Professor of Radiation Oncology, Associate Chair for Research & Director of the Division of Radiation & Cancer Biology in the Department of Radiation Oncology. He also heads the Radiation Biology Program in Stanford’s Cancer Center, and is Director of the Cancer Biology Interdisciplinary Graduate Program. Professor Giaccia was awarded an American Cancer Society Junior Faculty Research Award and the Michael Fry Award from the Radiation Research Society for his outstanding contributions on understanding the molecular mechanisms of resistance promoted by the tumor microenvironment. Additionally, he was the recipient of the 2013 ASTRO Gold Medal. Professor Giaccia has co-authored the sixth & seventh editions of the textbook, “Radiation Biology for the Radiologist,” with Professor Eric Hall from Columbia. In addition, he is currently the “Jack, Lulu and Sam Willson Professor in Cancer Biology” at Stanford University School of Medicine.
Associate Director for Basic Science, Stanford Cancer Institute (2012 - Present)
Associate Chair, Research, Department of Radiation Oncology, Stanford University School of Medicine (2011 - Present)
Leader, Molecular Therapeutics Program, Stanford Cancer Center (2011 - Present)
Honors & Awards
ASTRO Gold Medal, American Society of Therapeutic Radiology and Oncology (2013)
MERIT Award, NIH (2010)
Jack, Lulu and Sam Willson Endowed Professor of Cancer Biology, Stanford University (2006)
Virginia Logan Lecture, Thomas Jefferson University (2003)
John Yuhas Award (Excellence in Radiation Oncology, University of Pennsylvania), University of Pennsylvania (2000)
Michael Fry Research Awardee of the Radiation Research Society, Radiation Research Society (1997)
Howard Hughes Junior Faculty Award, Stanford University (1996)
American Cancer Society Junior Faculty Research Award, Stanford University (1995)
Boards, Advisory Committees, Professional Organizations
Editorial Board Member, International Journal of Radiation Biology (1995 - 2000)
Associate Editor, International Journal of Radiation Biology (2000 - 2009)
Associate Editor, Radiation Research, Official Journal of the Radiation Research Society (1998 - 2002)
Editorial Board, International Journal of Radiation Oncology, Biology, Physics (2000 - Present)
Foreign Associate Editor, Journal of Radiation Research, Official Journal of the Japan Radiation Research Society (2004 - Present)
Associate Editor, Cancer Biology and Therapy (2001 - Present)
Deputy Editor for Reviews and Senior Editor, Molecular Cancer Research (2002 - Present)
Associate Editor, Cancer Research (2003 - Present)
Editorial Advisory Board, Current Cancer Therapy Reviews (2005 - Present)
Ph.D., University of Pennsylvania, Pathology/Molecular Biology (1989)
B.A., Lafayette College, Biology (1980)
Current Research and Scholarly Interests
During the last five years, we have identified several small molecules that acted to kill VHL deficient renal cancer cells through a synthetic lethal screening approach. We published on one of the molecules (Cancer Cell 14: 90, 2008) that killed VHL deficient tumor and are performing screens to identify new therapeutics against other targets. Another major interest of my laboratory is in identifying hypoxia-induced genes involved in invasion and metastases. We have identified and characterized several genes that are induced by hypoxia and promote metastases in breast, ovarian, renal and head and neck cancer (Nature 440:1222, 2006, Cancer Cell 15:35, 2009) and are developing therapeutics against them. My group is also investigating how hypoxia regulates gene expression epigenetically through the regulation of histone demethylases and microRNAs.
Imaging and Biomarkers of Hypoxia in Solid Tumors
We hope to find safer, noninvasive methods for measuring tumor hypoxia. Hypoxia, meaning a lack of oxygen, has been associated strongly with a wide range of human cancers. Hypoxia occurs when tumor growth exceeds the ability of blood vessels to supply the tumor with oxygenated blood. It is currently understood that hypoxic tumors are more aggressive. Current methods for measuring hypoxia include invasive procedures such as tissue biopsy, or insertion of an electrode into the tumor. EF5-PET may be a non-invasive way to make this measurement.
Phase I Trial of Metabolic Reprogramming Therapy for Treatment of Recurrent Head and Neck Cancers
The purpose of this study is to study the effect of the drug DCA (dichloroacetate) on recurrent head and neck cancers. Part of this study will also use EF5 PET scan to study tumor hypoxia.
- Cancer Biology Journal Club
CBIO 280 (Aut, Win, Spr)
- Cellular Metabolism: An Emerging Hallmark of Cancer and Aging
BIOS 240 (Win)
- Molecular, Cellular, and Genetic Basis of Cancer
CBIO 241 (Aut)
- Program in Radiation Biology Seminar Series
RADO 244 (Aut, Win, Spr)
- The Basic Science of Radiation and Cancer Biology
RADO 202 (Spr)
Independent Studies (10)
- Directed Reading in Cancer Biology
CBIO 299 (Aut, Win, Spr, Sum)
- Directed Reading in Radiation Oncology
RADO 299 (Aut, Win, Spr, Sum)
- Early Clinical Experience in Radiation Oncology
RADO 280 (Aut, Win, Spr, Sum)
- Graduate Research
CBIO 399 (Aut, Win, Spr, Sum)
- Graduate Research
RADO 399 (Aut, Win, Spr, Sum)
- Medical Scholars Research
RADO 370 (Aut, Win, Spr, Sum)
- Out-of-Department Advanced Research Laboratory in Experimental Biology
BIO 199X (Sum)
- Readings in Radiation Biology
RADO 101 (Aut, Win, Spr, Sum)
- Teaching in Cancer Biology
CBIO 260 (Spr)
- Undergraduate Research
RADO 199 (Aut, Win, Spr, Sum)
- Directed Reading in Cancer Biology
Prior Year Courses
- Cancer Biology Journal Club
CBIO 280 (Aut, Win, Spr)
- Molecular, Cellular, and Genetic Basis of Cancer
CBIO 241 (Win)
- Radiation and Cancer Biology Seminar Series
RADO 244 (Aut, Win, Spr)
- Cancer Biology Journal Club
CBIO 280 (Aut, Win, Spr)
- Molecular, Cellular, and Genetic Basis of Cancer
CBIO 241 (Win)
- Principles of Cancer Systems Biology
CBIO 243 (Spr)
- Radiation and Cancer Biology Seminar Series
RADO 244 (Aut, Win, Spr)
- Cancer Biology Journal Club
CTGF is a therapeutic target for metastatic melanoma
2014; 33 (9): 1093-1100
Metastatic melanoma remains a devastating disease with a 5-year survival rate of less than five percent. Despite recent advances in targeted therapies for melanoma, only a small percentage of melanoma patients experience durable remissions. Therefore, it is critical to identify new therapies for the treatment of advanced melanoma. Here, we define connective tissue growth factor (CTGF) as a therapeutic target for metastatic melanoma. Clinically, CTGF expression correlates with tumor progression and is strongly induced by hypoxia through HIF-1 and HIF-2-dependent mechanisms. Genetic inhibition of CTGF in human melanoma cells is sufficient to significantly reduce orthotopic tumor growth, as well as metastatic tumor growth in the lung of severe combined immunodeficient (SCID) mice. Mechanistically, inhibition of CTGF decreased invasion and migration associated with reduced matrix metalloproteinase-9 expression. Most importantly, the anti-CTGF antibody, FG-3019, had a profound inhibitory effect on the progression of established metastatic melanoma. These results offer the first preclinical validation of anti-CTGF therapy for the treatment of advanced melanoma and underscore the importance of tumor hypoxia in melanoma progression.Oncogene advance online publication, 25 February 2013; doi:10.1038/onc.2013.47.
View details for DOI 10.1038/onc.2013.47
View details for Web of Science ID 000331933800003
View details for PubMedID 23435419
A novel aldehyde dehydrogenase-3 activator (Alda-89) protects submandibular gland function from irradiation without accelerating tumor growth.
Clinical cancer research
2013; 19 (16): 4455-4464
To determine the effect of Alda-89 (an ALDH3 activitor) on (1) the function of irradiated (RT) submandibular gland (SMG) in mice, (2) its toxicity profile and (3) its effect on the growth of head and neck cancer (HNC) in vitro and in vivo.Adult mice were infused with Alda-89 or vehicle before, during and after RT. Saliva secretion was monitored weekly. Hematology, metabolic profile and post-mortem evaluation for toxicity were examined at the time of sacrifice. Alda-89 or vehicle was applied to HNC cell lines in vitro, and SCID mice transplanted with HNC in vivo with or without radiation; HNC growth was monitored. The ALDH3A1 and ALDH3A2 protein expression was evaluated in 89 HNC patients and correlated to freedom from relapse (FFR) and overall survival (OS).Alda-89 infusion significantly resulted in more whole saliva production and a higher percentage of preserved acini after RT compared to vehicle control. There was no difference in the complete blood count, metabolic profile, and major organ morphology between the Alda-89 and vehicle groups. Compared to vehicle control, Alda-89 treatment did not accelerate HNC cell proliferation in vitro, nor did it affect tumor growth in vivo with or without RT. Higher expression of ALDH3A1 or ALDH3A2 was not significantly associated with worse FFR or OS in either HPV-positive or HPV-negative group.Alda-89 preserves salivary function after RT without affecting HNC growth or causing measurable toxicity in mice. It is a promising candidate to mitigate RT-related xerostomia.
View details for DOI 10.1158/1078-0432.CCR-13-0127
View details for PubMedID 23812668
- A low-carb diet kills tumor cells with a mutant p53 tumor suppressor gene: the Atkins diet suppresses tumor growth. Cell cycle 2013; 12 (5): 718-719
Regulation of Bone Marrow Angiogenesis by Osteoblasts during Bone Development and Homeostasis.
Frontiers in endocrinology
2013; 4: 85-?
Bone marrow is a highly heterogeneous and vascularized tissue. The various cell types populating the bone marrow extensively communicate with each other, and cell-to-cell cross talk is likely to be essential for proper bone development and homeostasis. In particular, the existence of osteogenesis and angiogenesis coupling has been recently proposed. Despite its high degree of vascularization, a gradient of oxygenation is present in the bone marrow, and the endosteal surface of cortical bone appears to be among the most hypoxic areas in the body. Oxygen (O2) is both an essential metabolic substrate and a regulatory signal that is in charge of a specific genetic program. An important component of this program is the family of transcription factors known as hypoxia-inducible factors (HIFs). In this Perspective, we will summarize our current knowledge about the role of the HIF signaling pathway in controlling bone development and homeostasis, and especially in regulating the crosstalk between osteoblasts, progenitor cells, and bone marrow blood vessels.
View details for DOI 10.3389/fendo.2013.00085
View details for PubMedID 23847596
The mitochondrial citrate transporter, CIC, is essential for mitochondrial homeostasis
2012; 3 (10): 1220-1235
Dysregulation of the pathways that preserve mitochondrial integrity hallmarks many human diseases including diabetes, neurodegeration, aging and cancer. The mitochondrial citrate transporter gene, SLC25A1 or CIC, maps on chromosome 22q11.21, a region amplified in some tumors and deleted in developmental disorders known as velo-cardio-facial- and DiGeorge syndromes. We report here that in tumor cells CIC maintains mitochondrial integrity and bioenergetics, protects from mitochondrial damage and circumvents mitochondrial depletion via autophagy, hence promoting proliferation. CIC levels are increased in human cancers and its inhibition has anti-tumor activity, albeit with no toxicity on adult normal tissues. The knock-down of the CIC gene in zebrafish leads to mitochondria depletion and to proliferation defects that recapitulate features of human velo-cardio-facial syndrome, a phenotype rescued by blocking autophagy. Our findings reveal that CIC maintains mitochondrial homeostasis in metabolically active, high proliferating tissues and imply that this protein is a therapeutic target in cancer and likely, in other human diseases.
View details for Web of Science ID 000312410800019
View details for PubMedID 23100451
Global alteration in gene expression profiles of deciduas from women with idiopathic recurrent pregnancy loss
MOLECULAR HUMAN REPRODUCTION
2012; 18 (9): 442-450
Recurrent pregnancy loss (RPL) occurs in ?5% of women. However, the etiology is still poorly understood. Defects in decidualization of the endometrium during early pregnancy contribute to several pregnancy complications, such as pre-eclampsia and intrauterine growth restriction (IUGR), and are believed to be important in the pathogenesis of idiopathic RPL. We performed microarray analysis to identify gene expression alterations in the deciduas of idiopathic RPL patients. Control patients had one antecedent term delivery, but were undergoing dilation and curettage for current aneuploid miscarriage. Gene expression differences were evaluated using both pathway and gene ontology (GO) analysis. Selected genes were validated using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). A total of 155 genes were found to be significantly dysregulated in the deciduas of RPL patients (>2-fold change, P < 0.05), with 22 genes up-regulated and 133 genes down-regulated. GO analysis linked a large percentage of genes to discrete biological functions, including immune response (23%), cell signaling (18%) and cell invasion (17.1%), and pathway analysis revealed consistent changes in both the interleukin 1 (IL-1) and IL-8 pathways. All genes in the IL-8 pathway were up-regulated while genes in the IL-1 pathway were down-regulated. Although both pathways can promote inflammation, IL-1 pathway activity is important for normal implantation. Additionally, genes known to be critical for degradation of the extracellular matrix, including matrix metalloproteinase 26 and serine peptidase inhibitor Kazal-type 1, were also highly up-regulated. In this first microarray approach to decidual gene expression in RPL patients, our data suggest that dysregulation of genes associated with cell invasion and immunity may contribute significantly to idiopathic recurrent miscarriage.
View details for DOI 10.1093/molehr/gas017
View details for Web of Science ID 000308243000003
View details for PubMedID 22505054
State of the Science: An Update on Renal Cell Carcinoma
MOLECULAR CANCER RESEARCH
2012; 10 (7): 859-880
Renal cell carcinomas (RCC) are emerging as a complex set of diseases that are having a major socioeconomic impact and showing a continued rise in incidence throughout the world. As the field of urologic oncology faces these trends, several major genomic and mechanistic discoveries are altering our core understanding of this multitude of cancers, including several new rare subtypes of renal cancers. In this review, these new findings are examined and placed in the context of the well-established association of clear cell RCC (ccRCC) with mutations in the von Hippel-Lindau (VHL) gene and resultant aberrant hypoxia inducible factor (HIF) signaling. The impact of novel ccRCC-associated genetic lesions on chromatin remodeling and epigenetic regulation is explored. The effects of VHL mutation on primary ciliary function, extracellular matrix homeostasis, and tumor metabolism are discussed. Studies of VHL proteostasis, with the goal of harnessing the proteostatic machinery to refunctionalize mutant VHL, are reviewed. Translational efforts using molecular tools to elucidate discriminating features of ccRCC tumors and develop improved prognostic and predictive algorithms are presented, and new therapeutics arising from the earliest molecular discoveries in ccRCC are summarized. By creating an integrated review of the key genomic and molecular biological disease characteristics of ccRCC and placing these data in the context of the evolving therapeutic landscape, we intend to facilitate interaction among basic, translational, and clinical researchers involved in the treatment of this devastating disease, and accelerate progress toward its ultimate eradication.
View details for DOI 10.1158/1541-7786.MCR-12-0117
View details for Web of Science ID 000308027300001
View details for PubMedID 22638109
- Blood and bones Osteoblastic HIF signaling regulates erythropoiesis CELL CYCLE 2012; 11 (12): 2221-2222
The HIF Signaling Pathway in Osteoblasts Directly Modulates Erythropoiesis through the Production of EPO
2012; 149 (1): 63-74
Osteoblasts are an important component of the hematopoietic microenvironment in bone. However, the mechanisms by which osteoblasts control hematopoiesis remain unknown. We show that augmented HIF signaling in osteoprogenitors results in HSC niche expansion associated with selective expansion of the erythroid lineage. Increased red blood cell production occurred in an EPO-dependent manner with increased EPO expression in bone and suppressed EPO expression in the kidney. In contrast, inactivation of HIF in osteoprogenitors reduced EPO expression in bone. Importantly, augmented HIF activity in osteoprogenitors protected mice from stress-induced anemia. Pharmacologic or genetic inhibition of prolyl hydroxylases1/2/3 in osteoprogenitors elevated EPO expression in bone and increased hematocrit. These data reveal an unexpected role for osteoblasts in the production of EPO and modulation of erythropoiesis. Furthermore, these studies demonstrate a molecular role for osteoblastic PHD/VHL/HIF signaling that can be targeted to elevate both HSCs and erythroid progenitors in the local hematopoietic microenvironment.
View details for DOI 10.1016/j.cell.2012.01.051
View details for Web of Science ID 000302235400010
View details for PubMedID 22464323
Prognostic and Predictive Significance of Plasma HGF and IL-8 in a Phase III Trial of Chemoradiation with or without Tirapazamine in Locoregionally Advanced Head and Neck Cancer
CLINICAL CANCER RESEARCH
2012; 18 (6): 1798-1807
Hepatocyte growth factor (HGF) is a hypoxia-induced secreted protein that binds to cMet and regulates interleukin (IL)-8 expression. We evaluated the role of circulating HGF and IL-8 as prognostic and predictive factors for efficacy of tirapazamine (TPZ), a hypoxic cell cytotoxin.Patients with stages III to IV head and neck cancer were randomized to receive radiotherapy with cisplatin (CIS) or CIS plus TPZ (TPZ/CIS). Eligibility for the substudy included plasma sample availability for HGF and IL-8 assay by ELISA and no major radiation deviations (N = 498). Analyses included adjustment for major prognostic factors. p16(INK4A) staining (human papillomavirus surrogate) was carried out on available tumors. Thirty-nine patients had hypoxia imaging with (18)F-fluoroazomycin arabinoside ((18)FAZA)-positron emission tomography.Elevated IL-8 level was associated with worse overall survival (OS) irrespective of treatment. There was an interaction between HGF and treatment arm (P = 0.053); elevated HGF was associated with worse OS in the control but not in the TPZ/CIS arm. Similar trends were observed in analyses restricted to p16(INK4A)-negative patients. Four subgroups defined by high and low HGF/IL-8 levels were examined for TPZ effect; the test for interaction with arm was P = 0.099. TPZ/CIS seemed to be beneficial for patients with high HGF and IL-8 but adverse for low HGF and high IL-8. Only HGF correlated with (18)FAZA tumor standard uptake value.IL-8 is an independent prognostic factor irrespective of treatment. There is an interaction between HGF and treatment arm. Certain subgroups based on IL-8/HGF levels seemed to do better with TPZ/CIS while others did worse, highlighting the complexity of hypoxia targeting in unselected patients.
View details for DOI 10.1158/1078-0432.CCR-11-2094
View details for Web of Science ID 000301672400037
View details for PubMedID 22383739
VEGF-independent cell-autonomous functions of HIF-1a regulating oxygen consumption in fetal cartilage are critical for chondrocyte survival
JOURNAL OF BONE AND MINERAL RESEARCH
2012; 27 (3): 596-609
Fetal growth plate cartilage is nonvascularized, and chondrocytes largely develop in hypoxic conditions. We previously found that mice lacking the hypoxia-inducible transcription factor HIF-1? in cartilage show massive death of centrally located, hypoxic chondrocytes. A similar phenotype was observed in mice with genetic ablation of either all or specifically the diffusible isoforms of vascular endothelial growth factor (VEGF), a prime angiogenic target of HIF-1?. Here, we assessed whether VEGF is a critical downstream component of the HIF-1?-dependent survival pathway in chondrocytes. We used a genetic approach to conditionally overexpress VEGF164 in chondrocytes lacking HIF-1?, evaluating potential rescuing effects. The effectiveness of the strategy was validated by showing that transgenic expression of VEGF164 in Col2-Cre;VEGF(f/f) mice stimulated angiogenesis in the perichondrium, fully corrected the excessive hypoxia of VEGF-deficient chondrocytes, and completely prevented chondrocyte death. Yet, similarly crossed double-mutant embryos lacking HIF-1? and overexpressing VEGF164 in the growth plate cartilage still displayed a central cell death phenotype, albeit slightly delayed and less severe compared with mice exclusively lacking HIF-1?. Transgenic VEGF164 induced massive angiogenesis in the perichondrium, yet this only partially relieved the aberrant hypoxia present in HIF-1?-deficient cartilage and thereby likely inflicted only a partial rescue effect. In fact, excessive hypoxia and failure to upregulate phosphoglycerate-kinase 1 (PGK1), a key enzyme of anaerobic glycolytic metabolism, were among the earliest manifestations of HIF-1? deficiency in cartilaginous bone templates, and reduced PGK1 expression was irrespective of transgenic VEGF164. These findings suggest that HIF-1? activates VEGF-independent cell-autonomous mechanisms to sustain oxygen levels in the challenged avascular cartilage by reducing oxygen consumption. Hence, regulation of the metabolic pathways by HIF-1? and VEGF-dependent regulation of angiogenesis coordinately act to maintain physiological cartilage oxygenation. We conclude that VEGF and HIF-1? are critical preservers of chondrocyte survival by ensuring an adequate balance between availability and handling of oxygen in developing growth cartilage.
View details for DOI 10.1002/jbmr.1487
View details for Web of Science ID 000300682500010
View details for PubMedID 22162090
Prognostic Significance of Plasma Osteopontin in Patients with Locoregionally Advanced Head and Neck Squamous Cell Carcinoma Treated on TROG 02.02 Phase III Trial
CLINICAL CANCER RESEARCH
2012; 18 (1): 301-307
High plasma osteopontin (OPN) levels have been reported to be an adverse prognostic factor in head and neck squamous cell carcinomas (HNSCC), correlate with tumor hypoxia, and be predictive of benefit from hypoxia-targeted therapy. We sought to confirm the prognostic and predictive significance of OPN in patients treated on a large international trial.Patients with stage III/IV HNSCC were randomized to receive definitive radiotherapy concurrently with cisplatin or cisplatin plus the hypoxic cell cytotoxin, tirapazamine (TPZ). Eligibility criteria for this prospective substudy included plasma sample availability for OPN assay by ELISA and absence of major radiation therapy deviations (N = 578). OPN concentrations were analyzed for overall survival (OS) and time to locoregional failure (TTLRF), adjusting for known prognostic factors. Additional analysis was carried out in patients with available tumor p16(INK4A) staining status.The median OPN level was 544 ng/mL (range: 7-2,640). High OPN levels were not associated with worse OS (relative HR, 1.03 for highest tertile) or TTLRF (relative HR 0.91 for highest tertile). There was no interaction between OPN and treatment arm for OS or TTLRF (P = 0.93 for OS; P = 0.87 for TTLRF). For the highest tertile the 2-year OS was 66% on control arm and 67% on TPZ arm (HR = 1.11, P = 0.67). Similarly for p16(INK4A) negative patients in the highest tertile, the 2-year OS was 61% on control arm and 63% on TPZ arm (HR = 1.05, P = 0.86).We found no evidence that high plasma OPN levels were associated with an adverse prognosis in HNSCC, or were predictive of benefit with hypoxia targeting therapy.
View details for DOI 10.1158/1078-0432.CCR-11-2295
View details for Web of Science ID 000298758900032
View details for PubMedID 22096023
Targeting GLUT1 and the Warburg Effect in Renal Cell Carcinoma by Chemical Synthetic Lethality
SCIENCE TRANSLATIONAL MEDICINE
2011; 3 (94)
Identifying new targeted therapies that kill tumor cells while sparing normal tissue is a major challenge of cancer research. Using a high-throughput chemical synthetic lethal screen, we sought to identify compounds that exploit the loss of the von Hippel-Lindau (VHL) tumor suppressor gene, which occurs in about 80% of renal cell carcinomas (RCCs). RCCs, like many other cancers, are dependent on aerobic glycolysis for ATP production, a phenomenon known as the Warburg effect. The dependence of RCCs on glycolysis is in part a result of induction of glucose transporter 1 (GLUT1). Here, we report the identification of a class of compounds, the 3-series, exemplified by STF-31, which selectively kills RCCs by specifically targeting glucose uptake through GLUT1 and exploiting the unique dependence of these cells on GLUT1 for survival. Treatment with these agents inhibits the growth of RCCs by binding GLUT1 directly and impeding glucose uptake in vivo without toxicity to normal tissue. Activity of STF-31 in these experimental renal tumors can be monitored by [(18)F]fluorodeoxyglucose uptake by micro-positron emission tomography imaging, and therefore, these agents may be readily tested clinically in human tumors. Our results show that the Warburg effect confers distinct characteristics on tumor cells that can be selectively targeted for therapy.
View details for DOI 10.1126/scitranslmed.3002394
View details for Web of Science ID 000293459700004
View details for PubMedID 21813754
Tumor Galectin-1 Mediates Tumor Growth and Metastasis through Regulation of T-Cell Apoptosis
2011; 71 (13): 4423-4431
Galectin-1 (Gal-1), a carbohydrate-binding protein whose secretion is enhanced by hypoxia, promotes tumor aggressiveness by promoting angiogenesis and T-cell apoptosis. However, the importance of tumor versus host Gal-1 in tumor progression is undefined. Here we offer evidence that implicates tumor Gal-1 and its modulation of T-cell immunity in progression. Comparing Gal-1-deficient mice as hosts for Lewis lung carcinoma cells where Gal-1 levels were preserved or knocked down, we found that tumor Gal-1 was more critical than host Gal-1 in promoting tumor growth and spontaneous metastasis. Enhanced growth and metastasis associated with Gal-1 related to its immunomodulatory function, insofar as the benefits of Gal-1 expression to Lewis lung carcinoma growth were abolished in immunodeficient mice. In contrast, angiogenesis, as assessed by microvessel density count, was similar between tumors with divergent Gal-1 levels when examined at a comparable size. Our findings establish that tumor rather than host Gal-1 is responsible for mediating tumor progression through intratumoral immunomodulation, with broad implications in developing novel targeting strategies for Gal-1 in cancer.
View details for DOI 10.1158/0008-5472.CAN-10-4157
View details for Web of Science ID 000292287300013
View details for PubMedID 21546572
SAR studies of 4-pyridyl heterocyclic anilines that selectively induce autophagic cell death in von Hippel-Lindau-deficient renal cell carcinoma cells
BIOORGANIC & MEDICINAL CHEMISTRY
2011; 19 (11): 3347-3356
We recently identified a class of pyridyl aniline thiazoles (PAT) that displayed selective cytotoxicity for von Hippel-Lindau (VHL) deficient renal cell carcinoma (RCC) cells in vitro and in vivo. Structure-activity relationship (SAR) studies were used to develop a comparative molecular field analysis (CoMFA) model that related VHL-selective potency to the three-dimensional arrangement of chemical features of the chemotype. We now report the further molecular alignment-guided exploration of the chemotype to discover potent and selective PAT analogues. The contribution of the central thiazole ring was explored using a series of five- and six-membered ring heterocyclic replacements to vary the electronic and steric interactions in the central unit. We also explored a positive steric CoMFA contour adjacent to the pyridyl ring using Pd-catalysed cross-coupling Suzuki-Miyaura, Sonogashira and nucleophilic displacement reactions to prepare of a series of aryl-, alkynyl-, alkoxy- and alkylamino-substituted pyridines, respectively. In vitro potency and selectivity were determined using paired RCC cell lines: the VHL-null cell line RCC4 and the VHL-positive cell line RCC4-VHL. Active analogues selectively induced autophagy in RCC4 cells. We have used the new SAR data to further develop the CoMFA model, and compared this to a 2D-QSAR method. Our progress towards realising the therapeutic potential of this chemotype as a targeted cytotoxic therapy for the treatment of RCC by exploiting the absence of the VHL tumour suppressor gene is reported.
View details for DOI 10.1016/j.bmc.2011.04.042
View details for Web of Science ID 000290955400005
View details for PubMedID 21561782
A central role for hypoxic signaling in cartilage, bone, and hematopoiesis.
Current osteoporosis reports
2011; 9 (2): 46-52
Hypoxic signaling plays an essential role in maintaining oxygen homeostasis and cell survival. Hypoxia-inducible transcription factors HIF-1 and HIF-2 are central mediators of the cellular response to hypoxia by regulating the expression of genes controlling metabolic adaptation, oxygen delivery, and survival in response to oxygen deprivation. Recent studies have identified an important role for HIF-1 and HIF-2 in the regulation of skeletal development, bone formation, and regeneration, as well as joint formation and homeostasis. In addition, overexpression of HIF-1 and HIF-2 is clinically associated with osteosarcoma and osteoarthritis. Together, these findings implicate hypoxic signaling as a central regulator of bone biology and disease.
View details for DOI 10.1007/s11914-011-0047-2
View details for PubMedID 21360287
Harnessing synthetic lethal interactions in anticancer drug discovery
NATURE REVIEWS DRUG DISCOVERY
2011; 10 (5): 351-364
Unique features of tumours that can be exploited by targeted therapies are a key focus of current cancer research. One such approach is known as synthetic lethality screening, which involves searching for genetic interactions of two mutations whereby the presence of either mutation alone has no effect on cell viability but the combination of the two mutations results in cell death. The presence of one of these mutations in cancer cells but not in normal cells can therefore create opportunities to selectively kill cancer cells by mimicking the effect of the second genetic mutation with targeted therapy. Here, we summarize strategies that can be used to identify synthetic lethal interactions for anticancer drug discovery, describe examples of such interactions that are currently being investigated in preclinical and clinical studies of targeted anticancer therapies, and discuss the challenges of realizing the full potential of such therapies.
View details for DOI 10.1038/nrd3374
View details for Web of Science ID 000290017300017
View details for PubMedID 21532565
Hypoxia and Senescence: The Impact of Oxygenation on Tumor Suppression
MOLECULAR CANCER RESEARCH
2011; 9 (5): 538-544
Cellular senescence has emerged as a biological response to two major pathophysiological states of our being: cancer and aging. In the course of the transformation of a normal cell to a cancerous cell, senescence is frequently induced to suppress tumor development. In aged individuals, senescence is found in cells that have exhausted their replication potential. The similarity in these responses suggests that understanding how senescence is mediated can provide insight into both cancer and aging. One environmental factor that is implicated in both of these states is tissue hypoxia, which increases with aging and can inhibit senescence. Hypoxia is particularly important in normal physiology to maintain the stem cell niche; but at the same time, hypoxic inhibition of an essential tumor suppressor response can theoretically contribute to cancer initiation.
View details for DOI 10.1158/1541-7786.MCR-11-0065
View details for Web of Science ID 000290610600002
View details for PubMedID 21385881
VHL loss in renal cell carcinoma leads to up-regulation of CUB domain-containing protein 1 to stimulate PKC delta-driven migration
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2011; 108 (5): 1931-1936
A common genetic mutation found in clear cell renal cell carcinoma (CC-RCC) is the loss of the von Hippel-Lindau (VHL) gene, which results in stabilization of hypoxia-inducible factors (HIFs), and contributes to cancer progression and metastasis. CUB-domain-containing protein 1 (CDCP1) was shown to promote metastasis in scirrhous and lung adenocarcinomas as well as in prostate cancer. In this study, we established a molecular mechanism linking VHL loss to induction of the CDCP1 gene through the HIF-1/2 pathway in renal cancer. Also, we report that Fyn, which forms a complex with CDCP1 and mediates its signaling to PKC?, is a HIF-1 target gene. Mechanistically, we found that CDCP1 specifically regulates phosphorylation of PKC?, but not of focal adhesion kinase or Crk-associated substrate. Signal transduction from CDCP1 to PKC? leads to its activation, increasing migration of CC-RCC. Furthermore, patient survival can be stratified by CDCP1 expression at the cell surface of the tumor. Taken together, our data indicates that CDCP1 protein might serve as a therapeutic target for CC-RCC.
View details for DOI 10.1073/pnas.1011777108
View details for Web of Science ID 000286804700036
View details for PubMedID 21233420
- Lack of HIF-2 alpha in limb bud mesenchyme causes a modest and transient delay of endochondral bone development NATURE MEDICINE 2011; 17 (1): 25-26
Control of lysyl oxidase activity through site-specific deuteration of lysine
BIOORGANIC & MEDICINAL CHEMISTRY LETTERS
2011; 21 (1): 255-258
Lysyl oxidase (LOX) is implicated in several extracellular matrix related disorders, including fibrosis and cancer. Methods of inhibition of LOX in vivo include antibodies, copper sequestration and toxic small molecules such as ?-aminopropionitrile. Here, we propose a novel approach to modulation of LOX activity based on the kinetic isotope effect (KIE). We show that 6,6-d(2)-lysine is oxidised by LOX at substantially lower rate, with apparent deuterium effect on V(max)/K(m) as high as 4.35 ± 0.22. Lys is an essential nutrient, so dietary ingestion of D(2)Lys and its incorporation via normal Lys turnover suggests new approaches to mitigating LOX-associated pathologies.
View details for DOI 10.1016/j.bmcl.2010.11.018
View details for Web of Science ID 000285544400049
View details for PubMedID 21106372
- Basic Principles of Radiobiology Breast Surgical Techniques and Interdisciplinary Management 2011: 799-812
Hypoxia in Models of Lung Cancer: Implications for Targeted Therapeutics
CLINICAL CANCER RESEARCH
2010; 16 (19): 4843-4852
To efficiently translate experimental methods from bench to bedside, it is imperative that laboratory models of cancer mimic human disease as closely as possible. In this study, we sought to compare patterns of hypoxia in several standard and emerging mouse models of lung cancer to establish the appropriateness of each for evaluating the role of oxygen in lung cancer progression and therapeutic response.Subcutaneous and orthotopic human A549 lung carcinomas growing in nude mice as well as spontaneous K-ras or Myc-induced lung tumors grown in situ or subcutaneously were studied using fluorodeoxyglucose and fluoroazomycin arabinoside positron emission tomography, and postmortem by immunohistochemical observation of the hypoxia marker pimonidazole. The response of these models to the hypoxia-activated cytotoxin PR-104 was also quantified by the formation of ?H2AX foci in vitro and in vivo. Finally, our findings were compared with oxygen electrode measurements of human lung cancers.Minimal fluoroazomycin arabinoside and pimonidazole accumulation was seen in tumors growing within the lungs, whereas subcutaneous tumors showed substantial trapping of both hypoxia probes. These observations correlated with the response of these tumors to PR-104, and with the reduced incidence of hypoxia in human lung cancers relative to other solid tumor types.These findings suggest that in situ models of lung cancer in mice may be more reflective of the human disease, and encourage judicious selection of preclinical tumor models for the study of hypoxia imaging and antihypoxic cell therapies.
View details for DOI 10.1158/1078-0432.CCR-10-1206
View details for Web of Science ID 000282647900017
View details for PubMedID 20858837
Renal Oxygenation Suppresses VHL Loss-Induced Senescence That Is Caused by Increased Sensitivity to Oxidative Stress
MOLECULAR AND CELLULAR BIOLOGY
2010; 30 (19): 4595-4603
Loss of the VHL tumor suppressor is regarded as an initiating event in the development of clear-cell renal carcinoma. Surprisingly, loss of VHL induces senescence in mouse fibroblasts in vitro, a response that would restrict development of renal carcinoma in vivo. Typical in vitro cell culture levels of oxygen, however, are significantly higher than physiological levels of oxygen, which have been shown to abrogate senescence induced by many stimuli. Therefore, we investigated the oxygen dependence of VHL loss-induced senescence. Using mouse fibroblasts and primary renal epithelial cells in vitro, we found that VHL loss leads to senescence under atmospheric conditions (21% O(2)), partly through increasing p27 levels, but not under physiological oxygenation (2% to 5% O(2)), despite maintaining increased p27 expression. This suggests that VHL inactivation sensitizes cells to oxidative stress. In support of this concept, senescence following VHL loss depends on p53 activity, which decreases under the less stressful conditions of mild hypoxia. We confirmed these observations in vivo by treating kidney-specific VHL knockout animals with the potent oxidizer paraquat and observed a robust induction of cellular senescence. Together, these data demonstrate that in vivo oxygenation promotes tolerance of VHL loss in renal epithelia, which may promote the development of renal carcinoma.
