terms of age, sex distribution, race, or mean (± SD) plasma 25(OH)D concentration. Values for 25(OH)D were 32 ± 10, 30 ± 10, and 28 ± 8 ng/mL in NW-IS, OB-IS, and OB-IR groups, respectively. These concentrations were essentially identical when comparing IR with IS subjects matched for BMI or when comparing OB with NW subjects matched for SSPG. Concentrations of 25(OH)D ≤ 30 ng/mL were somewhat more common in OB subjects than in NW subjects (54% compared with 35%), but SSPG concentrations were not different within either the IR or IS groups when subgroups with 25(OH)D concentrations ≤ 30 or > 30 ng/mL were compared.In 78 individuals, 47% of whom were vitamin D deficient or insufficient (≤ 30 ng/mL), 25(OH)D concentrations did not vary with differences in insulin sensitivity (SSPG concentration) when matched for BMI (OB-IR compared with OB-IS). Similarly, when matched for SSPG concentrations, plasma 25(OH)D concentrations were not different in NW or OB individuals (NW-IS compared with OB-IS).

  View details for DOI 10.3945/ajcn.111.032060

  View details for Web of Science ID 000303140700010

  View details for PubMedID 22440850

  View details for PubMedCentralID PMC3325832

• The Role of the Vitamin D Receptor and ERp57 in Photoprotection by 1 alpha,25-Dihydroxyvitamin D-3 MOLECULAR ENDOCRINOLOGY Sequeira, V. B., Rybchyn, M. S., Tongkao-on, W., Gordon-Thomson, C., Malloy, P. J., Nemere, I., Norman, A. W., Reeve, V. E., Halliday, G. M., Feldman, D., Mason, R. S. 2012; 26 (4): 574-582

  Abstract

  UV radiation (UVR) is essential for formation of vitamin D(3), which can be hydroxylated locally in the skin to 1α,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)]. Recent studies implicate 1,25-(OH)(2)D(3) in reduction of UVR-induced DNA damage, particularly thymine dimers. There is evidence that photoprotection occurs through the steroid nongenomic pathway for 1,25-(OH)(2)D(3) action. In the current study, we tested the involvement of the classical vitamin D receptor (VDR) and the endoplasmic reticulum stress protein 57 (ERp57), in the mechanisms of photoprotection. The protective effects of 1,25-(OH)(2)D(3) against thymine dimers were abolished in fibroblasts from patients with hereditary vitamin D-resistant rickets that expressed no VDR protein, indicating that the VDR is essential for photoprotection. Photoprotection remained in hereditary vitamin D-resistant rickets fibroblasts expressing a VDR with a defective DNA-binding domain or a mutation in helix H1 of the classical ligand-binding domain, both defects resulting in a failure to mediate genomic responses, implicating nongenomic responses for photoprotection. Ab099, a neutralizing antibody to ERp57, and ERp57 small interfering RNA completely blocked protection against thymine dimers in normal fibroblasts. Co-IP studies showed that the VDR and ERp57 interact in nonnuclear extracts of fibroblasts. 1,25-(OH)(2)D(3) up-regulated expression of the tumor suppressor p53 in normal fibroblasts. This up-regulation of p53, however, was observed in all mutant fibroblasts, including those with no VDR, and with Ab099; therefore, VDR and ERp57 are not essential for p53 regulation. The data implicate the VDR and ERp57 as critical components for actions of 1,25-(OH)(2)D(3) against DNA damage, but the VDR does not require normal DNA binding or classical ligand binding to mediate photoprotection.

  View details for DOI 10.1210/me.2011-1161

  View details for Web of Science ID 000302139800005

  View details for PubMedID 22322599

  View details for PubMedCentralID PMC3327356

• Genetic Disorders and Defects in Vitamin D Action RHEUMATIC DISEASE CLINICS OF NORTH AMERICA Malloy, P. J., Feldman, D. 2012; 38 (1): 93-?

  Abstract

  The biochemical and genetic analysis of the VDR in patients with HVDRR has yielded important insights into the structure and function of the receptor in mediating 1,25(OH)2D3 action. Similarly, study of children affected by HVDRR continues to provide a more complete understanding of the biologic role of 1,25(OH)2D3 in vivo. A concerted investigative approach to HVDRR at the clinical, cellular, and molecular levels has proved valuable in gaining knowledge of the functions of the domains of the VDR and elucidating the detailed mechanism of action of 1,25(OH)2D3. These studies have been essential to promote the well-being of the families with HVDRR and in improving the diagnostic and clinical management of this rare genetic disease.

  View details for DOI 10.1016/j.rdc.2012.03.009

  View details for Web of Science ID 000304575500008

  View details for PubMedID 22525845

• The Role of Vitamin D in Cancer Prevention and Treatment RHEUMATIC DISEASE CLINICS OF NORTH AMERICA Krishnan, A. V., Trump, D. L., Johnson, C. S., Feldman, D. 2012; 38 (1): 161-?

  Abstract

  Considerable data described in the first part of this review suggest that there is a role for vitamin D in cancer therapy and prevention. Although the preclinical data are persuasive and the epidemiologic data intriguing, no well-designed clinical trial of optimal administration of vitamin D as a cancer therapy has ever been conducted. Had there been the opportunity and insight to develop calcitriol as any other cancer drug, the following studies would have been completed: 1. Definition of the MTD. 2. Definition of a phase II dose, as a single agent and in combination with cytotoxic agents. 3. Studies to define a biologically optimal dose. 4. Phase II (probably randomized phase II) studies of calcitriol alone and chemotherapy ± calcitriol. 5. Then, randomized phase III trials would be conducted and designed such that the only variable was the administration of calcitriol. Prerequisites 1 to 5 have not been completed for calcitriol. Preclinical data provide considerable rationale for continued development of vitamin D analogue-based cancer therapies. However, design of future studies should be informed by good clinical trials design principles and the mistakes of the past not repeated. Such studies may finally provide compelling data to prove whether or not there is a role for vitamin D analogues in cancer therapy.

  View details for DOI 10.1016/j.rdc.2012.03.014

  View details for Web of Science ID 000304575500013

  View details for PubMedID 22525850

• Vitamin D metabolism and action in the prostate: Implications for health and disease MOLECULAR AND CELLULAR ENDOCRINOLOGY Swami, S., Krishnan, A. V., Feldman, D. 2011; 347 (1-2): 61-69

  Abstract

  Prostate cancer (PCa) is the second most common cancer in men worldwide. Epidemiological, molecular, and cellular studies have implicated vitamin D deficiency as a risk factor for the development and/or progression of PCa. Studies using cell culture systems and animal models suggest that vitamin D acts to reduce the growth of PCa through regulation of cellular proliferation and differentiation. However, although preclinical studies provide a strong indication for anti-cancer activity, proof of therapeutic benefits in men is still lacking. The anti-proliferative and pro-differentiating properties of vitamin D have been attributed to calcitriol [1,25(OH)(2)D(3)], the hormonally active form of vitamin D, acting through the vitamin D receptor (VDR). Metabolism of vitamin D in target tissues is mediated by two key enzymes: 1α-hydroxylase (CYP27B1), which catalyzes the synthesis of calcitriol from 25(OH)D and 24-hydroxylase (CYP24), which catalyzes the initial step in the conversion of calcitriol to less active metabolites. Many factors affect the balance of calcitriol synthesis and catabolism and several maneuvers, like combination therapy of calcitriol with other drugs, have been explored to treat PCa and reduce its risk. The current paper is an overview addressing some of the key factors that influence the biological actions of vitamin D and its metabolites in the treatment and/or prevention of PCa.

  View details for DOI 10.1016/j.mce.2011.05.010

  View details for Web of Science ID 000297193000010

  View details for PubMedID 21664249

  View details for PubMedCentralID PMC3189327

• The role of vitamin D receptor mutations in the development of alopecia MOLECULAR AND CELLULAR ENDOCRINOLOGY Malloy, P. J., Feldman, D. 2011; 347 (1-2): 90-96

  Abstract

  Hereditary Vitamin D Resistant Rickets (HVDRR) is a rare disease caused by mutations in the vitamin D receptor (VDR). The consequence of defective VDR is the inability to absorb calcium normally in the intestine. This leads to a constellation of metabolic abnormalities including hypocalcemia, secondary hyperparathyroidism and hypophosphatemia that cause the development of rickets at an early age in affected children. An interesting additional abnormality is the presence of alopecia in some children depending on the nature of the VDR mutation. The data indicate that VDR mutations that cause defects in DNA binding, RXR heterodimerization or absence of the VDR cause alopecia while mutations that alter VDR affinity for 1,25(OH)(2)D(3) or disrupt coactivator interactions do not cause alopecia. The cumulative findings indicate that hair follicle cycling is dependent on unliganded actions of the VDR. Further research is ongoing to elucidate the role of the VDR in hair growth and differentiation.

  View details for DOI 10.1016/j.mce.2011.05.045

  View details for Web of Science ID 000297193000013

  View details for PubMedID 21693169

  View details for PubMedCentralID PMC3196847

• Hereditary Vitamin D-Resistant Rickets (HVDRR) Owing to a Heterozygous Mutation in the Vitamin D Receptor JOURNAL OF BONE AND MINERAL RESEARCH Malloy, P. J., Zhou, Y., Wang, J., Hiort, O., Feldman, D. 2011; 26 (11): 2710-2718

  Abstract

  Hereditary vitamin D-resistant rickets (HVDRR) is a rare autosomal recessive disease caused by mutations in the vitamin D receptor (VDR). Patients exhibit severe rickets and hypocalcemia. Heterozygous parents and siblings appear normal and exhibit no symptoms of the disease. We analyzed the VDR gene of a young girl who exhibited the clinical features of HVDRR without alopecia. The patient had clinical and radiographic features of rickets, hypocalcemia, and elevated serum concentrations of 1,25-dihydroxyvitamin D [1,25(OH)(2)D]. A single heterozygous missense mutation was found in the VDR gene that substituted glutamic acid with alanine at amino acid 420 (E420A). Sequencing of the girl's VDR cDNAs showed that the f/M1 allele contained the E420A mutation, whereas the F/M4 allele was completely normal. The girl's father, who was also heterozygous for the E420A mutation on the f/M1 allele, exhibited minor symptoms of vitamin D resistance. In contrast, the mother had no signs of the disease and had no mutations in her VDR gene. Both the girl and the father's skin fibroblasts showed resistance to 1,25(OH)(2)D(3) by their severely reduced induction of CYP24A1 gene expression. In transactivation assays, the E420A mutant VDR showed dominant-negative activity towards the wild-type VDR. This is the first report that we are aware of describing a patient with HVDRR caused by a single heterozygous missense mutation in the VDR gene. The E420A mutant appears to act in a dominant-negative fashion, silencing the wild-type VDR and resulting in an attenuated response to 1,25(OH)(2)D(3).

  View details for DOI 10.1002/jbmr.484

  View details for Web of Science ID 000296391600015

  View details for PubMedID 21812032

• Report of two unrelated patients with hereditary vitamin D resistant rickets due to the same novel mutation in the vitamin D receptor JOURNAL OF PEDIATRIC ENDOCRINOLOGY & METABOLISM Aljubeh, J. M., Wang, J., Al-Remeithi, S. S., Malloy, P. J., Feldman, D. 2011; 24 (9-10): 793-799

  Abstract

  Two unrelated patients found to have hereditary vitamin D resistant rickets (HVDRR) were admitted to our hospital.This article describes the diagnosis, management and molecular basis for their disease.Both patients had severe growth and motor developmental retardation, rickets with chest deformities and pulmonary abnormalities, but no alopecia. Both had hypocalcemia, secondary hyperparathyroidism and susceptibility to pulmonary infections. In both cases, good response with normalization of abnormal biochemistries and healing of rickets was achieved with IV calcium infusion. Subsequently, improvement was maintained with oral calcium. Both children harbored the same unique missense mutation in the vitamin D receptor (VDR) gene that substituted arginine with histidine at amino acid 274 (R274H) in the VDR ligand-binding domain (LBD). R274 is a contact point for the 1alpha-hydroxyl group of 1,25(OH)2D3, the active ligand for the VDR. Functional analyses of the R274H mutation revealed a 100-fold decrease in activity compared to wild-type VDR.We describe a novel missense mutation at R274H in the VDR gene that resulted in the HVDRR syndrome in two unrelated children. Vigorous treatment using IV calcium to normalize their hypocalcemia achieved dramatic improvement in these complex and severely ill patients.

  View details for DOI 10.1515/JPEM.2011.341

  View details for Web of Science ID 000297470000035

  View details for PubMedID 22145479

• The anti-cancer and anti-inflammatory actions of 1,25(OH)(2)D-3 BEST PRACTICE & RESEARCH CLINICAL ENDOCRINOLOGY & METABOLISM Vanoirbeek, E., Krishnan, A., Eelen, G., Verlinden, L., Bouillon, R., Feldman, D., Verstuyf, A. 2011; 25 (4): 593-604

  Abstract

  Various epidemiological studies have shown an aetiological link between vitamin D deficiency and cancer incidence. The active metabolite of vitamin D, 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], has potent anti-cancer activities both in vitro and in vivo. These anti-cancer effects are attained by regulating the transcription of numerous genes that are involved in different pathways to reduce tumorigenesis and are dependent on the cancer cell type. Besides reducing cell growth and inducing apoptosis, 1,25(OH)₂D₃ also inhibits angiogenesis and metastasis. Moreover, its potency to inhibit inflammation also contributes to its anti-tumoral activity. Here, we report the different ways in which 1,25(OH)₂D₃ interferes with the malignant processes that are activated in cancer cells.

  View details for DOI 10.1016/j.beem.2011.05.001

  View details for Web of Science ID 000295070000007

  View details for PubMedID 21872801

  View details for PubMedCentralID PMC3164534

• Calcium Plus Vitamin D Supplementation and the Risk of Nonmelanoma and Melanoma Skin Cancer: Post Hoc Analyses of the Women's Health Initiative Randomized Controlled Trial JOURNAL OF CLINICAL ONCOLOGY Tang, J. Y., Fu, T., Leblanc, E., Manson, J. E., Feldman, D., Linos, E., Vitolins, M. Z., Zeitouni, N. C., Larson, J., Stefanick, M. L. 2011; 29 (22): 3078-3084

  Abstract

  In light of inverse relationships reported in observational studies of vitamin D intake and serum 25-hydroxyvitamin D levels with risk of nonmelanoma skin cancer (NMSC) and melanoma, we evaluated the effects of vitamin D combined with calcium supplementation on skin cancer in a randomized placebo-controlled trial.Postmenopausal women age 50 to 79 years (N = 36,282) enrolled onto the Women's Health Initiative (WHI) calcium/vitamin D clinical trial were randomly assigned to receive 1,000 mg of elemental calcium plus 400 IU of vitamin D3 (CaD) daily or placebo for a mean follow-up period of 7.0 years. NMSC and melanoma skin cancers were ascertained by annual self-report; melanoma skin cancers underwent physician adjudication.Neither incident NMSC nor melanoma rates differed between treatment (hazard ratio [HR], 1.02; 95% CI, 0.95 to 1.07) and placebo groups (HR, 0.86; 95% CI, 0.64 to 1.16). In subgroup analyses, women with history of NMSC assigned to CaD had a reduced risk of melanoma versus those receiving placebo (HR, 0.43; 95% CI, 0.21 to 0.90; P(interaction) = .038), which was not observed in women without history of NMSC.Vitamin D supplementation at a relatively low dose plus calcium did not reduce the overall incidence of NMSC or melanoma. However, in women with history of NMSC, CaD supplementation reduced melanoma risk, suggesting a potential role for calcium and vitamin D supplements in this high-risk group. Results from this post hoc subgroup analysis should be interpreted with caution but warrant additional investigation.

  View details for DOI 10.1200/JCO.2011.34.5967

  View details for Web of Science ID 000293222200029

  View details for PubMedID 21709199

  View details for PubMedCentralID PMC3157967

• Synthesis and Biological Evaluation of 1 alpha,25-Dihydroxyvitamin D-3 Analogues Hydroxymethylated at C-26 JOURNAL OF MEDICINAL CHEMISTRY Regueira, M. A., Samanta, S., Malloy, P. J., Ordonez-Moran, P., Resende, D., Sussman, F., Munoz, A., Mourino, A., Feldman, D., Torneiro, M. 2011; 54 (11): 3950-3962

  Abstract

  We designed by docking and synthesized two novel analogues of 1α,25-dihydroxyvitamin D(3) hydroxymethylated at C-26 (2 and 3). The syntheses were carried out by the convergent Wittig-Horner approach via epoxide 12a as a common key intermediate. The antiproliferative and transactivation potency of the compounds was evaluated in colon and breast cancer cell lines. The analogues showed a similar but reduced activity compared to 1,25(OH)(2)D(3). Analogue 3 was more potent than analogue 2, and in some assays it exhibited potency similar to that of the natural ligand.

  View details for DOI 10.1021/jm200276y

  View details for Web of Science ID 000291082500021

  View details for PubMedID 21524075

• Inhibitory effects of calcitriol on the growth of MCF-7 breast cancer xenografts in nude mice: selective modulation of aromatase expression in vivo. Hormones & cancer Swami, S., Krishnan, A. V., Wang, J. Y., Jensen, K., Peng, L., Albertelli, M. A., Feldman, D. 2011; 2 (3): 190-202

  Abstract

  Calcitriol (1,25-dihydroxyvitamin D(3)), the hormonally active metabolite of vitamin D, exerts many anticancer effects in breast cancer (BCa) cells. We have previously shown using cell culture models that calcitriol acts as a selective aromatase modulator (SAM) and inhibits estrogen synthesis and signaling in BCa cells. We have now examined calcitriol effects in vivo on aromatase expression, estrogen signaling, and tumor growth when used alone and in combination with aromatase inhibitors (AIs). In immunocompromised mice bearing MCF-7 xenografts, increasing doses of calcitriol exhibited significant tumor inhibitory effects (~50% to 70% decrease in tumor volume). At the suboptimal doses tested, anastrozole and letrozole also caused significant tumor shrinkage when used individually. Although the combinations of calcitriol and the AIs caused a statistically significant increase in tumor inhibition in comparison to the single agents, the cooperative interaction between these agents appeared to be minimal at the doses tested. Calcitriol decreased aromatase expression in the xenograft tumors. Importantly, calcitriol also acted as a SAM in the mouse, decreasing aromatase expression in the mammary adipose tissue, while increasing it in bone marrow cells and not altering it in the ovaries and uteri. As a result, calcitriol significantly reduced estrogen levels in the xenograft tumors and surrounding breast adipose tissue. In addition, calcitriol inhibited estrogen signaling by decreasing tumor ERα levels. Changes in tumor gene expression revealed the suppressive effects of calcitriol on inflammatory and growth signaling pathways and demonstrated cooperative interactions between calcitriol and AIs to modulate gene expression. We hypothesize that cumulatively these calcitriol actions would contribute to a beneficial effect when calcitriol is combined with an AI in the treatment of BCa.

  View details for DOI 10.1007/s12672-011-0073-7

  View details for PubMedID 21686077

  View details for PubMedCentralID PMC3114631

• CALCITRIOL ACTIONS IN BREAST CANCER Swami, S., Krishnan, A., Feldman, D. INT INST ANTICANCER RESEARCH. 2011: 1493–93

  View details for Web of Science ID 000290292000065

• Two New Unrelated Cases of Hereditary 1,25-Dihydroxyvitamin D-Resistant Rickets with Alopecia Resulting from the Same Novel Nonsense Mutation in the Vitamin D Receptor Gene JOURNAL OF PEDIATRIC ENDOCRINOLOGY & METABOLISM Forghani, N., Lum, C., Krishnan, S., Wang, J., Wilson, D. M., Blackett, P. R., Malloy, P. J., Feldman, D. 2010; 23 (8): 843-850

  Abstract

  1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) an important regulator of bone homeostasis, mediates its actions by binding to the vitamin D receptor (VDR), a nuclear transcription factor. Mutations in the VDR cause the rare genetic disease hereditary vitamin D resistant rickets (HVDRR). In this study, we examined two unrelated young female patients who exhibited severe early onset rickets, hypocalcemia, and hypophosphatemia. Both patients had partial alopecia but with different unusual patterns of scant hair. Sequencing of the VDR gene showed that both patients harbored the same unique nonsense mutation that resulted in a premature stop codon (R50X). Skin fibroblasts from patient #1 were devoid of VDR protein and 1,25(OH)2D3 treatment of these cells failed to induce CYP24A1 gene expression, a marker of 1,25(OH)2D3 action. In conclusion, we identified a novel nonsense mutation in the VDR gene in two patients with HVDRR and alopecia. The mutation truncates the VDR protein and causes 1,25(OH)2D3 resistance.

  View details for PubMedID 21073129

• Vitamin D and breast cancer: Inhibition of estrogen synthesis and signaling 14th Workshop on Vitamin D Krishnan, A. V., Swami, S., Feldman, D. PERGAMON-ELSEVIER SCIENCE LTD. 2010: 343–48

  Abstract

  Calcitriol (1,25-dihydroxyvitamin D3), the hormonally active metabolite of vitamin D, inhibits the growth and induces the differentiation of many malignant cells including breast cancer (BCa) cells. Calcitriol exerts its anti-proliferative activity in BCa cells by inducing cell cycle arrest and stimulating apoptosis. Calcitriol also inhibits invasion, metastasis and tumor angiogenesis in experimental models of BCa. Our recent studies show additional newly discovered pathways of calcitriol action to inhibit the growth of BCa cells. Calcitriol suppresses COX-2 expression and increases that of 15-PGDH thereby reducing the levels and biological activity of prostaglandins (PGs). Calcitriol decreases the expression of aromatase, the enzyme that catalyzes estrogen synthesis selectively in BCa cells and the breast adipose tissue surrounding BCa, by a direct repression of aromatase transcription via promoter II as well as an indirect effect due to the reduction in the levels and biological activity of PGE2, which is a major stimulator of aromatase transcription through promoter II in BCa. Calcitriol down-regulates the expression of estrogen receptor alpha and thereby attenuates estrogen signaling in BCa cells including the proliferative stimulus provided by estrogens. We hypothesize that the inhibition of estrogen synthesis and signaling by calcitriol and its anti-inflammatory actions will play an important role in the use of calcitriol for the prevention and/or treatment of BCa.

  View details for DOI 10.1016/j.jsbmb.2010.02.009

  View details for Web of Science ID 000280600200077

  View details for PubMedID 20156557

• Photoprotection by 1 alpha,25-dihydroxyvitamin D and analogs: Further studies on mechanisms and implications for UV-damage 14th Workshop on Vitamin D Mason, R. S., Sequeira, V. B., Dixon, K. M., Gordon-Thomson, C., Pobre, K., Dilley, A., Mizwicki, M. T., Norman, A. W., Feldman, D., Halliday, G. M., Reeve, V. E. PERGAMON-ELSEVIER SCIENCE LTD. 2010: 164–68

  Abstract

  Ultraviolet (UV) irradiation causes DNA damage in skin cells, immunosuppression and photocarcinogenesis. 1alpha,25-dihydroxyvitamin D3 (1,25D) reduces UV-induced DNA damage in the form of cyclobutane pyrimidine dimers (CPD) in human keratinocytes in culture and in mouse and human skin. UV-induced immunosuppression is also reduced in mice by 1,25D, in part due to the reduction in CPD and a reduction in interleukin (IL-6. The cis-locked analog, 1alpha,25-dihydroxylumisterol3 (JN), which has almost no transactivating activity, reduces UV-induced DNA damage, apoptosis and immunosuppression with similar potency to 1,25D, consistent with a non-genomic signalling mechanism. The mechanism of the reduction in DNA damage in the form of CPD is unclear. 1,25D doubles nuclear expression of p53 compared to UV alone, which suggests that 1,25D facilitates DNA repair. Yet expression of a key DNA repair gene, XPG is not affected by 1,25D. Chemical production of CPD has been described. Incubation of keratinocytes with a nitric oxide donor, SNP, induces CPD in the dark. We previously reported that 1,25D reduced UV-induced nitrite in keratinocytes, similar to aminoguanidine, an inhibitor of nitric oxide synthase. A reduction in reactive nitrogen species has been shown to facilitate DNA repair, but in view of these findings may also reduce CPD formation via a novel mechanism.

  View details for DOI 10.1016/j.jsbmb.2010.03.082

  View details for Web of Science ID 000280600200035

  View details for PubMedID 20399269

• The Role of Vitamin D in Cancer Prevention and Treatment ENDOCRINOLOGY AND METABOLISM CLINICS OF NORTH AMERICA Krishnan, A. V., Trump, D. L., Johnson, C. S., Feldman, D. 2010; 39 (2): 401-?

  Abstract

  Calcitriol (1,25-dihydroxyvitamin D(3)), the hormonally active form of vitamin D, exerts growth inhibitory and prodifferentiating effects on many malignant cells and retards tumor growth in animal models. Calcitriol is being evaluated as an anticancer agent in several human cancers. The mechanisms underlying the anticancer effects of calcitriol include inhibition of cell proliferation, stimulation of apoptosis, suppression of inflammation, and inhibition of tumor angiogenesis, invasion, and metastasis. This review discusses some of the molecular pathways mediating these anticancer actions of calcitriol and the preclinical data in cell culture and animal models. The clinical trials evaluating the use of calcitriol and its analogues in the treatment of patients with cancer are described. The reasons for the lack of impressive beneficial effects in clinical trials compared with the substantial efficacy seen in preclinical models are discussed.

  View details for DOI 10.1016/j.ecl.2010.02.011

  View details for Web of Science ID 000279584200014

  View details for PubMedID 20511060

• Genetic Disorders and Defects in Vitamin D Action ENDOCRINOLOGY AND METABOLISM CLINICS OF NORTH AMERICA Malloy, P. J., Feldman, D. 2010; 39 (2): 333-?

  Abstract

  Two rare genetic diseases can cause rickets in children. The critical enzyme to synthesize calcitriol from 25-hydroxyvitamin D, the circulating hormone precursor, is 25-hydroxyvitamin D-1alpha-hydroxylase (1alpha-hydroxylase). When this enzyme is defective and calcitriol can no longer be synthesized, the disease 1alpha-hydroxylase deficiency develops. The disease is also known as vitamin D-dependent rickets type 1 or pseudovitamin D deficiency rickets. When the VDR is defective, the disease hereditary vitamin D-resistant rickets, also known as vitamin D-dependent rickets type 2, develops. Both diseases are rare autosomal recessive disorders characterized by hypocalcemia, secondary hyperparathyroidism, and early onset severe rickets. In this article, these 2 genetic childhood diseases, which present similarly with hypocalcemia and rickets in infancy, are discussed and compared.

  View details for DOI 10.1016/j.ecl.2010.02.004

  View details for Web of Science ID 000279584200009

  View details for PubMedID 20511055

  View details for PubMedCentralID PMC2879401

• The role of vitamin D and SLCO1B1*5 gene polymorphism in statin-associated myalgias. Dermato-endocrinology Linde, R., Peng, L., Desai, M., Feldman, D. 2010; 2 (2): 77-84

  Abstract

  Myalgias are the most common side effect of statin use and the commonest cause for discontinuing therapy. Vitamin D has known physiologic functions in muscle and vitamin D deficiency is known to cause myalgias, with its correction leading to disappearance of muscle symptoms. The 521T>C SLCO1B1*5 gene polymorphism decreasing function in the gene coding for a liver anion transporter that is responsible for statin uptake has been found to explain the majority of statin-associated muscle symptoms. Patients with statin-associated myalgias have been reported to improve with vitamin D supplementation. We therefore investigated (i) whether repletion of vitamin D in deficient patients with myalgias could lead to tolerance for subsequent statin therapy and (ii) whether vitamin D status modifies the effect of the SLCO1B1*5 genotype on myalgia risk. Using a retrospective cohort of 64 patients in whom 25-hydroxyvitamin D [25(OH)D] had been measured for any reason while on statin therapy, including 46 patients who consented to be genotyped, we found strong evidence showing that repletion of vitamin D in vitamin D deficient patients improved myalgias. Of 21 vitamin D deficient patients with intolerable statin-associated myalgias, 14 of 15 rechallenged with statins were subsequently symptomfree, with one patient experiencing mild and tolerable symptoms, far exceeding expected rates of acquired tolerability with no therapy (p = 0.01). In addition, while the SLCO1B1*5 genotype was associated with a three-fold increased risk of myalgias (p = 0.07), this risk was not found to differ by vitamin D status (p = 0.60).

  View details for DOI 10.4161/derm.2.2.13509

  View details for PubMedID 21547103

  View details for PubMedCentralID PMC3081682

• Molecular pathways mediating the anti-inflammatory effects of calcitriol: implications for prostate cancer chemoprevention and treatment ENDOCRINE-RELATED CANCER Krishnan, A. V., Feldman, D. 2010; 17 (1): R19-R38

  Abstract

  Calcitriol, the hormonally active form of vitamin D, exerts multiple anti-proliferative and pro-differentiating actions including cell cycle arrest and induction of apoptosis in many malignant cells, and the hormone is currently being evaluated in clinical trials as an anti-cancer agent. Recent research reveals that calcitriol also exhibits multiple anti-inflammatory effects. First, calcitriol inhibits the synthesis and biological actions of pro-inflammatory prostaglandins (PGs) by three mechanisms: i) suppression of the expression of cyclooxygenase-2, the enzyme that synthesizes PGs; ii) up-regulation of the expression of 15-hydroxyprostaglandin dehydrogenase, the enzyme that inactivates PGs; and iii) down-regulation of the expression of PG receptors that are essential for PG signaling. The combination of calcitriol and nonsteroidal anti-inflammatory drugs results in a synergistic inhibition of the growth of prostate cancer (PCa) cells and offers a potential therapeutic strategy for PCa. Second, calcitriol increases the expression of mitogen-activated protein kinase phosphatase 5 in prostate cells resulting in the subsequent inhibition of p38 stress kinase signaling and the attenuation of the production of pro-inflammatory cytokines. Third, calcitriol also exerts anti-inflammatory activity in PCa through the inhibition of nuclear factor-kappaB signaling that results in potent anti-inflammatory and anti-angiogenic effects. Other important direct effects of calcitriol as well as the consequences of its anti-inflammatory effects include the inhibition of tumor angiogenesis, invasion, and metastasis. We hypothesize that these anti-inflammatory actions, in addition to the other known anti-cancer effects of calcitriol, play an important role in its potential use as a therapeutic agent for PCa. Calcitriol or its analogs may have utility as chemopreventive agents and should be evaluated in clinical trials in PCa patients with early or precancerous disease.

  View details for DOI 10.1677/ERC-09-0139

  View details for Web of Science ID 000275663100036

  View details for PubMedID 19926709

• Tissue-Selective Regulation of Aromatase Expression by Calcitriol: Implications for Breast Cancer Therapy ENDOCRINOLOGY Krishnan, A. V., Swami, S., Peng, L., Wang, J., Moreno, J., Feldman, D. 2010; 151 (1): 32-42

  Abstract

  Aromatase, the enzyme that catalyzes estrogen synthesis, is critical for the progression of estrogen receptor-positive breast cancer (BCa) in postmenopausal women. We show that calcitriol, the hormonally active form of vitamin D, regulates the expression of aromatase in a tissue-selective manner. Calcitriol significantly decreased aromatase expression in human BCa cells and adipocytes and caused substantial increases in human osteosarcoma cells (a bone cell model exhibiting osteoblast phenotype in culture) and modest increases in ovarian cancer cells. Calcitriol administration to immunocompromised mice bearing human BCa xenografts decreased aromatase mRNA levels in the tumors and the surrounding mammary adipose tissue but did not alter ovarian aromatase expression. In BCa cells, calcitriol also reduced the levels of prostaglandins (PGs), major stimulators of aromatase transcription, by suppressing the expression of cyclooxygenase-2 (which catalyzes PG synthesis) and increasing that of 15-hydroxyprostaglandin dehydrogenase (which catalyzes PG degradation). The mechanism of aromatase down-regulation by calcitriol in BCa cells is therefore 2-fold: a direct repression of aromatase transcription via promoter II through the vitamin D-response elements identified in this promoter and an indirect suppression by reducing the levels of PGs. Combinations of calcitriol with three different aromatase inhibitors (AIs) caused enhanced inhibition of BCa cell growth. The combination of calcitriol and an AI may have potential benefits for BCa therapy. In addition to augmenting the ability of AIs to inhibit BCa growth, calcitriol acting as a selective aromatase modulator that increases aromatase expression in bone would reduce the estrogen deprivation in bone caused by the AIs, thus ameliorating the AI-induced side effect of osteoporosis.

  View details for DOI 10.1210/en.2009-0855

  View details for Web of Science ID 000272977700006

  View details for PubMedID 19906814

  View details for PubMedCentralID PMC2803154

• Hereditary 1,25-dihydroxyvitamin D-resistant rickets with alopecia resulting from a novel missense mutation in the DNA-binding domain of the vitamin D receptor MOLECULAR GENETICS AND METABOLISM Malloy, P. J., Wang, J., Srivastava, T., Feldman, D. 2010; 99 (1): 72-79

  Abstract

  The rare genetic recessive disease, hereditary vitamin D resistant rickets (HVDRR), is caused by mutations in the vitamin D receptor (VDR) that result in resistance to the active hormone 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3) or calcitriol). In this study, we examined the VDR from a young boy with clinical features of HVDRR including severe rickets, hypocalcemia, hypophosphatemia and partial alopecia. The pattern of alopecia was very unusual with areas of total baldness, adjacent to normal hair and regions of scant hair. The child failed to improve on oral calcium and vitamin D therapy but his abnormal chemistries and his bone X-rays normalized with intravenous calcium therapy. We found that the child was homozygous for a unique missense mutation in the VDR gene that converted valine to methionine at amino acid 26 (V26M) in the VDR DNA-binding domain (DBD). The mutant VDR was studied in the patient's cultured skin fibroblasts and found to exhibit normal [(3)H]1,25(OH)(2)D(3) binding and protein expression. However, the fibroblasts were unresponsive to treatment with high concentrations of 1,25(OH)(2)D(3) as demonstrated by their failure to induce CYP24A1 gene expression, a marker of 1,25(OH)(2)D(3) responsiveness. We recreated the V26M mutation in the WT VDR and showed that in transfected COS-7 cells the mutation abolished 1,25(OH)(2)D(3)-mediated transactivation. The mutant VDR exhibited normal ligand-induced binding to RXRalpha and to the coactivator DRIP205. However, the V26M mutation inhibited VDR binding to a consensus vitamin D response element (VDRE). In summary, we have identified a novel V26M mutation in the VDR DBD as the molecular defect in a patient with HVDRR and an unusual pattern of alopecia.

  View details for DOI 10.1016/j.ymgme.2009.09.004

  View details for Web of Science ID 000273758000011

  View details for PubMedID 19815438

  View details for PubMedCentralID PMC2794978

• Modulation of Vitamin D Receptor Activity by the Corepressor Hairless: Differential Effects of Hairless Isoforms ENDOCRINOLOGY Malloy, P. J., Wang, J., Jensen, K., Feldman, D. 2009; 150 (11): 4950-4957

  Abstract

  The vitamin D receptor (VDR) and its corepressor Hairless (HR) are thought to regulate key steps in the hair cycle because mutations in VDR or HR cause alopecia in humans and mice. Many mammalian cells express two major HR isoforms due to alternative splicing of exon 17. HR isoform-a encodes an 1189-amino acid protein (full-length HR), and isoform-b encodes an 1134-amino acid protein (HRDelta1072-1126). We demonstrated that both HR isoforms are expressed in primary human keratinocytes and in the human keratinocyte cell line HaCaT. In transfected COS-7 cells, the full-length HR repressed VDR-mediated transactivation. In contrast, HRDelta1072-1126 failed to suppress and even stimulated VDR-mediated transactivation. In coimmunoprecipitation, both HR isoforms interacted with the VDR, but only the full-length HR interacted with histone deacetylase 1 (HDAC1). Alanine mutagenesis of two conserved glutamic acids residues (E1100A/E1101A) encoded by exon 17 completely eliminated HR corepressor activity and interactions with HDAC1. When the two HR isoforms were coexpressed in COS-7 cells, the corepressor activity of the full-length HR was not antagonized by the HRDelta1072-1126 isoform. When transfected into HaCaT cells, the full-length HR inhibited endogenous CYP24A1 basal gene expression as well as 1,25-dihydroxyvitamin D3-stimulated CYP24A1 expression. HRDelta1072-1126 failed to suppress basal or 1,25-dihydroxyvitamin D3-stimulated CYP24A1 gene expression. In conclusion, we have demonstrated that both HR isoforms are expressed in keratinocytes and that the HRDelta1072-1126 isoform lacks corepressor activity and is unable to bind HDACs. HRDelta1072-1126 may function as a coactivator in some settings by inhibiting HDAC recruitment to the VDR transcriptional complex.

  View details for DOI 10.1210/en.2009-0358

  View details for Web of Science ID 000271007700015

  View details for PubMedID 19819974

  View details for PubMedCentralID PMC2775984

• Hereditary 1,25-Dihydroxyvitamin D-Resistant Rickets in a Pomeranian Dog Caused by a Novel Mutation in the Vitamin D Receptor Gene JOURNAL OF VETERINARY INTERNAL MEDICINE LeVine, D. N., Zhou, Y., Ghiloni, R. J., Fields, E. L., Birkenheuer, A. J., Gookin, J. L., Roberston, I. D., Malloy, P. J., Feldman, D. 2009; 23 (6): 1278-1283

  View details for DOI 10.1111/j.1939-1676.2009.0405.x

  View details for Web of Science ID 000271249400023

  View details for PubMedID 19909429

• Hereditary vitamin D resistant rickets: Identification of a novel splice site mutation in the vitamin D receptor gene and successful treatment with oral calcium therapy BONE Ma, N. S., Malloy, P. J., Pitukcheewanont, P., Dreimane, D., Geffner, M. E., Feldman, D. 2009; 45 (4): 743-746

  Abstract

  To study the vitamin D receptor (VDR) gene in a young girl with severe rickets and clinical features of hereditary vitamin D resistant rickets, including hypocalcemia, hypophosphatemia, partial alopecia, and elevated serum levels of 1,25-dihydroxyvitamin D.We amplified and sequenced DNA samples from blood from the patient, her mother, and the patient's two siblings. We also amplified and sequenced the VDR cDNA from RNA isolated from the patient's blood.DNA sequence analyses of the VDR gene showed that the patient was homozygous for a novel guanine to thymine substitution in the 5'-splice site in the exon 8-intron J junction. Analysis of the VDR cDNA using reverse transcriptase-polymerase chain reaction showed that exons 7 and 9 were fused, and that exon 8 was skipped. The mother was heterozygous for the mutation and the two siblings were unaffected.A novel splice site mutation was identified in the VDR gene that caused exon 8 to be skipped. The mutation deleted amino acids 303-341 in the VDR ligand-binding domain, which is expected to render the VDR non-functional. Nevertheless, successful outpatient treatment was achieved with frequent high doses of oral calcium.

