- Cancer > Hematology
- Cancer > Hematology > Hematologic Malignancies
- Cancer > Lymphoma
Deputy Director, Stanford Cancer Center (2005 - 2008)
Director, Stanford Cancer Institute (2008 - Present)
Honors & Awards
National Academy of Sciences, Institute of Medicine (Inducted 2001)
Fellow, American Association for the Advancement of Science (2015)
Member, Association of American Professors (1991)
Member, American Society for Clinical Investigation (to present)
President, American Society of Hematology (2001)
Mentorship Award, American Society of Hematology (2012)
Walter Hewlitt Physician Scientist Award, Stanford Department of Medicine (2012)
Residency:University of Washington Dept of Surgery (1973) WA
Medical Education:Harvard Medical School (1969) MA
Board Certification: Hematology, American Board of Internal Medicine (1978)
Fellowship:University Hospital Zurich (1973) Switzerland
Fellowship:University of Michigan Hospital (1977) MI
Board Certification: Internal Medicine, American Board of Internal Medicine (1973)
Internship:Harborview Medical Center (1970) WA
M.D., Harvard Medical School, Medicine (1969)
Current Research and Scholarly Interests
Beverly S. Mitchell, MD, is the Director of the Stanford Cancer Institute and is the George E. Becker Professor of Medicine at Stanford University. Before joining the Stanford faculty, Dr. Mitchell lead the Molecular Therapeutics Program at UNC Chapel Hill's Lineberger Comprehensive Cancer Center, where she also served as Associate Director for Translational Research and Chief of the Division of Hematology/Oncology.
She has authored over 130 peer-reviewed articles. She served as President of the American Society of Hematology (ASH) and was Chair of the Medical and Scientific Affairs Committee and Vice Chair for Medical and Scientific Affairs of the Leukemia and Lymphoma Society of America.
Dr. Mitchell's current research relates to the development of new therapies for hematologic malignancies. She is interested in preclinical proof of principle studies on mechanisms inducing cell death and on metabolic targets involving nucleic acid biosynthesis in malignant cells. Recent studies have focused on the role of nucleolar proteins in cellular stress responses, including that induced by reactive oxygen species. She is also interested in the regulation of ribosomal RNA synthesis in hematopoietic stem and progenitor cells and in the role of dysregulated synthesis in bone marrow failure syndromes. in addition, her laboratory is involved in the translation of these studies into scientifically designed clinical trials.
Independent Studies (8)
- Directed Reading in Cancer Biology
CBIO 299 (Aut, Win, Spr, Sum)
- Directed Reading in Medicine
MED 299 (Aut, Win, Spr, Sum)
- Early Clinical Experience in Medicine
MED 280 (Aut, Win, Spr, Sum)
- Graduate Research
CBIO 399 (Aut, Win, Spr, Sum)
- Graduate Research
MED 399 (Aut, Win, Spr, Sum)
- Medical Scholars Research
MED 370 (Aut, Win, Spr, Sum)
- Teaching in Cancer Biology
CBIO 260 (Spr)
- Undergraduate Research
MED 199 (Aut, Win, Spr, Sum)
- Directed Reading in Cancer Biology
Autophagy mediates proteolysis of NPM1 and HEXIM1 and sensitivity to BET inhibition in AML cells.
The mechanisms underlying activation of the BET pathway in AML cells remain poorly understood. We have discovered that autophagy is activated in acute leukemia cells expressing mutant nucleophosmin 1 (NPMc+) or MLL-fusion proteins. Autophagy activation results in the degradation of NPM1 and HEXIM1, two negative regulators of BET pathway activation. Inhibition of autophagy with pharmacologic inhibitors or through knocking down autophagy-related gene 5 (Atg5) expression increases the expression of both NPM1 and HEXIM1. The Brd4 inhibitors JQ1 and I-BET-151 also inhibit autophagy and increase NPM1 and HEXIM1 expression. We conclude that the degradation of NPM1 and HEXIM1 through autophagy in certain AML subsets contributes to the activation of the BET pathway in these cells.
View details for DOI 10.18632/oncotarget.12493
View details for PubMedID 27732946
- Expression and Role of the ErbB3-Binding Protein 1 in Acute Myelogenous Leukemic Cells CLINICAL CANCER RESEARCH 2016; 22 (13): 3320-3327
- Regulation of Ribosomal Gene Expression in Cancer JOURNAL OF CELLULAR PHYSIOLOGY 2015; 230 (6): 1181-1188
Regulation of ribosomal gene expression in cancer.
Journal of cellular physiology
2015; 230 (6): 1181-1188
The ability of a cell to undergo malignant transformation is both associated with and dependent on a concomitant increase in protein synthesis due to increased cell division rates and biosynthetic activities. Protein synthesis, in turn, depends upon the synthesis of ribosomes and thus ultimately on the transcription of ribosomal RNA by RNA polymerase I that occurs in the nucleolus. Enlargement of nucleoli has long been considered a hallmark of the malignant cell, but it is only recently that the rate of synthesis of rRNA in the nucleolus has been recognized as both a critical regulator of cellular proliferation and a potential target for therapeutic intervention. As might be expected, the factors regulating rRNA synthesis are both numerous and complex. It is the objective of this review to highlight recent advances in understanding how rRNA synthesis is perturbed in transformed mammalian cells and to consider the impact of these findings on the development of new approaches to the treatment of malignancies. In-depth analysis of the process of rRNA transcription itself may be found in several recently published reviews (Drygin et al., 2010, Annu Rev Pharmacol Toxicol 50:131-156; Bywater et al., 2013,Cancer Cell 22: 51-65; Hein et al., 2013,Trends Mol Med 19:643-654). J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
View details for DOI 10.1002/jcp.24854
View details for PubMedID 25336383
Regulation of ribosomal RNA synthesis in T cells: requirement for GTP and Ebp1.
2015; 125 (16): 2519-2529
Mycophenolic acid (MPA) is the active metabolite of Mycophenolate Mofeteil (MMF), an effective immunosuppressive drug. Both MPA and MMF are highly specific inhibitors of guanine nucleotide synthesis and of T cell activation. However, the mechanism by which guanine nucleotide depletion suppresses T cell activation is unknown. Depletion of GTP inhibits ribosomal RNA synthesis in T cells by inhibiting TIF-IA, a GTP-binding protein that recruits RNA Polymerase I to the ribosomal DNA promoter. TIF-IA-GTP binds the ErbB3 binding protein 1 (Ebp1) and together they enhance the transcription of proliferating cell nuclear antigen (PCNA). GTP binding by TIF-IA and Ebp1 phosphorylation by protein kinase C delta are both required for optimal PCNA expression. The PKC inhibitor Sotrastaurin markedly potentiates the inhibition of rRNA synthesis, PCNA expression, and T cell activation induced by MPA, suggesting that the combination of the two agents are more highly effective than either alone in inducing immunosuppression.
View details for DOI 10.1182/blood-2014-12-616433
View details for PubMedID 25691158
- Regulation of ribosomal RNA synthesis in T cells: requirement for GTP and Ebp1 BLOOD 2015; 125 (16): 2519-2529
Reversibility of Defective Hematopoiesis Caused by Telomere Shortening in Telomerase Knockout Mice.
2015; 10 (7)
Telomere shortening is common in bone marrow failure syndromes such as dyskeratosis congenita (DC), aplastic anemia (AA) and myelodysplastic syndromes (MDS). However, improved knowledge of the lineage-specific consequences of telomere erosion and restoration of telomere length in hematopoietic progenitors is required to advance therapeutic approaches. We have employed a reversible murine model of telomerase deficiency to compare the dependence of erythroid and myeloid lineage differentiation on telomerase activity. Fifth generation Tert-/- (G5 Tert-/-) mice with shortened telomeres have significant anemia, decreased erythroblasts and reduced hematopoietic stem cell (HSC) populations associated with neutrophilia and increased myelopoiesis. Intracellular multiparameter analysis by mass cytometry showed significantly reduced cell proliferation and increased sensitivity to activation of DNA damage checkpoints in erythroid progenitors and in erythroid-biased CD150hi HSC, but not in myeloid progenitors. Strikingly, Cre-inducible reactivation of telomerase activity restored hematopoietic stem and progenitor cell (HSPC) proliferation, normalized the DNA damage response, and improved red cell production and hemoglobin levels. These data establish a direct link between the loss of TERT activity, telomere shortening and defective erythropoiesis and suggest that novel strategies to restore telomerase function may have an important role in the treatment of the resulting anemia.
View details for DOI 10.1371/journal.pone.0131722
View details for PubMedID 26133370
Interaction of TIF-90 and filamin A in the regulation of rRNA synthesis in leukemic cells.
2014; 124 (4): 579-589
The transcription initiation factor I (TIF-IA) is an important regulator of the synthesis of ribosomal RNA (rRNA) through its facilitation of the recruitment of RNA polymerase I (Pol I) to the ribosomal DNA promoter. Activation of the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway, which occurs commonly in acute myelogenous leukemia, enhances rRNA synthesis through TIF-IA stabilization and phosphorylation. We have discovered that TIF-IA coexists with a splicing isoform, TIF-90, which is expressed preferentially in the nucleolus and at higher levels in proliferating and transformed hematopoietic cells. TIF-90 interacts directly with Pol I to increase rRNA synthesis as a consequence of Akt activation. Furthermore, TIF-90 binds preferentially to a 90-kDa cleavage product of the actin binding protein filamin A (FLNA) that inhibits rRNA synthesis. Increased expression of TIF-90 overcomes the inhibitory effect of this cleavage product and stimulates rRNA synthesis. Because activated Akt also reduces FLNA cleavage, these results indicate that activated Akt and TIF-90 function in parallel to increase rRNA synthesis and, as a consequence, cell proliferation in leukemic cells. These results provide evidence that the direct targeting of Akt would be an effective therapy in acute leukemias in which Akt is activated.
View details for DOI 10.1182/blood-2013-12-544726
View details for PubMedID 24850755
Akt activation enhances ribosomal RNA synthesis through casein kinase II and TIF-IA
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2013; 110 (51): 20681-20686
Transcription initiation factor I (TIF-IA) plays an essential role in regulating ribosomal RNA (rRNA) synthesis by tethering RNA polymerase I (Pol I) to the rDNA promoter. We have found that activated Akt enhances rRNA synthesis through the phosphorylation of casein kinase IIα (CK2α) on a threonine residue near its N terminus. CK2 in turn phosphorylates TIF-IA, thereby increasing rDNA transcription. Activated Akt also stabilizes TIF-IA, induces its translocation to the nucleolus, and enhances its interaction with Pol I. Treatment with AZD8055, an inhibitor of both Akt and mammalian target of rapamycin phosphorylation, but not with rapamycin, disrupts Akt-mediated TIF-IA stability, translocation, and activity. These data support a model in which activated Akt enhances rRNA synthesis both by preventing TIF-IA degradation and phosphorylating CK2α, which in turn phosphorylates TIF-IA. This model provides an explanation for the ability of activated Akt to promote cell proliferation and, potentially, transformation.
View details for DOI 10.1073/pnas.1313097110
View details for Web of Science ID 000328548600074
Reduced rRNA expression and increased rDNA promoter methylation in CD34(+) cells of patients with myelodysplastic syndromes
2012; 120 (24): 4812-4818
Myelodysplastic syndromes (MDS) are clonal disorders of hematopoietic stem cells characterized by ineffective hematopoiesis. The DNA-hypomethylating agents 5-azacytidine and 5-aza-2'-deoxycytidine are effective treatments for patients with MDS, increasing the time to progression to acute myelogenous leukemia and improving overall response rates. Although genome-wide increases in DNA methylation have been documented in BM cells from MDS patients, the methylation signatures of specific gene promoters have not been correlated with the clinical response to these therapies. Recently, attention has been drawn to the potential etiologic role of decreased expression of specific ribosomal proteins in MDS and in other BM failure states. Therefore, we investigated whether rRNA expression is dysregulated in MDS. We found significantly decreased rRNA expression and increased rDNA promoter methylation in CD34(+) hematopoietic progenitor cells from the majority of MDS patients compared with normal controls. Treatment of myeloid cell lines with 5-aza-2'-deoxycytidine resulted in a significant decrease in the methylation of the rDNA promoter and an increase in rRNA levels. These observations suggest that an increase in rDNA promoter methylation can result in decreased rRNA synthesis that may contribute to defective hematopoiesis and BM failure in some patients with MDS.
View details for DOI 10.1182/blood-2012-04-423111
View details for Web of Science ID 000313115300023
View details for PubMedID 23071274
- Effect of nucleophosmin1 haploinsufficiency on hematopoietic stem cells LEUKEMIA 2012; 26 (4): 853-855
Cyclopentenyl Cytosine Induces Senescence in Breast Cancer Cells through the Nucleolar Stress Response and Activation of p53
2011; 80 (1): 40-48
The induction of senescence has emerged as a potentially important contributor to the effects of chemotherapeutic agents against tumors. We have demonstrated that depletion of CTP induced by cyclopentenyl cytosine (CPEC; NSC 375575), a specific inhibitor of the enzyme CTP synthetase, induces irreversible growth arrest and senescence characterized by altered morphology and expression of senescence-associated β-galactosidase activity in MCF-7 breast cancer cells expressing wild-type p53. In contrast, differentiation in the absence of senescence resulted from CPEC treatment in MDA-MB-231 breast cancer cells that express a mutated p53. Both senescence of MCF-7 cells and differentiation of MDA-MB-231 cells were prevented by repletion of CTP through the cytidine salvage pathway. Senescence in MCF-7 cells was associated with a G(2)- and S-phase arrest, whereas differentiation in MDA-MB-231 cells was associated with arrest in G(1) phase at 5 days. Mechanistic studies revealed that CTP depletion induced a rapid translocation of nucleolar proteins, including nucleostemin and nucleolin into the nucleoplasm. This nucleolar stress response resulted in a sustained elevation of p53 and the p53 target genes, p21 and Mdm2, in cells with wild-type p53. Furthermore, short interfering RNA-induced knockdown of p53 in MCF-7 cells treated with CPEC prevented cellular senescence and increased apoptotic cell death. We conclude that CTP depletion and the resulting nucleolar stress response results in a senescence-like growth arrest through activation of p53, whereas cells with mutated p53 undergo differentiation or apoptotic cell death.
View details for DOI 10.1124/mol.110.070284
View details for Web of Science ID 000291717000005
View details for PubMedID 21464199
Reactive Oxygen Species Regulate Nucleostemin Oligomerization and Protein Degradation
JOURNAL OF BIOLOGICAL CHEMISTRY
2011; 286 (13): 11035-11046
Nucleostemin (NS) is a nucleolar-nucleoplasmic shuttle protein that regulates cell proliferation, binds p53 and Mdm2, and is highly expressed in tumor cells. We have identified NS as a target of oxidative regulation in transformed hematopoietic cells. NS oligomerization occurs in HL-60 leukemic cells and Raji B lymphoblasts that express high levels of c-Myc and have high intrinsic levels of reactive oxygen species (ROS); reducing agents dissociate NS into monomers and dimers. Exposure of U2OS osteosarcoma cells with low levels of intrinsic ROS to hydrogen peroxide (H(2)O(2)) induces thiol-reversible disulfide bond-mediated oligomerization of NS. Increased exposure to H(2)O(2) impairs NS degradation, immobilizes the protein within the nucleolus, and results in detergent-insoluble NS. The regulation of NS by ROS was validated in a murine lymphoma tumor model in which c-Myc is overexpressed and in CD34+ cells from patients with chronic myelogenous leukemia in blast crisis. In both instances, increased ROS levels were associated with markedly increased expression of NS protein and thiol-reversible oligomerization. Site-directed mutagenesis of critical cysteine-containing regions of nucleostemin altered both its intracellular localization and its stability. MG132, a potent proteasome inhibitor and activator of ROS, markedly decreased degradation and increased nucleolar retention of NS mutants, whereas N-acetyl-L-cysteine largely prevented the effects of MG132. These results indicate that NS is a highly redox-sensitive protein. Increased intracellular ROS levels, such as those that result from oncogenic transformation in hematopoietic malignancies, regulate the ability of NS to oligomerize, prevent its degradation, and may alter its ability to regulate cell proliferation.
View details for DOI 10.1074/jbc.M110.208470
View details for Web of Science ID 000288797100015
View details for PubMedID 21242306
Depletion of Guanine Nucleotides Leads to the Mdm2-Dependent Proteasomal Degradation of Nucleostemin
2009; 69 (7): 3004-3012
Nucleostemin is a positive regulator of cell proliferation and is highly expressed in a variety of stem cells, tumors, and tumor cell lines. The protein shuttles between the nucleolus and the nucleus in a GTP-dependent fashion. Selective depletion of intracellular guanine nucleotides by AVN-944, an inhibitor of the de novo purine synthetic enzyme, IMP dehydrogenase, leads to the rapid disappearance of nucleostemin protein in tumor cell lines, an effect that does not occur with two other nucleolar proteins, nucleophosmin or nucleolin. Endogenous nucleostemin protein is completely stabilized by MG132, an inhibitor of the 26S proteasome, as are the levels of expressed enhanced green fluorescent protein-tagged nucleostemin, both wild-type protein and protein containing mutations at the G(1) GTP binding site. Nutlin-3a, a small molecule that disrupts the binding of the E3 ubiquitin ligase, Mdm2, to p53, stabilizes nucleostemin protein in the face of guanine nucleotide depletion, as does siRNA-mediated knockdown of Mdm2 expression and overexpression of a dominant-negative form of Mdm2. Neither Doxorubicin nor Actinomycin D, which cause the release of nucleostemin from the nucleolus, results in nucleostemin degradation. We conclude that nucleostemin is a target for Mdm2-mediated ubiquitination and degradation when not bound to GTP. Because this effect does not occur with other chemotherapeutic agents, the induction of nucleostemin protein degradation in tumor cells by IMP dehydrogenase inhibition or by other small molecules that disrupt GTP binding may offer a new approach to the treatment of certain neoplastic diseases.
View details for DOI 10.1158/0008-5472.CAN-08-3413
View details for Web of Science ID 000264908100043
View details for PubMedID 19318567
Determinants of sensitivity of human T-cell leukemia CCRF-CEM cells to immucillin-H
2008; 32 (8): 1268-1278
Immucillin-H (BCX-1777, forodesine) is a transition state analogue and potent inhibitor of PNP that shows promise as a specific agent against activated human T-cells and T-cell leukemias. The immunosuppressive or antileukemic effects of Immucillin-H (ImmH) in cultured cells require co-administration with deoxyguanosine (dGuo) to attain therapeutic levels of intracellular dGTP. In this study we investigated the requirements for sensitivity and resistance to ImmH and dGuo. (3)H-ImmH transport assays demonstrated that the equilibrative nucleoside transporters (ENT1 and ENT2) facilitated the uptake of ImmH in human leukemia CCRF-CEM cells whereas (3)H-dGuo uptake was primarily dependent upon concentrative nucleoside transporters (CNTs). Analysis of lysates from ImmH-resistant CCRF-CEM-AraC-8D cells demonstrated undetectable deoxycytidine kinase (dCK) activity, suggesting that dCK and not deoxyguanosine kinase (dGK) was the rate-limiting enzyme for phosphorylation of dGuo in these cells. Examination of ImmH cytotoxicity in a hypoxanthine-guanine phosphoribosyltransferase (HGPRT)-deficient cell line CCRF-CEM-AraC-8C, demonstrated enhanced sensitivity to low concentrations of ImmH and dGuo. RT-PCR and sequencing of HGPRT from the HGPRT-deficient CCRF-CEM-AraC-8C cells identified an Exon 8 deletion mutation in this enzyme. Thus these studies show that specific nucleoside transporters are required for ImmH cytotoxicity and predict that ImmH may be more cytotoxic to 6-thioguanine (6-TG) or 6-thiopurine-resistant leukemia cells caused by HGPRT deficiency.
View details for DOI 10.1016/j.leukres.2007.12.015
View details for Web of Science ID 000256251500014
View details for PubMedID 18279955
- Guanine nucleotide depletion inhibits pre-ribosomal RNA synthesis and causes nucleolar proteins Leuk Res 2008; 32 (1): 131-141
The 5 '-nucleotidases as regulators of nucleotide and drug metabolism
PHARMACOLOGY & THERAPEUTICS
2005; 107 (1): 1-30
The 5'-nucleotidases are a family of enzymes that catalyze the dephosphorylation of nucleoside monophosphates and regulate cellular nucleotide and nucleoside levels. While the nucleoside kinases responsible for the initial phosphorylation of salvaged nucleosides have been well studied, many of the catabolic nucleotidases have only recently been cloned and characterized. Aside from maintaining balanced ribo- and deoxyribonucleotide pools, substrate cycles that are formed with kinase and nucleotidase activities are also likely to regulate the activation of nucleoside analogues, a class of anticancer and antiviral agents that rely on the nucleoside kinases for phosphorylation to their active forms. Both clinical and in vitro studies suggest that an increase in nucleotidase activity can inhibit nucleoside analogue activation and result in drug resistance. The physiological role of the 5'-nucleotidases will be covered in this review, as will the evidence that these enzymes can mediate resistance to nucleoside analogues.
View details for DOI 10.1016/j.pharmthera.2005.01.003
View details for Web of Science ID 000230279500001
View details for PubMedID 15963349
Phase I study of 506U78 administered on a consecutive 5-day schedule in children and adults with refractory hematologic malignancies
JOURNAL OF CLINICAL ONCOLOGY
2005; 23 (15): 3396-3403
A phase I study was conducted to determine the maximum-tolerated dose (MTD), toxicity profile, and pharmacokinetics of a novel purine nucleoside, nelarabine, a soluble prodrug of 9-beta-D-arabinosylguanine (araG; Nelarabine), in pediatric and adult patients with refractory hematologic malignancies.Between April 1994 and April 1997, 93 patients with refractory hematologic malignancies were treated with one to 16 cycles of study drug. Nelarabine was administered daily, as a 1-hour intravenous infusion for 5 consecutive days, every 21 to 28 days. First-cycle pharmacokinetic data, including plasma nelarabine and araG levels, were obtained on all patients treated. Intracellular phosphorylation of araG was studied in samples of leukemic blasts from selected patients.The MTDs were defined at 60 mg/kg/dose and 40 mg/kg/dose daily x 5 days in children and adults, respectively. Dose-limiting toxicity (DLT) was neurologic in both children and adults. Myelosuppression and other significant organ toxicities did not occur. Pharmacokinetic parameters were similar in children and adults. Accumulation of araGTP in leukemic blasts was correlated with cytotoxic activity. The overall response rate was 31%. Major responses were seen in patients with T-cell malignancies, with 54% of patients with T-lineage acute lymphoblastic leukemia achieving a complete or partial response after one to two courses of drug.Nelarabine is a novel nucleoside with significant cytotoxic activity against malignant T cells. DLT is neurologic. Phase II and III trials in patients with T-cell malignancies are encouraged.
View details for DOI 10.1200/JCO.2005.03.199
View details for Web of Science ID 000229352400017
View details for PubMedID 15908652
Synergy between imatinib and mycophenolic acid in inducing apoptosis in cell lines expressing Bcr-Abl
2005; 105 (8): 3270-3277
Bcr-Abl tyrosine kinase activity initiates a number of intracellular signaling cascades that result in leukemogenesis. Imatinib mesylate, a specific Bcr-Abl tyrosine kinase inhibitor, has been highly successful in the treatment of chronic myelogenous leukemia (CML). However, the emergence of imatinib resistance and the incomplete molecular response of a significant number of patients receiving this therapy have led to a search for combinations of drugs that will enhance the efficacy of imatinib. We have demonstrated that mycophenolic acid (MPA), a specific inosine monophosphate dehydrogenase (IMPDH) inhibitor that results in depletion of intracellular guanine nucleotides, is synergistic with imatinib in inducing apoptosis in Bcr-Abl-expressing cell lines. Studies of signaling pathways downstream of Bcr-Abl demonstrated that the addition of MPA to imatinib reduced the phosphorylation of both Stat5 and Lyn, a Src kinase family member. The phosphorylation of S6 ribosomal protein was also greatly reduced. These results demonstrate that inhibitors of guanine nucleotide biosynthesis may synergize with imatinib in reducing the levels of minimal residual disease in CML and lay the foundation for clinical trials in which IMPDH inhibitors are added to imatinib in patients who have suboptimal molecular responses to single agent therapy or who have progressive disease.