View details for DOI 10.1128/MCB.01618-09
View details for Web of Science ID 000281721400004
View details for PubMedID 20679489
AXL Is an Essential Factor and Therapeutic Target for Metastatic Ovarian Cancer
2010; 70 (19): 7570-7579
The receptor tyrosine kinase AXL is thought to play a role in metastasis; however, the therapeutic efficacy of an AXL-targeting agent remains largely untested in metastatic disease. In this study, we defined AXL as a therapeutic target for metastatic ovarian cancer. AXL is primarily expressed in metastases and advanced-stage human ovarian tumors but not in normal ovarian epithelium. Genetic inhibition of AXL in human metastatic ovarian tumor cells is sufficient to prevent the initiation of metastatic disease in vivo. Mechanistically, inhibition of AXL signaling in animals with metastatic disease results in decreased invasion and matrix metalloproteinase activity. Most importantly, soluble human AXL receptors that imposed a specific blockade of the GAS6/AXL pathway had a profound inhibitory effect on progression of established metastatic ovarian cancer without normal tissue toxicity. These results offer the first genetic validation of GAS6/AXL targeting as an effective strategy for inhibition of metastatic tumor progression in vivo. Furthermore, this study defines the soluble AXL receptor as a therapeutic candidate agent for treatment of metastatic ovarian cancer, for which current therapies are ineffective.
View details for DOI 10.1158/0008-5472.CAN-10-1267
View details for Web of Science ID 000282647700021
View details for PubMedID 20858715
Hypoxia-inducible Factor-1 Activation in Nonhypoxic Conditions: The Essential Role of Mitochondrial-derived Reactive Oxygen Species
MOLECULAR BIOLOGY OF THE CELL
2010; 21 (18): 3247-3257
Hypoxia-inducible factor-1 (HIF-1) is a key transcription factor for responses to low oxygen. Different nonhypoxic stimuli, including hormones and growth factors, are also important HIF-1 activators in the vasculature. Angiotensin II (Ang II), the main effecter hormone in the renin-angiotensin system, is a potent HIF-1 activator in vascular smooth muscle cells (VSMCs). HIF-1 activation by Ang II involves intricate mechanisms of HIF-1? transcription, translation, and protein stabilization. Additionally, the generation of reactive oxygen species (ROS) is essential for HIF-1 activation during Ang II treatment. However, the role of the different VSMC ROS generators in HIF-1 activation by Ang II remains unclear. This work aims at elucidating this question. Surprisingly, repression of NADPH oxidase-generated ROS, using Vas2870, a specific inhibitor or a p22(phox) siRNA had no significant effect on HIF-1 accumulation by Ang II. In contrast, repression of mitochondrial-generated ROS, by complex III inhibition, by Rieske Fe-S protein siRNA, or by the mitochondrial-targeted antioxidant SkQ1, strikingly blocked HIF-1 accumulation. Furthermore, inhibition of mitochondrial-generated ROS abolished HIF-1? protein stability, HIF-1-dependent transcription and VSMC migration by Ang II. A large number of studies implicate NADPH oxidase-generated ROS in Ang II-mediated signaling pathways in VSMCs. However, our work points to mitochondrial-generated ROS as essential intermediates for HIF-1 activation in nonhypoxic conditions.
View details for Web of Science ID 000281761300014
View details for PubMedID 20660157
Toxoplasma gondii Activates Hypoxia-inducible Factor (HIF) by Stabilizing the HIF-1 alpha Subunit via Type I Activin-like Receptor Kinase Receptor Signaling
JOURNAL OF BIOLOGICAL CHEMISTRY
2010; 285 (35): 26852-26860
Toxoplasma gondii is an intracellular protozoan parasite that can cause devastating disease in fetuses and immune-compromised individuals. We previously reported that the alpha subunit of the host cell transcription factor, hypoxia-inducible factor-1 (HIF-1), is up-regulated by infection and necessary for Toxoplasma growth. Under basal conditions, HIF-1alpha is constitutively expressed but rapidly targeted for proteasomal degradation after two proline residues are hydroxylated by a family of prolyl hydroxylases (PHDs). The PHDs are alpha-ketoglutarate-dependent dioxygenases that have low K(m) values for oxygen, making them important cellular oxygen sensors. Thus, when oxygen levels decrease, HIF-1alpha is not hydroxylated, and HIF-1 is activated. How Toxoplasma activates HIF-1 under normoxic conditions remains unknown. Here, we report that Toxoplasma infection increases HIF-1alpha stability by preventing HIF-1alpha prolyl hydroxylation. Infection significantly decreases PHD2 abundance, which is the key prolyl hydroxylase for regulating HIF-1alpha. The effects of Toxoplasma on HIF-1alpha abundance and prolyl hydroxylase activity require activin-like receptor kinase signaling. Finally, parasite growth is severely diminished when signaling from this family of receptors is inhibited. Together, these data indicate that PHD2 is a key host cell factor for T. gondii growth and represent a novel mechanism by which a microbial pathogen subverts host cell signaling and transcription to establish its replicative niche.
View details for DOI 10.1074/jbc.M110.147041
View details for Web of Science ID 000281172700016
View details for PubMedID 20581113
PHD2 in tumour angiogenesis
BRITISH JOURNAL OF CANCER
2010; 103 (1): 1-5
Originally identified as the enzymes responsible for catalysing the oxidation of specific, conserved proline residues within hypoxia-inducible factor-1alpha (HIF-1alpha), the additional roles for the prolyl hydroxylase domain (PHD) proteins have remained elusive. Of the four identified PHD enzymes, PHD2 is considered to be the key oxygen sensor, as knockdown of PHD2 results in elevated HIF protein. Several recent studies have highlighted the importance of PHD2 in tumourigenesis. However, there is conflicting evidence as to the exact role of PHD2 in tumour angiogenesis. The divergence seems to be because of the contribution of stromal-derived PHD2, and in particular the involvement of endothelial cells, vs tumour-derived PHD2. This review summarises our current understanding of PHD2 and tumour angiogenesis, focusing on the influences of PHD2 on vascular normalisation and neovascularisation.
View details for DOI 10.1038/sj.bjc.6605682
View details for Web of Science ID 000279374800001
View details for PubMedID 20461086
Hypoxia, inflammation, and the tumor microenvironment in metastatic disease
CANCER AND METASTASIS REVIEWS
2010; 29 (2): 285-293
Metastasis, the leading cause of cancer deaths, is an intricate process involving many important tumor and stromal proteins that have yet to be fully defined. This review discusses critical components necessary for the metastatic cascade, including hypoxia, inflammation, and the tumor microenvironment. More specifically, this review focuses on tumor cell and stroma interactions, which allow cell detachment from a primary tumor, intravasation to the blood stream, and extravasation at a distant site where cells can seed and tumor metastases can form. Central players involved in this process and discussed in this review include integrins, matrix metalloproteinases, and soluble growth factors/matrix proteins, including the connective tissue growth factor and lysyl oxidase.
View details for DOI 10.1007/s10555-010-9224-5
View details for Web of Science ID 000277411300006
View details for PubMedID 20393783
MiR-210-micromanager of the hypoxia pathway
TRENDS IN MOLECULAR MEDICINE
2010; 16 (5): 230-237
Hypoxia inducible factors (HIFs) regulate a variety of genes to prepare cells to adapt and survive under a hypoxic environment. Recently, microRNAs (miRNAs) have emerged as a new class of genes regulated by HIFs in response to hypoxia, of which miR-210 is the most consistently and predominantly upregulated miRNA. Functional studies have demonstrated that miR-210 is a versatile gene that regulates many aspects of hypoxia pathways, both in physiological and malignant conditions. Here, we summarize recent findings on the mechanism of hypoxia regulation of miR-210 expression and its multifaceted biological functions in normal physiological and malignant conditions, and discuss the challenges we face in elucidating the biological functions of miR-210 and exploring its potential use for therapeutics.
View details for DOI 10.1016/j.molmed.2010.03.004
View details for Web of Science ID 000278669500004
View details for PubMedID 20434954
Targeting cancer cells through autophagy for anticancer therapy
CURRENT OPINION IN CELL BIOLOGY
2010; 22 (2): 246-251
Autophagy is a cellular degradation process in which portions of the cell's cytoplasm and organelles are sequestered in a double-membrane bound vesicle called an autophagosome. Fusion of autophagosomes with lysosomes results in the formation of autolysosomes, where the proteins and organelles are degraded. This degradation pathway is induced under nutrient deprivation, metabolic stress or microenvironmental conditions to ensure energy balance, clearance of damaged proteins and adaptation to stress. Disruption of autophagy is involved in diverse human diseases including cancer. In particular, the regulation of autophagy in cancer cells is complex since it can enhance tumor cell survival in response to certain stresses, yet it can also act to suppress the initiation of tumor growth. Understanding the signaling pathways involved in the regulation of autophagy as well as the autophagy process itself represents new directions in the development of anticancer therapies. In this review, we discuss recent advances in our understanding the complexity of the autophagy process and the development of targeted therapies that modulate autophagy in cancer cells in the clinic.
View details for DOI 10.1016/j.ceb.2009.12.007
View details for Web of Science ID 000277845300019
View details for PubMedID 20056398
The RGD Domain of Human Osteopontin Promotes Tumor Growth and Metastasis through Activation of Survival Pathways
2010; 5 (3)
Human osteopontin (OPN), a known tumor associated protein, exists in different isoforms, whose function is unclear. It also possesses a RGD domain, which has been implicated in diverse function. Here, we use genetic approaches to systematically investigate the function of the RGD domain in different OPN isoforms on tumor progression and metastasis for 2 different solid tumor models.Using isoform-specific qRT-PCR, we found that OPN-A and B were the main isoforms overexpressed in evaluated human tumors, which included 4 soft tissue sarcomas, 24 lung and 30 head and neck carcinomas. Overexpression of either OPN-A or B in two different cell types promoted local tumor growth and lung metastasis in SCID mouse xenografts. However, expression of either isoform with the RGD domain either mutated or deleted decreased tumor growth and metastasis, and resulted in increased apoptosis by TUNEL staining. In vitro, whereas mutation of the RGD domain did not affect cell-cell adhesion, soft agar growth or cell migration, it increased apoptosis under hypoxia and serum starvation. This effect could be mitigated when the RGD mutant cells were treated with condition media containing WT OPN. Mechanistically, the RGD region of OPN inhibited apoptosis by inducing NF-kappaB activation and FAK phosphorylation. Inhibition of NF-kappaB (by siRNA to the p65 subunit) or FAK activation (by a inhibitor) significantly increased apoptosis under hypoxia in WT OPN cells, but not in RGD mutant cells.Unlike prior reports, our data suggest that the RGD domain of both OPN-A and B promote tumor growth and metastasis mainly by protecting cells against apoptosis under stressed conditions and not via migration or invasion. Future inhibitors directed against OPN should target multiple isoforms and should inhibit cell survival mechanisms that involve the RGD domain, FAK phosphorylation and NF-kappaB activation.
View details for DOI 10.1371/journal.pone.0009633
View details for Web of Science ID 000275328800027
View details for PubMedID 20224789
4-Pyridylanilinothiazoles That Selectively Target von Hippel-Lindau Deficient Renal Cell Carcinoma Cells by Inducing Autophagic Cell Death
JOURNAL OF MEDICINAL CHEMISTRY
2010; 53 (2): 787-797
Renal cell carcinomas (RCC) are refractory to standard therapy with advanced RCC having a poor prognosis; consequently treatment of advanced RCC represents an unmet clinical need. The von Hippel-Lindau (VHL) tumor suppressor gene is mutated or inactivated in a majority of RCCs. We recently identified a 4-pyridyl-2-anilinothiazole (PAT) with selective cytotoxicity against VHL-deficient renal cells mediated by induction of autophagy and increased acidification of autolysosomes. We report exploration of structure-activity relationships (SAR) around this PAT lead. Analogues with substituents on each of the three rings, and various linkers between rings, were synthesized and tested in vitro using paired RCC4 cell lines. A contour map describing the relative spatial contributions of different chemical features to potency illustrates a region, adjacent to the pyridyl ring, with potential for further development. Examples probing this domain validated this approach and may provide the opportunity to develop this novel chemotype as a targeted approach to the treatment of RCC.
View details for DOI 10.1021/jm901457w
View details for Web of Science ID 000273672100025
View details for PubMedID 19994864
- DNA Damage and Repair Textbook of Radiation Oncology, 3rd Edition 2010: 31-39
- Role of carcinoma-associated fibroblasts and hypoxia in tumor progression Diverse Effects of Hypoxia on Tumor Progression 2010: 31-45
Role of Carcinoma-Associated Fibroblasts and Hypoxia in Tumor Progression
DIVERSE EFFECTS OF HYPOXIA ON TUMOR PROGRESSION
2010; 345: 31-45
In recent years, a variety of experimental evidence has convincingly shown that progression of malignant tumors does not depend exclusively on cell-autonomous properties of the cancer cells, but can also be influenced by the tumor stroma. The concept that cancer cells are subjected to microenvironmental control has thus emerged as an important chapter in cancer biology. Recent findings have suggested an important role, in particular, for macrophages, endothelial cells, and cancer-associated fibroblasts (CAFs) in tumor growth and progression. Numerous lines of evidence indicate that the bone marrow is the source, at least in part, of these cells. This chapter summarizes our current knowledge of how bone marrow contributes to the tumor stroma, with particular emphasis on CAFs. The potential role of hypoxia in modulating the differentiation and activity of CAFs, and the therapeutical implications of targeting CAFs for anticancer therapy are discussed.
View details for DOI 10.1007/82_2010_73
View details for Web of Science ID 000282105100003
View details for PubMedID 20517716
- Prematastatic Niches Tumor Microenvironment 2010: 161-182
Regulation of the Histone Demethylase JMJD1A by Hypoxia-Inducible Factor 1 alpha Enhances Hypoxic Gene Expression and Tumor Growth
MOLECULAR AND CELLULAR BIOLOGY
2010; 30 (1): 344-353
The hypoxia-inducible transcription factors (HIFs) directly and indirectly mediate cellular adaptation to reduced oxygen tensions. Recent studies have shown that the histone demethylase genes JMJD1A, JMJD2B, and JARID1B are HIF targets, suggesting that HIFs indirectly influence gene expression at the level of histone methylation under hypoxia. In this study, we identify a subset of hypoxia-inducible genes that are dependent on JMJD1A in both renal cell and colon carcinoma cell lines. JMJD1A regulates the expression of adrenomedullin (ADM) and growth and differentiation factor 15 (GDF15) under hypoxia by decreasing promoter histone methylation. In addition, we demonstrate that loss of JMJD1A is sufficient to reduce tumor growth in vivo, demonstrating that histone demethylation plays a significant role in modulating growth within the tumor microenvironment. Thus, hypoxic regulation of JMJD1A acts as a signal amplifier to facilitate hypoxic gene expression, ultimately enhancing tumor growth.
View details for DOI 10.1128/MCB.00444-09
View details for Web of Science ID 000272569200028
View details for PubMedID 19858293
- Hypoxia, Gene Expression and Metastasis The Tumor Microenvironment 2010: 43-58
Pharmacologically Increased Tumor Hypoxia Can Be Measured by F-18-Fluoroazomycin Arabinoside Positron Emission Tomography and Enhances Tumor Response to Hypoxic Cytotoxin PR-104
CLINICAL CANCER RESEARCH
2009; 15 (23): 7170-7174
Solid tumors contain microenvironmental regions of hypoxia that present a barrier to traditional radiotherapy and chemotherapy, and this work describes a novel approach to circumvent hypoxia. We propose to overcome hypoxia by augmenting the effectiveness of drugs that are designed to specifically kill hypoxic tumor cells.We have constructed RKO colorectal tumor cells that express a small RNA hairpin that specifically knocks down the hypoxia-inducible factor 1a (HIF1a) transcription factor. We have used these cells in vitro to determine the effect of HIF1 on cellular sensitivity to the hypoxic cytotoxin PR-104, and its role in cellular oxygen consumption in response to the pyruvate dehydrogenase kinase inhibitor dichloroacetate (DCA). We have further used these cells in vivo in xenografted tumors to determine the role of HIF1 in regulating tumor hypoxia in response to DCA using (18)F-fluoroazomycin arabinoside positron emission tomography, and its role in regulating tumor sensitivity to the combination of DCA and PR-104.HIF1 does not affect cellular sensitivity to PR-104 in vitro. DCA transiently increases cellular oxygen consumption in vitro and increases the extent of tumor hypoxia in vivo as measured with (18)F-fluoroazomycin arabinoside positron emission tomography. Furthermore, we show that DCA-dependent alterations in hypoxia increase the antitumor activity of the next-generation hypoxic cytotoxin PR-104.DCA interferes with the HIF-dependent "adaptive response," which limits mitochondrial oxygen consumption. This approach transiently increases tumor hypoxia and represents an important method to improve antitumor efficacy of hypoxia-targeted agents, without increasing toxicity to oxygenated normal tissue.
View details for DOI 10.1158/1078-0432.CCR-09-1676
View details for Web of Science ID 000272363700011
View details for PubMedID 19920111
Inhibition of Histone Deacetylase Activity in Reduced Oxygen Environment Enhances the Osteogenesis of Mouse Adipose-Derived Stromal Cells
TISSUE ENGINEERING PART A
2009; 15 (12): 3697-3707
Recent studies suggest that oxygen tension has a great impact on the osteogenic differentiation capacity of mesenchymal cells derived from adipose tissue: reduced oxygen impedes osteogenesis. We have found that expansion of mouse adipose-derived stromal cells (mASCs) in reduced oxygen tension (10%) results in increased cell proliferation along with induction of histone deacetylase (HDAC) activity. In this study, we utilized two HDAC inhibitors (HDACi), sodium butyrate (NaB) and valproic acid (VPA), and studied their effects on mASCs expanded in various oxygen tensions (21%, 10%, and 1% O(2)). Significant growth inhibition was observed with NaB or VPA treatment in each oxygen tension. Osteogenesis was enhanced by treatment with NaB or VPA, particularly in reduced oxygen tensions (10% and 1% O(2)). Conversely, adipogenesis was decreased with treatments of NaB or VPA at all oxygen tensions. Finally, NaB- or VPA-treated, reduced oxygen tension-exposed (1% O(2)) ASCs were grafted into surgically created mouse tibial defects and resulted in significantly increased bone regeneration. In conclusion, HDACi significantly promote the osteogenic differentiation of mASCs exposed to reduced oxygen tension; HDACi may hold promise for future clinical applications of ASCs for skeletal regeneration.
View details for DOI 10.1089/ten.tea.2009.0213
View details for Web of Science ID 000272528400003
View details for PubMedID 19505250
Matrix Crosslinking Forces Tumor Progression by Enhancing Integrin Signaling
2009; 139 (5): 891-906
Tumors are characterized by extracellular matrix (ECM) remodeling and stiffening. The importance of ECM remodeling to cancer is appreciated; the relevance of stiffening is less clear. We found that breast tumorigenesis is accompanied by collagen crosslinking, ECM stiffening, and increased focal adhesions. Induction of collagen crosslinking stiffened the ECM, promoted focal adhesions, enhanced PI3 kinase (PI3K) activity, and induced the invasion of an oncogene-initiated epithelium. Inhibition of integrin signaling repressed the invasion of a premalignant epithelium into a stiffened, crosslinked ECM and forced integrin clustering promoted focal adhesions, enhanced PI3K signaling, and induced the invasion of a premalignant epithelium. Consistently, reduction of lysyl oxidase-mediated collagen crosslinking prevented MMTV-Neu-induced fibrosis, decreased focal adhesions and PI3K activity, impeded malignancy, and lowered tumor incidence. These data show how collagen crosslinking can modulate tissue fibrosis and stiffness to force focal adhesions, growth factor signaling and breast malignancy.
View details for DOI 10.1016/j.cell.2009.10.027
View details for Web of Science ID 000272169400014
View details for PubMedID 19931152
- Bioreductively Activated Lysyl Oxidase Inhibitors against Hypoxic Tumours CHEMMEDCHEM 2009; 4 (10): 1590-1594
Hypoxia-Inducible mir-210 Regulates Normoxic Gene Expression Involved in Tumor Initiation
2009; 35 (6): 856-867
Previous studies have suggested that the HIF transcription factors can both activate and inhibit gene expression. Here we show that HIF1 regulates the expression of mir-210 in a variety of tumor types through a hypoxia-responsive element. Expression analysis in primary head and neck tumor samples indicates that mir-210 may serve as an in vivo marker for tumor hypoxia. By Argonaute protein immunoprecipitation, we identified 50 potential mir-210 targets and validated randomly selected ones. The majority of these 50 genes are not classical hypoxia-inducible genes, suggesting mir-210 represses genes expressed under normoxia that are no longer necessary to adapt and survive in a hypoxic environment. When human head and neck or pancreatic tumor cells ectopically expressing mir-210 were implanted into immunodeficient mice, mir-210 repressed initiation of tumor growth. Taken together, these data implicate an important role for mir-210 in regulating the hypoxic response of tumor cells and tumor growth.
View details for DOI 10.1016/j.molcel.2009.09.006
View details for Web of Science ID 000270559100018
View details for PubMedID 19782034
Validation of Lysyl Oxidase As a Prognostic Marker for Metastasis and Survival in Head and Neck Squamous Cell Carcinoma: Radiation Therapy Oncology Group Trial 90-03
JOURNAL OF CLINICAL ONCOLOGY
2009; 27 (26): 4281-4286
To validate lysyl oxidase (LOX), a hypoxia-related protein, as a marker for metastasis in an independent head and neck cancer (HNC) patient group enrolled onto a prospective trial.We performed traditional immunohistochemical (IHC) staining and automated quantitative analysis (AQUA) for LOX expression in 66 HNC patients from one institution. We also performed AQUA staining for LOX in 306 of 1,113 patients treated on a phase III trial comparing four radiation fractionation schedules in locally advanced HNC (RTOG 90-03). Pretreatment characteristics and outcome were similar between patients with and without LOX assessment. We correlated AQUA LOX expression with time to metastasis (TTM), time to progression (TTP), and overall survival (OS).LOX expression from both staining methods predicted for TTM in the first 66 patients. Multivariate analysis, controlling for significant parameters including nodal stage and performance status, revealed tumor LOX expression, as a continuous variable, was an independent predictor for TTM (hazard ratio [HR], 1.21; 95% CI, 1.10 to 1.33; P = .0001), TTP (HR, 1.06; 95% CI, 1.02 to 1.10; P = .0069), and OS (HR, 1.04; 95% CI, 1.00 to 1.07; P = .0311) in RTOG 90-03 patients. This translates into a 259% increase in metastatic risk for a patient at the 75th percentile of LOX compared with one at the 25th percentile.AQUA LOX expression was strongly associated with increased metastasis, progression, and death in RTOG 90-03 patients. This study validates that LOX is a marker for metastasis and survival in HNC.
View details for DOI 10.1200/JCO.2008.20.6003
View details for Web of Science ID 000269652200010
View details for PubMedID 19667273
Bringing H2AX into the Angiogenesis Family
2009; 15 (6): 459-461
The cell's ability to sense and respond to DNA damage is critical to maintain homeostasis and prevent the development of cancer. Paradoxically, Economopoulou et al. recently reported that a DNA damage response protein, H2AX, promotes tumor growth and angiogenesis.
View details for DOI 10.1016/j.ccr.2009.05.004
View details for Web of Science ID 000266686500003
View details for PubMedID 19477424
Tumor Vasculature Is Regulated by PHD2-Mediated Angiogenesis and Bone Marrow-Derived Cell Recruitment
2009; 15 (6): 527-538
Sustained angiogenesis, through either local sprouting (angiogenesis) or the recruitment of bone marrow-derived cells (BMDCs) (vasculogenesis), is essential to the development of a tumor. How BMDCs are recruited to the tumor and their contribution to the tumor vasculature is poorly understood. Here, we demonstrate that both IL-8 and angiogenin contribute to the complementary pathways of angiogenesis and BMDC mobilization to increase tumor growth. These two factors are regulated by PHD2 in a HIF-independent but NF-kappaB-dependent manner. PHD2 levels are decreased in human cancers, compared with corresponding normal tissue, and correlate with an increase in mature blood vessels. Thus, PHD2 plays a critical role in regulating tumor angiogenesis.
View details for DOI 10.1016/j.ccr.2009.04.010
View details for Web of Science ID 000266686500010
View details for PubMedID 19477431
Aberrant regulation of pVHL levels by microRNA promotes the HIF/VEGF axis in CLL B cells
2009; 113 (22): 5568-5574
The molecular mechanism of autocrine regulation of vascular endothelial growth factor (VEGF) in chronic lymphocytic leukemia (CLL) B cells is unknown. Here, we report that CLL B cells express constitutive levels of HIF-1alpha under normoxia. We have examined the status of the von Hippel-Lindau gene product (pVHL) that is responsible for HIF-1alpha degradation and found it to be at a notably low level in CLL B cells compared with normal B cells. We demonstrate that the microRNA, miR-92-1, overexpressed in CLL B cells, can target the VHL transcript to repress its expression. We found that the stabilized HIF-1alpha can form an active complex with the transcriptional coactivator p300 and phosphorylated-STAT3 at the VEGF promoter and recruit RNA polymerase II. This is initial evidence that pVHL, without any genetic alteration, can be regulated by microRNA and explains the aberrant autocrine VEGF secretion in CLL.
View details for DOI 10.1182/blood-2008-10-185686
View details for Web of Science ID 000266634700026
View details for PubMedID 19336759
The Role of Tumor Cell-Derived Connective Tissue Growth Factor (CTGF/CCN2) in Pancreatic Tumor Growth
2009; 69 (3): 775-784
Pancreatic cancer is highly aggressive and refractory to existing therapies. Connective tissue growth factor (CTGF/CCN2) is a fibrosis-related gene that is thought to play a role in pancreatic tumor progression. However, CCN2 can be expressed in a variety of cell types, and the contribution of CCN2 derived from either tumor cells or stromal cells as it affects the growth of pancreatic tumors is unknown. Using genetic inhibition of CCN2, we have discovered that CCN2 derived from tumor cells is a critical regulator of pancreatic tumor growth. Pancreatic tumor cells derived from CCN2 shRNA-expressing clones showed dramatically reduced growth in soft agar and when implanted s.c. We also observed a role for CCN2 in the growth of pancreatic tumors implanted orthotopically, with tumor volume measurements obtained by positron emission tomography imaging. Mechanistically, CCN2 protects cells from hypoxia-mediated apoptosis, providing an in vivo selection for tumor cells that express high levels of CCN2. We found that CCN2 expression and secretion was increased in hypoxic pancreatic tumor cells in vitro, and we observed colocalization of CCN2 and hypoxia in pancreatic tumor xenografts and clinical pancreatic adenocarcinomas. Furthermore, we found increased CCN2 staining in clinical pancreatic tumor tissue relative to stromal cells surrounding the tumor, supporting our assertion that tumor cell-derived CCN2 is important for pancreatic tumor growth. Taken together, these data improve our understanding of the mechanisms responsible for pancreatic tumor growth and progression, and also indicate that CCN2 produced by tumor cells represents a viable therapeutic target for the treatment of pancreatic cancer.
View details for DOI 10.1158/0008-5472.CAN-08-0987
View details for Web of Science ID 000263048700010
View details for PubMedID 19179545
ATM Activation and Signaling under Hypoxic Conditions
MOLECULAR AND CELLULAR BIOLOGY
2009; 29 (2): 526-537
The ATM kinase has previously been shown to respond to the DNA damage induced by reoxygenation following hypoxia by initiating a Chk 2-dependent cell cycle arrest in the G(2) phase. Here we show that ATM is both phosphorylated and active during exposure to hypoxia in the absence of DNA damage, detectable by either comet assay or 53BP1 focus formation. Hypoxia-induced activation of ATM correlates with oxygen concentrations low enough to cause a replication arrest and is entirely independent of hypoxia-inducible factor 1 status. In contrast to damage-activated ATM, hypoxia-activated ATM does not form nuclear foci but is instead diffuse throughout the nucleus. The hypoxia-induced activity of both ATM and the related kinase ATR is independent of NBS1 and MRE11, indicating that the MRN complex does not mediate the DNA damage response to hypoxia. However, the mediator MDC1 is required for efficient activation of Kap1 by hypoxia-induced ATM, indicating that similarly to the DNA damage response, there is a requirement for MDC1 to amplify the ATM response to hypoxia. However, under hypoxic conditions, MDC1 does not recruit BRCA1/53BP1 or RNF8 activity. Our findings clearly demonstrate that there are alternate mechanisms for activating ATM that are both stress-specific and independent of the presence of DNA breaks.
View details for DOI 10.1128/MCB.01301-08
View details for Web of Science ID 000262045800020
View details for PubMedID 18981219
Hypoxia-Induced Lysyl Oxidase Is a Critical Mediator of Bone Marrow Cell Recruitment to Form the Premetastatic Niche
2009; 15 (1): 35-44
Tumor cell metastasis is facilitated by "premetastatic niches" formed in destination organs by invading bone marrow-derived cells (BMDCs). Lysyl oxidase (LOX) is critical for premetastatic niche formation. LOX secreted by hypoxic breast tumor cells accumulates at premetastatic sites, crosslinks collagen IV in the basement membrane, and is essential for CD11b+ myeloid cell recruitment. CD11b+ cells adhere to crosslinked collagen IV and produce matrix metalloproteinase-2, which cleaves collagen, enhancing the invasion and recruitment of BMDCs and metastasizing tumor cells. LOX inhibition prevents CD11b+ cell recruitment and metastatic growth. CD11b+ cells and LOX also colocalize in biopsies of human metastases. Our findings demonstrate a critical role for LOX in premetastatic niche formation and support targeting LOX for the treatment and prevention of metastatic disease.
View details for DOI 10.1016/j.ccr.2008.11.012
View details for Web of Science ID 000262334800007
View details for PubMedID 19111879
- Hypoxia-Mediated Signaling Pathways Handbook of Cell Signaling, 2nd Edition 2009; 3: 2241-2245
- Hypoxia, angiogenesis and oral cancer metastasis Oral Cancer Metastasis 2009: 299-321
- Ph.D. Training in Cancer Biology CANCER RESEARCH 2008; 68 (22): 9122-9124
Notch1 is an effector of Akt and hypoxia in melanoma development
JOURNAL OF CLINICAL INVESTIGATION
2008; 118 (11): 3660-3670
Melanomas are highly aggressive neoplasms resistant to most conventional therapies. These tumors result from the interaction of altered intracellular tumor suppressors and oncogenes with the microenvironment in which these changes occur. We previously demonstrated that physiologic skin hypoxia contributes to melanomagenesis in conjunction with Akt activation. Here we show that Notch1 signaling is elevated in human melanoma samples and cell lines and is required for Akt and hypoxia to transform melanocytes in vitro. Notch1 facilitated melanoma development in a xenograft model by maintaining cell proliferation and by protecting cells from stress-induced cell death. Hyperactivated PI3K/Akt signaling led to upregulation of Notch1 through NF-kappaB activity, while the low oxygen content normally found in skin increased mRNA and protein levels of Notch1 via stabilization of HIF-1alpha. Taken together, these findings demonstrate that Notch1 is a key effector of both Akt and hypoxia in melanoma development and identify the Notch signaling pathway as a potential therapeutic target in melanoma treatment.
View details for DOI 10.1172/JCI36157
View details for Web of Science ID 000260622600020
View details for PubMedID 18924608
Targeted therapy for the loss of von Hippel-Lindau in renal cell carcinoma - A novel molecule that induces autophagic cell death
2008; 4 (7): 944-946
Radiation and conventional cytotoxic chemotherapies are ineffective in treating renal cancer. Approximately 75 percent of renal cell carcinoma (RCC) is associated with an inactivation of the tumor suppressor gene von Hippel-Lindau (VHL). We exploited the possibility of targeting VHL-deficient RCC through synthetic lethality using a high-throughput screening approach. In this screen, STF-62247 was identified to be selectively toxic and growth inhibitory to renal cells lacking VHL. We recently demonstrated that the cytotoxicity of STF-62247 is due to dysregulated autophagy. Furthermore, the reduction of protein levels of essential autophagy pathway components such as Atg5, Atg7 and Atg9 reduces sensitivity of VHL-deficient cells to killing by STF-62247. Loss of proteins involved in Golgi trafficking sensitized RCC with wild-type VHL to killing by STF-62247, indicating a potential role for these proteins as a target of the compound. Our study supports the concept of using synthetic lethality to selectively kill VHL-deficient cells that represents a new type of targeted therapy for the treatment of RCC.
View details for Web of Science ID 000259829700018
View details for PubMedID 18769110
Targeting cancer cells by synthetic lethality
2008; 7 (19): 2987-2990
Standard cytotoxic agents for treating cancer were developed based on their effectiveness to kill rapidly dividing cells, not on their ability to selectively kill cancer cells and spare normal tissue. Much of contemporary cancer research is aimed at identifying specific molecular features of cancers to directly target tumor cells with the hope of reducing or eliminating unwanted side effects. Targeted therapy for the treatment of cancer can be divided into two main categories: monoclonal antibodies and small molecules. In this Perspective, we review the approach of synthetic lethality to target cancer, specifically renal cell carcinoma. The concept of synthetic lethality is used to describe a genetic interaction of two non-allelic and non-lethal genes that when mutated simultaneously results in cell death. Recently, we identified a compound, STF-62247, that functions in a synthetic lethal manner to the loss of VHL, a mutation found in the majority of renal cell carcinomas.
View details for Web of Science ID 000260263000010
View details for PubMedID 18818511
Short hairpin RNA interference therapy for ischemic heart disease
2008; 118 (14): S226-U289
During hypoxia, upregulation of hypoxia inducible factor-1 alpha transcriptional factor can activate several downstream angiogenic genes. However, hypoxia inducible factor-1 alpha is naturally degraded by prolyl hydroxylase-2 (PHD2) protein. Here we hypothesize that short hairpin RNA (shRNA) interference therapy targeting PHD2 can be used for treatment of myocardial ischemia and this process can be followed noninvasively by molecular imaging.PHD2 was cloned from mouse embryonic stem cells by comparing the homolog gene in human and rat. The best candidate shRNA sequence for inhibiting PHD2 was inserted into the pSuper vector driven by the H1 promoter followed by a separate hypoxia response element-incorporated promoter driving a firefly luciferase reporter gene. This construct was used to transfect mouse C2C12 myoblast cell line for in vitro confirmation. Compared with the control short hairpin scramble (shScramble) as control, inhibition of PHD2 increased levels of hypoxia inducible factor-1 alpha protein and several downstream angiogenic genes by >30% (P<0.01). Afterward, shRNA targeting PHD2 (shPHD2) plasmid was injected intramyocardially following ligation of left anterior descending artery in mice. Animals were randomized into shPHD2 experimental group (n=25) versus shScramble control group (n=20). Bioluminescence imaging detected plasmid-mediated transgene expression for 4 to 5 weeks. Echocardiography showed the shPHD2 group had improved fractional shortening compared with the shScramble group at Week 4 (33.7%+/-1.9% versus 28.4%+/-2.8%; P<0.05). Postmortem analysis showed increased presence of small capillaries and venules in the infarcted zones by CD31 staining. Finally, Western blot analysis of explanted hearts also confirmed that animals treated with shPHD2 had significantly higher levels of hypoxia inducible factor-1 alpha protein.This is the first study to image the biological role of shRNA therapy for improving cardiac function. Inhibition of PHD2 by shRNA led to significant improvement in angiogenesis and contractility by in vitro and in vivo experiments. With further validation, the combination of shRNA therapy and molecular imaging can be used to track novel cardiovascular gene therapy applications in the future.