  View details for DOI 10.1016/j.bone.2009.06.003

  View details for Web of Science ID 000270204700015

  View details for PubMedID 19523546

  View details for PubMedCentralID PMC2782671

• A Phase II Trial of Calcitriol and Naproxen in Recurrent Prostate Cancer 3rd International Symposium on Vitamin D Analogs in Cancer Prevention and Therapy Srinivas, S., Feldman, D. INT INST ANTICANCER RESEARCH. 2009: 3605–10

  Abstract

  Androgen-deprivation therapy is commonly used in patients with progressive prostate cancer (PCa), but can be associated with unpleasant side-effects. The objective of the study was to determine whether treatment with calcitriol and naproxen is effective in safely delaying the growth and progression of PCa in men with early recurrent disease.Patients with biochemical relapse after local therapy for prostate cancer were treated with high dose calcitriol (DN101, Novacea) (45 microg once per week) and naproxen (375 mg twice daily) for one year and followed with serum PSA levels as well as imaging studies.Twenty-one patients were enrolled in the trial. Four patients met criteria for progression, with a PSA doubling time (PSADT) that decreased while on therapy. Fourteen patients had a prolongation of PSADT compared to baseline.Combination therapy with weekly calcitriol and daily naproxen is well tolerated by most patients and prolongation of PSADT was achieved in 75% of patients.

  View details for PubMedID 19667155

• Unraveling Insulin-Like Growth Factor Binding Protein-3 Actions in Human Disease ENDOCRINE REVIEWS Jogie-Brahim, S., Feldman, D., Oh, Y. 2009; 30 (5): 417-437

  Abstract

  The IGF system plays critical roles in somatic growth in an endocrine fashion (somatomedin hypothesis) as well as proliferation and differentiation of normal and malignant cells in a paracrine/autocrine fashion. IGFBP-3 is known to modulate the actions of IGFs in circulation as well as the immediate extracellular environment. Interestingly, apart from the ability to inhibit or enhance IGF actions, IGFBP-3 also exhibits very clear, distinct biological effects independent of the IGF/IGF-I receptor axis. Over the past decade it has become widely appreciated that IGF/IGF-IR-independent actions of IGFBP-3 (antiproliferative and proapoptotic effects) contribute to improving the pathophysiology of a variety of human diseases, such as cancer, diabetes, and malnutrition. Recent studies have implicated interaction of IGFBP-3 with a variety of proteins or signaling cascades critical to cell cycle control and apoptosis; however, the actual mechanism of IGFBP-3 action is still unclear. This review reinforces the concept in support of the IGF/IGF-IR axis-independent actions of IGFBP-3 and delineates potential underlying mechanisms involved and subsequent biological significance, focusing in particular on functional binding partners and the clinical significance of IGFBP-3 in the assessment of cancer risk.

  View details for DOI 10.1210/er.2008-0028

  View details for Web of Science ID 000268768000001

  View details for PubMedID 19477944

  View details for PubMedCentralID PMC2819737

• Prostatic Soy Isoflavone Concentrations Exceed Serum Levels After Dietary Supplementation PROSTATE Gardner, C. D., Oelrich, B., Liu, J. P., Feldman, D., Franke, A. A., Brooks, J. D. 2009; 69 (7): 719-726

  Abstract

  The effects of soy isoflavones on prostate cancer may be concentration-dependent. The impact of soy supplementation on isoflavone concentrations in prostate tissues and serum remain unclear.To assess and compare concentrations of soy isoflavones in prostate tissue and serum among 19 men with prostate cancer who had elected to undergo radical prostatectomy.Participants were randomized to receive either daily soy supplements (82 mg/day aglycone equivalents) or placebos for 2 weeks (14 days) prior to surgery. Serum samples were obtained at the time of the surgery. Isoflavone concentrations were measured by HPLC/ESI-MS-MS.The median (25th, 75th percentile) total isoflavone concentration in the isoflavone-supplemented group was 2.3 micromol/L (1.2, 6.9) in the prostate tissue and 0.7 micromol/L (0.2, 1.2) in the serum. Total isoflavone concentrations in this group were an average of approximately 6-fold higher in prostate tissue compared to serum; the tissue versus serum ratio was significantly lower for genistein than daidzein, 4-fold versus 10-fold, P = 0.003. Tissue and serum levels of isoflavones among the placebo group were negligible with a few exceptions.The findings from the present study suggest that prostate tissue may have the ability to concentrate dietary soy isoflavones to potentially anti-carcinogenic levels.

  View details for DOI 10.1002/pros.20922

  View details for Web of Science ID 000265727700004

  View details for PubMedID 19180569

  View details for PubMedCentralID PMC2734961

• Inhibition of prostaglandin synthesis and actions by genistein in human prostate cancer cells and by soy isoflavones in prostate cancer patients INTERNATIONAL JOURNAL OF CANCER Swami, S., Krishnan, A. V., Moreno, J., Bhattacharya, R. S., Gardner, C., Brooks, J. D., Peehl, D. M., Feldman, D. 2009; 124 (9): 2050-2059

  Abstract

  Soy and its constituent isoflavone genistein inhibit the development and progression of prostate cancer (PCa). Our study in both cultured cells and PCa patients reveals a novel pathway for the actions of genistein, namely the inhibition of the synthesis and biological actions of prostaglandins (PGs), known stimulators of PCa growth. In the cell culture experiments, genistein decreased cyclooxygenase-2 (COX-2) mRNA and protein expression in both human PCa cell lines (LNCaP and PC-3) and primary prostate epithelial cells and increased 15-hydroxyprostaglandin dehydrogenase (15-PGDH) mRNA levels in primary prostate cells. As a result genistein significantly reduced the secretion of PGE(2) by these cells. EP4 and FP PG receptor mRNA were also reduced by genistein, providing an additional mechanism for the suppression of PG biological effects. Further, the growth stimulatory effects of both exogenous PGs and endogenous PGs derived from precursor arachidonic acid were attenuated by genistein. We also performed a pilot randomised double blind clinical study in which placebo or soy isoflavone supplements were given to PCa patients in the neo-adjuvant setting for 2 weeks before prostatectomy. Gene expression changes were measured in the prostatectomy specimens. In PCa patients ingesting isoflavones, we observed significant decreases in prostate COX-2 mRNA and increases in p21 mRNA. There were significant correlations between COX-2 mRNA suppression, p21 mRNA stimulation and serum isoflavone levels. We propose that the inhibition of the PG pathway contributes to the beneficial effect of soy isoflavones in PCa chemoprevention and/or treatment.

  View details for DOI 10.1002/ijc.24161

  View details for Web of Science ID 000264647600007

  View details for PubMedID 19127598

• Interaction of the Vitamin D Receptor with a Vitamin D Response Element in the Mullerian-Inhibiting Substance (MIS) Promoter: Regulation of MIS Expression by Calcitriol in Prostate Cancer Cells ENDOCRINOLOGY Malloy, P. J., Peng, L., Wang, J., Feldman, D. 2009; 150 (4): 1580-1587

  Abstract

  Calcitriol (1,25-dihydroxyvitamin D(3)) inhibits the growth of a variety of cancer cells including human prostate cancer. Müllerian-inhibiting substance (MIS) also exhibits antiproliferative and proapoptotic actions on multiple cancer cells including human prostate cancer. In this study, we investigated whether calcitriol regulated MIS expression in prostate cancer, an action that might contribute to its antiproliferative activity. We identified a 15-bp sequence, GGGTGAgcaGGGACA, in the MIS promoter that was highly similar to direct repeat 3-type vitamin D response elements (VDREs). The human MIS promoter containing the putative VDRE was cloned into a luciferase reporter vector. In HeLa cells transfected with the vitamin D receptor (VDR), MIS promoter activity was stimulated by calcitriol. Coexpression of steroidogenic factor 1, a key regulator of MIS, increased basal MIS promoter activity that was further stimulated by calcitriol. Mutation or deletion of the VDRE reduced calcitriol-induced transactivation. In addition, the MIS VDRE conferred calcitriol responsiveness to a heterologous promoter. In gel shift assays, VDR and retinoid X receptor bound to the MIS VDRE and the binding was increased by calcitriol. Chromatin immunoprecipitation assays showed that VDR and retinoid X receptor were present on the MIS promoter in prostate cancer cells. In conclusion, we demonstrated that MIS is a target of calcitriol action. MIS is up-regulated by calcitriol via a functional VDRE that binds the VDR. Up-regulation of MIS by calcitriol may be an important component of the antiproliferative actions of calcitriol in some cancers.

  View details for DOI 10.1210/en.2008-1555

  View details for Web of Science ID 000264442900005

  View details for PubMedID 19056816

  View details for PubMedCentralID PMC2659287

• Compound Heterozygous Mutations in the Vitamin D Receptor in a Patient With Hereditary 1,25-Dihydroxyvitamin D-Resistant Rickets With Alopecia JOURNAL OF BONE AND MINERAL RESEARCH Zhou, Y., Wang, J., Malloy, P. J., Dolezel, Z., Feldman, D. 2009; 24 (4): 643-651

  Abstract

  Hereditary vitamin D-resistant rickets (HVDRR) is a rare recessive genetic disorder caused by mutations in the vitamin D receptor (VDR). In this study, we examined the VDR in a young girl with clinical features of HVDRR including rickets, hypophosphatemia, and elevated serum 1,25(OH)(2)D. The girl also had total alopecia. Two mutations were found in the VDR gene: a nonsense mutation (R30X) in the DNA-binding domain and a unique 3-bp in-frame deletion in exon 6 that deleted the codon for lysine at amino acid 246 (DeltaK246). The child and her mother were both heterozygous for the 3-bp deletion, whereas the child and her father were both heterozygous for the R30X mutation. Fibroblasts from the patient were unresponsive to 1,25(OH)(2)D(3) as shown by their failure to induce CYP24A1 gene expression, a marker of 1,25(OH)(2)D(3) responsiveness. [(3)H]1,25(OH)(2)D(3) binding and immunoblot analysis showed that the patient's cells expressed the VDRDeltaK246 mutant protein; however, the amount of VDRDeltaK246 mutant protein was significantly reduced compared with wildtype controls. In transactivation assays, the recreated VDRDeltaK246 mutant was unresponsive to 1,25(OH)(2)D(3). The DeltaK246 mutation abolished heterodimerization of the mutant VDR with RXRalpha and binding to the coactivators DRIP205 and SRC-1. However, the DeltaK246 mutation did not affect the interaction of the mutant VDR with the corepressor Hairless (HR). In summary, we describe a patient with compound heterozygous mutations in the VDR that results in HVDRR with alopecia. The R30X mutation truncates the VDR, whereas the DeltaK246 mutation prevents heterodimerization with RXR and disrupts coactivator interactions.

  View details for DOI 10.1359/JBMR.081216

  View details for Web of Science ID 000264724700009

  View details for PubMedID 19049339

  View details for PubMedCentralID PMC2659515

• Inactivation of the Human Vitamin D Receptor by Caspase-3 ENDOCRINOLOGY Malloy, P. J., Feldman, D. 2009; 150 (2): 679-686

  Abstract

  Calcitriol actions are mediated by the vitamin D receptor (VDR), a nuclear transcription factor of the steroid-retinoid-thyroid nuclear receptor gene superfamily. Calcitriol inhibits the growth of many cells including cancer cells by inducing cell cycle arrest. In some cancer cell lines, calcitriol also induces apoptosis. In the LNCaP prostate cancer cell line, induction of apoptosis and caspase-3/7 activities by staurosporine (STS) abolished [(3)H]1,25-dihydroxy vitamin D(3) binding and VDR protein, suggesting that the VDR may be targeted for inactivation by caspases during apoptosis. A potential caspase-3 site (D(195)MMD(198)S) was identified in the human VDR ligand-binding domain. Mutations D195A, D198A, and S199A were generated in the putative capase-3 cleavage site. In transfected COS-7 cells, STS treatment resulted in the cleavage of the wild-type (WT) VDR and S199A mutant VDR but not the D195A or D198A mutants. In in vitro assays, the WT VDR and S199A mutant VDR were cleaved by caspase-3, although the D195A and D198A mutants were resistant to caspase-3. In vitro, the WT VDR was also cleaved by caspase-6 and caspase-7 and in extracts of STS-treated LNCaP cells. In STS-treated LNCaP cells and human skin fibroblasts, the proteasome inhibitor MG-132 protected the VDR caspase cleavage fragment from further degradation by the 26S proteasome. The rat VDR that does not contain the caspase-3 cleavage site was not cleaved in STS-treated COS-7 cells. In conclusion, our results demonstrate that the human VDR is a target of caspase-3 and suggest that activation of caspase-3 may limit VDR activity.

  View details for DOI 10.1210/en.2008-1217

  View details for Web of Science ID 000262851200015

  View details for PubMedID 18832097

  View details for PubMedCentralID PMC2646540

• Inhibition of prostaglandin synthesis and actions contributes to the beneficial effects of calcitriol in prostate cancer. Dermato-endocrinology Krishnan, A. V., Srinivas, S., Feldman, D. 2009; 1 (1): 7-11

  Abstract

  Our research is aimed at obtaining a better understanding of the molecular mechanisms of the anti-proliferative and cancer preventive effects of calcitriol with the goal of developing strategies to improve the treatment of prostate cancer (PCa). In PCa cells calcitriol inhibits the synthesis and biological actions of prostaglandins (PGs) by three actions: (i) the inhibition of the expression of cyclooxygenase-2 (COX-2), the enzyme that synthesizes PGs, (ii) the upregulation of the expression of 15-prostaglandin dehydrogenase (15-PGDH), the enzyme that inactivates PGs and (iii) decreasing the expression of EP and FP PG receptors that are essential for PG signaling. Since PGs have been shown to promote carcinogenesis and progression of multiple cancers, we hypothesize that the inhibition of the PG pathway contributes to the ability of calcitriol to prevent or inhibit PCa development and growth. We have shown that the combination of calcitriol and non-steroidal anti-inflammatory drugs (NSAIDs) result in a synergistic inhibition of the growth of PCa cell cultures and this combination therapy offers a potential therapeutic strategy. These findings led us to embark on a clinical trial combining the non-selective NSAID naproxen with calcitriol in men with early recurrent PCa. The results indicate that the combination of high dose weekly calcitriol with naproxen slows the rate of rise (doubling time) of PSA in most patients indicating the slowing of disease progression. Further studies are warranted to determine the role of this combination therapy in the management of recurrent PCa.

  View details for PubMedID 20046582

• Selenite treatment inhibits LAPC-4 tumor growth and prostate-specific antigen secretion in a xenograft model of human prostate cancer INTERNATIONAL JOURNAL OF RADIATION ONCOLOGY BIOLOGY PHYSICS Bhattacharyya, R. S., Husbeck, B., Feldman, D., Knox, S. J. 2008; 72 (3): 935-940

  Abstract

  Selenium compounds have known chemopreventive effects on prostate cancer. However selenite, an inorganic form of selenium, has not been extensively studied as a treatment option for prostate cancer. Our previous studies have demonstrated the inhibition of androgen receptor expression and androgen stimulated prostate-specific antigen (PSA) expression by selenite in human prostate cancer cell lines. In this study, we investigated the in vivo effects of selenite as a therapy to treat mice with established LAPC-4 tumors.Male mice harboring androgen-dependent LAPC-4 xenograft tumors were treated with selenite (2 mg/kg intraperitoneally three times per week) or vehicle for 42 days. In addition, androgen-independent LAPC-4 xenograft tumors were generated in female mice over 4 to 6 months. Once established, androgen-independent LAPC-4 tumor fragments were passaged into female mice and were treated with selenite or vehicle for 42 days. Changes in tumor volume and serum PSA levels were assessed.Selenite significantly decreased androgen-dependent LAPC-4 tumor growth in male mice over 42 days (p < 0.001). Relative tumor volume was decreased by 41% in selenite-treated animals compared with vehicle-treated animals. The inhibition of LAPC-4 tumor growth corresponded to a marked decrease in serum PSA levels (p < 0.01). In the androgen-independent LAPC-4 tumors in female mice, selenite treatment decreased tumor volume by 58% after 42 days of treatment (p < 0.001).These results suggest that selenite may have potential as a novel therapeutic agent to treat both androgen-dependent and androgen-independent prostate cancer.

  View details for DOI 10.1016/j.ijrobp.2008.07.005

  View details for Web of Science ID 000259894300043

  View details for PubMedID 18760546

• The role of insulin-like growth factor binding protein-3 in the growth inhibitory actions of androgens in LNCaP human prostate cancer cells INTERNATIONAL JOURNAL OF CANCER Peng, L., Wang, J., Malloy, P. J., Feldman, D. 2008; 122 (3): 558-566

  Abstract

  Insulin-like growth factor binding protein-3 (IGFBP-3), an antiproliferative and proapoptotic protein, has been shown to be upregulated by growth inhibitory concentrations of androgens in LNCaP human prostate cancer (PCa) cells, but the mechanism of regulation and the role of IGFBP-3 in the modulation of PCa cell proliferation are unknown. In this study, we have examined the effects of a range of concentrations of the synthetic androgen R1881 on IGFBP-3 expression and cell growth in LNCaP cells. We have also investigated the role of androgen-stimulated IGFBP-3 in androgen-induced growth inhibition. We show that low doses of R1881 stimulate LNCaP cell proliferation, but do not induce IGFBP-3 expression, whereas high doses of R1881 that inhibit cell growth, significantly increase expression of IGFBP-3. Importantly, we demonstrate that the combination of calcitriol and androgens not only synergistically upregulates IGFBP-3 expression but also inhibits cell growth better than either hormone alone. siRNA knockdown of IGFBP-3 expression partially reverses the growth inhibition by calcitriol and by androgens. Furthermore, we find that the growth inhibitory dose of R1881 leads to increases in the cyclin dependent kinase inhibitors (CDKIs), p21 and p27 as well as to G1 arrest. These changes can be blocked or partially reversed by IGFBP-3 siRNA, indicating that the induction of CDKIs is downstream of IGFBP-3. Our data suggest, for the first time, that IGFBP-3 is involved in the antiproliferative action of high doses of androgens partly through p21 and p27 pathways and that IGFBP-3 may contribute significantly to androgen-induced changes in LNCaP cell growth.

  View details for DOI 10.1002/ijc.23100

  View details for Web of Science ID 000252132900010

  View details for PubMedID 17918155

• Calcitriol as a chemopreventive and therapeutic agent in prostate cancer: Role of anti-inflammatory activity Conference on the Contemporary Diagnosis and Treatment of Vitamin D-Related Disorders Krishnan, A. V., Moreno, J., Nonn, L., Swami, S., Peehl, D. M., Feldman, D. AMER SOC BONE & MINERAL RES. 2007: V74–V80

  Abstract

  Calcitriol, the hormonally active form of vitamin D, inhibits the growth and development of several cancers. Inflammation has been implicated in the development and progression of many cancers, including prostate cancer (PCa). Recent research from our laboratory suggests that calcitriol exhibits anti-inflammatory actions that may contribute to its inhibitory effects in PCa. We found that calcitriol inhibits the synthesis and actions of pro-inflammatory prostaglandins (PGs) by three mechanisms: (1) inhibition of the expression of cyclooxygenase-2 (COX-2), the enzyme that synthesizes PGs, (2) induction of the expression of 15-prostaglandin dehydrogenase (15-PGDH), the enzyme that inactivates PGs, and (3) decreasing the expression of prostaglandin E and prostaglandin F PG receptors, which are the mediators of PG signaling. The combination of calcitriol and nonsteroidal anti-inflammatory drugs (NSAIDs) result in a synergistic inhibition of PCa cell growth and offers a potential therapeutic strategy. Acting on a separate anti-inflammatory pathway, calcitriol induces the expression of mitogen-activated protein kinase phosphatase 5 (MKP5), a member of a family of phosphatases that are negative regulators of MAP kinases, causing the selective dephosphorylation and inactivation of the stress-activated protein kinase p38. Because p38 activation may be both procarcinogenic and promote inflammation, this calcitriol action, especially coupled with the inhibition of the PG pathway, may contribute to the chemopreventive activity of calcitriol. We conclude that calcitriol exerts several anti-inflammatory actions in prostate cells, which contribute to its potential as a chemopreventive and therapeutic agent in PCa.

  View details for Web of Science ID 000253417900016

  View details for PubMedID 18290727

• Interactions of the vitamin D receptor with the corepressor hairless - Analysis of hairless mutants in atrichia with papular lesions JOURNAL OF BIOLOGICAL CHEMISTRY Wang, J., Malloy, P. J., Feldman, D. 2007; 282 (35): 25231-25239

  Abstract

  Atrichia with papular lesions (APL) and hereditary vitamin D-resistant rickets have a similar congenital hair loss disorder caused by mutations in hairless (HR) and vitamin D receptor (VDR) genes, respectively. HR is a VDR corepressor, and it has been hypothesized that VDR.HR suppress gene expression during specific phases of the hair cycle. In this study, we examined the corepressor activity of HR mutants (E583V, C622G, N970S, V1056M, D1012N, V1136D, and Q1176X) previously described as the molecular cause of APL as well as HR variants (P69S, C397Y, A576V, E591G, R620Q, T1022A) due to non-synonymous polymorphisms in the HR gene. We found that the corepressor activities of all but one of the pathogenic HR mutants were completely abolished. HR mutant E583V exhibited normal corepressor activity, suggesting that it may not be pathogenic. In co-immunoprecipitation assays, all of the pathogenic HR mutants bound VDR but exhibited reduced binding to histone deacetylase 1 (HDAC1), suggesting that the impaired corepressor activity is due in part to defective interactions with HDACs. The HR variants exhibited two classes of corepressor activity, those with normal activity (C397Y, E591G, R620Q) and those with partially reduced activity (P69S, A576V, T1022A). All of the variants interacted with VDR and HDAC1 with the exception of P69S, which was degraded. When coexpressed with VDR, all of the HR pathogenic mutants and variants increased the level of VDR protein, demonstrating that this function of HR was not impaired by these mutations. This study of HR mutations provides evidence for the molecular basis of APL.

  View details for DOI 10.1074/jbc.M702939200

  View details for Web of Science ID 000249014100008

  View details for PubMedID 17609203

• Vitamin D inhibition of the prostaglandin pathway as therapy for prostate cancer Conference on Vitamin D and Cancer - Current Dilemas/Future Needs Feldman, D., Krishnan, A., Moreno, J., Swami, S., Peehl, D. M., Srinivas, S. BLACKWELL PUBLISHING. 2007: S113–S115

  View details for PubMedID 17867384

• Potentiation of the growth-inhibitory effects of vitamin D in prostate cancer by genistein Conference on Vitamin D and Cancer - Current Dilemas/Future Needs Krishnan, A. V., Swami, S., Moreno, J., Bhattacharyya, R. B., Peehl, D. M., Feldman, D. BLACKWELL PUBLISHING. 2007: S121–S123

  View details for Web of Science ID 000248995500015

  View details for PubMedID 17867387

• A unique insertion/duplication in the VDR gene that truncates the VDR causing hereditary 1,25-dihydroxyvitamin D-resistant rickets without alopecia ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Malloy, P. J., Wang, J., Peng, L., Nayak, S., Sisk, J. M., Thompson, C. C., Feldman, D. 2007; 460 (2): 285-292

  Abstract

  Hereditary vitamin D resistant rickets (HVDRR) is caused by mutations in the vitamin D receptor (VDR). Here we describe a patient with HVDRR who also exhibited some hypotrichosis of the scalp but otherwise had normal hair and skin. A 102 bp insertion/duplication was found in the VDR gene that introduced a premature stop (Y401X). The patient's fibroblasts expressed the truncated VDR, but were resistant to 1,25(OH)2D3. The truncated VDR weakly bound [3H]-1,25(OH)2D3 but was able to heterodimerize with RXR, bind to DNA and interact with the corepressor hairless (HR). However, the truncated VDR failed to bind coactivators and was transactivation defective. Since the patient did not have alopecia or papular lesions of the skin generally found in patients with premature stop mutations this suggests that this distally truncated VDR can still regulate the hair cycle and epidermal differentiation possibly through its interactions with RXR and HR to suppress gene transactivation.

  View details for DOI 10.1016/j.abb.2006.09.027

  View details for Web of Science ID 000246310200018

  View details for PubMedID 17078924

  View details for PubMedCentralID PMC1892178

• Novel pathways that contribute to the anti-proliferative and chemopreventive activities of calcitriol in prostate cancer 13th Workshop on Vitamin D Krishnan, A. V., Moreno, J., Nonn, L., Malloy, P., Swami, S., Peng, L., Peehl, D. M., Feldman, D. PERGAMON-ELSEVIER SCIENCE LTD. 2007: 694–702

  Abstract

  Calcitriol, the hormonally active form of Vitamin D, inhibits the growth and development of many cancers through multiple mechanisms. Our recent research supports the contributory role of several new and diverse pathways that add to the mechanisms already established as playing a role in the actions of calcitriol to inhibit the development and progression of prostate cancer (PCa). Calcitriol increases the expression of insulin-like growth factor binding protein-3 (IGFBP-3), which plays a critical role in the inhibition of PCa cell growth by increasing the expression of the cell cycle inhibitor p21. Calcitriol inhibits the prostaglandin (PG) pathway by three actions: (i) the inhibition of the expression of cyclooxygenase-2 (COX-2), the enzyme that synthesizes PGs, (ii) the induction of the expression of 15-prostaglandin dehydrogenase (15-PGDH), the enzyme that inactivates PGs and (iii) decreasing the expression of EP and FP PG receptors that are essential for PG signaling. Since PGs have been shown to promote carcinogenesis and progression of multiple cancers, the inhibition of the PG pathway may add to the ability of calcitriol to prevent and inhibit PCa development and growth. The combination of calcitriol and non-steroidal anti-inflammatory drugs (NSAIDs) result in a synergistic inhibition of PCa cell growth and offers a potential therapeutic strategy. Mitogen activated protein kinase phosphatase 5 (MKP5) is a member of a family of phosphatases that are negative regulators of MAP kinases. Calcitriol induces MKP5 expression in prostate cells leading to the selective dephosphorylation and inactivation of the stress-activated kinase p38. Since p38 activation is pro-carcinogenic and is a mediator of inflammation, this calcitriol action, especially coupled with the inhibition of the PG pathway, contributes to the chemopreventive activity of calcitriol in PCa. Mullerian Inhibiting Substance (MIS) has been evaluated for its inhibitory effects in cancers of the reproductive tissues and is in development as an anti-cancer drug. Calcitriol induces MIS expression in prostate cells revealing yet another mechanism contributing to the anti-cancer activity of calcitriol in PCa. Thus, we conclude that calcitriol regulates myriad pathways that contribute to the potential chemopreventive and therapeutic utility of calcitriol in PCa.

  View details for DOI 10.1016/j.jsbmb.2006.12.051

  View details for Web of Science ID 000245826800094

  View details for PubMedID 17229571

• Calcitriol and genistein actions to inhibit the prostaglandin pathway: Potential combination therapy to treat prostate cancer International Research Conference on Food, Nutrition, and Cancer Swami, S., Krishnan, A. V., Moreno, J., Bhattacharyya, R. B., Peehl, D. M., Feldman, D. AMER SOC NUTRITIONAL SCIENCE. 2007: 205S–210S

  Abstract

  We present an overview of the prostaglandin (PG) pathway as a novel target for the treatment of prostate cancer (PCa) using a combination of calcitriol and genistein, both of which have known antiproliferative properties. Calcitriol inhibits the PG pathway in PCa cells in 3 separate ways: by decreasing cyclooxygenase-2 (COX-2) expression, stimulating 15-hydroxyprostaglandin dehydrogenase (15-PGDH) expression, and decreasing EP (PGE2) and FP (PGF(2alpha)) receptors. These actions of calcitriol result in reduced levels of biologically active PGE2, leading ultimately to growth inhibition of the PCa cells. We also demonstrate the advantages of using calcitriol in combination with genistein for the treatment of PCa. Genistein, a major component of soy, is a potent inhibitor of the activity of CYP24, the enzyme that initiates the degradation of calcitriol. This leads to increased half-life of bioactive calcitriol, thereby enhancing all of calcitriol's actions including those on the PG pathway. In addition to inhibiting CYP24 enzyme activity, genistein has its own independent actions on the PG pathway in PCa cells. Like calcitriol it inhibits COX-2 expression and activity, leading to decreased synthesis of PGE2. It also inhibits the EP and FP receptors, thereby reducing the biological function of PGE2. Thus, the combination of calcitriol and genistein acts additively to inhibit the PG pathway. Both calcitriol and genistein are relatively safe and have little toxicity associated with their intake. We postulate that the combination of calcitriol and genistein is an attractive therapeutic option for the treatment of PCa.

  View details for Web of Science ID 000243330800009

  View details for PubMedID 17182827

• Sex steroid hormones in young manhood and the risk of subsequent prostate cancer: a longitudinal study in African-Americans and Caucasians (United States) CANCER CAUSES & CONTROL Tsai, C. J., Cohn, B. A., Cirillo, P. M., Feldman, D., Stanczyk, F. Z., Whittemore, A. S. 2006; 17 (10): 1237-1244

  Abstract

  To investigate the relation of sex hormone levels in young adults to subsequent prostate cancer risk.From 1959 to 1967, the Child Health and Development Studies collected sera from 10,442 men (median age: 34 years) and followed them for a median of 32 years. In this analysis, we selected 119 African-Americans and 206 Caucasians diagnosed with prostate cancer during the follow-up period. Two prostate cancer-free men were chosen to match each prostate cancer case on race and birth year. We compared the levels of testosterone, estradiol, and sex hormone-binding globulin in cases to those of their matched controls using conditional logistic regression.There was no significant association between absolute levels of sex hormones in youth and prostate cancer risk in either race. However, among Caucasians, but not African-Americans, prostate cancer risk was positively associated with the ratio of total testosterone to total estradiol (odds ratio relating the fourth to the first quartile: 3.01; 95% confidence interval: 1.42-6.39).The association between testosterone to estradiol ratio and prostate cancer risk in young Caucasians is consistent with similar findings in older Caucasians. The absence of this association in African-Americans needs confirmation in other data involving larger numbers of African-Americans.

  View details for DOI 10.1007/s10552-006-0052-4

  View details for Web of Science ID 000242145700003

  View details for PubMedID 17111254

• Growth inhibitory concentrations of androgens up-regulate insulin-like growth factor binding protein-3 expression via an androgen response element in LNCaP human prostate cancer cells ENDOCRINOLOGY Peng, L., Malloy, P. J., Wang, J., Feldman, D. 2006; 147 (10): 4599-4607

  Abstract

  IGF binding protein-3 (IGFBP-3), the most abundant circulating IGF binding protein, inhibits cell growth and induces apoptosis by both IGF-I-dependent and -independent pathways. The ability of IGFBP-3 to inhibit tumor growth has been demonstrated in many cancers including prostate cancer (PCa). High concentrations of androgens, which inhibit the growth of the LNCaP human PCa cell line, have been shown to have both positive and negative effects on IGFBP-3 expression by different laboratories. To further explore the relationship between IGFBP-3 and androgens, we examined IGFBP-3 expression in LNCaP cells. We demonstrate that IGFBP-3 expression can be induced by 10 nm of the synthetic androgen R1881 or dihydrotestosterone. Transactivation assays show that the 6-kb IGFBP-3 promoter sequence directly responds to androgen treatment. In silico analysis identified a putative androgen response element (ARE) at -2,879/-2,865 in the IGFBP-3 promoter. A single point mutation in this ARE disrupted transactivation by R1881. Combining the data obtained from EMSA, chromatin immunoprecipitation and mutational analysis, we conclude that a novel functional ARE is present in the IGFBP-3 promoter that directly mediates androgen induction of IGFBP-3 expression. Furthermore, we found that the combination of androgens and calcitriol significantly potentiated the IGFBP-3 promoter activity, suggesting that enhanced induction of the expression of the endogenous IGFBP-3 gene may contribute to the greater inhibition of LNCaP cell growth by combined calcitriol and androgens. Because androgens are well known to stimulate PCa growth and androgen deprivation therapy causes PCa to regress, the stimulation by androgens of this antiproliferative and proapoptotic protein is paradoxical and raises interesting questions about the role of androgen-stimulated IGFBP-3 in PCa.

  View details for DOI 10.1210/en.2006-0560

  View details for Web of Science ID 000240737100010

  View details for PubMedID 16825320

• Inhibition of androgen receptor signaling by selenite and methylseleninic acid in prostate cancer cells: two distinct mechanisms of action MOLECULAR CANCER THERAPEUTICS Husbeck, B., Bhattacharyya, R. S., Feldman, D., Knox, S. J. 2006; 5 (8): 2078-2085

  Abstract

  The development of prostate cancer and its progression to a hormone-refractory state is highly dependent on androgen receptor (AR) expression. Recent studies have shown that the selenium-based compound methylseleninic acid (MSeA) can disrupt AR signaling in prostate cancer cells. We have found that selenite can inhibit AR expression and activity in LAPC-4 and LNCaP prostate cancer cells as well but through a different mechanism. On entering the cell, selenite consumes reduced glutathione (GSH) and generates superoxide radicals. Pretreatment with N-acetylcysteine, a GSH precursor, blocked the down-regulation of AR mRNA and protein expression by selenite and restored AR ligand binding and prostate-specific antigen expression to control levels. MSeA reacts with reduced GSH within the cell; however, N-acetylcysteine did not effect MSeA-induced down-regulation of AR and prostate-specific antigen. The superoxide dismutase mimetic MnTMPyP was also found to prevent the decrease in AR expression caused by selenite but not by MSeA. A Sp1-binding site in the AR promoter is a key regulatory component for its expression. Selenite decreased Sp1 expression and activity, whereas MSeA did not. The inhibition of Sp1 by selenite was reversed in the presence of N-acetylcysteine. In conclusion, we have found that selenite and MSeA disrupt AR signaling by distinct mechanisms. The inhibition of AR expression and activity by selenite occurs via a redox mechanism involving GSH, superoxide, and Sp1.

  View details for DOI 10.1158/1535-7163.MCT-06-0056

  View details for Web of Science ID 000240029400021

  View details for PubMedID 16928829

• Mechanisms of vitamin D-mediated growth inhibition in prostate cancer cells: Inhibition of the prostaglandin pathway 2nd International Symposium on Vitamin D Analogs in Cancer Prevention and Therapy Moreno, J., Krishnan, A. V., Peehl, D. M., Feldman, D. INT INST ANTICANCER RESEARCH. 2006: 2525–30

  Abstract

  Calcitriol (1,25-dihydroxyvitamin D3), the active form of vitamin D, promotes growth inhibition and differentiation in prostate cancer (PCa) cells. To unravel the molecular pathways of calcitriol actions, cDNA microarray analysis was used to identify novel calcitriol target genes including two that play key roles in the metabolism of prostaglandins (PGs), known stimulators of PCa growth and progression. Calcitriol significantly decreases the expression of the PG synthesizing cyclooxygenase-2 (COX-2) gene, while increasing that of PG inactivating 15-prostaglandin dehydrogenase (15-PGDH). Calcitriol also inhibits the expression of the PG receptors EP2 and FP. It reduces the levels of biologically active PGs and inhibits PG actions in PCa cells, thereby decreasing the proliferative stimulus of PGs. We postulate that the regulation of the PG pathway contributes to the growth inhibitory actions of calcitriol. We also propose that calcitriol can be combined with non-steroidal anti-inflammatory drugs (NSAIDs) that inhibit COX enzyme activity, as a potential therapeutic strategy in PCa.

  View details for Web of Science ID 000241391500003

  View details for PubMedID 16886660

• Fulvestrant (ICI 182,780) down-regulates androgen receptor expression and diminishes androgenic responses in LNCaP human prostate cancer cells MOLECULAR CANCER THERAPEUTICS Bhattacharyya, R. S., Krishnan, A. V., Swami, S., Feldman, D. 2006; 5 (6): 1539-1549

  Abstract

  The androgen receptor (AR) plays a key role in the development and progression of prostate cancer. Targeting the AR for down-regulation would be a useful strategy for treating prostate cancer, especially hormone-refractory or androgen-independent prostate cancer. In the present study, we showed that the antiestrogen fulvestrant [ICI 182,780 (ICI)] effectively suppressed AR expression in several human prostate cancer cells, including androgen-independent cells. In LNCaP cells, ICI (10 micromol/L) treatment decreased AR mRNA expression by 43% after 24 hours and AR protein expression by approximately 50% after 48 hours. We further examined the mechanism of AR down-regulation by ICI in LNCaP cells. ICI did not bind to the T877A-mutant AR present in the LNCaP cells nor did it promote proteasomal degradation of the AR. ICI did not affect AR mRNA or protein half-life. However, ICI decreased the activity of an AR promoter-luciferase reporter plasmid transfected into LNCaP cells, suggesting a direct repression of AR gene transcription. As a result of AR down-regulation by ICI, androgen induction of prostate-specific antigen mRNA and protein expression were substantially attenuated. Importantly, LNCaP cell proliferation was significantly inhibited by ICI treatment. Following 6 days of ICI treatment, a 70% growth inhibition was seen in androgen-stimulated LNCaP cells. These data show that the antiestrogen ICI is a potent AR down-regulator that causes significant inhibition of prostate cancer cell growth. Our study suggests that AR down-regulation by ICI would be an effective strategy for the treatment of all prostate cancer, especially AR-dependent androgen-independent prostate cancer.

  View details for DOI 10.1158/1535-7163.MCT-06-0065

  View details for Web of Science ID 000239029700019

  View details for PubMedID 16818513

• Phase II study evaluating oral triamcinolone in patients with androgen-independent prostate cancer UROLOGY Srinivas, S., Krishnan, A. V., Colocci, N., Feldman, D. 2006; 67 (5): 1001-1006

  Abstract

  To assess the effect of triamcinolone administration on the serum prostate-specific antigen (PSA) response and the time to progression in patients with androgen-independent prostate cancer (AIPC).Patients with AIPC were prospectively treated with oral triamcinolone 4 mg twice daily, and their serum PSA and cortisol levels were measured monthly. Patients with greater than 25% increases in serum PSA from baseline were considered to have progressive disease and were removed from the study. Those patients who had a decrease in serum PSA levels or stable disease continued in the study until disease progression. Bone scans were obtained every 12 weeks and at progression.Twenty-four patients with AIPC were treated from November 2002 to June 2004. A partial response with a more than 50% decrease in serum PSA level was seen in 29%. Another 21% achieved stable disease. No statistically significant difference was found in the time to progression in the partial responders and patients with stable disease. The median time to progression in both groups was 7.5 months. Treatment was well tolerated without any grade 3 or 4 toxicity.Oral triamcinolone was well tolerated by patients with AIPC, with 50% of the patients exhibiting a good response to therapy in terms of serum PSA level and time to progression.