View details for DOI 10.1182/blood-2004-10-3864
View details for Web of Science ID 000228344500046
View details for PubMedID 15604220
Phase 1 trial of the proteasome inhibitor bortezomib and pegylated liposomal doxorubicin in patients with advanced hematologic malignancies
2005; 105 (8): 3058-3065
Proteasome inhibitors, a novel class of chemotherapeutic agents, enhance the antitumor efficacy of anthracyclines in vitro and in vivo. We therefore sought to determine the maximum tolerated dose (MTD) and dose-limiting toxicities of bortezomib and pegylated liposomal doxorubicin (PegLD). Bortezomib was given on days 1, 4, 8, and 11 from 0.90 to 1.50 mg/m2 and PegLD on day 4 at 30 mg/m2 to 42 patients with advanced hematologic malignancies. Grade 3 or 4 toxicities in at least 10% of patients included thrombocytopenia, lymphopenia, neutropenia, fatigue, pneumonia, peripheral neuropathy, febrile neutropenia, and diarrhea. The MTD based on cycle 1 was 1.50 and 30 mg/m2 of bortezomib and PegLD, respectively. However, due to frequent dose reductions and delays at this level, 1.30 and 30 mg/m2 are recommended for further study. Pharmacokinetic and pharmacodynamic studies did not find significant drug interactions between these agents. Antitumor activity was seen against multiple myeloma, with 8 of 22 evaluable patients having a complete response (CR) or near-CR, including several with anthracycline-refractory disease, and another 8 having partial responses (PRs). One patient with relapsed/refractory T-cell non-Hodgkin lymphoma (NHL) achieved a CR, whereas 2 patients each with acute myeloid leukemia and B-cell NHL had PRs. Bortezomib/PegLD was safely administered in this study with promising antitumor activity, supporting further testing of this regimen.
View details for DOI 10.1182/blood-2004-07-2911
View details for Web of Science ID 000228344500018
View details for PubMedID 15626743
- ACCO: ASCO core curriculum outline JOURNAL OF CLINICAL ONCOLOGY 2005; 23 (9): 2049-2077
Effects of the IMP-dehydrogenase inhibitor, Tiazofurin, in bcr-abl positive acute myelogenous leukemia - Part I. In vivo studies
2004; 28 (11): 1125-1136
Six patients with bcr-abl positive AML or chronic myelogenous leukemia in blast crisis (CML-BC) were treated with the IMP-dehydrogenase (IMPDH) inhibitor, Tiazofurin, in a Phase-II trial. Tiazofurin was given by IV infusion (2200-2700 mg/m2 per day) for up to 10 days. Leukemia blasts rapidly disappeared from the circulation of patients during treatment, while mature myeloid cells in the marrow increased in number. Although these hematologic responses were transient, persisting less than 3-4 weeks, our findings confirm that Tiazofurin has anti-leukemia activity. This drug warrants further study in combination regimens with other chemotherapeutic agents for the treatment of bcr-abl positive AML and CML-BC.
View details for DOI 10.1016/j.leukres.2004.03.003
View details for Web of Science ID 000224222400003
View details for PubMedID 15380335
Role of estrogen receptor in the regulation of ecto-5 '-nucleotidase and adenosine in breast cancer
CLINICAL CANCER RESEARCH
2004; 10 (2): 708-717
The purpose is to understand the expression of ecto-5'-nucleotidase (eN), an adenosine producing enzyme with potential roles in angiogenesis, growth, and immunosuppression, in estrogen receptor (ER)-negative and -positive breast cancer.We investigated the regulation of eN expression at the mRNA and protein levels by alpha in a panel of breast cancer cell lines that differ in ER status and invasive and metastatic potential. We also determined rates of adenosine formation in cells with high and low eN expression and in ER+ cells treated with estradiol.ER-negative cells express high eN protein and mRNA levels and produce up to 104-fold more adenosine from AMP and ATP. Estradiol and antiestrogen treatments confirm that eN mRNA and protein expression and adenosine generation are negatively regulated through the ER. Endogenous expression of eN in ER- cells transfected with ERalpha and phorbol ester-induced eN expression in ER+ cells was strongly suppressed by estradiol, suggesting a dominant function of ER. Finally, an examination of 18 clinical breast cancer samples that were analyzed for both ER status and eN expression by Martin et al. (Cancer Res., 60: 2232-2238, 2000) revealed a significant inverse correlation between ER and eN status.Our results show for the first time that eN is negatively regulated by ERalpha in dominant fashion and suggests that eN expression and its generation of adenosine may relate to breast cancer progression. Additionally, increased expression of eN in a subset of ER-negative cells may serve as a novel marker for a subset of more aggressive breast carcinoma.
View details for Web of Science ID 000188801100037
View details for PubMedID 14760094
Role of human Pso4 in mammalian DNA repair and association with terminal deoxynucleotidyl transferase
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2003; 100 (19): 10746-10751
Terminal deoxynucleotidyl transferase (TdT; EC 184.108.40.206) adds nucleotides to DNA ends generated during V(D)J recombination that are subsequently processed by proteins involved in general double-strand break (DSB) repair pathways. We report an association between TdT and a 55-kDa protein in lymphoid cells. This protein, identified as hPso4, is a homolog of the protein encoded by the PS04/PRP19 gene in Saccharomyces cerevisiae that has pleiotropic functions in DNA recombination and error-prone repair. Purified hPso4 binds double-stranded DNA in a sequence-nonspecific manner but does not bind single-stranded DNA. hPso4 protein is induced 15- to 30-fold in cells by gamma radiation and chemical mutagens but not by UV treatment. Loss of hPso4 expression induced by siRNA results in accumulation of DSBs, apoptosis, and decreased cell survival after DNA damage. We conclude that hPso4 plays a major and previously undefined role in mammalian DNA DSB repair.
View details for DOI 10.1073/pnas.1631060100
View details for Web of Science ID 000185415300033
View details for PubMedID 12960389
Expression and Role of the ErbB3-Binding Protein 1 in Acute Myelogenous Leukemic Cells.
Clinical cancer research
2016; 22 (13): 3320-3327
The ErbB3 binding protein 1 (Ebp1) has been implicated in diverse cancers as having either oncogenic or tumor suppressor activities. The present study was undertaken to determine the effects of Ebp1 expression in AML cells and to determine the mechanisms by which Ebp1 promotes cell proliferation in these cells.The expression of Ebp1 was studied in mononuclear cells obtained from the peripheral blood of 54 patients with AML by Western blot. The effects of Ebp1 expression on Proliferating Cell Nuclear Antigen (PCNA) expression and cell proliferation was measured using Western Blot, immunoprecipitation, in vitro ubiquitination, and colony forming assays. The role of Ebp1 in promoting rRNA synthesis and cell proliferation was evaluated by measuring the level of pre-rRNA and the recruitment of Pol I to rDNA.Ebp1 is highly expressed in acute myeloid leukemia (AML) cells and regulates the level of ribosomal RNA (rRNA) synthesis by binding to RNA Polymerase I (Pol I) and enhancing the formation of the Pol I initiation complex. Ebp1 also increases the stability of proliferating cell nuclear antigen (PCNA) protein by preventing its interaction with Mdm2, for which it is a substrate.These results demonstrate an important role of Ebp1 in promoting cell proliferation in AML cells through the regulation of both rRNA synthesis and PCNA expression.
View details for DOI 10.1158/1078-0432.CCR-15-2282
View details for PubMedID 26813358
Selective Toxicity of Investigational Ixazomib for Human Leukemia Cells Expressing Mutant Cytoplasmic NPM1: Role of Reactive Oxygen Species.
Clinical cancer research
2016; 22 (8): 1978-1988
This study was performed to determine whether the investigational proteasome inhibitor ixazomib demonstrated selective anti-neoplastic activity against acute myelogenous leukemia cells expressing a mutated nucleophosmin-1 gene and to gain a better understanding of its mechanisms of action.The cytotoxic effects of ixazomib treatment were analyzed in human AML cell lines and primary AML samples expressing wild type or mutated NPM1 (NPMc+). The potential roles of oxidative stress in mediating cytotoxic activity were determined using flow cytometry, enzyme-based assays, and western blots.Apoptosis induced by ixazomib was abrogated by knock down of NPM1/NPMc+ expression using an inducible shRNA construct and enhanced by NPMc+ overexpression. Cytotoxicity was associated with superoxide generation and was reduced by the addition of the antioxidant N-acetylcysteine. AML cells expressing NPMc+ had significantly reduced levels of intracellular glutathione and NADPH associated with reduced antioxidant responses to drug treatment. Treatment of three patients with relapsed NPMc+ AML resulted in an anti-leukemic effect in one patient as demonstrated by a marked reduction of leukemic blasts in the peripheral blood. Efficacy was associated with superoxide generation, reduced glutathione levels, and reduced mRNA and protein expression of antioxidant effectors in responding cells.In this study a direct association was observed between NPMc+ expression in AML, reduced antioxidant responses, and enhanced sensitivity to an oral proteasome inhibitor that induces oxidative stress. These data suggest that intracellular determinants of antioxidant responses may be good predictors of therapeutic response to ixazomib.
View details for DOI 10.1158/1078-0432.CCR-15-1440
View details for PubMedID 26634271
Age and Genetics How Do Prognostic Factors at Diagnosis Explain Disparities in Acute Myeloid Leukemia?
AMERICAN JOURNAL OF CLINICAL ONCOLOGY-CANCER CLINICAL TRIALS
2015; 38 (2): 159-164
OBJECTIVES:: Survival disparities in acute myeloid leukemia (AML) among blacks and Hispanics have been described but not studied extensively in adults. Although younger age and cytogenetic profiles of t(8;21) and acute promyelocytic leukemia (APL) subtypes of AML are associated with improved survival, these factors have not been investigated by race. The purpose is to evaluate whether the observed survival differences for blacks and Hispanics with AML are attributable to older age at diagnosis or lower rates of favorable cytogenetic profiles at diagnosis. The hypothesis is that survival disparities for blacks and Hispanics with AML will be explained by older age at diagnosis and lower rates of favorable cytogenetics. METHODS:: Patients with AML were identified in the Surveillance Epidemiology and End Results database (1999 to 2008). Kaplan-Meier (KM) survival curves predicted survival by race/ethnicity, stratified by age. Cox proportional hazard models estimated mortality by race with adjustment for age, sex, year of diagnosis, t(8;21), and APL subtypes. RESULTS:: A total of 25,692 patients were included. Blacks and Hispanics were diagnosed at younger ages (younger than 61 y), and had higher rates of t(8;21) and APL compared with non-Hispanic whites (NHWs). The overall KM curve shows that NHWs had a worse survival compared with other races/ethnicities. However, when KM curves were stratified by age, blacks and Hispanics had worse survival in younger age categories (younger than 61 y). In multivariable models, black race was associated with an increased risk of death compared with NHWs (HR, 1.10; 95% CI, 1.04-1.16). Adjustment for t(8;21) and APL subtypes did not attenuate the disparity. CONCLUSIONS:: Despite younger age and higher prevalence of favorable cytogenetics at diagnosis, blacks and Hispanics have an increased mortality from AML compared with other racial/ethnic groups. Future studies should investigate other factors that may influence outcomes among minority populations.
View details for DOI 10.1097/COC.0b013e31828d7536
View details for Web of Science ID 000351770500007
View details for PubMedID 23608826
How do differences in treatment impact racial and ethnic disparities in acute myeloid leukemia?
Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology
2015; 24 (2): 344-349
We previously demonstrated disparate acute myelogenous leukemia (AML) survival for black and Hispanic patients; these differences persisted despite younger ages and higher prevalence of favorable cytogenetics in these groups. This study determined: (i) whether there are differences in treatment delivered to minorities, and (ii) how these differences affect outcomes in AML. We hypothesize that differences in treatment explain some proportion of survival disparities.We used California Cancer Registry data linked to hospital discharge abstracts for patients with AML (1998-2008). Logistic regression models estimated odds of treatment (chemotherapy and/or hematopoietic stem cell transplant) by race/ethnicity. Cox proportional hazard models estimated mortality by race after adjustment for treatment.We analyzed 11,084 records. Black race was associated with lower odds of chemotherapy [OR, 0.74; 95% confidence interval (CI), 0.61-0.91]. Black and Hispanic patients had decreased odds of transplant [(OR, 0.64; 95% CI, 0.46-0.87); (OR, 0.74; 95% CI, 0.62-0.89), respectively]. Black patients had increased hazard of mortality (HR, 1.14; 95% CI, 1.04-1.25) compared with whites. Adjustment for receipt of any treatment resulted in decreased mortality (HR, 1.09; 95% CI, 1.00-1.20) for black patients.AML treatment differences for black patients explain some proportion of the disparity. Future AML disparities studies should investigate socioeconomic and other characteristics.Study findings may better elucidate drivers of disparities in AML. Cancer Epidemiol Biomarkers Prev; 24(2); 344-9. ©2015 AACR.
View details for DOI 10.1158/1055-9965.EPI-14-0963
View details for PubMedID 25662426
- Effect of CAL-101, a PI3K delta inhibitor, on ribosomal rna synthesis and cell proliferation in acute myeloid leukemia cells BLOOD CANCER JOURNAL 2014; 4
Role of cysteine 288 in nucleophosmin cytoplasmic mutations: sensitization to toxicity induced by arsenic trioxide and bortezomib
2013; 27 (10): 1970-1980
Mutations in exon 12 of the NPM1 gene (NPMc+) define a distinct subset of acute myelogenous leukemias (AML) in which the NPMc+ protein localizes aberrantly to the leukemic cell cytoplasm. We have found that introduction of the most common NPMc+ variant into K562 and 32D cells sensitizes these cells to apoptosis induced by drugs such as bortezomib and arsenic trioxide that induce reactive oxygen species (ROS) formation and that cytotoxicity is prevented in the presence of N-acetyl-1-cysteine, a ROS scavenger. The substitution of tryptophan288 by cysteine occurs in the great majority of NPM1c+ mutations. Mutagenesis of C288 to alanine re-localizes NPMc+ from the cytoplasm to the nucleolus and attenuates the sensitivity of cells expressing this mutation to bortezomib and arsenic trioxide. Primary AML leukemic cells expressing NPMc+ are also significantly more sensitive than other AML cells to apoptosis induced by both drugs at pharmacologically achievable doses. We conclude that the presence of a cysteine moiety at position 288 results in the cytoplasmic localization of NPM1c+ and the increased sensitivity to bortezomib and arsenic trioxide. These data suggest that bortezomib and arsenic trioxide may have increased therapeutic efficacy in NPM1c+ leukemias.Leukemia accepted article preview online, 23 July 2013. doi:10.1038/leu.2013.222.
View details for DOI 10.1038/leu.2013.222
View details for Web of Science ID 000325642600003
View details for PubMedID 23877794
Sequential azacitidine plus lenalidomide combination for elderly patients with untreated acute myeloid leukemia.
2013; 98 (4): 591-596
There are limited treatment options for older patients with acute myeloid leukemia and prognosis of these patients remains poor, thereby warranting development of novel therapies. We evaluated the efficacy and safety of azacitidine in combination with lenalidomide as front-line therapy for older patients with acute myeloid leukemia. Patients ≥ 60 years of age with untreated acute myeloid leukemia received azacitidine 75 mg/m2 for 7 days followed by escalating doses of lenalidomide daily for 21 days starting on day 8 of each cycle every 6 weeks. Patients received continued therapy until disease progression, unacceptable toxicity, or completion of 12 cycles. Forty-two patients (median age, 74 years) were enrolled with equal distribution according to European LeukemiaNet risk. The overall response rate was 40% (rate of complete remission with or without complete recovery of blood counts = 28%). The median time to complete remission with or without complete recovery of blood counts was 12 weeks, and duration of this status was 28 weeks (range, 4 - >104 weeks). Therapy-related acute myeloid leukemia and a high score on the Hematopoietic Cell Transplantation Comorbidity Index were negative predictors of response. Early death was noted in 17% of patients. Grades ≥ 3 toxicities were uncommon and most adverse events were gastrointestinal, fatigue and myelosuppression. In conclusion, a sequential combination of azacitidine plus lenalidomide has clinical activity in older patients with acute myeloid leukemia, and further studies of this combination are underway.
View details for DOI 10.3324/haematol.2012.076414
View details for PubMedID 23242596
Understanding disparities in leukemia: a national study
CANCER CAUSES & CONTROL
2012; 23 (11): 1831-1837
Disparities in solid tumors have been well studied. However, disparities in hematologic malignancies have been relatively unexplored on population-based levels. The purpose of this study is to examine the relationship between race/ethnicity and acute leukemia mortality.All patients with acute leukemia [acute lymphoblastic leukemia (ALL) and acute myelogenous leukemia (AML)] were identified in the Surveillance Epidemiology and End Results database, 1999-2008. Kaplan-Meier curves were generated to reflect survival probabilities by race/ethnicity. Multivariable Cox proportional hazard models estimated hazard of mortality by race with adjustment for individual (age, gender, year of diagnosis) and select genetic factors.A total of 39,002 patients with acute leukemia were included in the study. Overall, there was a mortality disparity in acute leukemia for blacks (HR 1.17, p < 0.0001) and Hispanics (HR 1.13, p < 0.0001) compared with non-Hispanic whites. In stratified analysis, disparities in ALL were greater than AML; blacks (HR[ALL]1.45, p < 0.0001; HR[AML]1.12, p < 0.0011); Hispanics (HR[ALL]1.46, p < 0.0001; HR[AML]1.06, p < 0.0001). Adjustment for individual patient and select genetic factors did not explain disparities.Blacks and Hispanics suffer decreased survival in acute leukemia as compared to others. Further investigation is needed to understand the drivers of poor cancer outcomes in these populations.
View details for DOI 10.1007/s10552-012-0062-3
View details for Web of Science ID 000309671300009
View details for PubMedID 22971999
Safety, efficacy and biological predictors of response to sequential azacitidine and lenalidomide for elderly patients with acute myeloid leukemia
2012; 26 (5): 893-901
Acute myeloid leukemia (AML) is a disease of the elderly. Poor outcomes with standard therapies necessitate novel approaches. Outpatient regimens sufficiently potent and well tolerated to induce remissions and enable continuation therapy may be beneficial. In this phase-1 study, we determined the maximum tolerated dose (MTD) and the efficacy for sequential azacitidine and lenalidomide as remission induction and continuation therapy in elderly, previously untreated patients. We investigated the impact on global DNA methylation and bone marrow cytokines, and sought biological predictors of response. Eighteen patients were enrolled. The MTD was not reached. Median follow-up was 8.2 months (10.3 months for survivors). Common adverse events included fatigue, injection site reactions, constipation, nausea, pruritus and febrile neutropenia. Ten patients responded (56%), and the rate of complete remissions (CRs) or CRs with incomplete recovery of blood counts for evaluable patients was 44% (7/16). The median response duration was 6.2 months. DNA demethylation and changes in bone marrow cytokines were observed; responders had a unique cytokine profile and a trend towards lower methylation levels. Sequential azacitidine and lenalidomide was well tolerated with encouraging clinical and biological activity in previously untreated elderly AML patients. This trial is registered at ClinicalTrials.gov (NCT00890929).
View details for DOI 10.1038/leu.2011.294
View details for Web of Science ID 000303883500005
View details for PubMedID 22033493
Synthesis of 2 '-Deoxy-2 '-[F-18]Fluoro-9-beta-D-Arabinofuranosylguanine: a Novel Agent for Imaging T-Cell Activation with PET
MOLECULAR IMAGING AND BIOLOGY
2011; 13 (5): 812-818
9-(β-D-Arabinofuranosyl)guanine (AraG) is a guanosine analog that has a proven efficacy in the treatment of T-cell lymphoblastic disease. To test the possibility of using a radiofluorinated AraG as an imaging agent, we have synthesized 2'-deoxy-2'-[(18)F]fluoro-9-β-D-arabinofuranosylguanine ([(18)F]F-AraG) and investigated its uptake in T cells.We have synthesized [(18)F]F-AraG via a direct fluorination of 2-N-acetyl-6-O-((4-nitrophenyl)ethyl)-9-(3',5'-di-O-trityl-2'-O-trifyl-β-D-ribofuranosyl)guanine with [(18)F]KF/K.2.2.2 in DMSO at 85°C for 45 min. [(18)F]F-AraG uptake in both a CCRF-CEM leukemia cell line (unactivated) and activated primary thymocytes was evaluated.We have successfully prepared [(18)F]F-AraG in 7-10% radiochemical yield (decay corrected) with a specific activity of 0.8-1.3 Ci/μmol. Preliminary cell uptake experiments showed that both a CCRF-CEM leukemia cell line and activated primary thymocytes take up the [(18)F]F-AraG.For the first time to the best of our knowledge, [(18)F]F-AraG has been successfully synthesized by direct fluorination of an appropriate precursor of a guanosine nucleoside. This approach maybe also useful for the synthesis of other important positron emission tomography (PET) probes such as [(18)F]FEAU, [(18)F]FMAU, and [(18)F]FBAU which are currently synthesized by multiple steps and involve lengthy purification. The cell uptake studies support future studies to investigate the use of [(18)F]F-AraG as a PET imaging agent of T cells.
View details for DOI 10.1007/s11307-010-0414-x
View details for Web of Science ID 000295176200002
View details for PubMedID 20838911
- Impact of TET2 mutations on mRNA expression and clinical outcomes in MDS patients treated with DNA methyltransferase inhibitors HEMATOLOGICAL ONCOLOGY 2011; 29 (3): 157-160
- Tenascin C interacts with ecto-5'-nucleotidase (eN) and regulates adenosine generation in cancer cells Biochem Biophys Acta 2008; 1782 (1): 35-40
Guanine nucleotide depletion inhibits pre-ribosornal RNA synthesis and causes nucleolar disruption
2008; 32 (1): 131-141
Inosine monophosphate dehydrogenase (IMPDH) is a pivotal enzyme in the de novo pathway of guanine nucleotide biosynthesis. Inhibitors of this enzyme decrease intracellular guanine nucleotide levels by 50-80% and have potential as anti-neoplastic agents. Both mycophenolic acid (MPA) and AVN-944 are highly specific inhibitors of IMPDH that cause cell cycle arrest or apoptosis in lymphocytes and leukemic cell lines. We have examined the mechanisms by which these two agents cause cytotoxicity. Both MPA and AVN-944 inhibit the growth of K562 cells, and induce apoptosis in Raji B and CCRF-CEM T cells. Both compounds strikingly inhibit RNA synthesis within 2 h of exposure. Depletion of guanine nucleotides by MPA and AVN-944 also causes an early and near-complete reduction in levels of the 45S precursor rRNA synthesis and the concomitant translocation of nucleolar proteins including nucleolin, nucleophosmin, and nucleostemin from the nucleolus to the nucleoplasm. This efflux correlates temporally with the sustained induction of p53 in cell lines with wild-type p53. We conclude that inhibition of IMPDH causes a primary reduction in rRNA synthesis and secondary nucleolar disruption and efflux of nucleolar proteins that most likely mediate cell cycle arrest or apoptosis. The ability of AVN-944 to induce apoptosis in a number of leukemic cell lines supports its potential utility in the treatment of hematologic malignancies.