View details for DOI 10.1161/CIRCULATIONAHA.107.760785
View details for Web of Science ID 000259648600033
View details for PubMedID 18824759
(2R)-[(4-biphenylylsulfonyl)amino]-N-hydroxy-3-phenylpropionamide (BiPS), a matrix metalloprotease inhibitor, is a novel and potent activator of hypoxia-inducible factors
2008; 74 (1): 282-288
Hypoxia-inducible factors (HIFs) are unstable heterodimeric transcription factors and decisive elements for the transcriptional regulation of genes important in the adaptation to low-oxygen conditions. Hypoxia is the ubiquitous inducer of HIFs, stabilizing the alpha-subunit and permitting the formation of a functional HIF complex. Here, we identify (2R)-[(4-biphenylylsulfonyl)amino]-N-hydroxy-3-phenylpropionamide (BiPS), a commercially available metalloprotease-2 and -9 inhibitor, as a rapid and potent inducer of HIFs. We show that in different cell lines, BiPS induces the HIF-alpha subunit by inhibiting its degradation through stabilization of its labile oxygen-dependent degradation domain. This is achieved through the inhibition of HIF-1alpha hydroxylation. The HIF-1 complex, formed after BiPS treatment, is capable of DNA binding and activation of HIF target genes, including the expression of vascular endothelial growth factor. Because novel HIF activators have generated considerable interest in the possible treatment of different ischemic diseases, we believe that BiPS and derivative molecules could have strong therapeutic potential.
View details for DOI 10.1124/mol.108.045690
View details for Web of Science ID 000256889600029
View details for PubMedID 18424552
A molecule targeting VHL-deficient renal cell carcinoma that induces autophagy
2008; 14 (1): 90-102
Renal cell carcinomas (RCCs) are refractory to standard therapies. The von Hippel-Lindau (VHL) tumor suppressor gene is inactivated in 75% of RCCs. By screening for small molecules selectively targeting VHL-deficient RCC cells, we identified STF-62247. STF-62247 induces cytotoxicity and reduces tumor growth of VHL-deficient RCC cells compared to genetically matched cells with wild-type VHL. STF-62247-stimulated toxicity occurs in a HIF-independent manner through autophagy. Reduction of protein levels of essential autophagy pathway components reduces sensitivity of VHL-deficient cells to STF-62247. Using a yeast deletion pool, we show that loss of proteins involved in Golgi trafficking increases killing by STF-62247. Thus, we have found a small molecule that selectively induces cell death in VHL-deficient cells, representing a paradigm shift for targeted therapy.
View details for DOI 10.1016/j.ccr.2008.06.004
View details for Web of Science ID 000257655100012
View details for PubMedID 18598947
The metastasis-associated gene Prl-3 is a p53 target involved in cell-cycle regulation
2008; 30 (3): 303-314
The p53 tumor suppressor restricts tumorigenesis through the transcriptional activation of target genes involved in cell-cycle arrest and apoptosis. Here, we identify Prl-3 (phosphatase of regenerating liver-3) as a p53-inducible gene. Whereas previous studies implicated Prl-3 in metastasis because of its overexpression in metastatic human colorectal cancer and its ability to promote invasiveness and motility, we demonstrate here that Prl-3 is an important cell-cycle regulator. Consistent with a role in DNA damage-induced cell-cycle arrest, Prl-3 overexpression induces G(1) arrest downstream of p53 by triggering a PI3K-Akt-activated negative feedback loop. Surprisingly, attenuation of Prl-3 expression also elicits an arrest response, suggesting that basal level Prl-3 expression is pivotal for normal cell-cycle progression. Our findings highlight key dose-dependent functions of Prl-3 in both positive and negative regulation of cell-cycle progression and provide insight into Prl-3's role in cancer progression.
View details for DOI 10.1016/j.molcel.2008.04.002
View details for Web of Science ID 000255761200008
View details for PubMedID 18471976
Molecular Imaging of Hypoxia-Inducible Factor 1 alpha and von Hippel-Lindau Interaction in Mice
2008; 7 (3): 139-146
Tumor hypoxia plays a crucial role in tumorigenesis. Under hypoxia, hypoxia-inducible factor 1 alpha (HIF-1 alpha) regulates activation of genes promoting malignant progression. Under normoxia, HIF-1 alpha is hydroxylated on prolines 402 and 564 and is targeted for ubiquitin-mediated degradation by interacting with the von Hippel-Lindau protein complex (pVHL). We have developed a novel method of studying the interaction between HIF-1 alpha and pVHL using the split firefly luciferase complementation-based bioluminescence system in which HIF-1 alpha and pVHL are fused to amino-terminal and carboxy-terminal fragments of the luciferase, respectively. We demonstrate that hydroxylation-dependent interaction between the HIF-1 alpha and pVHL leads to complementation of the two luciferase fragments, resulting in bioluminescence in vitro and in vivo. Complementation-based bioluminescence is diminished when mutant pVHLs with decreased affinity for binding HIF-1 alpha are used. This method represents a new approach for studying interaction of proteins involved in the regulation of protein degradation.
View details for DOI 10.2310/7290.2008.00017
View details for Web of Science ID 000260954700004
View details for PubMedID 19123984
The role of hypoxia-inducible factors in tumorigenesis
CELL DEATH AND DIFFERENTIATION
2008; 15 (4): 678-685
Hypoxia-inducible factors (HIFs) are essential mediators of the cellular oxygen-signaling pathway. They are heterodimeric transcription factors consisting of an oxygen-sensitive alpha subunit (HIF-alpha) and a constitutive beta subunit (HIF-beta) that facilitate both oxygen delivery and adaptation to oxygen deprivation by regulating the expression of genes that control glucose uptake, metabolism, angiogenesis, erythropoiesis, cell proliferation, and apoptosis. In most experimental models, the HIF pathway is a positive regulator of tumor growth as its inhibition often results in tumor suppression. In clinical samples, HIF is found elevated and correlates with poor patient prognosis in a variety of cancers. In summary, HIF regulates multiple aspects of tumorigenesis, including angiogenesis, proliferation, metabolism, metastasis, differentiation, and response to radiation therapy, making it a critical regulator of the malignant phenotype.
View details for DOI 10.1038/cdd.2008.21
View details for Web of Science ID 000254041800010
View details for PubMedID 18259193
- Principles of Radiation Oncology Principles & Practice of Oncology, 8th Edition 2008: 307-327
Hypoxia-inducible factor-1 alpha stabilization in nonhypoxic conditions: Role of oxidation and intracellular ascorbate depletion
MOLECULAR BIOLOGY OF THE CELL
2008; 19 (1): 86-94
Hypoxia-inducible factor-1 (HIF-1) is a decisive element for the transcriptional regulation of many genes induced under low oxygen conditions. Under normal oxygen conditions, HIF-1alpha, the active subunit of HIF-1, is hydroxylated on proline residues by specific HIF prolyl-hydroxylases, leading to ubiquitination and degradation by the proteasome. In hypoxia, hydroxylation and ubiquitination are blocked and HIF-1alpha accumulates in cells. Recent studies have shown that in normal oxygen conditions G-protein-coupled receptor agonists, including angiotensin (Ang) II and thrombin, potently induce and activate HIF-1 in vascular smooth muscle cells. The current study identifies HIF-1alpha protein stabilization as a key mechanism for HIF-1 induction by Ang II. We show that hydroxylation on proline 402 is altered by Ang II, decreasing pVHL binding to HIF-1alpha and allowing HIF-1alpha protein to escape subsequent ubiquitination and degradation mechanisms. We show that HIF-1alpha stability is mediated through the Ang II-mediated generation of hydrogen peroxide and a subsequent decrease in ascorbate levels, leading to decreased HIF prolyl-hydroxylase activity and HIF-1alpha stabilization. These findings identify novel and intricate signaling mechanisms involved in HIF-1 complex activation and will lead to the elucidation of the importance of HIF-1 in different Ang II-related cell responses.
View details for DOI 10.1091/mbc.E07-06-0612
View details for Web of Science ID 000258847500009
View details for PubMedID 17942596
Age decreases endothelial progenitor cell recruitment through decreases in hypoxia-inducible factor 1 alpha stabilization during ischemia
2007; 116 (24): 2818-2829
Advanced age is known to impair neovascularization. Because endothelial progenitor cells (EPCs) participate in this process, we examined the effects of aging on EPC recruitment and vascular incorporation.Murine neovascularization was examined by use of an ischemic flap model, which demonstrated aged mice (19 to 24 months) had decreased EPC mobilization (percent mobilized 1.4+/-0.2% versus 0.4+/-0.1%, P<0.005) that resulted in impaired gross tissue survival compared with young mice (2 to 6 months). This decrease correlated with diminished tissue perfusion (P<0.005) and decreased CD31+ vascular density (P<0.005). Gender-mismatched bone marrow transplantation demonstrated significantly fewer chimeric vessels in aged mice (P<0.05), which confirmed a deficit in bone marrow-mediated vasculogenesis. Age had no effect on total EPC number in mice or humans. Reciprocal bone marrow transplantations confirmed that impaired neovascularization resulted from defects in the response of aged tissue to hypoxia and not from intrinsic defects in EPC function. We demonstrate that aging decreased hypoxia-inducible factor 1alpha stabilization in ischemic tissues because of increased prolyl hydroxylase-mediated hydroxylation (P<0.05) and proteasomal degradation. This resulted in a diminished hypoxia response, including decreased stromal cell-derived factor 1 (P<0.005) and vascular endothelial growth factor (P<0.0004). This effect can be reversed with the iron chelator deferoxamine, which results in hypoxia-inducible factor 1alpha stabilization and increased tissue survival.Aging impairs EPC trafficking to sites of ischemia through a failure of aged tissues to normally activate the hypoxia-inducible factor 1alpha-mediated hypoxia response.
View details for DOI 10.1161/CIRCULATIONAHA.107.715847
View details for Web of Science ID 000251542200009
View details for PubMedID 18040029
- Oxygen sensing and the DNA-damage response CURRENT OPINION IN CELL BIOLOGY 2007; 19 (6): 680-684
In vitro expansion of adipose-derived adult stromal cells in hypoxia enhances early chondrogenesis
2007; 13 (12): 2981-2993
Cartilage is an avascular tissue, and chondrocytes in vivo experience a severely hypoxic environment. Using a defined in vitro model of early chondrogenesis, we attempted to enrich for cells with an enhanced ability for chondrogenic differentiation by pre-exposure of mouse adipose-derived adult stromal cells (ADASs) to a hypoxic (2% oxygen) environment. ADASs were subsequently expanded in 2% or 21% oxygen environments, resulting in 2 groups of cells, and then early chondrogenic differentiation was induced at 21% oxygen tension using a 3-dimensional micromass culture system. ADAS chondrogenesis was assessed using Alcian Blue staining for proteoglycans and quantification of sulfated glycosaminoglycans. Osteogenesis of the 2 cell groups was also studied. Two percent oxygen tension profoundly increased the proliferation of ADASs. ADASs expanded in 2% oxygen tension exhibited enhanced early chondrogenic differentiation and diminished osteogenesis, suggesting that the reduced oxygen environment may favor chondroprogenitors. Gene expression analysis suggested that matrix metalloproteinase synthesis was inhibited in cells expanded in 2% oxygen. Furthermore, re-oxygenation of the 2% oxygen-expanded ADASs before differentiation did not significantly affect early chondrogenesis. Thus, priming ADASs with 2% oxygen may have selected for chondrogenic progenitors with an enhanced ability to survive and differentiate. This study is relevant for the future application of cell-based therapies involving cartilage tissue regeneration.
View details for DOI 10.1089/ten.2007.0050
View details for Web of Science ID 000251788400018
View details for PubMedID 17916040
Expression and prognostic significance of a panel of tissue hypoxia markers in head-and-neck squamous cell carcinomas
INTERNATIONAL JOURNAL OF RADIATION ONCOLOGY BIOLOGY PHYSICS
2007; 69 (1): 167-175
To investigate the expression pattern of hypoxia-induced proteins identified as being involved in malignant progression of head-and-neck squamous cell carcinoma (HNSCC) and to determine their relationship to tumor pO(2) and prognosis.We performed immunohistochemical staining of hypoxia-induced proteins (carbonic anhydrase IX [CA IX], BNIP3L, connective tissue growth factor, osteopontin, ephrin A1, hypoxia inducible gene-2, dihydrofolate reductase, galectin-1, IkappaB kinase beta, and lysyl oxidase) on tumor tissue arrays of 101 HNSCC patients with pretreatment pO(2) measurements. Analysis of variance and Fisher's exact tests were used to evaluate the relationship between marker expression, tumor pO(2), and CA IX staining. Cox proportional hazard model and log-rank tests were used to determine the relationship between markers and prognosis.Osteopontin expression correlated with tumor pO(2) (Eppendorf measurements) (p = 0.04). However, there was a strong correlation between lysyl oxidase, ephrin A1, and galectin-1 and CA IX staining. These markers also predicted for cancer-specific survival and overall survival on univariate analysis. A hypoxia score of 0-5 was assigned to each patient, on the basis of the presence of strong staining for these markers, whereby a higher score signifies increased marker expression. On multivariate analysis, increasing hypoxia score was an independent prognostic factor for cancer-specific survival (p = 0.015) and was borderline significant for overall survival (p = 0.057) when adjusted for other independent predictors of outcomes (hemoglobin and age).We identified a panel of hypoxia-related tissue markers that correlates with treatment outcomes in HNSCC. Validation of these markers will be needed to determine their utility in identifying patients for hypoxia-targeted therapy.
View details for DOI 10.1016/j.ijrobp.2007.01.071
View details for Web of Science ID 000248978300024
View details for PubMedID 17707270
Oxygen sensitivity of reporter genes: Implications for preclinical imaging of tumor hypoxia
2007; 6 (4): 219-228
Reporter gene techniques have been applied toward studying the physiologic phenomena associated with tumor hypoxia, a negative prognostic indicator. The purpose of this study was to assess the potential adverse effects of hypoxic conditions on the effectiveness of four commonly used reporter genes: Renilla luciferase, monomeric red fluorescent protein, thymidine kinase, and lacZ. Tumor-forming A375 cells expressing a trifusion reporter consisting of Renilla luciferase, monomeric red fluorescent protein, and thymidine kinase were subjected to decreasing oxygen tensions and assayed for reporter expression and activity. A375 cells expressing beta-galactosidase were similarly exposed to hypoxia, with activity of the reporter monitored by cleavage of the fluorescent substrate 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one)-beta-galactoside (DDAOG). Generation of signal in in vivo tumor models expressing bioluminescent or beta-galactosidase reporters were also examined over the course of hypoxic stresses, either by tumor clamping or the antivascular agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA). Our findings indicate that bioluminescent and fluorescent reporter activity are decreased under hypoxia despite minimal variations in protein production, whereas beta-galactosidase reporter activity per unit protein was unchanged. These results demonstrate that combining beta-galactosidase with the DDAOG optical probe may be a robust reporter system for the in vivo study of tumor hypoxia.
View details for DOI 10.2310/7290.2007.00017
View details for Web of Science ID 000249349100001
View details for PubMedID 17711777
Targeting the loss of the von Hippel-Lindau tumor suppressor gene in renal cell carcinoma cells
2007; 67 (12): 5896-5905
Late-stage clear cell renal carcinoma poses a formidable clinical challenge due to the high mortality rate associated with this disease. Molecular and genetic studies have identified functional loss of the von Hippel-Lindau (VHL) gene as a frequent and crucial event in the development of the malignant phenotype of clear cell renal carcinomas. Loss of VHL function thus represents a pathognomonic molecular defect for therapeutic exploitation. The objective of this study was to evaluate the possibility of targeting VHL loss through pharmacologic means. Chromomycin A3 (ChA3) was identified through in silico analysis of existing publicly available drug profiles from the National Cancer Institute as an agent that seemed to selectively target VHL-deficient clear cell renal carcinoma cells. Genotype-selective toxicity was first determined through short-term viability assays and then confirmed with clonogenic studies. Coculture of fluorescently labeled VHL-deficient and VHL-positive cells showed discriminate killing of the VHL-deficient cells with ChA3. Mechanistically, overexpression of hypoxia-inducible factor (HIF)-2alpha in VHL-positive clear cell renal carcinoma cells phenocopied loss of VHL with respect to ChA3 toxicity, establishing ChA3 as a HIF-dependent cytotoxin. This study shows the feasibility of selectively targeting the loss of the VHL tumor suppressor gene in clear cell renal carcinoma for potential clinical benefit and may have greater ramifications in the development of new targeted therapies for the treatment of cancer and other genetic diseases.
View details for DOI 10.1158/0008-5472.CAN-07-0604
View details for Web of Science ID 000247360100042
View details for PubMedID 17575159
Energy regulation: HIF MXles it up with the C-MYC powerhouse
2007; 12 (6): 845-846
The phenomenon of aerobic glycolysis observed in cancer cells has remained unexplained. In the May 2007 issue of Cancer Cell, Zhang and colleagues determine that the hypoxia-inducible factor family of transcriptional factors regulate mitochondrial biogenesis through inhibition of C-MYC.
View details for DOI 10.1016/j.devcel.2007.05.006
View details for Web of Science ID 000247119700004
View details for PubMedID 17543856
Hypoxia inducible factor-1 alpha deficiency affects chondrogenesis of adipose-derived adult stromal cells
2007; 13 (6): 1159-1171
Increased cartilage-related disease, poor regeneration of cartilage tissue, and limited treatment options have led to intense research in tissue engineering of cartilage. Adipose-derived adult stromal cells (ADAS) are a promising cell source for skeletal tissue engineering; understanding ADAS cellular signaling and chondrogenesis will advance cell-based therapies in cartilage repair. Chondrocytes are unique-they are continuously challenged by a hypoxic microenvironment. Hypoxia inducible factor-1-alpha (HIF-1alpha), a critical mediator of a cell's response to hypoxia, plays a significant role in chondrocyte survival, growth arrest, and differentiation. By using an established in vitro 3-dimensional micromass system, we investigated the role of HIF-1alpha in chondrogenesis. Targeted deletion of HIF-1alpha in ADAS substantially inhibited the chondrogenic pathway specifically. In marked contrast, deletion of HIF-1alpha did not affect osteogenic differentiation but enhanced adipogenic differentiation. This study demonstrates the critical and specific interplay between HIF-1alpha and chondrogenesis in vitro.
View details for DOI 10.1089/ten.2006.0265
View details for Web of Science ID 000247237600003
View details for PubMedID 17518738
Hypoxia, gene expression, and metastasis
CANCER AND METASTASIS REVIEWS
2007; 26 (2): 333-339
Hypoxia poses many problems to the treatment of cancer. Hypoxic tumors are more resistant to chemotherapy and radiation. In addition, hypoxia induces a number of genes responsible for increased invasion, aggressiveness, and metastasis of tumors. The augmented metastatic potential due to hypoxia-mediated gene expression is discussed in this section. Particular attention is given to recent studies of specific genes involved in the key steps of metastasis, including extracellular matrix interactions, migration, and proliferation.
View details for DOI 10.1007/s10555-007-9063-1
View details for Web of Science ID 000248300100012
View details for PubMedID 17458506
Hif-1 alpha regulates differentiation of limb bud mesenchyme and joint development
JOURNAL OF CELL BIOLOGY
2007; 177 (3): 451-464
Recent evidence suggests that low oxygen tension (hypoxia) may control fetal development and differentiation. A crucial mediator of the adaptive response of cells to hypoxia is the transcription factor Hif-1alpha. In this study, we provide evidence that mesenchymal condensations that give origin to endochondral bones are hypoxic during fetal development, and we demonstrate that Hif-1alpha is expressed and transcriptionally active in limb bud mesenchyme and in mesenchymal condensations. To investigate the role of Hif-1alpha in mesenchymal condensations and in early chondrogenesis, we conditionally inactivated Hif-1alpha in limb bud mesenchyme using a Prx1 promoter-driven Cre transgenic mouse. Conditional knockout of Hif-1alpha in limb bud mesenchyme does not impair mesenchyme condensation, but alters the formation of the cartilaginous primordia. Late hypertrophic differentiation is also affected as a result of the delay in early chondrogenesis. In addition, mutant mice show a striking impairment of joint development. Our study demonstrates a crucial, and previously unrecognized, role of Hif-1alpha in early chondrogenesis and joint formation.
View details for DOI 10.1083/jcb.200612023
View details for Web of Science ID 000246580000009
View details for PubMedID 17470636
Imaging tumoral hypoxia: Oxygen concentrations and beyond
2007; 21 (3): 368-376
The role of hypoxia as a key determinant of outcome for human cancers has encouraged efforts to noninvasively detect and localize regions of poor oxygenation in tumors. In this review, we will summarize existing and developing techniques for imaging tumoral hypoxia. A brief review of the biology of tumor oxygenation and its effect on tumor cells will be provided initially. We will then describe existing methods for measurement of tissue oxygenation status. An overview of emerging molecular imaging techniques based on radiolabeled hypoxic markers such as misonidazole or hypoxia-related genes and proteins will then be given, and the usefulness of these approaches toward targeting hypoxia directly will be assessed. Finally, we will evaluate the clinical potential of oxygen- and molecular-specific techniques for imaging hypoxia, and discuss how these methods will individually and collectively advance oncology.
View details for Web of Science ID 000253822400010
View details for PubMedID 17447439
Multiple factors affecting cellular redox status and energy metabolism modulate hypoxia-inducible factor prolyl hydroxylase activity in vivo and in vitro
MOLECULAR AND CELLULAR BIOLOGY
2007; 27 (3): 912-925
Prolyl hydroxylation of hypoxible-inducible factor alpha (HIF-alpha) proteins is essential for their recognition by pVHL containing ubiquitin ligase complexes and subsequent degradation in oxygen (O(2))-replete cells. Therefore, HIF prolyl hydroxylase (PHD) enzymatic activity is critical for the regulation of cellular responses to O(2) deprivation (hypoxia). Using a fusion protein containing the human HIF-1alpha O(2)-dependent degradation domain (ODD), we monitored PHD activity both in vivo and in cell-free systems. This novel assay allows the simultaneous detection of both hydroxylated and nonhydroxylated PHD substrates in cells and during in vitro reactions. Importantly, the ODD fusion protein is regulated with kinetics identical to endogenous HIF-1alpha during cellular hypoxia and reoxygenation. Using in vitro assays, we demonstrated that the levels of iron (Fe), ascorbate, and various tricarboxylic acid (TCA) cycle intermediates affect PHD activity. The intracellular levels of these factors also modulate PHD function and HIF-1alpha accumulation in vivo. Furthermore, cells treated with mitochondrial inhibitors, such as rotenone and myxothiazol, provided direct evidence that PHDs remain active in hypoxic cells lacking functional mitochondria. Our results suggest that multiple mitochondrial products, including TCA cycle intermediates and reactive oxygen species, can coordinate PHD activity, HIF stabilization, and cellular responses to O(2) depletion.
View details for DOI 10.1128/MCB.01223-06
View details for Web of Science ID 000243946000011
View details for PubMedID 17101781
HIF gene expression in cancer therapy
OXYGEN BIOLOGY AND HYPOXIA
2007; 435: 323-?
Tumor hypoxia is a feature common to almost all solid tumors due to malformed vasculature and inadequate perfusion. Tumor cells have evolved mechanisms that allow them to respond and adapt to a hypoxic microenvironment. The hypoxia-inducible transcription factor (HIF) family is comprised of oxygen-sensitive alpha (alpha) subunits that respond rapidly to decreased oxygen levels and oxygen-insensitive beta (beta) subunits. HIF binds to specific recognition sequences in the genome and increases the transcription of genes involved in a variety of metabolic and enzymatic pathways that are necessary for cells to respond to an oxygen-poor environment. The critical role of this family of transcriptional regulators in maintaining oxygen homeostasis is supported by multiple regulatory mechanisms that allow the cell to control the levels of HIF as well as its transcriptional activity. This review will focus on how the transcriptional activity of HIF is studied and how it can be exploited for cancer therapy.
View details for DOI 10.1016/S0076-6879(07)35016-7
View details for Web of Science ID 000251162300016
View details for PubMedID 17998061
HIF1 alpha delays premature senescence through the activation of MIF
GENES & DEVELOPMENT
2006; 20 (24): 3366-3371
Premature senescence in vitro has been attributed to oxidative stress leading to a DNA damage response. In the absence of oxidative damage that occurs at atmospheric oxygen levels, proliferation of untransformed cells continues for extended periods of time. We have investigated the role of the hypoxia-inducible factor 1alpha (HIF1alpha) transcription factor in preventing senescence in aerobic and hypoxic conditions. Using embryonic fibroblasts from a conditional HIF1alpha knockout mouse, we found that loss of HIF1alpha under aerobic conditions significantly accelerated the onset of cellular senescence, and decreased proliferation under hypoxia. Furthermore, we identify the macrophage migration inhibitory factor (MIF) as a crucial effector of HIF1alpha that delays senescence. Inhibition of MIF phenocopies loss of HIF1alpha. Our findings highlight a novel role for HIF1alpha under aerobic conditions, and identify MIF as a target responsible for this function.
View details for DOI 10.1101/gad.1471106
View details for Web of Science ID 000242996100004
View details for PubMedID 17142669
Plasma osteopontin is an independent prognostic marker for head and neck cancers
JOURNAL OF CLINICAL ONCOLOGY
2006; 24 (33): 5291-5297
To confirm the relationship between plasma osteopontin (OPN) levels and treatment outcomes in head and neck squamous cell carcinoma (HNSCC) patients in an expanded study.One hundred forty patients with newly diagnosed HNSCC were enrolled onto this study, 54 previously reported and 86 new patients. Pretreatment plasma OPN levels were assessed in all patients by an enzyme-linked immunosorbent assay method. OPN levels were correlated to treatment outcomes in the new group of patients. Detailed analyses were also performed on the relationship between OPN and tumor control rate, event-free survival (EFS), and postrelapse survival for the entire group.Using a previously defined cut off point of 450 ng/mL, there was a significant correlation between OPN and freedom-from-relapse (P = .047), overall survival (P = .019), and EFS (P = .023) in the new, independent patient cohort (n = 86). Sequence of event analyses using the entire group (N = 140) revealed that OPN was an independent prognostic factor for initial tumor control, EFS in those who have achieved tumor control, and postrelapse survival.In this expanded study, we were able to replicate the prognostic significance of OPN using a predefined cut off point in an independent patient group and demonstrated that plasma OPN is an independent prognostic marker for HNSCC.
View details for DOI 10.1200/JCO.2006.06.8627
View details for Web of Science ID 000242342800017
View details for PubMedID 17114663
Lysyl oxidase mediates hypoxic control of metastasis
2006; 66 (21): 10238-10241
Hypoxic cancer cells pose a great challenge to the oncologist because they are especially aggressive, metastatic, and resistant to therapy. Recently, we showed that elevation of the extracellular matrix protein lysyl oxidase (LOX) correlates with metastatic disease and is essential for hypoxia-induced metastasis. In an orthotopic rodent model of breast cancer, a small-molecule or antibody inhibitor of LOX abolished metastasis, offering preclinical validation of this enzyme as a therapeutic target.
View details for DOI 10.1158/0008-5472.CAN-06-3197
View details for Web of Science ID 000241781600003
View details for PubMedID 17079439
Hypoxia: Importance in tumor biology, noninvasive measurement by imaging, and value of its measurement in the management of cancer therapy
INTERNATIONAL JOURNAL OF RADIATION BIOLOGY
2006; 82 (10): 699-757
The Cancer Imaging Program of the National Cancer Institute convened a workshop to assess the current status of hypoxia imaging, to assess what is known about the biology of hypoxia as it relates to cancer and cancer therapy, and to define clinical scenarios in which in vivo hypoxia imaging could prove valuable.Hypoxia, or low oxygenation, has emerged as an important factor in tumor biology and response to cancer treatment. It has been correlated with angiogenesis, tumor aggressiveness, local recurrence, and metastasis, and it appears to be a prognostic factor for several cancers, including those of the cervix, head and neck, prostate, pancreas, and brain. The relationship between tumor oxygenation and response to radiation therapy has been well established, but hypoxia also affects and is affected by some chemotherapeutic agents. Although hypoxia is an important aspect of tumor physiology and response to treatment, the lack of simple and efficient methods to measure and image oxygenation hampers further understanding and limits their prognostic usefulness. There is no gold standard for measuring hypoxia; Eppendorf measurement of pO(2) has been used, but this method is invasive. Recent studies have focused on molecular markers of hypoxia, such as hypoxia inducible factor 1 (HIF-1) and carbonic anhydrase isozyme IX (CA-IX), and on developing noninvasive imaging techniques.This workshop yielded recommendations on using hypoxia measurement to identify patients who would respond best to radiation therapy, which would improve treatment planning. This represents a narrow focus, as hypoxia measurement might also prove useful in drug development and in increasing our understanding of tumor biology.
View details for DOI 10.1080/09553000601002324
View details for Web of Science ID 000242762100001
View details for PubMedID 17118889
Functional analysis of p53 binding under differential stresses
MOLECULAR AND CELLULAR BIOLOGY
2006; 26 (19): 7030-7045
Hypoxia and DNA damage stabilize the p53 protein, but the subsequent effect that each stress has on transcriptional regulation of known p53 target genes is variable. We have used chromatin immunoprecipitation followed by CpG island (CGI) microarray hybridization to identify promoters bound by p53 under both DNA-damaging and non-DNA-damaging conditions in HCT116 cells. Using gene-specific PCR analysis, we have verified an association with CGIs of the highest enrichment (> 2.5-fold) (REV3L, XPMC2H, HNRPUL1, TOR1AIP1, glutathione peroxidase 1, and SCFD2), with CGIs of intermediate enrichment (> 2.2-fold) (COX7A2L, SYVN1, and JAG2), and with CGIs of low enrichment (> 2.0-fold) (MYC and PCNA). We found little difference in promoter binding when p53 is stabilized by these two distinctly different stresses. However, expression of these genes varies a great deal: while a few genes exhibit classical induction with adriamycin, the majority of the genes are unchanged or are mildly repressed by either hypoxia or adriamycin. Further analysis using p53 mutated in the core DNA binding domain revealed that the interaction of p53 with CGIs may be occurring through both sequence-dependent and -independent mechanisms. Taken together, these experiments describe the identification of novel p53 target genes and the subsequent discovery of distinctly different expression phenomena for p53 target genes under different stress scenarios.
View details for DOI 10.1128/MCB.00322-06
View details for Web of Science ID 000240740200002
View details for PubMedID 16980608
- Hypoxia-inducible factor-1 and p53: Friends, acquaintances, or strangers? CLINICAL CANCER RESEARCH 2006; 12 (17): 5007-5009
The roles of Chk 1 and Chk 2 in hypoxia and reoxygenation
2006; 238 (2): 161-167
Both Chk 1 and Chk 2 are critically important checkpoint kinases. Chk 1 is an essential gene that is required for normal cell division and Chk 2 has been found to be mutated in an ever-growing list of human malignancies. Our recent studies indicate that both Chk 1 and Chk 2 have roles to play in the physiological stress of hypoxia/reoxygenation. Loss or inhibition of either kinase sensitizes cells to hypoxia/reoxygenation indicating that either or both could represent significant therapeutic targets.
View details for DOI 10.1016/j.canlet.2005.06.029
View details for Web of Science ID 000238904400001
View details for PubMedID 16085356
Checking in on hypoxia/reoxygenation
2006; 5 (12): 1304-1307
Hypoxia/reoxygenation is a physiological stress that activates the DNA damage pathway. Significantly, this pathway is initiated during hypoxia, in the absence of detectable DNA damage. Our most recent study determined that during hypoxia, Chk 2 is phosphorylated in an ATM-dependent manner. In addition to this finding, we found that components of the MRN complex were not required for Chk 2 phosphorylation during hypoxia/reoxygenation. Once activated, Chk 2 initiates a signaling cascade, which induces a cell cycle arrest in the G2 phase. Loss of the Chk 2-mediated arrest correlated with an increase in sensitivity to hypoxia/reoxygenation. In contrast, loss of a p53-mediated reoxygenation-induced G1 arrest does not correlate with increased sensitivity to hypoxia/reoxygenation.
View details for Web of Science ID 000238581200013
View details for PubMedID 16760660
Connective tissue growth factor-specific monoclonal antibody therapy inhibits pancreatic tumor growth and metastasis
2006; 66 (11): 5816-5827
Pancreatic cancer is highly aggressive and refractory to most existing therapies. Past studies have shown that connective tissue growth factor (CTGF) expression is elevated in human pancreatic adenocarcinomas and some pancreatic cancer cell lines. To address whether and how CTGF influences tumor growth, we generated pancreatic tumor cell lines that overexpress different levels of human CTGF. The effect of CTGF overexpression on cell proliferation was measured in vitro in monolayer culture, suspension culture, or soft agar, and in vivo in tumor xenografts. Although there was no effect of CTGF expression on proliferation in two-dimensional cultures, anchorage-independent growth (AIG) was enhanced. The capacity of CTGF to enhance AIG in vitro was linked to enhanced pancreatic tumor growth in vivo when these cells were implanted s.c. in nude mice. Administration of a neutralizing CTGF-specific monoclonal antibody, FG-3019, had no effect on monolayer cell proliferation, but blocked AIG in soft agar. Consistent with this observation, anti-CTGF treatment of mice bearing established CTGF-expressing tumors abrogated CTGF-dependent tumor growth and inhibited lymph node metastases without any toxicity observed in normal tissue. Together, these studies implicate CTGF as a new target in pancreatic cancer and suggest that inhibition of CTGF with a human monoclonal antibody may control primary and metastatic tumor growth.
View details for DOI 10.1158/0008-5472.CAN-06-0081
View details for Web of Science ID 000238003100038
View details for PubMedID 16740721
Genome-wide analysis of p53 under hypoxic conditions
MOLECULAR AND CELLULAR BIOLOGY
2006; 26 (9): 3492-3504
Hypoxia is an important nongenotoxic stress that modulates the tumor suppressor activity of p53 during malignant progression. In this study, we investigated how genotoxic and nongenotoxic stresses regulate p53 association with chromatin, p53 transcriptional activity, and p53-dependent apoptosis. We found that genotoxic and nongenotoxic stresses result in the accumulation and binding of the p53 tumor suppressor protein to the same cognate binding sites in chromatin. However, it is the stress that determines whether downstream signaling is mediated by association with transcriptional coactivators. In contrast to p53 induced by DNA-damaging agents, hypoxia-induced p53 has primarily transrepression activity. Using extensive microarray analysis, we identified families of repressed targets of p53 that are involved in cell signaling, DNA repair, cell cycle control, and differentiation. Following our previous study on the contribution of residues 25 and 26 to p53-dependent hypoxia-induced apoptosis, we found that residues 25-26 and 53-54 and the polyproline- and DNA-binding regions are also required for both gene repression and the induction of apoptosis by p53 during hypoxia. This study defines a new role for residues 53 and 54 of p53 in regulating transrepression and demonstrates that 25-26 and 53-54 work in the same pathway to induce apoptosis through gene repression.
View details for DOI 10.1128/MCB.26.9.3492-3504.2006
View details for Web of Science ID 000236993300013
View details for PubMedID 16611991
Lysyl oxidase is essential for hypoxia-induced metastasis
2006; 440 (7088): 1222-1226
Metastasis is a multistep process responsible for most cancer deaths, and it can be influenced by both the immediate microenvironment (cell-cell or cell-matrix interactions) and the extended tumour microenvironment (for example vascularization). Hypoxia (low oxygen) is clinically associated with metastasis and poor patient outcome, although the underlying processes remain unclear. Microarray studies have shown the expression of lysyl oxidase (LOX) to be elevated in hypoxic human tumour cells. Paradoxically, LOX expression is associated with both tumour suppression and tumour progression, and its role in tumorigenesis seems dependent on cellular location, cell type and transformation status. Here we show that LOX expression is regulated by hypoxia-inducible factor (HIF) and is associated with hypoxia in human breast and head and neck tumours. Patients with high LOX-expressing tumours have poor distant metastasis-free and overall survivals. Inhibition of LOX eliminates metastasis in mice with orthotopically grown breast cancer tumours. Mechanistically, secreted LOX is responsible for the invasive properties of hypoxic human cancer cells through focal adhesion kinase activity and cell to matrix adhesion. Furthermore, LOX may be required to create a niche permissive for metastatic growth. Our findings indicate that LOX is essential for hypoxia-induced metastasis and is a good therapeutic target for preventing and treating metastases.