  View details for DOI 10.1016/j.urology.2005.11.004

  View details for PubMedID 16698360

• Inhibition of p38 by vitamin D reduces interleukin-6 production in normal prostate cells via mitogen-activated protein kinase phosphatase 5: Implications for prostate cancer prevention by vitamin D CANCER RESEARCH Nonn, L., Peng, L. H., Feldman, D., Peehl, D. M. 2006; 66 (8): 4516-4524

  Abstract

  Although numerous studies have implicated vitamin D in preventing prostate cancer, the underlying mechanism(s) remains unclear. Using normal human prostatic epithelial cells, we examined the role of mitogen-activated protein kinase phosphatase 5 (MKP5) in mediating cancer preventive activities of vitamin D. Up-regulation of MKP5 mRNA by 1,25-dihydroxyvitamin-D3 (1,25D) was dependent on the vitamin D receptor. We also identified a putative positive vitamin D response element within the MKP5 promoter that associated with the vitamin D receptor following 1,25D treatment. MKP5 dephosphorylates/inactivates the stress-activated protein kinase p38. Treatment of prostate cells with 1,25D inhibited p38 phosphorylation, and MKP5 small interfering RNA blocked this effect. Activation of p38 and downstream production of interleukin 6 (IL-6) are proinflammatory. Inflammation and IL-6 overexpression have been implicated in the initiation and progression of prostate cancer. 1,25D pretreatment inhibited both UV- and tumor necrosis factor alpha-stimulated IL-6 production in normal cells via p38 inhibition. Consistent with inhibition of p38, 1,25D decreased UV-stimulated IL-6 mRNA stabilization. The ability of 1,25D to up-regulate MKP5 was maintained in primary prostatic adenocarcinoma cells but was absent in metastases-derived prostate cancer cell lines. The inability of 1,25D to regulate MKP5 in the metastasis-derived cancer cells suggests there may be selective pressure to eliminate key tumor suppressor functions of vitamin D during cancer progression. These studies reveal MKP5 as a mediator of p38 inactivation and decreased IL-6 expression by 1,25D in primary prostatic cultures of normal and adenocarcinoma cells, implicating decreased prostatic inflammation as a potential mechanism for prostate cancer prevention by 1,25D.

  View details for DOI 10.1158/0008-5472.CAN-05-3796

  View details for Web of Science ID 000236843200071

  View details for PubMedID 16618780

• Molecular mechanisms mediating the anti-proliferative effects of Vitamin D in prostate cancer Workshop on Vitamin D and Cancer Treatment and Prevention Moreno, J., Krishnan, A. V., Feldman, D. PERGAMON-ELSEVIER SCIENCE LTD. 2005: 31–36

  Abstract

  Calcitriol (1,25-dihydroxyvitamin D(3)) inhibits the growth and stimulates the differentiation of prostate cancer (PCa) cells. The effects of calcitriol are varied, appear to be cell-specific and result in growth arrest and stimulation of apoptosis. Our goal was to define the genes involved in the multiple pathways mediating the anti-proliferative effects of calcitriol in PCa. We used cDNA microarray analysis to identify calcitriol target genes involved in these pathways in both LNCaP human PCa cells and primary prostatic epithelial cells. Interestingly, two of the target genes that we identified play key roles in the metabolism of prostaglandins (PGs), which are known stimulators of PCa cell growth and progression. The expression of the PG synthesizing cyclooxygenase-2 (COX-2) gene was significantly decreased by calcitriol, while that of PG inactivating 15-prostaglandin dehydrogenase gene (15-PGDH) was increased. We postulate that this dual action of calcitriol would reduce the levels of biologically active PGs in PCa cells decreasing their proliferative stimulus and contribute to the growth inhibitory actions of calcitriol. In addition, we propose that calcitriol can be combined with non-steroidal anti-inflammatory drugs that inhibit COX activity, as a potential therapeutic strategy to improve the potency and efficacy of both drugs in the treatment of PCa.

  View details for DOI 10.1016/j.jsbmb.2005.06.012

  View details for Web of Science ID 000233543100006

  View details for PubMedID 16024246

• Genistein potentiates the growth inhibitory effects of 1,25-dihydroxyvitamin D-3 in DU145 human prostate cancer cells: Role of the direct inhibition of CYP24 enzyme activity MOLECULAR AND CELLULAR ENDOCRINOLOGY Swami, S., Krishnan, A. V., Peehl, D. M., Feldman, D. 2005; 241 (1-2): 49-61

  Abstract

  In a search for improved therapies for prostate cancer, we investigated the effect of genistein in combination with 1alpha-25-dihydroxyvitamin D3 [1,25(OH)2D3], on the growth of DU145 human prostate cancer cells. DU145 cells were very resistant to the growth inhibitory action of 1,25(OH)2D3 or genistein when administered individually. However, the combination caused a significant growth inhibition seen at lower concentrations of both agents. 1,25(OH)2D3 induces the expression of the CYP24 gene, which codes for the enzyme that initiates the catabolism of 1,25(OH)2D3. We showed for the first time that genistein at low doses (50-100 nM) directly inhibited CYP24 at the enzyme level. Addition of genistein to mitochondrial preparations inhibited CYP24 enzyme activity in a noncompetitive manner. CYP24 inhibition by genistein increased the half-life of 1,25(OH)2D3 thereby augmenting the homologous up-regulation of the vitamin D receptor (VDR) both at the mRNA and protein levels. Genistein co-treatment enhanced 1,25(OH)2D3-mediated transactivation of the vitamin D responsive reporters OC-Luc and OP-Luc transfected into DU145 cells. Consistent with the growth inhibition due to the combination treatment, significant changes in the expression of genes involved in growth arrest and apoptosis were seen. We conclude that genistein potentiates the antiproliferative actions of 1,25(OH)2D3 in DU145 cells by two mechanisms: (i) an increase in the half-life of 1,25(OH)2D3 due to the direct inhibition of CYP24 enzyme activity and (ii) an amplification of the homologous up-regulation of VDR. Together these two effects lead to a substantial enhancement of the cellular responses to the growth inhibitory and pro-apoptotic signaling by 1,25(OH)2D3.

  View details for DOI 10.1016/j.mce.2005.05.001

  View details for Web of Science ID 000232439700006

  View details for PubMedID 15955619

• Enhanced coactivator binding and transcriptional activation of mutant vitamin D receptors from patients with hereditary 1,25-dihydroxyvitamin D-resistant rickets by phosphorylation and vitamin D analogs JOURNAL OF BONE AND MINERAL RESEARCH Liu, Y., Shen, Q., Malloy, P. J., Soliman, E., Peng, X. R., Kim, S., PIKE, J. W., Feldman, D., Christakos, S. 2005; 20 (9): 1680-1691

  Abstract

  In this study, we report that the function of certain mutant VDRs from patients with hereditary HVDRR can at least be partially restored by phosphorylation and hexafluoro 1,25(OH)2D3 analogs. Our study provides new insights into mechanisms involved in enhancement of mutant VDR function.1,25-Dihydroxyvitamin D-resistant rickets (HVDRR) is a rare genetic disorder caused by inactivating mutations in the vitamin D receptor (VDR). In this study, we examined VDR from patients with HVDRR having mutations in the ligand-binding domain (F251C, I268T, H305Q, E420K). We examined methods of restoring transcriptional activity of these mutants and the mechanisms involved.Reporter gene transcriptional assays were used to examine the activation of mutant VDRs. Western-blot analysis, glutathione S-transferase (GST) pull-down assays, and chromatin immunoprecipitation (ChIP) assays were also used in this study.Using mutant VDRs, H305Q, F251C, I268T, and 10(-8) M 1,25(OH)2D3, only 10-30% of the activity of wildtype (WT) VDR in activating 24(OH)ase transcription was observed. The transcriptional response of mutant VDR mutants was significantly enhanced 2- to 3-fold by co-treatment of VDR mutant transfected COS-7 cells with 1,25(OH)2D3 and okadaic acid (OA; inhibitor of phosphatase; 50 nM). The H305Q mutant was the most responsive (90% of the response exhibited by WT VDR was restored). The E420K mutant was unresponsive to 1,25(OH)2D3 in the presence or absence of OA. The increased transcriptional response correlated with an increase in the interaction between DRIP205 and the mutant VDR. We further provide evidence that OA induces the phosphorylation of CREB-binding protein (CBP), indicating for the first time a correlation between phosphorylation of CBP and enhanced VDR function. Hexafluoro 1,25(OH)2D3 analogs (RO-26-2198 and RO-4383561) also resulted in at least a partial restoration of the transcriptional responsiveness of mutant VDRs I268T, F251C, and H305Q. Our data indicate that the enhanced potency of the hexafluoro analogs may be caused by increased DRIP205 and glucocorticoid receptor interacting protein 1 (GRIP-1) binding to VDRs and enhanced association of VDRs with DNA, as suggested by results of ChIP assays.Our study provides new insights into the mechanisms involved in the enhancement of VDR function by both phosphorylation and hexafluoro analogs and forms a basis for future study of vitamin D analogs or specifically designed kinase activity mediators as potential therapy for the treatment of selected patients with HVDRR.

  View details for DOI 10.1359/JBMR.050410

  View details for Web of Science ID 000231490200019

  View details for PubMedID 16059639

• Regulation of prostaglandin metabolism by calcitriol attenuates growth stimulation in prostate cancer cells CANCER RESEARCH Moreno, J., Krishnan, A. V., Swami, S., Nonn, L., Peehl, D. M., Feldman, D. 2005; 65 (17): 7917-7925

  Abstract

  Calcitriol exhibits antiproliferative and pro-differentiation effects in prostate cancer. Our goal is to further define the mechanisms underlying these actions. We studied established human prostate cancer cell lines and primary prostatic epithelial cells and showed that calcitriol regulated the expression of genes involved in the metabolism of prostaglandins (PGs), known stimulators of prostate cell growth. Calcitriol significantly repressed the mRNA and protein expression of prostaglandin endoperoxide synthase/cyclooxygenase-2 (COX-2), the key PG synthesis enzyme. Calcitriol also up-regulated the expression of 15-hydroxyprostaglandin dehydrogenase, the enzyme initiating PG catabolism. This dual action was associated with decreased prostaglandin E2 secretion into the conditioned media of prostate cancer cells exposed to calcitriol. Calcitriol also repressed the mRNA expression of the PG receptors EP2 and FP, providing a potential additional mechanism of suppression of the biological activity of PGs. Calcitriol treatment attenuated PG-mediated functional responses, including the stimulation of prostate cancer cell growth. The combination of calcitriol with nonsteroidal anti-inflammatory drugs (NSAIDs) synergistically acted to achieve significant prostate cancer cell growth inhibition at approximately 2 to 10 times lower concentrations of the drugs than when used alone. In conclusion, the regulation of PG metabolism and biological actions constitutes a novel pathway of calcitriol action that may contribute to its antiproliferative effects in prostate cells. We propose that a combination of calcitriol and nonselective NSAIDs might be a useful chemopreventive and/or therapeutic strategy in men with prostate cancer, as it would allow the use of lower concentrations of both drugs, thereby reducing their toxic side effects.

  View details for DOI 10.1158/0008-5472.CAN-05-1435

  View details for Web of Science ID 000231659500052

  View details for PubMedID 16140963

• Prostate specific antigen levels in young adulthood predict prostate cancer risk: Results from a cohort of black and white Americans JOURNAL OF UROLOGY Whittemore, A. S., Cirillo, P. M., Feldman, D., Cohn, B. A. 2005; 174 (3): 872-876

  Abstract

  Prostate specific antigen (PSA) is a serine protease produced by normal and malignant prostate epithelial cells. Serum PSA increases with age, due largely to age related increases in the prevalence of benign prostatic disease. Little is known about PSA distribution in young adulthood, when benign and malignant prostatic diseases are rare, or about how PSA within the normal range in youth relates to subsequent prostate cancer risk.We evaluated serum PSA and subsequent prostate cancer occurrence in a cohort of young black and white American men with a median age at blood draw of 34 years, who in 1959 to 1966 participated as the fathers of newborns enrolled in the Child Health and Development Study, and who were followed for several decades for prostate cancer. We examined associations between PSA in young adulthood and subsequent prostate cancer risk using a nested case-control design based on 119 black and 206 white cases with 2 control men matched to each case on race and year of birth.Prostate cancer risk increased with increasing PSA in black and white men. The OR comparing risk in the highest to lowest quartiles of PSA was 4.4 (95% CI 2.0 to 9.6) in black men and 3.5 (95% CI 2.0 to 6.1) in white men. ORs relating risk to PSA were higher when analysis was restricted to cases diagnosed before age 65 years.These findings suggest that PSA levels in young adulthood indicate increased risk of prostate cancer and, thus, they may be useful for targeting men for screening and early diagnosis.

  View details for DOI 10.1097/01.ju.0000169262.18000.8a

  View details for Web of Science ID 000231274700019

  View details for PubMedID 16093978

• Hereditary 1,25-dihydroxyvitamin D resistant rickets due to a mutation causing multiple defects in vitamin D receptor function ENDOCRINOLOGY Malloy, P. J., Xu, R., Peng, L. H., Peleg, S., Al-Ashwal, A., Feldman, D. 2004; 145 (11): 5106-5114

  Abstract

  Hereditary vitamin D-resistant rickets (HVDRR) is an autosomal recessive disease caused by mutations in the vitamin D receptor (VDR). We studied a young Saudi Arabian girl who exhibited the typical clinical features of HVDRR, but without alopecia. Analysis of her VDR gene revealed a homozygous T to C mutation in exon 7 that changed isoleucine to threonine at amino acid 268 (I268T). From crystallographic studies of the VDR ligand-binding domain, I268 directly interacts with 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] and is involved in the hydrophobic stabilization of helix H12. We recreated the I268T mutation and analyzed its effects on VDR function. In ligand binding assays, the I268T mutant VDR exhibited an approximately 5- to 10-fold lower affinity for [(3)H]1,25(OH)(2)D(3) compared with the wild-type (WT) VDR. The I268T mutant required approximately a 65-fold higher concentration of 1,25(OH)(2)D(3) to be equipotent in gene transactivation. Both retinoid X receptor heterodimerization and coactivator binding were reduced in the I268T mutant. Analogs of 1,25(OH)(2)D(3) have been proposed as potential therapeutics for patients with HVDRR. Interestingly, in protease sensitivity assays, treatment with the potent vitamin D analog, 20-epi-1,25(OH)(2)D(3), stabilized I268T mutant proteolytic fragments better than 1,25(OH)(2)D(3). Moreover, 20-epi-1,25(OH)(2)D(3) restored transactivation of the I268T mutant to levels exhibited by WT VDR treated with 1,25(OH)(2)D(3). In conclusion, we describe a novel mutation, I268T, in the VDR ligand-binding domain that alters ligand binding, retinoid X receptor heterodimerization, and coactivator binding. These combined defects in VDR function cause resistance to 1,25(OH)(2)D(3) action and result in the syndrome of HVDRR.

  View details for DOI 10.1210/en.2004-0080

  View details for PubMedID 15308610

• Interaction of nuclear receptor ligands with the vitamin D signaling pathway in prostate cancer JOURNAL OF STEROID BIOCHEMISTRY AND MOLECULAR BIOLOGY Peehl, D. M., Feldman, D. 2004; 92 (4): 307-315

  Abstract

  A number of hormonal ligands and/or the nuclear receptors that mediate their actions have been targeted for prostate cancer therapy. Androgens, the ligands for the androgen receptor (AR), are critical for the growth of prostate cancer. Inhibition of androgen production has been the mainstay of treatment for advanced prostate cancer for decades. Other more recently tested targets include retinoid receptors (RAR and RXR), glucocorticoid receptors (GR), estrogen receptors (ER) and peroxisome proliferator-activated receptors (PPAR). Calcitriol, acting through the Vitamin D receptor (VDR), has many tumor suppressive activities in the prostate, including inhibition of proliferation, induction of apoptosis and/or differentiation, and reduction of cellular invasion. Because of these properties, calcitriol and its less hypercalcemic analogs are being evaluated as agents to prevent or treat prostate cancer. Androgens, retinoids, glucocorticoids, estrogens and agonists of PPAR directly or indirectly impact Vitamin D signaling pathways, and vice versa. In order to design the most effective strategies to use calcitriol to prevent or treat prostate cancer, the interactions of other nuclear receptors and their ligands with the Vitamin D signaling pathway need to be considered.

  View details for DOI 10.1016/j.jsbmb.2004.10.006

  View details for Web of Science ID 000227022500010

  View details for PubMedID 15663994

• Molecular activity of 1,25-dihydroxyvitamin D-3 in primary cultures of human prostatic epithelial cells revealed by cDNA microarray analysis JOURNAL OF STEROID BIOCHEMISTRY AND MOLECULAR BIOLOGY Peehl, D. M., Shinghal, R., Nonn, L., Seto, E., Krishnan, A. V., Brooks, J. D., Feldman, D. 2004; 92 (3): 131-141

  Abstract

  1,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] exerts anti-proliferative, differentiating and apoptotic effects on prostatic cells. These activities, in addition to epidemiologic findings that link Vitamin D to prostate cancer risk, support the use of 1,25(OH)(2)D(3) for prevention or therapy of prostate cancer. The molecular mechanisms by which 1,25(OH)(2)D(3) exerts antitumor effects on prostatic cells are not well-defined. In addition, there is heterogeneity among the responses of various prostate cell lines and primary cultures to 1,25(OH)(2)D(3) with regard to growth inhibition, differentiation and apoptosis. To understand the basis of these differential responses and to develop a better model of Vitamin D action in the prostate, we performed cDNA microarray analyses of primary cultures of normal and malignant human prostatic epithelial cells, treated with 50 nM of 1,25(OH)(2)D(3) for 6 and 24 h. CYP24 (25-hydroxyvitamin D(3)-24-hydroxylase) was the most highly upregulated gene. Significant and early upregulation of dual specificity phosphatase 10 (DUSP10), validated in five additional primary cultures, points to inhibition of members of the mitogen-activated protein kinase (MAPK) superfamily as a key event mediating activity of 1,25(OH)(2)D(3) in prostatic epithelial cells. The functions of other regulated genes suggest protection by 1,25(OH)(2)D(3) from oxidative stress. Overall, these results provide new insights into the molecular basis of antitumor activities of Vitamin D in prostate cells.

  View details for DOI 10.1016/j.jsbmb.2004.07.003

  View details for Web of Science ID 000226009000003

  View details for PubMedID 15555907

• Risk of early-onset prostate cancer in relation to germ line polymorphisms of the vitamin D receptor CANCER EPIDEMIOLOGY BIOMARKERS & PREVENTION Oakley-Girvan, I., Feldman, D., Eccleshall, T. R., Gallagher, R. P., Wu, A. H., Kolonel, L. N., Halpern, J., Balise, R. R., West, D. W., Paffenbarger, R. S., Whittemore, A. S. 2004; 13 (8): 1325-1330

  Abstract

  Vitamin D inhibits prostate cancer cell growth, angiogenesis, and metastasis. These actions are mediated by the vitamin D receptor. We examined associations between prostate cancer risk and five polymorphisms in the VDR gene: four single nucleotide polymorphisms (FokI, BsmI, ApaI, and TaqI restriction sites) and the polyadenylic acid microsatellite. Specifically, we genotyped population-based samples of young African Americans (113 cases and 121 controls) and Whites (232 cases and 171 controls) and members of 98 predominantly White families with multiple cases of prostate cancer. Among Whites, there was no evidence for association between prostate cancer risk and alleles at any of the five polymorphic sites regardless of how the men were ascertained. Moreover, estimated five-locus haplotype frequencies were similar in White cases and controls. Among African Americans, prostate cancer risk was associated with homozygosity for the F allele at the FokI site (odds ratio 1.9, 95% confidence interval 1.0-3.3). In addition, estimated haplotype frequencies differed significantly (P < 0.01) between African American cases and controls. These findings need replication in other studies of African Americans. Homozygosity for the F allele at the FokI site is more prevalent in the African American population than in U.S. Whites. If the FokI association noted here were causal, this difference could account for some of the disease burden among African Americans and some of the excess risk in African Americans compared with Whites.

  View details for Web of Science ID 000223155500010

  View details for PubMedID 15298953

• Mechanisms of decreased Vitamin D 1 alpha-hydroxylase activity in prostate cancer cells MOLECULAR AND CELLULAR ENDOCRINOLOGY Ma, J. F., Nonn, L., Campbell, M. J., Hewison, M., Feldman, D., Peehl, D. M. 2004; 221 (1-2): 67-74

  Abstract

  Vitamin D 1alpha-hydroxylase (1alpha(OH)ase), which converts the circulating prohormone 25-hydroxyvitamin-D(3) (25(OH)D(3)) to the active 1alpha-25-dihydroxyvitamin-D(3) (1,25(OH)(2)D(3)), is present in normal prostatic epithelium. However, prostate cancer cells, both primary cultured cells and cell lines, have greatly decreased activity of 1alpha(OH)ase and are therefore resistant to the tumor suppressor activity of circulating 25(OH)D(3). We quantitated 1alpha(OH)ase mRNA and protein levels to investigate mechanism(s) responsible for decreased 1alpha(OH)ase enzymatic activity in prostate cancer. Prostate cancer cell lines had low 1alpha(OH)ase mRNA levels. Primary prostate cell cultures derived from normal and cancer tissues had equivalent levels of 1alpha(OH)ase RNA and protein. Equivalent 1alpha(OH)ase protein levels were observed in prostate tissue sections containing normal and malignant cells. The protein levels of hsc70, whose homolog intracellular Vitamin D binding protein (IDBP-1) facilitates delivery of 25(OH)D(3) to 1alpha(OH)ase in monkey cells, were equivalent in the normal and cancer cells. Equivalent activity in normal and cancer cells of Vitamin D 24-hydroxylase, a mitochondrial enzyme that also uses 25(OH)D(3) as a substrate, further ruled out lack of access to substrate as a basis for low activity of 1alpha(OH)ase in cancer cells. We conclude that diminished 1alpha(OH)ase activity in prostate cancer cell lines is through decreased gene expression, whereas decreased activity in primary cultures and tissues is post-translational.

  View details for DOI 10.1016/j.mce.2004.03.003

  View details for Web of Science ID 000222854100007

  View details for PubMedID 15223133

• A unique insertion/substitution in helix H1 of the vitamin D receptor ligand binding domain in a patient with hereditary 1,25-dihydroxyvitamin D-resistant rickets JOURNAL OF BONE AND MINERAL RESEARCH Malloy, P. J., Xu, R., Cattani, A., Reyes, M. L., Feldman, D. 2004; 19 (6): 1018-1024

  Abstract

  Hereditary vitamin D--resistant rickets (HVDRR) is a genetic disorder caused by mutations in the vitamin D receptor (VDR). In this study, we examined the VDR in a young boy who exhibited the typical clinical features of HVDRR but without alopecia.The patient's VDR was studied using cultured dermal fibroblasts, and the recreated mutant VDR was analyzed in transfected cells.The patient's fibroblasts were resistant to 1,25-dihydroxyvitamin D [1,25(OH)2D3], exhibiting only a slight induction of 24-hydroxylase gene expression when treated with 1 microM 1,25(OH)2D3 x [3H]1,25(OH)2D3 binding was absent in cell extracts from the patient's fibroblasts. Sequence analysis of the VDR gene uncovered a unique 5-bp deletion/8-bp insertion in exon 4. The mutation in helix HI of the ligand-binding domain deletes two amino acids (H141 and T142) and inserts three amino acids (L141, W142, and A143). In transactivation assays, the recreated mutant VDR was 1000-fold less active than the wildtype (WT) VDR. In glutathione S-transferase (GST) pull-down assays, the mutant VDR bound GST-retinoid X receptor (RXR) weakly in the absence of 1,25(OH)2D3; however, the binding did not increase with increasing concentrations of ligand. The mutant VDR did not bind to GST-vitamin D receptor interacting protein (DRIP) 205 at concentrations up to 1 microM 1,25(OH)2D3. We also examined effects of the three individual mutations on VDR transactivation. Only the insertion of A143 into the WT VDR disrupted VDR transactivation to the same extent observed with the natural mutation.We describe a novel insertion/substitution mutation in helix Hl of the VDR ligand-binding domain (LBD) that abolishes ligand binding and result in the syndrome of HVDRR. This is the first time an insertion/substitution has been found as the defect-causing HVDRR.

  View details for DOI 10.1359/JBMR.

  View details for PubMedID 15190891

• Analysis of vitamin D-regulated gene expression in LNCaP human prostate cancer cells using cDNA microarrays PROSTATE Krishnan, A. V., Shinghal, R., Raghavachari, N., Brooks, J. D., Peehl, D. M., Feldman, D. 2004; 59 (3): 243-251

  Abstract

  1,25-dihydroxyvitamin D(3) [1,25(OH)2D3] exerts growth inhibitory, pro-differentiating, and pro-apoptotic effects on prostate cells. To better understand the molecular mechanisms underlying these actions, we employed cDNA microarrays to study 1,25(OH)2D3-regulated gene expression in the LNCaP human prostate cancer cells.mRNA isolated from LNCaP cells treated with vehicle or 50 nM 1,25(OH)2D3 for various lengths of time were hybridized to microarrays carrying approximately 23,000 genes. Some of the putative target genes revealed by the microarray analysis were verified by real-time PCR assays.1,25(OH)2D3 most substantially increased the expression of the insulin-like growth factor binding protein-3 (IGFBP-3) gene. Our analysis also revealed several novel 1,25(OH)2D3-responsive genes. Interestingly, some of the key genes regulated by 1,25(OH)2D3 are also androgen-responsive genes. 1,25(OH)2D3 also down-regulated genes that mediate androgen catabolism.The putative 1,25(OH)2D3 target genes appear to be involved in a variety of cellular functions including growth regulation, differentiation, membrane transport, cell-cell and cell-matrix interactions, DNA repair, and inhibition of metastasis. The up-regulation of IGFBP-3 gene has been shown to be crucial in 1,25(OH)2D3-mediated inhibition of LNCaP cell growth. 1,25(OH)2D3 regulation of androgen-responsive genes as well as genes involved in androgen catabolism suggests that there are interactions between 1,25(OH)2D3 and androgen signaling pathways in LNCaP cells. Further studies on the role of these genes and others in mediating the anti-cancer effects of 1,25(OH)2D3 may lead to better approaches to the prevention and treatment of prostate cancer.

  View details for DOI 10.1002/pros.20006

  View details for Web of Science ID 000220910600003

  View details for PubMedID 15042599

• Identification of a functional vitamin D response element in the human insulin-like growth factor binding protein-3 promoter MOLECULAR ENDOCRINOLOGY Peng, L. H., Malloy, P. J., Feldman, D. 2004; 18 (5): 1109-1119

  Abstract

  1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] plays a critical role in maintaining calcium and phosphate homeostasis and bone formation but also exhibits antiproliferative activity on many cancer cells, including prostate cancer. We have shown that the antiproliferative actions of 1,25-(OH)2D3 in the LNCaP human prostate cancer cell line are mediated in part by induction of IGF binding protein-3 (IGFBP-3). The purpose of this study was to determine the molecular mechanism involved in 1,25-(OH)2D3 regulation of IGFBP-3 expression and to identify the putative vitamin D response element (VDRE) in the IGFBP-3 promoter. We cloned approximately 6 kb of the IGFBP-3 promoter sequence and demonstrated its responsiveness to 1,25-(OH)2D3 in transactivation assays. Computer analysis identified a putative VDRE between -3296/-3282 containing the direct repeat motif GGTTCA ccg GGTGCA that is 92% identical with the rat 24-hydroxylase distal VDRE. In EMSAs, the vitamin D receptor (VDR) showed strong binding to the putative IGFBP-3 VDRE in the presence of 1,25-(OH)2D3. Supershift assays confirmed the presence of VDR in the IGFBP-3 VDRE complex. Chromatin immunoprecipitation assay demonstrated that 1,25-(OH)2D3 recruited the VDR/retinoid X receptor heterodimer to the VDRE site in the natural IGFBP-3 promoter in intact cells. In transactivation assays, the putative VDRE coupled to a heterologous simian virus 40 promoter construct was induced 2-fold by 1,25-(OH)2D3. Mutations in the VDRE resulted in a loss of inducibility confirming the critical hexameric sequence. In conclusion, we have identified a functional VDRE in the distal region of the human IGFBP-3 promoter. The induction of IGFBP-3 by 1,25-(OH)2D3 appears to be directly mediated via VDR interaction with this VDRE.

  View details for DOI 10.1210/me.2003-0344

  View details for Web of Science ID 000221054400005

  View details for PubMedID 14963110

• Vitamin D growth inhibition of breast cancer cells: gene expression patterns assessed by cDNA microarray BREAST CANCER RESEARCH AND TREATMENT Swami, S., Raghavachari, N., Muller, U. R., Bao, Y. J., Feldman, D. 2003; 80 (1): 49-62

  Abstract

  1,25-Dihydroxyvitamin D3 [1,25(OH)2D3], the active metabolite of vitamin D, is a potent inhibitor of breast cancer cell growth. Although it is evident that 1,25(OH)2D3 inhibits growth of both estrogen receptor alpha-positive [ER alpha(+)] and -negative [ER alpha(-)] breast cancer cells, the cellular pathways contributing to these effects remain unclear. We studied the gene expression patterns in ER alpha(+) MCF-7 and ER alpha(-) MDA MB 231 human breast cancer cells following 1,25(OH)2D3 treatment, using cDNA expression arrays. Both cell lines showed a significant induction of the 1,25(OH)2D3-dependent 24-hydroxylase gene, a marker for the actions of 1,25(OH)2D3. In MCF-7 cells, 51 genes were up-regulated and 19 genes were down-regulated. The up-regulated genes encoded cell adhesion molecules, growth factors/modulators, steroid receptors/co-activators, cytokines, kinases and transcription factors. Of the up-regulated genes, 40% were implicated in cell cycle regulation and apoptosis and included cyclin G1 and cyclin I, p21-activated kinase-1 (PAK-1), p53, retinoblastoma like-2 [Rb2 (p130)], insulin-like growth factor binding protein-5 (IGFBP5) and caspases. Among the down-regulated genes were ER alpha, growth factors, cytokines and several kinases. Some of these results were confirmed by real-time PCR. In MDA MB 231 cells, 20 genes were up-regulated and 13 genes were down-regulated. Very few genes directly implicated in cell cycle regulation were up-regulated. The matrix metalloproteinases formed a major class of genes that were down-regulated in the MDA MB 231 cells. Seven genes were commonly up-regulated in both cell lines and these included transforming growth factor (TGFbeta2) and Rb2 (p130). In conclusion, the gene expression profiles of the two cell lines studied were different with a few overlapping genes suggesting that different cellular pathways might be regulated by 1,25(OH)2D3 to exert its growth inhibitory effects in ER alpha(+) and ER alpha(-) cells.

  View details for Web of Science ID 000183702500005

  View details for PubMedID 12889598

• Pathways mediating the growth-inhibitory actions of vitamin D in prostate cancer Conference on Nutritional Genomics and Proteomics in Cancer Prevention Peehl, D. M., Krishnan, A. V., Feldman, D. AMER SOC NUTRITIONAL SCIENCE. 2003: 2461S–2469S

  Abstract

  Vitamin D is emerging as an important dietary factor that affects the incidence and progression of many malignancies including prostate cancer. The active form of vitamin D, 1,25-dihydroxycholecalciferol [1,25(OH)(2)D(3)], inhibits the growth and stimulates the differentiation of prostate cancer cells. We have studied primary cultures of normal and cancer-derived prostatic epithelial cells as well as established human prostate cancer cell lines to elucidate the molecular pathways of 1,25(OH)(2)D(3) actions. These pathways are varied and appear to be cell specific. In LNCaP cells, 1,25(OH)(2)D(3) mainly causes growth arrest through the induction of insulin-like growth factor binding protein-3 and also stimulates apoptosis to a much smaller extent. We have used cDNA-microarray analyses to identify additional genes that are regulated by 1,25(OH)(2)D(3) and to raise novel therapeutic targets for use in the chemoprevention or treatment of prostate cancer. Less calcemic analogs of 1,25(OH)(2)D(3) that have more antiproliferative activity are being developed that will be more useful clinically. In target cells, 1,25(OH)(2)D(3) induces 24-hydroxylase, the enzyme that catalyzes its self inactivation. Cotreatment with 24-hydroxylase inhibitors enhances the antiproliferative activity of 1,25(OH)(2)D(3). The combination of other anticancer agents such as retinoids with vitamin D offers another promising therapeutic approach. A small clinical trial has shown that 1,25(OH)(2)D(3) can slow the rate of prostate-specific antigen increase in prostate cancer patients, which demonstrates proof of the concept that vitamin D or its analogs are clinically effective. Our research is directed at understanding the mechanisms of vitamin D action in prostate cells with the goal of developing chemoprevention and treatment strategies to improve prostate cancer therapy.

  View details for Web of Science ID 000184163200012

  View details for PubMedID 12840225

• The role of vitamin D and retinoids in controlling prostate cancer progression 11th International Congress on Hormonal Steroids/7th International Congress on Hormones and Cancer Peehl, D. M., Feldman, D. BIOSCIENTIFICA LTD. 2003: 131–40

  Abstract

  Prostate cancer is a leading cause of cancer-related deaths in many countries. Premalignant lesions and invasive cancer occur more frequently in the prostate than in any organ other than the skin. Yet, the incidence of clinically detected prostate cancer is much lower than the histopathological incidence. The slow growth of prostate cancer and the low incidence of clinically manifest disease in some geographical locations or racial/ethnic groups suggest that prostate cancer can be controlled, perhaps by dietary factors. Vitamin D and retinoids have emerged as leading candidates both to prevent and to treat prostate cancer. Many of the activities of these compounds, established from epidemiological studies, research with cell culture and animal models, and clinical trials, are consistent with tumor suppressor effects. However, retinoids may have additional tumor enhancer properties that balance or negate anti-cancer activity. This perhaps explains the overall lack of protective effects of vitamin A compounds against prostate cancer found in epidemiological studies, and the minimal efficacy of retinoids in clinical trials to treat prostate cancer. While current efforts focus on developing strategies to use vitamin D compounds to control prostate cancer, the possibility exists that prostate cancer cells may become resistant to tumor suppressor effects of vitamin D. Analyses of experimental model systems show that prostate cancer cells become less sensitive to vitamin D through loss of receptors or signaling molecules that mediate vitamin D's actions, or through changes in metabolic enzymes that synthesize or degrade vitamin D compounds. The potential promise of exploiting vitamin D to control prostate cancer is tempered by the possibility that prostate cancer, perhaps even at early stages, may develop mechanisms to escape tumor suppressor activities of vitamin D and/or retinoids.

  View details for Web of Science ID 000184151400003

  View details for PubMedID 12790775

• Inhibition of prostate cancer growth by vitamin D: Regulation of target gene expression JOURNAL OF CELLULAR BIOCHEMISTRY Krishnan, A. V., Peehl, D. M., Feldman, D. 2003; 88 (2): 363-371

  Abstract

  Prostate cancer (PCa) cells express vitamin D receptors (VDR) and 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) inhibits the growth of epithelial cells derived from normal, benign prostate hyperplasia, and PCa as well as established PCa cell lines. The growth inhibitory effects of 1,25(OH)(2)D(3) in cell cultures are modulated tissue by the presence and activities of the enzymes 25-hydroxyvitamin D(3) 24-hydroxylase which initiates the inactivation of 1,25(OH)(2)D(3) and 25-hydroxyvitamin D(3) 1alpha-hydroxylase which catalyses its synthesis. In LNCaP human PCa cells 1,25(OH)(2)D(3) exerts antiproliferative activity predominantly by cell cycle arrest through the induction of IGF binding protein-3 (IGFBP-3) expression which in turn increases the levels of the cell cycle inhibitor p21 leading to growth arrest. cDNA microarray analyses of primary prostatic epithelial and PCa cells reveal that 1,25(OH)(2)D(3) regulates many target genes expanding the possible mechanisms of its anticancer activity and raising new potential therapeutic targets. Some of these target genes are involved in growth regulation, protection from oxidative stress, and cell-cell and cell-matrix interactions. A small clinical trial has shown that 1,25(OH)(2)D(3) can slow the rate of prostate specific antigen (PSA) rise in PCa patients demonstrating proof of concept that 1,25(OH)(2)D(3) exhibits therapeutic activity in men with PCa. Further investigation of the role of calcitriol and its analogs for the therapy or chemoprevention of PCa is currently being pursued.

  View details for DOI 10.1002/jcb.10334

  View details for Web of Science ID 000180479100024

  View details for PubMedID 12520538

• A low-calcemic vitamin D analog (Ro 25-4020) inhibits the growth of LNCaP human prostate cancer cells with increased potency by producing an active 24-oxo metabolite (Ro 29-9970) 1st International Symposium on Vitamin D Analogs in Cancer Prevention and Therapy Swami, S., Zhao, X. Y., Sarabia, S., Siu-Caldera, M. L., Uskokovic, M., Reddy, S. G., Feldman, D. SPRINGER-VERLAG BERLIN. 2003: 349–352

  Abstract

  In this study, we have characterized a novel less-calcemic vitamin D analog Ro 25-4020 (1alpha, 25 dihydroxy-16-ene-5,6-trans-vitamin D3) and investigated the mechanisms underlying its enhanced growth inhibitory properties. We found that Ro 25-4020 (IC50 = 0.3 nM) exhibited greater inhibitory activity than 1,25(OH)2D3 (IC50 = 1 nM) on LNCaP human prostate cancer cell growth. However, Ro 25-4020 was tenfold less active than 1,25(OH)2D3 in receptor-binding assays, ligand-induced heterodimerization and transactivation assays using VDR. HPLC and GC-MS analyses revealed that 1,25(OH)2D3 is converted to a 24-hydroxy metabolite, which has been shown to be less potent than 1,25(OH)2D3. In contrast, Ro 25-4020 was converted to a major 24-oxo metabolite that was more stable. Ligand-binding assays reveal that both Ro 25-4020 and its 24-oxo metabolite have similar affinity for VDR. Synthetic 24-oxo-Ro 25-4020, however, inhibited LNCaP cell proliferation as potently as 1,25(OH)2D3 and was more potent in transactivation of two out of three vitamin D target genes tested. These results suggest that conversion of Ro 25-4020 into an active and more stable 24-oxo metabolite with longer half-life contributes significantly to its potent antiproliferative actions on the LNCaP cells.