View details for DOI 10.1016/j.leukres.2007.03.025
View details for Web of Science ID 000251892600019
View details for PubMedID 17462731
Guanine nucleotide depletion mediates translocation of nucleolar proteins, including RNA helicase A (DHX-9)
12th International Symposium on Purine and Pyrimidine Metabolism
TAYLOR & FRANCIS INC. 2008: 704–11
DHX-9, a member of the DEXH family of RNA helicases, unwinds dsRNA/dsDNA by ATP or GTP-dependent hydrolysis. We asked whether DHX-9 played a role in the GTP depletion-induced inhibition of rRNA synthesis and/or nucleolar disruption. MPA, a specific inhibitor of inosine monophosphate dehydrogenase (IMPDH), induced a rapid translocation of DHX-9 from the nucleolus to the nucleus. EGFP-tagged DHX-9 mutated at the GTP binding site also localized to the nucleus. However, knockdown of DHX-9 by siRNA did not inhibit the rRNA synthesis or cause the nucleolar disruption. Thus, DHX-9 translocation found with IMPDH inhibition does not mediate the inhibition of rRNA synthesis.
View details for DOI 10.1080/15257770802145132
View details for Web of Science ID 000257338600029
Nelarabine induces complete remissions in adults with relapsed or refractory T-lineage acute lymphoblastic leukemia or lymphoblastic lymphoma: Cancer and Leukemia Group B study 19801
2007; 109 (12): 5136-5142
Nelarabine (506U78) is a soluble pro-drug of 9-beta-D-arabinofuranosylguanine (ara-G), a deoxyguanosine derivative. We treated 26 patients with T-cell acute lymphoblastic leukemia (T-ALL) and 13 with T-cell lymphoblastic lymphoma (T-LBL) with nelarabine. All patients were refractory to at least one multiagent regimen or had relapsed after achieving a complete remission. Nelarabine was administered on an alternate day schedule (days 1, 3, and 5) at 1.5 g/m(2)/day. Cycles were repeated every 22 days. The median age was 34 years (range, 16-66 years); 32 (82%) patients were male. The rate of complete remission was 31% (95% confidence interval [CI], 17%, 48%) and the overall response rate was 41% (95% CI, 26%, 58%). The principal toxicity was grade 3 or 4 neutropenia and thrombocytopenia, occurring in 37% and 26% of patients, respectively. There was only one grade 4 adverse event of the nervous system, which was a reversible depressed level of consciousness. The median disease-free survival (DFS) was 20 weeks (95% CI, 11, 56), and the median overall survival was 20 weeks (95% CI, 13, 36). The 1-year overall survival was 28% (95% CI, 15%, 43%). Nelarabine is well tolerated and has significant antitumor activity in relapsed or refractory T-ALL and T-LBL.
View details for DOI 10.1182/blood-2006-11-056754
View details for Web of Science ID 000247360200020
View details for PubMedID 17344466
- Nelarabing induces complete remissions in adults with relapsed or refractory t-lineage acute lymphoblastic leukemia or lymphoblastic lymphoma: cancer and leukemia group B study 19801 Blood 2007; 109 (12): 5136-5142
Genetic and biochemical biomarkers of IMPDH inhibition in phase I dose escalation of AVN-944 for hematologic malignancies.
48th Annual Meeting of the American-Society-of-Hematology
AMER SOC HEMATOLOGY. 2006: 731A–732A
View details for Web of Science ID 000242440003380
Effect of lysophosphatidic acid acyltransferase-beta inhibition in acute leukemia
2006; 30 (8): 1027-1036
Phosphatidic acid (PA) is an important component of mammalian target of rapamycin (mTOR) signaling and in the recruitment of Raf to the cell membrane. PA can be produced by several mechanisms, including by a series of lysophosphatidic acid acyl transferases (LPAATs). LPAAT-beta is an isoform that is overexpressed in some human cancers and its inhibition has been investigated as a potential targeted cancer therapy. We report that LPAAT-protein and enzyme activity in acute leukemia cell lines and blasts from patient samples are equivalent to levels in normal mononuclear cells. Treatment with the LPAAT-beta inhibitor CT-32228 (Cell Therapeutics, Seattle, WA) uniformly induces apoptosis in multiple leukemia cell lines. In patient samples, however, apoptosis was variably induced by CT-32228 and appeared to be related to the degree of cellular proliferation. The growth inhibitory effect of CT-32228 on normal hematopoietic progenitors was more pronounced in cells induced to proliferate by growth factors. These data suggest that CT-32228 may have potential in the treatment of acute leukemias, but that efficacy is more directly related to the degree of cell proliferation rather than to the level of LPAAT-beta expression or activity.
View details for DOI 10.1016/j.leukres.2005.11.018
View details for Web of Science ID 000238936600017
View details for PubMedID 16488473
Regulation of the interaction of inosine monophosphate dehydrogenase with mycophenolic acid by GTP
JOURNAL OF BIOLOGICAL CHEMISTRY
2006; 281 (1): 206-212
Inosine monophosphate dehydrogenase (IMPDH), a rate-limiting enzyme in the de novo synthesis of guanine nucleotides, is a major therapeutic target. A prototypic uncompetitive inhibitor of IMPDH, mycophenolic acid (MPA), is the active form of mycophenolate mofeteil (CellCept), a widely used immunosuppressive drug. We have found that MPA interacts with intracellular IMPDH in vivo to alter its mobility on SDS-polyacrylamide gels. MPA also induces a striking conformational change in IMPDH protein in intact cells, resulting in the formation of annular aggregates of protein with concomitant inhibition of IMPDH activity. These aggregates are not associated with any known intracellular organelles and are reversible by incubating cells with guanosine, which repletes intracellular GTP, or with GTPgammaS. GTP also restores IMPDH activity. Treatment of highly purified IMPDH with MPA also results in the formation of large aggregates of protein, a process that is both prevented and reversed by the addition of GTP. Finally, GTP binds to IMPDH at physiologic concentrations, induces the formation of linear arrays of tetrameric protein, and prevents the aggregation of protein induced by MPA. We conclude that intracellular GTP acts as an antagonist to MPA by directly binding to IMPDH and reversing the conformational changes in the protein.
View details for DOI 10.1074/jbc.M507056200
View details for Web of Science ID 000234307200028
View details for PubMedID 16243838
- Effect of lysosphatidic acid acyltransferase-B inhibition in acute luekemia Leu Res 2006; 30 (8): 1027-1036
Regulation of equilibrative nucleoside uptake by protein kinase inhibitors
Joint 11th International and 9th European Symposium on Purines and Pyrimidines in Man
TAYLOR & FRANCIS INC. 2004: 1445–50
The uptake of nucleosides and nucleoside analogs into human leukemia K562 cells is facilitated by the equilibrative transporters ENT1 and ENT2. Incubation of K562 cells with a variety of protein kinase inhibitors inhibited the transport of both uridine (ARA-C) and cytidine (CPEC) analogs. These inhibitory effects were observed for a large number of kinase inhibitors including those against p38 MAPK, the EGF receptor kinase, protein kinase C, TOR and others. Thus these results suggest that the nucleoside transporters are unexpected targets for kinase inhibitors and may influence the design and application of combinatorial approaches of nucleoside analogs and kinase inhibitors in clinical applications.
View details for DOI 10.1081/NCN-200027667
View details for Web of Science ID 000225113700070
View details for PubMedID 15571274
On the molecular pathology of neurodegeneration in IMPDH1-based retinitis pigmentosa
HUMAN MOLECULAR GENETICS
2004; 13 (6): 641-650
Retinitis pigmentosa (RP), the hereditary degenerative disease of the photoreceptor neurons of the retina, probably represents the most prevalent cause of registered blindness amongst those of working age in developed countries. Mutations within the gene encoding inosine monophosphate dehydrogenase 1 (IMPDH1), the widely expressed rate-limiting enzyme of the de novo pathway of guanine nucleotide biosynthesis, have recently been shown to cause the RP10 form of autosomal dominant RP. We examined the expression of IMPDH1, IMPDH2 and HPRT transcripts, encoding enzymes of the de novo and salvage pathways of guanine nucleotide biosynthesis, respectively, in retinal sections of mice, the data indicating that the bulk of GTP within photoreceptors is generated by IMPDH1. Impdh1(-/-) null mice are shown here to display a slowly progressive form of retinal degeneration in which visual transduction, analysed by electroretinographic wave functions, becomes gradually compromised, although at 12 months of age most photoreceptors remain structurally intact. In contrast, the human form of RP caused by mutations within the IMPDH1 gene is a severe autosomal dominant degenerative retinopathy in those families that have been examined to date. Expression of mutant IMPDH1 proteins in bacterial and mammalian cells, together with computational simulations, indicate that protein misfolding and aggregation, rather than reduced IMPDH1 enzyme activity, is the likely cause of the severe phenotype experienced by human subjects. Taken together, these findings suggest that RP10 may represent an attractive target for therapeutic intervention, based upon a strategy combining simultaneous suppression of transcripts from normal and mutant IMPDH1 alleles with supplementation of GTP within retinal tissues.
View details for DOI 10.1093/hmg/ddh061
View details for Web of Science ID 000220073800007
View details for PubMedID 14981049
Targeted disruption of the inosine 5 '-monophosphate dehydrogenase type I gene in mice
MOLECULAR AND CELLULAR BIOLOGY
2003; 23 (18): 6702-6712
Inosine 5'-monophosphate dehydrogenase (IMPDH) is the critical, rate-limiting enzyme in the de novo biosynthesis pathway for guanine nucleotides. Two separate isoenzymes, designated IMPDH types I and II, contribute to IMPDH activity. An additional pathway salvages guanine through the activity of hypoxanthine-guanine phosphoribosyltransferase (HPRT) to supply the cell with guanine nucleotides. In order to better understand the relative contributions of IMPDH types I and II and HPRT to normal biological function, a mouse deficient in IMPDH type I was generated by standard gene-targeting techniques and bred to mice deficient in HPRT or heterozygous for IMPDH type II. T-cell activation in response to anti-CD3 plus anti-CD28 antibodies was significantly impaired in both single- and double-knockout mice, whereas a more general inhibition of proliferation in response to other T- and B-cell mitogens was observed only in mice deficient in both enzymes. In addition, IMPDH type I(-/-) HPRT(-/0) splenocytes showed reduced interleukin-4 production and impaired cytolytic activity after antibody activation, indicating an important role for guanine salvage in supplementing the de novo synthesis of guanine nucleotides. We conclude that both IMPDH and HPRT activities contribute to normal T-lymphocyte activation and function.
View details for DOI 10.1128/MCB.23.18.6702-6712.2003
View details for Web of Science ID 000185103900032
View details for PubMedID 12944494
- The proteasome - An emerging therapeutic target in cancer NEW ENGLAND JOURNAL OF MEDICINE 2003; 348 (26): 2597-2598
Induction of apoptosis in IL-3-dependent hematopoietic cell lines by guanine nucleotide depletion
2003; 101 (12): 4958-4965
Inosine 5'-monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme that catalyzes the conversion of IMP to xanthosine monophosphate (XMP) at the branch point of purine nucleotide biosynthesis, leading to the generation of guanine nucleotides. Inhibition of IMPDH results in the depletion of guanine nucleotides, prevents cell growth by G1 arrest, and induces cell differentiation in a cell-type-specific manner. The molecular and sensing mechanisms underlying these effects are not clear. We have examined the induction of apoptosis by mycophenolic acid (MPA), a specific IMPDH inhibitor, in interleukin-3 (IL-3)-dependent murine hematopoietic cell lines. MPA treatment, at clinically relevant doses, caused apoptosis in 32D myeloid cells and in FL5.12 and BaF3 pre-B cells in the ongoing presence of IL-3. Apoptosis was completely prevented by the addition of guanosine at time points up to 12 hours, after which caspase 3 activity increased and apoptosis was not reversible. MPA treatment caused marked down-regulation of the MAP kinase kinase/extracellular regulatory kinase (MEK/Erk) pathway at 3 hours while simultaneously increasing the phosphorylation of c-Jun kinase. In addition, MPA strongly down-regulated the mammalian target of rapamcyin (mTOR) pathway, as indicated by the decreased phosphorylation of p70 S6 kinase and of 4EBP1. Inhibition of either the mitogen-activated protein kinase (MAPK) or the mTOR pathway alone by standard pharmacologic inhibitors did not induce apoptosis in IL-3-dependent cells, whereas inhibition of both pathways simulated the effects of MPA treatment. These results indicate that IMPDH inhibitors may be effective in modulating signal transduction pathways in hematopoietic cells, suggesting their usefulness in chemotherapeutic regimens for hematologic malignancies.
View details for DOI 10.1182/blood-2002-06-2547
View details for Web of Science ID 000183481700055
View details for PubMedID 12609835
Inhibition of nucleoside transport by protein kinase inhibitors
JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
2003; 304 (2): 753-760
Recently we reported that the pyridinylimidazole class of p38 mitogen-activated protein (MAP) kinase inhibitors potently inhibited the facilitated transport of nucleosides and nucleoside analogs in K562 cells. These compounds competed with the binding of nitrobenzylthioinosine (NBMPR) to K562 cells, consistent with inhibition of the NBMPR-sensitive equilibrative transporter (ENT1). In this study we examined a large number of additional protein kinase inhibitors for their effects on nucleoside transport. We find that incubation of K562 cells with tyrosine kinase inhibitors (AG825, AG1517, AG1478, STI-571), protein kinase C (PKC) inhibitors (staurosporine, GF 109203X, R0 31-8220, arcyriarubin A), cyclin-dependent kinase inhibitors (roscovitine, olomoucine, indirubin-3'-monoxime), or rapamycin resulted in a dose-dependent reduction of intracellular uptake of [3H]uridine. In contrast, neither the MAP kinase kinase inhibitors (U0126, PD 98059) nor the phosphatidyl inositol-3 kinase inhibitors (wortmannin, LY 294002) affected this process. Furthermore, both transient uptake and prolonged [3H]thymidine incorporation in K562 cells were inhibited by protein kinase inhibitors, inactive analogs of kinase inhibitors (R0 31-6045, SB202474), and NBMPR, independently of effects on cell proliferation as determined by MTT assay. These studies demonstrate that a wide variety of protein kinase inhibitors affect nucleoside uptake through selective inhibition of nucleoside transporters, independently of kinase inhibition.
View details for DOI 10.1124/jpet.102.044214
View details for Web of Science ID 000180526600034
View details for PubMedID 12538831
Phase I trial of the proteasome inhibitor PS-341 in patients with refractory hematologic malignancies
42nd Annual Meeting of the American-Society-of-Hematology
AMER SOC CLINICAL ONCOLOGY. 2002: 4420–27
To determine the maximum-tolerated dose (MTD), dose-limiting toxicity (DLT), and pharmacodynamics (PD) of the proteasome inhibitor bortezomib (previously known as PS-341) in patients with refractory hematologic malignancies.Patients received PS-341 twice weekly for 4 weeks at either 0.40, 1.04, 1.20, or 1.38 mg/m(2), followed by a 2-week rest. The PD of PS-341 was evaluated by measurement of whole blood 20S proteasome activity.Twenty-seven patients received 293 doses of PS-341, including 24 complete cycles. DLTs at doses above the 1.04-mg/m(2) MTD attributed to PS-341 included thrombocytopenia, hyponatremia, hypokalemia, fatigue, and malaise. In three of 10 patients receiving additional therapy, serious reversible adverse events appeared during cycle 2, including one episode of postural hypotension, one systemic hypersensitivity reaction, and grade 4 transaminitis in a patient with hepatitis C and a substantial acetaminophen ingestion. PD studies revealed PS-341 induced 20S proteasome inhibition in a time-dependent manner, and this inhibition was also related to both the dose in milligrams per meter squared, and the absolute dose of PS-341. Among nine fully assessable patients with heavily pretreated plasma cell dyscrasias completing one cycle of therapy, there was one complete response and a reduction in paraprotein levels and/or marrow plasmacytosis in eight others. In addition, one patient with mantle cell lymphoma and another with follicular lymphoma had shrinkage of nodal disease.PS-341 was well tolerated at 1.04 mg/m(2) on this dose-intensive schedule, although patients need to be monitored for electrolyte abnormalities and late toxicities. Additional studies are indicated to determine whether incorporation of dose/body surface area yields a superior PD model to dosing without normalization. PS-341 showed activity against refractory multiple myeloma and possibly non-Hodgkin's lymphoma in this study, and merits further investigation in these populations.
View details for Web of Science ID 000179267200003
View details for PubMedID 12431963
Clinical course of thrombocytopenia in patients treated with imatinib mesylate for accelerated phase chronic myelogenous leukemia
AMERICAN JOURNAL OF HEMATOLOGY
2002; 71 (3): 184-190
We studied 28 patients with accelerated phase chronic myelogenous leukemia (CML) who were enrolled on the Novartis expanded access study 114. Diagnosis of accelerated phase CML was based on karyotypic evolution (n = 9) and hematologic criteria (n = 18). All patients were begun on 600 mg/day of imatinib mesylate. Dose reductions to 400 mg/day and then 300 mg/day were prescribed for an absolute neutrophil count (ANC) of <0.5/microl or a platelet count of <20,000/microl. Twenty-seven of the 28 patients continued treatment for a median of 34 weeks. Eleven patients developed thrombocytopenia following an average of 8.4 +/- 1.4 weeks of therapy. The onset of thrombocytopenia was associated with disease progression in one patient and a decline in bone marrow megakaryocytes in the other 10. Nine patients recovered to a platelet count of >20,000/microl after an average of 19.7 +/- 1.8 weeks. Patients who developed thrombocytopenia had a longer duration of disease (9.39 vs. 4.35 years; P < 0.01) and were more likely to be diagnosed with accelerated phase CML by hematologic criteria. Hematologic responses in patients with and without thrombocytopenia were comparable; however, 31.3% of patients without thrombocytopenia had a complete cytogenetic response compared to none of those with thrombocytopenia. Grade III-IV thrombocytopenia is common in accelerated phase CML and may be a marker for the inability to achieve cytogenetic response using single agent imatinib mesylate.
View details for DOI 10.1002/ajh.10215
View details for Web of Science ID 000178973900007
View details for PubMedID 12410573
Alemtuzumab in previously treated chronic lymphocytic leukemia patients who also had received fludarabine
42nd Annual Meeting of the American-Society-of-Hematology
AMER SOC CLINICAL ONCOLOGY. 2002: 3891–97
This phase II pilot study determined the efficacy and safety of alemtuzumab (Campath-1H; Burroughs Wellcome, United Kingdom) in patients with chronic lymphocytic leukemia (CLL), all of whom had previously received fludarabine and other chemotherapy regimens.Twenty-four patients were treated with intravenous alemtuzumab at six centers in the United States. The target dose of 30 mg over 2 hours, three times weekly, was administered for up to 16 weeks. Responses were evaluated by an independent panel of experts using 1996 National Cancer Institute-sponsored Working Group criteria. Safety assessments included analysis of lymphocyte subpopulations. Antimicrobial prophylaxis was not mandatory.Eight patients (33%) achieved a major response (all partial remissions), with a median time to response of 3.9 months (range, 1.6 to 5.3 months). The median duration of response was 15.4 months (range, 4.6 to >or= 38.0 months), the median time to disease progression was 19.6 months (range, 7.7 to >or= 42.0 months), and the median survival time was 35.8 months (range, 8.8 to >or= 47.1 months). Acute infusion-related events, mainly grades 1 and 2, were most common and most severe in the first week. Ten patients (eight nonresponders and two responders) experienced major infections on-study. Pneumocystis carinii pneumonia was reported in two patients on-study; neither had received prophylaxis. Median CD4+ and CD8+ counts decreased and then began to increase by the end of the study, with further recovery by 1-month follow-up. One of 53 samples obtained from 10 patients had a low titer of alemtuzumab antibodies.Alemtuzumab has significant activity in poor-prognosis, fludarabine-treated CLL patients. However, because of a relatively high incidence of opportunistic infections accompanying profound lymphopenia, future protocols should include mandatory prophylaxis.
View details for DOI 10.1200/JCO.2002.06.119
View details for Web of Science ID 000178215600018
View details for PubMedID 12228210
A77 1726 induces differentiation of human myeloid leukemia K562 cells by depletion of intracellular CTP pools
2002; 62 (3): 463-472
A77 1726 (LEF) is the active metabolite of leflunomide, a recently approved immunosuppressive agent. We examined the ability of LEF to induce differentiation of a human erythroleukemia (K562) cell line and show that LEF induces a dose- and time-dependent differentiation of these cells as characterized by growth inhibition, hemoglobin production, and erythroid membrane protein glycophorin A expression. This effect was dependent on depletion of the intracellular pyrimidine ribonucleotides (UTP and CTP), and preceded by a specific S-phase arrest of the cell cycle. Supplementation of the cultures with exogenous uridine restored intracellular UTP and CTP to normal levels and prevented the LEF-induced cell cycle block and differentiation of K562 cells. Interestingly, addition of cytidine alone blocked the LEF-induced differentiation of K562 cells but only restored the CTP pool. By contrast, neither deoxycytidine nor thymidine prevented the effects of LEF on these cells. Similarly, pyrimidine starvation of a cell line lacking the de novo pyrimidine pathway (G9c) resulted in an S-phase arrest that was reversed by the addition of cytidine. Thus these studies demonstrate an important role for CTP in regulating cell cycle progression and show that LEF is an effective inducer of tumor cell differentiation through depletion of this ribonucleotide.
View details for Web of Science ID 000177438900006
View details for PubMedID 12181422
Inhibition of nucleoside transport by p38 MAPK inhibitors
JOURNAL OF BIOLOGICAL CHEMISTRY
2002; 277 (32): 28364-28367
While investigating the ability of p38 MAPK to regulate cytarabine (Ara C)-dependent differentiation of erythroleukemia K562 cells, we observed effects that indicated that the imidazoline class of p38 MAPK inhibitors prevented nucleoside transport. Incubation of K562 cells with SB203580, SB203580-iodo, or SB202474, an analogue of SB203580 that does not inhibit p38 MAPK activity, inhibited the uptake of [3H]Ara C or [3H]uridine and the differentiation of K562 cells. Consistent with the effects of these compounds on the nitrobenzylthioinosine (NBMPR)-sensitive equilibrative nucleoside transporter (ENT1), incubation with SB203580 or SB203580-iodo eliminated the binding of [3H]NBMPR to K562 cells or membranes isolated from human erythrocytes. Furthermore, using a uridine-dependent cell type (G9c), we observed that SB203580 or SB203580-iodo efficiently inhibited the salvage synthesis of pyrimidine nucleotides in vivo. Thus these studies demonstrate that the NBMPR-sensitive equilibrative nucleoside transporters are novel and unexpected targets for the p38 MAPK inhibitors at concentrations typically used to inhibit protein kinases.
View details for DOI 10.1074/jbc.C200321200
View details for Web of Science ID 000177342600002
View details for PubMedID 12077112
Cyclosporin A and FK506 decrease adenosine kinase activity and adenosine uptake in T-lymphocytes
JOURNAL OF LABORATORY AND CLINICAL MEDICINE
2002; 140 (2): 84-91
Adenosine is a potent modulator of immune function, and adenosine kinase (AK), a rate-limiting enzyme for adenosine uptake and metabolism, is a potential mediator of adenosine regulation. We have found that adenosine uptake increased six- to 18-fold during T-lymphocyte activation. This increase correlated with an increase in AK activity but not in AK protein. The immunosuppressive drugs cyclosporin A (CsA) and FK506 inhibited both adenosine uptake and AK activity in a concentration-dependent manner. Among several nucleosides and bases, the inhibition of uptake was selective for adenosine. Immunosuppressive drug treatment also caused a twofold increase in the level of extracellular adenosine but not of inosine, suggesting that the effect is not related to the general toxicity of drugs. Inhibitors of calcineurin did not inhibit adenosine uptake, suggesting that this protein phosphatase does not mediate the effect. These data demonstrate that CsA and FK506 enhance adenosine concentrations in T-lymphocytes by way of a mechanism that involves AK inhibition.