View details for DOI 10.1038/nature04695
View details for Web of Science ID 000237080000052
View details for PubMedID 16642001
Effect of reduced oxygen tension on chondrogenesis and osteogenesis in adipose-derived mesenchymal cells
AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY
2006; 290 (4): C1139-C1145
Recent studies have demonstrated that adipose-derived mesenchymal cells (AMCs) offer great promise for cell-based therapies because of their ability to differentiate toward bone, cartilage, and fat. Given that cartilage is an avascular tissue and that mesenchymal cells experience hypoxia during prechondrogenic condensation in endochondral ossification, the goal of this study was to understand the influence of oxygen tension on AMC differentiation into bone and cartilage. In vitro chondrogenesis was induced using a three-dimensional micromass culture model supplemented with TGF-beta1. Collagen II production and extracellular matrix proteoglycans were assessed with immunohistochemistry and Alcian blue staining, respectively. Strikingly, micromasses differentiated in reduced oxygen tension (2% O(2)) showed markedly decreased chondrogenesis. Osteogenesis was induced using osteogenic medium supplemented with retinoic acid or vitamin D and was assessed with alkaline phosphatase activity and mineralization. AMCs differentiated in both 21 and 2% O(2) environments. However, osteogenesis was severely diminished in a low-oxygen environment. These data demonstrated that hypoxia strongly inhibits in vitro chondrogenesis and osteogenesis in AMCs.
View details for DOI 10.1152/ajpcell.00415.2005
View details for Web of Science ID 000236573300023
View details for PubMedID 16291817
Gene expression programs in response to hypoxia: Cell type specificity and prognostic significance in human cancers
2006; 3 (3): 395-409
Inadequate oxygen (hypoxia) triggers a multifaceted cellular response that has important roles in normal physiology and in many human diseases. A transcription factor, hypoxia-inducible factor (HIF), plays a central role in the hypoxia response; its activity is regulated by the oxygen-dependent degradation of the HIF-1alpha protein. Despite the ubiquity and importance of hypoxia responses, little is known about the variation in the global transcriptional response to hypoxia among different cell types or how this variation might relate to tissue- and cell-specific diseases.We analyzed the temporal changes in global transcript levels in response to hypoxia in primary renal proximal tubule epithelial cells, breast epithelial cells, smooth muscle cells, and endothelial cells with DNA microarrays. The extent of the transcriptional response to hypoxia was greatest in the renal tubule cells. This heightened response was associated with a uniquely high level of HIF-1alpha RNA in renal cells, and it could be diminished by reducing HIF-1alpha expression via RNA interference. A gene-expression signature of the hypoxia response, derived from our studies of cultured mammary and renal tubular epithelial cells, showed coordinated variation in several human cancers, and was a strong predictor of clinical outcomes in breast and ovarian cancers. In an analysis of a large, published gene-expression dataset from breast cancers, we found that the prognostic information in the hypoxia signature was virtually independent of that provided by the previously reported wound signature and more predictive of outcomes than any of the clinical parameters in current use.The transcriptional response to hypoxia varies among human cells. Some of this variation is traceable to variation in expression of the HIF1A gene. A gene-expression signature of the cellular response to hypoxia is associated with a significantly poorer prognosis in breast and ovarian cancer.
View details for DOI 10.1371/journal.pmed.0030047
View details for Web of Science ID 000236897500020
View details for PubMedID 16417408
An evaluation of tumor oxygenation and gene expression in patients with early stage non-small cell lung cancers
CLINICAL CANCER RESEARCH
2006; 12 (5): 1507-1514
To directly assess tumor oxygenation in resectable non-small cell lung cancers (NSCLC) and to correlate tumor pO2 and the selected gene and protein expression to treatment outcomes.Twenty patients with resectable NSCLC were enrolled. Intraoperative measurements of normal lung and tumor pO2 were done with the Eppendorf polarographic electrode. All patients had plasma osteopontin measurements by ELISA. Carbonic anhydrase-IX (CA IX) staining of tumor sections was done in the majority of patients (n = 16), as was gene expression profiling (n = 12) using cDNA microarrays. Tumor pO2 was correlated with CA IX staining, osteopontin levels, and treatment outcomes.The median tumor pO2 ranged from 0.7 to 46 mm Hg (median, 16.6) and was lower than normal lung pO2 in all but one patient. Because both variables were affected by the completeness of lung deflation during measurement, we used the ratio of tumor/normal lung (T/L) pO2 as a reflection of tumor oxygenation. The median T/L pO2 was 0.13. T/L pO2 correlated significantly with plasma osteopontin levels (r = 0.53, P = 0.02) and CA IX expression (P = 0.006). Gene expression profiling showed that high CD44 expression was a predictor for relapse, which was confirmed by tissue staining of CD44 variant 6 protein. Other variables associated with the risk of relapse were T stage (P = 0.02), T/L pO2 (P = 0.04), and osteopontin levels (P = 0.001).Tumor hypoxia exists in resectable NSCLC and is associated with elevated expression of osteopontin and CA IX. Tumor hypoxia and elevated osteopontin levels and CD44 expression correlated with poor prognosis. A larger study is needed to confirm the prognostic significance of these factors.
View details for DOI 10.1158/1078-0432.CCR-05-2049
View details for Web of Science ID 000235988000016
View details for PubMedID 16533775
DNA damage during reoxygenation elicits a Chk2-dependent checkpoint response
MOLECULAR AND CELLULAR BIOLOGY
2006; 26 (5): 1598-1609
Due to the abnormal vasculature of solid tumors, tumor cell oxygenation can change rapidly with the opening and closing of blood vessels, leading to the activation of both hypoxic response pathways and oxidative stress pathways upon reoxygenation. Here, we report that ataxia telangiectasia mutated-dependent phosphorylation and activation of Chk2 occur in the absence of DNA damage during hypoxia and are maintained during reoxygenation in response to DNA damage. Our studies involving oxidative damage show that Chk2 is required for G2 arrest. Following exposure to both hypoxia and reoxygenation, Chk2-/- cells exhibit an attenuated G2 arrest, increased apoptosis, reduced clonogenic survival, and deficient phosphorylation of downstream targets. These studies indicate that the combination of hypoxia and reoxygenation results in a G2 checkpoint response that is dependent on the tumor suppressor Chk2 and that this checkpoint response is essential for tumor cell adaptation to changes that result from the cycling nature of hypoxia and reoxygenation found in solid tumors.
View details for DOI 10.1128/MCB.26.5.1598-1609.2006
View details for Web of Science ID 000235602700003
View details for PubMedID 16478982
- Comment on: Osteopontin as toxic marker RADIOTHERAPY AND ONCOLOGY 2006; 78 (2): 230-230
The host cell transcription factor hypoxia-inducible factor 1 is required for Toxoplasma gondii growth and survival at physiological oxygen levels
2006; 8 (2): 339-352
Toxoplasma gondii is an obligate intracellular protozoan pathogen. We previously found that genes mediating cellular responses to hypoxia were upregulated in Toxoplasma -infected cells but not in cells infected with another intracellular pathogen, Trypanosoma cruzi. The inducible expression of these genes is controlled by the hypoxia-inducible factor 1 (HIF1) transcription factor, which is the master regulator of cells exposed to low oxygen. Because this response may be important for parasites to grow at physiological oxygen levels, we tested the hypothesis that HIF1 is important for Toxoplasma growth. Here, we demonstrate that Toxoplasma infection rapidly increased the abundance of the HIF1alpha subunit and activated HIF1 reporter gene expression. In addition, we found that Toxoplasma growth and survival was severely reduced in HIF1alpha knockout cells at 3% oxygen. While HIF1alpha was not required for parasite invasion, we determined that HIF1 was required for parasite cell division and organelle maintenance at 3% oxygen. These data indicate that Toxoplasma activates HIF1 and requires HIF1 for growth and survival at physiologically relevant oxygen levels.
View details for DOI 10.1111/j.1462-5822.2005.00628.x
View details for Web of Science ID 000234667200015
View details for PubMedID 16441443
Mutations in the PI3K/PTEN/TSC2 pathway contribute to mammalian target of rapamycin activity and increased translation under hypoxic conditions
2006; 66 (3): 1561-1569
Decreased oxygen causes a rapid inhibition of mRNA translation. An important regulatory mechanism of translational repression under hypoxic conditions involves inhibition of the mammalian target of rapamycin (mTOR). mTOR is a target of the phosphatase and tensin homologue detected on chromosome 10 (PTEN)/phosphatidylinositol 3-kinase/AKT/TSC2 pathway, a pathway that is frequently mutated in human cancers. Although hypoxia has been shown to inhibit mTOR activity, we show here that the hypoxia-induced inhibition of mTOR activity is attenuated in cells lacking TSC2 or PTEN, resulting in a higher translation rate even under hypoxic conditions. Comparison of mTOR inhibition by hypoxia alone or in combination with rapamycin showed that prolonged exposure to hypoxia was required to fully inhibit mTOR activity even in wild-type cells. Increased mTOR activity and protein synthesis did not translate into enhanced cell proliferation rates. However, lack of TSC2 resulted in a survival advantage when cells were exposed to hypoxia. Protection against hypoxia-induced cell death due to TSC2 deficiency is rapamycin-resistant, suggesting that TSC2 affects an apoptotic pathway. Tumors derived from TSC2 wild-type cells exhibited a growth delay compared with TSC2-deficient tumors, indicating that enhanced mTOR activity is advantageous in the initial phase of tumor growth. Therefore, failure to inhibit mTOR under oxygen-limiting conditions can be affected by upstream activating mutations and increases the survival and growth of hypoxic tumor cells.
View details for DOI 10.1158/0008-5472.CAN-05-3375
View details for Web of Science ID 000235095900040
View details for PubMedID 16452213
- Radiobiology for the Radiologist, Sixth Edition 2006
- Hypoxia in Cancer Apoptosis and Cancer Chemotherapy: From Cutting-edge Science to Novel Therapeutic Concepts 2006; 2: 777-798
Galectin-1: A link between tumor hypoxia and tumor immune privilege
JOURNAL OF CLINICAL ONCOLOGY
2005; 23 (35): 8932-8941
To identify a 15-KDa novel hypoxia-induced secreted protein in head and neck squamous cell carcinomas (HNSCC) and to determine its role in malignant progression.We used surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS) and tandem MS to identify a novel hypoxia-induced secreted protein in FaDu cells. We used immunoblots, real-time polymerase chain reaction (PCR), and enzyme-linked immunoabsorbent assay to confirm the hypoxic induction of this secreted protein as galectin-1 in cell lines and xenografts. We stained tumor tissues from 101 HNSCC patients for galectin-1, CA IX (carbonic anhydrase IX, a hypoxia marker) and CD3 (a T-cell marker). Expression of these markers was correlated to each other and to treatment outcomes.SELDI-TOF studies yielded a hypoxia-induced peak at 15 kDa that proved to be galectin-1 by MS analysis. Immunoblots and PCR studies confirmed increased galectin-1 expression by hypoxia in several cancer cell lines. Plasma levels of galectin-1 were higher in tumor-bearing severe combined immunodeficiency (SCID) mice breathing 10% O2 compared with mice breathing room air. In HNSCC patients, there was a significant correlation between galectin-1 and CA IX staining (P = .01) and a strong inverse correlation between galectin-1 and CD3 staining (P = .01). Expression of galectin-1 and CD3 were significant predictors for overall survival on multivariate analysis.Galectin-1 is a novel hypoxia-regulated protein and a prognostic marker in HNSCC. This study presents a new mechanism on how hypoxia can affect the malignant progression and therapeutic response of solid tumors by regulating the secretion of proteins that modulate immune privilege.
View details for DOI 10.1200/JCO.2005.02.0206
View details for Web of Science ID 000234026500004
View details for PubMedID 16219933
The hypoxic microenvironment of the skin contributes to Akt-mediated melanocyte transformation
2005; 8 (6): 443-454
Constitutive activation of Akt characterizes a high percentage of human melanomas and represents a poor prognostic factor of the disease. We show that Akt transforms melanocytes only in a hypoxic environment, which is found in normal skin. The synergy between Akt and hypoxia is HIF1alpha mediated. Inhibition of HIF1alpha decreases Akt transformation capacity in hypoxia and tumor growth in vivo, while overexpression of HIF1alpha allows anchorage-independent growth in normoxia and development of more aggressive tumors. Finally, we show that mTOR activity is necessary to maintain the transformed phenotype by sustaining HIF1alpha activity. Taken together, these findings demonstrate that Akt hyperactivation and HIF1alpha induction by normally occurring hypoxia in the skin significantly contribute to melanoma development.
View details for DOI 10.1016/j.ccr.2005.11.005
View details for Web of Science ID 000234164200004
View details for PubMedID 16338658
Hypoxia upregulates osteopontin expression in NIH-3T3 cells via a ras-activated enhancer
2005; 24 (43): 6555-6563
Osteopontin (OPN) is a secreted phosphoglycoprotein that has been linked to tumor progression and survival in several solid tumors, including head and neck cancers. Previous studies showed that OPN expression is induced by tumor hypoxia, and its plasma levels can serve as a surrogate marker for tumor hypoxia and treatment outcome in head and neck cancer patients. In this study, we investigate the transcriptional mechanism by which hypoxia enhances OPN expression. We found that OPN is induced in head and neck squamous cell carcinoma (HNSCC) cell lines and in NIH3T3 cells by hypoxia at both mRNA and protein levels in a time-dependent manner. Actinomycin D chase experiments showed that hypoxic induction of OPN was not due to increased mRNA stability. Deletion analyses of the mouse OPN promoter regions indicated that a ras-activated enhancer (RAE) located at -731 to -712 relative to the transcription start site was essential for hypoxia-enhanced OPN transcription. Using electrophoretic mobility shift assays with the RAE DNA sequence, we found that hypoxia induced sequence-specific DNA-binding complexes. Furthermore, hypoxia and ras exposure resulted in an additive induction of OPN protein and mRNA levels that appeared to be mediated by the RAE. Induction of OPN through the RAE element by hypoxia is mediated by an Akt-kinase signaled pathway as decreasing Akt levels with dominant negative constructs resulted in inhibition of OPN induction by hypoxia. Taken together, these results have identified a new hypoxia responsive transcriptional enhancer that is regulated by Akt signaling.
View details for DOI 10.1038/sj.onc.1208800
View details for Web of Science ID 000232204100009
View details for PubMedID 16007184
Coordinate regulation of the oxygen-dependent degradation domains of hypoxia-inducible factor 1 alpha
MOLECULAR AND CELLULAR BIOLOGY
2005; 25 (15): 6415-6426
Oxygen-dependent proteolysis is the primary means of regulating the hypoxia-inducible factor (HIF) family of transcription factors. The alpha-subunit of HIF factor 1 (HIF-1) contains two highly conserved oxygen-dependent degradation domains (402 ODD and 564 ODD), each of which includes a proline that is hydroxylated in the presence of oxygen, allowing the von Hippel-Lindau (VHL) E3 ubiquitin ligase to interact and target HIF-1alpha to the proteasome for degradation. Mutation of either proline is sufficient to partially stabilize HIF-1alpha under conditions of normoxia, but the specific contributions of each hydroxylation event to the regulation of HIF-1alpha are unknown. Here we show that the two ODDs of HIF-1alpha have independent yet interactive roles in the regulation of HIF-1alpha protein turnover, with the relative involvement of each ODD depending on the levels of oxygen. Using hydroxylation-specific antibodies, we found that under conditions of normoxia proline 564 is hydroxylated prior to proline 402, and mutation of proline 564 results in a significant reduction in the hydroxylation of proline 402. Mutation of proline 402, however, has little effect on the hydroxylation of proline 564. To determine whether the more rapid hydroxylation of the proline 564 under conditions of normoxia is due to a preference for the particular sequence surrounding proline 564 or for that site within the protein, we exchanged the degradation domains within the full-length HIF-1alpha protein. In these domain-swapping experiments, prolyl hydroxylase domain 1 (PHD1) and PHD2 preferentially hydroxylated the proline located in the site of the original 564 ODD, while PHD3 preferred the proline 564 sequence, regardless of its location. At limiting oxygen tensions, we found that proline 402 exhibits an oxygen-dependent decrease in hydroxylation at higher oxygen tensions relative to proline 564 hydroxylation. These results indicate that hydroxylation of proline 402 is highly responsive to physiologic changes in oxygen and, therefore, plays a more important role in HIF-1alpha regulation under conditions of hypoxia than under conditions of normoxia. Together, these findings demonstrate that each hydroxylated proline of HIF-1alpha has a distinct activity in controlling HIF-1alpha stability in response to different levels of oxygenation.
View details for DOI 10.1128/MCB.25.15.6415-6426.2005
View details for Web of Science ID 000230684400011
View details for PubMedID 16024780
A noninvasive approach for assessing tumor hypoxia in xenografts: Developing a urinary marker for hypoxia
2005; 65 (14): 6151-6158
Tumor hypoxia modifies the efficacy of conventional anticancer therapy and promotes malignant tumor progression. Human chorionic gonadotropin (hCG) is a glycoprotein secreted during pregnancy that has been used to monitor tumor burden in xenografts engineered to express this marker. We adapted this approach to use urinary beta-hCG as a secreted reporter protein for tumor hypoxia. We used a hypoxia-inducible promoter containing five tandem repeats of the hypoxia-response element (HRE) ligated upstream of the beta-hCG gene. This construct was stably integrated into two different cancer cell lines, FaDu, a human head and neck squamous cell carcinoma, and RKO, a human colorectal cancer cell line. In vitro studies showed that tumor cells stably transfected with this plasmid construct secrete beta-hCG in response to hypoxia or hypoxia-inducible factor 1alpha (HIF-1alpha) stabilizing agents. The hypoxia responsiveness of this construct can be blocked by treatment with agents that affect the HIF-1alpha pathways, including topotecan, 1-benzyl-3-(5'-hydroxymethyl-2'-furyl)indazole (YC-1), and flavopiridol. Immunofluorescent analysis of tumor sections and quantitative assessment with flow cytometry indicate colocalization between beta-hCG and 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide (EF5) and beta-hCG and pimonidazole, two extrinsic markers for tumor hypoxia. Secretion of beta-hCG from xenografts that contain these stable constructs is directly responsive to changes in tumor oxygenation, including exposure of the animals to 10% O2 and tumor bed irradiation. Similarly, urinary beta-hCG levels decline after treatment with flavopiridol, an inhibitor of HIF-1 transactivation. This effect was observed only in tumor cells expressing a HRE-regulated reporter gene and not in tumor cells expressing a cytomegalovirus-regulated reporter gene. The 5HRE beta-hCG reporter system described here enables serial, noninvasive monitoring of tumor hypoxia in a mouse model by measuring a urinary reporter protein.
View details for Web of Science ID 000230633400024
View details for PubMedID 16024616
Angiogenesis is required for successful bone induction during distraction osteogenesis
JOURNAL OF BONE AND MINERAL RESEARCH
2005; 20 (7): 1114-1124
The role of angiogenesis during mechanically induced bone formation is incompletely understood. The relationship between the mechanical environment, angiogenesis, and bone formation was determined in a rat distraction osteogenesis model. Disruption of either the mechanical environment or endothelial cell proliferation blocked angiogenesis and bone formation. This study further defines the role of the mechanical environment and angiogenesis during distraction osteogenesis.Whereas successful fracture repair requires a coordinated and complex transcriptional program that integrates mechanotransductive signaling, angiogenesis, and osteogenesis, the interdependence of these processes is not fully understood. In this study, we use a system of bony regeneration known as mandibular distraction osteogenesis (DO) in which a controlled mechanical stimulus promotes bone induction after an osteotomy and gradual separation of the osteotomy edges to examine the relationship between the mechanical environment, angiogenesis, and osteogenesis.Adult Sprague-Dawley rats were treated with gradual distraction, gradual distraction plus the angiogenic inhibitor TNP-470, or acute distraction (a model of failed bony regeneration). Animals were killed at the end of distraction (day 13) or at the end of consolidation (day 41) and examined with muCT, histology, and immunohistochemistry for angiogenesis and bone formation (n = 4 per time-point per group). An additional group of animals (n = 6 per time-point per group) was processed for microarray analysis at days 5, 9, 13, 21, and 41.Either TNP-470 administration or disruption of the mechanical environment prevented normal osteogenesis and resulted in a fibrous nonunion. Subsequent analysis of the regenerate showed an absence of angiogenesis by gross histology and immunohistochemical localization of platelet endothelial cell adhesion molecule in the groups that failed to heal. Microarray analysis revealed distinct patterns of expression of genes associated with osteogenesis, angiogenesis, and hypoxia in each of the three groups. Our findings confirm the interdependence of the mechanical environment, angiogenesis, and osteogenesis during DO, and suggest that induction of proangiogenic genes and the proper mechanical environment are both necessary to support new vasculature for bone induction in DO.
View details for DOI 10.1359/JBMR.050301
View details for Web of Science ID 000230134100005
View details for PubMedID 15940364
The role of p53 in hypoxia-induced apoptosis
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
2005; 331 (3): 718-725
Hypoxia represents one of the most physiologically relevant stresses, having significant roles in both normal development and malignant progression. Exposure to severe hypoxia leads to the accumulation of p53 which can in turn lead to rapid apoptosis. In contrast to the response to DNA-damaging agents, hypoxia-induced p53 has little or no transcriptional transactivation capabilities and instead seems to function primarily as a transrepressor in order to induce apoptosis.
View details for Web of Science ID 000229135900007
View details for PubMedID 15865928
Anoxia is necessary for tumor cell toxicity caused by a low-oxygen environment
2005; 65 (8): 3171-3178
Cells exposed to oxygen deprivation in vitro have been shown to reduce proliferation and/or engage in programmed cell death. There is considerable controversy in the literature as to the role of hypoxia-inducible factor-1 (HIF-1) and HIF-1 target genes in initiating these responses. We therefore examined the oxygen dependence and the role of the hypoxia-responsive transcription factor HIF-1 in making the cellular death decision. Oxygen concentrations as low as 0.5% did not alter the growth of HIF-1-proficient or HIF-1-deficient murine fibroblasts, or human tumor cells, despite the appropriate induction of HIF-1 target genes. Severe hypoxia (<0.01% oxygen) did induced apoptosis, resulting in decreased colony formation, chromatin condensation, DNA fragmentation, and caspase activation but also independent of HIF1alpha status. Transcriptional induction of HIF-1-dependent genes putatively involved in cell death like BNip3 and BNip3L was therefore disassociated from hypoxia-dependent toxicity. Likewise, forced overexpression of a nondegradable form of HIF-1alpha in several human tumor cell lines was not sufficient to induce apoptosis under normoxic conditions. Taken together, these findings indicate that additional molecular events are triggered by anoxia in a HIF-1-independent manner, and these changes are necessary for cell death observed in low-oxygen environments.
View details for Web of Science ID 000228424800029
View details for PubMedID 15833847
Adaptive myogenesis under hypoxia
MOLECULAR AND CELLULAR BIOLOGY
2005; 25 (8): 3040-3055
Previous studies have indicated that myoblasts can differentiate and repair muscle injury after an ischemic insult. However, it is unclear how hypoxia or glucose deprivation in the ischemic microenvironment affects myoblast differentiation. We have found that myogenesis can adapt to hypoxic conditions. This adaptive mechanism is accompanied by initial inhibition of the myoD, E2A, and myogenin genes followed by resumption of their expression in an oxygen-dependent manner. The regulation of myoD transcription by hypoxia is correlated with transient deacetylation of histones associated with the myoD promoter. It is noteworthy that, unlike the differentiation of other cell types such as preadipocytes or chondroblasts, the effect of hypoxia on myogenesis is independent of HIF-1, a ubiquitous regulator of transcription under hypoxia. While myogenesis can also adapt to glucose deprivation, the combination of severe hypoxia and glucose deprivation found in an ischemic environment results in pronounced loss of myoblasts. Our studies indicate that the ischemic muscle can be repaired via the adaptive differentiation of myogenic precursors, which depends on the levels of oxygen and glucose in the ischemic microenvironment.
View details for DOI 10.1128/MCB.25.8.3040-3055.2005
View details for Web of Science ID 000228138500015
View details for PubMedID 15798192
OS-9 interacts with hypoxia-inducible factor 1 alpha and prolyl hydroxylases to promote oxygen-dependent degradation of HIF-1 alpha
2005; 17 (4): 503-512
Hypoxia-inducible factor 1 (HIF-1) functions as a master regulator of oxygen homeostasis in metazoan species. HIF-1 mediates changes in gene transcription in response to changes in cellular oxygenation. The half-life of the HIF-1alpha subunit is determined by oxygen-dependent prolyl hydroxylation, which is required for binding of the von Hippel-Lindau protein (VHL), the recognition component of an E3 ubiquitin ligase that targets HIF-1alpha for ubiquitination and degradation. Here, we demonstrate that OS-9, the protein product of a widely expressed gene, interacts with both HIF-1alpha and HIF-1alpha prolyl hydroxylases. OS-9 gain-of-function promotes HIF-1alpha hydroxylation, VHL binding, proteasomal degradation of HIF-1alpha, and inhibition of HIF-1-mediated transcription. OS-9 loss-of-function caused by RNA interference increases HIF-1alpha protein levels, HIF-1-mediated transcription, and VEGF mRNA expression under nonhypoxic conditions. These data indicate that OS-9 is an essential component of a multiprotein complex that regulates HIF-1alpha levels in an O2-dependent manner.
View details for DOI 10.1016/j.molcel.2005.01.011
View details for Web of Science ID 000227143400007
View details for PubMedID 15721254
The p53(QS) transactivation-deficient mutant shows stress-specific apoptotic activity and induces embryonic lethality
2005; 37 (2): 145-152
The role of transcriptional activation in p53 function is highly controversial. To define this role in vivo, we generated a Trp53 knock-in construct encoding a protein carrying mutations of two residues that are crucial for transactivation (L25Q,W26S). Here we show that these mutations have selective effects on the biological functions of p53. Although its ability to activate various p53 target genes is largely compromised, the p53(QS) protein retains the ability to transactivate the gene Bax. The ability of the p53(QS) mutant protein to elicit a DNA damage-induced G1 cell cycle-arrest response is also partially impaired. p53(QS) has selective defects in its ability to induce apoptosis: it is completely unable to activate apoptosis in response to DNA damage, is partially unable to do so when subjected to serum deprivation and retains substantial apoptotic activity upon exposure to hypoxia. These findings suggest that p53 acts through distinct, stimulus-specific pathways to induce apoptosis. The importance of the biological activity of p53(QS) in vivo is underscored by our finding that expression of p53(QS), which cannot bind mdm2, induces embryonic lethality. Taken together, these results suggest that p53 has different mechanisms of action depending on specific contextual cues, which may help to clarify the function of p53 in preventing cancer.
View details for DOI 10.1038/ng1498
View details for Web of Science ID 000226690100020
View details for PubMedID 15654339
Identification of hypoxia-regulated proteins in head and neck cancer by proteomic and tissue array profiling
2004; 64 (20): 7302-7310
Hypoxia within solid tumors decreases therapeutic efficacy, and identification of hypoxia markers may influence the choice of therapeutic modality. Here, we used a proteomic approach to identify hypoxia-regulated proteins and validated their use as endogenous indicators of tumor hypoxia. Using two-dimensional gel electrophoresis and PowerBlot (antibody-based array), we identified a group of 20 proteins that are increased >/=1.5-fold during hypoxia. The majority of these proteins such as IkappaB kinase beta (IKKbeta), MKK3b, highly expressed in cancer (HEC), density-regulated protein 1, P150(glued), nuclear transport factor 2, binder of ARL 2, Paxillin, and transcription termination factor I have not been previously reported to be hypoxia inducible. The increase in these proteins under hypoxia was mediated through posttranscriptional mechanisms. We additionally characterized the role of IKKbeta, a regulator of the nuclear factor-kappaB transcription factor, during hypoxia. We demonstrated that IKKbeta mediates cell survival during hypoxia and is induced in a variety of squamous cell carcinoma cell lines. Furthermore, we showed that IKKbeta expression from tumor specimens correlated with tumor oxygenation in patients with head and neck squamous cell carcinomas. These data suggest that IKKbeta is a novel endogenous marker of tumor hypoxia and may represent a new target for anticancer therapy.
View details for Web of Science ID 000224522200021
View details for PubMedID 15492250
JunD reduces tumor angiogenesis by protecting cells from oxidative stress
2004; 118 (6): 781-794
Reactive oxygen species (ROS) are implicated in the pathophysiology of various diseases, including cancer. In this study, we show that JunD, a member of the AP-1 family of transcription factors, reduces tumor angiogenesis by limiting Ras-mediated production of ROS. Using junD-deficient cells, we demonstrate that JunD regulates genes involved in antioxidant defense, H2O2 production, and angiogenesis. The accumulation of H2O2 in junD-/- cells decreases the availability of FeII and reduces the activity of HIF prolyl hydroxylases (PHDs) that target hypoxia-inducible factors-alpha (HIFalpha) for degradation. Subsequently, HIF-alpha proteins accumulate and enhance the transcription of VEGF-A, a potent proangiogenic factor. Our study uncovers the mechanism by which JunD protects cells from oxidative stress and exerts an antiangiogenic effect. Furthermore, we provide new insights into the regulation of PHD activity, allowing immediate reactive adaptation to changes in O2 or iron levels in the cell.
View details for Web of Science ID 000223992400013
View details for PubMedID 15369676
Transient changes in oxygen tension inhibit osteogenic differentiation and Runx2 expression in osteoblasts
JOURNAL OF BIOLOGICAL CHEMISTRY
2004; 279 (38): 40007-40016
Vascular disruption following bony injury results in a hypoxic gradient within the wound microenvironment. Nevertheless, the effects of low oxygen tension on osteogenic precursors remain to be fully elucidated. In the present study, we investigated in vitro osteoblast and mesenchymal stem cell differentiation following exposure to 21% O(2) (ambient oxygen), 2% O(2) (hypoxia), and <0.02% O(2) (anoxia). Hypoxia had little effect on osteogenic differentiation. In contrast, short-term anoxic treatment of primary osteoblasts and mesenchymal precursors inhibited in vitro bone nodule formation and extracellular calcium deposition. Cell viability assays revealed that this effect was not caused by immediate or delayed cell death. Microarray profiling implicated down-regulation of the key osteogenic transcription factor Runx2 as a potential mechanism for the anoxic inhibition of differentiation. Subsequent analysis revealed not only a short-term differential regulation of Runx2 and its targets by anoxia and hypoxia, but a long-term inhibition of Runx2 transcriptional and protein levels after only 12-24 h of anoxic insult. Furthermore, we present evidence that Runx2 inhibition may, at least in part, be because of anoxic repression of BMP2, and that restoring Runx2 levels during anoxia by pretreatment with recombinant BMP2 rescued the anoxic inhibition of differentiation. Taken together, our findings indicate that brief exposure to anoxia (but not 2% hypoxia) down-regulated BMP2 and Runx2 expression, thus inhibiting critical steps in the osteogenic differentiation of pluripotent mesenchymal precursors and committed osteoblasts.
View details for DOI 10.1074/jbc.M403715200
View details for Web of Science ID 000223791500098
View details for PubMedID 15263007
The biology of hypoxia: the role of oxygen sensing in development, normal function, and disease
GENES & DEVELOPMENT
2004; 18 (18): 2183-2194
The ability to sense and respond to changes in oxygen is essential for the survival of prokaryotic and eukaryotic organisms. Oxygen-sensing mechanisms have been developed to maintain cell and tissue homeostasis, as well as to adapt to the chronic low-oxygen conditions found in diseases such as cancer. This report on the first Keystone Meeting on the Biology of Hypoxia will summarize our current understanding of key genes and pathways involved in oxygen sensing that are required for normal development and that are dysregulated in disease states. It will also comment on future directions for this exciting field.
View details for DOI 10.1101/gad.1243304
View details for Web of Science ID 000223835400001
View details for PubMedID 15371333
Inhibition of ATR leads to increased sensitivity to hypoxia/reoxygenation
2004; 64 (18): 6556-6562
The transient opening and closing of tumor vasculature result in periods of severe oxygen deprivation (hypoxia) followed by reoxygenation. This exerts a positive selective pressure for cells that have lost their sensitivity to killing by reduced oxygen levels. These cells are effectively resistant to hypoxia-induced apoptosis and conventional therapeutic approaches. Here we show hypoxia-induced S-phase arrest results in regions of single-stranded DNA in stalled replication forks and signals the activation of ATR. S-phase cells represent the most sensitive phase of the cell cycle to the stress of hypoxia/reoxygenation. Loss of ATR or inhibition of ATR kinase activity results in a further loss of reproductive viability in S-phase cells when exposed to hypoxic conditions followed by reoxygenation but has little effect on the inhibition of DNA synthesis. This is, at least in part, mediated via Chk1 signaling because loss of Chk1 also results in increased sensitivity to hypoxia/reoxygenation. The observed decrease in reproductive survival is in part because of the accumulation of DNA damage in S-phase cells during hypoxia exposure in the absence of full ATR activity. Therefore, ATR acts to protect stalled replication forks during hypoxia exposure. In conclusion, ATR and Chk1 play critical roles in the cellular response to hypoxia/reoxygenation, and inhibitors of ATR and Chk1 represent new hypoxic cell cytotoxins.
View details for Web of Science ID 000224089700031
View details for PubMedID 15374968
Developmental response to hypoxia
2004; 18 (12): 1348-1365
Molecular mechanisms underlying fetal growth restriction due to placental insufficiency and in utero hypoxia are not well understood. In the current study, time-dependent (3 h-11 days) changes in fetal tissue gene expression in a rat model of in utero hypoxia compared with normoxic controls were investigated as an initial approach to understand molecular events underlying fetal development in response to hypoxia. Under hypoxic conditions, litter size was reduced and IGFBP-1 was up-regulated in maternal serum and in fetal liver and heart. Tissue-specific, distinct regulatory patterns of gene expression were observed under acute vs. chronic hypoxic conditions. Induction of glycolytic enzymes was an early event in response to hypoxia during organ development; consistently, tissue-specific induction of calcium homeostasis-related genes and suppression of growth-related genes were observed, suggesting mechanisms underlying hypoxia-related fetal growth restriction. Furthermore, induction of inflammation-related genes in placentas exposed to long-term hypoxia (11 days) suggests a mechanism for placental dysfunction and impaired pregnancy outcome accompanying in utero hypoxia.
View details for DOI 10.1096/fj.03-1377com
View details for Web of Science ID 000224243200054
View details for PubMedID 15333578
Timeline - Radiation oncology: a century of achievements
NATURE REVIEWS CANCER
2004; 4 (9): 737-U15
Over the twentieth century the discipline of radiation oncology has developed from an experimental application of X-rays to a highly sophisticated treatment of cancer. Experts from many disciplines - chiefly clinicians, physicists and biologists - have contributed to these advances. Whereas the emphasis in the past was on refining techniques to ensure the accurate delivery of radiation, the future of radiation oncology lies in exploiting the genetics or the microenvironment of the tumour to turn cancer from an acute disease to a chronic disease that can be treated effectively with radiation.
View details for DOI 10.1038/nrc1451
View details for Web of Science ID 000223657800018
View details for PubMedID 15343280
XBP1 is essential for survival under hypoxic conditions and is required for tumor growth
2004; 64 (17): 5943-5947
Hypoxia within solid tumors is a major determinant of outcome after anticancer therapy. Analysis of gene expression changes during hypoxia indicated that unfolded protein response genes were one of the most robustly induced groups of genes. In this study, we investigated the hypoxic regulation of X-box binding protein (XBP1), a major transcriptional regulator of the unfolded protein response. Hypoxia induced XBP1 at the transcriptional level and activated splicing of its mRNA, resulting in increased levels of activated XBP1 protein. After exposure to hypoxia, apoptosis increased and clonogenic survival decreased in XBP1-deficient cells. Loss of XBP1 severely inhibited tumor growth due to a reduced capacity for these transplanted tumor cells to survive in a hypoxic microenvironment. Taken together, these studies directly implicate XBP1 as an essential survival factor for hypoxic stress and tumor growth.