  View details for Web of Science ID 000184907200024

  View details for PubMedID 12899533

• Vitamin D receptor start codon polymorphism (FokI) and prostate cancer progression CANCER EPIDEMIOLOGY BIOMARKERS & PREVENTION Xu, Y., Shibata, A., McNeal, J. E., Stamey, T. A., Feldman, D., Peehl, D. M. 2003; 12 (1): 23-27

  Abstract

  Vitamin D plays an important role in cell growth and differentiation and is proposed to protect against cancer initiation and/or progression. The vitamin D receptor (VDR) has a thymine/cytosine (T/C) polymorphism located in the first of two potential start (ATG) codons that can be detected by a RFLP using the endonuclease FokI. The C variant, which lacks the first ATG, results in a shorter VDR and is referred to as the F allele. The T variant (f allele) initiates at the first ATG. We examined the association of the VDR FokI genotype with histopathological characteristics and prognosis of prostate cancer among 191 mostly Caucasian subjects who had undergone radical prostatectomy between 1984 and 1992. The frequencies of the FF, Ff, and ff genotypes were 41%, 38%, and 21%, respectively. Subjects with the ff genotype had a lower mean percentage of Gleason grade 4/5 cancer (30.3%) than subjects with the FF or Ff genotypes (42.8% and 43.8%, respectively; P = 0.015 by t test for ff versus FF + Ff). The data suggest that the presence of an F allele increased the risk of being diagnosed with more aggressive cancer because higher percentage of Gleason grade 4/5 is associated with worse prognosis. The age-adjusted risk of prostate-specific antigen failure was lower for the ff genotype than for the FF genotype by Cox proportional hazards analysis but did not achieve statistical significance (hazard ratio = 0.76; 95% confidence interval, 0.44-1.32). This risk reduction disappeared after further adjustment for percentage of Gleason grade 4/5, cancer volume, and preoperative serum prostate-specific antigen level (hazard ratio = 1.03; 95% confidence interval, 0.58-1.85). In conclusion, the ff genotype was associated with less aggressive histopathological findings than Ff or FF genotypes. Additional studies with a larger sample size and investigation of the functional significance of the FokI polymorphism in prostate cancer cells are warranted.

  View details for PubMedID 12540499

• The role of vitamin D in prostate cancer 1st International Symposium on Vitamin D Analogs in Cancer Prevention and Therapy Krishnan, A. V., Peehl, D. M., Feldman, D. SPRINGER-VERLAG BERLIN. 2003: 205–221

  Abstract

  Prostate cancer (PCa) cells harbor receptors for vitamin D (VDR) as well as androgens (AR). 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] increases AR expression and enhances androgen actions linking the two receptor systems. 1,25(OH)2D3 exhibits antiproliferative activity in both AR-positive and AR-negative PCa cells. Less calcemic analogs of 1,25(OH)2D3, with more antiproliferative activity, are being developed and will be more useful clinically. The mechanisms underlying differential analog activity are being investigated. In target cells, 1,25(OH)2D3 induces 24-hydroxylase, the enzyme that catalyzes its self-inactivation. Co-treatment with 24-hydroxylase inhibitors enhances the antiproliferative activity of calcitriol. Primary cultures of normal or cancer-derived prostatic epithelial cells express 1alpha-hydroxylase, the enzyme that catalyzes the synthesis of 1,25(OH)2D3, the levels being much lower in the cancer-derived cells and in PCa cell lines. This finding raises the possibility of using 25-hydroxyvitamin D3 [25(OH)D3] as a chemopreventive agent in PCa. In LNCaP human PCa cells, 1,25(OH)2D3 and its analogs exert antiproliferative activity predominantly by cell cycle arrest, but also induce apoptosis, although to a much lesser degree. Growth arrest is mediated by induction of IGF binding protein-3 (IGFBP-3), which in turn increases the expression of the cell cycle inhibitor p21, leading to growth arrest. Other actions of 1,25(OH)2D3 in PCa cells include promotion of pro-differentiation effects and inhibition of tumor cell invasion, metastasis and angiogenesis. Combination therapy with retinoids, other anticancer agents or 24-hydroxylase inhibitors augments the inhibitory activity of 1,25(OH)2D3 in PCa and provides another effective approach in PCa treatment. Small clinical trials have shown that 1,25(OH)2D3 can slow the rate of prostate specific antigen (PSA) rise in PCa patients, demonstrating proof of concept that 1,25(OH)2D3 or its analogs will be clinically effective in PCa therapy. Current research involves further investigation of the role of 1,25(OH)2D3 and its analogs for the therapy or chemoprevention of PCa.

  View details for Web of Science ID 000184907200015

  View details for PubMedID 12899524

• Hereditary 1,25-Dihydroxyvitamin D-resistant rickets. Endocrine development Malloy, P. J., Feldman, D. 2003; 6: 175-199

  View details for PubMedID 12964432

• A novel nonsense mutation in the ligand binding domain of the vitamin D receptor causes hereditary 1,25-dihydroxyvitamin D-resistant rickets MOLECULAR GENETICS AND METABOLISM Malloy, P. J., Zhu, W. J., Bouillon, R., Feldman, D. 2002; 77 (4): 314-318

  Abstract

  Hereditary 1,25-dihydroxyvitamin D resistant rickets (HVDRR) is a genetic disorder most often caused by mutations in the vitamin D receptor (VDR). In this report, we present our findings on a young girl who exhibited the typical clinical features of HVDRR with early onset rickets, hypocalcemia, secondary hyperparathyroidism, and elevated serum concentrations of alkaline phosphatase and 1,25-dihydroxyvitamin D [1,25(OH)(2)D(3)]. The patient also had total body alopecia. Fibroblasts from the patient were cultured for analysis of the VDR structure and function. In [3H]1,25(OH)(2)D(3) binding assays, no significant specific binding to the VDR was observed in cytosols from the patient's fibroblasts. The patient's fibroblast were also totally resistant to high doses of 1,25(OH)(2)D(3) as demonstrated by their failure to induce expression of the 24-hydroxylase gene, a marker of 1,25(OH)(2)D(3) activity. DNA sequence analysis of the VDR gene uncovered a unique C to T mutation in exon 8. The mutation changed the codon for glutamine to a premature stop codon at amino acid 317 (Q317X). Restriction enzyme analysis showed that the patient was homozygous for the mutation. Both parents were heterozygous for the mutant allele. In conclusion, we have identified a novel mutation in the VDR, Q317X, as the molecular defect in a patient with HVDRR. The Q317X mutation deletes 110 amino acids of the ligand-binding domain of the VDR and results in the loss of [3H]1,25(OH)(2)D(3) binding and target gene transactivation.

  View details for Web of Science ID 000179793800007

  View details for PubMedID 12468277

• A novel mutation in helix 12 of the vitamin D receptor impairs coactivator interaction and causes hereditary 1,25-dihydroxyvitamin D-resistant rickets without alopecia MOLECULAR ENDOCRINOLOGY Malloy, P. J., Xu, R., Peng, L. H., Clark, P. A., Feldman, D. 2002; 16 (11): 2538-2546

  Abstract

  Hereditary vitamin D-resistant rickets (HVDRR) is a genetic disorder most often caused by mutations in the vitamin D receptor (VDR). The patient in this study exhibited the typical clinical features of HVDRR with early onset rickets, hypocalcemia, secondary hyperparathyroidism, and elevated serum concentrations of alkaline phosphatase and 1,25-dihydroxyvitamin D [1,25-(OH)(2)D(3)]. The patient did not have alopecia. Assays of the VDR showed a normal high affinity low capacity binding site for [(3)H]1,25-(OH)(2)D(3) in extracts from the patient's fibroblasts. However, the cells were resistant to 1,25-dihydroxyvitamin D action as demonstrated by the failure of the patient's cultured fibroblasts to induce the 24-hydroxylase gene when treated with either high doses of 1,25-(OH)(2)D(3) or vitamin D analogs. A novel point mutation was identified in helix H12 in the ligand-binding domain of the VDR that changed a highly conserved glutamic acid at amino acid 420 to lysine (E420K). The patient was homozygous for the mutation. The E420K mutant receptor recreated by site-directed mutagenesis exhibited many normal properties including ligand binding, heterodimerization with the retinoid X receptor, and binding to vitamin D response elements. However, the mutant VDR was unable to elicit 1,25-(OH)(2)D(3)-dependent transactivation. Subsequent studies demonstrated that the mutant VDR had a marked impairment in binding steroid receptor coactivator 1 (SRC-1) and DRIP205, a subunit of the vitamin D receptor-interacting protein (DRIP) coactivator complex. Taken together, our data indicate that the mutation in helix H12 alters the coactivator binding site preventing coactivator binding and transactivation. In conclusion, we have identified the first case of a naturally occurring mutation in the VDR (E420K) that disrupts coactivator binding to the VDR and causes HVDRR.

  View details for DOI 10.1210/me.2002-0152

  View details for PubMedID 12403843

• Preclinical activity of ketoconazole in combination with calcitriol or the vitamin D analogue EB 1089 in prostate cancer cells JOURNAL OF UROLOGY Peehl, D. M., Seto, E., Hsu, J. Y., Feldman, D. 2002; 168 (4): 1583-1588

  Abstract

  Ketoconazole is a general inhibitor of P450 enzymes, of which some are necessary for androgen biosynthesis and the metabolism of vitamin D compounds. We tested the growth inhibitory activity of ketoconazole combined with 1,25-dihydroxyvitamin D3 (calcitriol) and with the vitamin D analogue EB 1089 in a preclinical model of prostate cancer.Clonal assays with primary cultures of human prostatic cancer cells were performed to test anti-proliferative effects of ketoconazole alone or in combination with calcitriol or EB 1089. Enzyme substrate reactions were done to determine whether the ability of ketoconazole to potentiate the activity of calcitriol or EB 1089 was due to the inhibition of 25-hydroxyvitamin D3-24-hydroxylase (24-hydroxylase), the enzyme that initiates conversion of active vitamin D compounds to inactive products.Ketoconazole, calcitriol and EB 1089 each inhibited the growth of prostatic cancer cells. In combination 0.1 microg./ml. ketoconazole potentiated growth inhibitory activity of calcitriol 50-fold and EB 1089 10-fold. Induction of 24-hydroxylase by calcitriol or EB 1089 was partially blocked by this level of ketoconazole.Combination therapy with ketoconazole and calcitriol or EB 1089 may enhance antitumor activities of vitamin D compounds for prostate cancer and alleviate side effects of vitamin D deficiency that are likely associated with ketoconazole therapy.

  View details for DOI 10.1097/01.ju.0000030158.18335.84

  View details for Web of Science ID 000178187800086

  View details for PubMedID 12352462

• A glucocorticoid-responsive mutant androgen receptor exhibits unique ligand specificity: Therapeutic implications for androgen-independent prostate cancer ENDOCRINOLOGY Krishnan, A. V., Zhao, X. Y., Swami, S., Brive, L., Peehl, D. M., Ely, K. R., Feldman, D. 2002; 143 (5): 1889-1900

  Abstract

  The cortisol/cortisone-responsive AR (AR(ccr)) has two mutations (L701H and T877A) that were found in the MDA PCa human prostate cancer cell lines established from a castrated patient whose metastatic tumor exhibited androgen-independent growth. Cortisol and cortisone bind to the AR(ccr) with high affinity. In the present study, we characterized the structural determinants for ligand binding to the AR(ccr). Our data revealed that many of the C17, C19, and C21 circulating steroids, at concentrations that are found in vivo, functioned as effective activators of the AR(ccr) but had little or no activity via the wild-type AR or GRalpha. Among the synthetic glucocorticoids tested, dexamethasone activated both GRalpha and AR(ccr), whereas triamcinolone was selective for GRalpha. In MDA PCa 2b cells, growth and prostate-specific antigen production were stimulated by potent AR(ccr) agonists such as cortisol or 9alpha-fluorocortisol but not by triamcinolone (which did not bind to or activate the AR(ccr)). Of the potential antagonists tested, bicalutamide (casodex) and GR antagonist RU38486 showed inhibitory activity. We postulate that corticosteroids provide a growth advantage to prostate cancer cells harboring the promiscuous AR(ccr) in androgen-ablated patients and contribute to their transition to androgen-independence. We predict that triamcinolone, a commonly prescribed glucocorticoid, would be a successful therapeutic agent for men with this form of cancer, perhaps in conjunction with the antagonist casodex. We hypothesize that triamcinolone administration would inhibit the hypothalamic-pituitary-adrenal axis, thus suppressing endogenous corticosteroids, which stimulate tumor growth. Triamcinolone, by itself, would not activate the AR(ccr) or promote tumor growth but would provide glucocorticoid activity essential for survival.

  View details for Web of Science ID 000175170500037

  View details for PubMedID 11956172

• Rationale for combination ketoconazole/ vitamin D treatment of prostate cancer. Urology Peehl, D. M., Seto, E., Feldman, D. 2001; 58 (2): 123-126

  Abstract

  The high rate of progression of prostate cancer after androgen deprivation therapy mandates that new strategies be developed. Adjuvant therapy combined with androgen deprivation may slow or prevent progression. Ketoconazole plus calcitriol therapy is an example of 1 such a combination with a mechanistic basis for synergistic activity. Ketoconazole is commonly used as a second-line androgen deprivation therapy. This imidazole derivative is an inhibitor of P-450 enzymes, including those involved in steroidogenesis. Other P-450 enzymes that are inhibited by ketoconazole include 1alpha-hydroxylase and 24-hydroxylase, which metabolize vitamin D. Growth inhibition of prostate cancer cells by vitamin D depends on levels of the active metabolite, 1,25-dihydroxyvitamin D(3) (calcitriol). The enzyme 24-hydroxylase converts calcitriol to less active products. The inhibition of 24-hydroxylase by ketoconazole maintains the magnitude and duration of response to calcitriol. Combined ketoconazole/calcitriol therapy might therefore potentiate the antitumor activity of calcitriol. Because androgen-independent prostate cancer cells often remain responsive to growth inhibition by calcitriol, it is also possible that calcitriol would slow or prevent development of androgen-independent cancer growth. Another consideration is that ketoconazole blocks 1alpha-hydroxylase activity, which is the key enzyme that creates calcitriol in the body. Therefore, patients receiving ketoconazole therapy are likely to be deficient in vitamin D. The detrimental consequences of vitamin D deficiency in these patients would also be alleviated by the addition of calcitriol to the therapeutic regimen.

  View details for PubMedID 11502466

• Rationale for combination ketoconazole/vitamin D treatment of prostate cancer 1st International Conference on Newer Approaches to Androgen Deprivation Therapy in Prostate Cancer Peehl, D. M., Seto, E., Feldman, D. ELSEVIER SCIENCE INC. 2001: 123–26

  Abstract

  The high rate of progression of prostate cancer after androgen deprivation therapy mandates that new strategies be developed. Adjuvant therapy combined with androgen deprivation may slow or prevent progression. Ketoconazole plus calcitriol therapy is an example of 1 such a combination with a mechanistic basis for synergistic activity. Ketoconazole is commonly used as a second-line androgen deprivation therapy. This imidazole derivative is an inhibitor of P-450 enzymes, including those involved in steroidogenesis. Other P-450 enzymes that are inhibited by ketoconazole include 1alpha-hydroxylase and 24-hydroxylase, which metabolize vitamin D. Growth inhibition of prostate cancer cells by vitamin D depends on levels of the active metabolite, 1,25-dihydroxyvitamin D(3) (calcitriol). The enzyme 24-hydroxylase converts calcitriol to less active products. The inhibition of 24-hydroxylase by ketoconazole maintains the magnitude and duration of response to calcitriol. Combined ketoconazole/calcitriol therapy might therefore potentiate the antitumor activity of calcitriol. Because androgen-independent prostate cancer cells often remain responsive to growth inhibition by calcitriol, it is also possible that calcitriol would slow or prevent development of androgen-independent cancer growth. Another consideration is that ketoconazole blocks 1alpha-hydroxylase activity, which is the key enzyme that creates calcitriol in the body. Therefore, patients receiving ketoconazole therapy are likely to be deficient in vitamin D. The detrimental consequences of vitamin D deficiency in these patients would also be alleviated by the addition of calcitriol to the therapeutic regimen.

  View details for Web of Science ID 000170422300033

• A novel inborn error in the ligand-binding domain of the vitamin D receptor causes hereditary vitamin D-resistant rickets MOLECULAR GENETICS AND METABOLISM Malloy, P. J., Zhu, W. J., Zhao, X. Y., Pehling, G. B., Feldman, D. 2001; 73 (2): 138-148

  Abstract

  Mutations in the vitamin D receptor (VDR) cause hereditary vitamin D-resistant rickets (HVDRR), an autosomal recessive disease resulting in target organ resistance to 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)]. In this report, we describe the clinical case and molecular basis of HVDRR in an Asian boy exhibiting the typical clinical features of the disease including alopecia. Using cultured dermal fibroblasts from the patient, 1,25(OH)(2)D(3) resistance was demonstrated by a shift in the dose response required for 25-hydroxyvitamin D-24-hydroxylase (24-hydroxylase) mRNA induction. Western blot showed that the cells express a normal size VDR but contained reduced levels of receptor compared to normal cells. At 24 degrees C, the affinity of the patient's VDR for [(3)H]1,25(OH)(2)D(3) was 50-fold lower than the VDR in normal fibroblasts. Sequence analysis identified a unique T to G missense mutation in exon 6 that changed phenylalanine to cysteine at amino acid 251 (F251C). The recreated F251C mutant VDR showed reduced transactivation activity using a 24-hydroxylase promoter-luciferase reporter. Maximal transactivation activity exhibited by the WT VDR was not achieved by the mutant VDR even when the cells were treated with up to 10(-6) M 1,25(OH)(2)D(3). However, the transactivation activity was partially rescued by addition of RXRalpha. In the yeast two-hybrid system and GST-pull-down assays, high concentrations of 1,25(OH)(2)D(3) were needed to promote F251C mutant VDR binding to RXRalpha, indicating defective heterodimerization. In conclusion, a novel mutation was identified in the VDR LBD that reduces VDR abundance and its affinity for 1,25(OH)(2)D(3) and interferes with RXRalpha heterodimerization resulting in the syndrome of HVDRR.

  View details for Web of Science ID 000169323700003

  View details for PubMedID 11386849

• Insulin-like growth factor binding protein-3 mediates 1 alpha,25-dihydroxyvitamin D-3 growth inhibition in the LNCaP prostate cancer cell line through p21/WAF1 JOURNAL OF UROLOGY Boyle, B. J., Zhao, X. Y., Cohen, P., Feldman, D. 2001; 165 (4): 1319-1324

  Abstract

  We determined that insulin-like growth factor binding protein 3 (IGFBP-3) induction by 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) is a necessary component of 1,25-(OH)(2)D(3) mediated growth inhibition of the LNCaP human prostate cancer cell line. In addition, induction of the cyclin dependent kinase inhibitory protein p21/WAF/CIP1 by 1,25-(OH)(2)D(3) is mediated by IGFBP-3.Induction of IGFBP-3 by 1,25-(OH)(2)D(3) was determined by enzyme-linked immunosorbent assay for IGFBP-3 protein and by Northern blot analysis for IGFBP-3 messenger (m) RNA. Growth assays for LNCaP cells were determined by measuring DNA content. The contribution of IGFBP-3 toward 1,25-(OH)(2)D(3) mediated growth inhibition was determined by adding either antisense oligonucleotides or immuno-neutralizing antibodies to culture media of growth assays. Regulation of p21/WAF/CIP1 was determined by Western blot analysis.Adding 1,25-(OH)(2)D(3) to LNCaP prostate cancer cells demonstrated that 1,25-(OH)(2)D(3) significantly up-regulated IGFBP-3 at the mRNA and protein levels in these cells approximately 3-fold over control levels. Also, adding IGFBP-3 protein to LNCaP cell growth medium inhibited LNCaP cell growth. Interestingly adding IGFBP-3 antisense oligonucleotides or antibodies directed toward IGFBP-3 abolished the growth inhibitory actions of 1,25-(OH)(2)D(3), indicating that this effect is IGFBP-3 dependent. Furthermore, to connect the mechanisms of IGFBP-3 and 1,25-(OH)(2)D(3) mediated growth inhibition we demonstrated that IGFBP-3 up-regulates the expression of p21/WAF1 protein to approximately 2-fold over the control level. Adding an IGFBP-3 immuno-neutralizing antibody completely prevented the 1,25-(OH)(2)D(3) induced up-regulation of p21/WAF1.1,25-(OH)(2)D(3) up-regulates IGFBP-3 in the LNCaP cell line at the mRNA and protein levels. The growth inhibitory action of 1,25-(OH)(2)D(3) on LNCaP cells depends on active IGFBP-3, as evidenced by the loss of growth inhibition induced by IGFBP-3 antisense oligonucleotide and immuno-neutralization experiments. A possible connection between IGFBP-3 and 1,25-(OH)(2)D(3) lies in the cyclin dependent kinase inhibitory protein p21/WAF1 since IGFBP-3 and 1,25-(OH)(2)D(3) each up-regulate this protein and both inhibit LNCaP cell growth. Therefore, we hypothesize that the mechanism of action by which IGFBP-3 and 1,25-(OH)(2)D(3) induce growth inhibition is the induction of p21/WAF1 because IGFPB-3 immuno-neutralizing antibodies completely abrogate the 1,25-(OH)(2)D(3) mediated up-regulation of p21/WAF1 and growth inhibition.

  View details for Web of Science ID 000167503700095

  View details for PubMedID 11257709

• Reduced 1 alpha-hydroxylase activity in human prostate cancer cells correlates with decreased susceptibility to 25-hydroxyvitamin D-3-induced growth inhibition CANCER RESEARCH Hsu, J. Y., Feldman, D., McNeal, J. E., Peehl, D. M. 2001; 61 (7): 2852-2856

  Abstract

  Evidence from epidemiological, molecular, and genetic studies suggests a role for vitamin D in the development and/or progression of prostate cancer. In experimental models and clinical trials, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] was shown to exert antiproliferative, prodifferentiating, and antimetastatic/invasive effects on prostatic epithelial cells. Because the direct clinical application of 1,25(OH)2D3 is limited by the major side effect of hypercalcemia, we investigated the potential therapeutic utility of its less calcemic precursor, 25-hydroxyvitamin D3 [25(OH)D3], which is converted locally within the prostate to 1,25(OH)2D3 by 1alpha-hydroxylase. Quantification of 1alpha-hydroxylase activity in human prostatic epithelial cells by enzyme-substrate reaction analyses revealed a significantly decreased activity in cells derived from adenocarcinomas compared with cells derived from normal tissues or benign prostatic hyperplasia (BPH). In growth assays, we found that 25(OH)D3 inhibited growth of normal or BPH cells similarly to 1,25(OH)2D3. In contrast, in primary cultures of cancer cells and established cell lines, the antiproliferative action of 25(OH)D3 was significantly less pronounced than that of 1,25(OH)2D3. Our results indicate that growth inhibition by 25(OH)D3 depends on endogenous 1alpha-hydroxylase activity, and that this activity is deficient in prostate cancer cells. This finding has ramifications for both the prevention and therapy of prostate cancer with vitamin D compounds.

  View details for Web of Science ID 000167967100012

  View details for PubMedID 11306457

• Estradiol inhibits glucocorticoid receptor expression and induces glucocorticoid resistance in MCF-7 human breast cancer cells JOURNAL OF STEROID BIOCHEMISTRY AND MOLECULAR BIOLOGY Krishnan, A. V., Swami, S., Feldman, D. 2001; 77 (1): 29-37

  Abstract

  Our study has shown that treatment of MCF-7 human breast cancer cells with 17-beta estradiol (E(2)) produced significant decreases in glucocorticoid receptor (GR) concentrations and GR mRNA levels. E(2) pre-treatment of MCF-7 cells stably transfected with the GR responsive pMTV-CAT reporter (MCF-7-MTV cells), caused significant attenuation of dexamethasone (DEX)-induced chloramphenicol acetyl transferase (CAT). In MCF-7 cells transiently transfected with [(GRE)(3)-Luc] reporter plasmid, E(2) pre-treatment significantly suppressed DEX-induced luciferase, which was abolished by the estrogen receptor antagonist ICI 182,780. We examined the effect of chronic E(2) treatment as well as E(2) withdrawal on GR function and abundance. MCF-7-MTV cells were treated with vehicle (control) or E(2) for up to 16 days. A third group received E(2) for 5 days followed by E(2) withdrawal from day 6 to 16. Chronic E(2) treatment almost totally abrogated DEX-induced CAT and reduced GR to very low levels. Interestingly, in the group subjected to E(2) withdrawal, neither the DEX response nor GR abundance recovered and reached control values suggesting that the estrogen mediated suppression is long lasting and could not be easily reversed. The E(2) induced resistance to glucocorticoid action may be of potential clinical significance in a number of settings including breast cancer, neuroendocrine response to stress and osteoporosis and could possibly contribute to the differences in glucocorticoid responsiveness among patients.

  View details for Web of Science ID 000169515600004

  View details for PubMedID 11358672

• The role of vitamin D in prostate cancer 1st International Conference on Chemistry and Biology of Vitamin D Analogs Zhao, X. Y., Feldman, D. ELSEVIER SCIENCE INC. 2001: 293–300

  Abstract

  Prostate cancer is the second leading cause of cancer deaths in men in the United States. Developing new treatment strategies is critical to improving the health of men. This article will be a general review of the field with a focus on research from our laboratory. Our research has focused on four areas in which we have pursued the possible use of 1alpha,25(OH)(2)D(3) and its analogs to treat prostate cancer: 1) The ability of 1alpha,25(OH)(2)D(3) to up-regulate androgen receptors in LNCaP human prostate cancer cells. The implications of this finding on 1alpha,25(OH)(2)D(3)'s ability to inhibit cell growth in vivo are unclear at present.2) The reasons for an inability of 1alpha,25(OH)(2)D(3) to inhibit DU 145 prostate cancer cell growth were explored. We found that combination of an imidazole drug, Liarozole, with 1alpha,25(OH)(2)D(3) was capable of inhibiting DU 145 cell growth.3) A number of low-calcemic vitamin D analogs exhibit potent anti-proliferative activity on prostate cancer cells. We have developed a novel approach using the yeast two-hybrid system to screen for potent analogs.4) The results of a clinical trial of 1alpha,25(OH)(2)D(3) treatment of patients with early recurrent prostate cancer. We provide preliminary evidence that 1alpha,25(OH)(2)D(3) may be effective in slowing the rate of PSA rise in selected cases of prostate cancer. In conclusion, we believe that 1alpha,25(OH)(2)D(3) has a role in the treatment and/or prevention strategies being developed for prostate cancer. However, to increase antiproliferative potency without increasing side-effects, the use of less calcemic analogs appears to be the most reasonable approach.

  View details for Web of Science ID 000167537700019

  View details for PubMedID 11179737

• A new enzyme-linked immunosorbant assay for the measurement of human vitamin D receptor BONE Swami, S., Sarabia, S. F., Diamandis, A., Mistry, J., Khosravi, J., Feldman, D. 2001; 28 (3): 319-326

  Abstract

  The hormonal actions of 1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] are mediated by its cognate receptor protein, the vitamin D receptor (VDR). Despite the growing importance of the VDR system as a modulator of cell growth and differentiation, convenient assays for quantitative measurement of VDR are not readily available, and [(3)H]1,25(OH)(2)D(3) ligand binding assays remain the standard method. In this paper, we present data to validate and characterize the usefulness of a new VDR enzyme-linked immunosorbant assay (ELISA) kit developed for the measurement of VDR in biological samples. In this assay, samples are added to microtitration wells coated with anti-VDR antibody and incubated with a second anti-VDR antibody that is biotinylated. The antibody receptor complex is then detected with streptavidin-labeled horseradish peroxidase followed by incubation with a chromogenic substrate, tetramethylbenzidine. The assay was found to be sensitive and accurate for measurements of VDR and compared favorably with the conventional radioligand binding assay (RBA). The interassay variation ranged from 5% to 25% and the intraassay variation was less than 5%. The ELISA presents several advantages over existing methodology, including the use of nonradioactive detection systems, lower protein and sample volume requirements, as well as convenience and speed. The assay can be completed in as short a time as 3 h, avoiding overnight incubations. Data are also presented to demonstrate the ability of the ELISA to detect both occupied and unoccupied VDR, making it a valuable research tool in settings where 1,25(OH)(2)D(3) is present. However, the ELISA, as currently formulated, is only useful for the detection of human VDR.

  View details for Web of Science ID 000167798200012

  View details for PubMedID 11248664

• 1 alpha,25-dihydroxyvitamin D-3 inhibits prostate cancer cell growth by androgen-dependent and androgen-independent mechanisms ENDOCRINOLOGY Zhao, X. Y., Peehl, D. M., Navone, N. M., Feldman, D. 2000; 141 (7): 2548-2556

  Abstract

  We recently reported that 1alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] inhibits the growth of the LNCaP human prostate cancer cell line by an androgen-dependent mechanism. In the present study we examined the actions and interactions of 1,25-(OH)2D3 and the androgen 5alpha-dihydrotestosterone (DHT) on two new human prostate cancer cell lines (MDA), MDA PCa 2a and MDA PCa 2b. Scatchard analyses revealed that both cell lines express high affinity vitamin D receptors (VDRs) with a binding affinity (Kd) for [3H]1,25-(OH)2D3 of 0.1 nM. However, the MDA cell lines contain low affinity androgen receptors (ARs) with a Kd of 25 nM for [3H]DHT binding. This is 50-fold lower than the AR in LNCaP cells (Kd = 0.5 nM). Their response to DHT is greatly reduced; 2a cells do not respond to 100 nM DHT, and 2b cells show a modest response at that high concentration. 1,25-(OH)2D3 causes significant growth inhibition in both MDA cell lines, greater (for 2b cells) or lesser (for 2a cells) than that in the LNCaP cell line. Moreover, 1,25-(OH)2D3 significantly up-regulates AR messenger RNA in all three cell lines, as shown by Northern blot analysis. The growth inhibitory effect of 1,25-(OH)2D3 on LNCaP cells is blocked by the pure antiandrogen, Casodex, as we previously reported. However, Casodex (at 1 microM) did not block the antiproliferative activity of 1,25-(OH)2D3 in MDA cells. In conclusion, the growth inhibitory action of 1,25-(OH)2D3 in the MDA cell lines appears to be androgen independent, whereas the actions of 1,25-(OH)2D3 in LNCaP cells are androgen dependent. Most importantly, the MDA cell lines, derived from a bone metastasis of human prostate carcinoma, remain sensitive to 1,25-(OH)2D3, a finding relevant to the therapeutic application of vitamin D and its low calcemic analogs in the treatment of advanced prostate cancer.

  View details for Web of Science ID 000088386200032

  View details for PubMedID 10875257

• 1,25-dihydroxyvitamin D-3 decreases human prostate cancer cell adhesion and migration MOLECULAR AND CELLULAR ENDOCRINOLOGY Sung, V., Feldman, D. 2000; 164 (1-2): 133-143

  Abstract

  1,25-Dihydroxyvitamin-D3 [1,25(OH)2D3], the active hormonal metabolite of vitamin D, acts through a specific nuclear receptor to inhibit proliferation and promote differentiation of several tumor cell types including the LNCaP, DU145 and PC-3 prostate cancer cell lines as well as primary prostate tumor lines. 1,25(OH)2D3 can also decrease invasion of breast and prostate cancer cell lines in vitro. We confirm this latter finding in the DU145 and PC-3 prostate cancer cell lines, and further show that 1,25(OH)2D3 inhibits overall invasion, cell adhesion and migration to the basement membrane matrix protein laminin. These changes appear to be due in part to a 1,25(OH)2D3-induced decrease in expression of alpha6 and beta4 integrins, both of which are receptors for laminin and associated with increased migration and invasion of prostate cancer cells in vitro. Blocking function of these particular integrins with antibodies inhibits both adhesion and migration of the cells. Collectively, these data demonstrate that 1,25(OH)2D3, in addition to decreasing proliferation of tumor cells, can also inhibit prostate cancer cell invasion through modulation of select cell surface adhesion molecules.

  View details for Web of Science ID 000088848400014

  View details for PubMedID 11026565

• Vitamin D, parathyroid hormone, and calcium: A complex regulatory network AMERICAN JOURNAL OF MEDICINE Feldman, D. 1999; 107 (6): 637-639

  View details for Web of Science ID 000084119900017

  View details for PubMedID 10625035

• Two mutations identified in the androgen receptor of the new human prostate cancer cell line MDA PCa 2a JOURNAL OF UROLOGY Zhao, X. Y., Boyle, B., Krishnan, A. V., Navone, N. M., Peehl, D. M., Feldman, D. 1999; 162 (6): 2192-2199

  Abstract

  We have characterized the androgen receptor (AR) in a new human prostate cancer cell line, MDA PCa 2a, that has recently been established from a bone metastasis of a patient whose cancer exhibited androgen-independent growth.Androgen responsiveness of these cells was assessed by measuring the effect of DHT and R1881 on cell growth and PSA secretion. Scatchard analysis was used to characterize the affinity and abundance of AR protein. Using a PCR based strategy, genomic DNA of the entire coding region of AR gene was sequenced to identify possible mutations.These cells express abundant AR (Nmax = 685 +/- 149 fmol./mg. protein), but the AR binding affinity (Kd) for DHT is only 25 nM, approximately 50-fold lower affinity than the mutated AR in LNCaP prostate cancer cells (Kd = 0.5 nM) or the wildtype AR in MCF-7 breast cancer cells (Kd = 0.4 nM). Two mutations, L701H and T877A, were identified in the ligand binding domain of the AR gene. Compared with LNCaP cells, the new cell line is significantly less responsive to DHT and R1881 as well as to other androgens such as testosterone, androstenedione, and DHEA. Similar to LNCaP cells, the ligand specificity of the AR in MDA PCa 2a cells appears to be relaxed and non-androgens such as progesterone and estradiol act as agonists although with less potency than in LNCaP cells. Interestingly, in the absence of androgens, the new cell line expresses 15-fold higher baseline levels of PSA than LNCaP.Two mutations were identified in the AR gene of the MDA PCa 2a cell line that are likely responsible for the decreased androgen sensitivity and altered ligand specificity observed in these cells. Thus, this new cell line with partial androgen responsiveness and PSA expression can serve as a functionally relevant model system of bone metastatic prostate cancer, and can be used to investigate the role of AR mutations in prostate cancer and its progression to androgen independence.

  View details for Web of Science ID 000083639500119

  View details for PubMedID 10569618

• PPAR gamma agonists enhance human vascular endothelial adhesiveness by increasing ICAM-1 expression BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Chen, N. G., Sarabia, S. F., Malloy, P. J., Zhao, X. Y., Feldman, D., Reaven, G. M. 1999; 263 (3): 718-722

  Abstract

  Early atherosclerotic lesions are characterized by increased monocyte adhesion to the overlying endothelium. Oxidized LDL (oxLDL) stimulates the adhesion of human monocytes to endothelial cells, in part, by increasing expression of ICAM-1. However, the cellular role of oxLDL in endothelial adhesiveness is not well understood. The peroxisome proliferator-activated receptor gamma (PPARgamma), a member of the nuclear receptor superfamily, is expressed in vascular endothelial cells. Whether it can be activated by a synthetic ligand, troglitazone, as well as by natural ligands, oxLDL and its lipid components (i.e., 9- and 13-HODE), has not yet been explored. This study was undertaken to determine whether PPARgamma is expressed in ECV304 human vascular endothelial cells and if so to define the biological effects of its activation by these agonists. Our results demonstrate that PPARgamma mRNA is expressed in ECV304 cells, and transfected cells with a PPARE luciferase construct respond to these agonists. In addition, ligand-dependent PPARgamma activation increased ICAM-1 protein expression and enhanced adherence of monocytes to ECV304 cells by two- to threefold. These findings suggest that the PPARgamma signaling pathway might contribute to the atherogenicity of oxLDL in vascular endothelial cells.

  View details for Web of Science ID 000083018100023

  View details for PubMedID 10512746

• Liarozole acts synergistically with 1 alpha,25-dihydroxyvitamin D-3 to inhibit growth of DU 145 human prostate cancer cells by blocking 24-hydroxylase activity ENDOCRINOLOGY Ly, L. H., Zhao, X. Y., Holloway, L., Feldman, D. 1999; 140 (5): 2071-2076

  Abstract

  1Alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] inhibits the proliferation of many cancer cells in culture, but not the aggressive human prostate cancer cell line DU 145. We postulated that the 1,25-(OH)2D3-resistant phenotype in DU 145 cells might result from the high levels of expression of 25-hydroxyvitamin D-24-hydroxylase (24-hydroxylase) induced by treatment with 1,25-(OH)2D3. As this P450 enzyme initiates 1,25-(OH)2D3 catabolism, we presumed that a high level of enzyme induction could limit the effectiveness of the 1,25-(OH)2D3 antiproliferative action. To examine this hypothesis we explored combination therapy with liarozole fumarate (R85,246), an imidazole derivative currently in trials for prostate cancer therapy. As imidizole derivatives are known to inhibit P450 enzymes, we postulated that this drug would inhibit 24-hydroxylase activity, increasing the 1,25-(OH)2D3 half-life, thereby enhancing 1,25-(OH)2D3 antiproliferative effects on DU 145 cells. Cell growth was assessed by measurement of viable cells using the MTS assay. When used alone, neither 1,25-(OH)2D3 (1-10 nM) nor liarozole (1-10 microM) inhibited DU 145 cell growth. However, when added together, 1,25-(OH)2D3 (10 nM)/liarozole (1 microM) inhibited growth 65% after 4 days of culture. We used a TLC method to assess 24-hydroxylase activity and demonstrated that liarozole (1-100 microM) inhibited this P450 enzyme in a dose-dependent manner. Moreover, liarozole treatment caused a significant increase in 1,25-(OH)2D3 half-life from 11 to 31 h. In addition, 1,25-(OH)2D3 can cause homologous up-regulation of the vitamin D receptor (VDR), and in the presence of liarozole, this effect was amplified, thus enhancing 1,25-(OH)2D3 activity. Western blot analyses demonstrated that DU 145 cells treated with 1,25-(OH)2D3/liarozole showed greater VDR up-regulation than cells treated with either drug alone. In summary, our data demonstrate that liarozole augments the ability of 1,25-(OH)2D3 to inhibit DU 145 cell growth. The mechanism appears to be due to inhibition of 24-hydroxylase activity, leading to increased 1,25-(OH)2D3 half-life and augmentation of homologous up-regulation of VDR. We raise the possibility that combination therapy using 1,25-(OH)2D3 and liarozole or other inhibitors of 24-hydroxylase, both in nontoxic doses, might serve as an effective treatment for prostate cancer.