View details for DOI 10.1067/mlc.2002.125798
View details for Web of Science ID 000178086700004
View details for PubMedID 12228764
Association of DNA polymerase mu (pol mu) with Ku and ligase IV: Role for pol mu in end-joining double-strand break repair
MOLECULAR AND CELLULAR BIOLOGY
2002; 22 (14): 5194-5202
Mammalian DNA polymerase mu (pol mu) is related to terminal deoxynucleotidyl transferase, but its biological role is not yet clear. We show here that after exposure of cells to ionizing radiation (IR), levels of pol mu protein increase. pol mu also forms discrete nuclear foci after IR, and these foci are largely coincident with IR-induced foci of gammaH2AX, a previously characterized marker of sites of DNA double-strand breaks. pol mu is thus part of the cellular response to DNA double-strand breaks. pol mu also associates in cell extracts with the nonhomologous end-joining repair factor Ku and requires both Ku and another end-joining factor, XRCC4-ligase IV, to form a stable complex on DNA in vitro. pol mu in turn facilitates both stable recruitment of XRCC4-ligase IV to Ku-bound DNA and ligase IV-dependent end joining. In contrast, the related mammalian DNA polymerase beta does not form a complex with Ku and XRCC4-ligase IV and is less effective than pol mu in facilitating joining mediated by these factors. Our data thus support an important role for pol mu in the end-joining pathway for repair of double-strand breaks.
View details for DOI 10.1128/MCB.22.14.5194-5202.2002
View details for Web of Science ID 000176448400021
View details for PubMedID 12077346
Arabinosylguanine is phosphorylated by both cytoplasmic deoxycytidine kinase and mitochondrial deoxyguanosine kinase
2002; 62 (11): 3100-3105
The prodrug of 9-beta-D-arabinosylguanine (ara-G), nelarabine, demonstrated efficacy against T-cell acute lymphoblastic leukemia, and its effectiveness correlated with the accumulation of the triphosphate form (ara-GTP). Although in vitro investigations using purified deoxycytidine kinase (dCK) or deoxyguanosine kinase (dGK) suggested that ara-G is a substrate for both enzymes, controversy exists in regard to the role of these enzymes in whole cells. In this work, we used a CEM mutant cell line containing low endogenous levels of dGK and deficient in dCK (dCK-) to assess the role of these kinases in ara-G phosphorylation. Using a retroviral vector system, we infected the dCK- mutant cell line to obtain cell lines with overexpression of dCK (dCK+) or dGK (dGK+). Only the dCK+ cell line phosphorylated 1-beta-D-arabinofuranosylcytosine (used as a substrate for dCK) in a cell-free system; phosphorylation of this compound by dGK+ was below the limit of detection. Again in in vitro assays, the dCK-and dCK+ cell lines phosphorylated dGuo to similar levels (0.91 +/- 0.15 and 0.93 +/- 0.19 pmol/mg/min, respectively), whereas dGK+ phosphorylated dGuo more efficiently (150 pmol at 60 min). When ara-G was used as a substrate in a cell-free system, the maximum accumulation of phosphorylated product was observed in dGK+ extracts at low ara-G levels (10 microM) and in dCK+ extracts at high concentrations of ara-G (100 microM). Thus, both dCK and dGK can phosphorylate ara-G, but at low ara-G concentrations, dGK seems to predominate, whereas at higher ara-G concentrations, dCK seems to be the preferred enzyme. In whole-cell systems after a 3-h incubation with 10 microM ara-G, both dCK+ and dGK+ cells accumulated ara-GTP; however, the levels were significantly (P = 0.0008) higher in dGK+ cells. In contrast, at 100 microM ara-G, intracellular ara-GTP accumulated to similar levels (P = 0.5529) in these cell types; 25 +/- 3.7 microM in dCK+, and 27.8 +/- 2.7 microM in the dGK+ cells. These results from whole-cell experiments are consistent with those from the cell-free system and strongly suggest that ara-G is phosphorylated by both kinases, and at low substrate concentrations, dGK is preferred enzyme. Evaluation of the expression of each of these kinases in primary leukemia cells may reveal a biochemical basis for the pharmacological differences in the accumulation of ara-GTP.
View details for Web of Science ID 000176038500019
View details for PubMedID 12036920
Tumor uptake and elimination of 2 ',2 '-difluoro-2 '-deoxycytidine (gemcitabine) after deoxycytidine kinase gene transfer: Correlation with in vivo tumor response
CLINICAL CANCER RESEARCH
2001; 7 (10): 3263-3268
We hypothesized that tumor uptake and elimination of 2',2'-difluoro-2'-deoxycytidine/2',2'-difluoro-2'-deoxycytidine 5'-triphosphate (dFdCyd/dFdCTP) would be altered after dCK gene transfer and that this change would result in an enhanced cytotoxic effect. To test this hypothesis, we examined dFdCyd/dFdCTP uptake and clearance in HT-29 human colon carcinoma xenografts in nude mice by high-performance liquid chromatography (HPLC) and fluorine-19 magnetic resonance spectroscopy (F-19 MRS).HT-29 tumors were grown from cells infected with either the retroviral vector alone (LNPO-LacZ) or vector containing the dCK gene (LNPO-dCK). HPLC and F-19 MRS analyses were performed after a single 160 mg/kg i.p. injection of dFdCyd. Tumor response was determined in animals receiving a similar dosing schedule of dFdCyd.HPLC experiments revealed an increased tumor accumulation of dFdCTP in xenografts overexpressing dCK compared with wild-type controls (P < or = 0.05). dFdCTP in the dCK-infected tumors was easily identified at 24 h postinjection. Conversely, no dFdCTP could be detected in the control xenografts 14 h postinjection. Subsequent F-19 MRS experiments confirmed an altered uptake, revealing a 2.5-fold greater accumulation of dFdCyd/dFdCTP in the dCK xenografts. Whereas a modest tumor growth delay was observed in the wild-type tumors receiving dFdCyd, dCK xenografts demonstrated a marked tumor growth delay following treatment (P < or = 0.05).These data support the hypothesis that increased expression of dCK cDNA in HT-29 xenografts results in an enhanced dFdCTP accumulation and prolonged elimination kinetics, and ultimately a potentiated in vivo tumor response to dFdCyd. Related to these effects, changes in the overall tumor metabolism of dFdCyd/dFdCTP was detectable by noninvasive F-19 MRS. These data are relevant to future preclinical and clinical studies evaluating dCK gene transfer and dFdCyd therapy.
View details for Web of Science ID 000171574600043
View details for PubMedID 11595723
Human cytosolic 5 '-nucleotidase I - Characterization and role in nucleoside analog resistance
JOURNAL OF BIOLOGICAL CHEMISTRY
2001; 276 (13): 10498-10504
Nucleoside analogs are important in the treatment of hematologic malignancies, solid tumors, and viral infections. Their metabolism to the triphosphate form is central to their chemotherapeutic efficacy. Although the nucleoside kinases responsible for the phosphorylation of these compounds have been well described, the nucleotidases that may mediate drug resistance through dephosphorylation remain obscure. We have cloned and characterized a novel human cytosolic 5'-nucleotidase (cN-I) that potentially may have an important role in nucleoside analog metabolism. It is expressed at a high level in skeletal and heart muscle, at an intermediate level in pancreas and brain, and at a low level in kidney, testis, and uterus. The recombinant cN-I showed high affinity toward dCMP and lower affinity toward AMP and IMP. ADP was necessary for maximal catalytic activity. Expression of cN-I in Jurkat and HEK 293 cells conferred resistance to 2-chloro-2'-deoxyadenosine, with a 49-fold increase in the IC(50) in HEK 293 and a greater than 400-fold increase in the IC(50) in Jurkat cells. Expression of cN-I also conferred a 22-fold increase in the IC(50) to 2',3'-difluorodeoxycytidine in HEK 293 cells and an 82-fold increase in the IC(50) to 2',3'-dideoxycytidine in Jurkat cells. These data indicate that cN-I may play an important role in the regulation of physiological pyrimidine nucleotide pools and may also alter the therapeutic efficacy of certain nucleoside analogs.
View details for Web of Science ID 000167996400118
View details for PubMedID 11133996
Inhibition of T lymphocyte activation in mice heterozygous for loss of the IMPDH II gene
JOURNAL OF CLINICAL INVESTIGATION
2000; 106 (4): 599-606
Inosine 5'-monophosphate dehydrogenase (IMPDH) is the rate-limiting enzyme in the de novo synthesis of guanine nucleotides, which are also synthesized from guanine by a salvage reaction catalyzed by the X chromosome-linked enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT). Since inhibitors of IMPDH are in clinical use as immunosuppressive agents, we have examined the consequences of knocking out the IMPDH type II enzyme by gene targeting in a mouse model. Loss of both alleles of the gene encoding this enzyme results in very early embryonic lethality despite the presence of IMPDH type I and HPRT activities. Lymphocytes from IMPDH II(+/-) heterozygous mice are normal with respect to subpopulation distribution and respond normally to a variety of mitogenic stimuli. However, mice with an IMPDH II(+/-), HPRT(-/o) genotype demonstrate significantly decreased lymphocyte responsiveness to stimulation with anti-CD3 and anti-CD28 antibodies and show a 30% mean reduction in GTP levels in lymphocytes activated by these antibodies. Furthermore, the cytolytic activity of their T cells against allogeneic target cells is significantly impaired. These results demonstrate that a moderate decrease in the ability of murine lymphocytes to synthesize guanine nucleotides during stimulation results in significant impairment in T-cell activation and function.
View details for Web of Science ID 000088913500017
View details for PubMedID 10953035
Pharmacokinetics of nelarabine and 9-beta-D-arabinofuranosyl guanine in pediatric and adult patients during a phase I study of nelarabine for the treatment of refractory hematologic malignancies
JOURNAL OF CLINICAL ONCOLOGY
2000; 18 (5): 995-1003
To characterize the pharmacokinetics of nelarabine (506U78), the water-soluble prodrug of 9-beta-D-arabinofuranosyl guanine (ara-G), and ara-G in pediatric and adult patients with refractory hematologic malignancies. Ara-G is phosphorylated within leukemic cells to form ara-G triphosphate (ara-GTP), which acts to terminate DNA chain elongation, resulting in cell death.The pharmacokinetics of nelarabine and/or ara-G were evaluated in 71 patients (25 pediatric and 46 adult patients) on the first day of therapy. Blood was collected at specified times for the determination of plasma nelarabine and ara-G concentrations.There were no statistically significant differences in the pharmacokinetics of nelarabine between any of the groups of patients. The harmonic mean half-life (t1/2) of nelarabine in pediatric and adult patients was 14.1 minutes and 16.5 minutes, respectively. The maximum concentrations (C(max)) of ara-G occurred at or near the end of the nelarabine infusion. The C(max) of ara-G ranged from 11.6 micromol/L to 308.7 micromol/L at nelarabine doses of 5 to 75 mg/kg and was linearly related to the nelarabine dose. No statistically significant differences were noted for the pharmacokinetic parameter estimates of ara-G between adult male and female patients. In children versus adults, the dose-normalized C(max), time of the C(max), and the steady-state volume of distribution of ara-G were similar. However, the clearance of ara-G was higher in pediatric patients (0.312 L.h(-1).kg(-1)) as compared with adult patients (0. 213 L.h(-1).kg(-1)) (P <.001). The t1/2 of ara-G was shorter in pediatric patients as compared with adult patients (2.1 hours v 3.0 hours; P <.01).Nelarabine is an effective prodrug of ara-G, allowing systemic concentrations of ara-G that result in clinical activity.
View details for Web of Science ID 000085586000008
View details for PubMedID 10694549
Association of terminal deoxynucleotidyl transferase with Ku
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1999; 96 (24): 13926-13931
Terminal deoxynucleotidyl transferase (TdT) catalyzes the addition of nucleotides at the junctions of rearranging Ig and T cell receptor gene segments, thereby generating antigen receptor diversity. Ku is a heterodimeric protein composed of 70- and 86-kDa subunits that binds DNA ends and is required for V(D)J recombination and DNA double-strand break (DSB) repair. We provide evidence for a direct interaction between TdT and Ku proteins. Studies with a baculovirus expression system show that TdT can interact specifically with each of the Ku subunits and with the heterodimer. The interaction between Ku and TdT is also observed in pre-T cells with endogenously expressed proteins. The protein-protein interaction is DNA independent and occurs at physiological salt concentrations. Deletion mutagenesis experiments reveal that the N-terminal region of TdT (131 amino acids) is essential for interaction with the Ku heterodimer. This region, although not important for TdT polymerization activity, contains a BRCA1 C-terminal domain that has been shown to mediate interactions of proteins involved in DNA repair. The induction of DSBs in Cos-7 cells transfected with a human TdT expression construct resulted in the appearance of discrete nuclear foci in which TdT and Ku colocalize. The physical association of TdT with Ku suggests a possible mechanism by which TdT is recruited to the sites of DSBs such as V(D)J recombination intermediates.
View details for Web of Science ID 000083872900061
View details for PubMedID 10570175
Tissue-specific regulation of the ecto-5 '-nucleotidase promoter - Role of the cAMP response element site in mediating repression by the upstream regulatory region
JOURNAL OF BIOLOGICAL CHEMISTRY
1999; 274 (32): 22705-22712
We have isolated the 5' region of the ecto-5'-nucleotidase (low K(m) 5'-NT) gene and established that a 969-base pair (bp) fragment confers cell-specific expression of a CAT reporter gene that correlates with the expression of endogenous ecto-5'-NT mRNA and enzymatic activity. A 768-bp upstream negative regulatory region has been identified that conferred lymphocyte-specific negative regulation in a heterologous system with a 244-bp deoxycytidine kinase core promoter. DNase I footprinting identified several protected areas including Sp1, Sp1/AP-2, and cAMP response element (CRE) binding sites within the 201-bp core promoter region and Sp1, NRE-2a, TCF-1/LEF-1, and Sp1/NF-AT binding sites in the upstream regulatory region. Whereas the CRE site was essential in mediating the negative activity of the upstream regulatory region in Jurkat but not in HeLa cells, mutation of the Sp1/AP-2 site decreased promoter activity in both cell lines. Electrophoretic mobility shift assay analysis of proteins binding to the CRE site identified both ATF-1 and ATF-2 in Jurkat cells. Finally, phorbol 12-myristate 13-acetate increased the activity of both the core and the 969-bp promoter fragments, and this increase was abrogated by mutations at the CRE site. In summary, we have identified a tissue-specific regulatory region 5' of the ecto-5'-NT core promoter that requires the presence of a functional CRE site within the basal promoter for its suppressive activity.
View details for Web of Science ID 000081868400083
View details for PubMedID 10428853
Nucleotide pool imbalance and adenosine deaminase deficiency induce alterations of N-region insertions during V(D)J recombination
JOURNAL OF CLINICAL INVESTIGATION
1999; 103 (6): 833-841
Template-independent nucleotide additions (N regions) generated at sites of V(D)J recombination by terminal deoxynucleotidyl transferase (TdT) increase the diversity of antigen receptors. Two inborn errors of purine metabolism, deficiencies of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP), result in defective lymphoid development and aberrant pools of 2'-deoxynucleotides that are substrates for TdT in lymphoid precursors. We have asked whether selective increases in dATP or dGTP pools result in altered N regions in an extrachromosomal substrate transfected into T-cell or pre-B-cell lines. Exposure of the transfected cells to 2'-deoxyadenosine and an ADA inhibitor increased the dATP pool and resulted in a marked increase in A-T insertions at recombination junctions, with an overall decreased frequency of V(D)J recombination. Sequence analysis of VH-DH-JH junctions from the IgM locus in B-cell lines from ADA-deficient patients demonstrated an increase in A-T insertions equivalent to that found in the transfected cells. In contrast, elevation of dGTP pools, as would occur in PNP deficiency, did not alter the already rich G-C content of N regions. We conclude that the frequency of V(D)J recombination and the composition of N-insertions are influenced by increases in dATP levels, potentially leading to alterations in antigen receptors and aberrant lymphoid development. Alterations in N-region insertions may contribute to the B-cell dysfunction associated with ADA deficiency.
View details for Web of Science ID 000079203100010
View details for PubMedID 10079104
ATP and phosphate reciprocally affect subunit association of human recombinant High Km 5 '-nucleotidase - Role for the C-terminal polyglutamic acid tract in subunit association and catalytic activity
EUROPEAN JOURNAL OF BIOCHEMISTRY
1999; 259 (3): 851-858
IMP-specific, High Km 5'-nucleotidase (EC 220.127.116.11) is an ubiquitous enzyme, the activity of which is highly regulated by substrate, ATP, and inorganic phosphate. The cDNA encoding this enzyme has recently been cloned and found to contain a unique stretch of nine glutamic and four aspartic acid residues at the C-terminus. To study the effects of this acidic tail, and of ATP and inorganic phosphate on enzyme function, we generated several structural modifications of the 5'-nucleotidase cDNA, expressed the corresponding proteins in Escherichia coli and compared their molecular and kinetic properties. As with the enzyme purified from human placenta, all recombinant proteins were activated by ATP and inhibited by inorganic phosphate. Although the S0.5-values were higher, the specific activities of the purified protein variants (except that truncated at the C-terminus) were similar. The molecular mass of the full-length enzyme subunit has been estimated at 57.3 kDa and the molecular mass of the native protein, as determined by gel-filtration chromatography, was estimated to be 195 kDa. Increasing the concentration of NaCl to 0.3 M promoted oligomerization of the protein and the formation of aggregates of 332 kDa. ATP induced further oligomerization to 715 kDa, while inorganic phosphate reduced the estimated molecular mass to 226 kDa. In contrast to the truncation of 30 amino acids at the N-terminus, which did not alter enzyme properties, the removal of the polyglutamic/aspartic acid tail of 13 residues at the C-terminus caused profound kinetic and structural changes, including a 29-fold decrease in specific activity and a significant increase in the sensitivity to inhibition by inorganic phosphate in the presence of AMP. Structurally, there was a dramatic loss of the ability to form oligomers at physiological salt concentration which was only partially restored by the addition of NaCl or ATP. These data suggest an important function of the polyglutamic acid tract in the process of association and dissociation of 5'-nucleotidase subunits.
View details for Web of Science ID 000078737000037
View details for PubMedID 10092873
The use of the lectin Helix pomatia agglutinin (HPA) as a prognostic indicator and as a tool in cancer research
HISTOLOGY AND HISTOPATHOLOGY
1999; 14 (1): 217-226
Progress in treatment for cancer has enabled extension of the disease-free interval, and of the quality of life for patients, but there has been very little improvement in overall survival rates. The main reason for this has been the ineffectiveness of current therapies to kill all the cancer cells once they have spread to distant sites to form metastatic deposits. One marker which has proved to be useful in identifying those cancers which have the potential to spread is the lectin Helix pomatia agglutinin (HPA). In clinical studies, HPA binding to primary tumours in tissue sections has been of prognostic value in breast, colon and gastric cancer, while no prognostic significance for HPA could be detected in tumours of the head and neck. These studies hence indicate that HPA is best suited to recognise a glycotope on adenocarcinomas. In several studies, HPA reactivity is equal or superior to other classical markers of metastatic potential. Since HPA is a marker of prognosis at the level of individual tumour cells, human tumour cell lines were screened for their HPA positivity. When transplanted into severe combined immunodeficient (scid) mice, HPA positive human breast and colon cancer cells metastasised while HPA negative cancer cell lines in general did not. In order to define HPA binding glycotopes at the molecular level, isolated cell membrane glycoproteins were exposed to labelled HPA on nitrocellulose membranes after Western blotting procedure. The majority of the isolated cell membrane glycoproteins bound HPA indicating that not a single HPA binding glycoprotein exists, which is associated with the metastatic phenotype. Functional investigations using the human/scid mouse chimeras will aid in the identification of those HPA positive glycoproteins which are functionally involved in the metastatic cascade.
View details for Web of Science ID 000078017300023
View details for PubMedID 9987666
Compound GW506U78 in refractory hematologic malignancies: Relationship between cellular pharmacokinetics and clinical response
JOURNAL OF CLINICAL ONCOLOGY
1998; 16 (11): 3607-3615
In vitro investigations with arabinosylguanine (ara-G) demonstrated potent cytotoxicity to T-lymphoblastoid cell lines. The goals of the present study were to evaluate GW506U78, a prodrug of ara-G, against human hematologic malignancies and to determine its pharmacokinetics in plasma and cells.During a phase I multicenter trial of GW506U78, 26 patients were treated at M.D. Anderson Cancer Center (MDACC). Daily doses between 20 and 60 mg/kg were administered for 5 days. Parallel plasma and cellular pharmacokinetic studies were conducted.Complete (n=5) or partial remission (n=5) was achieved in T-cell acute lymphoblastic leukemia (T-ALL), T-lymphoid blast crisis, T-lymphoma, and B-cell chronic lymphocytic leukemia (B-CLL) (n=13). In contrast, patients with B-ALL, B-lymphoma, acute myelogenous leukemia (AMI), or T-CLL did not respond. Peak plasma concentrations of GW506U78 and ara-G were dose-dependent. The elimination of GW506U78 (half-life [t1/2]=17 minutes) was faster than the elimination of ara-G (t1/2=3.7 hours). Median peak concentrations of ara-GTP were 23, 42, 85, and 93 micromol/L at 20, 30, 40, and 60 mg/kg, respectively. T-lymphoblasts accumulated significantly (P=.0008) higher peak arabinsylguanosine triphosphate (ara-GTP) (median, 140 micromol/L; n=7) compared with other diagnoses (median, 50 micromol/L; n=9) and normal mononuclear cells (n=3). The ara-GTP elimination was slow in all diagnoses (median, > 24 hours). Responders accumulated significantly (P=.0005) higher levels of ara-GTP (median, 157 micromol/L) compared with patients who failed to respond (median, 44 micromol/L).GW506U78 is an effective prodrug and a potent agent for hematologic malignancies with major efficacy in T-cell diseases. The pharmacokinetics of ara-GTP in leukemia cells are strongly correlated with clinical responses to GW506U78.
View details for Web of Science ID 000076742000020
View details for PubMedID 9817282
Effects of guanine nucleotide depletion on cell cycle progression in human T lymphocytes
1998; 91 (8): 2896-2904
Depletion of guanine nucleotide pools after inhibition of inosine monophosphate dehydrogenase (IMPDH) potently inhibits DNA synthesis by arresting cells in G1 and has been shown to induce the differentiation of cultured myeloid and erythroid cell lines, as well as chronic granulocytic leukemic cells after blast transformation. Inhibitors of IMPDH are also highly effective as immunosuppressive agents. The mechanism underlying these pleiotropic effects of depletion of guanine nucleotides is unknown. We have examined the effects of mycophenolic acid (MPA), a potent IMPDH inhibitor, on the cell cycle progression of activated normal human T lymphocytes. MPA treatment resulted in the inhibition of pRb phosphorylation and cell entry into S phase. The expression of cyclin D3, a major component of the cyclin-dependent kinase (CDK) activity required for pRb phosphorylation, was completely abrogated by MPA treatment of T cells activated by interleukin-2 (IL-2) and leucoagglutinin (PHA-L), whereas the expression of cyclin D2, CDK6, and CDK4 was more mildly attenuated. The direct kinase activity of a complex immunoprecipitated with anti-CDK6 antibody was also inhibited. In addition, MPA prevented the IL-2-induced elimination of p27(Kip1), a CDK inhibitor, and resulted in the retention of high levels of p27(Kip1) in IL-2/PHA-L-treated T cells bound to CDK2. These results indicate that inhibition of the de novo synthesis of guanine nucleotides blocks the transition of normal peripheral blood T lymphocytes from G0 to S phase in early- to mid-G1 and that this cell cycle arrest results from inhibition of the induction of cyclin D/CDK6 kinase and the elimination of p27(Kip1) inhibitory activity.