View details for Web of Science ID 000223603200007
View details for PubMedID 15342372
Hypoxic gene expression and metastasis
CANCER AND METASTASIS REVIEWS
2004; 23 (3-4): 293-310
Solid tumors possess malformed vasculature that results in the exposure of tumor cells to a low oxygen environment. Tumor hypoxia has been demonstrated in human and mouse tumors through the use of oxygen microelectrodes, hypoxic specific biomarkers, specific transcriptional changes induced by hypoxia, and secreted proteins. While many elegant experiments have demonstrated that hypoxia enhances metastatic potential, it is still unknown what mechanisms are involved in this enhancement. In this review, we discuss the clinical and basic science studies that support an important role for hypoxia in increasing the metastatic potential of tumor cells by promoting tissue remodeling, inducing angiogenesis and reducing apoptosis. Particular emphasis is given to recent findings that provide insight to the role of hypoxia in the metastatic process.
View details for Web of Science ID 000222017400009
View details for PubMedID 15197330
The role of ATM and ATR in the cellular response to hypoxia and re-oxygenation
2004; 3 (8-9): 1117-1122
ATM and ATR are stress-response kinases which respond to a variety of insults including ionizing radiation, replication arrest, ultraviolet radiation and hypoxia/re-oxygenation. Hypoxia occupies a unique niche in the study of both ATR- and ATM-mediated checkpoint pathways. Hypoxia is a physiologically significant stress that occurs in virtually all solid tumors and differs from most other stresses in that it does not induce DNA damage. Previous studies have indicated that hypoxia provides a unique way to induce ATR in response to inhibition of DNA replication. During tumor expansion hypoxia is inevitably followed by periods of re-oxygenation which in vitro has been shown to induce significant levels of DNA damage and an ATM response. Therefore both ATR and ATM have a role to play in hypoxia/re-oxygenation.
View details for DOI 10.1016/j.dnarep.2004.03.035
View details for Web of Science ID 000223650700038
View details for PubMedID 15279800
The use of plasma surface-enhanced laser desorption/ionization time-of-flight mass spectrometry proteomic patterns for detection of head and neck squamous cell cancers
CLINICAL CANCER RESEARCH
2004; 10 (14): 4806-4812
Our study was undertaken to determine the utility of plasma proteomic profiling using surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometry for the detection of head and neck squamous cell carcinomas (HNSCCs).Pretreatment plasma samples from HNSCC patients or controls without known neoplastic disease were analyzed on the Protein Biology System IIc SELDI-TOF mass spectrometer (Ciphergen Biosystems, Fremont, CA). Proteomic spectra of mass:charge ratio (m/z) were generated by the application of plasma to immobilized metal-affinity-capture (IMAC) ProteinChip arrays activated with copper. A total of 37356 data points were generated for each sample. A training set of spectra from 56 cancer patients and 52 controls were applied to the "Lasso" technique to identify protein profiles that can distinguish cancer from noncancer, and cross-validation was used to determine test errors in this training set. The discovery pattern was then used to classify a separate masked test set of 57 cancer and 52 controls. In total, we analyzed the proteomic spectra of 113 cancer patients and 104 controls.The Lasso approach identified 65 significant data points for the discrimination of normal from cancer profiles. The discriminatory pattern correctly identified 39 of 57 HNSCC patients and 40 of 52 noncancer controls in the masked test set. These results yielded a sensitivity of 68% and specificity of 73%. Subgroup analyses in the test set of four different demographic factors (age, gender, and cigarette and alcohol use) that can potentially confound the interpretation of the results suggest that this model tended to overpredict cancer in control smokers.Plasma proteomic profiling with SELDI-TOF mass spectrometry provides moderate sensitivity and specificity in discriminating HNSCC. Further improvement and validation of this approach is needed to determine its usefulness in screening for this disease.
View details for Web of Science ID 000222840700027
View details for PubMedID 15269156
- Stem cell differentiation NATURE BIOTECHNOLOGY 2004; 22 (7): 804-805
The hypoxic tumor microenvironment and gene expression
SEMINARS IN RADIATION ONCOLOGY
2004; 14 (3): 207-214
Solid tumors are not static entities but are constantly responding to environmental signals as they grow and develop. One mechanism by which they respond to the adverse conditions of the tumor microenvironment is through coordinated changes in gene expression. The synchronized turning of genes on and off leads to biologic adaption to the adverse oxygen-poor environment. Because tumor hypoxia can be found in almost every solid tumor, it represents one of the most pervasive microenvironmental stresses that can impact malignant progression and therapeutic response. Interestingly, tumors that exhibit robust induction of hypoxia-responsive gene expression networks show a clinically more aggressive natural history. The contribution of hypoxia-responsive gene networks to malignant response is currently under investigation. An understanding of the coordinated functions of hypoxia induced and repressed genes can lead to a better understanding of the clinical significance of the hypoxic tumor phenotype.
View details for DOI 10.1016/j.semradonc.2004.04.007
View details for Web of Science ID 000223088200003
View details for PubMedID 15254863
Deletion of Vhlh in chondrocytes reduces cell proliferation and increases matrix deposition during growth plate development
2004; 131 (10): 2497-2508
The von Hippel Lindau tumor suppressor protein (pVHL) is a component of a ubiquitin ligase that promotes proteolysis of the transcription factor hypoxia-inducible-factor 1alpha (HIF1alpha), the key molecule in the hypoxic response. We have used conditional inactivation of murine VHL (Vhlh) in all cartilaginous elements to investigate its role in endochondral bone development. Mice lacking Vhlh in cartilage are viable, but grow slower than control littermates and develop a severe dwarfism. Morphologically, Vhlh null growth plates display a significantly reduced chondrocyte proliferation rate, increased extracellular matrix, and presence of atypical large cells within the resting zone. Furthermore, stabilization of the transcription factor HIF1alpha leads to increased expression levels of HIF1alpha target genes in Vhlh null growth plates. Lastly, newborns lacking both Vhlh and Hif1a genes in growth plate chondrocytes display essentially the same phenotype as Hif1a null single mutant mice suggesting that the Vhlh null phenotype could result, at least in part, from increased activity of accumulated HIF1alpha. This is the first study reporting the novel and intriguing findings that pVHL has a crucial role in endochondral bone development and is necessary for normal chondrocyte proliferation in vivo.
View details for DOI 10.1242/dev.01138
View details for Web of Science ID 000222127700025
View details for PubMedID 15128677
Topical treatment with inhibitors of the phosphatidylinositol 3 '-kinase/akt and raf/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase pathways reduces melanoma development in severe combined immunodericient mice
2004; 64 (7): 2552-2560
Topical treatment with inhibitors of the phosphatidylinositol 3'-kinase/Akt and Raf/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase pathways inhibited the growth of TPras transgenic melanomas in severe combined immunodeficient mice, blocked invasive behavior, and reduced angiogenesis. The inhibitor Ly294002, which is specific for phosphatidylinositol 3'-kinase, effectively reduced melanoma cell growth both in vitro and in vivo. Both Ly294002 and U0126, a mitogen-activated protein kinase kinase 1/2 inhibitor, reduced invasion, which correlated with reduction of the metalloproteinase matrix metalloproteinase 2. Tumor angiogenesis was disrupted through inhibition of vascular endothelial growth factor production from the tumor cells and antiangiogenic effects on endothelial cells. Observations with TPras melanoma cells that express dominant negative Deltap85 or kinase-inactive Raf(301) supported the specificity of the phenomena observed with the chemical inhibitors. These studies demonstrate that topical treatment targeting Ras effectors is efficacious, without systemic toxicities, and may prove to be useful in treating and preventing the progression of cutaneous melanoma.
View details for Web of Science ID 000220586500035
View details for PubMedID 15059911
Distinct aerobic and hypoxic mechanisms of HIF-alpha regulation by CSN5
GENES & DEVELOPMENT
2004; 18 (7): 739-744
Mammalian oxygen homeostasis is dependent on the HIF family of transcription factors. The CSN subunit, CSN5, binds both the CODD of HIF-1 alpha and the pVHL tumor suppressor. High CSN5 expression generates a pVHL-independent form of CSN5 that stabilizes HIF-1 alpha aerobically by inhibiting HIF-1 alpha prolyl-564 hydroxylation. Aerobic CSN5 association with HIF-1 alpha occurs independently of the CSN holocomplex, leading to HIF-1 alpha stabilization independent of Cullin 2 deneddylation. CSN5 weakly associates with HIF-1 alpha under hypoxia, but is required for optimal hypoxia-mediated HIF-1 alpha stabilization. These results indicate that CSN5 regulates aerobic as well as hypoxic HIF-1 alpha stability by different mechanisms during oncogenesis.
View details for DOI 10.1101/gad.1180104
View details for Web of Science ID 000220923100002
View details for PubMedID 15082527
Dead cells don't form tumors - HIF-dependent cytotoxins
2004; 3 (2): 160-163
Elimination or reduction of tumor burden is the primary goal of cancer therapy. Strategies to achieve this goal with the fewest adverse effects to the patient are an area of intense investigation. Elevated protein levels of hypoxia-inducible factor (HIF) are commonly found in solid tumors, while rarely found in healthy tissue. Numerous studies have suggested that HIF activity is essential for the development of solid tumors. Thus, inhibition of HIF represents an attractive therapeutic target for eradicating tumors. The search for small molecules that target and inhibit HIF activity is currently underway. We propose an alternate approach: to directly target and kill HIF-activated tumor cells. This approach is advantageous in that cells with activated HIF will be eliminated directly. Specific elimination of HIF-activated cells represents a potential mechanism for inhibiting tumor growth, with the potential advantage of sparing the patient of the normal tissue toxicity associated with current treatment options.
View details for Web of Science ID 000222361200020
View details for PubMedID 14712081
- Oxygen-dependent regulation of adipogenesis OXYGEN SENSING 2004; 381: 387-?
Comparison of hypoxia-induced replication arrest with hydroxyurea and aphidicolin-induced arrest
MUTATION RESEARCH-FUNDAMENTAL AND MOLECULAR MECHANISMS OF MUTAGENESIS
2003; 532 (1-2): 205-213
Severe levels of hypoxia (oxygen concentrations of less that 0.02%) have been shown to induce a rapid S-phase arrest. The mechanism behind hypoxia-induced S-phase arrest is unclear, we show here that it was not mediated by a shortage of nucleosides and was not dependent on p53, p21 or Hif 1alpha status. The drugs aphidicolin and hydroxyurea both induce rapid replication arrest and have been used throughout the literature to study the ATR-mediated response to stalled replication. We have shown previously that hypoxia induces ATR-dependent phosphorylation of p53, Chk1 and histone H2AX. Using comet-assays to detect DNA-damage we found that both aphidicolin and hydroxyurea induced significant levels of DNA-damage while hypoxia did not. Here we show that like aphidicolin and hydroxyurea, hypoxia induces phosphorylation of Nbs1 at serine 343 and Rad17 serine 645. Hypoxia-dependent phosphorylation of Nbs1 and Rad17 was ATM-independent and therefore likely to be a result of the ATR kinase activity. In contrast, p53 was phosphorylated differentially in response to the three treatments considered here. p53 was phosphorylated at serine 15 in response to all three treatments but was only phosphorylated at serine 20 in response to the drug treatments. We propose that treatment with either aphidicolin or hydroxyurea leads to not only replication arrest but also DNA-damage and therefore both ATM and ATR-mediated signaling. In contrast replication arrest induced by severe hypoxia is sensed exclusively through ATR, with ATM only having a role to play after re-oxygenation.
View details for DOI 10.1016/j.mrfmmm.2003.08.017
View details for Web of Science ID 000187355500014
View details for PubMedID 14643437
Therapeutic exploitation of the physiological and molecular genetic alterations in head and neck cancer
CLINICAL CANCER RESEARCH
2003; 9 (12): 4287-4295
Despite improvements in the diagnosis and management of head and neck squamous cell carcinomas, there has been minimal increase in the long-term survival in these patients over the last 30 years. Treatment intensification with concurrent chemoradiotherapy has been shown to increase survival and improve organ preservation over radiotherapy alone in patients with locally advanced tumor; however, at a cost of increased long-term toxicity. Recent advances in molecular technology have ushered in a new age of targeted therapy, which holds promise for a better outcome for these patients with potentially less normal tissue toxicity. Some of the new approaches aim to specifically inhibit tumor growth and metastasis by targeting the tumor microenvironment or vasculature, whereas others focus on specific protein or signal transduction pathways. This review will summarize these new molecular and physiological based strategies that can be used for both treatment and chemoprevention of head and neck squamous cell carcinoma.
View details for Web of Science ID 000185830700001
View details for PubMedID 14555497
HIF-1 as a target for drug development
NATURE REVIEWS DRUG DISCOVERY
2003; 2 (10): 803-811
Sensing and responding to fluxes in oxygen tension is perhaps the single most important variable in physiology, and animal tissues have developed a number of essential mechanisms to cope with the stress of low physiological oxygen levels, or hypoxia. Among these coping mechanisms is the response mediated by the hypoxia-inducible transcription factor, or HIF-1. HIF-1 is an essential component in changing the transcriptional repertoire of tissues as oxygen levels drop, and could prove to be a very important target for drug development, as treatments evolve for diseases, such as cancer, heart disease and stroke, in which hypoxia is a central aspect.
View details for DOI 10.1038/nrd1199
View details for Web of Science ID 000185920400013
View details for PubMedID 14526383
Investigating hypoxic tumor physiology through gene expression patterns
2003; 22 (37): 5907-5914
Clinical evidence shows that tumor hypoxia is an independent prognostic indicator of poor patient outcome. Hypoxic tumors have altered physiologic processes, including increased regions of angiogenesis, increased local invasion, increased distant metastasis and altered apoptotic programs. Since hypoxia is a potent controller of gene expression, identifying hypoxia-regulated genes is a means to investigate the molecular response to hypoxic stress. Traditional experimental approaches have identified physiologic changes in hypoxic cells. Recent studies have identified hypoxia-responsive genes that may define the mechanism(s) underlying these physiologic changes. For example, the regulation of glycolytic genes by hypoxia can explain some characteristics of the Warburg effect. The converse of this logic is also true. By identifying new classes of hypoxia-regulated gene(s), we can infer the physiologic pressures that require the induction of these genes and their protein products. Furthermore, these physiologically driven hypoxic gene expression changes give us insight as to the poor outcome of patients with hypoxic tumors. Approximately 1-1.5% of the genome is transcriptionally responsive to hypoxia. However, there is significant heterogeneity in the transcriptional response to hypoxia between different cell types. Moreover, the coordinated change in the expression of families of genes supports the model of physiologic pressure leading to expression changes. Understanding the evolutionary pressure to develop a 'hypoxic response' provides a framework to investigate the biology of the hypoxic tumor microenvironment.
View details for DOI 10.1038/sj.onc.1206703
View details for Web of Science ID 000185086100017
View details for PubMedID 12947397
- HIF-alpha, a gender independent transcription factor CLINICAL CANCER RESEARCH 2003; 9 (7): 2391-2393
Hypoxia-induced gene expression occurs solely through the action of hypoxia-inducible factor 1 alpha (HIF-1 alpha): Role of cytoplasmic trapping of HIF-2 alpha
MOLECULAR AND CELLULAR BIOLOGY
2003; 23 (14): 4959-4971
The hypoxia-inducible factors 1alpha (HIF-1alpha) and 2alpha (HIF-2alpha) have extensive structural homology and have been identified as key transcription factors responsible for gene expression in response to hypoxia. They play critical roles not only in normal development, but also in tumor progression. Here we report on the differential regulation of protein expression and transcriptional activity of HIF-1alpha and -2alpha by hypoxia in immortalized mouse embryo fibroblasts (MEFs). We show that oxygen-dependent protein degradation is restricted to HIF-1alpha, as HIF-2alpha protein is detected in MEFs regardless of oxygenation and is localized primarily to the cytoplasm. Endogenous HIF-2alpha remained transcriptionally inactive under hypoxic conditions; however, ectopically overexpressed HIF-2alpha translocated into the nucleus and could stimulate expression of hypoxia-inducible genes. We show that the factor inhibiting HIF-1 can selectively inhibit the transcriptional activity of HIF-1alpha but has no effect on HIF-2alpha-mediated transcription in MEFs. We propose that HIF-2alpha is not a redundant transcription factor of HIF-1alpha for hypoxia-induced gene expression and show evidence that there is a cell type-specific modulator(s) that enables selective activation of HIF-1alpha but not HIF-2alpha in response to low-oxygen stress.
View details for DOI 10.1128/MCB.23.14.4959-4971.2003
View details for Web of Science ID 000183958100020
View details for PubMedID 12832481
ATR/ATM targets are phosphorylated by ATR in response to hypoxia and ATM in response to reoxygenation
JOURNAL OF BIOLOGICAL CHEMISTRY
2003; 278 (14): 12207-12213
The ATR kinase phosphorylates both p53 and Chk1 in response to extreme hypoxia (oxygen concentrations of less than 0.02%). In contrast to ATR, loss of ATM does not affect the phosphorylation of these or other targets in response to hypoxia. However, hypoxia within tumors is often transient and is inevitably followed by reoxygenation. We hypothesized that ATR activity is induced under hypoxic conditions because of growth arrest and ATM activity increases in response to the oxidative stress of reoxygenation. Using the comet assay to detect DNA damage, we find that reoxygenation induced significant amounts of DNA damage. Two ATR/ATM targets, p53 serine 15 and histone H2AX, were both phosphorylated in response to hypoxia in an ATR-dependent manner. These phosphorylations were then maintained in response to reoxygenation-induced DNA damage in an ATM-dependent manner. The reoxygenation-induced p53 serine 15 phosphorylation was inhibited by the addition of N-acetyl-l-cysteine (NAC), indicating that free radical-induced DNA damage was mediated by reactive oxygen species. Taken together these data implicate both ATR and ATM as critical roles in the response of hypoxia and reperfusion in solid tumors.
View details for DOI 10.1074/jbc.M212360200
View details for Web of Science ID 000182015700064
View details for PubMedID 12519769
Identification of osteopontin as a prognostic plasma marker for head and neck squamous cell carcinomas
CLINICAL CANCER RESEARCH
2003; 9 (1): 59-67
Tumor hypoxia modifies treatment efficacy and promotes tumor progression. Here, we investigated the relationship between osteopontin (OPN), tumor pO(2), and prognosis in patients with head and neck squamous cell carcinomas (HNSCC).We performed linear discriminant analysis, a machine learning algorithm, on the NCI-60 cancer cell line microarray expression database to identify a gene profile that best distinguish cell lines with high Von-Hippel Lindau (VHL) gene expression, an important regulator of hypoxia-related genes, from those with low expression. Plasma OPN levels in 15 volunteers, 31 VHL patients, and 54 HNSCC patients were quantitatively measured by ELISA. The relationships between plasma OPN levels, tumor pO(2) as measured by the Eppendorf microelectrode, freedom from relapse (FFR), and survival in HNSCC patients were evaluated.Microarray analysis indicated that OPN gene expression inversely correlated with that of VHL. These findings were confirmed by Northern blot analysis. ELISA studies and Western blot in a HNSCC cell line demonstrated that hypoxia exposure resulted in increased OPN secretion. Patients with VHL syndrome had significantly higher plasma OPN levels than healthy volunteers. Plasma OPN level inversely correlated with tumor pO(2) (P = 0.003, r = -0.42). OPN levels correlated with clinical outcomes. The 1-year FFR and survival rates were 80 and 100%, respectively, for patients with OPN levels
450 ng/ml (P = 0.002 and 0.0005). Multivariate analysis revealed that OPN was an independent predictor for FFR and survival.Plasma OPN levels appeared to correlate with tumor hypoxia in HNSCC patients and may serve as noninvasive tests to identify patients at high risk for tumor recurrence.
View details for Web of Science ID 000180430600008
View details for PubMedID 12538452
Tumor deprivation of oxygen and tumor suppressor gene function.
Methods in molecular biology (Clifton, N.J.)
2003; 223: 485-504
View details for PubMedID 12777746
- Hypoxia-Mediated Signaling Pathways Handbook of Cell Signaling 2003; 3: 277-282
Role of prolyl hydroxylation in oncogenically stabilized hypoxia-inducible factor-1 alpha
JOURNAL OF BIOLOGICAL CHEMISTRY
2002; 277 (42): 40112-40117
Stabilization of the hypoxia-inducible factor-1 (HIF-1) protein is essential for its role as a regulator of gene expression under low oxygen conditions. Here, employing a novel hydroxylation-specific antibody, we directly show that proline 564 of HIF-1alpha and proline 531 of HIF-2alpha are hydroxylated under normoxia. Importantly, HIF-1alpha Pro-564 and HIF-2alpha Pro-531 hydroxylation is diminished with the treatment of hypoxia, cobalt chloride, desferrioxamine, or dimethyloxalyglycine, regardless of the E3 ubiquitin ligase activity of the von Hippel-Lindau (VHL) tumor suppressor gene. Furthermore, in VHL-deficient cells, HIF-1alpha Pro-564 and HIF-2alpha Pro-531 had detectable amounts of hydroxylation following transition to hypoxia, indicating that the post-translational modification is not reversible. The introduction of v-Src or RasV12 oncogenes resulted in the stabilization of normoxic HIF-1alpha and the loss of hydroxylated Pro-564, demonstrating that oncogene-induced stabilization of HIF-1alpha is signaled through the inhibition of prolyl hydroxylation. Conversely, a constitutively active Akt oncogene stabilized HIF-1alpha under normoxia independently of prolyl hydroxylation, suggesting an alternative mechanism for HIF-1alpha stabilization. Thus, these results indicate distinct pathways for HIF-1alpha stabilization by different oncogenes. More importantly, these findings link oncogenesis with normoxic HIF-1alpha expression through prolyl hydroxylation.
View details for DOI 10.1074/jbc.M206922200
View details for Web of Science ID 000178662500133
View details for PubMedID 12186875
Regulation of hypoxia-inducible factor 1 alpha expression and function by the mammalian target of rapamycin
MOLECULAR AND CELLULAR BIOLOGY
2002; 22 (20): 7004-7014
Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric transcription factor containing an inducibly expressed HIF-1alpha subunit and a constititutively expressed HIF-1beta subunit. Under hypoxic conditions, the HIF-1alpha subunit accumulates due to a decrease in the rate of proteolytic degradation, and the resulting HIF-1alpha-HIF-1beta heterodimers undergo posttranslational modifications that promote transactivation. Recent studies suggest that amplified signaling through phosphoinositide 3-kinase, and its downstream target, mTOR, enhances HIF-1-dependent gene expression in certain cell types. In the present study, we have explored further the linkage between mTOR and HIF-1 in PC-3 prostate cancer cells treated with hypoxia or the hypoxia mimetic agent, CoCl(2). Pretreatment of PC-3 cells with the mTOR inhibitor, rapamycin, inhibited both the accumulation of HIF-1alpha and HIF-1-dependent transcription induced by hypoxia or CoCl(2). Transfection of these cells with wild-type mTOR enhanced HIF-1 activation by hypoxia or CoCl(2), while expression of a rapamycin-resistant mTOR mutant rendered both HIF-1alpha stabilization and HIF-1 transactivating function refractory to inhibition by rapamycin. Studies with GAL4-HIF-1alpha fusion proteins pinpointed the oxygen-dependent degradation domain as a critical target for the rapamycin-sensitive, mTOR-dependent signaling pathway leading to HIF-1alpha stabilization by CoCl(2). These studies position mTOR as an upstream activator of HIF-1 function in cancer cells and suggest that the antitumor activity of rapamycin is mediated, in part, through the inhibition of cellular responses to hypoxic stress.
View details for DOI 10.1128/MCB.22.20.7004-7014.2002
View details for Web of Science ID 000178266400007
View details for PubMedID 12242281
Epigenetic changes in tumor Fas levels determine immune escape and response to therapy
2002; 2 (2): 139-148
Epigenetic regulation of gene expression significantly influences cell growth and differentiation. Here we show that epigenetic silencing of Fas determines tumor growth in vivo and apoptotic sensitivity in vitro. In established tumors with epigenetically repressed Fas, restoration of Fas activity either by transfection of fas or treatment with Trichostatin A (TSA), an inhibitor of histone deacetylase, suppresses tumor growth and restores chemosensitivity. The TSA-dependent chemosensitivity and tumor growth control require both tumor Fas and the host NK (natural killer) cell functions. This work demonstrates the importance of epigenetic modification of Fas in tumor progression and immune evasion, and emphasizes the essential interplay between Fas and innate immunity in the control of chemoresistant tumors.
View details for Web of Science ID 000178352600009
View details for PubMedID 12204534
- Antiangiogenic therapy and p53 SCIENCE 2002; 297 (5581)
Effects of perillyl alcohol on melanoma in the TPras mouse model
CANCER EPIDEMIOLOGY BIOMARKERS & PREVENTION
2002; 11 (6): 573-579
This study evaluates the chemopreventive effects of topically applied perillyl alcohol on the development of melanoma in TPras transgenic mice. Our strategy was to target critical pathways in the development of melanoma, in particular, the ras pathway. Ras has been shown in our experimental mouse model, as well as others, to be important in the development and maintenance of melanomas. Perillyl alcohol (POH), a naturally occurring monoterpene, inhibits the isoprenylation of small G protein, including Ras. POH (10 mM) was applied to the shaved dorsal skin of TPras mice starting 1 week before five treatments of dimethylbenz[a]anthracene (50 microg) and was continued for 38 weeks. We observed a delay in the appearance of tumors and a 25-35% reduction in melanoma incidence. POH treatment of melanoma cells in vitro reduced the levels of detectable Ras protein and inhibits the activation of downstream targets, mitogen-activated protein kinases and Akt. POH only minimally induced apoptosis in this system. Pretreatment but not post-treatment of the melanoma cells with POH, however, markedly reduced levels of UV-induced reactive oxygen species. These studies suggest that POH inhibition of the Ras signaling pathway may be an effective target for chemoprevention of melanoma.
View details for Web of Science ID 000176142600010
View details for PubMedID 12050099
Inhibition of PPAR gamma 2 gene expression by the HIF-1-regulated gene DEC1/Stra13: A mechanism for regulation of adipogenesis by hypoxia
2002; 2 (3): 331-341
Cellular differentiation involves transcriptional responses to environmental stimuli. Adipocyte differentiation is inhibited under hypoxic conditions, indicating that oxygen (O(2)) is an important physiological regulator of adipogenesis. Hypoxia inhibits PPAR gamma 2 nuclear hormone receptor transcription, and overexpression of PPAR gamma 2 or C/EBP beta stimulates adipogenesis under hypoxia. Mouse embryonic fibroblasts deficient in hypoxia-inducible transcription factor 1 alpha (HIF-1 alpha) are refractory to hypoxia-mediated inhibition of adipogenesis. The HIF-1-regulated gene DEC1/Stra13, a member of the Drosophila hairy/Enhancer of split transcription repressor family, represses PPAR gamma 2 promoter activation and functions as an effector of hypoxia-mediated inhibition of adipogenesis. These data indicate that an O(2)-sensitive signaling mechanism regulates adipogenesis. Thus, agents that regulate HIF-1 activity or O(2) sensing may be used to inhibit adipogenesis and control obesity.
View details for Web of Science ID 000175387900012
View details for PubMedID 11879638
Hypoxia links ATR and p53 through replication arrest
MOLECULAR AND CELLULAR BIOLOGY
2002; 22 (6): 1834-1843
Previous studies have demonstrated that phosphorylation of human p53 on serine 15 contributes to protein stabilization after DNA damage and that this is mediated by the ATM family of kinases. However, cellular exposure to hypoxia does not induce any detectable level of DNA lesions compared to ionizing radiation, and the oxygen dependency of p53 protein accumulation differs from that of HIF-1, the hypoxia-inducible transcription factor. Here we show that, under severe hypoxic conditions, p53 protein accumulates only in S phase and this accumulation correlates with replication arrest. Inhibition of ATR kinase activity substantially reduces hypoxia-induced phosphorylation of p53 protein on serine 15 as well as p53 protein accumulation. Thus, hypoxia-induced cell growth arrest is tightly linked to an ATR-signaling pathway that is required for p53 modification and accumulation. These studies indicate that the ATR kinase plays an important role during tumor development in responding to hypoxia-induced replication arrest, and hypoxic conditions could select for the loss of key components of ATR-dependent checkpoint controls.
View details for DOI 10.1128/MCB.22.6.1834-1843.2002
View details for Web of Science ID 000174035500020
View details for PubMedID 11865061
Anticancer efficacy of systemically delivered anaerobic bacteria as gene therapy vectors targeting tumor hypoxia/necrosis
2002; 9 (4): 291-296
A major obstacle in cancer gene therapy is selective tumor delivery. Previous studies have suggested that genetically engineered anaerobes of the genus Clostridium might be gene therapy vectors because of their ability to proliferate selectively in the hypoxic/necrotic regions common to solid tumors. However, the tumor colonization efficiency of the strain previously used was insufficient to produce any antitumor effect. Here we describe for the first time the successful transformation of C. sporogenes, a clostridial strain with the highest reported tumor colonization efficiency, with the E. coli cytosine deaminase (CD) gene and show that systemically injected spores of these bacteria express CD only in the tumor. This enzyme can convert the nontoxic prodrug 5-fluorocytosine (5-FC) to the anticancer drug 5-fluorouracil (5-FU). Furthermore, systemic delivery of 5-FC into mice previously injected with CD-transformed spores of C. sporogenes produced greater antitumor effect than maximally tolerated doses of 5-FU. Since most human solid tumors have hypoxic and necrotic areas this vector system has considerable promise for tumor-selective gene therapy.
View details for DOI 10.1038/sj/gt/3301659
View details for Web of Science ID 000174314000007
View details for PubMedID 11896468
Hypoxia-inducible regulation of a prodrug-activating enzyme for tumor-specific gene therapy
2002; 4 (1): 40-48
Previous studies have suggested that tumor hypoxia could be exploited for cancer gene therapy. Using hypoxia-responsive elements derived from the human vascular endothelial growth factor gene, we have generated vectors expressing a bacterial nitroreductase (NTR) gene that can activate the anticancer prodrug CB1954. Stable transfectants of human HT1080 tumor cells with hypoxia-inducible vectors were established with G418 selection. Hypoxic induction of NTR protein correlated with increased sensitivity to in vitro exposure of HT1080 cells to the prodrug. Growth delay assays were performed with established tumor xenografts derived from the same cells to detect the in vivo efficacy of CB1954 conversion to its cytotoxic form. Significant antitumor effects were achieved with intraperitoneal injections of CB1954 both in tumors that express NTR constitutively or with a hypoxia-inducible promoter. In addition, respiration of 10% 02 increased tumor hypoxia in vivo and enhanced the antitumor effects. Taken together, these results demonstrate that hypoxia-inducible vectors may be useful for tumor-selective gene therapy, although the problem of delivery of the vector to the tumors, particularly to the hypoxic cells in the tumors, is not addressed by these studies.
View details for Web of Science ID 000173412000006
View details for PubMedID 11922390
- Genetic Basis of Altered Responsiveness of Cancer Cells to Their Microenvironment Oncogene Directed Therapies 2002: 113-129
- Hypoxia and Cell Cycle Cell Cycle Checkpoints and Cancer 2002: 143-154
Hypoxia regulates insulin-like growth factor-binding protein 1 in human fetal hepatocytes in primary culture: Suggestive molecular mechanisms for in utero fetal growth restriction caused by uteroplacental insufficiency
JOURNAL OF CLINICAL ENDOCRINOLOGY & METABOLISM
2001; 86 (6): 2653-2659
Intrauterine growth restriction (IUGR) can be a consequence of decreased uterine blood flow (uteroplacental insufficiency) and maternal and fetal hypoxia. Insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) are key elements in fetal growth. IGF-I is a major growth promoter in utero. IGFBP-1 is primarily made in the liver, and it mostly inhibits IGF actions at the cellular level. IGFBP-1 is elevated in the fetal circulation of human and animal pregnancies complicated by IUGR caused by placental insufficiency and in utero hypoxia and is believed to restrict fetal growth by sequestering IGFs. In this study, we developed a protocol to establish highly pure primary cultures of human fetal hepatocytes in vitro and investigated their expression of IGFBP-1 messenger RNA (mRNA) and protein and the effects of hypoxia on their expression of IGFBP-1 mRNA and protein. Hepatocytes were isolated from second-trimester human fetal livers (n = 7) and purified by Percoll gradient centrifugation. Hepatocyte cultures were characterized by immunocytochemistry and were compared with hepatocytes in situ in human fetal liver tissue, by immunohistochemistry, using specific antibodies and indirect immunofluorescence. Cultures consisted primarily (>90%) of cells positive for cytokeratin 18, fibrinogen, and IGFBP-1, with less than 2% vascular cells and less than 8% macrophages. Identification of isolated hepatocytes was further confirmed by morphology. Hepatocytes were cultured in defined medium, and Northern analysis revealed expression of a 1.5-kb IGFBP-1 mRNA transcript in hepatocytes cultured under normoxic conditions, for 24 h, that did not increase in steady-state levels after 48 h in culture. Under hypoxic conditions (2% O(2)), IGFBP-1 mRNA expression increased 3- to 4-fold, compared with normoxic controls. Cells cultured under 10% O(2) did not demonstrate an increase in IGFBP-1 mRNA levels. IGFBP-1 protein in conditioned medium (CM) was measured by immunoradiometric assay and increased 3- to 4-fold under hypoxic (2% O(2)), compared with normoxic, conditions. Western ligand blot analysis of CM revealed the presence of IGFBP-1, IGFBP-2, IGFBP-3, and IGFBP-4. IGFBP-1 was the most abundant IGFBP in CM, and densitometric analysis revealed a 2.5-fold increase in IGFBP-1 under hypoxic, compared with normoxic, conditions, supporting the immunoradiometric assay results. A 3-fold increase in IGFBP-3 mRNA, but not other IGFBPs, was noted under hypoxic, compared with normoxic, conditions. This study demonstrates that human fetal hepatocytes can be cultured in defined medium, as primary cultures with high purity, and that they express IGFBP-1 mRNA and secrete IGFBP-1 protein in vitro. In addition, the data demonstrate that hypoxia up-regulates fetal hepatocyte IGFBP-1 mRNA steady-state levels and protein, with this being the major IGFBP derived from the fetal hepatocyte. The data support a role for the fetal liver as a source of elevated circulating levels of IGFBP-1 in fetuses with in utero hypoxia and IUGR.
View details for Web of Science ID 000169412000049
View details for PubMedID 11397868
Hypoxia activates a platelet-derived growth factor receptor/phosphatidylinositol 3-kinase/Akt pathway that results in glycogen synthase kinase-3 inactivation
2001; 61 (6): 2429-2433
Hypoxia initiates numerous intracellular signaling pathways important in regulating cell proliferation, differentiation, and death. In this study, we investigated the pathway that hypoxia uses to activate Akt and inactivate glycogen synthase kinase-3 (GSK-3), two proteins the functions of which are important in cell survival and energy metabolism. Severe hypoxia (0.01% oxygen) initiated a signaling cascade by inducing the tyrosine phosphorylation of the platelet-derived growth factor (PDGF) receptor within 1 h of treatment and increasing receptor association with the p85 subunit of phosphatidylinositol 3-kinase (PI 3-K). Hypoxia-induced signaling also resulted in PI 3-K-dependent phosphorylation of Akt on Ser-473, a modification of Akt that is important for its activation. This activation of Akt by hypoxia was substantially diminished in cells that possessed mutations in their PDGF receptor-PI 3-K interaction domain. In addition, Akt activation by hypoxia was resistant to treatment with the growth factor receptor poison suramin but was sensitive to treatment with the PI 3-K inhibitor wortmannin. Activation of Akt by hypoxia resulted in the phosphorylation of GSK-3alpha and GSK-3beta at Ser-9 and Ser-21, two well-documented Akt phosphorylation sites, respectively, that are inactivating modifications of each GSK-3 isoform. In support of the phosphorylation data, GSK-3 activity was significantly reduced under hypoxia. In conclusion, we propose that hypoxia activates a growth factor receptor/PI 3-K/Akt cascade that leads to GSK-3 inactivation, a pathway that can impact cell survival, proliferation, and metabolism.