  View details for Web of Science ID 000079801200015

  View details for PubMedID 10218956

• Vitamin D resistance AMERICAN JOURNAL OF MEDICINE Malloy, P. J., Feldman, D. 1999; 106 (3): 355-370

  View details for Web of Science ID 000079310300014

  View details for PubMedID 10190386

• Induction of androgen receptor by 1 alpha,25-dihydroxyvitamin D-3 and 9-cis retinoic acid in LNCaP human prostate cancer cells ENDOCRINOLOGY Zhao, X. Y., Ly, L. H., Peehl, D. M., Feldman, D. 1999; 140 (3): 1205-1212

  Abstract

  We have recently shown that 1alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] inhibits proliferation of LNCaP cells, an androgen-responsive human prostate cancer cell line. Also, 1,25-(OH)2D3 increases androgen receptor (AR) abundance and enhances cellular responses to androgen in these cells. In the current study, we have investigated the mechanism by which 1,25-(OH)2D3 regulates AR gene expression and the involvement of AR in the 1,25-(OH)2D3- and 9-cis retinoic acid (RA)-mediated growth inhibition of LNCaP cells. Northern blot analyses demonstrated that the steady-state messenger RNA (mRNA) level of AR was significantly increased by 1,25-(OH)2D3 in a dose-dependent manner. Time-course experiments revealed that the increase of AR mRNA by 1,25-(OH)2D3 exhibited delayed kinetics. In response to 1,25-(OH)2D3, AR mRNA levels were first detected to rise at 8 h and reached a maximal induction of 10-fold over the untreated control at 48 h; the effect was sustained at 72 h. Furthermore, the induction of AR mRNA by 1,25-(OH)2D3 was completely abolished by incubation of cells with cycloheximide, a protein synthesis inhibitor. 1,25-(OH)2D3 was unable to induce expression of an AR promoter-luciferase reporter. Together, these findings indicate that the stimulatory effect of 1,25-(OH)2D3 on AR gene expression is indirect. Western blot analyses showed an increase of AR protein in 1,25-(OH)2D3-treated cells. This increased expression of AR was followed by 1,25-(OH)2D3-induced inhibition of growth in LNCaP cells. Similar to 1,25-(OH)2D3, 9-cis RA also induced AR mRNA expression, and the effect of both hormones was additive. Moreover, 1,25-(OH)2D3 and 9-cis RA acted synergistically to inhibit LNCaP cell growth. These antiproliferative effects of 1,25-(OH)2D3 and 9-cis RA, alone or in combination, were blocked by the pure AR antagonist, Casodex. In conclusion, our results demonstrate that growth inhibition of LNCaP cells by 1,25-(OH)2D3 and 9-cis RA is mediated by an AR-dependent mechanism and preceded by the induction of AR gene expression. This finding, that differentiating agents such as vitamin D and A derivatives are potent inducers of AR, may have clinical implications in the treatment of prostate cancer.

  View details for Web of Science ID 000078733700025

  View details for PubMedID 10067845

• The vitamin D receptor gene start codon polymorphism: A functional analysis of FokI variants 19th Annual Meeting of the American-Society-for-Bone-and-Mineral-Research Gross, C., Krishnan, A. V., Malloy, P. J., Eccleshall, T. R., Zhao, X. Y., Feldman, D. WILEY-BLACKWELL. 1998: 1691–99

  Abstract

  The vitamin D receptor (VDR) gene contains a start codon polymorphism (SCP) which is three codons upstream of a second start site (ATG). The SCP genotype can be determined with the restriction enzyme FokI, where "f" indicates the presence of the restriction site and the first ATG, while "F" indicates its absence. Recent evidence suggests that the ff genotype is correlated with lower bone mineral density (BMD) in some populations. The SCP results in alternate VDRs that differ structurally, with the F variant (F-VDR) being three amino acids shorter than the f variant (f-VDR). To determine whether there are functional differences between the f-VDR and the F-VDR, we studied the two VDR forms expressed in COS-7 cells. The proteins were distinguishable from one another on Western blots by their different mobilities, confirming the larger size of f-VDR. Ligand binding studies showed no significant differences between the affinities of the two VDR forms for [3H]-1,25-dihydroxyvitamin D3 ([3H]-1,25(OH)2D3) (Kd = 131+/-78 pM, f-VDR; Kd = 237+/-190 pM, F-VDR; p = 0.24); however, a 2-fold difference in affinity can not be discriminated by this method. There were no differences in the abilities of the two receptor forms to bind DNA as determined by electrophoretic mobility shift assays. The ability of the two VDR forms to transactivate target genes was investigated using three different vitamin D responsive luciferase reporter constructs: 24-hydroxylase, osteocalcin, and osteopontin. In these transactivation experiments, 1,25(OH)2D3 dose-response (0.1-10 nM) curves revealed that the ED50 values for transactivation were indistinguishable between the two VDR forms. Additionally, cultured human fibroblasts with FF, Ff, and ff genotypes had similar sensitivity to 1,25(OH)2D3 with respect to the induction of 24-hydroxylase mRNA. In summary, we were unable to detect significant differences in ligand affinity, DNA binding, or transactivation activity between f-VDR and F-VDR forms. We must emphasize, however, that the sensitivity of the methods used limits our ability to detect minor differences in VDR affinity and function. In conclusion, we cannot define a mechanism whereby the SCP in the VDR might contribute to population differences in BMD.

  View details for Web of Science ID 000076526500008

  View details for PubMedID 9797477

• Hereditary 1,25-dihydroxyvitamin D-resistant rickets due to an opal mutation causing premature termination of the vitamin D receptor JOURNAL OF BONE AND MINERAL RESEARCH Zhu, W. J., Malloy, P. J., Delvin, E., Chabot, G., Feldman, D. 1998; 13 (2): 259-264

  Abstract

  Mutations in the vitamin D receptor (VDR) gene have been shown to cause hereditary vitamin D-resistant rickets (HVDRR). The patient in this study is a young French-Canadian boy with no known consanguinity in his family. The child exhibited the clinical characteristics of HVDRR with early onset rickets, hypocalcemia, secondary hyperparathyroidism, and elevated 1,25-dihydroxyvitamin D (1,25(OH)2D) levels as well as total alopecia. Fibroblasts were cultured from a skin biopsy of the patient and used to assess the VDR. Northern blot analysis showed that a normal size VDR transcript was expressed; however, [3H]1,25(OH)2D3-binding levels were very low and Western blot analysis failed to detect any VDR protein. Total resistance to 1,25(OH)2D3 was demonstrated by the failure of the cultured fibroblasts to induce the transcription of the 25-hydroxyvitamin D-24-hydroxylase gene when treated with high concentrations of 1,25(OH)2D3. Analysis of the DNA sequence revealed a unique C to T base change corresponding to nucleotide 218 of the VDR cDNA. This single base substitution changes the codon for arginine (CGA) to an opal stop codon (TGA), resulting in the truncation of the VDR at amino acid 30. The Arg30stop mutation prematurely terminates translation and deletes 398 amino acids including most of the zinc fingers as well as the entire ligand-binding domain. Restriction fragment length polymorphism analysis of a DdeI restriction site created by the mutation showed that the parents were heterozygous for the mutant allele. In conclusion, the Arg30stop mutation truncates the VDR and leads to a hormone-resistant condition which is the molecular basis of HVDRR in this patient.

  View details for Web of Science ID 000071831900012

  View details for PubMedID 9495519

• Vitamin D receptor gene polymorphisms: Analysis of ligand binding and hormone responsiveness in cultured skin fibroblasts BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Gross, C., Musiol, I. M., Eccleshall, T. R., Malloy, P. J., Feldman, D. 1998; 242 (3): 467-473

  Abstract

  Recent reports have suggested that polymorphisms in the gene encoding the vitamin D receptor (VDR) determine a portion of the genetic contribution to bone mineral density (BMD). Individuals homozygous for the allele lacking the Bsm I restriction site in the intron between exons 8 and 9 (BB genotype) have been found to have lower BMD than individuals homozygous for the allele having the Bsm I site (bb genotype). Interestingly, this polymorphism has also been associated with prostate cancer risk. The observed changes in BMD and prostate cancer risk might be due to an alteration in the function or abundance of the VDR leading to differential responsiveness of target cells to the action of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. To test this hypothesis, we cultured dermal fibroblasts from donors with BB, Bb, and bb genotypes and determined the level of VDR expression and the cellular responsiveness to 1,25(OH)2D3 treatment. VDR abundance, affinity for [3H]1,25(OH)2D3, and VDR mRNA levels were not detectably different in BB cells compared to bb cells. Moreover, equal expression of both VDR gene alleles was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) on mRNA from Bb fibroblasts. Fibroblast responsiveness to 1,25(OH)2D3, assessed by induction of 24-hydroxylase mRNA, was similar between BB and bb cell types in dose-response experiments. Although there were individual variations in the parameters we measured, there were no detectable or consistent differences in mean values from our small sample of cultured dermal fibroblasts. In conclusion, we did not detect significant differences in VDR properties or cellular responsiveness to 1,25(OH)2D3 that correlated with VDR genotype. Our findings suggest that these polymorphisms do not affect VDR function, but rather may be a marker for a nearby gene that is responsible for the genotype-associated variation in osteoporosis and prostate cancer risk.

  View details for Web of Science ID 000071824800002

  View details for PubMedID 9464239

• Lack of correlation between start codon polymorphism of the vitamin D receptor gene and bone mineral density in premenopausal French women: The OFELY study JOURNAL OF BONE AND MINERAL RESEARCH Eccleshall, T. R., Garnero, P., Gross, C., Delmas, P. D., Feldman, D. 1998; 13 (1): 31-35

  Abstract

  Previous studies have demonstrated an association between bone mineral density (BMD) and a start codon polymorphism (SCP) of the vitamin D receptor (VDR) gene in pre- and postmenopausal Caucasian and Japanese women. The SCP can be determined by a restriction fragment length polymorphism defined by the FokI restriction endonuclease. VDR alleles containing the FokI site are denoted by f and alleles lacking the site by F. In this study, the association between BMD and the SCP was examined in a group of 174 premenopausal French women who previously had been studied for a relationship between BMD and the VDR BsmI polymorphism. The SCP genotypes of the French women were FF 40%, Ff 44%, and ff 16% and they were independent of the BsmI genotype. BMD was measured by dual-energy X-ray absorptiometry at the lumbar spine, proximal femur, forearm, and total body. In contrast to previous reports, there was no association of BMD with SCP genotype in this group of Caucasian women at any site. We also measured several biochemical indices of calcium homeostasis and bone turnover. We found no statistically significant associations between SCP genotype and calcium, parathyroid hormone, or vitamin D levels. There was a 33.5% higher level of the skeletal resorption marker N-telopeptides of type I collagen in the women with the ff genotype when compared with women with the FF genotype (p = 0.004). Other bone turnover markers failed to show an association with SCP genotype. In summary, the SCP genotype may not be associated with reduced BMD in all geographical or ethnic populations.

  View details for Web of Science ID 000071209800005

  View details for PubMedID 9443787

• 1 alpha 25-dihydroxyvitamin D-3 actions in LNCaP human prostate cancer cells are androgen-dependent ENDOCRINOLOGY Zhao, X. Y., Ly, L. H., Peehl, D. M., Feldman, D. 1997; 138 (8): 3290-3298

  Abstract

  We and others have recently shown that 1alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] significantly inhibits cell proliferation and increases secretion of prostate-specific antigen (PSA) in LNCaP cells, an androgen-responsive human prostate cancer cell line. The present study was designed to investigate the possible interactions between 1,25-(OH)2D3 and androgens in the regulation of LNCaP cellular function. LNCaP cell growth was dose-dependently inhibited by 1,25-(OH)2D3 (60% inhibition at 10 nM) when cells were cultured in medium supplemented with FBS (FBS medium). 1,25-(OH)2D3-treated cells showed a 5-fold increase in PSA secretion, similar to the increase seen in dihydrotestosterone (DHT)-treated cells. In combination, 1,25-(OH)2D3 and DHT synergistically enhanced PSA secretion 22-fold. This synergistic effect was even greater when cells were cultured in medium supplemented with charcoal-stripped serum (CSS medium), where endogenous steroids are substantially depleted. Under these conditions, 1,25-(OH)2D3 and DHT together stimulated PSA secretion up to 50-fold over the untreated control. Radioligand binding assays and Western blot analyses showed that the androgen receptor (AR) content was increased significantly by 1,25-(OH)2D3 at 48 h. Furthermore, the steady-state mRNA level of AR was up-regulated approximately 2-fold by 1,25-(OH)2D3 at 24 h. When cells were grown in CSS medium, 1,25-(OH)2D3 alone no longer inhibited cell growth or induced PSA secretion. Titration experiments revealed that the addition of DHT at 1 nM to the medium restored the antiproliferative activity of 1,25-(OH)2D3. Conversely, an antiandrogen, Casodex, completely blocked 1,25-(OH)2D3 antiproliferative and PSA stimulation activities when cells were cultured in FBS medium. In conclusion, these results demonstrate that the antiproliferative and PSA induction activities of 1,25-(OH)2D3 in LNCaP cells are dependent upon androgen action and that AR up-regulation by 1,25-(OH)2D3 likely contributes to the synergistic actions of 1,25-(OH)2D3 and DHT in these cells.

  View details for Web of Science ID A1997XL84200028

  View details for PubMedID 9231780

• The vitamin D receptor start codon polymorphism (FokI) and bone mineral density in premenopausal American black and white women JOURNAL OF BONE AND MINERAL RESEARCH Harris, S. S., Eccleshall, T. R., Gross, C., DAWSONHUGHES, B., Feldman, D. 1997; 12 (7): 1043-1048

  Abstract

  This study examines the association between bone mineral density (BMD) and a start codon polymorphism (SCP) at the translation initiation site of the vitamin D receptor (VDR) gene. The thymine/cytosine (T/C) polymorphism in the first of two start (ATG) codons can be detected by a restriction fragment length polymorphism (RFLP) using the endonuclease FokI, which recognizes ATG as part of its restriction site. F indicates absence of the first ATG and a VDR that is shorter by three amino acids. The FokI genotype was determined in 154 premenopausal American women (72 black and 82 white) who were 20-40 years old. BMD of the total body, femoral neck, and lumbar spine were measured by dual-energy X-ray absorptiometry. The distribution of the SCP genotypes differed significantly by race (p < 0.001): 4% of blacks versus 18% of whites were ff homozygous and 65% of blacks versus 37% of whites were FF homozygous. There was no statistically significant interaction between race and SCP genotype in analyses of BMD at any skeletal site. In the group as a whole, the ff women had femoral neck BMD that was 7.4% lower than that of the FF women. The ff white women had total body BMD values that were 4.3% lower and femoral neck values that were 12.1% lower than FF white women. Total body and femoral neck BMD did not differ significantly by genotype in black women, and spine BMD did not differ by genotype in either race. Addition of the SCP genotype to analysis of covariance models comparing BMD of the black and white women reduced estimated differences in femoral neck BMD between the two groups by about 35%. In conclusion, the SCP polymorphism, detected with the endonuclease FokI, appears to influence peak bone density, particularly at the femoral neck. Racial differences in its distribution may explain some of the racial difference in femoral neck BMD.

  View details for Web of Science ID A1997XE89900008

  View details for PubMedID 9200003

• Analysis of vitamin D analog-induced heterodimerization of vitamin D receptor with retinoid X receptor using the yeast two-hybrid system MOLECULAR ENDOCRINOLOGY Zhao, X. Y., Eccleshall, T. R., Krishnan, A. V., Gross, C., Feldman, D. 1997; 11 (3): 366-378

  Abstract

  Several synthetic analogs of 1 alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] are potent inducers of cellular differentiation and inhibitors of cell growth, yet they are less calcemic than 1,25-(OH)2D3 itself. The mechanisms by which these vitamin D analogs elicit a different profile of cellular activities than 1,25-(OH)2D3 are not fully understood. We propose that the analogs bind to the vitamin D receptor (VDR) to produce a conformational change that is more or less constrained than that induced by 1,25-(OH)2D3. This conformational change determines the extent of the VDR-retinoid X receptor (RXR) heterodimerization which, in turn, determines the interaction with other factors that specify the selectivity and magnitude of gene transactivation. We used the yeast two-hybrid system to evaluate a series of six vitamin D analogs for their ability to induce VDR-RXR heterodimerization. The VDR-RXR interaction was elicited by the analogs in a concentration-dependent manner. To evaluate how this activity compared with other known steps in 1,25-(OH)2D3 action, we also measured the ability of the same six analogs to bind to VDR, to enhance the binding of VDR-RXR to DNA, to transactivate a vitamin D-response element-reporter construct, and to inhibit proliferation in mammalian cells. Our results indicate that, for most analogs, the level of transcriptional activation correlates well with the strength of VDR-RXR heterodimerization in intact cells. We conclude that the yeast two-hybrid system provides a useful means to investigate heterodimerization potency and that this property contributes significantly to the overall pattern of analog activity. The yeast two-hybrid system, being an intact cell assay and easy to perform, may be a useful supplement to the conventional assays employed to screen vitamin D analogs.

  View details for Web of Science ID A1997WK71000011

  View details for PubMedID 9058382

• Vitamin D receptor polymorphisms, bone mineral density, and bone metabolism in postmenopausal Mexican-American women JOURNAL OF BONE AND MINERAL RESEARCH McClure, L., Eccleshall, T. R., Gross, C., Villa, M. L., Lin, N., Ramaswamy, V., Kohlmeier, L., Kelsey, J. L., Marcus, R., Feldman, D. 1997; 12 (2): 234-240

  Abstract

  Common polymorphisms in the vitamin D receptor (VDR) gene have been shown to correlate with bone mineral density (BMD). However, attempts to replicate the original findings in other populations have yielded variable results. These disparities may reflect ethnic or environmental differences in the expression of the VDR effect upon BMD. We examined a relatively ethnically homogeneous group of 103 healthy postmenopausal Caucasian women of Mexican descent living in Northern California. We determined the VDR genotype and measured the BMD at the lumbar spine and femoral neck by dual-energy X-ray absorptiometry, as well as several biochemical indices of mineral metabolism. The prevalence of the BB genotype, associated in previous studies with the lowest BMD, was 8% and highly linked to the tt genotype. Absolute and age-adjusted BMD at both hip and spine showed a trend toward lower BMD in the BB, AA, and tt genotypes, but this trend did not achieve statistical significance. There were no consistent intergroup differences in change in BMD over 2 years of follow-up, nor in mean serum concentrations of 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D, osteocalcin, or total urinary pyridinolines. Intact parathyroid hormone concentrations were significantly higher in subjects with the AA genotype, with a trend toward higher values in those with the BB and tt genotypes as well. Our data suggest that there may be a decrease in BMD associated with the B, A, and t alleles, but the intergroup difference in BMD is 0.2-0.5 standard deviations (SD) at the lumbar spine and 0.3 SD at the femoral neck, decreases that are smaller than previously reported. Given the relatively low prevalence of the BB/tt genotype in Mexican-American Caucasians, a larger sample would be required to detect a significant association between VDR alleles and differences in BMD of the magnitude suggested by our data. We conclude that a genotype effect of this magnitude, if present, would be clinically relevant, but the impact on BMD is too small to detect with statistical significance in a study of this size.

  View details for Web of Science ID A1997WG80900010

  View details for PubMedID 9041055

• Hereditary vitamin D resistant rickets caused by a novel mutation in the vitamin D receptor that results in decreased affinity for hormone and cellular hyporesponsiveness JOURNAL OF CLINICAL INVESTIGATION Malloy, P. J., Eccleshall, T. R., Gross, C., VANMALDERGEM, L., Bouillon, R., Feldman, D. 1997; 99 (2): 297-304

  Abstract

  Mutations in the vitamin D receptor (VDR) result in target organ resistance to 1alpha,25-dihydroxyvitamin D [1,25(OH)2D3], the active form of vitamin D, and cause hereditary 1,25-dihydroxyvitamin D resistant rickets (HVDRR). We analyzed the VDR of a patient who exhibited three genetic diseases: HVDRR, congenital total lipodystrophy, and persistent mullerian duct syndrome. The patient was treated with extremely high dose calcitriol (12.5 microg/d) which normalized serum calcium and improved his rickets. Analysis of [3H]1,25(OH)2D3 binding in the patient's cultured fibroblasts showed normal abundance of VDR with only a slight decrease in binding affinity compared to normal fibroblasts when measured at 0 degrees C. The patient's fibroblasts demonstrated 1,25(OH)2D3-induction of 24-hydroxylase mRNA, but the effective dose was approximately fivefold higher than in control cells. Sequence analysis of the patient's VDR gene uncovered a single point mutation, H305Q. The recreated mutant VDR was transfected into COS-7 cells where it was 5 to 10-fold less responsive to 1,25(OH)2D3 in gene transactivation. The mutant VDR had an eightfold lower affinity for [3H]1,25(OH)2D3 than the normal VDR when measured at 24 degrees C. RFLP demonstrated that the patient was homozygous for the mutation while the parents were heterozygous. In conclusion, we describe a new ligand binding domain mutation in the VDR that causes HVDRR due to decreased affinity for 1,25(OH)2D3 which can be effectively treated with extremely high doses of hormone.

  View details for Web of Science ID A1997WE31100019

  View details for PubMedID 9005998

  View details for PubMedCentralID PMC507797

• 1,25-dihydroxyvitamin D-3 induction of nerve growth factor in L929 mouse fibroblasts: Effect of vitamin D receptor regulation and potency of vitamin D-3 analogs ENDOCRINOLOGY Musiol, I. M., Feldman, D. 1997; 138 (1): 12-18

  Abstract

  Decreased nerve growth factor (NGF) synthesis in the hippocampus and reduced nerve growth factor receptor immunoreactivity in CH1-4 basal forebrain areas have been implicated in neurodegeneration. Vitamin D receptors (VDR) have been located in brain areas affected by neurodegenerative diseases. 1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3], the active form of vitamin D, has been shown to induce NGF in L929 mouse fibroblasts and rat hippocampus. In the present study we analyzed the VDR in L929 cells, which we used as a model system. We studied the regulation of VDR abundance and the ability of 1,25-(OH)2D3 to induce NGF synthesis. Scatchard analysis of [3H]1,25-(OH)2D3 binding showed the VDR concentration to be 173 fmol/mg protein and the affinity to be 0.12 nM. VDR was localized to nuclei of L929 cells by immunocytochemistry. Treatment of cells with forskolin (FSK; 50 microM), which activates the cAMP-protein kinase A pathway, resulted in an 8- to 10-fold up-regulation of VDR by 6 h, and VDR remained elevated at 24 h, as we have reported for other cells. NGF secretion was measured in serum-free conditioned medium using a double sided enzyme-linked immunosorbent assay. 1,25-(OH)2D3 treatment (0.1 pM to 10 nM) for 24 h increased the NGF concentration 2- to 3-fold, an effect that plateaued at 1 nM 1,25-(OH)2D3. VDR up-regulation by FSK pretreatment augmented the NGF response to 1,25-(OH)2D3 2-fold compared to that in vehicle-pretreated cells for a total 6-fold increase compared to basal NGF levels. The vitamin D analogs EB-1089 and 22-oxacalcitriol, which have been found to be less calcemic than 1,25-(OH)2D3, also induced NGF synthesis. The effects of these analogs were further enhanced by prior up-regulation of VDR with FSK. In conclusion, we have characterized the VDR in L929 cells and shown that 1,25-(OH)2D3 and its less calcemic analogs induce NGF. Furthermore, up-regulation of VDR abundance enhanced NGF induction. These effects of 1,25-(OH)2D3 and its analogs via VDR to regulate NGF synthesis may have significance for the eventual treatment of neurodegenerative diseases that are caused by decreased NGF production.

  View details for Web of Science ID A1997VZ82400004

  View details for PubMedID 8977379

• Vitamin D. Academic Press, Inc., San Diego D. Feldman, F. Glorieux, J.W. Pike, editors. 1997
• The presence of a polymorphism at the translation initiation site of the vitamin D receptor gene is associated with low bone mineral density in postmenopausal Mexican-American women JOURNAL OF BONE AND MINERAL RESEARCH Gross, C., Eccleshall, T. R., Malloy, P. J., Villa, M. L., Marcus, R., Feldman, D. 1996; 11 (12): 1850-1855

  Abstract

  We examined the association of bone mineral density (BMD) with a polymorphism in the gene encoding the vitamin D receptor (VDR) that causes a change in the predicted protein sequence. The polymorphism results from a C-to-T transition and creates an initiation codon (ATG) three codons proximal to a downstream start site. The polymorphism can be defined by a restriction fragment length polymorphism (RFLP) using the restriction endonuclease FokI. The presence of a FokI site, designated f, allows protein translation to initiate from the first ATG. The allele lacking the site (designated F), initiates from a second ATG site. Thus, translation products from these alleles are predicted to differ by three amino acids with the f variant elongated. In a group of 100 postmenopausal Mexican-American Caucasian women, subjects with the ff genotype (15% of the study population) had a 12.8% lower BMD at the lumbar spine than FF subjects (37% of the population) (p = 0.01). Heterozygote (Ff) subjects (48% of the population) had an intermediate BMD. This association between BMD and genotype was not apparent at the femoral neck or forearm. Over a 2-year follow-up period, a decrease in BMD at the femoral neck was greater in ff compared with FF subjects (-4.7% vs. -0.5%, p = 0.005). This trend was not apparent at the lumbar spine or forearm. There were no differences between genotype groups in measurements of 25-hydroxyvitamin D (25(OH)D), calcitriol, parathyroid hormone (PTH), osteocalcin, or urinary pyridinolines. We conclude that the FokI polymorphism of the VDR gene correlates significantly with decreased BMD at the lumbar spine and with an increased rate of bone loss at the hip in ff subjects. We emphasize that these initial data should be interpreted with caution but that the utility of this polymorphism as a genetic marker to determine BMD and osteoporosis risk warrants further study in larger populations with subjects of diverse ethnic backgrounds.

  View details for Web of Science ID A1996VY56200003

  View details for PubMedID 8970885

• A novel mutation in the deoxyribonucleic acid-binding domain of the vitamin D receptor causes hereditary 1,25-dihydroxyvitamin D-resistant rickets JOURNAL OF CLINICAL ENDOCRINOLOGY & METABOLISM Lin, N. U., Malloy, P. J., Sakati, N., ALASHWAL, A., Feldman, D. 1996; 81 (7): 2564-2569

  Abstract

  Mutations in the vitamin D receptor (VDR) result in hereditary 1,25-dihydroxyvitamin D3-resistant rickets (HVDRR), an autosomal recessive disease caused by target organ resistance to the action of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. In this study, we investigated the molecular basis of HVDRR in a child from Saudi Arabia who was previously shown to be resistant to 1,25-(OH)2D3 action, but whose cultured skin fibroblasts exhibited normal [3H]1,25-(OH)2D3 binding. Using the PCR, exons 2 and 3 of the VDR gene that encode the DNA-binding region of the receptor were amplified and sequenced. A novel point mutation at nucleotide 252 in exon 2 of the VDR was identified. This missense mutation (GGC to GAC) resulted in the conversion of glycine to aspartic acid at amino acid position 46 (G46D), located at the base of the first zinc finger. This single base change was introduced into wild-type VDR complementary DNA by site-directed mutagenesis, and the mutant VDR was then expressed in COS-1 cells. The expressed mutant VDR displayed a normal binding affinity (Kd = 1.2 x 10(-10) mol/L) for [3H]1,25-(OH)2D3 as determined by Scatchard analysis. However, the mutant VDR was shown to have reduced binding affinity for DNA by DNA-cellulose chromatography. In COS-7 cells cotransfected with a vitamin D response element-chloramphenicol acetyltransferase reporter construct and the mutant VDR complementary DNA expression vector, the mutant VDR was unable to activate gene transcription in cells treated with up to 100 nmol/L 1,25-(OH)2D3. Restriction fragment length polymorphism analysis using MwoI restriction digests of exon 2 demonstrated that the affected child is homozygous for the mutation, whereas the child's father is heterozygous and a carrier of the defective allele. In conclusion, a new mutation was identified in exon 2 of the VDR gene. This mutation, which occurs in the first zinc finger of the DNA-binding domain of the receptor, blocks 1,25-(OH)2D3 action and leads to the syndrome of HVDRR.

  View details for Web of Science ID A1996UW84200028

  View details for PubMedID 8675579

• Parathyroid hormone-related protein (PTHrP) is an epidermal growth factor-regulated secretory product of human prostatic epithelial cells PROSTATE Cramer, S. D., Peehl, D. M., Edgar, M. G., Wong, S. T., Deftos, L. J., Feldman, D. 1996; 29 (1): 20-29

  Abstract

  Parathyroid hormone-related protein (PTHrP) has previously been shown to be expressed in human prostatic tissue and in prostatic cancer cell lines. In the present study, PTHrP immunoreactivity was detected in the glandular epithelium of normal prostate and benign prostatic hyperplasia (BPH), as well as in prostatic adenocarcinoma (CaP). Epithelial cell cultures derived from normal, BPH, and CaP tissues were also stained by antibodies against PTHrP, and northern analysis revealed multiple transcripts of PTHrP in the cellular RNA. PTHrP (1-34) was measurable by radioimmunoassay (RIA) in media conditioned by the prostatic epithelial cell cultures, and PTHrP accumulated in conditioned media during a 72 hr time course. Addition of complete growth medium to starved cells resulted in increased PTHrP mRNA levels by 1 hr, with maximal stimulation at 8-24 hr. Several individual factors contained in the complete growth medium were tested for their ability to regulate PTHrP expression. Epidermal growth factor (EGF) was the major inducer of PTHrP expression, while cholera toxin, bovine pituitary extract, hydrocortisone, and insulin had minimal or no effect on PTHrP transcript levels. Since each of these factors is growth stimulatory, the unique ability of EGF to induce PTHrP is apparently unrelated to mitogenicity. 1,25-Dihydroxyvitamin D3[1,25(OH)2D3], an inhibitor of PTHrP expression in several other cell types, had no effect on steady-state levels of PTHrP mRNA expressed by epithelial cells in complete growth medium, although prostate cells have vitamin D receptors and are responsive to 1,25(OH)2D3 in other ways. Our results indicate that PTHrP expression is not confined to the neuroendocrine cells of the human prostate and that our culture system can be used as a model to investigate the role of PTHrP in the prostate.

  View details for Web of Science ID A1996UX88000003

  View details for PubMedID 8685051

• Simian virus 40-, but not human papillomavirus-, transformation of prostatic epithelial cells results in loss of growth-inhibition by 1,25-dihydroxyvitamin D-3 INTERNATIONAL JOURNAL OF ONCOLOGY Gross, C., Skowronski, R. J., Plymate, S. R., Rhim, J. S., Peehl, D. M., Feldman, D. 1996; 8 (1): 41-47

  Abstract

  In addition to its well known calcemic actions, 1,25-dihydroxyvitamin D-3 [1,25(OH)(2)D] exhibits differentiating and antiproliferative effects in several types of cancer cells. 1,25(OH)(2)D receptors (VDR) as well as 1,25(OH)(2)D-mediated growth-inhibition have been demonstrated in human prostate cancer cell lines. In order to further develop model systems for the study of 1,25(OH)(2)D action and to elucidate the mechanism of growth-inhibition, we studied several human prostate cell lines immortalized with either simian virus 40 (SV40) or human papillomavirus type 18 (HPV). The SV40-transformed cell lines P69SV40-T and P153SV40-T were not growth-inhibited by 1,25(OH)(2)D at concentrations as high as 100 nM, whereas the HPV-transformed cells PZ-HPV-7 and CA-HPV-10 were growth-inhibited. All cell lines expressed VDR, and VDR mRNA was demonstrated by Northern blot analysis. All cells exhibited induction of 24-hydroxylase mRNA, a 1,25(OH)(2)D responsive gene, after 1,25(OH)(2)D treatment. In an attempt to understand the apparent dissociation of 1,25(OH)(2)D actions in the SV40-transformed cells, we turned to the human prostate cancer cell line DU 145. These cells, like the SV40-transformed cells, are not growth-inhibited but demonstrate induction of 24-hydroxylase mRNA after 1,25(OH)(2)D treatment. DU 145 cells contain a mutated retinoblastoma gene (Rb) which contributes to their uncontrolled growth, analogous to the disruption of Rb by SV40 and HPV. We compared DU,145 cells to DU 145 cells transfected with normal Rb (DU 145/Rb). Similar to DU 145, DU 145/Rb cells were not growth-inhibited by 1,25(OH)(2)D, while 24-hydroxylase mRNA was induced. These results suggest that divergent pathways mediate the growth-inhibitory effect of 1,25(OH)(2)D and its induction of 24-hydroxylase. It also appears that the antiproliferative effect of 1,25(OH)(2)D is mediated by an Rb-independent mechanism.

  View details for Web of Science ID A1996TM02400006

  View details for PubMedID 21544329

• ESTROGENS IN UNEXPECTED PLACES - POSSIBLE IMPLICATIONS FOR RESEARCHERS AND CONSUMERS Symposium on Estrogens in the Environment, III - Global Health Implications Feldman, D., Krishnan, A. US DEPT HEALTH HUMAN SCIENCES PUBLIC HEALTH SCIENCE. 1995: 129–133

  Abstract

  Estrogenic activity originating in unexpected places was encountered on three occasions during an investigation of whether Saccharomyces cerevisiae synthesized estrogens. In each instance, estradiol found in the conditioned yeast culture medium originated from an exogenous source and was not synthesized by the yeast. In the first instance, yeast grown in the laboratory showed a time-dependent increase in estradiol in the conditioned medium. However, the culture medium supplement Bacto-peptone was found to contain large amounts of estrone. When added to yeast cultures in the form of YPD medium (yeast extract, Bacto-peptone, and dextrose), S. cerevisiae converted the estrone to estradiol leading to the accumulation of estradiol over time. In the second instance, commercially purchased S. cerevisiae grown in a molasses medium exhibited substantial amounts of estradiol. However, corn and beet molasses contained sufficient estrone and estradiol to account for the findings. As in the first instance, the yeast converted the estrone into estradiol. In the third instance, autoclaving culture medium in polycarbonate plastic flasks was found to cause an estrogenic substance to be added to the medium, whether yeast were present or not. It was determined that the autoclaving process leached bisphenol-A (BPA) out of the polycarbonate plastic. BPA was shown to bind to estrogen receptors and to induce estrogenic activity, including stimulation of MCF-7 breast cancer-cell proliferation and induction of the expression of progesterone receptors. The three instances highlight potential problems for investigators who might inadvertently add estrogens to experimental systems confounding their results. The BPA findings raise concerns about the possible addition of this estrogenic molecule to the food supply since polycarbonate plastic is used in myriad applications in the packaging of food and beverages. Although we are unaware of the substantial contamination of food products with BPA, we believe this possibility should be carefully investigated.

  View details for Web of Science ID A1995TG78200024

  View details for PubMedID 8593858

  View details for PubMedCentralID PMC1518886

• ESTROGEN-BINDING PROTEIN IN CANDIDA-ALBICANS - ANTIBODY DEVELOPMENT AND CELLULAR-LOCALIZATION BY ELECTRON IMMUNOCYTOCHEMISTRY MICROBIOLOGY-UK Zhao, X., Malloy, P. J., Ardies, C. M., Feldman, D. 1995; 141: 2685-2692

  Abstract

  Candida albicans, the most common fungal pathogen of humans, possesses an oestrogen (estrogen)-binding protein (EBP) that binds oestrogens with high affinity and specificity. The gene that encodes the EBP (CaEBP1) has been cloned and sequenced and shown to be structurally related to the old yellow enzyme from Saccharomyces cerevisiae. Here, we report the purification and the subcellular localization of the EBP from C. albicans. Using ion-exchange chromatography and an oestradiol affinity column, the EBP was purified from a strain of C. albicans (strain 422) which was selected because it constitutively expressed elevated levels of the binding protein. The purified protein displayed a subunit molecular mass of approximately 46 kDa when examined by denaturing gel electrophoresis, which is consistent with the size estimated from the sequence of the cloned CaEBP1 gene. An immunoaffinity column, prepared using a polyclonal antisera generated against EBP, depleted the oestrogen-binding activity from C. albicans cell extracts. Western blot analysis showed that the antisera specifically recognized the EBP from C. albicans. The antibodies also recognized the protein when the cloned CaEBP1 gene was expressed in S. cerevisiae and did not cross react with S. cerevisiae proteins. Using electron microscopy and antigen detection by immunogold staining, the EBP appeared to be primarily associated with vacuoles. However, when overexpressed in S. cerevisiae, the EBP was found diffusely throughout the cell. In conclusion, the EBP has been purified from C. albicans and antibodies generated against the protein were used to demonstrate that EBP is found associated with vacuoles in C. albicans.

  View details for Web of Science ID A1995TB87300041

  View details for PubMedID 7582028

• Suramin, hydrocortisone, and retinoic acid modify inhibitory effects of 1,25-dihydroxyvitamin D(3) on prostatic epithelial cells. Urologic oncology Peehl, D. M., Wong, S. T., Cramer, S. D., Gross, C., Feldman, D. 1995; 1 (5): 188-194

  Abstract

  The proliferation of prostatic epithelial cells is regulated by the complex interplay of numerous growth-stimulatory and growth-inhibitory factors. 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] has recently been identified as a potent inhibitor of the growth of prostatic epithelial cells. Epidemiologic studies indicate that vitamin D deficiency may be a risk factor for the development of clinical prostate cancer, possibly due to increased growth and reduced differentiation of prostatic cells in an environment with decreased 1,25(OH)(2)D(3). The application of vitamin D or analogs in chemotherapy against prostate and other cancers is being explored by several investigators. In order to use vitamin D most efficaciously in a clinical setting, it may be beneficial to learn more about the interaction of 1,25(OH)(2)D(3) with other factors that regulate prostatic epithelial cellular growth. In this study, we examined the effect of the proliferative status of cultured cells on their ability to respond to 1,25(OH)(2)D(3), and found that minimally proliferative cells were equally as responsive to 1,25(OH)2D3 as actively dividing cells. We noted no apparent interaction of 1,25(OH)(2)D(3) with epidermal growth factor, insulin-like growth factor, cholera toxin, or transforming growth factor-β, but we did find synergistic inhibitory effects of 1,25(OH)(2)D(3) with suramin and retinoic acid. Perhaps most noteworthy was the dramatic increase in potency of 1,25(OH)(2)D(3) that occurred upon deletion of hydrocortisone from the culture medium. Our in vitro studies indicate that combination therapy of vitamin D analogs with suramin, vitamin A analogs, or anti-glucocorticoids might be considered for prostate cancer.