View details for Web of Science ID 000073012000032
View details for PubMedID 9531600
Lectin histochemistry of human leukaemic mast cells (HMC-1) transplanted into severe combined immunodeficient (scid) mice
1998; 100 (1): 1-9
It is difficult to isolate and impossible to propagate human mast cells in tissue culture. As an alternative to the use of human differentiated mast cells, a human leukaemic mast cell line (HMC-1), which can be propagated in vitro, has been employed in a number of studies. Carbohydrate binding proteins, lectins, have been used to characterise the terminal sugar residues of human mast cells in situ. The aim of the present study is to characterise the lectin binding sites of HMC-1 cells transplanted into severe combined immunodeficient (scid) mice. Lectins specific for the complex carbohydrates, neuraminic acid and N-acetylglucosamine residues showed generally a strong uniform binding pattern, whereas mannose and glucose specific yielded lectins a greater heterogeneity. This glycotope expression pattern has some similarities with those of human mast cells in situ, and therefore HMC-1 cells grown in scid mice constitute a valuable model system for the study of carbohydrate expression in human mast cells.
View details for Web of Science ID 000072279800001
View details for PubMedID 9542577
- Effects of guanine nucleotide depletion on cell cycle progression in human T lymphocytes Blood 1998: 2896-2904
Inosine-5 '-monophosphate dehydrogenase: Regulation of expression and role in cellular proliferation and T lymphocyte activation
PROGRESS IN NUCLEIC ACID RESEARCH AND MOLECULAR BIOLOGY, VOL 61
1998; 61: 181-209
Guanine nucleotide synthesis is essential for the maintenance of normal cell growth and function, as well as for cellular transformation and immune responses. The expression of two genes encoding human inosine-5'-monophosphate dehyrogenase (IMPDH) type I and type II results in the translation of catalytically indistinguishable enzymes that control the rate-limiting step in the de novo synthesis of guanine nucleotides. Cellular IMPDH activity is increased more than 10-fold in activated peripheral blood T lymphocytes and is attributable to the increased expression of both the type I and type II enzymes. In contrast, abrogation of cellular IMPDH activity by selective inhibitors prevents T lymphocyte activation and establishes a requirement for elevated IMPDH activity in T lymphocytic responses. In order to assess the molecular mechanisms governing the expression of the IMPDH type I and type II genes in resting and activated peripheral blood T lymphocytes, we have cloned the human IMPDH type I and type II genes and characterized their genomic organization and their respective 5'-flanking regions. Both genes contain 14 highly conserved exons that vary in size from 49 to 207 base pairs. However, the intron structures are completely divergent, resulting in disparities in gene length (18 kilobases for type I and 5.8 kilobases for type II). In addition, the 5'-regulatory sequences are highly divergent; expression of the IMPDH type I gene is controlled by three distinct promoters in a tissue specific manner while the type II gene is regulated by a single promoter and closely flanked in the 5' region by a gene of unknown function. The conservation of the IMPDH type I and type II coding sequence in the presence of highly divergent 5'-regulatory sequences points to a multifactorial control of enzyme expression and suggests that tissue-specific and/or developmentally specific regulation of expression may be important. Delineation of these regulatory mechanisms will aid in the elucidation of the signaling events that ultimately lead to the synthesis of guanine nucleotides required for cellular entry into S phase and the initiation of DNA replication.
View details for Web of Science ID 000079436100005
View details for PubMedID 9752721
Regulation of inosine-5'-monophosphate dehydrogenase type II gene expression in human T cells - Role for a novel 5' palindromic octamer sequence
JOURNAL OF BIOLOGICAL CHEMISTRY
1997; 272 (36): 22913-22923
Expression of the gene encoding human inosine- 5'-monophosphate dehydrogenase (IMPDH) type II, an enzyme catalyzing the rate-limiting step in the generation of guanine nucleotides, is increased more than 10-fold in activated peripheral blood T lymphocytes and is required for T cell activation. We have examined the 5'-regulatory sequences that are important for the transcriptional regulation of this gene in T cells. DNase I mapping of genomic DNA identified a hypersensitive element near the transcription initiation site. Fine mapping by in vivo footprinting demonstrated five transcription factor binding sites that are occupied in both resting and activated peripheral blood T lymphocytes; these are tandem CRE motifs, a Sp1 site, an overlapping Egr-1/Sp1 site, and a novel palindromic octamer sequence (POS). The tandem CRE and POS sites are of major functional importance as judged by mutational and electrophoretic mobility shift analyses. These data provide evidence that expression of the human IMPDH type II gene is predominantly regulated by the nuclear factors ATF-2 and an as yet unidentified POS-binding protein. Additional major protein-DNA interactions do not occur within the promoter region after T lymphocyte activation, indicating a requirement for additional protein-protein interactions and/or post-translational modifications of pre-bound transcription factors to account for the observed increase in IMPDH type II gene expression.
View details for Web of Science ID A1997XV49200076
View details for PubMedID 9278455
Cellular delivery of nucleoside diphosphates: A prodrug approach
1997; 14 (6): 706-712
This study is concerned with cellular delivery/generation of 2'-azido-2'-deoxyuridine and -deoxycytidine diphosphate (N3UDP or N3CDP), potent inhibitors of ribonucleotide reductase. It characterizes the phosphorylation steps involved in the conversion of 2'-azido-2'-deoxyuridine (N3Urd) and 2'-azido-2'-deoxycytidine (N3Cyd) to the corresponding diphosphates and explores a prodrug approach in cellular delivery of the inhibitor which circumvents the requirement of deoxynucleoside kinases.Cell growth of CHO and 3T6 cells of known deoxycytidine kinase level was determined in the presence of N3Urd and N3Cyd. Activity of ribonucleotide reductase was determined in the presence of the azidonucleosides as well as their mono- or di-phosphates in a Tween 80-containing permeabilizing buffer. A prodrug of 5'-monophosphate of N3Urd was prepared and its biological activity was evaluated with CHO cells as well as with cells transfected with deoxycytidine kinase.N3Urd failed to inhibit the growth of both cell lines, while N3Cyd was active against 3T6 cells and moderately active against CHO cells. These results correlate with the deoxycytidine kinase levels found in the cells. Importance of the kinase was further established with the finding that the nucleoside analogs were inactive as reductase inhibitors in a permeabilized cell assay system while their mono- and di-phosphates were equally active. The prodrug was active in cell growth inhibition regardless of the deoxycytidine kinase level.The azidonucleosides become potent inhibitors of the reductase by two sequential phosphorylation steps. The present study indicates that the first step to monophosphate is rate-limiting, justifying a prodrug approach with the monophosphate.
View details for Web of Science ID A1997XH04100003
View details for PubMedID 9210185
Adenosine metabolism during phorbol myristate acetate-mediated induction of HL-60 cell difference - Changes in expression pattern of adenosine kinase, adenosine deaminase, and 5'-nucleotidase
JOURNAL OF IMMUNOLOGY
1997; 158 (10): 4947-4952
Adenosine has potent immunosuppressive activity. Since the source of adenosine and the mechanism of its release in the immune system is largely unknown and may vary according to cell type, we have evaluated the relationship between adenosine metabolism and the enzymatic activities and mRNA levels of adenosine-metabolizing enzymes in myeloid and lymphoid cell lines. Induction of HL-60 cell differentiation along the macrophage lineage by PMA resulted in a reduction in the activities of adenosine deaminase (ADA), adenosine kinase (AK), and inosine monophosphate-specific cytosolic 5'-nucleotidase and an elevation of ecto-5'-nucleotidase (ecto-5'-NT). These changes were accompanied by an elevation of ecto-5'-NT mRNA and a decrease in ADA and AK mRNAs in a time-dependent fashion. Comparison of AK and ADA mRNA levels in several other leukemic cell lines revealed generally similar responses to PMA with much stronger suppression in immature T cells than in B cells. The metabolism of adenosine either through phosphorylation (AK) or deamination (ADA) was reduced in PMA-stimulated cells. Furthermore, the cumulative changes in enzyme expression resulted in a 2.5-fold increase in intracellular adenosine formation in PMA-stimulated cells. The inhibition of AK by 5'-iodotubercidin further increased adenosine formation by 6-fold over that in untreated cells. In accord with the increase in ecto-5'-NT activity, extracellular AMP dephosphorylation increased dramatically, but there was no increase in extracellular ATP degradation. These results indicate that a coordinated shift in adenosine-metabolizing enzyme levels during PMA-induced HL-60 cell differentiation is accompanied by a decrease in adenosine uptake and an increase in adenosine release.
View details for Web of Science ID A1997WX98700053
View details for PubMedID 9144513
Regulation of the human inosine monophosphate dehydrogenase type I gene - Utilization of alternative promoters
JOURNAL OF BIOLOGICAL CHEMISTRY
1997; 272 (7): 4458-4466
Catalysis of guanine nucleotide formation from IMP in the de novo purine synthetic pathway is carried out by two isoforms of the enzyme inosine monophosphate dehydrogenase (IMPDH) that are catalytically indistinguishable but are encoded by separate genes. In order to assess the potential for cell type-specific expression of IMPDH activity, we have characterized the IMPDH type I gene and identified three major RNA transcripts that are differentially expressed from three different promoters. A 4.0-kilobase pair (kb) mRNA containing 1.3 kb of 5'-untranslated region is expressed in activated peripheral blood lymphocytes and to a far lesser extent in cultured tumor cell lines. The P1 promoter that regulates the transcription of this mRNA has a high degree of sequence identity to an Alu repetitive sequence. A transcript of 2.7 kb is found in a subset of the tumor cell lines examined, whereas a 2.5-kb mRNA species is universally expressed and is the prevalent mRNA in most cell lines and tissues. The relative strengths of the three promoter regions and the effects of variable extents of 5'-flanking sequence on the P3 promoter differ in Jurkat T, as compared with Raji B lymphoid cell lines, demonstrating a complex cell type-specific transcriptional regulation of IMPDH type I gene expression.
View details for Web of Science ID A1997WH01900086
View details for PubMedID 9020170
Retroviral transfer of deoxycytidine kinase into tumor cell lines enhances nucleoside toxicity
1996; 56 (10): 2343-2347
Deoxycytidine kinase (dCK) phosphorylates a number of nucleoside analogues that are useful in the treatment of various malignancies. Although the level of dCK activity in malignant cells is thought to correlate with chemotherapeutic response, no direct data are available to support this assumption. We have tested this hypothesis by infecting three tumor cell lines, MCF-7, HT-29, and H1437, with the retroviral vector LNPO containing either dCK or LacZ cDNA and measuring the corresponding sensitivity to nucleoside analogues. DCK activity was increased by 1.7-, 2.3-, and 16-fold in MCF-7, HT-29, and H1437 cells, respectively. Northern and Western blots demonstrated a similar increase in mRNA and protein levels. As a result of dCK expression, MCF-7 cells demonstrated a 2.5-fold increase in drug sensitivity to 1-beta-D-arabinofuranosylcytosine (AraC) and 2-chloro-2'-deoxyadenosine (CdA). HT-29 cells had a 7-fold increase in sensitivity to AraC, CdA, and 2-fluoro-9-beta-D-arabinofuranosyladenine, whereas H1437 cells demonstrated a 20- to 106-fold increase. For all three drugs, there was a linear relationship between dCK activity in clonally derived cell lines and IC50s. These data demonstrate a direct effect of dCK activity on drug sensitivity in cell lines. Because many tumors have relatively low levels of dCK, it is possible that dCK gene transfer will be a useful adjunct to the treatment of these malignancies.
View details for Web of Science ID A1996UK18700021
View details for PubMedID 8625309
Viral vector transduction of the human deoxycytidine kinase cDNA sensitizes glioma cells to the cytotoxic effects of cytosine arabinoside in vitro and in vivo
1996; 2 (5): 567-573
Cytosine arabinoside (ara-C) is a cytidine analog that incorporates into replicating DNA and induces lethal DNA strand breaks. Although ara-C is a potent antitumor agent for hematologic malignancies, it has only minimal activity against most solid tumors. The rate-limiting step in intracellular ara-C activation is phosphorylation of the prodrug by deoxycytidine kinase (dCK). The present results demonstrate that both retroviral and adenoviral vector-mediated transduction of the dCK cDNA results in marked sensitization of glioma cells lines to the cytotoxic effects of ara-C in vitro. We also demonstrate that ara-C treatment of established intradermal and intracerebral gliomas transduced with dCK results in significant antitumor effects in vivo. These data suggest that viral vector transduction of the dCK gene followed by treatment with ara-C represents a new chemosensitization strategy for cancer gene therapy.
View details for Web of Science ID A1996UJ23000041
View details for PubMedID 8616717
Cloning of human adenosine kinase cDNA: Sequence similarity to microbial ribokinases and fructokinases
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1996; 93 (3): 1232-1237
Adenosine kinase catalyzes the phosphorylation of adenosine to AMP and hence is a potentially important regulator of extracellular adenosine concentrations. Despite extensive characterization of the kinetic properties of the enzyme, its primary structure has never been elucidated. Full-length cDNA clones encoding catalytically active adenosine kinase were obtained from lymphocyte, placental, and liver cDNA libraries. Corresponding mRNA species of 1.3 and 1.8 kb were noted on Northern blots of all tissues examined and were attributable to alternative polyadenylylation sites at the 3' end of the gene. The encoding protein consists of 345 amino acids with a calculated molecular size of 38.7 kDa and does not contain any sequence similarities to other well-characterized mammalian nucleoside kinases, setting it apart from this family of structurally and functionally related proteins. In contrast, two regions were identified with significant sequence identity to microbial ribokinase and fructokinases and a bacterial inosine/guanosine kinase. Thus, adenosine kinase is a structurally distinct mammalian nucleoside kinase that appears to be akin to sugar kinases of microbial origin.
View details for Web of Science ID A1996TU64000051
View details for PubMedID 8577746
Inosine monophosphate dehydrogenase expression: Transcriptional regulation of the type I and type II genes
36th Symposium on Regulation of Enzyme Activity and Synthesis in Normal and Neoplastic Tissues
PERGAMON PRESS LTD. 1996: 75–84
Inosine 5'-monophosphate dehydrogenase (IMPDH) is an essential rate-limiting enzyme in the de novo guanine nucleotide synthetic pathway that catalyzes the conversion of IMP to XMP. Enzyme activity is accounted for by the expression of two distinct but closely related genes termed IMPDH I and II. Increased IMPDH activity has been linked to both cellular proliferation and neoplastic transformation and generally ascribed to an increase in the expression of the type II gene. We have characterized the type I and type II genes and identified elements important in the transcriptional regulation of both genes. The type II IMPDH gene contains a 466 bp 5' flanking region spanning the translation start site that contains several transcription factor binding sites and mediates increased transcription of a CAT reporter gene in peripheral blood T lymphocytes when these cells are induced to proliferate. The single functional IMPDH type I gene contains exon-intron boundaries and exon structures that are nearly identical to those in the type II gene. In contrast to the type II gene, however, it contains two putative promoter sites, each with the potential for transcriptional regulation. We conclude that these two genes most probably arose from an early gene duplication event and that their highly conserved structures and differential regulation at the transcriptional level argue strongly for a significant role for each gene in cellular metabolism, growth, and differentiation.
View details for Web of Science ID A1996BG03K00005
View details for PubMedID 8869741
- Transcriptional regulationof the IMPDH type I and II genes Adv Enzyme Regul 1996: 75-84
- A 73 year-old woman with anemia and thrombocytopenia J Am Ger Soc 1996: 723-726
RECOMBINANT HUMAN INOSINE MONOPHOSPHATE DEHYDROGENASE TYPE-I AND TYPE-II PROTEINS - PURIFICATION AND CHARACTERIZATION OF INHIBITOR BINDING
1995; 49 (9): 1323-1329
Inosine monophosphate dehydrogenase (IMPDH) activity results from the expression of two separate genes, and the resulting proteins (type I and type II) are 84% identical at the amino acid level. Although the type II mRNA is expressed at higher levels in proliferating cells, both mRNAs, and by extrapolation both proteins, are present in normal and malignant cells. Since IMPDH is an important target for the development of drugs with both chemotherapeutic and immunosuppressive activity, we have compared the kinetic and physical properties of the two human enzymes expressed in and purified from Escherichia coli. Type I and II IMPDH had kcat values of 1.8 and 1.4 sec-1, respectively, with Km values for IMP of 14 and 9 microM and Km values for NAD of 42 and 32 microM. The two enzymes were inhibited competitively by the immunosuppressive agent mizoribine 5'-monophosphate (MMP) with Ki values of 8 and 4 nM and inhibited uncompetitively by mycophenolic acid with Ki values of 11 and 6 nM. The association of MMP to either isozyme, as monitored by fluorescence quenching, was relatively slow with kon values of 3-8 x 10(4) M-1 sec-1 and koff values of 3 x 10(-4) sec-1 (half-lives of 36-43 min). Thus, MMP is a potent, tight-binding competitive inhibitor that does not discriminate between the two IMPDH isozymes.
View details for Web of Science ID A1995QZ29800018
View details for PubMedID 7763314
GTP DEPLETION INDUCED BY IMP DEHYDROGENASE INHIBITORS BLOCKS RNA-PRIMED DNA-SYNTHESIS
1995; 47 (5): 948-955
Inhibitors of IMP dehydrogenase (EC 18.104.22.168), including mizoribine (Bredinin) and mycophenolic acid, have significant antitumor and immunosuppressive activities. Studies were aimed at determining the mechanism by which intracellular GTP depletion induced by these agents results in inhibition of DNA synthesis. Incubation of human CEM leukemia cells for 2 hr with IC50 concentrations of either mizoribine (4 microM) or mycophenolic acid (0.5 microM) reduced cellular GTP levels an average of 68% or 58%, respectively, compared with the levels in control cells. Under similar conditions, mizoribine and mycophenolic acid decreased the amount of [3H]adenosine incorporated into primer RNA by 75% and 70%, respectively, relative to the untreated controls, but had no significant effect on total RNA synthesis. Repletion of the guanine nucleotide pools by coincubation of CEM cells with guanosine plus 8-aminoguanosine prevented both the inhibition of primer RNA synthesis and the inhibition of tumor cell growth induced by these agents. Additional studies demonstrated that GTP depletion alone was capable of directly inducing inhibition of primer RNA synthesis. Primer RNA synthesis was inhibited an average of 84% in whole-cell lysates that lacked GTP but contained all remaining ribo- and deoxyribonucleoside triphosphates. On an M13 DNA template, RNA-primed DNA synthesis catalyzed by the purified complex of DNA primase (EC 22.214.171.124) and DNA polymerase alpha (EC 126.96.36.199) was decreased an average of 70% in the absence of GTP, compared with synthesis in the presence of 0.5 mM GTP. These results provide evidence that mizoribine and mycophenolic acid inhibit DNA replication by inducing GTP depletion, which suppresses the synthesis of RNA-primed DNA intermediates.
View details for Web of Science ID A1995QX97900009
View details for PubMedID 7746281
CHARACTERIZATION OF THE DEOXYCYTIDINE KINASE PROMOTER IN HUMAN LYMPHOBLAST CELL-LINES
JOURNAL OF CLINICAL INVESTIGATION
1995; 95 (4): 1660-1668
Deoxycytidine kinase (dCK) phosphorylates 2'-deoxycytidine, as well as the purine deoxyribonucleosides and a number of nucleoside analogues that are important in the chemotherapy of leukemias. The enzyme is highly expressed in the thymus relative to other tissues and may play an important role in the T cell depletion associated with adenosine deaminase and purine nucleoside phosphorylase deficiencies. To characterize the dCK promoter region and to determine whether it mediates higher levels of gene expression in T lymphoblasts, we have analyzed a 700-bp genomic fragment encompassing 548 bp of 5' flanking region for functional activity and for transcription factor binding using T and B lymphoblast cell lines and nuclear extracts. The regions of the promoter that were defined as important to its function include a 5' GC box, and E box, a 3' GC box, and an E2F site. The transcription factor Sp1 binds to both GC boxes, activating at the 5' site but repressing at the 3' site. MLTF/USF activates transcription through the E box, whereas E2F activates through the E2F site, but binds weakly to this site in vitro and does not appear to mediate cell cycle-specific expression of dCK in vivo. No significant differences in promoter activity or transcription factor binding were observed between Jurkat T and Raji B lymphoblasts. The promoter of the dCK gene is thus regulated by a number of ubiquitously expressed transcription factors. DCK expression in cultured lymphoblast cell lines is not solely a function of the T or B lineage derivation.
View details for Web of Science ID A1995QQ59600032
View details for PubMedID 7706474
CHARACTERIZATION OF THE HUMAN INOSINE-5'-MONOPHOSPHATE DEHYDROGENASE TYPE-II GENES
JOURNAL OF BIOLOGICAL CHEMISTRY
1995; 270 (12): 6808-6814
Inosine-5'-monophosphate dehydrogenase (IMPDH) activity and mRNA levels are induced up to 15-fold upon mitogenic or antigenic stimulation of human peripheral blood T lymphocytes. This increase in IMPDH activity is required for cellular proliferation and has been associated with malignant transformation. We have cloned the human IMPDH type II gene and show that it contains 14 exons and is approximately 5.8 kilobases in length. Exons vary in size from 49 to 207 base pairs and introns from 73 to 1065 base pairs. The transcription start site was mapped to a position 50 nucleotides upstream of the translation initiation site. The 5'-flanking region consisting of 463 base pairs upstream of the translation initiation site confers induced transcription and differential regulation upon a chloramphenicol acetyltransferase reporter gene when transfected into Jurkat T cells and human peripheral blood T lymphocytes, respectively. DNase I footprinting analysis using Jurkat T cell nuclear extract identified four protected regions in the promoter which coincide with consensus transcription factor binding sites for the nuclear factors AP2, ATF, CREB, Egr-1, Nm23, and Sp1. These findings suggest that several of these nuclear factors may play a critical role in the regulation of IMPDH type II gene expression during T lymphocyte activation.
View details for Web of Science ID A1995QQ85500058
View details for PubMedID 7896827
ASSIGNMENT OF THE HUMAN TYPE-I IMP DEHYDROGENASE GENE (IMPDH1) TO CHROMOSOME 7Q31.3-Q32
1994; 24 (1): 179-181
Two phage lambda clones that contain the 5' portion of the human type I inosine 5'-monophosphate dehydrogenase (IMPDH, EC 188.8.131.52) gene were isolated. Both polymerase chain reaction analysis of a panel of human-mouse and human-hamster cell somatic hybrids using primers specific for the type I IMPDH gene and fluorescence in situ hybridization with metaphase human chromosomes using type I IMPDH genomic DNA as probes indicate that the type I IMPDH gene (symbol IMPDH1) is located on chromosome 7. Sequential GTG-banding was performed to assign the band location of the type I IMPDH gene to chromosome 7q31.3-q32.
View details for Web of Science ID A1994PT47300024
View details for PubMedID 7896275
HEREDITARY OVEREXPRESSION OF ADENOSINE-DEAMINASE IN ERYTHROCYTES - STUDIES IN ERYTHROID CELL-LINES AND TRANSGENIC MICE
1994; 84 (7): 2346-2353
Overexpression of adenosine deaminase (ADA) in red blood cells is characterized by a marked, tissue-specific increase in levels of structurally normal ADA mRNA and enzymatic activity in the erythrocytes of affected individuals, leading to adenosine triphosphate (ATP) depletion and hemolytic anemia. This autosomal dominant trait is linked to the ADA gene. To investigate the molecular mechanism responsible for this disorder, we examined relative reporter gene activity using constructs containing 10.6 kb of 5' flanking sequence and 12.3 kb of the first intron of the ADA gene from the normal and mutant alleles. No differences in chloramphenicol acetyltransferase (CAT) activity were found in transient transfection experiments using erythroleukemia cell lines. Transgenic mice containing the ADA constructs expressed CAT in the appropriate tissue-specific fashion, with 10(2)- to 10(4)-fold higher activity in the thymus. However, CAT activities in erythrocytes and bone marrow of mice containing high transgene copy numbers did not differ between the normal and mutant alleles. These results indicate that the mutation responsible for ADA overexpression is unlikely to reside in the 5' and promoter regions or in the regulatory regions of the first intron. It is possible that the erythroid-specific overexpression of ADA results from a mutation at some distance from the gene or requires an interaction of a proximal mutation with more distal DNA elements.