View details for Web of Science ID 000167697500017
View details for PubMedID 11289110
- Tumor hypoxia, the physiological link between Trousseau's syndrome (carcinoma-induced coagulopathy) and metastasis CANCER RESEARCH 2001; 61 (3): 795-798
Regulation of p53 by hypoxia: Dissociation of transcriptional repression and apoptosis from p53-dependent transactivation
MOLECULAR AND CELLULAR BIOLOGY
2001; 21 (4): 1297-1310
Hypoxic stress, like DNA damage, induces p53 protein accumulation and p53-dependent apoptosis in oncogenically transformed cells. Unlike DNA damage, hypoxia does not induce p53-dependent cell cycle arrest, suggesting that p53 activity is differentially regulated by these two stresses. Here we report that hypoxia induces p53 protein accumulation, but in contrast to DNA damage, hypoxia fails to induce endogenous downstream p53 effector mRNAs and proteins. Hypoxia does not inhibit the induction of p53 target genes by ionizing radiation, indicating that p53-dependent transactivation requires a DNA damage-inducible signal that is lacking under hypoxic treatment alone. At the molecular level, DNA damage induces the interaction of p53 with the transcriptional activator p300 as well as with the transcriptional corepressor mSin3A. In contrast, hypoxia primarily induces an interaction of p53 with mSin3A, but not with p300. Pretreatment of cells with an inhibitor of histone deacetylases that relieves transcriptional repression resulted in a significant reduction of p53-dependent transrepression and hypoxia-induced apoptosis. These results led us to propose a model in which different cellular pools of p53 can modulate transcriptional activity through interactions with transcriptional coactivators or corepressors. Genotoxic stress induces both kinds of interactions, whereas stresses that lack a DNA damage component as exemplified by hypoxia primarily induce interaction with corepressors. However, inhibition of either type of interaction can result in diminished apoptotic activity.
View details for Web of Science ID 000166651800030
View details for PubMedID 11158315
p21(Cip1) and p27(Kip1) regulate cell cycle reentry after hypoxic stress but are not necessary for hypoxia-induced arrest
MOLECULAR AND CELLULAR BIOLOGY
2001; 21 (4): 1196-1206
We investigated the role of the cyclin-dependent kinase inhibitors p21(Cip1) and p27(Kip1) in cell cycle regulation during hypoxia and reoxygenation. While moderate hypoxia (1 or 0.1% oxygen) does not significantly impair bromodeoxyuridine incorporation, at very low oxygen tensions (0.01% oxygen) DNA replication is rapidly shut down in immortalized mouse embryo fibroblasts. This S-phase arrest is intact in fibroblasts lacking the cyclin kinase inhibitors p21(Cip1) and p27(Kip1), indicating that these molecules are not essential elements of the arrest pathway. Hypoxia-induced arrest is accompanied by dephosphorylation of pRb and inhibition of cyclin-dependent kinase 2, which results in part from inhibitory phosphorylation. Interestingly, cells lacking the retinoblastoma tumor suppressor protein also display arrest under hypoxia, suggesting that pRb is not an essential mediator of this response. Upon reoxygenation, DNA synthesis resumes by 3.5 h and reaches aerobic levels by 6 h. Cells lacking p21, however, resume DNA synthesis more rapidly upon reoxygenation than wild-type cells, suggesting that this inhibitor may play a role in preventing premature reentry into the cell cycle upon cessation of the hypoxic stress. While p27 null cells did not exhibit rapid reentry into the cell cycle, cells lacking both p21 and p27 entered S phase even more aggressively than those lacking p21 alone, revealing a possible secondary role for p27 in this response. Cdk2 activity is also restored more rapidly in the double-knockout cells when returned to normoxia. These studies reveal that restoration of DNA synthesis after hypoxic stress, but not the S phase arrest itself, is regulated by p21 and p27.
View details for Web of Science ID 000166651800021
View details for PubMedID 11158306
Genetic determinants that influence hypoxia-induced apoptosis.
Novartis Foundation symposium
2001; 240: 115-128
The p53 tumour suppressor gene is capable of activating both death receptor and mitochondrial-signalled forms of apoptotic cell death in response to diverse stimuli. Studies have suggested that impairment of the mitochondrial-signalled Apaf/caspase 9 pathway and not the death receptor Fas pathway results in almost complete resistance to apoptotic cell death induced by a low oxygen environment. However, it is unclear how p53 signals the activation of this pathway and whether it is through already identified p53 effector genes such as the pro-apoptotic gene bax, or through novel effectors such as BNIP-3/BNIP-3L. Comparison of cell lines genetically matched at the bax, cytochrome c, apaf, caspase 9 and caspase 3 loci indicated that except for bax, all of these genes were essential for hypoxia induced apoptosis both in cell culture and in transplanted tumours. These data imply that cytochrome c plays a pivotal role in signalling cell death by apoptosis under hypoxic conditions, and that the release of cytochrome c is independent of both Bax and p53. In contrast to cytochrome c, p53 modulates the magnitude of apoptosis under hypoxic conditions, but in itself is not required for the activation of the caspase cascade.
View details for PubMedID 11727925
p53 promotes selection for Fas-mediated apoptotic resistance
2000; 60 (16): 4638-4644
Although p53 inactivation is implicated as a mechanism to explain diminished apoptotic response, it is clear that tumor cells that possess transcriptionally functional p53 can also be resistant to diverse apoptotic stimuli. We hypothesize that oncogenic activation and DNA damage are sufficient stimuli to increase the p53-dependent transcription of Fas and thereby establish a situation in which cell to cell contact could be a selective pressure to either lose p53 function or inactivate components of the Fas death pathway. Examination of genetically matched tumor cell lines that possessed either wild-type or null p53 loci indicated that cells possessing functional p53 increased their surface levels of Fas and Fas ligand (FasL) in response to DNA damage. In contrast, stress induced by changes in the tumor microenvironment such as decreased oxygen did not up-regulate Fas or FasL. Cells with wild-type p53 underwent Fas-mediated killing in the presence of either FasL-expressing killer cells or activating Fas antibodies, whereas cells in which p53 was deleted or inactivated were protected from such killing. Furthermore, Fas and FasL expression and induction became transcriptionally repressed in transformed cells with wild-type p53 with increasing passage, whereas other p53 downstream targets and functions, such as p21 inducibility and cell cycle arrest, remained intact. Repression of the Fas locus could be reverted by treatment with the histone deacetylase inhibitor trichostatin A. These results support a model of tumor progression in which oncogenic transformation drives tumor cells to lose either p53 or their Fas sensitivity as a means of promoting their survival and evade immune surveillance.
View details for Web of Science ID 000088961100055
View details for PubMedID 10969818
Regulation of insulin-like growth factor-binding protein-1 by nitric oxide under hypoxic conditions
JOURNAL OF CLINICAL ENDOCRINOLOGY & METABOLISM
2000; 85 (8): 2714-2721
Nitric oxide (NO) is believed to play an important, but as yet undefined, role in regulating hypoxia inducible gene expression. Recently, we have reported evidence suggesting that the human insulin-like growth factor-binding protein-1 (IGFBP-1) gene is directly regulated by hypoxia through the hypoxia-inducible factor-1 pathway. The goal of the current study was to investigate NO regulation of hypoxic induction of IGFBP-1 gene expression using HepG2 cells, a model system of hepatic gene expression. We report that a NO generator, sodium nitroprusside, significantly diminishes hypoxic activation of IGFBP-1 protein and messenger ribonucleic acid expression. Furthermore, these effects are independent of guanylate cyclase/ cGMP signaling, as two different inhibitors, LY 83583, a specific inhibitor of guanylate cyclase, and KT 5823, a protein kinase G inhibitor, had no effect on IGFBP-1 induction by hypoxia. Hypoxic induction of a reporter gene containing four tandemly ligated hypoxia response elements was completely blocked by sodium nitroprusside, but not by 8-bromo-cGMP, an analog ofcGMP. These results suggest that NO blocks hypoxic induction of IGFBP-1 by a guanylate cyclase/ cGMP-independent pathway, possibly at the level of oxygen sensing. The impaired hypoxia regulation of IGFBP-1 by nitric oxide may play a key role in the hyperinduction of IGFBP-1 observed in pathophysiological conditions such as fetal hypoxia and preeclampsia where dysregulation of NO has been observed.
View details for Web of Science ID 000165784500013
View details for PubMedID 10946870
p53 checkpoint-defective cells are sensitive to X rays, but not hypoxia
EXPERIMENTAL CELL RESEARCH
2000; 258 (1): 82-91
X-ray-induced damage leads to cell-cycle "checkpoint" arrest by p53-dependent induction of the cyclin-dependent kinase inhibitor p21 (Waf1/Cip1/Sdi1). Human tumor cells that lack this response fail to arrest after exposure to DNA-damaging agents, undergo multiple rounds of endoreduplicative DNA synthesis, and eventually commit to an apoptotic cell death. Since low oxygen tension can also induce p53 protein accumulation, and can lead to cell-cycle arrest or apoptosis, we examined the expression of p21 in tumor cells under normoxic and hypoxic conditions. In a survey of cells, mRNA for the p21 gene was induced two- to threefold in response to hypoxia in a seemingly p53-independent manner. We therefore examined genetically matched cells that differ in their p21 and p53 status for response to ionizing radiation and hypoxia. We found that both p21-deficient and p53-deficient cells exhibit an increase in chromosome instability, an increased level of apoptosis, and a failure to arrest after exposure to ionizing radiation. However, cells that lack either p21 or p53 exhibit no increase in chromosome instability or elevated apoptosis and still arrest in response to hypoxia. Thus, the mechanism responsible for the differential response to either hypoxia or X rays presumably lies in the control of cell-cycle progression in response to stress and its dependence on p21. Since the loss of a DNA-damage-dependent checkpoint does not sensitize cells to killing by stresses that elicit a DNA-damage-independent checkpoint, targeting the function of p21 pharmacologically will not kill tumor cells in situ in the absence of a DNA damage signal.
View details for DOI 10.1006/excr.2000.4928
View details for Web of Science ID 000088156200009
View details for PubMedID 10912790
Opposing effects of hypoxia on expression of the angiogenic inhibitor thrombospondin 1 and the angiogenic inducer vascular endothelial growth factor
CLINICAL CANCER RESEARCH
2000; 6 (7): 2941-2950
Tumor angiogenesis, the development of new blood vessels during malignant progression, is a regulated process that has both genetic and physiological controls. Physiologically, angiogenesis is stimulated by decreases in tissue oxygenation (i.e., hypoxia). We investigated the effect of hypoxia on the expression of two angiogenic factors reported to be genetically regulated by the p53 tumor suppressor gene: (a) the angiogenic inhibitor thrombospondin 1 (TSP-1); and (b) the angiogenic inducer vascular endothelial growth factor (VEGF). Analysis of rodent cells that differ in their p53 genotype (p53+/+ or p53-/-) indicated that in vitro exposure to hypoxia simultaneously suppressed TSP-1 and induced VEGF expression, regardless of the p53 genotype. On transformation of these cells with E1A and oncogenic H-ras, the basal level of TSP-1 expression was strongly diminished, whereas that of VEGF could still be induced by hypoxia. Consistent with these in vitro findings, sections of tumors derived from the transformed p53+/+ and p53-/- cells showed that VEGF protein overlapped with regions of hypoxia, whereas TSP-1 protein was below the limits of detection in tumor tissue. Using a panel of normal/immortalized and transformed human cells, it was found that the ability of hypoxia to inhibit TSP-1 expression depends on the cell type and/or the degree of transformation. In contrast, VEGF expression was induced by hypoxia in all of the human cell types examined. Together, these findings suggest that hypoxic and oncogenic signals could interact in the tumor microenvironment to inhibit TSP-1 and induce VEGF expression, promoting the switch to the angiogenic phenotype.
View details for Web of Science ID 000088147500047
View details for PubMedID 10914744
Development of a hypoxia-responsive vector for tumor-specific gene therapy
2000; 7 (6): 493-498
We are developing new gene therapy vectors whose expression is selectively activated by hypoxia, a unique feature of human solid tumors. As vascular endothelial growth factor (VEGF) is upregulated by hypoxia, such regulatory mechanisms would enable us to achieve hypoxia-inducible expression of therapeutic genes. Constructs with five copies of hypoxia-responsive elements (HREs) derived from the 5'-untranslated region (UTR) of the human VEGF showed excellent transcriptional activation at low oxygen tension relevant to tumor hypoxia. In an attempt to achieve higher responsiveness, various combinations of HREs and promoters were examined. In addition, we also investigated whether the 3' UTR of the VEGF gene would confer increased post-transcriptional mRNA stability under hypoxic conditions. However, despite increases in the hypoxic/aerobic ratio of luciferase activity, gene expression with 3' UTR was lower due to mRNA destabilization by AU-rich elements (AREs). Thus, we found no benefit from the inclusion of the 3' UTR in our vectors. Of all the vectors tested, the combination of 5HRE and a CMV minimal promoter exhibited hypoxia responsiveness (over 500-fold) to the similar level to the intact CMV promoter. We propose that this vector would be useful for tumor selective gene therapy.
View details for Web of Science ID 000085785500006
View details for PubMedID 10757022
Caveolin 1-mediated regulation of receptor tyrosine kinase-associated phosphatidylinositol 3-kinase activity by ceramide
MOLECULAR AND CELLULAR BIOLOGY
2000; 20 (5): 1507-1514
Previous studies have indicated that proapoptotic stresses downregulate the phosphatidylinositol 3-kinase [PI(3)K]/Akt survival pathway via the activation of acid-sphingomyelinase (A-SMase) and ceramide production. Ceramide induces apoptosis and inhibits PI(3)K activity without altering expression, association, or phosphorylation of receptors, adapter proteins, or PI(3)K subunits. PI(3)K inhibition by ceramide is associated with recruitment of caveolin 1 to PI(3)K-associated receptor complexes within lipid raft microdomains. Overexpression of caveolin 1 alone is sufficient to alter PI(3)K activity and sensitizes fibroblasts to ceramide-induced cell death. Most importantly, antisense expression of caveolin 1 dramatically reduces ceramide-induced PI(3)K deregulation and results in a loss-of-function stress response similar to that in A-SMase-deficient cells. Stress-induced recruitment of caveolin 1 to receptor complexes was found to be dependent on A-SMase since cell lines deficient in A-SMase did not exhibit caveolin 1 association with PI(3)K receptor complexes. Thus, a genetic link between A-SMase activation and caveolin 1-induced inhibition of PI(3)K activity exists. These results led us to propose that stress-induced changes in raft microdomains lead to altered receptor tyrosine kinase signal transduction through the modulation of caveolin 1 by ceramide.
View details for Web of Science ID 000085342200005
View details for PubMedID 10669728
Candidate genes for the hypoxic tumor phenotype
2000; 60 (4): 883-887
In this study, we have analyzed changes induced by hypoxia at the transcriptional level of genes that could be responsible for a more aggressive phenotype. Using a series of DNA array membranes, we identified a group of hypoxia-induced genes that included plasminogen activator inhibitor-1 (PAI-1), insulin-like growth factor-binding protein 3 (IGFBP-3), endothelin-2, low-density lipoprotein receptor-related protein (LRP), BCL2-interacting killer (BIK), migration-inhibitory factor (MIF), matrix metalloproteinase-13 (MMP-13), fibroblast growth factor-3 (FGF-3), GADD45, and vascular endothelial growth factor (VEGF). The induction of each gene was confirmed by Northern blot analysis in two different squamous cell carcinoma-derived cell lines. We also analyzed the kinetics of PAI-1 induction by hypoxia in more detail because it is a secreted protein that may serve as a useful molecular marker of hypoxia. On exposure to hypoxia, there was a gradual increase in PAI-1 mRNA between 2 and 24 h of hypoxia followed by a rapid decay after 2 h of reoxygenation. PAI-1 levels were also measured in the serum of a small group of head and neck cancer patients and were found to correlate with the degree of tumor hypoxia found in these patients.
View details for Web of Science ID 000085503100022
View details for PubMedID 10706099
Loss of PTEN facilitates HIF-1-mediated gene expression
GENES & DEVELOPMENT
2000; 14 (4): 391-396
In glioblastoma-derived cell lines, PTEN does not significantly alter apoptotic sensitivity or cause complete inhibition of DNA synthesis. However, in these cell lines PTEN regulates hypoxia- and IGF-1-induced angiogenic gene expression by regulating Akt activation of HIF-1 activity. Restoration of wild-type PTEN to glioblastoma cell lines lacking functional PTEN ablates hypoxia and IGF-1 induction of HIF-1-regulated genes. In addition, Akt activation leads to HIF-1alpha stabilization, whereas PTEN attenuates hypoxia-mediated HIF-1alpha stabilization. We propose that loss of PTEN during malignant progression contributes to tumor expansion through the deregulation of Akt activity and HIF-1-regulated gene expression.
View details for Web of Science ID 000085571300001
View details for PubMedID 10691731
Epigenetic regulation of gene expression in cervical cancer cells by the tumor microenvironment
CLINICAL CANCER RESEARCH
2000; 6 (2): 480-487
Evidence is accumulating that the adverse tumor microenvironment both modifies the malignant progression of tumor cells and contributes to chemotherapy and radiation resistance. We hypothesized that some of the effects on malignant progression are mediated through the transcriptional regulation of genes responsive to the stresses of the microenvironment, such as low oxygen or low glucose conditions. To determine epigenetic changes in gene expression that were consistent with that hypothesis, we used an in vitro subtractive hybridization method, representational difference analysis, to identify hypoxia-induced cDNAs from cultured human cervical epithelial cells. We identified 12 induced genes: two novel genes (HIG1 and HIG2), three genes known to be hypoxia-inducible (tissue factor, GAPDH, thioredoxin), and seven genes not previously identified as hypoxia-inducible [HNRNP(a1), ribosomal L7, annexin V, lipocortin 2, Ku(70), PRPP synthase, and acetoacetyl-CoA thiolase]. In cultured cells, HIG1 and HIG2 expression is induced by hypoxia and by glucose deprivation, but their expression is not induced by serum deprivation, UV, or ionizing radiation. The putative HIG1 and HIG2 open reading frames are expressed in cells, as confirmed by epitope tagging. In addition, tumor xenografts derived from human cervical cancer cells display increased expression of HIG1 and HIG2 when they are deprived of oxygen. Taken together, these data suggest a coordinated transcriptional response of eukaryotic cells to microenvironmental stresses found in the solid tumor.
View details for Web of Science ID 000085502600023
View details for PubMedID 10690527
Genetically modified clostridium for gene therapy of tumors.
Methods in molecular medicine
2000; 35: 413-418
Many murine and human tumors contain hypoxic or necrotic regions in which the oxygen tension is abnormally low. For example,>50% of primary tumors of the breast, cervix, and head and neck contain areas that are hypoxic. Because hypoxic regions are not present in normal tissue, this provides the potential for selectively targeting gene therapy to tumor cells.
View details for DOI 10.1385/1-59259-086-1:413
View details for PubMedID 21390820
Hypoxia induces p53 accumulation through MDM2 down-regulation and inhibition of E6-mediated degradation
1999; 59 (24): 6046-6051
Hypoxia, a result of DNA-damaging agents such as ionizing radiation, induces the nuclear accumulation of the p53 tumor suppressor protein. However, unlike the effect in ionizing radiation, hypoxia readily induces the nuclear accumulation of p53 in HPV E6-infected cells. In HPV-infected cells, a key regulator of p53 protein levels is the E6 oncoprotein. In association with the endogenous cellular protein E6-associated protein (E6AP), E6 can accelerate the degradation of p53 under aerobic conditions. To better define the mechanism of p53 induction in E6-infected cells by hypoxia, we studied the expression and association of E6 and E6AP with p53 in vivo. We found that hypoxia did not alter the protein levels of E6 or E6AP as compared with those found under aerobic growth conditions, indicating that protein inhibition of E6 or E6AP alone is not sufficient to explain the increased accumulation of p53 under hypoxic conditions. However, p53 did fail to coprecipitate with E6AP under hypoxia, indicating that hypoxia uncouples the interaction of p53 with E6 and E6AP. We also present evidence to indicate that hypoxia decreases the expression of the endogenous cellular regulator of p53 protein, the human MDM2 protein, resulting in an inhibition of p53 export from the nucleus to the cytoplasm for degradation. Taken together, these results suggest that the hypoxic induction of p53 is attributable to the down-regulation of MDM2 protein levels and uncoupling of p53 from its interaction with the E6/E6AP complex.
View details for Web of Science ID 000084530400007
View details for PubMedID 10626788
A p53 and apoptotic independent role for p21(waf1) in tumour response to radiation therapy
1999; 18 (47): 6540-6545
Loss of p21 in human cancer cells results in checkpoint failure, induction of polyploidy and subsequent apoptosis following DNA damage. Tumours in immunodeficient mice derived from cells lacking p21 are also more sensitive to ionizing radiation than their wild-type counterparts. Abrogation of p53 in the p21+/+ parental cells results in an in vitro phenotype that is indistinguishable from that of the p21 knockout cells. Thus, the in vitro phenotype resulting from loss of p21 is consistent with its well-established role in the p53/p21 damage response pathway. However, despite the similar in vitro phenotype, p21+/+ cells with abrogated p53 show no evidence of the sensitivity observed in the p21-/- cells when grown as tumours in immunodeficient mice. The increased radio-sensitization stabilization of p21-/- tumours is also unrelated to the increase in apoptosis observed in these tumours following radiation treatment. Apoptosis in the p21-/- tumours was significantly reduced by expression of bcl-2 without any corresponding change in the overall response of the tumour. Similarly, abrogation of p53 in the p21+/+ tumours substantially increased radiation-induced apoptosis within the tumours without increasing their radiation sensitivity. Dissociation of these in vivo and in vitro phenotypes indicates that p21 participates in a novel in vivo specific damage response pathway that is distinct from its role in the p53 pathway, and therefore that it may be an effective therapeutic target for cancer therapy.
View details for Web of Science ID 000083709200017
View details for PubMedID 10597257
Hypoxic microenvironment within an embryo induces apoptosis and is essential for proper morphological development
1999; 60 (4): 215-225
Recent studies have suggested the importance of hypoxia-inducible transcription factors in development, yet the questions of whether hypoxia actually exists in a developing embryo in vivo and, if so, what role it plays in development remain unanswered. In this study, we directly demonstrate that regions of hypoxia, most prominently the hindbrain, otic vesicle, and first branchial arch, exist in a gestational day (GD) 11 rat embryo grown in utero. We also show that varying the oxygen environment of an embryo affects its morphological development. Rat embryos which were grown at 45% oxygen from GD 9-11 showed gross morphological abnormalities, including defective cranial neural tube closure, incomplete otic vesicle invagination, and abnormal somite formation and embryo turning. These embryos, in addition, exhibited reduced cell death. On the other hand, embryos which were grown at 5% oxygen during the same period were stunted in overall growth, yet morphologically normal, and displayed prominent areas of apoptosis. In this study, we propose that embryonic development, like tumor development, requires two different but interactive sets of signals. One set exists in the genetic program for development; the other set arises from changes in the microenvironment of the embryo. Therefore, it is the interplay between these two sets of cues that drives normal embryonic development. The requirement for hypoxia to activate apoptotic cell death is but one example of such interactions.
View details for Web of Science ID 000082964400006
View details for PubMedID 10508975
Hypoxia and cAMP stimulate vascular endothelial growth factor (VEGF) in human endometrial stromal cells: Potential relevance to menstruation and endometrial regeneration
JOURNAL OF CLINICAL ENDOCRINOLOGY & METABOLISM
1999; 84 (6): 2245-2248
The human female reproductive tract shows unique cycle-specific changes in vascularization. Vascular endothelial growth factor (VEGF) is a specific vascular endothelial mitogen which is produced by human endometrium and is known to be regulated by steroid hormones. Vasoconstriction during menstruation leads to endometrial hypoxia, a possible stimulus for angiogenesis. In the current study we tested the hypothesis that hypoxia and cAMP, a known stimulus for endometrial decidualization, can induce VEGF in human endometrial stromal cells. Decidualized as well as non decidualized stromal cells from 6 patients were exposed to normoxia (20% oxygen) and hypoxia (2% oxygen) for up to 72h. VEGF levels were assessed by Northern analysis using a 605 bp BamHI fragment of the human VEGF cDNA, and hybridization signals were normalized to levels of 18S RNA. VEGF protein was determined by ELISA. Hypoxia stimulated VEGF mRNA in decidualized stromal cells by 10.2 fold at 48h compared to normoxic controls. VEGF protein increased 10 fold by 48h and increased further to 13 fold at 72h. In the presence of 2% oxygen VEGF mRNA in nondecidualized endometrial stromal cells was increased 1.2-8 fold between 2 and 72h of treatment. VEGF protein also increased 1.2-9 fold in this time period. cAMP regulated both VEGF mRNA and protein in non decidualized stromal cells. VEGF mRNA increased 2-4 fold in 2-72h and protein production showed a 2-6 fold increase. VEGF was seen to be regulated by both cAMP and hypoxia in an additive manner. These results demonstrate that both non-decidualized and decidualized endometrial stromal cells respond to hypoxia with increasing levels of VEGF. 8Br-cAMP, which is shown to increase VEGF levels in endometrial cells per se, has an additive effect on VEGF production under hypoxic conditions. This effect may have physiologic and pathophysiologic relevance during the process of menstruation and in post menstrual endometrial repair and angiogenesis.
View details for Web of Science ID 000080674000090
View details for PubMedID 10372740
Mitogen-activated protein kinase phosphatase-1 (MKP-1) expression is induced by low oxygen conditions found in solid tumor microenvironments - A candidate MKP for the inactivation of hypoxia-inducible stress-activated protein kinase/c-Jun N-terminal protein kinase activity
JOURNAL OF BIOLOGICAL CHEMISTRY
1999; 274 (18): 12890-12897
Pathophysiological hypoxia is an important modulator of gene expression in solid tumors and other pathologic conditions. We observed that transcriptional activation of the c-jun proto-oncogene in hypoxic tumor cells correlates with phosphorylation of the ATF2 transcription factor. This finding suggested that hypoxic signals transmitted to c-jun involve protein kinases that target AP-1 complexes (c-Jun and ATF2) that bind to its promoter region. Stress-inducible protein kinases capable of activating c-jun expression include stress-activated protein kinase/c-Jun N-terminal protein kinase (SAPK/JNK) and p38 members of the mitogen-activated protein kinase (MAPK) superfamily of signaling molecules. To investigate the potential role of MAPKs in the regulation of c-jun by tumor hypoxia, we focused on the activation SAPK/JNKs in SiHa human squamous carcinoma cells. Here, we describe the transient activation of SAPK/JNKs by tumor-like hypoxia, and the concurrent transcriptional activation of MKP-1, a stress-inducible member of the MAPK phosphatase (MKP) family of dual specificity protein-tyrosine phosphatases. MKP-1 antagonizes SAPK/JNK activation in response to diverse environmental stresses. Together, these findings identify MKP-1 as a hypoxia-responsive gene and suggest a critical role in the regulation of SAPK/JNK activity in the tumor microenvironment.
View details for Web of Science ID 000080056800098
View details for PubMedID 10212278
Apaf-1 and caspase-9 in p53-dependent apoptosis and tumor inhibition
1999; 284 (5411): 156-159
The ability of p53 to promote apoptosis in response to mitogenic oncogenes appears to be critical for its tumor suppressor function. Caspase-9 and its cofactor Apaf-1 were found to be essential downstream components of p53 in Myc-induced apoptosis. Like p53 null cells, mouse embryo fibroblast cells deficient in Apaf-1 and caspase-9, and expressing c-Myc, were resistant to apoptotic stimuli that mimic conditions in developing tumors. Inactivation of Apaf-1 or caspase-9 substituted for p53 loss in promoting the oncogenic transformation of Myc-expressing cells. These results imply a role for Apaf-1 and caspase-9 in controlling tumor development.
View details for Web of Science ID 000079509000057
View details for PubMedID 10102818
Activation of metallothionein gene expression by hypoxia involves metal response elements and metal transcription factor-1
1999; 59 (6): 1315-1322
Metallothioneins (MTs) are a family of stress-induced proteins with diverse physiological functions, including protection against metal toxicity and oxidants. They may also contribute to the regulation of cellular proliferation, apoptosis, and malignant progression. We reported previously that the human (h)MT-IIA isoform is induced in carcinoma cells (A431, SiHa, and HT29) exposed to low oxygen, conditions commonly found in solid tumors. The present study demonstrates that the genes for hMT-IIA and mouse (m)MT-I are transcriptionally activated by hypoxia through metal response elements (MREs) in their proximal promoter regions. These elements bind metal transcription factor-1 (MTF-1). Deletion and mutational analyses of the hMT-IIA promoter indicated that the hMRE-a element is essential for basal promoter activity and for induction by hypoxia, but that other elements contribute to the full transcriptional response. Functional studies of the mMT-I promoter demonstrated that at least two other MREs (mMRE-d and mMRE-c) are responsive to hypoxia. Multiple copies of either hMRE-a or mMRE-d conferred hypoxia responsiveness to a minimal MT promoter. Mouse MT-I gene transcripts in fibroblasts with targeted deletions of both MTF-1 alleles (MTF-1(-/-); dko7 cells) were not induced by zinc and showed low responsiveness to hypoxia. A transiently transfected MT promoter was unresponsive to hypoxia or zinc in dko7 cells, but inductions were restored by cotransfecting a mouse MTF-1 expression vector. Electrophoretic mobility shift assays detected a specific protein-DNA complex containing MTF-1 in nuclear extracts from hypoxic cells. Together, these results demonstrate that hypoxia activates MT gene expression through MREs and that this activation involves MTF-1.
View details for Web of Science ID 000079125200025
View details for PubMedID 10096565
- Multiple roles for myc deregulation in oncogenesis DNA Alternations in Cancer: Genetic and Epigenetic Changes 1999: 105-119
- Chemotherapy, tumor microenvironment and apoptosis Apoptosis and Cancer Chemotherapy 1999: 223-240
- The influence of tumor hypoxia on malignant progression Tumor Hypoxia: Pathophysiology, Clinical Significance and Therapeutic Perspectives 1999: 115-124
Highly conserved RNA sequences that are sensors of environmental stress
MOLECULAR AND CELLULAR BIOLOGY
1998; 18 (12): 7371-7382
The putative function of highly conserved regions (HCRs) within 3' untranslated regions (3'UTRs) as regulatory RNA sequences was efficiently and quantitatively assessed by using modular retroviral vectors. This strategy led to the identification of HCRs that alter gene expression in response to oxidative or mitogenic stress. Databases were screened for UTR sequences of >100 nucleotides that had retained 70% identity over more than 300 million years of evolution. The effects of 10 such HCRs on a standard reporter mRNA or protein were studied. To this end, we developed a modular retroviral vector that can allow for a direct comparison of the effects of different HCRs on gene expression independent of their gene-intrinsic 5'UTR, promoter, protein coding region, or poly(A) sequence. Five of the HCRs tested decreased mRNA steady-state levels 2- to 10-fold relative to controls, presumably by altering mRNA stability. One HCR increased translation, and one decreased translation. Elevated mitogen levels caused four HCRs to increase protein levels twofold. One HCR increased protein levels fourfold in response to hypoxia. Although nonconserved UTR sequences may also have a role, these results provide evidence that sequences that are highly conserved during evolution are good candidates for RNA motifs with posttranscriptional regulatory functions in gene expression.
View details for Web of Science ID 000077099700049
View details for PubMedID 9819424
- The complexity of p53 modulation: emerging patterns from divergent signals GENES & DEVELOPMENT 1998; 12 (19): 2973-2983
Hypoxia stimulates insulin-like growth factor binding protein 1 (IGFBP-1) gene expression in HepG2 cells: A possible model for IGFBP-1 expression in fetal hypoxia
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1998; 95 (17): 10188-10193
IGFBP-1 is elevated in fetuses with long-term, chronic hypoxia and intrauterine growth restriction. We investigated the hypothesis that hypoxia regulates IGFBP-1 in the human fetus in vivo and IGFBP-1 gene expression and protein in vitro. Umbilical artery IGFBP-1 levels (mean +/- SEM) from term babies with respiratory acidosis (acute hypoxia), normal babies, and those with mixed respiratory/metabolic acidosis (more profound and prolonged hypoxia) were measured using an immunoradiometric assay. IGFBP-1 levels were similar in normal (n = 12) and acutely hypoxic (n = 6) babies (189.1 +/- 71.8 vs. 175.8 +/- 45.9 ng /ml, respectively, P = 0.789). However, with more profound and prolonged hypoxia (n = 19), IGFBP-1 levels were markedly elevated (470.6 +/- 80.0 ng /ml, P = 0.044). To investigate IGFBP-1 regulation by hypoxia in vitro, HepG2 cells were incubated under hypoxia (pO2 = 2%) and normoxia (pO2 = 20%). IGFBP-1 protein and mRNA increased 8- and 12-fold, respectively, under hypoxic conditions. Hypoxia did not affect protein or mRNA levels of IGFBP-2 or -4. IGFBP-5 and -6 mRNAs, undetectable in control cells, were not induced by hypoxia, whereas minimally expressed IGFBP-3 mRNA increased twofold. Investigation into IGFBP-1 gene structure revealed three potential consensus sequences for the hypoxia response element (HRE) in the first intron. To investigate functionality, a 372-bp fragment of IGFBP-1 intron 1, containing putative HREs, was placed 5' to a heterologous hsp70 promoter in a plasmid using luciferase as a reporter gene. Under hypoxia, reporter gene activity increased up to 30-fold. Mutations in the middle HRE abolished reporter activity in response to hypoxia, suggesting that this HRE is functional in the IGFBP-1 hypoxia response. Cotransfection of HRE reporter genes with a constitutively expressing hypoxia-inducible factor 1 plasmid in HepG2 cells resulted in a fourfold induction of reporter activity, suggesting a role for hypoxia-inducible factor 1 in hypoxia induction of IGFBP-1 gene expression. These data support the hypothesis that hypoxia regulation of IGFBP-1 may be a mechanism operating in the human fetus to restrict insulin-like growth factor-mediated growth in utero under conditions of chronic hypoxia and limited substrate availability.
View details for Web of Science ID 000075475200085
View details for PubMedID 9707622
Heat shock protein 72 modulates pathways of stress-induced apoptosis
JOURNAL OF BIOLOGICAL CHEMISTRY
1998; 273 (27): 17147-17153
The resistance to stress-induced apoptosis conferred by the thermotolerant state or by exogenous expression of HSP72 was measured in mouse embryo fibroblasts. The induction of thermotolerance protects cells from heat, tumor necrosis factor alpha (TNFalpha), and ceramide-induced apoptosis but not from ionizing radiation. Because the development of thermotolerance is associated with increased levels of heat shock proteins, we determined whether constitutive expression of one of the major inducible heat shock proteins, HSP72, could also protect cells from stress-induced apoptosis. Cells expressing constitutive HSP72 were shown to have significantly reduced levels of apoptosis after heat, TNFalpha, and ceramide but not after ionizing radiation. Activation of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) was found to be strongly inhibited in thermotolerant cells after heat shock but not after other stresses. Cells that constitutively express HSP72 did not demonstrate decreased SAPK/JNK activation after any of these stresses. Thus, factors other than HSP72 that are induced in the thermotolerant state are able to reduce activation of SAPK/JNK after heat stress. Notably, the level of activation of SAPK/JNK did not correlate with the amount of apoptosis detected after different stresses. Constitutive HSP72 expression inhibited poly(ADP-ribose) polymerase cleavage in cells after heat shock and TNFalpha but not after ceramide or ionizing radiation. The results suggest either that SAPK/JNK activation is not required for apoptosis in mouse embryo fibroblasts or that HSP72 acts downstream of SAPK/JNK. Furthermore, the data support the concept that caspase activity, which can be down-regulated by HSP72, is a crucial step in stress-induced apoptosis. Based on data presented here and elsewhere, we propose that the heat shock protein family can be classified as a class of anti-apoptotic genes, in addition to the Bcl-2 and inhibitor of apoptosis protein families of genes.