  View details for PubMedID 21224116

• REGULATION OF 1,25-DIHYDROXYVITAMIN-D-3 RECEPTORS BY PARATHYROID-HORMONE IN OSTEOBLASTIC CELLS - ROLE OF 2ND-MESSENGER PATHWAYS ENDOCRINOLOGY Krishnan, A. V., Cramer, S. D., Bringhurst, F. R., Feldman, D. 1995; 136 (2): 705-712

  Abstract

  The regulation of vitamin D receptor (VDR) abundance in MC3T3-E1 mouse osteoblasts and UMR 106-01 rat osteosarcoma cells by rat PTH 1-34, human PTH-related protein 1-34, and agents that activate specific signal transduction pathways was studied. Treatment of these cells with forskolin (FSK) caused up-regulation of VDR, whereas treatment with phorbol esters suppressed VDR levels. PTH or PTH-related protein treatment induced a 2- to 3-fold increase in VDR, which was equivalent to that elicited by FSK in UMR 106-01 cells but less than the FSK-induced increase (approximately 8-fold) in MC3T3-E1 cells. PTH treatment of MC3T3-E1 cells resulted in an approximately 3-fold increase in VDR levels with maximum stimulation occurring at 10(-9) M PTH after 4 h of treatment. In UMR 4-7 cells, a subclone of UMR 106-01 cells that express cAMP resistance due to regulated expression of a mutant form of the type 1 regulatory subunit of the cAMP-dependent protein kinase A (PKA), the up-regulation of VDR abundance due to FSK and PTH treatment was mostly prevented. Pretreatment of MC3T3-E1 cells with staurosporine, an inhibitor of PKC, resulted in an approximately 3-fold increase in basal VDR levels but did not enhance the PTH-mediated up-regulation of VDR. Collectively, these data suggest that the increase in VDR abundance observed in these target cells is mainly due to the activation of the PKA signal transduction pathway. Treatment of UMR 106-01 cells with PTH for 4 h before exposure of the cells to 1,25-dihydroxyvitamin D3 resulted in a 2-fold increase in the induction of 25-hydroxyvitamin D3-24 hydroxylase messenger RNA. Thus, exposure of target cells to PTH augments their response to 1,25-dihydroxyvitamin D3 due to up-regulation of VDR abundance.

  View details for Web of Science ID A1995QD58600043

  View details for PubMedID 7835303

• ACTIONS OF VITAMIN-D-3 ANALOGS ON HUMAN PROSTATE-CANCER CELL-LINES - COMPARISON WITH 1,25-DIHYDROXYVITAMIN-D-3 ENDOCRINOLOGY Skowronski, R. J., Peehl, D. M., Feldman, D. 1995; 136 (1): 20-26

  Abstract

  Data from epidemiological studies has suggested that vitamin D deficiency may promote prostate cancer, although the mechanism is not understood. We have previously demonstrated the presence of vitamin D receptors (VDR) in three human prostate carcinoma cell lines (LNCaP, PC-3, and DU-145) as well as in primary cultures of stromal and epithelial cells derived from normal and malignant prostate tissues. We have also shown that 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] can elicit an antiproliferative action in these cells. In the present study we compared the biological actions of 1,25-(OH)2D3 to those of a series of natural vitamin D3 metabolites and several synthetic analogs of vitamin D3 known to exhibit less hypercalcemic activity in vivo. In ligand binding competition experiments, we demonstrated the following order of potency in displacing [3H]1,25-(OH)2D3 from VDR: EB-1089 > 1,25-(OH)2D3 > MC-903 > 1,24,25-(OH)3D3 > 22-oxacalcitriol (OCT) > 1 alpha,25-dihydroxy-16-enecholecalciferol (Ro24-2637) > 25-hydroxyvitamin D3, with EB-1089 being approximately 2-fold more potent than the native hormone. No competitive activity was found for 25-hydroxy-16,23-diene-cholecalciferol. When compared for ability to inhibit proliferation of LNCaP cells, MC-903, EB-1089, OCT, and Ro24-2637 exhibited 4-, 3-, and 2-fold greater inhibitory activity than 1,25-(OH)2D3. Interestingly, although OCT and Ro24-2637 exhibit, respectively, 10 and 14 times lower affinity for VDR than 1,25-(OH)2D3, both compounds inhibited the proliferation of LNCaP cells with a potency greater than that of the native hormone. The relative potency of vitamin D3 metabolites and analogs to inhibit cell proliferation correlated well with the ability of these compounds to stimulate prostate-specific antigen secretion by LNCaP cells as well as with their potency to induce the 25-hydroxyvitamin D3-24-hydroxylase messenger RNA transcript in PC-3 cells. In conclusion, these results demonstrate that synthetic analogs of vitamin D3, known to exhibit reduced calcemic activity, can elicit antiproliferative effects and other biological actions in LNCaP and PC-3 cell lines. It is noteworthy that although binding to VDR is critical for 1,25-(OH)2D3 action, the analog data indicate that additional factors significantly contribute to the magnitude of the biological response. Finally, the strong antiproliferative effects of several synthetic analogs known to exhibit less calcemic activity than 1,25-(OH)2D3 suggest that these compounds potentially may be useful as an additional therapeutic option for the treatment of prostate cancer.

  View details for Web of Science ID A1995QD58400004

  View details for PubMedID 7530193

• Vitamin D and prostate cancer 5th Annual Conference of the American-Institute-for-Cancer-Research on Diet and Cancer - Molecular Mechanisms of Interactions Feldman, D., Skowronski, R. J., Peehl, D. M. PLENUM PRESS DIV PLENUM PUBLISHING CORP. 1995: 53–63

  Abstract

  Our findings demonstrate the presence of VDR in various human prostate cancer cell lines and in primary cultures derived from normal, BPH and prostate cancer. In addition, 1,25-D induced several bioresponses in these cells including growth inhibition and PSA stimulation. Based on examples in many different malignant cells as well as our data in prostate cells, that vitamin D is anti-proliferative and promotes cellular maturation, it seem clear that vitamin D must be viewed as an important cellular modulator of growth and differentiation if addition to its classical role as regulator of calcium homeostasis. In this respect, vitamin D has the potential to have beneficial actions on various malignancies including prostate cancer. Its ultimate role in prostate cancer remains to be determined, but 1,25-D may prove useful in chemoprevention and/or differentiation therapy. We believe the data currently available provide the basis for an optimistic view on the possible use of vitamin D to treat prostate cancer in patients and that further investigation is clearly warranted to better define its potential therapeutic utility.

  View details for Web of Science ID A1995BD36X00005

  View details for PubMedID 7645428

• ACTH-INDEPENDENT MASSIVE BILATERAL ADRENAL DISEASE (AIMBAD) - A SUBTYPE OF CUSHINGS-SYNDROME WITH MAJOR DIAGNOSTIC AND THERAPEUTIC IMPLICATIONS EUROPEAN JOURNAL OF ENDOCRINOLOGY Lieberman, S. A., Eccleshall, T. R., Feldman, D. 1994; 131 (1): 67-73

  Abstract

  A 49-year-old man with classic manifestations of Cushing's syndrome had undetectable levels of ACTH, lack of suppression of hypercortisolism with dexamethasone in doses of 2, 8, or 16 mg per day, bilaterally enlarged adrenal glands on MRI, and bilateral adrenal uptake of iodocholesterol. Preoperative treatment with ketoconazole lowered blood pressure and serum cortisol and produced symptoms of steroid withdrawal. Bilateral adrenalectomy revealed massively enlarged adrenal glands (left: 199 g, right: 93 g). Sequencing of the gene encoding the stimulatory G protein, GS alpha, did not show either of two activating mutations previously reported in patients with McCune-Albright syndrome or acromegaly. Twenty-three previous cases of Cushing's syndrome due to ACTH-independent massive bilateral adrenal disease (AIMBAD) have been reported. AIMBAD may cause confusion in the differential diagnosis of Cushing's syndrome as endocrine testing suggests a unilateral, ACTH-independent process while adrenal imaging demonstrates bilateral abnormalities. Bilateral adrenalectomy is curative and appears to carry little risk of Nelson's syndrome. The pathogenesis of AIMBAD appears to be heterogeneous, as recent reports have demonstrated GIP-mediated hypercortisolism and familial AIMBAD. Transition from Cushing's disease to ACTH-independence is not supported by the available data. Future cases of AIMBAD should be investigated carefully to further elucidate the pathogenesis of this disorder.

  View details for Web of Science ID A1994NY88600010

  View details for PubMedID 8038906

• INHIBITION OF ALDOSTERONE SYNTHESIS IN RAT ADRENAL-CELLS BY NICOTINE AND RELATED CONSTITUENTS OF TOBACCO-SMOKE ENDOCRINOLOGY Skowronski, R. J., Feldman, D. 1994; 134 (5): 2171-2177

  Abstract

  Previous work has shown that nicotine and related constituents of tobacco smoke inhibit selected P450 enzymes in the glucocorticoid and sex steroid synthetic pathways. Because aldosterone synthesis is also cytochrome P450 dependent, we hypothesized a similar inhibitory action on aldosterone production. In this study we examined the effects of nicotine, anabasine (a related alkaloid), and cotinine (the major metabolite of nicotine) on in vitro aldosterone synthesis. Freshly isolated rat adrenal cells were assayed for corticosterone and aldosterone production in the basal state and after stimulation with ACTH or angiotensin-II (ANG-II). The addition of nicotine, anabasine, and cotinine in concentrations up to 100 microM did not inhibit stimulated corticosterone production. However, there was a potent dose-dependent inhibitory action of all three tobacco compounds on aldosterone production. The relative inhibitory potency was: cotinine > anabasine > nicotine. When employed at a concentration of 100 microM, the three compounds inhibited ACTH-stimulated aldosterone synthesis by 75%, 44%, and 21%, respectively. ANG-II-stimulated aldosterone synthesis was inhibited by 92%, 78%, and 62%, respectively. The plasma cotinine concentration range attained in tobacco smokers is between 1-10 microM. When tested with [3H]corticosterone and [3H]progesterone as exogenous substrates, 1-10 microM cotinine caused a significant dose-dependent inhibition of ACTH- and ANG-II-stimulated aldosterone synthesis. Cotinine substantially blocked the conversion of corticosterone to 18-hydroxycorticosterone, implicating the 18-hydroxylase or corticosterone 18-methyloxidase-I (CMO-I) step as the major site of inhibition. In summary, our results indicate that tobacco compounds cause direct and specific inhibition of aldosterone synthesis, primarily at the CMO-I step. This enzymatic blockade would be expected to result in activation of the renin-angiotensin system in vivo. We postulate that chronic stimulation of the renin-angiotensin system by this mechanism might contribute to the cardiovascular damage that occurs with long term tobacco use.

  View details for Web of Science ID A1994NG46700028

  View details for PubMedID 8156919

• HEREDITARY 1-ALPHA,25-DIHYDROXYVITAMIN-D RESISTANT RICKETS RESULTING FROM A MUTATION IN THE VITAMIN-D RECEPTOR DEOXYRIBONUCLEIC ACID-BINDING DOMAIN JOURNAL OF CLINICAL ENDOCRINOLOGY & METABOLISM Malloy, P. J., Weisman, Y., Feldman, D. 1994; 78 (2): 313-316

  Abstract

  Hereditary 1 alpha,25-dihydroxyvitamin D-resistant rickets (HVDRR) is a genetic disease that results from mutations in the gene encoding the vitamin D receptor (VDR). In this study of two siblings showing classical features of HVDRR, cultured dermal fibroblasts were used to characterize their VDR and assess responsiveness to 1,25-dihydroxyvitamin D3 treatment. The VDR displayed normal affinity and binding capacity for [3H]1,25-dihydroxyvitamin D3; however, the cells failed to exhibit induction of 25-hydroxyvitamin D 24-hydroxylase activity when treated with hormone. A decreased affinity of liganded VDR for DNA cellulose suggested that the defect was localized to the DNA-binding domain. Exons 2 and 3 of the VDR gene, which encode the two zinc fingers in the DNA-binding domain, were amplified and sequenced by polymerase chain reaction. Both siblings exhibited a G to A missense mutation (CGG to CAG) in exon 3, which results in the replacement of Arg77 by Gln at the base of the second zinc finger. This mutation has been described previously in two unrelated cases of HVDRR by Sone et al. It is unclear at this time whether these kindreds might be distantly related and, therefore, harbor the same mutation, or whether this represents a mutational hot spot in the VDR gene.

  View details for Web of Science ID A1994MW71700015

  View details for PubMedID 8106618

• CANDIDA-ALBICANS ESTROGEN-BINDING PROTEIN GENE ENCODES AN OXIDOREDUCTASE THAT IS INHIBITED BY ESTRADIOL PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Madani, N. D., Malloy, P. J., RODRIGUEZPOMBO, P., Krishnan, A. V., Feldman, D. 1994; 91 (3): 922-926

  Abstract

  Candida albicans, the most common fungal pathogen of humans, possesses an estrogen-binding protein (EBP) that binds mammalian estrogens with high affinity. We report here the cloning and complete nucleotide sequence of a gene encoding a C. albicans EBP. Amino acid sequences obtained from cyanogen bromide fragments of purified EBP were used to design oligonucleotide primers for PCR. An 800-bp product was amplified and used to screen a C. albicans genomic library. A clone was isolated containing an insert with an open reading frame of 1221 nt capable of encoding a protein with 407 amino acids and having a calculated molecular mass of 46,073 Da, the estimated size of EBP. The cloned gene, expressed in Escherichia coli as a lacZ fusion protein, demonstrated high-affinity binding for estradiol and a competition profile comparable to C. albicans wild-type EBP. Northern blots of C. albicans RNA revealed a single transcript of approximately 1600 nt, whereas Southern blots identified three hybridizing fragments. Computer searches of data bases showed that EBP shares a 46% amino acid identity with the old yellow enzyme, an oxidoreductase from Saccharomyces cerevisiae, but was unrelated to the human estrogen receptor as previously speculated. In addition, a 51-amino acid region of EBP is highly conserved among a group of flavoproteins including old yellow enzyme. Expressed EBP was shown to exhibit oxidoreductase activity that could be inhibited by 17 beta-estradiol in vitro. In conclusion, the EBP from C. albicans has no evident homology to the mammalian steroid receptor superfamily but appears to be a member of a recently identified family of flavoproteins.

  View details for Web of Science ID A1994MV27800021

  View details for PubMedID 8302868

  View details for PubMedCentralID PMC521425

• REGULATION OF VITAMIN-D RECEPTOR ABUNDANCE AND RESPONSIVENESS DURING DIFFERENTIATION OF HT-29 HUMAN COLON CANCER-CELLS ENDOCRINOLOGY Zhao, X., Feldman, D. 1993; 132 (4): 1808-1814

  Abstract

  We have studied the effects of 1,25-dihydroxyvitamin D [1,25-(OH)2D3] on cellular differentiation in the HT-29 human colon cancer cell line. Our aim was to evaluate the regulation of 1,25-dihydroxyvitamin D receptor (VDR) abundance and hormone responsiveness during the transition of rapidly proliferating to differentiated cells. Differentiation was induced by three means: cells were cultured in galactose-supplemented medium without glucose (GAL), grown on Matrigel-coated surfaces (MTG), or treated with 1,25(OH)2D3. Cell proliferation, assessed by [3H]thymidine incorporation, was equivalently inhibited by treatment with 1,25(OH)2D3, GAL or MTG. Differentiation was assessed by the induction of amino-oligo peptidase activity which was low in the proliferating cells. Following treatment with 1,25(OH)2D3, or growth in GAL or on MTG, amino-oligo peptidase activity increased 8- to 9-fold. The abundance of VDR measured by [3H]1,25(OH)2D3 binding, decreased to half without significant change in affinity, in cells differentiated by all three means compared to proliferating cells. Northern blot analyses of differentiated cells showed decreased steady-state levels of VDR messenger RNA (mRNA), indicating that all three treatments similarly decreased the abundance of VDR, at least in part, at the mRNA level. When exposed to 1,25(OH)2D3, the proliferating cells exhibited homologous up-regulation of VDR as well as the induction of 24-hydroxylase mRNA; the differentiated cells failed to exhibit both of these biological responses. Our findings demonstrate that 1,25(OH)2D3, GAL and MTG treatment all inhibit HT-29 cell proliferation and stimulate differentiation. Postproliferative differentiation achieved by the three approaches was associated with decreased VDR abundance, loss of VDR homologous up-regulation, and development of hormone unresponsiveness to 1,25(OH)2D3.

  View details for Web of Science ID A1993KV62900053

  View details for PubMedID 8384998

• CLONING AND EXPRESSION OF THE GENE FROM CANDIDA-ALBICANS THAT ENCODES A HIGH-AFFINITY CORTICOSTEROID-BINDING PROTEIN PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Malloy, P. J., Zhao, X., Madani, N. D., Feldman, D. 1993; 90 (5): 1902-1906

  Abstract

  We have previously demonstrated the presence of a corticosteroid-binding protein (CBP) in Candida albicans and speculated on its homology to the glucocorticoid receptor. To explore this relationship further, we cloned the CBP gene. Our strategy employed sequencing enzymatically derived peptide fragments from purified CBP and using this information to synthesize degenerate oligonucleotide primers for use in the PCR. A 117-bp fragment amplified from C. albicans DNA was then used to screen a genomic library. Hybridizing clones were isolated, and DNA sequencing revealed an open reading frame of 1467 bp which encoded a protein with a molecular weight of 55,545. Southern analysis demonstrated that the gene was present at a unique locus within chromosome R of the Candida genome, while Northern analysis showed that the gene was expressed in C. albicans as a 1.8-kb transcript. CBP was over-expressed in Saccharomyces cerevisiae, and it exhibited an apparent dissociation constant (Kd) of 7 nM for [3H]corticosterone and displayed a steroid hormone binding profile comparable to that of the native protein. Searches of the data banks revealed little overall similarity to other cloned genes. However, the amino acid sequence contains a dinucleotide-binding fingerprint. In conclusion, we have cloned the gene encoding the CBP from C. albicans and have shown that the expressed protein has the properties of the native CBP. A comparison of the cloned gene to members of the steroid-thyroid-retinoic acid receptor gene superfamily showed that CBP is unrelated to these hormone receptors.

  View details for Web of Science ID A1993KP97900057

  View details for PubMedID 8446606

  View details for PubMedCentralID PMC45988

• CHRONIC ATYPICAL SEIZURE DISORDER AND CATARACTS DUE TO DELAYED DIAGNOSIS OF PSEUDOHYPOPARATHYROIDISM WESTERN JOURNAL OF MEDICINE FAIG, J. C., Kalinyak, J., Marcus, R., Feldman, D. 1992; 157 (1): 64-65

  View details for Web of Science ID A1992JD51600012

  View details for PubMedID 1413750

  View details for PubMedCentralID PMC1021912

• CYCLIC ADENOSINE-3', 5'-MONOPHOSPHATE UP-REGULATES 1,25-DIHYDROXYVITAMIN-D3 RECEPTOR GENE-EXPRESSION AND ENHANCES HORMONE ACTION MOLECULAR ENDOCRINOLOGY Krishnan, A. V., Feldman, D. 1992; 6 (2): 198-206

  Abstract

  We have previously shown that the abundance of vitamin D receptors (VDR) in cultured cells is increased by mitogens such as serum and growth factors, whereas activation of protein kinase-C (PK-C) causes inhibition of VDR gene expression. This study examines the effect of the cAMP-activated protein kinase-A (PK-A) second messenger system on VDR abundance and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] action. Elevation of intracellular cAMP levels in NIH-3T3 mouse fibroblasts by forskolin or (Bu)2cAMP caused a substantial (8- to 12-fold) increase in VDR abundance, as measured by ligand binding and Western blot analysis. The time course of the forskolin effect on VDR expression was complex. An early rise in VDR abundance occurred at 4 h, followed by a decrease and then a broad secondary rise at 18 h. At the mRNA level, forskolin caused a rapid rise in VDR transcripts after 1 h of exposure, a peak at 2 h, followed by a decline and a subsequent increase at 15 h. Activation of PK-C with the phorbol ester phorbol myristate acetate abolished the forskolin-induced increase in VDR protein and mRNA abundance. NIH-3T3 cells were stably transfected with phOC-CAT, a plasmid carrying a human osteocalcin promoter fragment containing the vitamin D response element fused to the reporter gene chloramphenicol acetyl transferase (CAT). 1,25-(OH)2D3 treatment of transfected cells induced a dose-dependent increase in CAT activity. Up- or down-regulation of VDR in these transfected cells by forskolin or phorbol myristate acetate pretreatment, respectively, resulted in corresponding enhancement or attenuation of 1,25-(OH)2D3-inducible CAT activity.(ABSTRACT TRUNCATED AT 250 WORDS)

  View details for Web of Science ID A1992HH58300006

  View details for PubMedID 1314957

• CLONING AND CHARACTERIZATION OF THE GENE ENCODING THE ADP-RIBOSYLATION FACTOR IN CANDIDA-ALBICANS GENE DENICH, K. T., Malloy, P. J., Feldman, D. 1992; 110 (1): 123-128

  Abstract

  We have cloned and sequenced the gene (ARF) encoding the ADP-ribosylation factor (ARF) of Candida albicans. The gene contains an open reading frame of 537 nucleotides (nt) that codes for a protein with an Mr of 20,259. The C. albicans ARF gene is 67-70% identical at the nt level to other ARF sequences including those of humans; the deduced amino acid sequence of C. albicans ARF shows a 78-83% identity and 89-92% similarity to the other ARFs. Southern analysis of C. albicans genomic DNA suggested the presence of a second ARF gene. The presence of multiple ARF genes is a consistent finding among the other organisms previously shown to have ARFs.

  View details for Web of Science ID A1992HE64700019

  View details for PubMedID 1544569

• STIMULATION OF 1,25-DIHYDROXYVITAMIN D3 RECEPTOR GENE-EXPRESSION IN CULTURED-CELLS BY SERUM AND GROWTH-FACTORS JOURNAL OF BONE AND MINERAL RESEARCH Krishnan, A. V., Feldman, D. 1991; 6 (10): 1099-1107

  Abstract

  The abundance of 1,25-dihydroxyvitamin D3 receptors (VDR) in bone cells has been shown to vary in direct relation to the rate of cell proliferation. In this study we further explored this upregulation of VDR as it relates to the mitogenic response using NIH-3T3 mouse fibroblasts and MCF-7 human breast cancer cells as model systems. Serum and growth factors, such as EGF, high concentrations of insulin (2 microM), and IGF-I, were mitogenic and stimulated the proliferation of both cells types. These factors also caused significant increases in VDR levels as measured by ligand binding assays, which preceded the rise in cell proliferation rate measured by [3H]thymidine incorporation. Serum and growth factors increased the abundance of VDR but did not affect the concentrations of other steroid receptors in MCF-7 cells. Mouse cells have been reported to have several VDR mRNA transcripts. Our northern blot analysis revealed three mRNA species at approximately 7.5, 4.4, and 3 kb of which the 4.4 kb species was the most prominent and the 7.5 kb the least. Serum and growth factor stimulation of quiescent 3T3 cells led to significant increases in all the transcripts, suggesting that the upregulation occurs at the level of VDR mRNA expression. A time course analysis of serum stimulation in 3T3 cells showed that the mRNA species reached peak levels 4 h after serum addition. When serum stimulation was carried out in the presence of the protein synthesis inhibitor cycloheximide, the 3 kb transcript as well as the 7.5 kb transcript were superinduced but the stimulation of the 4.4 kb transcript was inhibited.(ABSTRACT TRUNCATED AT 250 WORDS)

  View details for Web of Science ID A1991GN79200010

  View details for PubMedID 1665676

• EFFECTS OF DEXMEDETOMIDINE, A NOVEL IMIDAZOLE SEDATIVE-ANESTHETIC AGENT, ON ADRENAL STEROIDOGENESIS - INVIVO AND INVITRO STUDIES ANESTHESIA AND ANALGESIA Maze, M., Virtanen, R., Daunt, D., BANKS, S. J., Stover, E. P., Feldman, D. 1991; 73 (2): 204-208

  Abstract

  Inhibition of steroidogenesis may be produced perioperatively by imidazole compounds, such as the hypnotic agent etomidate, with potentially serious consequences for patient morbidity and mortality. Dexmedetomidine, ([+]4-[1-[2,3-dimethylphenyl]-ethyl]-1H-imidazole), another imidazole compound with anesthetic like properties, is now being used perioperatively. Therefore, we investigated the effects of dexmedetomidine on steroidogenesis as well as on binding to glucocorticoid receptors in a series of in vitro and in vivo animal studies. The effect of dexmedetomidine, 10(-8)-10(-3) M, on adrenocorticotrophic hormone (ACTH) stimulated release of corticosterone was assessed in isolated rat adrenal cells. To characterize dexmedetomidine interactions with the glucocorticoid receptor, dexmedetomidine's ability to compete for [3H]dexmethasone binding sites was studied in renal tubular cells. The effect of dexmedetomidine, 80 micrograms/kg subcutaneously, on ACTH-stimulated release of cortisol was studied in separate cohorts of dogs at various time intervals during and after anesthesia was given. To compare the inhibitory effects of etomidate and dexmedetomidine on steroidogenesis, ACTH-stimulated release of cortisol was studied in dogs treated with anesthetic doses of either dexmedetomidine (80 micrograms/kg IV) or etomidate (1 mg/kg IV). Finally, dogs were given dexmedetomidine by continuous subcutaneous infusion for 7 days at sedative doses after which their cortisol response to ACTH was determined. At dexmedetomidine concentrations greater than 10(-7) M, a dose-dependent inhibition of corticosterone release was detected in response to ACTH stimulation in vitro. At these high dexmedetomidine concentrations, [3H]dexamethasone binding was not affected. In the in vivo dog experiments, basal cortisol levels decreased and the cortisol response to ACTH was blunted 3 h after dexmedetomidine administration.(ABSTRACT TRUNCATED AT 250 WORDS)

  View details for Web of Science ID A1991FX91000015

  View details for PubMedID 1649559

• ACTIVATION OF PROTEIN-KINASE-C INHIBITS VITAMIN-D RECEPTOR GENE-EXPRESSION MOLECULAR ENDOCRINOLOGY Krishnan, A. V., Feldman, D. 1991; 5 (4): 605-612

  Abstract

  The abundance of 1,25-dihydroxyvitamin D3 receptors (VDR) in cultured cells has been shown to vary in direct relation to the rate of cell proliferation. This study examines the question of whether the growth-factor mediated up-regulation of VDR is due to direct modulation of VDR gene expression or is secondary to the stimulation of cell cycle events. Mitogenic agents, such as basic fibroblast growth factor and phorbol esters, were found to cause significant decreases in VDR abundance, while substantially stimulating proliferation of NIH-3T3 cells. Potent phorbol esters, such as phorbol myristate acetate (PMA) and phorbol-12,13-dibutyrate, whose biological actions have been shown to be mediated through the activation of protein kinase-C, down-regulated VDR in a time- and dose-dependent manner. An inactive phorbol ester, 4 alpha-phorbol-12,13-didecanoate, which does not activate protein kinase-C, did not alter VDR levels. Desensitization of protein kinase-C by prolonged exposure of cells to phorbol esters eliminated the PMA-mediated down-regulation of VDR. Staurosporine, an inhibitor of protein kinase-C, blocked the actions of PMA. Oleoyl acetyl glycerol, a synthetic diacyl glycerol, and A23187, a calcium ionophore, were both able to suppress VDR abundance alone and were additive in combination. The results suggest that activation of the protein kinase-C pathway and elevation of intracellular Ca2+ lead to significant down-regulation of VDR. The inhibitory effect of PMA appears to be exerted at the level of VDR mRNA expression. Northern blot analysis revealed significant decreases in steady state levels of VDR mRNA species that qualitatively corresponded to the decrease in VDR protein concentration seen on a Western blot.(ABSTRACT TRUNCATED AT 250 WORDS)

  View details for Web of Science ID A1991FK11400018

  View details for PubMedID 1656239

• GENETIC-DEFECTS OF THE 1,25-DIHYDROXYVITAMIN-D3 RECEPTOR 4TH SWISS WORKSHOP OF METHODOLOGY IN RECEPTOR RESEARCH Hughes, M. R., Malloy, P. J., OMALLEY, B. W., PIKE, J. W., Feldman, D. MARCEL DEKKER INC. 1991: 699–716

  Abstract

  Target organ resistance to steroid hormone action is known to produce clinical disorders ranging from testicular feminization in the case of androgen resistance to hypocalcemic vitamin D-resistant ricets (HVDRR) in the case of 1,25-dihydroxyvitamin D3. The etiologic basis of these disorders is thought to be genetic mutations in the gene encoding receptors for these hormones. We investigated this possibility by analyzing the vitamin D receptor (VDR) protein, mRNA, and DNA from patients with HVDRR. This autosomal recessive disease of children is characterized by early onset rickets, hypocalcemia, hyperparathyroidism, and elevated levels of 1,25-(OH)2D3. Cells from patients fall into three general classes of molecular defects: (i) decreased or absent hormone binding; (ii) decreased affinity of VDR for DNA, or; (iii) defective nuclear translocation or retention. Analysis of the DNA and/or mRNA from these cells has identified missense mutations in the DNA binding (zinc finger) domain and a nonsense mutation in the steroid binding domain of VDR. The mutations were individually recreated in wild type VDR and the expressed mutant protein behaved biochemically identically to the patient receptor. Further studies have shown that the receptor is unable to interact with the specific hormone response element (HRE) of the osteocalcin gene and activate appropriate transcription. Rapid diagnostic genotyping of these mutations is possible with either restriction digestion or allele-specific oligonucleotide hybridization. Analysis of these naturally occurring, disease producing mutations of a gene regulatory protein should provide insight into the key amino acid residues of the protein and the mechanism by which steroids modulate gene transcription.

  View details for Web of Science ID A1991FK03000053

  View details for PubMedID 1653354

• HEREDITARY 1,25-DIHYDROXYVITAMIN-D RESISTANT RICKETS - MOLECULAR-BASIS AND IMPLICATIONS FOR THE ROLE OF 1,25(OH)2D3 IN NORMAL PHYSIOLOGY MOLECULAR AND CELLULAR ENDOCRINOLOGY Feldman, D., Malloy, P. J. 1990; 72 (3): C57-C62

  View details for Web of Science ID A1990EA17800001

  View details for PubMedID 1963156

• MUTANT VITAMIN-D RECEPTORS WHICH CONFER HEREDITARY RESISTANCE TO 1,25-DIHYDROXYVITAMIN-D3 IN HUMANS ARE TRANSCRIPTIONALLY INACTIVE INVITRO JOURNAL OF BIOLOGICAL CHEMISTRY Sone, T., Scott, R. A., Hughes, M. R., Malloy, P. J., Feldman, D., OMALLEY, B. W., PIKE, J. W. 1989; 264 (34): 20230-20234

  Abstract

  Hereditary 1,25-dihydroxyvitamin D3-resistant rickets is a human syndrome that arises as a result of heterogeneous molecular defects in the vitamin D3 receptor. Recent studies have identified single unique point mutations within the second or third exons that encode the DNA-binding domain of the vitamin D receptor (VDR) gene in two families with this syndrome. In the experiments reported here, these mutations were introduced into the normal VDR cDNA by site-directed mutagenesis and the mutant products evaluated for hormone, nuclear, and DNA-binding characteristics. Each mutant VDR was expressed in COS-1 cells at equivalent levels, and saturation analysis of cell cytosol revealed normal affinity for the 1,25-dihydroxyvitamin D3 hormone. Incubation of transfected cells with radiolabeled hormone followed by lysis and extraction suggests a lowered salt dependence for solubilization of the mutant VDR. Concomitantly, mutant receptors exhibited reduced affinity for immobilized calf thymus DNA. While cotransfection of the wild type receptor together with a vitamin D-inducible (osteocalcin) chloramphenicol acetyltransferase reporter gene construction in CV-1 cells resulted in strong induction by 1,25-dihydroxyvitamin D3, neither mutant receptor was capable of directing significant activity either as a function of receptor or hormone concentration. These data suggest that the unique point mutations identified in each of these two families are responsible not only for the phenotype originally ascribed to the abnormal receptor but also severely compromise each protein's ability to activate transcription.

  View details for Web of Science ID A1989CB33400016

  View details for PubMedID 2555349

• STEROID-METABOLISM AS A MECHANISM OF ESCAPE FROM PROGESTERONE-MEDIATED GROWTH-INHIBITION IN TRICHOPHYTON-MENTAGROPHYTES JOURNAL OF BIOLOGICAL CHEMISTRY Clemons, K. V., Stover, E. P., Schar, G., STATHIS, P. A., Chan, K., Tokes, L., Stevens, D. A., Feldman, D. 1989; 264 (19): 11186-11192

  Abstract

  It has been shown by us and others that progesterone inhibits the growth of Trichophyton mentagrophytes and that the organism escapes from this inhibition over time. We report here studies which show that escape from growth inhibition is related to the enzymatic transformation of progesterone to polar metabolites. Isolation and identification of the progesterone metabolites confirm the production of 15 alpha-hydroxyprogesterone. In addition, three other metabolites were isolated. Two of these were determined to be 1-dehydroprogesterone and 11 alpha-hydroxyprogesterone. The third metabolite was a 1-dehydro-hydroxyprogesterone, but the location of the hydroxyl group could not be determined unequivocally. Studies using authentic 15 alpha-hydroxyprogesterone, 1-dehydroprogesterone, and 11 alpha-hydroxyprogesterone reveal that these derivatives are significantly less inhibitory to the growth of T. mentagrophytes than progesterone. Pretreatment of organisms with progesterone augments the rate of metabolism and enhances escape. We have described previously a progesterone-binding protein (PBP) in cytoplasmic extracts of T. mentagrophytes and hypothesized that progesterone mediates growth inhibition by binding to the PBP of this organism. The relative binding affinity that progesterone and its metabolites display for PBP correlates with the relative growth inhibitory potency of these compounds. These results suggest that metabolism of progesterone to more polar and less inhibitory compounds, which exhibit lower affinity for PBP, is the mechanism of escape from progesterone-mediated inhibition of growth in this organism.

  View details for Web of Science ID A1989AC81700042

  View details for PubMedID 2738064

• SEQUENCE AND EXPRESSION OF HUMAN MYOSIN ALKALI LIGHT CHAIN ISOFORMS MOLECULAR AND CELLULAR BIOCHEMISTRY Wade, R., Feldman, D., Gunning, P., Kedes, L. 1989; 87 (2): 119-136

  Abstract

  In order to initiate the study of the functional differences between myosin alkali light chain isoforms and to investigate the mechanisms of their differential expression, we have isolated cDNA clones for two human alkali light chain isoforms. Here we report DNA sequence and RNA blotting analyses that demonstrate that these cDNAs represent transcripts encoding human MLC3F and MLC1Sb. The sequence of the human MLC1Sb cDNA offers the first fully characterized example of a slow-fiber skeletal muscle alkali light chain isoform from any species. The sequence analysis of these two cDNAs allows an examination of evolutionarily conserved features of mammalian alkali light chain genes. Examination of the genomic organization of the human alkali light chain isoform genes revealed that, in contrast with some strains of mice, both are single copy genes. RNA blot analysis conclusively demonstrates that the human skeletal muscle MLC1Sb gene is also expressed in the heart ventricle but not the atria. In addition, we examined the expression of alkali light chain isoforms during the in vitro differentiation of a variety of human and rodent myogenic cells and found striking variation in the pattern of alkali light chain isoform gene expression in different myogenic cells.

  View details for Web of Science ID A1989U846300003

  View details for PubMedID 2475760

• CHARACTERIZATION OF AN ESTROGEN-BINDING PROTEIN IN THE YEAST CANDIDA-ALBICANS ENDOCRINOLOGY Skowronski, R., Feldman, D. 1989; 124 (4): 1965-1972

  Abstract

  An estrogen-binding protein (EBP) has been identified and characterized in the cytosol of the pathogenic yeast Candida albicans. Binding of [3H]estradiol was found to be optimal at pH 7.4 in the presence of 0.3 M KCl and was linearly related to protein concentration. Binding was very rapid, reaching maximal levels in about 30 min, and was reversible with a dissociation rate constant of 13.2 +/- 1.7 x 10(-4) sec-1. EBP binding was destroyed by treatment with proteolytic enzymes and by high temperatures. Scatchard analysis of the [3H]estradiol equilibrium binding data of C. albicans (strain stn-1) yielded an apparent dissociation constant of 12.3 +/- 2.1 nM and a maximal binding capacity of 753 +/- 145 fmol/mg protein. Binding competition experiments showed very high specificity and stereoselectivity of EBP, demonstrating the following order of potency in displacing [3H]estradiol: 17 beta-estradiol greater than estrone greater than estriol greater than 17 alpha-estradiol. Negligible competitive potency was found for other mammalian steroid hormones, diethylstilbestrol, tamoxifen, or fungal hormones. The abundance of EBP was 4- to 10-fold higher during the early logarithmic growth phase of yeast cells than during the stationary phase. The molecular size of EBP, measured by Sephacryl S-200 gel exclusion chromatography, yielded a Stokes radius of approximately 29 A. Sucrose density gradient sedimentation showed a sedimentation coefficient (S2020,W) of 4, with no ionic dependent aggregation of the [3H]estradiol-EBP complex. The apparent mol wt of the EBP is approximately 46,000, with an axial ratio of 1, indicating the symmetrical shape of the molecule. In summary, in addition to the previously described corticosterone-binding protein, a separate high affinity, stereospecific, estrogen-selective binder has been demonstrated in the cytosol of C. albicans.