View details for Web of Science ID A1994PK12500037
View details for PubMedID 7919352
STRUCTURAL-ANALYSIS OF THE DEOXYCYTIDINE KINASE GENE IN PATIENTS WITH ACUTE MYELOID-LEUKEMIA AND RESISTANCE TO CYTOSINE-ARABINOSIDE
1994; 8 (5): 780-785
Deficiency of deoxycytidine kinase (dCK) activity represents one possible cause of resistance to cytosine arabinoside (ara-C). Mutations of the dCK gene have recently been shown to be responsible for dCK deficiency and increased resistance in vitro. In order to define the relevance of this mechanism in vivo, we analyzed the dCK gene in 16 adult patients with relapsed/refractory acute myeloid leukemia (AML) and clinical resistance to standard-dose and/or high-dose ara-C. Southern blot analysis using genomic DNA from peripheral blood or bone marrow samples containing > or = 70% leukemic blasts and agarose gel electrophoresis of cDNA obtained by RT-PCR did not reveal gross rearrangements of the dCK gene. Sequencing of the dCK coding region showed point mutations in seven patients. Besides two silent mutations (or RFLPs) in codon 42 and 86, base pair mutations resulting in amino acid replacements were found in five patients affecting codon 20, 93, 98, 99, and 154, respectively. dCK cDNA clones from three patients with > or = 50% of sequenced clones revealing the specific base pair alteration were bacterially expressed in E. coli and analyzed for dCK activity. Normal enzyme activity was found in two patients (codon 20 and 98), and a complete loss of activity in one patient (codon 99). We conclude that structural alteration of the coding region of the dCK gene represents one possible mechanism for ara-C resistance in vivo, but, considering the frequency of this event, other mechanisms may play a more important role for clinical resistance to ara-C in patients with AML.
View details for Web of Science ID A1994NP11900010
View details for PubMedID 7514246
EFFECTS OF HUMAN T-LYMPHOCYTE ACTIVATION ON INOSINE MONOPHOSPHATE DEHYDROGENASE EXPRESSION
JOURNAL OF IMMUNOLOGY
1994; 152 (3): 984-991
Inosine monophosphate dehydrogenase (IMPDH) catalyzes the first step in the formation of guanine ribonucleotides from inosine monophosphate and the activity of this enzyme appears to be essential for cell proliferation. Inhibitors of IMPDH have been demonstrated to be effective immunosuppressive agents and to inhibit T cell activation in vitro. IMPDH activity results from the expression of two different genes (types I and II) that encode protein subunits of identical size with 84% amino acid identity. To determine the relative contribution of the expression of these two genes to T cell activation, we have examined the effects of T cell stimulation on IMPDH activity, mRNA levels, and protein. The stimulation of isolated peripheral blood CD28+ T cells with PMA and ionomycin causes a 15-fold increase in IMPDH activity over a 72-h period. This is associated with a 10-fold increase in type II mRNA levels at 48 h. Type I mRNA is expressed at very low levels in resting T cells, but increases 10-fold by 24 h after stimulation. The type I cDNA probe also detects a second larger mRNA species of 4.0 kb that is not detectable in a variety of normal tissues or in a panel of leukemic cell lines. RNase protection assays using RNA probes corresponding to the entire coding region of the type I enzyme reveal a single protected fragment, demonstrating that the 4.0-kb message is the result of alternate splicing in the 5' or 3' untranslated regions or the use of an alternative polyadenylation site. Western blot analysis demonstrates a concomitant increase in total IMPDH protein on T cell activation, although posttranslational modifications do not allow the distinction between type I and type II on isoelectric focusing gels. We conclude that the induction of both type I and type II IMPDH contribute significantly to the T cell proliferative response. Both enzymes therefore should be considered important targets for immunosuppressive therapy.
View details for Web of Science ID A1994MV63000003
View details for PubMedID 7905505
- Regulation of low Km (Ecto-) 5'-nucleotidase gene expression in leukemic cells 8th International Symposium on Purine and Pyrimidine Metabolism in Man PLENUM PRESS DIV PLENUM PUBLISHING CORP. 1994: 683–687
HEREDITARY OVEREXPRESSION OF ADENOSINE-DEAMINASE IN ERYTHROCYTES - EVIDENCE FOR A CIS-ACTING MUTATION
AMERICAN JOURNAL OF HUMAN GENETICS
1993; 53 (4): 889-893
Overexpression of adenosine deaminase (ADA) in red blood cells is inherited as an autosomal dominant trait and causes hemolytic anemia. The increased ADA activity in erythrocytes is due to an increase in steady-state levels of ADA mRNA of normal sequence. Increased ADA mRNA may be due to a cis-acting mutation which results in increased transcription or a loss of down-regulation during erythroid differentiation. Alternatively, it is possible that the mutation is in a trans-acting factor which interacts with normal ADA transcriptional elements to cause overexpression in red blood cells. To discriminate between a cis-acting and a trans-acting mutation, we took advantage of a highly polymorphic TAAA repeat located at the tail end of an Alu repeat approximately 1.1 kb upstream of the ADA gene. Using PCR to amplify this region, we identified five different alleles in 19 members of the family. All 11 affected individuals had an ADA allele with 12 TAAA repeats, whereas none of the 8 normal individuals did. We conclude that this disorder results from a cis-acting mutation in the vicinity of the ADA gene.
View details for Web of Science ID A1993LY62700013
View details for PubMedID 8213817
TYPE-I INOSINE MONOPHOSPHATE DEHYDROGENASE - EVIDENCE FOR A SINGLE FUNCTIONAL GENE IN MAMMALIAN-SPECIES
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1993; 195 (2): 897-901
Human inosine monophosphate dehydrogenase activity is the result of the expression of two independent but closely related genes, termed IMPDH type I and type II. We have documented the presence of multiple, processed pseudogenes of type I IMPDH in human and Rhesus monkey genomic DNA, as well as a single functional gene encoding low levels of type I mRNA in human brain, heart, kidney and placenta. Single copy genes for each IMPDH isoenzyme were also found in rat, mouse, dog, cow, and chicken DNA and distinct mRNA species for type I and type II were identified by Northern blots in mouse and hamster RNA. Northern blot analysis of chicken RNA revealed a single mRNA species that hybridized to human IMPDH type I and II probes. These data document the high degree of evolutionary conservation of these two genes among mammals.
View details for Web of Science ID A1993LW46900055
View details for PubMedID 7690562
IMP DEHYDROGENASE INHIBITORS AS IMMUNOMODULATORS
ANNALS OF THE NEW YORK ACADEMY OF SCIENCES
1993; 685: 217-224
IMP dehydrogenase is a key enzyme in the de novo pathway of purine biosynthesis and is responsible for catalyzing the first step in the formation of guanine ribonucleotides from inosine monophosphate. Mizoribine, an immunosuppressive agent in wide-spread clinical use in Japan, has been demonstrated to inhibit this enzyme. We have investigated the effects of mizoribine on human T cell activation. Stimulation of purified human peripheral blood T lymphocytes with phorbol ester and ionomycin leads to a five-fold increase in guanine ribonucleotide levels over 72 hours. The addition of mizoribine to these cultures at concentrations that are achieved in vivo leads to a dose-dependent inhibition of proliferation and concomitant 50% decrease in guanine ribonucleotide levels, an effect that is reversible with the addition of guanosine, which repletes the GTP pool. Similar effects are seen with direct stimulation via the CD3/T cell receptor complex. Inhibition of proliferation occurs at the G1/S interface of the cell cycle and is additive to that produced by cyclosporine. In order to determine whether inhibition of IMP dehydrogenase is a common mechanism of immunosuppression for drugs such as azathioprine and 6-mercaptopurine that interfere with purine biosynthesis, we compared the effects of these agents on the metabolism of purified T lymphocytes. The results of these studies demonstrate that mizoribine and mycophenolic acid, a highly specific inhibitor of IMP dehydrogenase, inhibit proliferation directly by the depletion of guanine ribonucleotides; 6-mercaptopurine, on the other hand, has a mixed effect on adenine and guanine ribonucleotide pools, whereas azathioprine inhibits proliferation by a mechanism completely independent of its effects on the purine metabolic pathway. We conclude from these studies that inhibitors of IMP dehydrogenase have potential as specific immunosuppressive agents.
View details for Web of Science ID A1993LL56700026
View details for PubMedID 8103312
GENOMIC STRUCTURE AND CHROMOSOMAL LOCALIZATION OF THE HUMAN DEOXYCYTIDINE KINASE GENE
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1993; 90 (2): 431-434
Deoxycytidine kinase (NTP:deoxycytidine 5'-phosphotransferase, EC 184.108.40.206) is an enzyme that catalyzes phosphorylation of deoxyribonucleosides and a number of nucleoside analogs that are important in antiviral and cancer chemotherapy. Deficiency of this enzyme activity is associated with resistance to these agents, whereas increased enzyme activity is associated with increased activation of such compounds to cytotoxic nucleoside triphosphate derivatives. To characterize the regulation of expression of this gene, we have isolated genomic clones encompassing its entire coding and 5' flanking regions and delineated all the exon/intron boundaries. The gene extends over more than 34 kilobases on chromosome 4 and the coding region is composed of 7 exons ranging in size from 90 to 1544 base pairs (bp). The 5' flanking region is highly G+C-rich and contains four regions that are potential Sp1 binding sites. A 697-bp fragment encompassing 386 bp of 5' upstream region, the 250-bp first exon, and 61 bp of the first intron was demonstrated to promote chloramphenicol acetyltransferase activity in a T-lymphoblast cell line and to have > 6-fold greater activity in a Jurkat T-lymphoblast than in a Raji B-lymphoblast cell line. Our data suggest that these 5' sequences may contain elements that are important for the tissue-specific differences in deoxycytidine kinase expression.
View details for Web of Science ID A1993KH51600015
View details for PubMedID 8421671
REGULATION OF HUMAN DEOXYCYTIDINE KINASE EXPRESSION
33RD SYMP ON REGULATION OF ENZYME ACTIVITY AND SYNTHESIS IN NORMAL AND NEOPLASTIC TISSUES
PERGAMON PRESS LTD. 1993: 61–68
The human deoxycytidine kinase gene is a single copy gene and is comprised of seven exons that are spread over more than 34 kb of the genome. The 5'-flanking region is highly G/C rich and does not contain CAAT or TATA boxes. This region, when cloned into a recorder gene construct containing the chloramphenicol acetyltransferase gene, is capable of mediating CAT activity in human lymphoid cell lines and appears to have greater activity in human T, as compared to B, lymphoblast cell lines. The expression of the gene at the mRNA level does not appear to be cell-cycle regulated in that the levels of mRNA in human peripheral blood T lymphocytes remain constant as the cells progress from a resting to a proliferating state. Since this enzyme catalyzes the conversion of a number of chemotherapeutic agents to their corresponding monophosphate form and is thus essential for their activation, it will be important to define further the genetic elements which regulate the expression of this gene.
View details for Web of Science ID A1993BY41V00006
RESISTANCE TO 1-BETA-D-ARABINOFURANOSYLCYTOSINE IN HUMAN T-LYMPHOBLASTS MEDIATED BY MUTATIONS WITHIN THE DEOXYCYTIDINE KINASE GENE
1992; 52 (9): 2389-2393
We have recently identified a complementary DNA clone which encodes the complete amino acid sequence for 2'-deoxycytidine kinase (dCK), the enzyme required for the initial phosphorylation of several deoxyribonucleosides and their analogues that are widely used as chemotherapeutic and antiviral agents. In order to identify the molecular basis for dCK deficiency in two clonal T-lymphoblast cell lines generated by virtue of their resistance to 1-beta-D-arabinofuranosylcytosine (ara-C-8D) or to 2',3'-dideoxycytidine (ddC50), we have cloned and sequenced their dCK complementary DNAs. The ara-C-8D cell line contained two identifiable mutations: (a) a 115-base pair deletion within the coding region, corresponding to the fifth exon of the gene and presumably resulting from a splice site mutation; and (b) a G to A point mutation that substitutes glutamic acid for glycine within the ATP-binding domain of the protein. Expression of each protein in Escherichia coli demonstrated a complete loss of catalytic activity and, in the case of the deletion, a proteolytic degradation product of the altered protein. The substitution of a negatively charged amino acid within the ATP-binding domain resulted in loss of enzyme activity with all nucleoside triphosphates tested. The ddC50 cell line contained a single identifiable structural gene mutation in all clones sequenced resulting in the substitution of arginine for glutamine at amino acid 156 of the protein. This mutation markedly diminished the catalytic activity of the expressed protein with the three substrates, deoxycytosine, deoxyadenosine, and deoxyguanosine. On the basis of the presence of a single point mutation and a marked reduction in dCK mRNA in this cell line, we postulate that the second allele either is not expressed or is expressed at extremely low levels. We conclude that cellular resistance to the toxicity of 1-beta-D-arabinofuranosylcytosine and dideoxycytidine in these cell lines is mediated by specific mutations within the dCK gene. Further elucidation of structural genes alterations in dCK-deficient cells will facilitate a more detailed understanding of the functional domains of this complex enzyme.
View details for Web of Science ID A1992HR29000003
View details for PubMedID 1568208
COMPARISON OF THE EFFECTS OF MIZORIBINE WITH THOSE OF AZATHIOPRINE, 6-MERCAPTOPURINE, AND MYCOPHENOLIC-ACID ON LYMPHOCYTE-T PROLIFERATION AND PURINE RIBONUCLEOTIDE METABOLISM
1992; 41 (4): 671-676
The immunosuppressive drug mizoribine has been demonstrated to inhibit T lymphocyte proliferation by depleting these cells of guanine ribonucleotides as a consequence of inhibiting the enzyme inosine monophosphate (IMP) dehydrogenase. Because the immunosuppressive agents azathioprine and 6-mercaptopurine (6MP) are both converted to the IMP analog 6-thio-IMP, we postulated that these drugs might inhibit T cell activation and/or proliferation by a similar mechanism. Incubation of isolated peripheral blood T cells with either mizoribine or the selective IMP dehydrogenase inhibitor mycophenolic acid caused a dose-dependent inhibition of T cell proliferation, which was reversible with the addition of 50 microM guanosine to replete guanine ribonucleotide pools. In contrast, guanosine exacerbated the inhibition of proliferation induced by azathioprine and restored proliferation at IC50 concentrations of 6MP by only 10%. Complete restoration of proliferation in the presence of 6MP, but not azathioprine, was achieved with the addition of adenine. The inhibitory effects of azathioprine, as well as those of mizoribine, 6MP, and mycophenolic acid, were identical in cells stimulated with antibody to the T cell receptor and in cells stimulated with phorbol ester and ionomycin. We conclude from these studies that mizoribine selectively inhibits guanine ribonucleotide formation in purified T cells, whereas the effect of 6MP appears to be more dependent on adenine ribonucleotide depletion. Azathioprine, on the other hand, inhibits proliferation by a mechanism independent of purine ribonucleotide depletion. None of these agents inhibits T cell proliferation by interfering with signal transduction mediated by the T cell receptor. Inhibition of guanine ribonucleotide biosynthesis appears to be a novel and perhaps more selective mechanism of inhibiting T cell proliferative responses after T cell activation.
View details for Web of Science ID A1992HP40500012
View details for PubMedID 1569921
GUANINE RIBONUCLEOTIDE DEPLETION INHIBITS T-CELL ACTIVATION - MECHANISM OF ACTION OF THE IMMUNOSUPPRESSIVE DRUG MIZORIBINE
JOURNAL OF CLINICAL INVESTIGATION
1991; 87 (3): 940-948
The immunosuppressive drug, mizoribine, has been used to prevent rejection of organ allografts in humans and in animal models. Based on studies in cell lines, mizoribine has been postulated to be an inhibitor of inosine monophosphate (IMP) dehydrogenase (EC220.127.116.11), a pivotal enzyme in the formation of guanine ribonucleotides from IMP. To further characterize the mechanism of action of this drug, we studied the effect of mizoribine on human peripheral blood T cells stimulated with alloantigen, anti-CD3 MAb, or pharmacologic mitogens. Mizoribine (1-50 micrograms/ml) was able to inhibit T cell proliferation by 10-100% in a dose-dependent fashion to all stimuli tested. Measurements of purine ribonucleotide pools by HPLC showed that mizoribine led to a decrease in intracellular GTP levels, and that repletion of GTP reversed its antiproliferative effects. We also examined sequential events occurring after T cell stimulation. Early events in T cell activation, as assessed by steady-state mRNA levels of c-myc, IL-2, c-myb, histone, and cdc2 kinase, as well as surface IL-2 receptor expression, were unaffected. However, cell cycle analysis revealed decreased numbers of cells in S, G2, and M phases, and showed that the G1/S block was reversed with GTP repletion. These data indicate that mizoribine has an effect on T cell proliferation by a mechanism distinct from that of cyclosporine or corticosteroids, and therefore may be useful in combination immunosuppressive regimens.
View details for Web of Science ID A1991FB17100024
View details for PubMedID 1999502
CLONING AND EXPRESSION OF HUMAN DEOXYCYTIDINE KINASE CDNA
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1991; 88 (4): 1531-1535
Deoxycytidine (dCyd) kinase is required for the phosphorylation of several deoxyribonucleosides and certain nucleoside analogs widely employed as antiviral and chemotherapeutic agents. Detailed analysis of this enzyme has been limited, however, by its low abundance and instability. Using oligonucleotides based on primary amino acid sequence derived from purified dCyd kinase, we have screened T-lymphoblast cDNA libraries and identified a cDNA sequence that encodes a 30.5-kDa protein corresponding to the subunit molecular mass of the purified protein. Expression of the cDNA in Escherichia coli results in a 40-fold increase in dCyd kinase activity over control levels. In dCyd kinase-deficient murine L cells, transfection with dCyd kinase cDNA in a mammalian expression vector produces a 400-fold increase over control in dCyd phosphorylating activity. The expressed enzyme has an apparent Km of 1.0 microM for dCyd and is also capable of phosphorylating dAdo and dGuo. Northern blot analysis reveals a single 2.8-kilobase mRNA expressed in T lymphoblasts at 5- to 10-fold higher levels than in B lymphoblasts, and decreased dCyd kinase mRNA levels are present in T-lymphoblast cell lines resistant to arabinofuranosylcytosine and dideoxycytidine. These findings document that this cDNA encodes the T-lymphoblast dCyd kinase responsible for the phosphorylation of dAdo and dGuo as well as dCyd and arabinofuranosylcytosine.
View details for Web of Science ID A1991EY61700093
View details for PubMedID 1996353
Guanine ribonucleotide depletion inhibits T cell activation.
Advances in experimental medicine and biology
1991; 309B: 293-296
View details for PubMedID 1781385
- Development of an in vitro model system to study the physiologic effects and toxicities of immunosuppressants Alternative Methods in Toxicology 1991; 8: 55-61
CD11C (LEU-M5) EXPRESSION CHARACTERIZES A B-CELL CHRONIC LYMPHOPROLIFERATIVE DISORDER WITH FEATURES OF BOTH CHRONIC LYMPHOCYTIC-LEUKEMIA AND HAIRY-CELL LEUKEMIA
1990; 76 (11): 2360-2367
Chronic lymphocytic leukemia (CLL) and hairy cell leukemia (HCL) are two common chronic lymphoproliferative disorders, each having characteristic clinical, morphologic, and immunologic features. Phenotypically, CD5 reactivity in CLL and CD11c (Leu-M5) reactivity in HCL have characterized these two leukemias among B-cell disorders. In this study, we report 14 cases of a novel chronic lymphoproliferative disorder characterized by lymphocytosis and CD11c expression, but morphologically similar to CLL. The patients' ages ranged from 46 to 81 years (median 62). Eleven had palpable splenomegaly, five with markedly enlarged spleens; only one patient had generalized lymphadenopathy. The white blood cell count ranged from 5.2 to 131.0 x 10(9)/L (median 20.8). The morphologic diagnosis in all cases was CLL, with the cells usually having abundant cytoplasm. No morphologic features, of hairy cells were evident; tartrate-resistant acid phosphatase cytochemistry was negative in all cases. Bone marrow biopsies were available in 8 of 14. Four showed focal nodular infiltrates and two had diffuse infiltrates similar to CLL; two showed only minimal interstitial involvement. All cases expressed multiple B-cell markers, and 12 of 14 had monoclonal surface immunoglobulin. The leukemic cells of all cases strongly expressed CD11c, while CD5 was expressed in 7 of 14; only 1 of the 14 cases expressed the lymph node homing receptor, Leu-8. This unique group of leukemias appears to represent the malignant transformation of lymphocytes arising from a stage of lymphocyte differentiation between that found in typical cases of CLL and that of HCL. CD11c is known to have an important function in cellular adhesion and may be important in determining the pattern of lymphocyte tissue distribution found in this group of patients.
View details for Web of Science ID A1990EK72700029
View details for PubMedID 1701669
MUTATIONS INDUCED AT THE HYPOXANTHINE-GUANINE PHOSPHORIBOSYLTRANSFERASE LOCUS OF HUMAN T-LYMPHOBLASTS BY PERTURBATIONS OF PURINE DEOXYRIBONUCLEOSIDE TRIPHOSPHATE POOLS
1990; 50 (15): 4566-4571
Chronic perturbations of intracellular deoxyribonucleoside triphosphate (dNTP) pools have been associated with a mutator phenotype and increased mutation rates at several genetic loci. We have examined the specific effects of transient pharmacological purine dNTP pool perturbations on mutations induced at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus in a cultured human T-lymphoblast cell line. Incubation of CEM cells with 50 microM 2'-deoxyguanosine for 6 h increased intracellular dGTP levels 43-fold and induced a 40-fold increase in mutation frequency at the HPRT locus. Six-h incubations with 5, 10, and 20 microM 2'-deoxyadenosine increased dATP pools 4.8-, 8-, and 14.5-fold, respectively, with 59-, 34-, and 43-fold increases in HPRT mutant fractions. In contrast, 24-h incubations with hydroxyurea at concentrations which inhibited cell growth to similar extents did not induce HPRT mutations. Sequencing of HPRT complementary DNA derived from mutant cell lines revealed that the mutations induced by transient purine dNTP pool perturbations exhibited no significant misincorporation of the nucleotide in excess or next-nucleotide effect, and were similar in nature and location to spontaneous HPRT mutations. We conclude that mutations caused by transient purine dNTP pool elevations in these dividing cells are most likely induced by inhibition of DNA repair processes.
View details for Web of Science ID A1990DP43100020
View details for PubMedID 2369732
DIFFERENTIAL METABOLISM OF 9-BETA-D-ARABINOFURANOSYLGUANINE IN HUMAN-LEUKEMIC CELLS
1989; 49 (23): 6498-6502
9-beta-D-Arabinofuranosylguanine (araG) is a nucleoside analogue that elicits cytotoxicity through the intracellular accumulation of its 5'-triphosphate, araGTP, araG is selectively toxic to cultured T-lymphoblasts due to their ability to accumulate higher levels of the cytotoxic metabolite, araGTP, relative to B- and null lymphoblastoid cells. In an effort to determine whether this selectivity may occur in leukemic cells in vivo, we have investigated the metabolism of araG in MOLT-4 T-lymphoblasts. MGL-8 B-lymphoblasts, HL-60 promyelocytes, and HUT-102 mature T-cells and compared it to that in freshly isolated leukemic cells from patients. MOLT-4 T-lymphoblasts were 50- to 380-fold more sensitive to growth inhibition with araG and accumulated 80-fold higher levels of araGTP than any of the other cell lines studied. Incubation of peripheral blood from patients with leukemia with araG for 4 h demonstrated that T-acute lymphocytic leukemia cells accumulated significantly higher median levels of araGTP than did acute myelogenous leukemia or chronic lymphocytic leukemia cells (187 versus 72 and 31 pmol of araGTP per 10(7) cells, respectively), araGTP accumulation was not dependent on the rate of degradation of araG during the incubation. In contrast, araG did not exhibit similar selective growth inhibition, nor did the accumulation of 1-beta-D-arabinofuranosylcytosine 5'-triphosphate in the freshly isolated leukemic cells differ significantly among T-acute lymphocytic leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, and non-T-, non-B-cell acute lymphocytic leukemia cells. These results demonstrate that the selective metabolism of araG observed in cultured cell lines was representative of the metabolism in freshly isolated leukemic cells. Furthermore, degradation of araG did not limit the accumulation of araGTP in the leukemic cells. These results indicate that araG may be valuable as a selectively acting chemotherapeutic agent in T-lymphoblastic malignancies.