View details for Web of Science ID 000074545200072
View details for PubMedID 9642282
- Tumor hypoxia and the cell cycle: Implications for malignant progression and response to therapy CANCER JOURNAL 1998; 4 (4): 218-223
Inhibition of the anti-apoptotic PI(3)K/Akt/Bad pathway by stress
GENES & DEVELOPMENT
1998; 12 (13): 1941-1946
The initiation of apoptosis often transpires in the presence of agents that regulate cell survival. This study evaluated the effects of stress-induced ceramide on the anti-apoptotic activity of the phosphoinositide-3 kinase [PI(3)K] pathway. PI(3)K activity is directly down-regulated by stress-induced ceramide in a dose-dependent manner with rapid kinetics and high specificity. Ceramide inhibition of PI(3)K is dependent on acid-sphingomyelinase. Down-regulation of PI(3)K by ceramide results in inhibition of the kinase Akt and decreased phosphorylation of the death effector Bad. Thus, ceramide levels could act as a general apoptotic rheostat controlling cell survival by regulating PI(3)K anti-apoptotic effector mechanisms.
View details for Web of Science ID 000074700100002
View details for PubMedID 9649498
The E6 and E7 genes of human papilloma virus-type 16 protect primary astrocyte cultures from injury
1998; 795 (1-2): 10-16
Many oncogenes are implicated in the regulation of apoptosis as well as in control of the cell cycle, and several have been shown to protect cells from injury. We tested whether the expression of human papillomavirus type 16 genes E6 and E7 could protect primary astrocytes from injury. Retroviral vectors were used to express E6, E7, or E6E7 in primary murine astrocyte cultures. Astrocytes expressing E6E7 suffered less than half the injury seen in controls after exposure to 400 microM H2O2. When we compared astrocytes expressing only E6 or E7, cells expressing E7 alone were protected to a greater extent and from more severe injury than those expressing E6. E6E7, E6 and E7 all provided protection from 30 h glucose deprivation, but again E7 provided the best protection, reducing injury to less than a third of that seen in controls. Expression of E7 alone decreases vulnerability to both hydrogen peroxide and glucose deprivation injury while E6 alone primarily decreases glucose deprivation injury. These results demonstrate that expression of human papillomavirus oncogenes can reduce the vulnerability of astrocytes to oxidative stress and nutrient deprivation.
View details for Web of Science ID 000074806500002
View details for PubMedID 9622584
Direct evidence that apoptosis enhances tumor responses to fractionated radiotherapy
1998; 58 (9): 1779-1784
Currently, the contribution of cellular apoptotic sensitivity to tumor response after radiation therapy remains controversial. To address this issue, the survival of Rat-1 fibroblasts containing a 4-hydroxytamoxifen-regulated c-Myc allele, c-MycER (T. D. Littlewood et al., Nucleic Acids Res., 23: 1686-1690, 1995), after single and fractionated doses of radiation was investigated. This model system allows pharmacological regulation of apoptosis sensitivity in the same cells in vitro and as xenograft tumors derived from these cells in vivo (G. I. Evan et al., Cell, 69: 119-128, 1992; R. M. Alarcon et al., Cancer Res., 56: 4315-4319, 1996). Activating c-MycER in vitro resulted in marked sensitization of Rat-1 fibroblasts to the effects of both single-dose and fractionated irradiation as measured by the induction of apoptosis and clonogenic survival. Overexpression of the antiapoptosis protein Bcl-2 suppressed the induction of apoptosis and increased clonogenic survival in cells with activated c-Myc after single-dose and fractionated radiation. Systemic time-release implant delivery of 4-hydroxytamoxifen to severe combined immunodeficient mice bearing Rat-1-MycER tumors over the course of either single-dose (10 Gy) or fractionated (five fractions of 2 Gy) radiotherapy resulted in prolonged tumor growth delay relative to identical tumors from mice that received placebo implants. Furthermore, tumors derived from Rat-1-MycER cells that overexpressed Bcl-2 exhibited shorter tumor growth delays relative to similarly treated Rat-1-MycER tumors. The length of tumor growth delay after single-dose or fractionated radiotherapy strongly correlated with the extent of radiation-induced apoptosis in the xenograft tumors as measured by terminal deoxynucleotidyl transferase-mediated nick end labeling. These in vivo results provide direct evidence that increasing the sensitivity of tumor cells to die by apoptosis increases the efficacy of fractionated radiotherapy by reducing tumor cell clonogenic survival.
View details for Web of Science ID 000073344700003
View details for PubMedID 9581811
The unique physiology of solid tumors: Opportunities (and problems) for cancer therapy
1998; 58 (7): 1408-1416
The physiology of solid tumors differs from that of normal tissues in a number of important aspects, the majority of which stem from differences between the two vasculatures. Compared with the regular, ordered vasculature of normal tissues, blood vessels in tumors are often highly abnormal, distended capillaries with leaky walls and sluggish flow. Tumor growth also requires continuous new vessel growth, or angiogenesis. These physiological differences can be problems for cancer treatment; for example, hypoxia in solid tumors leads to resistance to radiotherapy and to some anticancer drugs. However, these differences can also be exploited for selective cancer treatment. Here we review four such areas that are under active investigation: (a) hypoxia-selective cytotoxins take advantage of the unique low oxygen tension in the majority of human solid tumors. Tirapazamine, a drug in the final stages of clinical trials, is one of the more promising of these agents; (b) leaky tumor blood vessels can be exploited using liposomes that have been sterically stabilized to have a long intravascular half-life, allowing them to selectively accumulate in solid tumors; (c) the tumor microenvironment is a stimulus to angiogenenesis, and inhibition of angiogenesis can be a powerful anticancer therapy not susceptible to acquired drug resistance; and (d) we discuss attempts to use gene therapy activated either by the low oxygen environment or by necrotic regions of tumors.
View details for Web of Science ID 000072780200020
View details for PubMedID 9537241
Acidosis reduces neuronal apoptosis
1998; 9 (5): 875-879
Acidosis is a well established concomitant of tissue ischemia. Acidosis in the pH range 6.0-7.0 is seen in cerebral ischemia and within solid tumors. Extracellular acidosis of pH 6.0 and 6.4 provided essentially complete protection from 48 h serum deprivation induced apoptotic death of cultured primary murine neurons. We tested the effect of p53 using transformed mouse embryo fibroblasts of either p53+/+ or p53-/- genotype. Both were markedly protected from serum deprivation by acidity. Hypoxia induced fibroblast injury was also reduced at pH 6.8. Lower pH resulted in a shift from apoptotic to necrotic morphology after 42 h hypoxia. Acidosis reduces apoptosis of both normal and transformed cells, irrespective of p53 status.
View details for Web of Science ID 000072808900021
View details for PubMedID 9579683
p53 mediates apoptosis induced by c-Myc activation in hypoxic or gamma irradiated fibroblasts
CELL DEATH AND DIFFERENTIATION
1998; 5 (2): 141-147
Deregulated c-Myc expression leads to a cellular state where proliferation and apoptosis are equally favored depending on the cellular microenvironment. Since the apoptotic sensitivity of many cells is influenced by the status of the p53 tumor suppressor gene, we investigated whether the induction of apoptosis by DNA damage or non-genotoxic stress are also influenced by the p53 status of cells with altered c-Myc activity. Rat-1 fibroblasts expressing a conditional c-Myc allele (c-MycER), were transfected to express an antisense RNA complimentary to p53 mRNA. Expression of antisense p53 RNA decreased p53 protein levels and delayed p53 accumulation following c-Myc activation. Under hypoxic or low serum conditions, cells expressing antisense p53 were substantially more resistant to c-Myc-induced apoptosis than were control cells. c-Myc activation also sensitized Rat-1 cells to radiation-induced apoptosis. Rat-1 cells expressing antisense p53 RNA were more resistant to apoptosis induced by the combined effects of c-Myc activation and gamma irradiation. In a similar manner, apoptosis induced by c-Myc in serum starved, hypoxic or gamma irradiated fibroblasts was also inhibited by Bcl-2. These data indicate that p53 is involved in c-Myc-mediated apoptosis under a variety of stresses which may influence tumor growth, evolution and response to therapy.
View details for Web of Science ID 000071647200003
View details for PubMedID 10200458
- Cancer therapy and tumor physiology SCIENCE 1998; 279 (5347): 12-13
Transformed cells require continuous activity of RNA polymerase II to resist oncogene-induced apoptosis
MOLECULAR AND CELLULAR BIOLOGY
1997; 17 (12): 7306-7316
Studies have indicated that deregulated oncogene expression can result in either programmed cell death or proliferation, depending on the cellular microenvironment. However, little is known about whether oncogenic signals in themselves are able to activate a cellular apoptotic program. We have tested the hypothesis that oncogenic signals in the absence of gene expression are sufficient to induce cell death, which would indicate that constitutive expression of antiapoptotic genes is necessary for maintenance of the transformed state. Using two highly specific RNA polymerase (RNAP) II inhibitors, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) and alpha-amanitin, which inhibit RNAP II function by two distinct mechanisms, we found that inhibition of gene expression substantially increased apoptosis in a time- and dose-dependent manner in p53+/+- and p53(-/-)-transformed mouse embryonic fibroblasts and in HeLa cells, demonstrating that this type of apoptosis does not require wild-type p53. Engineered expression of an alpha-amanitin resistance RNAP II gene rendered cells resistant to induction of apoptosis by alpha-amanitin without affecting their sensitivity to DRB, indicating that alpha-amanitin induces apoptosis solely by inhibiting RNAP II function and not by a nonspecific mechanism. DRB-induced apoptosis was independent of the cell cycle or ongoing DNA replication, since DRB induced similar levels of apoptosis in asynchronous cells and cells synchronized by collection at mitosis. Inhibition of RNAP II in untransformed cells like Rat-1 or human AG1522 fibroblasts resulted not in apoptosis but in growth arrest. In contrast, deregulated expression of c-Myc in Rat-1 cells dramatically increased their sensitivity to DRB, directly demonstrating that apoptosis following inhibition of RNAP II function is greatly enhanced by oncogenic expression. The requirement for RNAP II function to prevent oncogene-induced apoptosis implies the need for the constitutive expression of an antiapoptotic gene(s) to maintain the transformed state. The differential sensitivities of untransformed and transformed cells to induction of apoptosis by transcriptional inhibition, coupled with the finding that this type of apoptosis is independent of p53 status, suggest that inhibition of RNAP II may be exploited therapeutically for the design of successful antitumor agents.
View details for Web of Science ID A1997YG44100055
View details for PubMedID 9372962
Induction of vascular endothelial growth factor by hypoxia is modulated by a phosphatidylinositol 3-kinase Akt signaling pathway in Ha-ras-transformed cells through a hypoxia inducible factor-1 transcriptional element
1997; 90 (9): 3322-3331
Tumor angiogenesis, the development of new blood vessels, is a highly regulated process that is controlled genetically by alterations in oncogene and tumor suppressor gene expression and physiologically by the tumor microenvironment. Previous studies indicate that the angiogenic switch in Ras-transformed cells may be physiologically promoted by the tumor microenvironment through the induction of the angiogenic mitogen, vascular endothelial growth factor (VEGF). In this report, we show Ras-transformed cells do not use the downstream effectors c-Raf-1 or mitogen activated protein kinases (MAPK) in signaling VEGF induction by hypoxia as overexpression of kinase-defective alleles of these genes does not inhibit VEGF induction under low oxygen conditions. In contrast to the c-Raf-1/MAP kinase pathway, hypoxia increases phosphatidylinositol 3-kinase (PI 3-kinase) activity in a Ras-dependent manner, and inhibition of PI 3-kinase activity genetically and pharmacologically results in inhibition of VEGF induction. We propose that hypoxia modulates VEGF induction in Ras-transformed cells through the activation of a stress inducible PI 3-kinase/Akt pathway and the hypoxia inducible factor-1 (HIF-1) transcriptional response element.
View details for Web of Science ID A1997YD10800006
View details for PubMedID 9345014
Loss of p21(Waf1/Cip1) sensitizes tumors to radiation by an apoptosis-independent mechanism
1997; 57 (21): 4703-4706
Cellular checkpoints are important mediators of the response of normal cells following genotoxic damage, and interruption of these checkpoints is a common feature of many solid tumors. Although the effects of loss in checkpoint function in tumor cells are well understood in terms of cell cycle control, there is little information on their role in determining treatment efficacy in vivo. We have examined both the in vitro and in vivo responses of isogenic lines differing only in the p53-transactivated checkpoint gene, p21Waf1/Cip1. When assayed in vitro, loss of p21 in human colon tumor cells results in a selective induction of apoptosis [Waldman, T., et al., Nature (Lond.), 381: 713-716, 1996.] but no difference in the clonogenic survival. However, when grown as xenografts and irradiated in situ, p21-deficient tumors were significantly more sensitive to radiation as assessed both by clonogenic survival and by regrowth of the tumors following treatment. These data indicate that loss of p21 results in increased sensitivity to killing by ionizing radiation that is independent of the induction of apoptosis and cell cycle arrest but that is specific to cells when they are grown as a solid tumor. These results have important implications for assessing both the genetic determinants of sensitivity to anticancer agents and efficacy of anticancer agents.
View details for Web of Science ID A1997YD35800009
View details for PubMedID 9354425
Selection of human cervical epithelial cells that possess reduced apoptotic potential to low-oxygen conditions
1997; 57 (19): 4200-4204
Since human papillomavirus (HPV) infection is strongly associated with cervical neoplasia and tumor hypoxia has prognostic significance in human cervical carcinomas, we examined the relationship between hypoxia and apoptosis in human cervical epithelial cells expressing high-risk HPV type 16 oncoproteins. In vitro, hypoxia stimulated both p53 induction and apoptosis in primary cervical epithelial cells infected with the HPV E6 and E7 genes but not in cervical fibroblasts infected with E6 and E7. Furthermore, cell lines derived from HPV-associated human cervical squamous cell carcinomas were substantially less sensitive to apoptosis induced by hypoxia, indicating that these cell lines have acquired additional genetic alterations that reduced their apoptotic sensitivity. Although the process of long-term cell culturing resulted in selection for subpopulations of HPV oncoprotein-expressing cervical epithelial cells with diminished apoptotic potential, the exposure of cells to hypoxia greatly accelerated the selection process. These results provide evidence for the role of hypoxia-mediated selection of cells with diminished apoptotic potential in the progression of human tumors and can in part explain why cervical tumors that possess low pO2 values are more aggressive.
View details for Web of Science ID A1997XZ01100013
View details for PubMedID 9331075
Anaerobic bacteria as a gene delivery system that is controlled by the tumor microenvironment
1997; 4 (8): 791-796
A fundamental obstacle in gene therapy for cancer treatment is the specific delivery of an anticancer gene product to a solid tumor. Although several strategies exist to control gene expression once a vector is directly introduced into a tumor, as yet no systemic delivery system exists that specifically targets solid tumors. Nonpathogenic, obligate anaerobic bacteria of the genus Clostridium have been used experimentally as anticancer agents because of their selective growth in the hypoxic regions of solid tumors after systemic application. In this report we further describe a novel approach to cancer gene therapy in which genetically engineered clostridia are used as tumor-specific vectors for the delivery of antitumor genes. We have introduced into a strain of C. beijerinckii the gene for an E. coli nitroreductase known to activate the nontoxic prodrug CB 1954 to a toxic anticancer drug. Nitroreductase produced by these clostridia enhanced the killing of tumor cells in vitro by CB 1954, by a factor of 22. To demonstrate the specificity of this approach for tumor targeting, we intravenously injected the inactive spore form of C. beijerinckii, which upon transition to a reproductive state will express the E. coli nitroreductase gene. Nitroreductase activity was detectable in 10 of 10 tumors during the first 5 days after intravenous injection of inactive clostridial spores, indicating a rapid transition from spore to reproductive state. Tumors harboring clostridial spores which did not possess the E. coli nitroreductase gene were devoid of nitroreductase activity. Most importantly, E. coli nitroreductase protein was not found in a large survey of normal mouse tissues following intravenous injection of nitroreductase containing clostridia, strongly suggesting that obligate anaerobic bacteria such as clostridia can be utilized as highly specific gene delivery vectors for cancer therapy.
View details for Web of Science ID A1997XP16200007
View details for PubMedID 9338007
Protease inhibitor TPCK represses Ha-ras (Val12) transformation and nuclear factor-kappa B activation
INTERNATIONAL JOURNAL OF ONCOLOGY
1997; 10 (5): 895-900
Certain chymotrypsin-like protease inhibitors such as TPCK exhibit a well described anti-tumorigenic activity by an as yet undescribed mechanism. One potential cellular target for TPCK in transformed cells is the ms-inducible NF-kappa B family of transcription factors. We therefore used TPCK to examine the physiologic role of NF-kappa B during Ha-ras induced transformation, independent of another major downstream effector of Ha-ras, AP-1. Using a conditionally transformed NIH3T3 cell line, we found that TPCK (but not the control inhibitor TLME) inhibited the anchorage-independent growth of Ha-ras transformed cells, but not their anchorage-dependent growth on plastic tissue culture dishes. Likewise, TPCK reduced the ability of Ha-ras to stimulate DNA synthesis in growth factor depleted cells, but not the ability of serum to stimulate DNA synthesis in the same growth factor depleted cells. Gel shift analysis and reporter gene expression indicated that TPCK blocked Ha-ras-induced NF-kappa B activity, while only having minimal effects on Ha-ras-induced AP-1 activity. TPCK is therefore able to Inhibit the Ha-ras transformed phenotype of cells by inhibiting the transcriptional activity of NF-kappa B, while having little effect upon transcriptional activity of AP-1.
View details for Web of Science ID A1997WW11900003
View details for PubMedID 21533459
- Protease Inhibitor TPCK represses Ha-ras (Val 12) transformation and nuclear factor-κ B activation Int J Oncology 1997; 10: 895-900
The DNA damage response in DNA-dependent protein kinase-deficient SCID mouse cells: Replication protein A hyperphosphorylation and p53 induction
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1996; 93 (24): 13825-13830
Severe combined immunodeficient (SCID) mice display an increased sensitivity to ionizing radiation compared with the parental, C.B-17, strain due to a deficiency in DNA double-strand break repair. The catalytic subunit of DNA-dependent protein kinase (DNA-PKCS) has previously been identified as a strong candidate for the SCID gene. DNA-PK phosphorylates many proteins in vitro, including p53 and replication protein A (RPA), two proteins involved in the response of cells of DNA damage. To determine whether p53 and RPA are also substrates of DNA-PK in vivo following DNA damage, we compared the response of SCID and MO59J (human DNA-PKcs-deficient glioblastoma) cells with their respective wild-type parents following ionizing radiation. Our findings indicate that (i) p53 levels are increased in SCID cells following ionizing radiation, and (ii) RPA p34 is hyperphosphorylated in both SCID cells and MO59J cells following ionizing radiation. The hyperphosphorylation of RPA p34 in vivo is concordant with a decrease in the binding of RPA to single-stranded DNA in crude extracts derived from both C.B-17 and SCID cells. These results suggest that DNA-PK is not the only kinase capable of phosphorylating RPA. We conclude that the DNA damage response involving p53 and RPA is not associated with the defect in DNA repair in SCID cells and that the physiological substrate(s) for DNA-PK essential for DNA repair has not yet been identified.
View details for Web of Science ID A1996VV46700068
View details for PubMedID 8943020
Modulation of c-myc activity and apoptosis in vivo
1996; 56 (19): 4315-4319
We have developed an animal tumor model system to study the effects of c-Myc activation on apoptosis induction in vivo. Tumors were generated in SCID mice from Rat-1 fibroblasts that constitutively express an inactive c-Myc-estrogen receptor fusion protein (T.D. Littlewood et al, Nucleic Acids Res., 23: 1686 -1690, 1995), which is activated in vivo by the administration of 4-hydroxytamoxifen in time release pellets. We demonstrate that activation of c-Myc results in a substantial increase in the number of apoptotic tumor cells and that this apoptosis is predominant in regions of tumor hypoxia. c-Myc-induced apoptosis of hypoxic cells is inhibited in tumors that overexpress the human Bcl-2 protein. Bcl-2, however, does not prevent p53 protein accumulation or the down-regulation of the cyclin-cdk inhibitor p27 protein following c-Myc activation by 4-hydroxytamoxifen. This result suggests that Bcl-2 does not affect c-Myc function directly but acts downstream of c-Myc to inhibit apoptosis. We propose that the ability of activated c-Myc to enhance cellular proliferation might contribute to the genesis of early neoplasms that are held in check by the alternate ability of c-Myc to induce apoptosis of cells that have outgrown their supply of oxygen or other factors associated with hypoxic regions of solid tumors. Secondary genetic lesions downstream of c-Myc that suppress the apoptotic potential of tumor cells, such as Bcl-2 overexpression, might play an important role in the malignant progression of these tumors because they would disrupt the balance between apoptosis and proliferation initiated by c-Myc deregulation.
View details for Web of Science ID A1996VK30700006
View details for PubMedID 8813114
Oncogenic transformation and hypoxia synergistically act to modulate vascular endothelial growth factor expression
1996; 56 (15): 3436-3440
Hypoxia can select for cells that have lost their apoptotic potential, thereby making them resistant to adverse conditions. However, long-term survival of transformed cells which have diminished apoptotic sensitivity when exposed to low oxygen conditions would require the activation of their angiogenic program to compensate for an insufficient oxygen supply. In this report, we show that the activity (of oncogenic Ha-ras, either constitutively or transiently, enhances the induction of the angiogenic mitogen, vascular endothelial growth factor (VEGF), by hypoxia. Analysis of the 5' flanking region of the VEGF promoter indicates that a HIF-1-like sequence is to promote a 15-fold increase in reporter gene activity in Ha-ras-transformed cells when exposed to hypoxia, whereas mutations in the same site totally inhibited VEGF induction. Under low oxygen conditions, VEGF induction is inhibited in cells expressing a mutant inhibitory allele of Ha-ras (RasN17), indicating a direct role for Ras in modulating VEGF activity. We propose that the angiogenic switch in Ras-transformed cells may be physiologically promoted by the tumor microenvironment through VEGF induction.
View details for Web of Science ID A1996UZ28800011
View details for PubMedID 8758908
Early responses of SiHa human squamous carcinoma cells to hypoxic signals: Evidence of parallel activation of NF-kappa B and AP-1 transcriptional complexes
INTERNATIONAL JOURNAL OF ONCOLOGY
1996; 8 (5): 875-882
We compared the responses of c-Jun/AP-1 transcriptional complexes with those of NF-kappa B, an established hypoxia-inducible transcriptional complex, in hypoxic SiHa human squamous carcinoma cells. We observed that NF-kappa B was activated rapidly, while AP-1 activation was detectable only after prolonged hypoxia. However, in parallel with NF-kappa B activation, hypoxia induced a protein kinase activity that could phosphorylate the transactivation domain of the ATF-2 transcription factor in vitro. Taken together, these experiments indicate that NF-kappa B can rapidly transduce hypoxic signals through increased DNA-binding and transactivation activities, whereas specific AP-1 (ATF-2/c-Jun) complexes may be activated under the same hypoxic conditions by a stress-reponsive MAPK pathway.
View details for Web of Science ID A1996UF76100006
View details for PubMedID 21544440
Mechanism of heat shock protein 72 induction in primary cultured astrocytes after oxygen-glucose deprivation
1996; 18 (1): 64-72
Induction of stress proteins is thought to be important in the protection of cells from a variety of environmental insults including heat, hypoxia and ischemia. The aim of this study was to compare the mechanism of induction of heat shock protein 72 (HSP72) in primary cultures of murine cortical astrocytes by heat and combined oxygen-glucose deprivation (OGD), a model of in vitro ischemia. 35S-methionine labeling and immunoblotting showed increased HSP72 synthesis and accumulation lasting for up to 24 h following heat or OGD. Heat induced a markedly greater amount of HSP72 mRNA and protein than did OGD. We then sought evidence of heat shock transcription factor-1 (HSF-1) activation. An increase in apparent molecular weight of nuclear HSF-1 after heat or OGD was observed, consistent with increased phosphorylation. To seek an explanation of the difference between heat and OGD as inducers of HSP72 we examined the binding activity of HSP72 + 73 to other proteins. More cellular protein was found to co-immunoprecipitate with HSP72 + 73, and more HSP72 + 73 was found in the pellet fraction after heat shock compared to OGD. These results suggest that HSP72 induction is regulated in astrocytes at least in part at the level of HSF activation, by both heat and OGD. Reduced availability of free HSP72 + 73 in heated cells could be responsible for the greater magnitude of HSP72 induction after heat compared to OGD.
View details for Web of Science ID A1996TW49400013
View details for PubMedID 8714540
Anaerobic bacteria as a delivery system for cancer gene therapy: In vitro activation of 5-fluorocytosine by genetically engineered clostridia
1996; 3 (2): 173-178
Certain species of anaerobic bacteria have been shown to localise and germinate specifically in the hypoxic regions of tumours, resulting in tumour lysis. We propose an innovative approach to cancer gene therapy in which genetically engineered anaerobic bacteria of the genus Clostridium are used to achieve tumour-specific gene delivery. Our strategy involves enzyme/prodrug therapy, in which the Escherichia coli enzyme cytosine deaminase is used to convert the non-toxic prodrug 5-fluorocytosine to the active chemotherapeutic agent 5-fluorouracil. The E. coli gene encoding cytosine deaminase has been cloned into a clostridial expression vector and transformed into Clostridium beijerinckii, resulting in constitutive expression of cytosine deaminase and significant levels of active enzyme in the bacterial medium. When added to an in vitro clonogenic survival assay, supernatant from clostridia expressing cytosine deaminase increased the sensitivity of murine EMT6 carcinoma cells to 5-fluorocytosine approximately 500-fold. This high level of prodrug activation, combined with the specificity of clostridia for hypoxic regions of tumours, indicates a potential use in cancer gene therapy.
View details for Web of Science ID A1996TZ78700010
View details for PubMedID 8867865
Hypoxia-mediated selection of cells with diminished apoptotic potential in solid tumours
1996; 379 (6560): 88-91
Apoptosis is a genetically encoded programme of cell death that can be activated under physiological conditions and may be an important safeguard against tumour development. Regions of low oxygen (hypoxia) and necrosis are common features of solid tumours. Here we report that hypoxia induces apoptosis in oncogenically transformed cells and that further genetic alterations, such as loss of the p53 tumour-suppressor gene or overexpression of the apoptosis-inhibitor protein Bcl-2, substantially reduce hypoxia-induced cell death. Hypoxia also selects for cells with defects in apoptosis, because small numbers of transformed cells lacking p53 overtake similar cells expressing wild-type p53 when treated with hypoxia. Furthermore, highly apoptotic regions strongly correlate with hypoxic regions in transplanted tumours expressing wild-type p53, whereas little apoptosis occurs in hypoxic regions of p53-deficient tumours. We propose that hypoxia provides a physiological selective pressure in tumours for the expansion of variants that have lost their apoptotic potential, and in particular for cells acquiring p53 mutations.
View details for Web of Science ID A1996TN21600062
View details for PubMedID 8538748
Hypoxic Stress Proteins: Survival of the Fittest.
Seminars in radiation oncology
1996; 6 (1): 46-58
The importance of tumor microenvironmental in malignant progression has been largely ignored. Tumor cells protect themselves from changes in the microenvironment due to a decrease in nutrients and oxygen by reducing macromolecular synthesis and inducing genes that will promote angiogenesis and tissue remodeling. This ability of transformed cells to survive fluctuations in oxygen tensions is clinically important, as tumors with high hypoxic fractions respond poorly to many forms of cancer therapy. While it is commonly accepted that the decrease in molecular oxygen in hypoxic cells makes them more refractory to killing by agents such as ionizing radiation which use oxygen radical formation, the cessation of division and loss of apoptotic (cell suicide) potential in hypoxic cells are also important in their resistance to killing by radiotherapy and chemotherapy. In this chapter, we will discuss hypoxia-induced stress proteins in regards to three clinically relevant end points: inhibition of cell proliferation, induced of apoptosis, and regulation of genes modulating angiogenesis. Although these three end points may seem unrelated, in fact, they are intimately linked with each other in the cellular response to hypoxia and malignant progression. One of the goals of this review is to inform both clinician and scientist of these interrelationships and discuss how hypoxia selects for tumors that are clonal expansions of cells that have lost their apoptotic ability and have switched to a proangiogenic phenotype.
View details for PubMedID 10717161
- Early response of SiHa human squamous carcinoma cells to hypoxic signals: Evidence of parallel activation of NF-kB and AP-1 transcriptional complexes Int J Oncol 1996; 8: 875-882
New directions for radiation biology research in cancer of the uterine cervix.
Journal of the National Cancer Institute. Monographs
A simplified model for tumorigenesis, locoregional growth, and metastases is proposed for carcinoma of the cervix. With the use of this model, four potential areas for future directions for radiobiologic-clinical research are identified. The first area concerns the influence of human papillomavirus infection and p53 mutations on tumor biology, with particular reference to radiosensitivity and metastatic potential. Research in this area should be most fruitful. The second area focuses on the influence of hypoxia on clinical outcome in carcinoma of the cervix. The use of selective hypoxic cell toxins (e.g., tirapazamine) for phase II testing in hypoxic tumors is recommended. The third area concerns the development and clinical confirmation of assays for the prediction of intrinsic tumor radiosensitivity (e.g., surviving fraction after 2 Gy) and normal tissue radiosensitivity. The need exists for more rapid assays so that their results can be available prior to institution of therapy. The influence of the intrinsic radiosensitivity of normal tissues (especially in patients who are heterozygotes for ataxia-telangiectasia and patients with autoimmune disease) may permit identification of those at increased risk for complications so that alternative, less toxic treatment can be allocated. The fourth area for additional study concerns the influence of both intrinsic (c-myc amplification, matrix metalloproteinase levels) and extrinsic factors (fever, immunosuppression) on the development of distant metastases. Such investigations will permit identification of patients at high risk of developing distant metastases so that adjuvant treatments (e.g., chemotherapy or metalloproteinase inhibitors) can be explored. It is believed that future clarification of our proposed model will lead to other worthwhile areas for therapeutic intervention.
View details for PubMedID 9023843
MITOGEN-ACTIVATED PROTEIN-KINASE ACTS AS A NEGATIVE REGULATOR OF THE HEAT-SHOCK RESPONSE IN NIH3T3 CELLS
1995; 55 (23): 5512-5519
Exposure of NIH3T3 cells to elevated temperatures induces the phosphorylation and activation of mitogen-activated protein (MAP) kinases [or extracellular signal-regulated kinases (ERKs)]. To investigate the significance of MAP kinase activation by heat shock, we examined the effect of inhibiting the activity of MAP kinase on heat shock protein 70 (hsp 70) expression. Overexpression of a dominant inhibitory mutant of ERK1, but not ERK2, in heat-shocked cells increased hsp70 reporter gene activity, suggesting that ERK1 acts as a repressor of hsp70 gene expression. Increases in ERK1 activity through treatment of cells with sodium vanadate (SV), an inhibitor of the dual-specificity MAP kinase phosphatase 1 (PAC1), resulted in increased phosphorylation of the heat shock transcription factor-1 (HSF-1) in unheated cells, delayed the activation of HSF-1 by heat shock, and inhibited the induction of hsp 70 by heat shock. Furthermore, the induction of thermotolerance was reduced significantly in cells that increased ERK1 activity by SV pretreatment. Immune complex kinase assays of heat shocked or SV-pretreated cells indicated that HSF-1 is a potential in vivo substrate for ERK1 phosphorylation. Taken together, these results suggest that agents that modulate MAP kinase act as negative regulators of the heat shock response in mammalian cells by modulating HSF-1 activity and hsp 70 expression.
View details for Web of Science ID A1995TG21100007
View details for PubMedID 7585624
ATTENUATED RESPONSE OF P53 AND P21 IN PRIMARY CULTURES OF HUMAN PROSTATIC EPITHELIAL-CELLS EXPOSED TO DNA-DAMAGING AGENTS
1995; 55 (17): 3726-3731
The multifocal origin of prostate cancer suggests a pan-organ defect in a tumor suppressor pathway. Although structural mutations in the p53 gene have been implicated in late-stage prostate cancer, little is known about the p53 response to genotoxic stress in normal human prostatic epithelial cells from which adenocarcinomas originate. We found that the majority (10 of 12) of epithelial cell cultures derived from histologically normal tissues of radical prostatectomy specimens failed to exhibit p53 accumulation in response to ionizing radiation. Epithelial cell cultures derived from benign prostatic hyperplasia and a primary prostatic adenocarcinoma also failed to accumulate p53 in response to ionizing radiation. In contrast, cultures of prostatic stromal cells derived from normal, benign prostatic hyperplasia, or adenocarcinoma tissues exhibited a 3-9-fold induction of p53 within 1-3 h after irradiation. Since p53 regulates a cell cycle checkpoint through the induction of the cyclin-cdk inhibitor p21, we examined p21 accumulation and cell cycle arrest following exposure to ionizing radiation. With one exception, epithelial cells that did not display increased p53 or p21 induction did not demonstrate a significant G1-S arrest in response to ionizing radiation, whereas stromal cells that accumulated p53 and p21 exhibited a large cell cycle arrest. These results indicate a functional difference between the DNA damage response of epithelial and stromal prostatic cells and suggest a possible mechanism for the increased susceptibility of prostatic epithelial cells to accumulate genetic alterations.
View details for Web of Science ID A1995RR73900010
View details for PubMedID 7543816
HYPOXIC ACTIVATION OF NUCLEAR FACTOR-KAPPA-B IS MEDIATED BY A RAS AND RAF SIGNALING PATHWAY AND DOES NOT INVOLVE MAP KINASE (ERK1 OR ERK2)
1994; 54 (20): 5273-5279
We have previously shown that hypoxia causes the activation of nuclear factor-kappa B (NF-kappa B), and the phosphorylation of its inhibitory subunit, I kappa B alpha, on tyrosine residues. With the use of dominant negative mutants of Ha-Ras and Raf-1, we investigated some of the early signaling events leading to the activation of NF-kappa B by hypoxia. Both dominant negative alleles of Ha-Ras and Raf-1 inhibited NF-kappa B induction by hypoxia, suggesting that the hypoxia-induced pathway of NF-kappa B induction is dependent on Ras and Raf-1 kinase activity. Furthermore, although conditions of low oxygen can also activate mitogen-activated protein kinases (ERK1 and ERK2), these kinases do not appear to be involved in regulating NF-kappa B by low oxygen conditions, as dominant negative mutants of mitogen-activated protein kinase do not inhibit NF-kappa B activation by hypoxia. Since Ras and Raf-1 have been previously shown to work downstream from membrane-associated tyrosine kinases such as Src, we determined if the Src membrane-associated kinase was also activated by low oxygen conditions. We detected an increase in Src proto-oncogene activity within 15-30 min of cellular exposure to hypoxia. We postulate that Src activation by hypoxia may be one of the earliest events that precedes Ras activation in the signaling cascade which ultimately leads to the phosphorylation and dissociation of the inhibitory subunit of NF-kappa B, I kappa B alpha.
View details for Web of Science ID A1994PL55800007
View details for PubMedID 7923153
MODULATION OF POTASSIUM CHANNELS BY PROTEIN-TYROSINE KINASE INHIBITORS
JOURNAL OF CELLULAR PHYSIOLOGY
1994; 161 (1): 142-148
Exposure of non-excitatory cells to the tyrosine kinase (PTK) inhibitors, genistein, herbimycin A, and tyrphostin, induced at least two families of K+ currents. The first, a TEA-insensitive slow-inactivating K+ current, is induced within 3 min following treatment with 140 mM genistein or 100 nM herbimycin A. The second current, a TEA-sensitive delayed rectifier, is induced within 30 min following treatment with 50 mM genistein or 10 nM herbimycin A. Currents with similar biophysical and pharmacological characteristics are induced in these cells following exposure to ionizing radiation. The radiation-induced currents are inhibited by pretreatment with the free radical scavenger, N-Acetyl L-Cysteine, or by pretreatment with the protein kinase C inhibitor, staurosporine; those induced by PTK inhibitors are not. The latter, therefore, do not appear to be mediated through free radicals or require serine/threonine phosphorylation for activation. Once the channels are activated by the PTK inhibitors, phosphorylation of the channel at serine/threonine residues results in slower inactivation of the induced current. We propose that protein tyrosine phosphorylation of the K+ channel protein itself or of a factor that interacts with it maintains the K+ channels of non-excitatory cells in a closed state. Following exposure to ionizing radiation, free radical-induced activation of serine/threonine kinase(s) results in phosphorylation of the channel and/or inactivation of a tyrosine kinase that in turn leads to activation of the K+ channels.