  View details for Web of Science ID A1989T815900050

  View details for PubMedID 2647470

• ABNORMAL BINDING OF VITAMIN-D RECEPTORS TO DEOXYRIBONUCLEIC-ACID IN A KINDRED WITH VITAMIN D-DEPENDENT RICKETS, TYPE-II JOURNAL OF CLINICAL ENDOCRINOLOGY & METABOLISM Malloy, P. J., Hochberg, Z., PIKE, J. W., Feldman, D. 1989; 68 (2): 263-269

  Abstract

  Vitamin D-dependent rickets, type II, is a hereditary disease that results from target organ resistance to the action of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. We describe here a family (designated G) with a defect in the DNA-binding domain of the 1,25-(OH)2D receptor (VDR) manifested by normal steroid binding and decreased VDR affinity for DNA. The phenotypically normal parents are heterozygous, expressing both normal and defective forms of VDR. The affected children in this family had early-onset rickets, alopecia, hypocalcemia, and elevated serum 1,25-(OH)2D3 levels. The VDR of cultured skin fibroblasts of the affected children (G1 and G2) as well as the parents (G3 and G4) bound [3H]1,25-(OH)2D3 normally (Kd = 2-3 X 10(-11) mol/L; maximal number of binding sites = 20-40 fmol/mg protein). The cells from G1 and G2 were resistant to 1,25-(OH)2D3 action, as measured by induction of 24-hydroxylase activity, while the cells from G3 and G4 responded normally. Western blot analysis using the anti-VDR monoclonal antibody 9A7 showed that hypertonic extracts of fibroblasts from both affected children (G1 and G2) and their parents (G3 and G4) had immunoreactive bands of 48K, identical to the size of the VDR in normal cells. The VDR from G1 and G2 eluted as a single peak from DNA-cellulose columns at a lower salt concentration (0.1 mol/L) than that from normal subjects (0.2 mol/L), indicating an apparent decreased affinity for DNA. Fibroblast VDR from G3 and G4 each eluted from DNA-cellulose columns as two peaks, the normal peak (0.2 mol/L) and the abnormal peak (0.1 mol/L), which was found in G1 and G2. Western blot analysis of the 0.1 and 0.2 mol/L KCl peak fractions also confirmed that VDR was present in only the 0.1 mol/L fraction and not the 0.2 mol/L fraction from G1. However, VDR was present in both fractions from G3. In summary, this vitamin D-dependent rickets, type II, kindred has a defect in the DNA-binding domain of VDR. The parents are phenotypically normal and express both the normal and defective VDR alleles, as demonstrated by both DNA-cellulose chromatography and Western blot analysis. The affected children are resistant to 1,25-(OH)2D3 action and are homozygous for the defective VDR.

  View details for Web of Science ID A1989R947500004

  View details for PubMedID 2537329

• Human vitamin D receptor mutations: identification of molecular defects in hypocalcemic vitamin D resistant rickets. Advances in experimental medicine and biology Hughes, M., MALLOY, P., Kieback, D., McDonnell, D., Feldman, D., PIKE, J. W., O'Malley, B. 1989; 255: 491-503

  View details for PubMedID 2618873

• DERMATOPHYTE-HORMONE RELATIONSHIPS - CHARACTERIZATION OF PROGESTERONE-BINDING SPECIFICITY AND GROWTH-INHIBITION IN THE GENERA TRICHOPHYTON AND MICROSPORUM JOURNAL OF CLINICAL MICROBIOLOGY Clemons, K. V., Schar, G., Stover, E. P., Feldman, D., Stevens, D. A. 1988; 26 (10): 2110-2115

  Abstract

  We reported previously that Trichophyton mentagrophytes contains a cytoplasmic macromolecule which specifically binds progesterone. Progesterone is also an effective inhibitor of growth of the fungus. We report here studies which characterize more fully the specific binding properties and the functional responses of T. mentagrophytes and taxonomically related fungi to a series of mammalian steroid hormones. Scatchard analysis of [3H]progesterone binding in both the + and - mating types of Arthroderma benhamiae and in Microsporum canis revealed a single class of binding sites with approximately the same affinity as that in T. mentagrophytes (Kd, 1 X 10(-7) to 2 X 10(-7) M). Trichophyton rubrum had a protein with a higher binding affinity (Kd, 1.6 X 10(-8) M). Characterization of the [3H]progesterone-binding sites in T. mentagrophytes showed the binder to be a protein which was destroyed by trypsin and heating to 56 degrees C. Previous examination of the steroid-binding specificity in T. mentagrophytes had demonstrated that deoxycorticosterone (DOC) and dihydrotestosterone (DHT) were effective competitors for [3H]progesterone binding. Expansion of this study to include other competitors revealed that R5020 (a synthetic progestin), androstenedione, and dehydroepiandosterone possessed relative binding affinities which were 20, 11, and 9% of that of progesterone, respectively. Other ligands tested were less effective. Competition studies for the binder in M. canis resulted in similar findings: DOC and DHT were effective competitors for [3H]progesterone binding. The growth of A. benhamiae + and -, M. canis, and T. rubrum were all inhibited by progesterone in a dose-responsive manner, with 50% inhibition achieved at concentrations of 9.8 x 10(-6), 1.2 x 10(-5), 1.5 x 10(-5), and 2.7 x 10(-6) M. respectively,.

  View details for Web of Science ID A1988Q160000035

  View details for PubMedID 3182998

  View details for PubMedCentralID PMC266826

• MODULATION OF 1,25-DIHYDROXYVITAMIN-D3 RECEPTOR-BINDING AND ACTION BY SODIUM-BUTYRATE IN CULTURED PIG-KIDNEY CELLS (LLC-PK1) JOURNAL OF BONE AND MINERAL RESEARCH Costa, E. M., Feldman, D. 1987; 2 (2): 151-159

  Abstract

  We have studied the effects of sodium butyrate (SB) on 1,25(OH)2D3 receptors in the pig kidney cell line (LLC-PK1). In this system, we have shown that 1,25(OH)2D3 induction of 25-hydroxyvitamin D3-24-hydroxylase (24-hydroxylase) activity is dependent on the level of 1,25(OH)2D3 receptors. Treatment of confluent cells with SB (5 mM/48 h) caused an approximately 50% decrease in total receptors, whereas the affinity for 1,25(OH)2D3 was unchanged. At 5 mm, the action of SB on these receptors required more than 24 h to be detected. The effect of the decrease in receptors on the functional response to the hormone was studied by measuring the 1,25(OH)2D3 induction of 24-hydroxylase activity after treatment with SB. The induction of 24-hydroxylase activity at higher doses of hormone paralleled the reduction in receptors and was diminished by 25-50%. At low doses of hormone, the cells appear to be more sensitive to 1,25(OH)2D3 induction, exhibiting an unexplained increase in 24-hydroxylase activity compared to cells not exposed to SB. An additional effect of SB was also noted: SB decreased cell proliferation and inhibited [3H]thymidine incorporation by 75% when added to cells prior to confluence. At confluence, SB caused a less drastic effect on protein and DNA synthesis. Therefore, most binding experiments were conducted at confluence when the SB effect on cell proliferation was less. Other short chain fatty acids in addition to SB were also tested. The action to decrease 1,25(OH)2D3 receptors was more specific upon exposure to SB. We have previously demonstrated up-regulation of 1,25(OH)2D3 receptors in LLC-PK1 cells after treatment with various vitamin D metabolites.(ABSTRACT TRUNCATED AT 250 WORDS)

  View details for Web of Science ID A1987H019300010

  View details for PubMedID 2843001

• KETOCONAZOLE AND OTHER IMIDAZOLE DERIVATIVES AS INHIBITORS OF STEROIDOGENESIS ENDOCRINE REVIEWS Feldman, D. 1986; 7 (4): 409-420

  View details for Web of Science ID A1986F067800003

  View details for PubMedID 3536461

• PROGESTERONE BINDING AND INHIBITION OF GROWTH IN TRICHOPHYTON-MENTAGROPHYTES INFECTION AND IMMUNITY Schar, G., Stover, E. P., Clemons, K. V., Feldman, D., Stevens, D. A. 1986; 52 (3): 763-767

  Abstract

  Specific binding of [3H]progesterone to cytosol of Trichophyton mentagrophytes was demonstrated. Scatchard analysis of [3H]progesterone binding showed a single class of binding sites with a dissociation constant of 9.5 X 10(-8) [corrected] +/- 2.4 X 10(-8) M (standard deviation) and a maximal binding capacity of 4,979 +/- 3,489 fmol/mg of cytosol protein. Deoxycorticosterone and dihydrotestosterone competitively inhibited binding by 50% at molar ratios of 10:1 and 20:1, respectively. Other steroid hormones that were tested had minimal activity, indicating binding specificity. Steroid hormone actions in T. mentagrophytes were examined in growth studies. Growth was assessed by determination of cellular ATP content. Progesterone inhibited growth in a dose-responsive manner, with a 50% inhibition concentration of 5.5 X 10(-6) M. Partial recovery from inhibition occurred after 24 to 48 h; inhibition could be enhanced by dividing the amount of added progesterone every 24 h. In the same rank order as was their relationship to each other and progesterone in binding studies, deoxycorticosterone and dihydrotestosterone were less effective inhibitors; other steroid hormones that were tested showed no consistent effect. We hypothesize that the binder described, acting as a hormone receptor, is the molecular site of action for the functional effect of the hormone. The functional effect may be related to the observed resistance of females to dermatophytosis.

  View details for Web of Science ID A1986C485300021

  View details for PubMedID 3710585

  View details for PubMedCentralID PMC260924

• DEXAMETHASONE INCREASES 1,25-DIHYDROXYVITAMIN-D3 RECEPTOR LEVELS AND AUGMENTS BIORESPONSES IN RAT OSTEOBLAST-LIKE CELLS ENDOCRINOLOGY Chen, T. L., Hauschka, P. V., Feldman, D. 1986; 118 (3): 1119-1126

  Abstract

  Glucocorticoids increase the level of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] receptors in primary cultures of rat calvarial osteoblast-like (OB) cells. The present study investigated how this dexamethasone (DEX) up-regulation of 1,25-(OH)2D3 receptors modulates three 1,25-(OH)2D3 bioresponses: inhibition of collagen synthesis, stimulation of osteocalcin synthesis, and induction of 25-hydroxyvitamin D3-24-hydroxylase activity. Pretreatment of OB cells with 13 nM DEX for 24 h doubled the 1,25-(OH)2D3 receptor level without changing receptor affinity for 1,25-(OH)2D3 to study bioresponses. After DEX treatment to increase the 1,25-(OH)2D3 receptor level, the magnitude and sensitivity of all three 1,25-(OH)2D3 bioresponses were enhanced. The maximal 1,25-(OH)2D3 inhibition of collagen synthesis was increased by DEX pretreatment compound to control values: 30 to 50% (1 day treatment) and 50 to 70% (2 day treatment). The sensitivity to 1,25-(OH)2D3, as measured by reduction of the half-maximal inhibitory dose (ED50), was increased 50%. This potentiation of 1,25-(OH)2D3 inhibitory action on collagen synthesis was still evident after correction for the inhibitory effect on collagen synthesis by DEX alone. The maximal stimulation of osteocalcin by 1,25-(OH)2D3 was also enhanced from 2- to 3-fold in controls to over 4- to 5-fold by DEX pretreatment. Similarly, the ED50 of the response was reduced 50%. For the induction of 25-hydroxyvitamin D3-24-hydroxylase activity, DEX doubled the enzyme activity over that seen with 1,25-(OH)2D3 alone, but only slightly affected the sensitivity of the enzyme induction. In conclusion, after DEX up-regulation of 1,25-(OH)2D3 receptor levels, there was a general potentiation of 1,25-(OH)2D3 bioresponses in rat OB cells. However, the detailed patterns of the augmented responses were different for each of the three biological functions we studied.

  View details for Web of Science ID A1986A106300033

  View details for PubMedID 3004896

• THE EFFECTS OF 1,25-DIHYDROXYVITAMIN-D3 AND DEXAMETHASONE ON RAT OSTEOBLAST-LIKE PRIMARY-CELL CULTURES - RECEPTOR OCCUPANCY AND FUNCTIONAL EXPRESSION PATTERNS FOR 3 DIFFERENT BIORESPONSES ENDOCRINOLOGY Chen, T. L., Hauschka, P. V., CABRALES, S., Feldman, D. 1986; 118 (1): 250-259

  Abstract

  The effects of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and dexamethasone to regulate collagen and osteocalcin synthesis and induction of 25-hydroxyvitamin D3-24-hydroxylase (24-hydroxylase) activity were studied in rat osteoblast-like cell primary cultures. In this culture system, the basal levels of collagen and osteocalcin synthesis increased with rising cell density in culture. At maximal doses, both 1,25-(OH)2D3 (8.1 nM) and dexamethasone (130 nM) reduced collagen synthesis to about 50% of the control levels, 1,25-(OH)2D3 affected osteocalcin synthesis in a biphasic manner: stimulatory at low doses, which peaked near 0.33 nM to reach 3- to 5-fold the basal level, followed by a gradual return to the basal level at higher concentrations. Dexamethasone had only a slight stimulatory effect on osteocalcin. 1,25-(OH)2D3 also induced 24-hydroxylase activity in rat osteoblast-like cells, while dexamethasone had no effect on the enzyme. Induction of enzyme activity achieved a 4- to 6-fold rise, but required higher concentrations of 1,25-(OH)2D3 to achieve maximal levels (16 nM). The half-maximal doses (ED50) of 1,25-(OH)2D3 required for each bioresponse were different. The approximate ED50 for the inhibition of collagen synthesis was near the Kin (0.4 nM; apparent dissociation constant of receptor nuclear internalization), while the ED50 for osteocalcin synthesis (0.08 nM) was below the Kin, and the ED50 for 24-hydroxylase induction (20 nM) was greater than the Kin. The ED50 for dexamethasone on collagen synthesis (20 nM) was about 5-fold higher than the Kin (4 nM) of dexamethasone receptor binding. The potencies of various vitamin D3 metabolites in all three functional responses followed their abilities to compete for the 1,25-(OH)2D3 receptor, indicating that these actions were 1,25-(OH)2D3 receptor mediated. In summary, these studies explored bone cell bioresponses to 1,25-(OH)2D3 and dexamethasone and examined the relationship between receptor occupancy and functional expression. Each action exhibited a different dose-response pattern, implying that different levels of control are required for each individual response.

  View details for Web of Science ID A1986AWR7000041

  View details for PubMedID 3000737

• ESTRADIOL-BINDING PROTEINS FROM MYCELIAL AND YEAST-FORM CULTURES OF PARACOCCIDIOIDES-BRASILIENSIS INFECTION AND IMMUNITY Stover, E. P., Schar, G., Clemons, K. V., Stevens, D. A., Feldman, D. 1986; 51 (1): 199-203

  Abstract

  Paracoccidioides brasiliensis, the etiologic agent of paracoccidioidomycosis, causes disease much more frequently in men than it does in women, suggesting that the hormonal milieu of the host might influence P. brasiliensis pathogenicity. We recently demonstrated that cytosol from yeast cultures of P. brasiliensis contains a high-affinity, low-capacity, tritiated 17 beta-estradiol [( 3H]estradiol)-binding protein. Estradiol and, to a lesser degree, diethylstilbestrol (DES), inhibited the transformation of P. brasiliensis cultures from the mycelial to the yeast form, an event critical to the establishment of infection. Our current studies demonstrated a somewhat higher affinity (apparent dissociation constant [Kd], approximately equal to 6 to 12 nM) of the estrogen-binding protein for [3H]estradiol than was previously described for yeast cytosol. The presence of both high- and low-affinity estrogen-binding sites in yeast-form P. brasiliensis cytosol was detected after warming the cytosol to 37 degrees C. The high-affinity protein was labile to further heating (56 degrees C), although the low-affinity protein was stable. Additional experiments demonstrated the presence of an estrogen-binding protein in cytosol prepared from mycelial-form P. brasiliensis. This estrogen-binding protein had a slightly lower affinity for [3H]estradiol (Kd approximately equal to 13 nM), and its cytosol contained somewhat fewer binding sites (approximately equal to 78 fmol/mg of protein) than did yeast-form P. brasiliensis cytosol. Of particular interest was the finding that DES, a weak competitor for [3H]estradiol binding in yeast cytosol, displaced [3H]estradiol from the mycelial-form binding moiety. DES had a 50- to 100-fold-lower affinity for the [3H]estradiol-binding protein than did estradiol, consistent with its lower bioactivity in the mycelial-to-yeast-form transformation studies. The current results lend further support to our hypothesis that endogenous estrogens in the host, acting through the cytosol binding protein in the fungus, inhibit mycelial-to-yeast-form transformation, thus explaining the resistance of women to paracoccidioidomycosis.

  View details for Web of Science ID A1986AXB5200031

  View details for PubMedID 3079729

  View details for PubMedCentralID PMC261086

• CHARACTERIZATION OF AN ESTROGEN-BINDING PROTEIN IN THE YEAST SACCHAROMYCES-CEREVISIAE JOURNAL OF BIOLOGICAL CHEMISTRY Burshell, A., STATHIS, P. A., Do, Y., Miller, S. C., Feldman, D. 1984; 259 (6): 3450-3456

  Abstract

  This paper further characterizes the estrogen-binding protein we have described in the cytosol of the yeast Saccharomyces cerevisiae. [3H]Estradiol was used as the radioprobe, and specific binding of cytosol fractions was measured by chromatography on Sephadex minicolumns. Other 3H-steroids did not exhibit specific binding. [3H]Estradiol binding was destroyed by treatment with trypsin, but not RNase, DNase, or phospholipase; N-ethylmaleimide substantially decreased the binding. The yeast did not metabolize estradiol added to the medium, and extraction and chromatography of the bound moiety showed it to be unmetabolized estradiol. Scatchard analysis of cytosol from both a and alpha mating types as well as the a/alpha diploid cell revealed similar binding properties: an apparent dissociation constant or Kd(25 degrees) for [3H]estradiol of 1.6-1.8 nM and a maximal binding capacity or Nmax of approximately 2000-2800 fmol/mg of cytosol protein. Gel exclusion chromatography on Sephacryl S-200 and high performance liquid chromatography suggested a Stokes radius of approximately 30 A. Sucrose gradient centrifugation showed a sedimentation coefficient of approximately 5 S, and the complex did not exhibit ionic dependent aggregation. The estrogen binder in S. cerevisiae differed in its steroidal specificities from classical mammalian estrogen receptors in rat uterus. 17 beta-Estradiol was the best competitor, 17 alpha-estradiol had about 5% the activity, and diethylstilbestrol exhibited negligible binding affinity as did tamoxifen, nafoxidine, and the zearalenones. In summary, a high affinity, stereospecific, steroid-selective binding protein has been demonstrated in the cytosol of the simple yeast S. cerevisiae. We speculate that this molecule may represent a primitive hormone receptor system, possibly for an estrogen-like message molecule.

  View details for Web of Science ID A1984SJ39800013

  View details for PubMedID 6368545

• MODULATION OF PTH-STIMULATED CYCLIC-AMP IN CULTURED RODENT BONE-CELLS - THE EFFECTS OF 1,25(OH)2 VITAMIN-D3 AND ITS INTERACTION WITH GLUCOCORTICOIDS CALCIFIED TISSUE INTERNATIONAL Chen, T. L., Feldman, D. 1984; 36 (5): 580-585

  Abstract

  Parathyroid hormone (PTH)-stimulated cyclic adenosine monophosphate (cAMP) in rat osteoblastlike (OB) cells has been shown to be modulated by steroid hormones; glucocorticoids are known to increase the level, while the effects of 1,25(OH)2D3 are inhibitory. In the present study, we found that the PTH-stimulated cAMP responses are similar in neonatal mouse and fetal rat OB cells. Dexamethasone (0.13-13 nM) augmented PTH-stimulated cAMP in both species. Mouse cells showed a higher maximal response to dexamethasone (100% increment) than rat cells (60-70% increment) with similar sensitivity to dexamethasone (ED50 approximately 1.0 nm). On the other hand, 1,25(OH)2D3 decreased PTH-stimulated cAMP, but the effect required pharmacological levels of hormone; mouse cells responded at a lower dose (1.3 nM) and were more sensitive than rat cells (responded at 13 nM) to 1,25(OH)2D3 treatment. Introduction of physiological concentrations of 1,25(OH)2D3 (0.013-1.3 nm) in addition to dexamethasone (13 nM) resulted in a synergistic enhancement of PTH-stimulated cAMP in rat cells. In contrast, a dose-dependent antagonistic effect was observed in mouse cells. In summary, our findings demonstrate species and concentration-dependent differences in hormonal responses to 1,25(OH)2D3 and a complex interplay among PTH, dexamethasone, and 1,25(OH)2D3.

  View details for Web of Science ID A1984TW89500011

  View details for PubMedID 6098354

• CHARACTERIZATION OF INSULIN-LIKE GROWTH FACTOR-I RECEPTORS ON CULTURED RAT BONE-CELLS - REGULATION OF RECEPTOR CONCENTRATION BY GLUCOCORTICOIDS ENDOCRINOLOGY Bennett, A., Chen, T., Feldman, D., Hintz, R. L., Rosenfeld, R. G. 1984; 115 (4): 1577-1583

  Abstract

  A specific receptor for insulin-like growth factor I (IGF-I) has been demonstrated in cultured fetal rat osteoblast-like bone cells. Specific binding of [125I]IGF-I to bone cells incubated at 15 C reached a steady state by 5 h. Half-maximal inhibition of [125I]IGF-I binding by unlabeled IGF-I was observed at 7 ng/ml. Multiplication-stimulating activity, insulin, and proinsulin were less effective than unlabeled IGF-I in competing for receptor occupancy. Scatchard analysis showed a curvilinear plot, with a Ka similar to that observed in human fibroblasts. Incubation of cell monolayers with glucocorticoids resulted in a concentration-dependent increase in [125I]IGF-I binding. This increase in [125I]IGF-I binding was dependent on cell density. After a 2-day exposure to dexamethasone, no increase in binding was observed in sparsely plated cells; however, an increase in binding was observed after 3 days in culture (log phase) and was maximal by 5 days (peak log phase). These data indicate that rat bone cells possess a specific receptor for IGF-I with binding characteristics similar to those reported in human fibroblasts, and that IGF-I receptor concentrations are increased by exposure to glucocorticoids. A role for glucocorticoids and IGF-I in rat bone proliferation is suggested by these findings.

  View details for Web of Science ID A1984TK43600050

  View details for PubMedID 6090106

• DISTRIBUTION OF A CORTICOSTEROID-BINDING PROTEIN IN CANDIDA AND OTHER FUNGAL GENERA JOURNAL OF GENERAL MICROBIOLOGY Loose, D. S., Stevens, D. A., Schurman, D. J., Feldman, D. 1983; 129 (AUG): 2379-2385

  Abstract

  Using [3H]corticosterone as a probe, corticosteroid-binding protein (CBP) was detected in eight out of eight isolates of Candida albicans, of both A and B serotypes. The apparent dissociation constant (Kd) in the various isolates ranged between 8 and 19 nM; the binding capacity varied from 122 to over 2400 fmol (mg cytosol protein)-1. There was no correlation between the amount or affinity of CBP and isolate virulence for murine hosts. Further analysis revealed demonstrable CBP in six out of six Candida species other than C. albicans. One isolate of C. tropicalis has been identified which fails to bind [3H]corticosterone. Saccharomyces cerevisiae, Neurospora crassa and Paracoccidioides brasiliensis also failed to bind [3H]corticosterone. Preliminary attempts were made to determine functions mediated by CBP in Candida, but in vitro growth, phase conversion and glucose oxidation by Candida were unaffected by the addition of a variety of steroid hormones. These data indicate that the presence of CBP in Candida does not correlate with either virulence or serotype. The physiological significance of CBP remains to be determined.

  View details for Web of Science ID A1983RF21400007

• REGULATION OF 1,25(OH)2VITAMIN-D3 RECEPTOR CONTENT IN CULTURED LLC-PK1 KIDNEY-CELLS LIMITS HORMONAL RESPONSIVENESS BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Hirst, M., Feldman, D. 1983; 116 (1): 121-127

  Abstract

  Receptor content in cultured kidney (LLC-PK1) cells was found to be modulated following the introduction of a culture medium change, declining to 40% of control values at 18 h. Scatchard analysis indicated that the reduced 1,25(OH)2-[3H]D3 nuclear binding we detected was due to decreased abundance of receptors (3811 vs 1619 sites/cell) with no change in the Kd (0.4-0.5 nM). Cells with reduced receptors exhibited diminished ability to respond to 1,25(OH)2D3 as measured by induction of 25(OH)vitamin D-24-hydroxylase activity. There was a close coupling between decreased receptor levels and diminished hormone responsiveness. The data suggest the absence of "spare" receptors and that receptor abundance is a limiting factor in cell responsiveness to 1,25(OH)2D3.

  View details for Web of Science ID A1983RN09300018

  View details for PubMedID 6315000

• KETOCONAZOLE BINDS TO GLUCOCORTICOID RECEPTORS AND EXHIBITS GLUCOCORTICOID ANTAGONIST ACTIVITY IN CULTURED-CELLS JOURNAL OF CLINICAL INVESTIGATION Loose, D. S., Stover, E. P., Feldman, D. 1983; 72 (1): 404-408

  Abstract

  We have recently found that ketoconazole inhibits adrenal steroidogenesis; in this paper we investigated whether imidazole antimycotic drugs additionally interact with glucocorticoid receptor sites in target tissues. Our approach was to assess the ability of three drugs: ketoconazole, clotrimazole, and RS 49910, to inhibit [3H]dexamethasone binding to hepatoma tissue culture (HTC) cell cytosol. The results indicated dose-dependent, competitive displacement of [3H]dexamethasone binding that was in the potency sequence: clotrimazole greater than ketoconazole greater than RS 49910. We then examined the functional response of this binding by measuring tyrosine aminotransferase (TAT) activity in HTC cells. The antimycotics did not exhibit TAT agonist activity and inhibition of basal enzyme levels was not detected. However, the drugs were potent antagonists of dexamethasone-induced TAT activity and the effect was temporally reversible. This antagonist activity was in the same sequence and closely correlated with the binding potency of the three drugs. We conclude that ketoconazole and other imidazole antimycotic drugs possess glucocorticoid antagonist activity by virtue of occupancy of glucocorticoid receptor sites in target tissues.

  View details for Web of Science ID A1983RA29700043

  View details for PubMedID 6135709

  View details for PubMedCentralID PMC1129197

• EFFECTS OF 1-ALPHA,25-DIHYDROXYVITAMIN-D3 AND GLUCOCORTICOIDS ON THE GROWTH OF RAT AND MOUSE OSTEOBLAST-LIKE BONE-CELLS CALCIFIED TISSUE INTERNATIONAL Chen, T. L., CONE, C. M., Feldman, D. 1983; 35 (6): 806-811

  Abstract

  The effects of 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) and its interaction with glucocorticoids to regulate bone cell growth were studied in osteoblast-like (OH) cell cultures. Owing to our earlier findings that species difference and cell density at the time of treatment modified hormonal responses, comparisons were made between rat and mouse cells and sparse and dense cultures. 1,25(OH)2D3 inhibited cell proliferation in both species regardless of cell density. The magnitude of inhibition was larger in mouse cells, but the sensitivity to 1,25(OH)2D3 was the same for both species. Other metabolites, 25(OH)D3 and 24R,25(OH)2D3, were greater than 100-fold less potent than 1,25(OH)2D3 even in serum-free medium, which is similar to their ratio of affinity for the 1,25(OH)2D3 receptor. Dexamethasone, as previously shown, inhibited sparse and dense mouse cell cultures and sparse rat cell cultures while stimulating dense rat cell cultures to grow. The inhibitory actions of 1,25(OH)2D3 were not additive to the inhibitory dexamethasone effects. However, 1,25(OH)2D3 addition resulted in attenuation of the stimulatory effect of dexamethasone. These responses to 1,25(OH)2D3 and dexamethasone were dependent on cell density and not selective attachment of certain cell types at either plating density. In conclusion, the findings demonstrated that 1,25(OH)2D3 exerts an inhibiting action on both mouse and rat bone cell proliferation. This effect must be reconciled with the in vivo beneficial actions of 1,25(OH)2D3 on bone metabolism. Also, the likelihood of decreased cell number must be considered when biochemical activities are assessed after vitamin D treatment in vitro.

  View details for Web of Science ID A1983RK33800019

  View details for PubMedID 6689138

• ESTRADIOL BINDS TO A RECEPTOR-LIKE CYTOSOL BINDING-PROTEIN AND INITIATES A BIOLOGICAL RESPONSE IN PARACOCCIDIODES-BRASILIENSIS PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES Loose, D. S., Stover, E. P., Restrepo, A., Stevens, D. A., Feldman, D. 1983; 80 (24): 7659-7663

  Abstract

  Paracoccidioidomycosis, a disease caused by Paracoccidioides brasiliensis, which is endemic to Latin America, is much more common in men than women, suggesting a role for hormonal factors. We recently showed that two other yeasts possess steroid binding proteins and postulated that these receptor-like molecules represented a mechanism by which the hormonal milieu of the host might influence an infecting pathogen. Therefore, we examined P. brasiliensis for a sex steroid binding protein. Because tritiated steroids rapidly dissociated from the other fungal binding proteins, we developed a fast binding method with Sephadex G-50 microcolumns speeded by centrifugation. This method detected specific binding of [3H]estradiol in P. brasiliensis cytosol. Other tritiated steroid hormones, including testosterone and corticosterone, failed to exhibit specific binding. Scatchard analysis of [3H]estradiol binding showed an apparent dissociation constant (Kd) of 1.7 X 10(-8) M and a maximal binding capacity (Nmax) of 235 fmol/mg of protein. Susceptibility to trypsin indicated the binding site was protein in nature. The protein had a Stokes radius of approximately equal to 32 A by HPLC exclusion column and a sedimentation coefficient of 4.4 S by sucrose gradient, consistent with an apparent Mr of approximately equal to 60,000. Competition experiments revealed that estrone, estriol, and progesterone had 25% of the affinity of estradiol, whereas diethylstilbestrol, androgens, and corticosteroids had low affinity. Investigation of steroid hormone actions in P. brasiliensis indicated that estradiol inhibited the fungal transformation from mycelial form to yeast form, the initial step of infection. This suppressive effect was dose-dependent and not found with testosterone. We hypothesize that endogenous estrogens in the host, acting through the cytosol binding protein in the fungus, inhibit mycelial-to-yeast transformation, thus explaining the resistance of women to paracoccidioidomycosis.

  View details for Web of Science ID A1983RY77900060

  View details for PubMedID 6584880

  View details for PubMedCentralID PMC534400

• 1-ALPHA,25-DIHYDROXYVITAMIN-D3 RECEPTORS IN CULTURED RAT OSTEOBLAST-LIKE CELLS - GLUCOCORTICOID TREATMENT INCREASES RECEPTOR CONTENT JOURNAL OF BIOLOGICAL CHEMISTRY Chen, T. L., CONE, C. M., MOREYHOLTON, E., Feldman, D. 1983; 258 (7): 4350-4355

  Abstract

  The direct actions of 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) in target cells are initiated by the binding of hormone to specific receptor sites. The aims of this study were to characterize the receptor for 1,25(OH)2D3 in cultured rat osteoblast-like (OB) cells and to determine whether these receptors are regulated by either the culture cycle itself or glucocorticoid treatment. The 1,25(OH)2D3 receptor in rat OB cells exhibited the same apparent binding affinity (Kd = 0.1 nM) and sedimentation coefficient (3.2 S) as mouse OB cells and receptors in other target organs. However, the receptor concentration in rat OB cells was substantially lower than mouse OB cells (approximately 20%). The concentration of receptors in rat OB cells did not show a correlation with the rate of DNA synthesis and therefore did not exhibit an endogenous rhythm in receptor concentration as was previously seen in mouse OB cells. Also in contrast to mouse cells, where glucocorticoids caused a decrease in receptor level, dexamethasone induced a marked increase in receptor binding throughout the culture cycle. This increase was due to an increase in receptor number with no change in receptor affinity. The change was glucocorticoid-specific, dose-dependent with half-maximal stimulation occurring between 1.3 and 13 nM and exhibited a latent period of at least 4 h. The independence of receptor concentration from DNA synthesis rate was established by assessing receptors after stimulating cell proliferation with epidermal growth factor and inhibiting it with hydroxyurea. Neither treatment altered basal 1,25(OH)2D3 receptor concentration or prevented the marked increase in receptor levels elicited by dexamethasone. We conclude that although the biochemical characteristics of 1,25(OH)2D3 receptors are indistinguishable in rat and mouse OB cells, there are genuine species differences in the regulation of the receptor number as it relates to DNA synthesis rate and response to glucocorticoids.

  View details for Web of Science ID A1983QJ80800053

  View details for PubMedID 6300083

• 1,25-DIHYDROXYVITAMIN-D3 RECEPTORS IN HUMAN EPITHELIAL CANCER CELL-LINES CANCER RESEARCH Colston, K., Colston, M. J., FIELDSTEEL, A. H., Feldman, D. 1982; 42 (3): 856-859

  Abstract

  Specific, high-affinity cytosolic receptors for 1,25-dihydroxyvitamin D3 have been demonstrated in five human cancer cell lines. The cell lines were derived from tumors of breast, lung, cervix, and melanotic and amelanotic melanomas. Binding affinity (Kd) of the receptors for 1,25-dihydroxyvitamin D3 were all approximately 0.2 nM, and receptor content ranged from 21 to 174 fmol/mg cytosol protein. The receptors from all five cell lines sedimented at 3.2S on sucrose density gradients and exhibited preferential affinity for 1,25-dihydroxyvitamin D3 compared to other vitamin D metabolites.

  View details for Web of Science ID A1982ND85800012

  View details for PubMedID 6895862

• 1,25-DIHYDROXYVITAMIN-D3 RECEPTORS AND FUNCTIONS IN CULTURED PIG-KIDNEY CELLS (LLC PK1) - REGULATION OF 24,25-DIHYDROXYVITAMIN-D3 PRODUCTION JOURNAL OF BIOLOGICAL CHEMISTRY Colston, K., Feldman, D. 1982; 257 (5): 2504-2508

  Abstract

  Although 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) regulates the renal metabolism of 25-hydroxyvitamin D3 (25-OH-D3), the mechanism is not well understood. The established pig kidney cell line, LLC PK1, was used to study this feedback regulation. These cells possess a receptor for 1,25-(OH)2D3 with a sedimentation coefficient of 3.2 S. Scatchard analysis of 1,25-(OH)2D3 binding to cell cytosol yielded an apparent Kd of 0.12 nM and an Nmax of 26 fmol/mg of cytosol protein. LLC PK1 cells respond to 1,25-(OH)2D3 by changes in the metabolism of 25-OH-D3. When incubated with 25-OH-[3H]D3, homogenates of untreated cells did not produce detectable 1,25-(OH)2[3H]D3 or 24,25-(OH)2[3H]D3. However, after treatment of cell monolayers with 1,25-(OH)2D3 for 8 h, homogenates converted substantial 25-OH-[3H]D2 substrate to 24,25-(OH)2[3H]D3. The appearance of this 24-hydroxylase activity in response to 1,25-(OH)2D3 was time- and dose-dependent. Half-maximal levels of enzyme activity were achieved with 0.13 nM 1,25-(OH)2D3, a concentration almost identical to the Kd of the 1,25-(OH)2D3 receptor. The stimulation of 24-hydroxylase activity was shown to be an induction event; treatment of monolayers with 13 nM 1,25-(OH)2D3 for a 4-h pulse was sufficient to induce maximal activity assayed at h. The presence of the transcriptional inhibitor, actinomycin D, during the 4-h pulse abolished the induction of 24-hydroxylase activity. These results demonstrate for the first time the presence of both 1,25-(OH)2D3 receptors and stimulation of 24-hydroxylase activity within the same established mammalian kidney cell line.

  View details for Web of Science ID A1982NE46000063

  View details for PubMedID 6277901

• GLUCOCORTICOID REGULATION OF 1,25(OH)2-VITAMIN-D3 RECEPTORS IN CULTURED MOUSE BONE-CELLS JOURNAL OF BIOLOGICAL CHEMISTRY Chen, T. L., CONE, C. M., MOREYHOLTON, E., Feldman, D. 1982; 257 (22): 13564-13569

  View details for Web of Science ID A1982PQ99300065

  View details for PubMedID 6292196

• CHARACTERIZATION OF A UNIQUE CORTICOSTERONE-BINDING PROTEIN IN CANDIDA-ALBICANS JOURNAL OF BIOLOGICAL CHEMISTRY Loose, D. S., Feldman, D. 1982; 257 (9): 4925-4930

  Abstract

  This paper further characterizes a protein we have demonstrated in Candida albicans which has the ability to bind corticosterone and related steroid hormones. Fungal cells are disrupted and cytosol is incubated with [3H]corticosterone for 3 h at which time peak steady state binding is achieved. Bound hormone is separated from free using Sephadex G-50 minicolumns or dextran-coated charcoal. Binding was found to be a linear function of protein concentration. The bound hormone co-migrates with authentic corticosterone in thin layer chromatographic systems indicating no metabolism of the radioprobe. Scatchard analysis of the binding in the pseudohyphal form of C. albicans yielded values of 6.3 nM for the Kd and a binding capacity of about 650 fmol/mg of cytosol protein; both determinations are comparable to our findings in the yeast form of this organism. A series of sterols were tested for their ability to displace [3H]corticosterone from the yeast binder, and the results show that the binder is remarkably selective and stereo specific. Physical-chemical studies show the binder to be degraded at high temperatures and that binding is destroyed by trypsin and sulfhydryl blockers. The protein sediments at 4 S on sucrose gradients and does not exhibit ionic dependent aggregation. The molecular weight is estimated to be approximately 43,000 daltons by gel chromatography. We hypothesize that this intracellular protein may represent a primitive form of either the mammalian glucocorticoid receptor or the plasma corticosteroid-binding globulin.

  View details for Web of Science ID A1982NN57300043

  View details for PubMedID 7040389

• GLUCOCORTICOID REGULATION OF 1,25(OH)2VITAMIN-D3 RECEPTORS - DIVERGENT EFFECTS ON MOUSE AND RAT INTESTINE ENDOCRINOLOGY Hirst, M., Feldman, D. 1982; 111 (4): 1400-1402

  Abstract

  We have investigated the possibility that glucocorticoids alter responsiveness to vitamin D by regulating the 1,25(OH)2D3 receptor in rat intestine. In contrast to the mouse where glucocorticoids caused receptors to decline, rats treated with glucocorticoids showed a substantial increase ( approximately 50%) in the number of intestinal 1,25(OH)2D3 receptors. This resulted from an increase in receptor content with no change in affinity for 1,25(OH)2D3. Receptor stimulation was even greater in vitamin D-deplete rats. Moreover, adrenalectomy led to a significant decline in receptor number. Although the properties of the receptor are similar in rat and mouse intestine, the divergent response to glucocorticoids emphasizes major differences between species in the regulation of 1,25(OH)2D3 receptor number.