View details for Web of Science ID A1989CA66300008
View details for PubMedID 2819707
ASSOCIATION OF A MATURE B-CELL LEUKEMIA WITH A 4P+ CHROMOSOMAL ABNORMALITY - DERIVATION AND CHARACTERIZATION OF A CELL-LINE
1989; 3 (9): 643-647
We have found a single 4p+ chromosomal abnormality, 46,XX, -4, +der(4)t(3;4)(q13.3;p16), in a patient with an unusual B cell leukemia of mature phenotype characterized by a high white cell count, tartrate-resistant acid phosphatase-positive malignant cells, splenic white pulp proliferation, and a serum IgM monoclonal gammopathy. The malignant cells were characterized by surface expression of CD19 (B4), CD20 (B1), IgM, IgD, kappa, and HLA-DR. They were weakly positive for CD21 (B2) and negative for CD25 (interleukin-2 receptor). The malignant cells also showed clonal rearrangement of the immunoglobulin heavy chain and kappa light chain genes. A cell line, designated HCLW-3B, was derived from unstimulated peripheral blood obtained during the leukemic phase and was found to contain the same 4p+ chromosomal abnormality as well as genomic sequences of the Epstein-Barr virus nuclear antigen. A somatic cell hybrid constructed from HCLW-3B containing the derivative chromosome 4 was used to confirm that chromosome 3q was the source of the translocated material. The availability of a cell line which is clonally derived from the patient's circulating leukemia cells should permit further characterization of this translocation at the molecular level.
View details for Web of Science ID A1989AM53300006
View details for PubMedID 2548046
ERYTHROCYTE ADENOSINE-DEAMINASE OVERPRODUCTION IN HEREDITARY HEMOLYTIC-ANEMIA
1989; 74 (1): 448-453
A marked tissue-specific increase in erythrocyte adenosine deaminase (ADA) activity is associated with an autosomal dominantly inherited hemolytic anemia. We investigated the molecular basis of ADA overproduction by studying reticulocyte ADA mRNA from affected individuals. Analysis of proband reticulocyte ADA cDNA clones revealed normal sequence. RNase mapping demonstrated that the amount of ADA mRNA in affected reticulocytes was greater than the amount in normal B lymphoblasts, whereas ADA mRNA was undetectable in normal reticulocytes. The 5'- and 3'-untranslated regions of reticulocyte and B-lymphoblast ADA mRNAs from affected individuals were structurally indistinguishable from those of normal B lymphoblasts. Northern blot analysis performed under stringent hybridization and washing conditions confirmed a markedly increased amount of reticulocyte ADA mRNA in affected individuals as compared with controls. We conclude that the RBC-specific overexpression of ADA in this disorder occurs at the mRNA level.
View details for Web of Science ID A1989AE78100063
View details for PubMedID 2752123
KINETIC-PROPERTIES AND INHIBITION OF HUMAN LYMPHOBLAST-T DEOXYCYTIDINE KINASE
JOURNAL OF BIOLOGICAL CHEMISTRY
1989; 264 (16): 9359-9364
The kinetic properties of 50,000-fold purified cultured human T lymphoblast (MOLT-4) deoxycytidine kinase were examined. The reaction velocity had an absolute requirement for magnesium. Maximal activity was observed at pH 6.5-7.0 with Mg:ATP for 1:1. High concentrations of free Mg2+ or free ATP were inhibitory. Double reciprocal plots of initial velocity studies yielded intersecting lines for both deoxycytidine and MgATP2-. dCMP was a competitive inhibitor with respect to deoxycytidine and ATP. ADP was a competitive inhibitor with respect to ATP and a mixed inhibitor with respect to deoxycytidine. dCTP, an important end product, is a very potent inhibitor and was a competitive inhibitor with respect to deoxycytidine and a non-competitive inhibitor with respect to ATP. TTP reversed dCTP inhibition. The data suggest that (a) MgATP2- is the true substrate of deoxycytidine kinase; (b) the kinetic mechanism of deoxycytidine kinase is consistent with rapid equilibrium random Bi Bi; (c) deoxycytidine kinase may be regulated by its product ADP and its end product dCTP as well as the availability of deoxycytidine. While many different nucleotides potently inhibit deoxycytidine kinase, their low intracellular concentrations make their regulatory role less important.
View details for Web of Science ID A1989U866500045
View details for PubMedID 2542307
HUMAN T-LYMPHOBLAST DEOXYCYTIDINE KINASE - PURIFICATION AND PROPERTIES
1989; 28 (1): 114-123
Previous observations present tremendous variations in the properties of deoxycytidine kinase. To clarify the properties and physiologic role of deoxycytidine kinase, we have undertaken its purification. Deoxycytidine kinase was purified from cultured human T-lymphoblasts (MOLT-4) to 90% purity with an estimated specific activity of 8 mumol min-1 (mg of protein)-1. The purification procedure included ammonium sulfate precipitation, Superose-12 HPLC gel filtration chromatography, DE-52 ion-exchange chromatography, AMP-Sepharose 4B affinity chromatography, and dCTP-Sepharose-4B affinity chromatography. Deoxyguanosine, deoxyadenosine, and cytidine phosphorylating activities copurified with deoxycytidine kinase to final specific activities of 7.2, 13.5, and 4 mumol min-1 (mg of protein)-1, respectively. The enzyme is very unstable at low protein concentration and is stabilized by storage at -85 degrees C with 1 mg/mL bovine serum albumin, 20% glycerol (v/v), 200 mM potassium chloride, and 25 mM dithiothreitol. The molecular weight was 60,000, and the Stokes radius was 32 A by gel filtration chromatography. The subunit molecular weight was 30,500. This enzyme had apparent Km values of 1.5, 430, 500, 450, and 40 microM for deoxycytidine, deoxyguanosine, deoxyadenosine, cytidine, and cytosine arabinoside, respectively. The pH optimum ranged from 6.5 to 9.0. Mg2+ and Mn2+ were the preferred divalent cations. ATP, GTP, dGTP, ITP, dITP, TTP, and XTP were substrates for the enzymes. Our study indicates that deoxycytidine kinase is a dimer with two subunits and has phosphorylating activity for deoxyguanosine, deoxyadenosine, cytidine, and cytosine arabinoside. This highly purified enzyme will facilitate the study of its regulation and phosphorylation of anticancer or antiviral nucleoside analogues.
View details for Web of Science ID A1989R737300017
View details for PubMedID 2539852
Autosomal dominant hemolytic anemia and adenosine deaminase overproduction.
Advances in experimental medicine and biology
1989; 253A: 493-497
View details for PubMedID 2624229
- Identification of an Apa I polymorphism within the human adenosine deaminase gene Nucl Acids Res 1989: 3626
Properties of highly purified human T lymphoblast (MOLT-4) deoxycytidine kinase.
Advances in experimental medicine and biology
1989; 253B: 87-92
View details for PubMedID 2558550
Erythrocyte-specific overproduction of adenosine deaminase: molecular genetic studies.
Progress in clinical and biological research
1989; 319: 55-64
A kindred with an autosomal dominant form of chronic hemolytic anemia has been found to have a 40- to 70-fold elevation in erythrocyte adenosine deaminase (ADA) activity in association with depletion of red blood cell (RBC) ATP pools. ADA activities in B lymphoblasts, skin fibroblasts, and granulocytes were normal. There were no alterations in the kinetic properties of partially purified proband ADA. We have shown by Western blot analysis that the elevation in ADA activity is accompanied by a corresponding increase in the amount of immunoreactive ADA protein. Southern blot analysis of proband DNA ruled out gene amplification and revealed no gross insertions, deletions, or rearrangements in the ADA gene. Northern blot analysis demonstrated a marked increase in the amount of ADA mRNA in proband and sibling reticulocytes compared to high reticulocyte controls. ADA mRNA levels in B lymphoblasts from the proband, sibling, and GM558 cell line were normal. Cloning and sequencing of proband reticulocyte cDNA revealed normal ADA mRNA sequence. No polymorphisms were detected among the seven clones studied. RNase mapping of the 5'- and 3'-non-coding sequences confirmed the quantitative increase in reticulocyte ADA mRNA and verified that these regions were normal in length and sequence. Southern blot analysis of DNA from four affected and three unaffected family members revealed two restriction fragment length polymorphisms (RFLPs) which segregate with the ADA allele from the unaffected grandfather. Both RFLPs are present in the unaffected grandchild and absent in the affected grandchild. These findings are consistent with a cis- mutation within the ADA gene, but they do not rule out a trans- mutation affecting some non-ADA regulatory factor. We conclude that erythrocyte-specific ADA overproduction is associated with increased amounts of structurally normal ADA mRNA. This increase may result from either increased transcription of the ADA gene or altered post-transcriptional processing resulting in increased stability of the RNA transcript. Further elucidation of the defect should provide valuable insights into the normal tissue-specific regulation of the ADA gene and the mechanisms by which erythroid cells regulate gene expression during differentiation.
View details for PubMedID 2622927
GENETIC EXPRESSION OF ADENOSINE-DEAMINASE IN HUMAN LYMPHOID MALIGNANCIES
1987; 69 (5): 1376-1380
Adenosine deaminase (ADA) is an enzyme in the purine catabolic pathway that has been used as an enzymatic marker of T cell lymphoblastic malignancies due to its high specific activity in thymocytes and immature T cells. We have investigated whether the level of ADA activity in lymphoid leukemic cells correlates with the amount of ADA-specific RNA and/or immunoreactive protein in these cells as an initial step toward characterizing the nature of the genetic regulation of ADA expression during differentiation. We have found a good correlation between the steady state levels of ADA-specific RNA and ADA-immunoreactive protein in T lymphoblastic leukemic cell lines, mature T cell lines, a B lymphoblast cell line, and leukemic cells directly isolated from four patients with acute lymphoblastic leukemia and three patients with chronic lymphocytic leukemia. Southern blot analysis of DNA from these cells shows no evidence for differences in ADA gene copy number or gene rearrangement to account for the variability in ADA expression. We conclude that levels of ADA in lymphoid leukemic cells are directly related to the amount of ADA-specific mRNA present. These findings imply that ADA expression in leukemic cells reflects either the transcriptional activity of the ADA gene or the stability of ADA mRNA in these cells.
View details for Web of Science ID A1987H128300016
View details for PubMedID 3567360
TRANSCRIPTIONAL REGULATION OF THE C-MYC PROTOONCOGENE BY 1,25-DIHYDROXYVITAMIN-D3 IN HL-60 PROMYELOCYTIC LEUKEMIA-CELLS
JOURNAL OF BIOLOGICAL CHEMISTRY
1987; 262 (9): 4104-4108
Exposure of HL-60 promyelocytic leukemia cells to calcitriol results in a decrease in steady-state levels of c-myc mRNA and induces cellular differentiation. We have asked whether calcitriol has a direct effect on the transcription of the c-myc gene. 1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) decreased RNA elongation in a nuclear run-off transcription assay by 4 h after treatment. In the continuous presence of 1,25-(OH)2D3, HL-60 cell transcription of c-myc was decreased by 38% at 4 h and was abolished by 48 h. In contrast, the transcription of beta-actin was not affected by 1,25-(OH)2D3 treatment. The rate of transcription of c-myc and beta-actin was proportional to the number of nuclei and to time. Furthermore, specific hybridization of c-myc and beta-actin RNA was a linear function of RNA input. After a 48-h treatment, the c-myc/beta-actin ratio was decreased by 80-100% at [32P]RNA inputs ranging from 2 to 20 X 10(6) cpm/ml. These data temporally correlate inhibition of c-myc transcription with decreases in the steady-state levels of c-myc mRNA as assessed by Northern blot analysis. We conclude that the effect of 1,25-(OH)2D3 on c-myc expression occurs at the transcriptional level.
View details for Web of Science ID A1987G578600030
View details for PubMedID 2435730
ELEVATED ADENOSINE-DEAMINASE ACTIVITY AND HEREDITARY HEMOLYTIC-ANEMIA - EVIDENCE FOR ABNORMAL TRANSLATIONAL CONTROL OF PROTEIN-SYNTHESIS
JOURNAL OF CLINICAL INVESTIGATION
1987; 79 (3): 1001-1005
We have investigated the molecular basis of the marked elevation in erythrocyte adenosine deaminase (ADA) activity in a kindred with hereditary hemolytic anemia. Red cell ADA-specific activity was verified to be 70- to 100-fold normal levels. Western blots demonstrated a corresponding increase in erythrocyte ADA-specific immunoreactive protein. Analysis of genomic DNA revealed no evidence for amplification or major structural changes in the ADA gene. ADA-specific messenger RNA (mRNA) from proband reticulocytes was comparable in size and amount to mRNA from control reticulocytes. Translation of proband poly A+ reticulocyte mRNA in a rabbit reticulocyte lysate system and immunoprecipitation of 35S-labeled protein products with anti-ADA antibody yielded a band of approximately 42,000 apparent mol wt that was absent in translation products from control reticulocyte mRNAs. These data suggest that the increased ADA activity in red cells in this disorder results from the increased translation of an aberrant ADA mRNA.
View details for Web of Science ID A1987G242500045
View details for PubMedID 3029177
- Molecular basis for elevated adenosine deaminase activity in a kindred with hemolytic anemia J Clin Invest 1987: 1001-1005
Characterization of arabinosylguanine resistance in a lymphoblastoid cell line.
Advances in experimental medicine and biology
1986; 195: 605-609
View details for PubMedID 3094330
Z-NUCLEOTIDE ACCUMULATION IN ERYTHROCYTES FROM LESCH-NYHAN PATIENTS
JOURNAL OF CLINICAL INVESTIGATION
1985; 76 (6): 2416-2419
5-Amino-4-imidazolecarboxamide riboside 5'-monophosphate (ZMP) is an intermediate in the purine de novo synthetic pathway that may be further metabolized to inosine 5'-monophosphate, degraded to the corresponding nucleoside (5-amino-4-imidazole-carboxamide riboside; Z-riboside), or phosphorylated to the corresponding 5'-triphosphate (ZTP). Accumulation of ZTP in microorganisms has been associated with depletion of folate intermediates that are necessary for the conversion of ZMP to inosine 5'-monophosphate and has been postulated to play a regulatory role in cellular metabolism. We have shown the presence of Z-nucleotides in erythrocytes derived from five individuals with the Lesch-Nyhan syndrome. Erythrocyte folate levels were within the normal range, although guanosine triphosphate levels were significantly reduced below those in normal controls (P less than 0.01). A small amount of Z-nucleotide accumulation was also found in one individual with partial deficiency of the enzyme hypoxanthine guanine phosphoribosyltransferase and in two individuals with other disorders of purine overproduction. In contrast, no Z-nucleotides were detected in 13 normal controls or in three individuals with hyperuricemia on allopurinol therapy. We conclude that Z-nucleotide formation may result from markedly increased rates of de novo purine biosynthesis. It is possible that metabolites of these purine intermediates may play a role in the pathogenesis of the Lesch-Nyhan syndrome.
View details for Web of Science ID A1985AWY8500050
View details for PubMedID 4077987
- BIOCHEMICAL CONSEQUENCES OF ADENOSINE-DEAMINASE INHIBITION INVIVO - DIFFERENTIAL-EFFECTS IN ACUTE AND CHRONIC T-CELL LEUKEMIA ANNALS OF THE NEW YORK ACADEMY OF SCIENCES 1985; 451 (OCT): 129-137
DIFFERENTIAL METABOLISM OF DEOXYRIBONUCLEOSIDES BY LEUKEMIC T-CELLS OF IMMATURE AND MATURE PHENOTYPE
BRITISH JOURNAL OF HAEMATOLOGY
1985; 61 (1): 125-134
Experimental evidence has indicated that T lymphoblasts are more sensitive to deoxynucleoside toxicity than are B lymphoblasts. These data have led to the use of purine enzyme inhibitors as selective chemotherapeutic drugs in the treatment of T cell malignancies ranging from T cell acute lymphoblastic leukaemia to cutaneous T cell lymphomas. We have compared the toxicities of 2'-deoxyadenosine, 2'-deoxyguanosine, and thymidine for T cell lines derived from patients with T cell acute lymphoblastic leukaemia with those for mature T cell lines derived from patients with cutaneous T cell leukaemia/lymphoma. We have found that both deoxynucleosides are far less toxic to the mature T cell lies than to T lymphoblasts and that the mature cells accumulate much lower amounts of dATP and dGTP when exposed to deoxyadenosine and deoxyguanosine, respectively. Similar studies performed on peripheral blood cells from patients with T cell leukaemias of mature phenotype and on peripheral blood T cells demonstrate similar low amounts of deoxynucleotide accumulation. Measurements of the activities of several purine metabolizing enzymes that participate in deoxynucleoside phosphorylation or degradation do not reveal differences which would explain the toxicity of deoxynucleosides for immature, as compared to mature, T cells. We conclude that deoxynucleoside metabolism in leukaemic T cells varies with their degree of differentiation. These observations may be relevant to the design of chemotherapeutic regimes for T cell malignancies.
View details for Web of Science ID A1985ARF6300014
View details for PubMedID 2996581
- Differential metabolism of deoxyribonucleosides by leukaemic T cells of immature and mature phenotype Br J Haemat 1985: 125-134
DIFFERENTIAL METABOLISM OF GUANINE NUCLEOSIDES BY HUMAN LYMPHOID-CELL LINES
PROCEEDINGS OF THE SOCIETY FOR EXPERIMENTAL BIOLOGY AND MEDICINE
1985; 179 (4): 427-431
Deficiency of the enzyme purine nucleoside phosphorylase is associated with a specific depletion of T cells which is presumably mediated by its substrate, 2'-deoxyguanosine. Inhibitors of this enzyme are therefore being developed as potential immunosuppressive agents. We have compared the effects of 8-aminoguanosine, a competitive inhibitor of purine nucleoside phosphorylase, on the metabolism of 2'-deoxyguanosine by human T lymphoblasts, B lymphoblasts, and mature T-cell lines. 8-Aminoguanosine markedly potentiates the accumulation of dGTP in T lymphoblasts, but results in increased GTP levels in B lymphoblasts and mature T cells. GTP accumulation is associated with ATP depletion of a magnitude similar to that seen with an inhibitor of de novo purine biosynthesis, but does not result in inhibition of either DNA or RNA synthesis. In contrast, direct inhibition of de novo purine biosynthesis sharply decreased the incorporation of [3H]uridine into both DNA and RNA. We conclude that the mechanism of cell damage resulting from prolonged accumulation of GTP appears to involve more than inhibition of de novo purine biosynthesis and consequent ATP depletion. Perturbations in guanine nucleotide pools resulting from partial inhibition of purine nucleoside phosphorylase activity in vivo could result in cellular toxicity not limited to the target T cell population.
View details for Web of Science ID A1985APH5700004
View details for PubMedID 3875100
METABOLISM AND SELECTIVE CYTO-TOXICITY OF 9-BETA-D-ARABINOFURANOSYLGUANINE IN HUMAN-LYMPHOBLASTS
1985; 45 (3): 1008-1014
The selective toxicity of purine deoxyribonucleosides for T-lymphoblasts appears to be mediated by the selective accumulation of the corresponding deoxyribonucleoside triphosphates in these cells. This finding has led to a search for deoxyribonucleoside analogues which may have clinical utility in T-cell lymphoproliferative disorders. 9-beta-D-arabinofuranosylguanine (ara-G) is a 2'-deoxyguanosine analogue which is 70-fold more inhibitory to the growth of T- than of B-lymphoblasts. It is less potent than ara-C but far more selective in its cytotoxic effect on T-cells. ara-G is not significantly degraded by purine nucleoside phosphorylase (EC 18.104.22.168) activity in T-lymphoblasts and is metabolized to 9-beta-D-arabinofuranosylguanine 5'-triphosphate. The accumulation of this metabolite directly correlates with inhibition of DNA but not of RNA or protein synthesis. MOLT-4 T-lymphoblasts were selected for ara-G resistance, and six clones were identified which exhibited a major degree of resistance to 2'-deoxyguanosine but little or none to ara-C. Further characterization of clone 24B3 revealed a 600-fold increase in ara-G resistance, a 36-fold increase in 2'-deoxyguanosine resistance, and only a 4-fold increase in 1-beta-D-arabinofuranosylcytosine resistance. The 24B3 cell line accumulated less than 10% of 9-beta-D-arabinofuranosylguanine 5'-triphosphate and 2'-deoxyguanosine 5'-triphosphate from the corresponding nucleosides as compared to wild-type MOLT-4 cells; in contrast, 1-beta-D-arabinofuranosylcytosine 5'-triphosphate accumulation was approximately 30% of control values. Thus, ara-G differs from 1-beta-D-arabinofuranosylcytosine in its selectivity for cultured T-lymphoblasts and may be of use as a chemotherapeutic or immunosuppressive agent.
View details for Web of Science ID A1985ACK7700009
View details for PubMedID 3971358
EFFECTS OF GUANINE RIBONUCLEOTIDE ACCUMULATION ON THE METABOLISM AND CELL-CYCLE OF HUMAN LYMPHOID-CELLS
1985; 45 (10): 4940-4945
A deficiency of purine nucleoside phosphorylase activity is associated with marked depletion of T-lymphocytes which is felt to be mediated by accumulation and further metabolism of the purine nucleoside phosphorylase substrate, 2'-deoxyguanosine. Human T-lymphoblasts incubated in the presence of 2'-deoxyguanosine and the purine nucleoside phosphorylase inhibitor 8-aminoguanosine accumulate deoxyguanosine 5'-triphosphate whereas B-lymphoblasts and mature T4+-cell lines accumulate GTP under identical conditions. We have compared the effects of guanine ribo- and deoxyribonucleotide accumulation on the metabolism and cell cycle of the respective cell lines. Deoxyguanosine 5'-triphosphate elevations in T-lymphoblasts are associated with inhibition of [3H]uridine incorporation into DNA and a complete block at the G1-S interface of the cell cycle. In contrast 3- to 5-fold increases in guanosine 5'-triphosphate pools in B-lymphoblasts and mature T-cell lines do not inhibit [3H]uridine incorporation into DNA or RNA but do cause a pronounced slowing in the progression of cells through S phase. B-lymphoblasts deficient in the salvage enzyme hypoxanthine guanine phosphoribosyltransferase do not accumulate guanosine 5'-triphosphate from 2'-deoxyguanosine and progress normally through the cell cycle, demonstrating a requirement for guanine salvage to inhibit cell growth. Guanine ribonucleotide accumulation was also associated with inhibition of de novo purine biosynthesis and a moderate decline in adenine nucleotide pools but not with inhibition of protein synthesis or alterations in basal levels of 3':5'-cyclic adenosine monophosphate or 3':5'-cyclic guanosine monophosphate. We conclude that the accumulation of guanine ribonucleotides by actively cycling human lymphoid cells is associated with an increase in S-phase cells and inhibition of growth. This effect is distinctly different from that produced by 2'-deoxyguanosine 5'-triphosphate and should be taken into account in pharmacological studies with 2'-deoxyguanosine and its analogues.
View details for Web of Science ID A1985ARH6200040
View details for PubMedID 2411392
- PLASMA-MEMBRANE 5'-NUCLEOTIDASE AND OTHER PURINE ENZYMES IN MURINE LYMPHOCYTES ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 1984; 165: 129-132
- Erythrocyte ATP a dATP measurements predict toxicity following 2'-deoxycoformycin therapy Cancer Treat Symp 1984: 67-74
2'-DEOXYGUANOSINE TOXICITY FOR B-LYMPHOID AND MATURE T-LYMPHOID CELL-LINES IS MEDIATED BY GUANINE RIBONUCLEOTIDE ACCUMULATION
JOURNAL OF CLINICAL INVESTIGATION
1984; 74 (5): 1640-1648
Inherited deficiency of the enzyme purine nucleoside phosphorylase (PNP) results in selective and severe T lymphocyte depletion which is mediated by its substrate, 2'-deoxyguanosine. This observation provides a rationale for the use of PNP inhibitors as selective T cell immunosuppressive agents. We have studied the relative effects of the PNP inhibitor 8-aminoguanosine on the metabolism and growth of lymphoid cell lines of T and B cell origin. We have found that 2'-deoxyguanosine toxicity for T lymphoblasts is markedly potentiated by 8-aminoguanosine and is mediated by the accumulation of deoxyguanosine triphosphate. In contrast, the growth of T4+ mature T cell lines and B lymphoblast cell lines is inhibited by somewhat higher concentrations of 2'-deoxyguanosine (ID50 20 and 18 microM, respectively) in the presence of 8-aminoguanosine without an increase in deoxyguanosine triphosphate levels. Cytotoxicity correlates instead with a three- to fivefold increase in guanosine triphosphate (GTP) levels after 24 h. Accumulation of GTP and growth inhibition also result from exposure to guanosine, but not to guanine at equimolar concentrations. B lymphoblasts which are deficient in the purine salvage enzyme hypoxanthine guanine phosphoribosyltransferase are completely resistant to 2'-deoxyguanosine or guanosine concentrations up to 800 microM and do not demonstrate an increase in GTP levels. Growth inhibition and GTP accumulation are prevented by hypoxanthine or adenine, but not by 2'-deoxycytidine. 8-Aminoguanosine appears to effectively inhibit extracellular PNP activity; thus, it prolongs the extracellular half-life of 2'-deoxyguanosine and guanosine, but does not completely inhibit intracellular PNP activity in these lymphoid cells. As a result, 2'-deoxyguanosine and guanosine are phosphorolyzed and actively salvaged within the cell, accounting for the accumulation of GTP. Partial inhibition of PNP activity in vivo, therefore, may lead to nonselective cellular toxicity by a mechanism independent of dGTP accumulation.
View details for Web of Science ID A1984TS19400010
View details for PubMedID 6334099
PURINE METABOLIZING ENZYMES AS PREDICTORS OF LYMPHOBLAST SENSITIVITY TO DEOXYADENOSINE
JOURNAL OF LABORATORY AND CLINICAL MEDICINE
1984; 104 (3): 414-424
The toxicity of low concentrations of 2'-deoxyadenosine for T-lymphoblasts and certain null lymphoblasts has been attributed to the decreased degradation of the deoxynucleotides formed from deoxyadenosine in these cells. Low activities of the ectoenzymes ecto-5'-nucleotidase and ecto-ATPase have each been associated with deoxyadenosine sensitivity and dATP accumulation in human T-lymphoblasts. We studied a B-lymphoblast cell line, NC-37, which lacks detectable ecto-5'-nucleotidase and ecto-ATPase activities, but which is otherwise easily distinguishable from T-lymphoblasts by its low adenosine deaminase activity and its pattern of reactivity with monoclonal antibodies to cell surface antigens (Bl and IgM positive). The NC-37 B cells were completely analogous to other B-lymphoblast lines with high ectonucleotidase activities in their relative resistance to deoxyadenosine toxicity and low rates of dATP accumulation. This resistance could not be accounted for by lower rates of deoxyadenosine phosphorylating activity. Cytoplasmic nucleotidase activity in crude extracts from the NC-37 line was similar to that in other B-lymphoblasts with regard to both substrate specificity and optimal pH. We conclude that low ectonucleotidase activities are not etiologically associated with the accumulation of deoxynucleotides by human lymphoblasts, although they may serve as markers of deoxyadenosine sensitivity in certain malignant lymphoid cells.
View details for Web of Science ID A1984TG95500013
View details for PubMedID 6147383
ALTERATIONS IN ERYTHROCYTE ADENINE-NUCLEOTIDE POOLS RESULTING FROM 2'-DEOXYCOFORMYCIN THERAPY
1983; 43 (3): 1409-1414
2'-Deoxycoformycin, a tight-binding adenosine deaminase inhibitor, was administered to 11 adult patients with refractory lymphoproliferative diseases. Total doses ranged from 1.0 to 13.5 mg/kg. Inhibition of lymphoblast adenosine deaminase was obtained in all cases and tumor cytoreduction was noted in eight of ten cases, but no clinically meaningful remissions were obtained. Major toxicities occurred in five patients and included pulmonary edema, renal failure, central nervous system toxicity, hypotension, and death. Toxicity prevented retreatment in several cases in which marked cytoreduction occurred. Deoxyadenosine triphosphate accumulated to a variable extent in the red blood cells of all patients, and a reciprocal decrease in erythrocyte adenosine triphosphate was noted in all cases but one. All patients who suffered major organ toxicity had red blood cell deoxyadenosine triphosphate/adenosine triphosphate ratios greater than 1.3. These data suggest that the degree of replacement of adenosine triphosphate by deoxyadenosine triphosphate in erythrocytes reflects the biochemical milieu which may result in systemic toxicity following treatment with 2'-deoxycoformycin.
View details for Web of Science ID A1983QC83800077
View details for PubMedID 6600652
DEOXYRIBONUCLEOSIDE TRIPHOSPHATE ACCUMULATION BY LEUKEMIC-CELLS
1983; 62 (2): 419-424
The toxicity of the deoxyribonucleosides, 2'-deoxyadenosine, 2'-deoxyguanosine, and thymidine, for human T lymphoblasts is mediated by the accumulation of the corresponding deoxyribonucleoside triphosphate (dATP, dGTP, or dTTP, respectively). We have examined whether leukemic cells of non-T-cell origin are capable of accumulating deoxyribonucleotides in culture and whether this capability correlates with the activities of purine metabolizing enzymes in these cells. We have found that non-T, non-B acute lymphoblastic leukemia cells with low ecto-5'-nucleotidase and high adenosine deaminase activities increase their dATP pools by greater than tenfold when exposed to deoxyadenosine and an inhibitor of adenosine deaminase in culture. Cells from 2 of 9 patients with chronic lymphocytic leukemia and 4 of 11 patients with acute nonlymphoblastic leukemia achieved similar elevations in dATP, but there was no relationship between dATP accumulation and adenosine deaminase, purine nucleoside phosphorylase, or ecto-5'-nucleotidase activities. Treatment of four individuals with acute lymphoblastic leukemia with the adenosine deaminase inhibitor, 2'-deoxycoformycin, resulted in elevations in plasma deoxyadenosine concentrations and in increments in lymphoblast dATP levels that were similar to those measured in lymphoblasts cultured with deoxyadenosine and deoxycoformycin prior to treatment. In vitro incubations of leukemic cells with deoxyribonucleosides may provide a rational basis for the use of these compounds as chemotherapeutic agents.
View details for Web of Science ID A1983RC87400028
View details for PubMedID 6603241
S-ADENOSYLHOMOCYSTEINE CATABOLISM AND BASIS FOR ACQUIRED-RESISTANCE DURING TREATMENT OF T-CELL ACUTE LYMPHOBLASTIC-LEUKEMIA WITH 2'-DEOXYCOFORMYCIN ALONE AND IN COMBINATION WITH 9-BETA-D-ARABINOFURANOSYLADENINE
1983; 43 (7): 3451-3458
A patient with refractory T-cell acute lymphoblastic leukemia was treated with eight courses of the adenosine deaminase inhibitor, 2'-deoxycoformycin (dCF), over a 5-month period. After developing resistance to dCF, he responded to treatment with the combination of dCF and 9-beta-D-arabinofuranosyladenine (ara-A). We monitored the levels in plasma and urine of adenosine, 2'-deoxyadenosine, and ara-A as well as the accumulation of their nucleotide derivatives in erythrocytes and circulating lymphoblasts. We also monitored the activities of adenosine deaminase and S-adenosylhomocysteine (AdoHcy) hydrolase and the concentrations of AdoHcy and S-adenosylmethionine in lymphoblasts. Production of 2'-deoxyadenosine was related to both the duration of dCF infusion and the magnitude of cytolysis that occurred during treatment: much more 2'-deoxyadenosine was produced by dCF infusion when disease was active than by the same infusion given during remission. Resistance to dCF was associated with a decrease of greater than 90% in the amount of deoxyadenosine 5'-triphosphate accumulated by circulating lymphoblasts. Infusion of dCF resulted in increases of up to 20-fold in the concentration of AdoHcy in circulating lymphoblasts, causing a decrease in the S-adenosylmethionine:AdoHcy ratio (the "methylation index") from a pretreatment value of greater than 40:1 to less than 4:1. This ratio decreased to 2.5:1 during combined treatment with dCF and ara-A, which caused nearly complete inactivation of lymphoblast AdoHcy hydrolase. Decline in the methylation index was accompanied by inhibition of the methylation of newly synthesized lymphoblast RNA. Impaired ability to catabolize AdoHcy may have contributed to the cytolytic responses to dCF and ara-A, as well as to hepatic and central nervous system toxicity associated with their combined use.
View details for Web of Science ID A1983QV62900075
View details for PubMedID 6601986
ADENOSINE-DEAMINASE DEFICIENCY WITH NORMAL IMMUNE FUNCTION - AN ACIDIC ENZYME MUTATION
JOURNAL OF CLINICAL INVESTIGATION
1983; 72 (2): 483-492
In most instances, marked deficiency of the purine catabolic enzyme adenosine deaminase results in lymphopenia and severe combined immunodeficiency disease. Over a 2-yr period, we studied a white male child with markedly deficient erythrocyte and lymphocyte adenosine deaminase activity and normal immune function. We have documented that (a) adenosine deaminase activity and immunoreactive protein are undetectable in erythrocytes, 0.9% of normal in lymphocytes, 4% in cultured lymphoblasts, and 14% in skin fibroblasts; (b) plasma adenosine and deoxyadenosine levels are undetectable and deoxy ATP levels are only slightly elevated in lymphocytes and in erythrocytes; (c) no defect in deoxyadenosine metabolism is present in the proband's cultured lymphoblasts; (d) lymphoblast adenosine deaminase has normal enzyme kinetics, absolute specific activity, S20,w, pH optimum, and heat stability; and (e) the proband's adenosine deaminase exhibits a normal apparent subunit molecular weight but an abnormal isoelectric pH. In contrast to the three other adenosine deaminase-deficient healthy subjects who have been described, the proband is unique in demonstrating an acidic, heat-stable protein mutation of the enzyme that is associated with less than 1% lymphocyte adenosine deaminase activity. Residual adenosine deaminase activity in tissues other than lymphocytes may suffice to metabolize the otherwise lymphotoxic enzyme substrate(s) and account for the preservation of normal immune function.
View details for Web of Science ID A1983RC91600008
View details for PubMedID 6603477
- Deficiencies of adenosine deaminase and purine nucleoside phosphorylase: Implimcations for immunopharmacology Med Gr Rounds 1982: 85-93
- Adult T-cell leukaemia-lymphoma with unusual phenotype The Lancet (Letter) 1982: 1273-1274
INHIBITION OF PURINE NUCLEOSIDE PHOSPHORYLASE BY 8-AMINOGUANOSINE - SELECTIVE TOXICITY FOR T-LYMPHOBLASTS
1981; 214 (4525): 1137-1139
The guanosine analog 8-aminoguanosine is an effective inhibitor of the purine degradative enzyme purine nucleoside phosphorylase, both in vitro and in intact lymphoid cells. In a human lymphoblast tissue culture system, 8-aminoguanosine, in combination with low concentrations of 2'-deoxyguanosine, causes toxicity toward T cells but not B cells. The selective T cell toxicity correlates with increased accumulation of deoxyguanosine triphosphate in the treated T lymphoblasts.
View details for Web of Science ID A1981MR19800027
View details for PubMedID 6795718
INHIBITION OF ADENOSINE-DEAMINASE ACTIVITY RESULTS IN CYTO-TOXICITY TO T-LYMPHOBLASTS INVIVO
1980; 56 (3): 556-559
We have treated a patient with refractory T-cell acute lymphoblastic leukemia with 2'-deoxycoformycin, a potent inhibitor of the enzyme adenosine deaminase. Inhibition of adenosine deaminase activity resulted in (1) an abrupt rise in plasma deoxyadenosine, but not adenosine, concentrations; (2) accumulation of deoxyadenosine triphosphate by lymphoblasts; (3) inhibition of the enzyme S-adenoylhomocysteine hydrolase; and (4) rapid lysis of the leukemic cells. The patient died suddenly 3 days after therapy was discontinued, and postmortem examination revealed a complete absence of leukemic cells in all organs. Pharmacologic inhibition of adenosine deaminase activity can result in the lysis of T lymphoblasts in vivo, and this effect appears to be mediated by deoxyadenosine.
View details for Web of Science ID A1980KG49500034
View details for PubMedID 6967747
PURINOGENIC IMMUNODEFICIENCY DISEASES - CLINICAL-FEATURES AND MOLECULAR MECHANISMS
ANNALS OF INTERNAL MEDICINE
1980; 92 (6): 826-831
Deficiencies of two enzymes that catalyze sequential reactions in the purine catabolic pathway have been causally associated with immunodeficiency states. Adenosine deaminase (ADA) deficiency results in severe combined immunodeficiency disease, while purine nucleoside phosphorylase (PNP) deficiency results in an isolated T-cell defect. Recent work in this area has provided major new insights into the molecular pathology of these syndromes. Deoxyadenosine and deoxyguanosine, substrates that accumulate in ADA and deoxyguanosine, substrates that accumulate in ADA and PNP deficiency, respectively, appear to be selectively phosphorylated by lymphoid cells to the corresponding deoxynucleoside triphosphate, resulting in inhibition of DNA synthesis in these cells. Both deoxynucleosides are far more toxic to cultured T lymphoblasts than to B lymphoblasts. Adenosine and deoxyadenosine may have additional lymphotoxic effects mediated by inhibition of essential methylation reactions. These observations help to explain the immunologic manifestations of ADA and PNP deficiency. Perhaps more important, they lay the foundation for the use of deoxynucleosides or enzyme inhibitors, or both, as selective immunosuppressive and chemotherapeutic agents.
View details for Web of Science ID A1980JW13400019
View details for PubMedID 6247948
MULTIPLE-MYELOMA - CURRENT CONCEPTS IN DIAGNOSIS AND MANAGEMENT
MEDICAL CLINICS OF NORTH AMERICA
1980; 64 (4): 729-742
The basic pathologic process in multiple myeloma is the neoplastic proliferation of a single clone of plasma cells. Although the events which trigger autonomous cell growth are not well understood, the secretion of an M component, a serum or urinary immunoglobulin molecule or a light chain fragment by the vast majority of myeloma cells has provided a biologic marker which has greatly facilitated the study of this disease Some of the more recent clinical concepts which have evolved from studies on the plasma cell and the immunoglobulin molecule are discussed.
View details for Web of Science ID A1980KA54200009
View details for PubMedID 6995732
ATP DEPLETION AS A CONSEQUENCE OF ADENOSINE-DEAMINASE INHIBITION IN MAN
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES
1980; 77 (10): 6157-6161
Hereditary deficiency of the enzyme adenosie deaminase (adenosine aminohydrolase, EC 22.214.171.124) results in an immunodeficiency syndrome characterized by a marked reduction in circulating lymphocytes. We have administered 2'-deoxycoformycin, a potent inhibitor of adenosine deaminase, to a patient with a lymphoproliferative malignancy. The clinical consequences of pharmacologic inhibition of adenosine deaminase activity included an abrupt decrease in the lymphocyte count, abnormalities of renal and hepatic function, and hemolytic anemia. The plasma concentrations of adenosine and deoxyadenosine rose to peak values of 13 microM and 5 microM, respectively, and erythrocyte dATP levels increased to 110 pmol/10(6) cells over 9 days. There was a corresponding decrease in erythrocyte ATP levels from 128 to < 6 pmol/10(6) cells. A similar profound reductin in ATP occurred in the erythrocytes of a second patient. The rapid and unexpected depletion of ATP associated with dATP accumulation may account, at least in part, for the toxicity associated with 2'-deoxycoformycin administration. The inverse relationship of ATP and dATP raises major questions about the control of energy metabolism in erythrocytes.
View details for Web of Science ID A1980KN96400112
View details for PubMedID 6969403
- AN ASSAY OF DEOXYADENOSINE AND ADENOSINE IN HUMAN-PLASMA BY HPLC BIOCHEMICAL MEDICINE 1980; 24 (2): 179-184
Treatment of acute lymphoblastic leukemia with the adenosine deaminase inhibitor 2'-deoxycoformycin.
Advances in experimental medicine and biology
1979; 122B: 347-350
View details for PubMedID 317568
TREATMENT OF ACUTE LYMPHOBLASTIC-LEUKEMIA WITH 2'-DEOXYCOFORMYCIN - CLINICAL AND BIOCHEMICAL CONSEQUENCES OF ADENOSINE-DEAMINASE INHIBITION
CANCER TREATMENT REPORTS
1979; 63 (11-1): 1949-1952
View details for Web of Science ID A1979JG46600041
Possible role for 5'-nucleotidase in deoxyadenosine selective toxicity to cultured human lymphoblasts.
Advances in experimental medicine and biology
1979; 122B: 243-259
View details for PubMedID 232631
BIOCHEMICAL BASIS FOR DIFFERENTIAL DEOXYADENOSINE TOXICITY TO LYMPHOBLAST-T AND LYMPHOBLAST-B - ROLE FOR 5'-NUCLEOTIDASE
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1979; 76 (5): 2434-2437
Deoxyadenosine metabolism was investigated in cultured human cells to elucidate the biochemical basis for the sensitivity of T lymphoblasts and the resistance of B lymphoblasts to deoxyadenosine toxicity. T lymphoblasts have a 20-to 45-fold greater capacity to synthesize deoxyadenosine nucleotides than B lymphoblasts at deoxyadenosine concentrations of 50--300 micron. During the synthesis of dATP, T lymphoblasts accumulate large quantities of dADP, whereas B lymphoblasts do not accumulate dADP. Enzymes affecting deoxyadenosine nucleotide synthesis were assayed in these cells. No substantial differences were evident in activities of deoxyadenosine kinase (ATP: deoxyadenosine 5'-phosphotransferase, EC 126.96.36.199) or deoxyadenylate kinase [ATP:(d)AMP phosphotransferase, EC 188.8.131.52]. The activity of 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 184.108.40.206) was increased 44-fold for AMP and 7-fold for dAMP in B lymphoblasts. A model for the regulation of deoxyadenosine nucleotide synthesis by 5'-nucleotidase activity is proposed on the basis of the observations.
View details for Web of Science ID A1979GW30700076
View details for PubMedID 221924
Molecular mechanism(s) of deoxyribonucleoside toxicity in T-lymphoblasts.
Advances in experimental medicine and biology
1979; 122B: 265-270
View details for PubMedID 317565
PURINOGENIC IMMUNODEFICIENCY DISEASES - DIFFERENTIAL EFFECTS OF DEOXYADENOSINE AND DEOXYGUANOSINE ON DNA-SYNTHESIS IN HUMAN T-LYMPHOBLASTS
JOURNAL OF CLINICAL INVESTIGATION
1979; 64 (5): 1475-1484
Deoxyadenosine and deoxyguanosine are toxic to human lymphoid cells in culture and have been implicated in the pathogenesis of the immunodeficiency states associated with adenosine deaminase and purine nucleoside phosphorylase deficiency, respectively. We have studied the relative incorporation of several labeled nucleosides into DNA and into nucleotide pools to further elucidate the mechanism of deoxyribonucleoside toxicity. In the presence of an inhibitor of adenosine deaminase [erythro-9-(2-hydroxy-3-nonyl)adenine [EHNA], 5 muM], deoxyadenosine (1-50 muM) progressively decreased the incorporation of thymidine, uridine, and deoxyuridine into DNA, but did not affect uridine incorporation into RNA. This decrease in DNA synthesis was associated with increasing dATP and decreasing dCTP pools. Likewise, incubation of cells with deoxyguanosine caused an elevation of dGTP, depletion of dCTP, and inhibition of DNA synthesis. To test the hypothesis that dATP and dGTP accumulation inhibit DNA synthesis by inhibiting the enzyme ribonucleotide reductase, simultaneous rates of incorporation of [(3)H]uridine and [(14)C]thymidine into DNA were measured in the presence of deoxyadenosine plus EHNA or deoxyguanosine, and in the presence of hydroxyurea, a known inhibitor of ribonucleotide reductase. Hydroxyurea (100 muM) and deoxyguanosine (10 muM) decreased the incorporation of [(3)H]uridine but not of [(14)C]thymidine into DNA; both compounds also substantially increased [(3)H]cytidine incorporation into the ribonucleotide pool while reducing incorporation into the deoxyribonucleotide pool. In contrast, deoxyadenosine plus EHNA did not show this differential inhibition of [(3)H]uridine incorporation into DNA, and the alteration in [(3)H]cytidine incorporation into nucleotide pools was less impressive. These data show an association between accumulation of dATP or dGTP and a primary inhibition of DNA synthesis, and they provide support for ribonucleotide reductase inhibition as the mechanism responsible for deoxyguanosine toxicity. Deoxyadenosine toxicity, however, appears to result from another, or perhaps a combination of, molecular event(s).
View details for Web of Science ID A1979HS54100040
View details for PubMedID 115901
PURINOGENIC IMMUNODEFICIENCY DISEASES - SELECTIVE TOXICITY OF DEOXYRIBONUCLEOSIDES FOR T-CELLS
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1978; 75 (10): 5011-5014
Deoxyadenosine at low concentrations and in the presence of an inhibitor of adenosine deaminase (adenosine aminohydrolase, EC 220.127.116.11) is markedly toxic to lymphoblast cell lines of T cell origin but does not impair growth of B cell lines. Deoxyguanosine is also more toxic for T lymphoblasts. In the presence of deoxyadenosine or deoxyguanosine, elevation of the corresponding deoxyribonucleoside triphosphate (dATP or dGTP) occurs in T cell, but not in B cell, lines. The addition of deoxycytidine or dipyridamole results in lower dATP and dGTP levels and prevents deoxyribonucleoside toxicity. These findings provide a molecular basis for the immunodeficiency observed in individuals with several inborn errors of purine metabolism.
View details for Web of Science ID A1978FU61400084
View details for PubMedID 311004
- Uridine diphosphate galactose 4'-epimerase deficiency Helv Paediat Acta 1976; IV (31): 441-454
- Granulocyte and erythroid colony forming cells from rat marrow: Studies of physical and cell cycle characteristics and competition for expression In Erythropoiesis, Nakao K, Fisher J, Pakaku F (Eds) University Park Press 1975
- Reversal of UDP-galactose 4-epimerase deficiency of human leukocytes in culture Proc Nat Acad Sci (USA) 1975: 5026-5030
- EPIDEMIOLOGICAL ASPECTS OF NEOPLASTIC DISORDERS IN ISRAELI MIGRANT POPULATION .V. LYMPHOMAS JOURNAL OF THE NATIONAL CANCER INSTITUTE 1969; 42 (3): 375-?
- The effect of phenylthiourea on melanogenesis of Fundulus Heteroclitus Biological Bulletin 1966: 398-399
- SEX CHROMATIN DETERMINATIONS IN SELECTED CASES OF DEVELOPMENTAL SEX ABNORMALITIES WITH AN ASSESSMENT OF RESULTS ACTA CYTOLOGICA 1963; 7 (3): 151-?