  View details for Web of Science ID A1982PH18000054

  View details for PubMedID 6288361

• AN ESTROGEN-BINDING PROTEIN AND ENDOGENOUS LIGAND IN SACCHAROMYCES-CEREVISIAE - POSSIBLE HORMONE RECEPTOR SYSTEM SCIENCE Feldman, D., Do, Y., Burshell, A., Stathis, P., Loose, D. S. 1982; 218 (4569): 297-298

  Abstract

  A protein macromolecule in the cytosol of the unicellular eukaryotic yeast Saccharomyces cerevisiae selectively binds the vertebrate estrogen hormone 17 beta-estradiol with high affinity. Lipid extracts of the yeast cells or the conditioned growth medium yield a substance that can bind competitively to the tritiated estradiol-binding sites in the yeast and to mammalian estrogen receptors. These findings suggest that the binding protein may be a primitive hormone receptor and that the lipid-extractable substance represents the endogenous ligand.

  View details for Web of Science ID A1982PK07200037

  View details for PubMedID 6289434

• REGULATION OF 1,25-DIHYDROXYVITAMIN-D3 RECEPTORS IN CULTURED MOUSE BONE-CELLS - CORRELATION OF RECEPTOR CONCENTRATION WITH THE RATE OF CELL-DIVISION JOURNAL OF BIOLOGICAL CHEMISTRY Chen, T. L., Feldman, D. 1981; 256 (11): 5561-5566

  View details for Web of Science ID A1981LS97800043

  View details for PubMedID 6263882

• A CORTICOSTEROID BINDING-PROTEIN AND ENDOGENOUS LIGAND IN C-ALBICANS INDICATING A POSSIBLE STEROID-RECEPTOR SYSTEM NATURE Loose, D. S., Schurman, D. J., Feldman, D. 1981; 293 (5832): 477-479

  View details for Web of Science ID A1981MJ81500050

  View details for PubMedID 7031475

• ORGAN DISTRIBUTION OF THE CYTOPLASMIC 1,25-DIHYDROXYCHOLECALCIFEROL RECEPTOR IN VARIOUS MOUSE-TISSUES ENDOCRINOLOGY Colston, K., Hirst, M., Feldman, D. 1980; 107 (6): 1916-1922

  View details for Web of Science ID A1980KS42800038

  View details for PubMedID 6253281

• HETEROGENEITY OF GLUCOCORTICOID BINDERS - A HIGH-AFFINITY TRIAMCINOLONE ACETONIDE BINDER IN BOVINE SERUM ENDOCRINOLOGY Do, Y. S., Feldman, D. 1980; 107 (5): 1370-1375

  Abstract

  While investigating glucocorticoid-binding proteins in bovine tissues, a new binder was found in fresh bovine serum which exhibited high affinity for certain synthetic glucocorticoids. This serum binder was characterized using [3H]triamcinolone acetonide (TA) as the ligand. On sucrose gradients, the [3H]TA peak sedimented at 8S, which was easily distinguishable from the [3H]cortisol-transcortin peak at 4S. Unlike the tissue receptor, which showed ionically dependent transformation from 8S in equilibrium or formed from 4S, the serum binder sedimented at 8S in both hypo- and hypertonic gradients. Binding properties were evaluated employing sephadex G-50 chromatography to separate bound from free steroid. Scatchard analysis of specific [3H]TA binding data revealed a straight line. The apparent equilibrium dissociation constant (Kd) was 7.8 +/- 0.7 X 10(-8) M, and the binding capacity was 772 +/- 70 fmol/mg serum protein. Hormonal specificity was determined by a competitive binding assay and revealed the following sequence: TA (100%) > betamethasone (47%) > triamcinolone (33%) > dexamethasone (2%) = cortisol = progesterone; aldosterone, estradiol, and testosterone exhibited negligible competitive activity. The serum binder was very stable, withstanding heating to 37 C for 60 min and long term storage in the frozen state. However, binding was significantly destroyed by trypsin. The binder was absent from fresh samples of chicken, mouse, rat, rabbit, dog, monkey, and human sera and frozen horse and porcine sera, but was clearly present in commercially available frozen calf, fetal calf, and lamb sera. At this time, we are unable to define the function of this binder, although its existence in both ovine and bovine sera suggests a possible role in ruminants. However, since bovine serum is routinely employed in tissue culture studies, the presence of this glucocorticoid binder might significantly influence many experiments.

  View details for Web of Science ID A1980KN60700017

  View details for PubMedID 6775927

• DEMONSTRATION OF GLUCOCORTICOID RECEPTORS IN THE ADRENAL-CORTEX - EVIDENCE FOR A DIRECT DEXAMETHASONE SUPPRESSIVE EFFECT ON THE RAT ADRENAL-GLAND ENDOCRINOLOGY Loose, D. S., Do, Y. S., Chen, T. L., Feldman, D. 1980; 107 (1): 137-146

  Abstract

  The adrenal cortex was evaluated for the presence of glucocorticoid receptors and functions. Substantial binding of [3H]dexamethasone was observed in aminoglutethimide-treated, hypophysectomized, and intact rats. Further studies demonstrated binding in cultured bovine adrenocortical cells and in Y-1 cells, a cloned murine cell line of adrenal cortical origin. Scatchard analysis of specific binding data in cytosol from hypophysectomized rats revealed an apparent Kd of approximately 15 nM and a receptor content (Nmax) of 123 fmol/mg cytosol protein. Analysis of Y-1 cell cytosol showed a Kd of approximately 17 nM and Nmax of 190 fmol/mg protein. The binding site in hypophysectomized rats had the following steroid specificities: high affinity for dexamethasone, corticosterone, and progesterone; moderate affinity for 11 beta-cortisol, and low affinity for testosterone, estradiol, pregnenolone, and 11 alpha-cortisol. Sedimentation in sucrose density gradients revealed 8S binding peaks in cytosols prepared from intact rat adrenal glands, Y-1 cells, and cultured bovine adrenocortical cells. Time- and temperature-dependent nuclear uptake of [3H]dexamethasone in Y-1 cells was demonstrated. In vivo treatment of hypophysectomized rats with dexamethasone significantly enhanced the rate of adrenal atrophy. ACTH stimulation tests in hypophysectomized rats showed a decreased corticosterone response in dexamethasone-treated rats compared to that in control animals. However, in vitro, there was no evidence for an effect of dexamethasone on ACTH-stimulated corticosterone production. The data indicate that the adrenal cortex possesses a high affinity binding site that fulfills the criteria for a glucocorticoid receptor. Glucocorticoid administration enhances adrenal atrophy and impairs adrenal function. We speculate that this action contributes to the suppressive effect of glucocorticoids on the pituitary-adrenal axis.

  View details for Web of Science ID A1980JX72200021

  View details for PubMedID 6247134

• NUCLEAR TRANSLOCATION OF THE 1,25-DIHYDROXYCHOLECALCIFEROL RECEPTOR IN MOUSE KIDNEY JOURNAL OF BIOLOGICAL CHEMISTRY Colston, K., Feldman, D. 1980; 255 (16): 7510-7513

  Abstract

  We have recently demonstrated that cytosol from mouse kidney contains a receptor-like binding protein for 1,25-dihydroxycholecalciferol (1,25-(OH)2D3). The present studies were undertaken to investigate whether this component undergoes nuclear translocation, thus adding support to the contention that this binding moiety represents a receptor for 1,25-(OH)2D3. Kidney slices from vitamin D-deficient mice were incubated with 1,25-(OH)2[3H]D3 and specific binding was assessed in cytosol and 0.3 M KCl extracts of isolated nuclei. The appearance of specific nuclear-bound 1,25-(OH)2[3H]D3 exhibited a saturation time course and the increase in nuclear binding was accompanied by a concomitant decrease in cytoplasmic binding such that, by 120 min, approximately 80% of specifically bound 1,25-(OH)2[3H]D3 was associated with the nuclear fraction and approximately 20% remained in the cytoplasmic compartment. The nuclear transfer was found to be temperature-dependent and specific for the dihydroxy metabolite. Both the cytoplasmic and nuclear receptors sedimented at 3.2 S in 0.3 M KCl sucrose density gradients. However, under low salt conditions, the cytoplasmic receptor was transformed to yield an approximately 5.4 S peak while the nuclear receptor sedimented unchanged t 3.2 S. In summary, the cytoplasmic 1,25-(OH)2D3 receptor in mammalian kidney undergoes temperature-dependent nuclear translocation and the cytoplasmic and nuclear forms of the receptor can be distinguished by their sedimentation properties.

  View details for Web of Science ID A1980KF60600003

  View details for PubMedID 6249801

• Characterization of a cytoplasmic receptor-like binder for 1 alpha, 25-dihydroxycholecalciferol in rat intestinal mucosa. journal of biological chemistry Feldman, D., McCain, T. A., HIRST, M. A., Chen, T. L., Colston, K. W. 1979; 254 (20): 10378-10384

  View details for PubMedID 489600

• RECEPTOR-LIKE BINDING MACROMOLECULE FOR "1-ALPHA,25-DIHYDROXYCHOLECALCIFEROL IN CULTURED MOUSE BONE-CELLS JOURNAL OF BIOLOGICAL CHEMISTRY Chen, T. L., HIRST, M. A., Feldman, D. 1979; 254 (16): 7491-7494

  Abstract

  1alpha, 25-Dihydroxycholecalciferol (1,25-(OH)2D3), the active form of vitamin D, like other steroid hormones, initiates its action by binding to cytoplasmic receptors in target cells. Although the 1,25-(OH)2D3 receptor has been well studied in intestine, little information beyond sucrose gradient analyses is presently available from mammalian bone. We, therefore, employed primary cultures of mouse calvarial cells to characterize the mammalian receptor in bone. A hypertonic molybdate-containing buffer was found to protect receptor binding. On hypertonic sucrose gradients, the 1,25-(OH)2-[3H]D3 binder sedimented at 3.2 S. Scatchard analysis of specific 1,25-(OH)2[3H]D3 binding sites at 0 degrees C yielded an apparent Kd of 0.26 nM and an Nmax of 75 fmol/mg of cytosol protein. Competitive binding experiments revealed the receptor to prefer 1,25-(OH)2D3 greater than 25-(OH)-D3 = 1 alpha-(OH)-D3 greater than 24R,25-(OH)2D3; vitamin D3, dihydrotachysterol, sex steroids, and glucocorticoids exhibited negligible binding. As shown in other systems, the receptor could be distinguished from a 25-(OH)-[3H]D3 binder which sedimented at approximately 6 S. In summary, cultured mouse calvarial cells possess a macromolecule with receptor-like properties. This system appears to be an ideal model for the investigation of 1,25-(OH)2D3 receptor binding and action in mammalian bone.

  View details for Web of Science ID A1979HJ62800009

  View details for PubMedID 468767

• GLUCOCORTICOID AND ESTROGEN REGULATION OF CORTICOSTEROID-BINDING GLOBULIN PRODUCTION BY RAT-LIVER AMERICAN JOURNAL OF PHYSIOLOGY Feldman, D., Mondon, C. E., Horner, J. A., Weiser, J. N. 1979; 237 (6): E493-E499

  Abstract

  We have recently demonstrated that rat liver can synthesize and secrete corticosteroid-binding globulin (CBG). The present study extends these observations and examines the hormonal regulation of hepatic CBG production. Male rats were pretreated by adrenalectomy and/or glucocorticoid or estrogen administration and the rate of CBG production was measured in vitro. The production rates were assessed by the generation of specific corticosterone binding sites in both a liver-slice preparation and in an isolated perfused liver. The two techniques showed qualitatively similar results. Adrenalectomy enhanced and glucorticoid administration inhibited the rate of CBG production and secretion. Pretreatment of male rats with estradiol stimulated the rate of CBG production. The production rates were 20- to 40-fold higher in the perfused liver demonstrating its superiority over the liver-slice system. The livers from intact rats secreted CBG binding sites at a rate of approximately 18 pmol/g liver per hours, generating an estimated 20% of the total CBG content of a rat each day. The possible clinical implications of the therapeutic use of glucocorticoids that bind to CBG, yet inhibit CBG production, are discussed.

  View details for Web of Science ID A1979HY31000002

  View details for PubMedID 517646

• CHARACTERIZATION OF A CYTOPLASMIC RECEPTOR-LIKE BINDER FOR "1-ALPHA,25-DIHYDROXYCHOLECALCIFEROL IN RAT INTESTINAL-MUCOSA JOURNAL OF BIOLOGICAL CHEMISTRY Feldman, D., McCain, T. A., HIRST, M. A., Chen, T. L., Colston, K. W. 1979; 254 (20): 378-384

  View details for Web of Science ID A1979HS35200072

• DEMONSTRATION OF A 1,25-DIHYDROXYCHOLECALCIFEROL CYTOPLASMIC RECEPTOR-LIKE BINDER IN MOUSE KIDNEY JOURNAL OF CLINICAL ENDOCRINOLOGY & METABOLISM Colston, K. W., Feldman, D. 1979; 49 (5): 798-800

  Abstract

  Isolated mouse renal tubule cells have been employed to demonstrate the presence of a specific high affinity cytoplasmic binding protein for 1,25-dihydroxycholecalciferol (1,25(OH)2D3) in kidney. This receptor-like macromolecule sedimented at 3.2 S in hypertonic sucrose density gradients. Scatchard analysis of [3H]1,25-(OH)2D3 binding at O C revealed an apparent Kd of 0.2 nM and a concentration of binding sites of 50 fmol/mg cytosol protein. In competition experiments, the binder exhibited a low affinity for other vitamin D3 metabolites; the order of potency was 1,25(OH)2D3 greater than 250HD3 greater than u alpha OHD3 greater than 24R,25(OH)2D3. The sedimentation properties, binding affinity, and specificity of this 1,25(OH)2D3 binding protein are strikingly similar to the receptors in rat intestine, mouse bone, and human intestine. The demonstration of a renal receptor-like binder adds further support to the concept that the kidney is a 1,25(OH)2D3 target organ.

  View details for Web of Science ID A1979HT13500025

  View details for PubMedID 489720

• RECEPTOR MEDIATED GLUCOCORTICOID INHIBITION OF PROTEIN-SYNTHESIS IN ISOLATED BONE-CELLS JOURNAL OF STEROID BIOCHEMISTRY AND MOLECULAR BIOLOGY Choe, J., Stern, P., Feldman, D. 1978; 9 (3): 265-271

  View details for Web of Science ID A1978EZ52400012

  View details for PubMedID 651351

• EVIDENCE THAT BROWN ADIPOSE-TISSUE IS A GLUCOCORTICOID TARGET ORGAN ENDOCRINOLOGY Feldman, D. 1978; 103 (6): 2091-2097

  View details for Web of Science ID A1978FZ32000016

  View details for PubMedID 218803

• IS GLUCOCORTICOID RECEPTOR IDENTICAL IN VARIOUS TARGET ORGANS JOURNAL OF STEROID BIOCHEMISTRY AND MOLECULAR BIOLOGY Feldman, D., Funder, J., Loose, D. 1978; 9 (2): 141-145

  View details for Web of Science ID A1978EU22000009

  View details for PubMedID 305978

• BINDING OF SOME NON-STEROIDAL ANTI-INFLAMMATORY DRUGS TO GLUCOCORTICOID RECEPTORS INVITRO BIOCHEMICAL PHARMACOLOGY Feldman, D. 1978; 27 (8): 1187-1191

  View details for Web of Science ID A1978FL03000016

  View details for PubMedID 697918

• 19-NOR DEOXYCORTICOSTERONE (19-NOR DOC) - MINERALOCORTICOID RECEPTOR AFFINITY HIGHER THAN ALDOSTERONE, ELECTROLYTE ACTIVITY LOWER ENDOCRINOLOGY Funder, J. W., Mercer, J., Ingram, B., Feldman, D., Wynne, K., Adam, W. R. 1978; 103 (4): 1514-1517

  Abstract

  By screening urine extracts from rats with adrenal regeneration hypertension, Gomez-Sanchez et al. found a steroid, subsequently identified as 19-nor DOC, with high affinity for tritiated aldosterone (3HA) binding sites in rat kidney cytosol. We here report studies on the affinity of authentic 19-nor DOC for mineralocorticoid receptors, its binding in plasma and its activity in the rat urinary mineralocorticoid assay. When kidney slices from adrenalectomized rats were incubated in protein-free buffer with 3HA, 19-nor DOC consistently competed better (approximately 140%) for 3HA binding sites than did equivalent concentrations of non-radioactive aldosterone. Under identical conditions, save for the inclusion of 20% adrenalectomized rat plasma in the incubation medium, 19-nor DOC shows only approximately 40% the potency of aldosterone in displacing 3HA. Determination of renal binding of 3HA after injection of 3HA +/- aldosterone +/- 19-nor DOC in vivo similarly shows 19-nor DOC to be approximately one third as potent a competitor for 3HA binding sites as aldosterone. In the rat urinary bioassay, 19-nor DOC shows no antagonist activity when injected with aldosterone; in the absence of aldosterone, 19-nor DOC acts as a mineralocorticoid agonist, with an apparent potency 10-30% that of aldosterone. Conclusions of the study are therefore (i) at a molecular level, 19-nor DOC has a higher affinity than aldosterone for mineralocorticoid receptors, (ii) in vivo, its potency in terms of receptor occupancy is markedly lower than that of aldosterone, due to higher levels of plasma binding, (iii) in effector terms, 19-nor DOC is a full agonist without antagonist activity.

  View details for Web of Science ID A1978FR17900065

  View details for PubMedID 744165

• GLUCOCORTICOID RECEPTORS IN ADIPOSE-TISSUE ENDOCRINOLOGY Feldman, D., Loose, D. 1977; 100 (2): 398-405

  Abstract

  Although adipose tissue appears to be a target organ for glucocorticoid hormones, previous studies have failed to detect glucocorticoid receptors in this tissue. In the present study, the addition of thioglycerol and trasylol to the homogenization medium provided an enuironment in which receptors were successfully demonstrated. [3H]Dexamethasone binding studies were carried out at 0 C in cytosol from various adipose tissues of adrenalectomized rats and bound hormone was separated from free by Sephadex chromtography. Despite the presence of protein protective agents, receptor binding decayed significantly over 24 h but appeared stable from 1 to 5 h. Epididymal fat pad cytosol had an apparent Kdiss at 0 C for dexamethasone of approximately 6 nM and a binding capacity of approximately 200 fmol per mg protein. To prove that the receptors were located in fat cells and not in surrounding connective tissue, isolated adipocytes were prepared by collagenase digestion and receptors were demonstrable in the cytosol from these cells as well. The affinity of series of steroids for the receptor was in the sequence: dexamethasone greater than corticosterone greater than progesterone greater than aldosterone greater than cortexolone greater than testosterone greater than estradiol. Receptors of roughly the same affinity but somewhat fewer binding sites on the basis of cytosol protein were also found on other fat depots including peri-renal, peri-scrotal and popliteal. Of interest is the fact that interscapular brown fat and human subcutaneous fat also possessed similar these receptors, the higher competitive capacity of dexamethasone indicated that the binding was to glucorticoid rather than mineralocorticoid receptors. The data suggest that fat cells contain glucocorticoid receptors which are similar to those seen in other glucocorticoid targets. Presumably these receptors mediate the effects of glucorticoids on adipose tissue.

  View details for Web of Science ID A1977CU48800017

  View details for PubMedID 832631

• GLUCOCORTICOID RECEPTORS AND REGULATION OF PHOSPHOENOLPYRUVATE CARBOXYKINASE ACTIVITY IN RAT-KIDNEY AND ADIPOSE-TISSUE AMERICAN JOURNAL OF PHYSIOLOGY Feldman, D. 1977; 233 (3): E147-E151

  View details for Web of Science ID A1977DW94400011

  View details for PubMedID 910900

• GLUCOCORTICOID RECEPTORS AND INHIBITION OF BONE CELL-GROWTH IN PRIMARY CULTURE ENDOCRINOLOGY Chen, T. L., ARONOW, L., Feldman, D. 1977; 100 (3): 619-628

  Abstract

  We have previously identified glucocorticoid binding proteins in cytosol of cells dispersed from fetal rat calvaria by collagenase digestion. The present study, employing primary culture of these cells, provides further evidence that these binding proteins represent glucocorticoid receptors. [3H]Dexamethasone bound to cytoplasmic extracts of cultured cells with an apparent Kdiss of 6.8 nM and exhibited approximately 8500 binding sites/cell. Nuclear translocation of [3H]dexamethasone was demonstrated with approximately 50% of bound steroid extractable from the nuclear pellet after incubation at 37 C; little nuclear transfer occurred at 0 C. The specificity of these binding site was characterized by competition studies with other steroids in whole cells, the order of affinities being: triamcinolone acetonide greater than dexamethasone greater than progesterone greater than cortisol greater than corticosterone = cortexolone. Non-glucocorticoids except progesterone competed only poorly. Sedimentation analysis of [3H]dexamethasone-protein complexes on sucrose gradients revealed a cytoplasmic peak of 6.5 S in salt-free gradients and 3.8 S in 0.3 M KCl gradients. Dexamethasone addition to the culture medium resulted in a dose-dependent inhibition of cell growth with approximately 40% reduction in cell number at 13 nM. That this inhibition was receptor mediated was substantiated by the partial blockade of the dexamethasone effect in the presence 1.3 microM progesterone. Functionally, dexamethasone inhibits the growth of these cells. These data provide evidence for receptor mediated inhibitory effects of glucocorticoids directly at the level of the bone cell.

  View details for Web of Science ID A1977CX43700002

  View details for PubMedID 401016

• INTRINSIC MINERALOCORTICOID AGONIST ACTIVITY OF SOME NONSTEROIDAL ANTI-INFLAMMATORY DRUGS - POSTULATED MECHANISM FOR SODIUM RETENTION JOURNAL OF CLINICAL INVESTIGATION Feldman, D., COUROPMITREE, C. 1976; 57 (1): 1-7

  Abstract

  Because some nonsteroidal anti-inflammatory drugs (NSAID) induce salt and water retention and exhibit other steroid-like actions, studies were performed to ascertain whether these drugs possess intrinsic mineralocorticoid agonist activity. In vitro competitive binding assays utilizing tissue from adrenalectomized rats demonstrated that some NSAID can displace [3H]-aldosterone from renal cytoplasmic mineralocorticoid receptors. Displacement potency for these sites was in the sequence: aldosterone greater than spironolactone greater than phenylbutazone (PBZ) greater than aspirin (ASA) greater than indomethacin (IDM). Concentration ratios required to obtain significant displacement of [3H]aldosterone were high but clearly within the therapeutic range for PBZ and ASA but not IDM. The analogues oxyphenbutazone (OBZ) and sodium salicylate (SS) were similar in binding activity to PBZ and ASA, respectively. Lineweaver-Burk analysis revealed that the inhibition of [3H]aldosterone binding was competitive in nature. In addition, PBZ was shown to prevent the nuclear binding of [3H]aldosterone. In vivo injection of PBZ and ASA resulted in competition for [3H]aldosterone renal binding comparable to the in vitro studies. Administration of PBZ and OBZ to adrenalectomized rats resulted in significant salt retention whereas ASA and SS did not differ significantly from controls. Salt retention elicited by PBZ and OBZ was inhibited by spironolactone, a competitive mineralocorticoid antagonist. These data suggest that, despite nonsteroidal structures, PBZ and OBZ induce salt retention via a receptor-mediated mineralocorticoid pathway analogous to aldosterone action.

  View details for Web of Science ID A1976BB98200001

  View details for PubMedID 173739

  View details for PubMedCentralID PMC436618

• AGONIST AND ANTI-MINERALOCORTICOID ACTIVITIES OF SPIROLACTONES AMERICAN JOURNAL OF PHYSIOLOGY SAKAUYE, C., Feldman, D. 1976; 231 (1): 93-97

  Abstract

  To investigate the mechanism of action of antimineralocorticoids, a series of spirolactone analogues was evaluated for both mineralocorticoid antagonist and agonist activity. Antagonist activity was assessed by inhibition of aldosterone stimulated sodium transport employing toad bladder short-circuit current (SCC) measurements. Agonist activity was assessed in the same system by the direct effect of spirolactones on SCC. Opening of the gamma-lactone ring of a spirolactone dramatically decreased antagonist activity in the compound studied. Several C-7 chi-substitutions resulted in either enhanced or diminished activity, whereas deletion of the C-10 methyl group (i.e., 19-nor compound) had only minimal effects on antagonist potency. Agonist activity was demonstrable for three of the analogues studied: the 19-nor compound, and those containing a C-7 chi-substitution with a carboxyl isopropyl ester or a C-6-7 cyclopropyl linkage. The functional activity in toad bladder was compared to previous measurements of the relative binding affinity of the same spirolactones for mineralocorticoid receptors in rat kidney. Although there was some correlation between binding to rat kidney receptors and antagonist activity in the toad bladder, the results did not coincide in the case of the three spirolactones that possessed partial agonist activity. Some of the discrepancy may have resulted from differences between mammalian and amphibian receptors; however, intrinsic agonist activity limits antagonist potency and thus may cause a divergence between binding and functional studies limited to antagonist activity alone. Binding affinity, although indicative of total activity, fails to distinguish agonist from antagonist potency.

  View details for Web of Science ID A1976CC86000017

  View details for PubMedID 183514

• 16 BETA-HYDROXYDEHYDROEPIANDROSTERONE - DICHOTOMY BETWEEN RENAL RECEPTOR-BINDING AND URINARY ELECTROLYTE ACTIVITY ENDOCRINOLOGY Funder, J. W., Robinson, J. A., Feldman, D., WYNNE, K. N., Adam, W. R. 1976; 99 (2): 619-628

  Abstract

  Excessive production of 16beta-hydroxydehydroepiandrosterone (16beta-OH-DHEA) has been suggested as a cause of low-renin essential hypertension. The mineralocorticoid effect of 16beta-OH-DHEA was reported to be one-fortieth that of aldostereone in the rat bioassay. Using kidney slices from adrenalectomized rats, the affinity of 16beta-OH-DHEA and a series of related compounds for mineralocorticoid receptors has been determined. In studies done at both 4 C and 37 C, the affinity of 16beta-OHDHEA for mineralocorticoid receptors was found to be less than 0.1% that of aldosterone (P less than 0.01). Various related steroids and/or potential metabolites similarly showed negligible affinity for the aldosterone receptor. In addition, In addition, 16beta-OHDHEA showed no significant affinity for renal dexamethasone-binding sites (Type II glucocorticoid receptors), corticosterone-binding sites (Type III glucocorticoid receptors), dihydrotestosterone binding sites, or estradiol binding sites. In in vivo competition experiments, the concurrent administration of 50 mug deoxycorticosterone reduced (3H)aldosterone binding to 20-30% of control levels; 50 mug 16beta-OH-DHEA did not compete for (3H)aldosterone binding sites. In in vivo bioassay electrolyte excretion was found-in contrast to that of aldosterone-to be variable. Within a given group, certain rats reproducibly responded to 16beta-OH-DHEA by sodium retention and kaliuresis; in others no response was observed. In vitro binding studies comparing "responders" with "non-responders" demonstrated that in neither group did 16beta-OH-DHEA have significant affinity for renal mineralocorticoid receptors. Accordingly, the mechanism whereby 16beta-OHDHEA produces changes in urinary electrolyte excretion appears independent of classical mineralocorticoid effector mechanisms. The conditions under which this effect is seen await eludication.

  View details for Web of Science ID A1976CA03000036

  View details for PubMedID 182463

• CYTOPLASMIC GLUCOCORTICOID BINDING-PROTEINS IN BONE-CELLS ENDOCRINOLOGY Feldman, D., Dziak, R., Koehler, R., Stern, P. 1975; 96 (1): 29-36

  Abstract

  The binding of 3H-dexamethasone (3H-DM) was studied in the cytoplasmic fraction of bone cells isolated from fetal rat calvaria by enzymatic digestion. An high-affinity glucocorticoid binding protein resembling those described in other target tissues was demonstrated. Scatchard analysis revealed a single class of binding sites with an apparent dissociation constant for 3H-DM (0C) of 7 x 10-9M and a concentration of binding sites of 0.11 pmoles/mg cytosol protein. The number of cytoplasmic binding sites per cell was calculated at 6,000 which is probably an underestimate due to occupancy of some sites by endogenous steroids. The binding sites appeared protein in nature since incubation with pronase destroyed 100% of the binding. Nuclear transfer was demonstrated in a reconstituted system utilizing bone cytosol as donor and liver nuclei as the acceptor. Competitive binding analysis revealed corticosterone to be equivalent to DM in binding affinity; progesterone was 75% as potent as DM. Aldosterone and SC-26304 (a spirolactone analogue) had, respectively, 1

  View details for Web of Science ID A1975V132300005

  View details for PubMedID 1167354

• ROLE OF HORMONE RECEPTORS IN ACTION OF ADRENAL STEROIDS ANNUAL REVIEW OF MEDICINE Feldman, D. 1975; 26: 83-90

  View details for Web of Science ID A1975AB11100009

  View details for PubMedID 167655

• MECHANISM OF ACTION OF SPIROLACTONES CLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY Funder, J. W., MARVER, D., STEWART, J., Feldman, D., Edelman, I. S. 1975: 99-101

  View details for Web of Science ID A1975AN10400017

  View details for PubMedID 1183096

• AUTORADIOGRAPHIC LOCALIZATION OF CORTICOSTERONE RECEPTORS (TYPE 3) TO COLLECTING TUBULE OF RAT-KIDNEY ENDOCRINOLOGY Strum, J. M., Feldman, D., Taggart, B., MARVER, D., Edelman, I. S. 1975; 97 (3): 505-516

  Abstract

  Recently, a class of receptors exhibiting high affinity for corticosterone was described in rat kidney (Feldman, D. et al., Endocrinology 92: 1429, 1973). These receptor sites exhibited negligible affinity for dexamethasone and aldosterone and were designated Type III to distinguish them from sites having high affinity for aldosterone (Type I), and sites with high affinity for dexamethasone and corticosterone (Type II). To visually localize Type III sites in the kidney and demonstrate whether or not they represent intracellular steroid receptors, we used an autoradiographic procedure for diffusible substances. Male adrenalectomized rats were injected intravenously with the following combination of steroids per 100 g body weight: 4 x 10(-9) mol [3H]corticosterone, 4 x 10(-9) mol unlabeled aldosterone, and 4 x 10(-9) mol unlabeled dexamethasone. To differentiate "nonspecific" binding, each experimental animal was paired with a control animal that received the same steroids plus 250-fold unlabeled corticosterone. At 3 min, 10 min, and 30 min, kidneys were removed, cut into quadrants, and frozen in isopentane cooled by liquid nitrogen. For autoradiography, 4 mum frozen sections were cut, pressed into contact with emulsion precoated slides at -30 C, melted and simultaneously dried under a jet of dry nitrogen gas, and exposed at 4 C for 2 to 6 weeks. At all three time intervals, silver grains representing [3H]corticosterone binding sites, were concentrated over collecting tubules, only in the outer medulla and cortex (those in the inner medulla and papilla were not labeled). In the labeled segments of the nephron, some of the cells showed an apparent high ratio of cytoplasmic to nuclear grains and in others nuclear labeling was more prominent. A small population of cells within labeled collecting tubules (possibly dark cells) were not labeled. Although no function can yet be ascribed to Type III receptors in the kidney, they may represent an important steroid-mediated renal mechanism.

  View details for Web of Science ID A1975AQ28500001

  View details for PubMedID 170062

• STEROIDAL 21-DIAZO KETONES - PHOTOGENERATED CORTICOSTEROID RECEPTOR LABELS BIOCHEMISTRY Wolff, M. E., Feldman, D., CATSOULACOS, P., Funder, J. W., Hancock, C., Amano, Y., Edelman, I. S. 1975; 14 (8): 1750-1759

  Abstract

  The 21-diazo derivatives of 9 alpha-fluoro- and 9 alpha-bromo-21 deoxycorticosterone, 21-deoxycorticosterone, and progesterone were synthesized for use as photoaffinity labels for corticosteroid receptors. In the isolated toad bladder system, 9 alpha-bromo-21-diazo-21-deoxycorticosterone was as active as d-aldosterone and more active than 9 alpha-fluoro-cortisol in augmenting active Na+ transport. The activities of 21-diazoprogesterone and progesterone were equal; both were much less potent than d-aldosterone, however. These results indicate that the 21-diazo derivatives had significant functional activity in the toad bladder system. The rat kidney slice system was used to estimate the relative affinities of the diazo steroids for aldosterone receptor sites by competition experiments. At 100-fold excess of competitor to [3-H]aldosterone, the order of affinities was 9 alpha-fluoro-21-diazo-21-deoxycorticosterone greater than 9 alpha-bromo-21-diazo-21-deoxycorticosterone greater than 21-diazoprogesterone. Moreover, 9 alpha-bromo-21-diazo-21-deoxycorticosterone reduced binding of [3-H]aldosterone to cytoplasmic and nuclear forms of the receptor proportionately. On the basis of competition for [3-H]corticosterone binding, presumably to corticosteroid-binding globulin (CBG), the order of affinities was 21-diazo-21-deoxycorticosterone greater than 21-diazoprogesterone greater than 9 alpha-bromo-21-diazo-21-deoxycorticosterone. These findings indicate that 21-diazo steroids may be suitable as photogenerated affinity labels for mineralocorticoid receptors. The tritiated derivative, [1,2-3-H]-9 alpha-bromo-21-diazo-21-deoxycorticosterone (specific activity 25 Ci/mol) was synthesized and used in model experiments on photogenerated covalent binding to rat plasma proteins. Irradiation with uv light resulted in binding of [1,2-3-H]-9 alpha-bromo-21-diazo-21-deoxycorticosterone to plasma proteins, that was resistant to extraction with methylene dichloride and did not exchange with unlabeled corticosterone. The diazocorticosteroids, therefore, may have the requisite functional and selectivity properties for photoaffinity labeling of corticosteroid-binding proteins. Further studies are needed, however, to assure that photogenerated labeling with these steroids was site specific.

  View details for Web of Science ID A1975W280600030

  View details for PubMedID 164894

• MINERALOCORTICOID RECEPTORS AND 18-HYDROXYDEOXYCORTICOSTERONE BINDING IN KIDNEY OF SPONTANEOUSLY HYPERTENSIVE RAT ENDOCRINOLOGY Feldman, D. 1974; 94 (4): 1185-1188

  View details for Web of Science ID A1974S712000040

  View details for PubMedID 4818778

• MOLECULAR MODIFICATIONS OF ANTI-ALDOSTERONE COMPOUNDS - EFFECTS ON AFFINITY OF SPIRONOLACTONES FOR RENAL ALDOSTERONE RECEPTORS BIOCHEMICAL PHARMACOLOGY Funder, J. W., Feldman, D., HIGHLAND, E., Edelman, I. S. 1974; 23 (10): 1493-1501

  View details for Web of Science ID A1974S932500006

  View details for PubMedID 4367952

• RENAL ALDOSTERONE RECEPTORS - STUDIES WITH [H-3] ALDOSTERONE AND ANTI-MINERALOCORTICOID [H-3] SPIROLACTONE (SC-26304) PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA MARVER, D., STEWART, J., Funder, J. W., Feldman, D., Edelman, I. S. 1974; 71 (4): 1431-1435

  Abstract

  In vivo, a spirolactone (SC-26304) inhibited the effects of aldosterone on urinary K(+):Na(+) ratios and the binding of [(3)H]aldosterone to renal cytoplasmic and nuclear receptors. Cytoplasmic binding of [(3)H]aldosterone and [(3)H]spirolactone (SC-26304) was similar in magnitude and involved the same set of sites. Under three sets of conditions-(i) in the intact rat, (ii) in kidney slices, and (iii) in reconstitution studies (mixing prelabeled cytoplasm with either purified renal nuclei or chromatin), [(3)H]spirolactone (SC-26304) did not yield specific nuclear complexes in contrast to the reproducible generation of these complexes with [(3)H]aldosterone. In glycerol density gradients, cytoplasmic [(3)H]aldosterone receptor complexes sedimented at 8.5 S and 4 S in low concentrations of salt and at 4.5 S in high concentrations of salt. Cytoplasmic [(3)H]spirolactone (SC-26304) receptor complexes sedimented at 3 S in low concentrations of salt and 4 S in high concentrations of salt. These results are discussed in terms of an allosteric model of the receptor system.

  View details for Web of Science ID A1974S853700062

  View details for PubMedID 4364539

  View details for PubMedCentralID PMC388243

• ROLES OF PLASMA BINDING AND RECEPTOR SPECIFICITY IN MINERALOCORTICOID ACTION OF ALDOSTERONE ENDOCRINOLOGY Funder, J. W., Feldman, D., Edelman, I. S. 1973; 92 (4): 994-1004

  View details for Web of Science ID A1973P370500005

  View details for PubMedID 4686329

• GLUCOCORTICOID RECEPTORS IN RAT-KIDNEY - BINDING OF TRITIATED-DEXAMETHASONE ENDOCRINOLOGY Funder, J. W., Feldman, D., Edelman, I. S. 1973; 92 (4): 1005-1013

  View details for Web of Science ID A1973P370500006

  View details for PubMedID 4686302

• EVIDENCE FOR A NEW CLASS OF CORTICOSTERONE RECEPTORS IN RAT-KIDNEY ENDOCRINOLOGY Feldman, D., Funder, J. W., Edelman, I. S. 1973; 92 (5): 1429-1441

  View details for Web of Science ID A1973P589000022

  View details for PubMedID 4348651

• BINDING OF 18-HYDROXYDEOXYCORTICOSTERONE AND 18-HYDROXYCORTICOSTERONE TO MINERALOCORTICOID AND GLUCOCORTICOID RECEPTORS IN RAT-KIDNEY ENDOCRINOLOGY Feldman, D., Funder, J. W. 1973; 92 (5): 1389-1396

  View details for Web of Science ID A1973P589000016

  View details for PubMedID 4348649

• SPECIFIC ALDOSTERONE BINDING IN RAT-KIDNEY AND PAROTID JOURNAL OF STEROID BIOCHEMISTRY Funder, J. W., Feldman, D., Edelman, I. S. 1972; 3 (2): 209-?

  View details for Web of Science ID A1972M048200014

  View details for PubMedID 4651322

• SUBCELLULAR MECHANISMS IN ACTION OF ADRENAL STEROIDS AMERICAN JOURNAL OF MEDICINE Feldman, D., Edelman, I. S., Funder, J. W. 1972; 53 (5): 545-?

  View details for Web of Science ID A1972N963900003

  View details for PubMedID 5079762