View details for Web of Science ID A1994PK53000016
View details for PubMedID 7929599
HYPOXIA INDUCES ACCUMULATION OF P53 PROTEIN, BUT ACTIVATION OF A G(1)-PHASE CHECKPOINT BY LOW-OXYGEN CONDITIONS IS INDEPENDENT OF P53 STATUS
MOLECULAR AND CELLULAR BIOLOGY
1994; 14 (9): 6264-6277
It has been convincingly demonstrated that genotoxic stresses cause the accumulation of the tumor suppressor gene p53. One important consequence of increased p53 protein levels in response to DNA damage is the activation of a G1-phase cell cycle checkpoint. It has also been shown that G1-phase cell cycle checkpoints are activated in response to other stresses, such as lack of oxygen. Here we show that hypoxia and heat, agents that induce cellular stress primarily by inhibiting oxygen-dependent metabolism and denaturing proteins, respectively, also cause an increase in p53 protein levels. The p53 protein induced by heat is localized in the cytoplasm and forms a complex with the heat shock protein hsc70. The increase in nuclear p53 protein levels and DNA-binding activity and the induction of reporter gene constructs containing p53 binding sites following hypoxia occur in cells that are wild type for p53 but not in cells that possess mutant p53. However, unlike ionizing radiation, the accumulation of cells in G1 phase by hypoxia is not strictly dependent on wild-type p53 function. In addition, cells expressing the human papillomavirus E6 gene, which show increased degradation of p53 by ubiquitination and fail to accumulate p53 in response to DNA-damaging agents, do increase their p53 levels following heat and hypoxia. These results suggest that hypoxia is an example of a "nongenotoxic" stress which induces p53 activity by a different pathway than DNA-damaging agents.
View details for Web of Science ID A1994PC75000068
View details for PubMedID 8065358
THE HUMAN HA-RAS ONCOGENE INDUCES GENOMIC INSTABILITY IN MURINE FIBROBLASTS WITHIN ONE-CELL CYCLE
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1994; 91 (11): 5124-5128
Many human tumors contain an activating mutation in one of the ras protooncogenes. Additionally, these tumor cells are often heteroploid and characterized by chromosome breaks and rearrangements that are consequences of the genomic instability that is thought to contribute to tumor progression. The concurrence of ras mutations and genomic instability in tumors prompted us to ask whether selective induction of an activated Ha-ras gene could render a genome unstable. The NIH 3T3 cells used in this study contained mutant p53 genes and carried a selectively inducible activated (EJ) Ha-ras transgene under the control of bacterial lactose regulatory elements. When stably transfected cells were induced to express activated Ha-ras by isopropyl beta-D-thiogalactoside administration, there was a marked increase in the number of gross chromosomal aberrations including acentric fragments, multicentric chromosomes, and double minutes, which occurred within the time frame of a single cell cycle from the time of induction. To confirm that these aberrations occurred within the first cell cycle after mutant Ha-ras induction, the cells were arrested in G1 phase by serum depletion and, subsequently, released by administration of isopropyl beta-D-thiogalactoside or serum. The mitoses from cells released with isopropyl beta-D-thiogalactoside contained a 3-fold elevation in the fraction of chromosomes containing aberrations compared to mitoses from parallel cell cultures that were released with serum. Thus, the induction of activated Ha-ras gene expression in these cells results in genomic instability that can be detected as aberrant chromosomes at the next mitosis.
View details for Web of Science ID A1994NN21400109
View details for PubMedID 8197195
ANALYSIS OF HSF-1 PHOSPHORYLATION IN A549 CELLS TREATED WITH A VARIETY OF STRESSES
INTERNATIONAL JOURNAL OF HYPERTHERMIA
1994; 10 (3): 371-379
In the absence of stress, heat shock transcription factor-1 (HSF-1) exists as a monomer. After the treatment of cells with variety of stresses, HSF-1 forms a trimer and binds to the heat shock element (HSE), a motif consisting of three consecutive NGAAN sequences located in an inverted orientation upstream of the heat shock genes. HSF-1 is then phosphorylated causing transactivation of heat shock mRNAs. Treatment of cells with some of the stresses has been shown to increase HSF binding to HSE without detectably increasing the synthesis of heat shock mRNAs. Here we used antibody specific to HSF-1 to detect its phosphorylation status following exposure of A549, a human lung carcinoma cell line to a variety of stresses in order to correlate HSF-1 phosphorylation with its transactivation ability. Our studies show that HSF-1 is phosphorylated following heat shock (43 degrees C for 1 h), hypoxia (5 h exposure to 0.02% oxygen), 8% ethanol (1 h exposure at 37 degrees C), or 200 microM sodium arsenite (1 h exposure at 37 degrees C). All such stresses have previously been shown to increase the synthesis of heat shock proteins (hsps). However, there are no detectable increases in HSF-1 phosphorylation after the treatment of cells with X-irradiation (2-8 Gy) or 100 microM canavanine, an amino acid analogue (1 h exposure at 37 degrees C). Treatment of cells with X-irradiation increases HSF binding to HSE without increasing the synthesis of hsps, while treatment of cells with canavanine has been shown to increase the synthesis of hsps.(ABSTRACT TRUNCATED AT 250 WORDS)
View details for Web of Science ID A1994NP47600009
View details for PubMedID 7930803
HYPOXIA CAUSES THE ACTIVATION OF NUCLEAR FACTOR KAPPA-B THROUGH THE PHOSPHORYLATION OF I-KAPPA-B-ALPHA ON TYROSINE RESIDUES
1994; 54 (6): 1425-1430
The response of mammalian cells to stress is controlled by transcriptional regulatory proteins such as nuclear factor kappa B (NF-kappa B) to induce a wide variety of early response genes. In this report, we show that exposure of cells to hypoxia (0.02% O2) results in I kappa B alpha degradation, increased NF-kappa B DNA binding activity, and transactivation of a reporter gene construct containing two NF-kappa B DNA binding sites. Pretreatment of cells with protein tyrosine kinase inhibitors and the dominant negative allele of c-Raf-1 (Raf 301) inhibited I kappa B alpha degradation, NF-kappa B binding, and transactivation of kappa B reporter constructs by hypoxia. To demonstrate a direct link between changes in the phosphorylation pattern of I kappa B alpha with NF-kappa B activation, we immunoprecipitated I kappa B alpha after varying times of hypoxic exposure and found that its tyrosine phosphorylation status increased during hypoxic exposure. Inhibition of the transfer of tyrosine phosphoryl groups onto I kappa B alpha prevented I kappa B alpha degradation and NF-kappa B binding. In comparison to other activators of NF-kappa B such as phorbol myristate acetate or tumor necrosis factor, we did not detect changes in the tyrosine phosphorylation status of I kappa B alpha following treatment with either of these agents. These results suggest that tyrosine phosphorylation of I kappa B alpha during hypoxia is an important proximal step which precedes its dissociation and degradation from NF-kappa B.
View details for Web of Science ID A1994NA88700009
View details for PubMedID 8137243
INCREASED CYTOTOXICITY OF CHRONIC HYPOXIC CELLS BY MOLECULAR INHIBITION OF GRP78 INDUCTION
INTERNATIONAL JOURNAL OF RADIATION ONCOLOGY BIOLOGY PHYSICS
1994; 28 (3): 661-666
To develop a molecular strategy of increasing cytotoxicity of chronically hypoxic cells by inhibiting Glucose Regulated Protein 78 kDal (GRP78) induction.A mutant nonGRP78 inducing cell line (78WO) was developed from its parent (DG44) by overexpressing antisense GRP78 mRNA. Following exposure to varying durations of hypoxia, Northern and Western blot analysis were used to characterize the amount of GRP78 expression both at the RNA and protein level. Hypoxia was achieved by placing cells in specially designed hypoxic chambers which were subjected to successive rounds of evacuation and flushing with 95% CO2/N2 to reduce the oxygen in the environment to 0.02% oxygen. After treatment with hypoxia, cells were assayed for colony forming ability.GRP78 mRNA and protein induction following exposure to hypoxia was 3-4 fold lower in the 78WO cell line than in the parental DG44 cell line. Furthermore, it was observed that there was no difference in the cytotoxicity of 78WO and DG44 cells after 10 h of hypoxia. However, after 15 h of hypoxia, the survival of 78WO cells decreased by 1 log and after 20 h of hypoxia, the survival of 78WO decreased by another log.These results show that stress protein induction is important for cellular survival to chronic hypoxia and that inhibition of GRP78 induction may represent a novel therapeutic strategy by selectively sensitizing chronically hypoxic cells within solid tumors.
View details for Web of Science ID A1994NA83600012
View details for PubMedID 8113109
CHROMOSOMAL RADIOSENSITIVITY AT INTRACHROMOSOMAL TELOMERIC SITES
GENES CHROMOSOMES & CANCER
1993; 8 (1): 8-14
Telomeric DNA is composed of highly conserved sequences which are present at the termini of chromosomes as well as at intrachromosomal locations. Here, we studied a Chinese hamster ovary (CHO) cell line, BL-10, with highly stable amplified telomeric DNA at the termini as well as at intrachromosomal locations. We show that intrachromosomal or interstitial telomeric sites in this cell line and in another CHO cell line, HA-I, are radiosensitive in that they are more prone to breakage than would be expected based on the percentage of the genome composed of telomeric sequences. The frequency of breakage at interstitial telomeric sites is 4.3 to 8.3 times higher than that in the CHO genome overall. These conclusions are reached by both conventional cytogenetic analysis of two CHO cell lines which have the same survival rates after exposure to ionizing radiation, and by use of double fluorescence in situ hybridization (FISH) with a pan-telomere-specific probe and a CHO chromosome-specific library in the same metaphase cells after irradiation.
View details for Web of Science ID A1993LU38000002
View details for PubMedID 7691162
ACTIVATION OF POTASSIUM CHANNELS BY HYPOXIA AND REOXYGENATION IN THE HUMAN LUNG ADENOCARCINOMA CELL-LINE A549
JOURNAL OF CELLULAR PHYSIOLOGY
1993; 156 (2): 341-347
Active oxygen species are generated in cells during pathophysiologic conditions such as inflammation and postischemic reperfusion. If oxygen radical scavengers are added before reperfusion, then the magnitude of injury is reduced. We investigated whether free radicals generated following exposure to hypoxia and reoxygenation activate voltage-dependent K+ ion channels in tumor cells in vitro. Using the technique of whole cell voltage clamping, we recorded currents from two families of potassium (K+) channels that were activated following reoxygenation. One of these groups possessed the electrophysical characteristics of a tetraethylammonium (TEA)-sensitive delayed rectifier channel and the other possessed characteristics of a Tea-insensitive slow inactivating channel. We present evidence which suggests that K+ channels are activated following reoxygenation but not during the hypoxia phase. The K+ currents decayed with time following reoxygenation. The decay characteristics of the K+ currents depended on the duration and level of hypoxia to which the cells were exposed. To determine whether activation of K+ channels by reoxygenation was initiated by free radicals, we pretreated cells with N-Acetyl L-Cysteine (NAC), a free radical scavenger, and found that this pretreatment abolished the currents induced by reoxygenation. We also present evidence that free radicals do not directly act on the channel itself, but activate a protein kinase which, in turn, activates the K+ channels. Taken together, these results indicate that one of the early responses to oxidative stress is the activation of K+ currents.
View details for Web of Science ID A1993LP82200016
View details for PubMedID 8344990
HEAT SENSITIVITY OF BLEOMYCIN-SENSITIVE CHO DERIVATIVES IS NOT DUE TO IMPROPER INITIALIZATION OF HEAT-SHOCK RESPONSE
INTERNATIONAL JOURNAL OF HYPERTHERMIA
1993; 9 (2): 275-284
We have investigated whether differences in the heat shock response exist between CHO and three bleomycin-sensitive, heat-sensitive CHO derivatives. The binding of heat shock factor (HSF) in response to heat and varying concentrations of bleomycin in the four cell lines was examined using a gel shift assay and a synthetic heat shock element (HSE). Heat (45 degrees C, 10 min) and exposure to 1 micrograms/ml bleomycin for 1 h at 37 degrees C induced similar levels of HSF binding in all four cell lines. We also examined if bleomycin dose and the length of recovery from bleomycin treatment affected the induction of HSF binding. The level of activated HSF binding to HSE was higher in cells treated with low doses (1 ng/ml) of bleomycin than in cells treated with 1 or 25 micrograms/ml bleomycin. The amount of activated HSF was directly proportional to the time elapsed since bleomycin treatment. Our results therefore indicate no difference between CHO and its bleomycin-sensitive derivatives in the ability to initiate the heat shock response as determined by the production of activated HSF in response to either heat or bleomycin. We conclude that the intrinsic thermosensitivity of these cell lines is not related to the early response to heat shock, but either occurs later in the pathway or is unrelated to events after heating.
View details for Web of Science ID A1993KR12400008
View details for PubMedID 7682247
POTASSIUM-CHANNEL ACTIVATION IN RESPONSE TO LOW-DOSES OF GAMMA-IRRADIATION INVOLVES REACTIVE OXYGEN INTERMEDIATES IN NONEXCITATORY CELLS
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1993; 90 (3): 908-912
Active oxygen species are generated during pathophysiologic conditions such as inflammation and ionizing radiation exposure. We tested the hypothesis that an early cellular event in response to these species involves regulation of ion channels. We exposed cells to gamma-irradiation or treated them with hydrogen peroxide, xanthine/xanthine oxidase, or [3H]thymidine and then monitored channel activity by the technique of whole-cell voltage clamping. Recordings showed that both normal and tumor cells exhibit an increase in K+ currents after treatment with radiation, H2O2, and xanthine/xanthine oxidase but not with high specific activity [3H]thymidine, suggesting that the signal for K+ channel activation originates at the cell membrane. A single noncytotoxic dose of 10 cGy induced measurable levels of K+ currents, suggesting that the induction of currents regulates biochemical changes in response to stress. To test whether channel activity is sensitive to active oxygen species, we pretreated cells with N-acetyl-L-cysteine (NAC) to increase cellular pools of free radical scavengers before radiation. In NAC-pretreated cells, K+ channel activation by gamma-irradiation was abolished. It has previously been shown that protein kinase C (PKC) is activated by ionizing radiation and can regulate K+ channels in some cells. However, the effect of radiation on induction of K+ channel activity was independent of PKC, since cells chronically exposed to phorbol esters still produced K+ currents after radiation. These results suggest that an early cellular response to oxidative stress is the activation of K+ channels.
View details for Web of Science ID A1993KK81300028
View details for PubMedID 8430104
ANALYSIS OF RESTRICTION ENZYME-INDUCED CHROMOSOMAL-ABERRATIONS BY FLUORESCENCE IN-SITU HYBRIDIZATION
ENVIRONMENTAL AND MOLECULAR MUTAGENESIS
1993; 22 (1): 26-33
Fluorescence in situ hybridization and Giemsa staining of metaphase chromosomes were used to determine the relative frequencies of symmetric exchange aberrations (translocations) and asymmetric exchange aberrations (rings, dicentrics, and polycentrics) after exposure of human lymphoblastoid cells to restriction enzymes or X-rays. The yield of symmetric exchanges was determined with the use of chromosome-specific probes for human chromosomes 2 or 4, which were hybridized to metaphase chromosomes from cells exposed to the enzymes PvuII, SacI, or XbaI or 3 or 5 Gy of X-rays. The yield of asymmetric exchanges was determined in Giemsa-stained metaphase chromosomes from the same enzyme-treated or irradiated cell population. About 1.5- to 3-fold more symmetric than asymmetric exchanges were induced after restriction enzyme treatment. However, after X-ray treatment the yield of dicentrics relative to the yield of reciprocal translocations was close to the expected 1:1 ratio.
View details for Web of Science ID A1993LP83100004
View details for PubMedID 8393403
DIFFERENTIAL EXPRESSION OF PROTEIN-KINASE-C EPSILON-PROTEIN IN LUNG-CANCER CELL-LINES BY IONIZING-RADIATION
BRITISH JOURNAL OF CANCER
1992; 66 (5): 844-849
The effect of ionising radiation on the regulation of gene and protein expression is complex. This study focuses on the translational regulational of the epsilon isoform of protein kinase C by ionising radiation. We found that protein kinase C epsilon is rapidly increased in the human lung adenocarcinoma cell line A549 following irradiation. Western blots showed increased accumulation of this protein at doses as low as 75 cGy after 15 min post irradiation. Maximal induction (11-fold over unirradiated cells) of PKC epsilon occurred at 150 cGy within 1 h after treatment by X-rays in A549 cells. The increased levels of PKC epsilon protein after X-rays does not require de novo protein or RNA synthesis, suggesting that this increase is post-translationally controlled. In contrast to A549 cells PKC epsilon levels in the large cell lung carcinoma cell line NCI H661 were not induced by radiation. In the small cell lung carcinoma cell line NCI N417, PKC epsilon was also not induced but a higher molecular weight PKC epsilon protein, suggestive of phosphorylation, appeared at 2 h after irradiation. The variation in induction or phosphorylation of PKC epsilon by ionising radiation in the cell lines tested in this study suggested that no clear correlation existed between intrinsic radiation sensitivity and PKC epsilon induction. To determine whether PKC epsilon does play a role in cell survival to irradiation, we used the protein kinase inhibitor staurosporin to decrease PKC activity and found that staurosporin sensitised cells to killing by ionising radiation. Pulsed field gel electrophoresis, however, indicated that DNA double-strand break repair was not decreased, suggesting that PKC epsilon is modifying the fidelity of rejoining and not the overall magnitude of repair. The regulation of PKC by ionising radiation will be discussed with respect to the biological consequences of gene induction by DNA damage agents.
View details for Web of Science ID A1992JW71300014
View details for PubMedID 1329908
THE USE OF ASYMMETRIC-FIELD INVERSION GEL-ELECTROPHORESIS TO PREDICT TUMOR-CELL RADIOSENSITIVITY
RADIOTHERAPY AND ONCOLOGY
1992; 24 (4): 231-238
The success of a predictive assay for radiotherapy relies on the use of one or more tumor cell traits that equate with tumor radioresistance or radiosensitivity. These traits can be divided into intrinsic (genetic) and extrinsic (epi-genetic) factors. Most probably, a tumor's response to radiotherapy will be influenced by both of these sets of traits. Radiobiological analysis of cultured cells derived from explanted tumors of head and neck patients has shown that in vitro survival of tumor cells is not the only factor affecting tumor radiocurability. Two possible reasons are the high degree of selection involved in growing the cells in vitro and the inability to assess the contribution of the cell-cell contact effect with cultured cells. A possible means of overcoming both of these problems would be an assessment of the radiosensitivity of the cell population immediately after removal from the tumor. Since a good correlation exists between intrinsic cellular radioresistance and DNA double-strand break repair (DSBR) as assayed by the Neutral Elution technique , we have investigated the feasibility of using asymmetric field inversion gel electrophoresis (AFIGE) in identifying resistant tumor cells in vitro. AFIGE has several advantages over neutral elution in that it is faster (approximately 60-80 samples can be run on the same agarose gel) and, most importantly, one can visualize DNA damage and repair by staining the DNA with ethidium bromide.(ABSTRACT TRUNCATED AT 250 WORDS)
View details for Web of Science ID A1992JN42500004
View details for PubMedID 1410578
ACTIVATION OF THE HEAT-SHOCK TRANSCRIPTION FACTOR BY HYPOXIA IN NORMAL AND TUMOR-CELL LINES INVIVO AND INVITRO
INTERNATIONAL JOURNAL OF RADIATION ONCOLOGY BIOLOGY PHYSICS
1992; 23 (4): 891-897
Cells exposed to hypoxia increase their synthesis of a specific set of proteins called oxygen regulated proteins. Recently, three of these proteins have been identified as hemoxygenase, Glucose Regulated Protein 78 kilodaltons and Glucose Regulated Protein 94 kilodaltons. In contrast, reoxygenation from hypoxic conditions increases the synthesis of the heat shock proteins. Although the molecular signals required for regulation of both sets of proteins by hypoxia and reoxygenation are still under investigation, it is known that their expression is regulated at the transcriptional level. This finding suggests that these stresses work either singularly or together to control the activation of nuclear transcription factors which bind distinct regulatory sequences in the promoter region of these genes. One possible nuclear transcription factor which could act as a transcriptional regulator for both hypoxia and reoxygenation gene transcription is the heat shock transcription factor. In this report, we focused on the kinetics of HSF activation by hypoxia in normal and tumor cell lines of murine and human origins. In cell culture, both the normal diploid cell line AG1522 and the tumor cell line JSQ-3 possess the same kinetics of HSF activation (binding to the heat shock element) by hypoxia, with maximal induction at or after 3 hr. We have also shown that the activation of HSF occurs in the SCCVII tumor in vivo without clamping, but not in SCCVII cells grown in monolayers. When SCCVII tumors are dissociated and allowed to reoxygenate in cell culture, HSF binding decreased in 5 hr, and was undetectable after 18 hr. Furthermore, one human tumor biopsy tested for the presence of hypoxia by both the pO2 histograph (Eppendorf, Germany) and HSF binding showed good agreement for both techniques. These results suggest that HSF binding may be a useful marker for monitoring the tumor hypoxia.
View details for Web of Science ID A1992JD82800027
View details for PubMedID 1618682
THE HYPERSENSITIVITY OF THE CHINESE-HAMSTER OVARY VARIANT BL-10 TO BLEOMYCIN KILLING IS DUE TO A LACK OF GLUTATHIONE S-TRANSFERASE-ALPHA ACTIVITY
1991; 51 (16): 4463-4469
As a means to understand the fundamental mechanisms of bleomycin cell killing, we previously isolated 19 bleomycin-sensitive mutants which represent at least six genetically distinct complementation groups (T.D. Stamato, B. Peters, P. Patil, N. Denko, R. Weinstein, and A. Giaccia. Cancer Res., 47: 1588-1592, 1987). One class of mutants represented by the cell line BL-10 displays only hypersensitivity to killing by bleomycin in both acute (16 h) and chronic treatments but no sensitivity to killing by other DNA-damaging agents. Complementation studies between this mutant and human fibroblasts suggested that the human gene which corrects the defect of BL-10 rested on human chromosome 6. It has been reported that the gene for human glutathione S-transferase (GST) alpha also resides on chromosome 6. Measurements of selenium-independent peroxidase (alpha-GST + glutathione peroxidase) activity in wild-type Chinese hamster ovary (CHO) cells, using cumene hydrogen peroxide as a substrate, gave a value of 112 nmol of glutathione oxidized/min/mg protein compared with 88.1 nmol of glutathione oxidized/min/mg protein for BL-10. Measurement of the selenium-dependent peroxidase activity, using H2O2 as a substrate, resulted in 65.9 nmol of reduced glutathione oxidized/min/mg protein in CHO and 81.5 nmol of reduced glutathione oxidized/min/mg protein for BL-10. In other words, BL-10 cells did not exhibit a difference in their ability to metabolize both substrates in contrast to CHO cells. This indicates that BL-10 possesses little alpha-GST activity. Transfection of BL-10 cells with a mammalian expression vector containing the alpha-GST gene increases the survival of BL-10 to bleomycin and does not increase the bleomycin resistance of two other bleomycin mutants which lie in different genetic complementation groups. These data strongly implicate a role for alpha-GST in the resistance of cells to bleomycin.
View details for Web of Science ID A1991GA54300054
View details for PubMedID 1714344
BIOCHEMICAL-EVIDENCE FOR 2 DIFFERENT MECHANISMS FOR BLEOMYCIN-INDUCED CELL KILLING
1991; 263 (2): 69-75
Using pulsed-field gel electrophoresis, we have measured the ability of two bleomycin-sensitive mutants, XR-1 and BL-10, to repair DNA double-strands breaks (DSB). XR-1 was originally isolated by its hypersensitivity to killing with ionizing radiation, but we have also shown that it is sensitive to killing with bleomycin. In contrast, BL-10 was isolated by its extreme sensitivity to killing with bleomycin, and it is not cross-sensitive to other DNA breaking agents. A 1-h treatment of bleomycin induces a similar number of DNA double-strand breaks in XR-1, BL-10 and CHO cells. However, XR-1 is unable to repair bleomycin-induced DNA double-strand breaks, whereas BL-10 possesses the same kinetics of repair as parental CHO. These data lead us to conclude that at least two mechanisms of killing exist for bleomycin; one of them is DNA DSB-dependent, and the other seems to be DNA DSB-independent.
View details for Web of Science ID A1991FT58100002
View details for PubMedID 1710776
LOSS OF THE INTRINSIC HEAT-RESISTANCE OF HUMAN-CELLS AND CHANGES IN MR 70,000 HEAT-SHOCK PROTEIN EXPRESSION IN HUMAN X HAMSTER HYBRIDS
1991; 51 (10): 2636-2641
Since mammalian cells vary widely in their intrinsic thermoresistance, we have investigated the genetic basis underlying this phenomenon in human and rodent cell lines. Typically, human cells are considerably more resistant to killing by heat than rodent cell lines. To determine whether the heat-resistant phenotype is dominant or recessive and to locate the chromosome(s) bearing determinants for heat resistance, we have prepared hybrids of heat-resistant human HT1080 cells and heat-sensitive Chinese hamster ovary (CHO) cells to test their response to heat. For both mass hybrid cultures and individual clones, the heat response of the hybrids was similar to that of the CHO parent. Analysis by in situ hybridization revealed the presence of five to 20 human chromosomes per cell in the mass hybrids and four to eight intact chromosomes plus some fragments in individual clones isolated from the hybrid cell population. A similar result was obtained using a different human cell line, AG1522. These data suggest that heat resistance is a recessive trait. Consistent with this conclusion are the results from a study of a fusion of HT1080 to a CHO mutant, BL-10, which was found to be hypersensitive to heat-induced killing. These hybrids had a normal CHO heat response and not the more heat-resistant phenotype of HT1080 cells. Two hybrid clones, H2 and H4, from the HT1080/BL-10 fusion were studied in more detail. Both clones possess similar amounts of Mr 70,000 heat shock protein (HSP70), despite the fact that H4 contains three human chromosomes (Nos. 6, 14, and 21) which carry HSP70 genes while H2 contains only one (chromosome 6). Both hybrid cell lines have the same response to heat. Although we found a wide range of sensitivities to heat, all cell lines contained a similar amount of constitutive HSP70, suggesting that HSP70 levels per se are not the critical determinant of intrinsic heat resistance.
View details for Web of Science ID A1991FK91000022
View details for PubMedID 2021941
THE USE OF FLUORESCENCE INSITU HYBRIDIZATION COMBINED WITH PREMATURE CHROMOSOME CONDENSATION FOR THE IDENTIFICATION OF CHROMOSOME-DAMAGE
BRITISH JOURNAL OF CANCER
1991; 63 (4): 517-521
The technique of fusing mitotic cells to interphase cells, thereby producing condensation of the chromosomes of the interphase cell (so-called 'premature chromosome condensation' or PCC), has allowed detection of the initial number of chromosome breaks and their repair following ionising radiation. However, the difficulty and tedium of scoring all the chromosome fragments, as well as the inability to readily detect exchange aberrations, has limited the use of PCC. We describe here the use of the recently developed technique of fluorescence in situ hybridisation with whole chromosome libraries to stain individual human chromosomes (also called 'chromosome painting') with the PCC's and show that this overcomes most of the limitations with the analysis of PCC's. First, by focusing on a single chromosome, scoring of breaks in the target chromosome is easy and rapid and greatly expands the radiation dose range over which the PCC technique can be used. Second, it allows the easy recognition of exchange type aberrations. A number of new applications of this technology, such as predicting the radiosensitivity of human tumours in situ, are feasible.
View details for Web of Science ID A1991FH59600011
View details for PubMedID 2021536
SCID MUTATION IN MICE CONFERS HYPERSENSITIVITY TO IONIZING-RADIATION AND A DEFICIENCY IN DNA DOUBLE-STRAND BREAK REPAIR
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1991; 88 (4): 1394-1397
C.B-17 severe combined immunodeficient (scid) mice carry the scid mutation and are severely deficient in both T cell- and B cell-mediated immunity, apparently as a result of defective V(D)J joining of the immunoglobulin and T-cell receptor gene elements. In the present studies, we have defined the tissue, cellular, and molecular basis of another characteristic of these mice: their hypersensitivity to ionizing radiation. Bone marrow stem cells, intestinal crypt cells, and epithelial skin cells from scid mice are 2- to 3-fold more sensitive when irradiated in situ than are congenic BALB/c or C.B-17 controls. Two independently isolated embryo fibroblastic scid mouse cell lines display similar hypersensitivities to gamma-rays. In addition, these cell lines are sensitive to cell killing by bleomycin, which also produces DNA strand breaks, but not by the DNA crosslinking agent mitomycin C or UV irradiation. Measurement of the rejoining of gamma-ray-induced DNA double-strand breaks by pulsed-field gel electrophoresis indicates that these animals are defective in this repair system. This suggests that the gamma-ray sensitivity of the scid mouse fibroblasts could be the result of reduced repair of DNA double-strand breaks. Therefore, a common factor may participate in both the repair of DNA double-strand breaks as well as V(D)J rejoining during lymphocyte development. This murine autosomal recessive mutation should prove extremely useful in fundamental studies of radiation-induced DNA damage and repair.
View details for Web of Science ID A1991EY61700064
View details for PubMedID 1996340
HUMAN CHROMOSOME-5 COMPLEMENTS THE DNA DOUBLE-STRAND BREAK-REPAIR DEFICIENCY AND GAMMA-RAY SENSITIVITY OF THE XR-1 HAMSTER VARIANT
AMERICAN JOURNAL OF HUMAN GENETICS
1990; 47 (3): 459-469
XR-1 is a Chinese hamster ovary (CHO) cell mutant which is unusually sensitive to killing by gamma rays in the G1 portion of the cell cycle but has nearly normal resistance to gamma-ray damage in late S phase. The cell-cycle sensitivity correlates with the mutant's inability to repair DNA double-strand breaks (DSBs) produced by ionizing radiation and restriction enzymes. We have previously shown in somatic cell hybrids of XR-1 cells and human fibroblasts that the XR-1 mutation is a recessive mutation. In this study, using somatic cell hybrids formed between XR-1 and human fibroblasts, we map the human complementing gene to chromosome 5 by chromosome-segregation analysis. This gene biochemically restores the hamster defect to wild-type levels of gamma-ray and bleomycin resistance as well as restoring its proficiency to repair DNA DSBs, suggesting that a single gene is responsible for the XR-1 phenotype. We have tentatively assigned the name XRCC4 (X-ray-complementing Chinese hamster gene 4) to this human gene until its biochemical function in repair is discovered.
View details for Web of Science ID A1990DX22300011
View details for PubMedID 1697445
USE OF FLUORESCENT INSITU HYBRIDIZATION TO DETECT CHROMOSOMAL REARRANGEMENTS IN SOMATIC-CELL HYBRIDS
GENES CHROMOSOMES & CANCER
1990; 2 (3): 248-251
In situ hybridization of hamster/human hybrids with biotinylated human genomic DNA has revealed that human chromosomal DNA can integrate into the hamster genome and is not always cytologically detectable. This finding helps to explain why discordancy can arise in gene mapping by failing to recognize small pieces of foreign DNA in the rodent genome. Fluorescent in situ hybridization allows one to locate these fragments in rodent chromosomes visually and possibly to identify their chromosome of origin.
View details for Web of Science ID A1990ED57700013
View details for PubMedID 2078516
INCREASED SENSITIVITY TO KILLING BY RESTRICTION ENZYMES IN THE XR-1 DNA DOUBLE-STRAND BREAK REPAIR-DEFICIENT MUTANT
1990; 236 (1): 67-76
Repair or misrepair of DNA double-strand breaks (DSBs) is critical in determining cellular survival after gamma-irradiation. In this report, we focus on the cellular and biochemical consequences of restriction enzyme induced DSBs in wild-type Chinese hamster ovary (CHO) cells and the DNA DSB repair-defective mutant XR-1. We find that XR-1 possesses reduced cellular survival after the introduction of restriction enzymes that produce either cohesive or blunt ends. XR-1's sensitivity to killing by restriction enzymes strongly mimics its response to gamma-rays. Using pulsed field electrophoresis, we find that for each enzyme, similar numbers of DNA DSBs are being introduced in both cell lines. The simplest explanation for the increased sensitivity to restriction enzymes in the mutant is that the biochemical defect in XR-1 is not confined to the repair of ionizing radiation induced ends, but extends to DSBs that possess ligatable 3'-hydroxyl and 5'-phosphate ends as well.
View details for Web of Science ID A1990DQ16900009
View details for PubMedID 2164147
MEASUREMENT OF THE RELATIVE PROPORTION OF SYMMETRICAL AND ASYMMETRICAL CHROMOSOME-TYPE INTERCHANGES INDUCED BY GAMMA-RADIATION IN HUMAN HAMSTER HYBRID-CELLS
1990; 123 (1): 105-107
Using hamster-human hybrid cells and methods for differential staining of the hamster and human chromosomes, the relative proportion of induced chromosome-type symmetrical and asymmetrical interchange aberrations was measured after a 6.5-Gy dose of gamma radiation. The ratio of these aberration types was not significantly different from 1:1, i.e., the proportion of such interchanges that are symmetrical is very near 0.5, in agreement with the conclusion of others (J.A. Heddle, Genetics 52, 1329-1334, 1965; M. Holmberg and J. Jonasson, Hereditas 74, 57-68, 1973; J.R.K. Savage and D.G. Papworth, Mutat. Res. 95, 7-18, 1983). This proportion is important because virtually all estimates of radiation-induced chromosome-type exchange aberrations are based on measurement of the easily observed but unstable and lethal asymmetrical types, while some of the biological effects of concern from the point of view of oncogenesis and mutagenesis are thought to result from production of stable and nonlethal symmetrical types.
View details for Web of Science ID A1990DN66500016
View details for PubMedID 2371375
GENETIC AND BIOCHEMICAL-CHARACTERIZATION OF THE CHO-UV-1 MUTANT DEFECTIVE IN POSTREPLICATION RECOVERY OF DNA
1990; 50 (8): 2356-2362
The CHO-UV-1 mutant, a Chinese hamster ovary cell with defective postreplication recovery of DNA, is 2- to 4-fold more sensitive than its wild-type counterpart (CHO-77256) to the lethal effects of ethylating agents and UV radiation; it is also hypersensitive (10- to 20-fold) to some DNA-methylating and -cross-linking agents. We studied the CHO-UV-1 mutant further to define its phenotype in terms of DNA damage induction and repair, methyltransferase activity, and effects of caffeine on mutational and lethal responses. Both wild-type and CHO-UV-1 cells incurred similar levels and types of damage when exposed to UV radiation, N-methyl-N'-nitro-N-nitrosoguanidine, or N-methyl-N-nitrosourea. The rate and extent of repair of Micrococcus luteus endonuclease-sensitive sites after UV irradiation or treatment with N-methyl-N'-nitro-N-nitrosoguanidine were also equivalent in these two cell types. Twenty % of the initial endonuclease-sensitive sites induced in either cell line remained at 18 h after UV irradiation; approximately 8% of the sites after N-methyl-N'-nitro-N-nitrosoguanidine exposure were present in both parental and CHO-UV-1 cells after a 17-h repair period. Moreover, the ability of CHO-UV-1 to resynthesize and ligate DNA during excision repair was similar to that of its parent. Neither CHO-UV-1 nor CHO-77256 had appreciable levels of O6-methylguanine-DNA methyltransferase activity which ameliorates the cytotoxicity of alkylating agents. Caffeine, a known inhibitor of postreplication repair, decreased the frequency of mutation induction at the hypoxanthine-guanine phosphoribosyltransferase locus by 40-55% in CHO-77256 but not in CHO-UV-1. These results rule out defective excision repair as a factor in the hypersensitivity of the CHO-UV-1 mutant to DNA-damaging agents. Hence, this cell line appears to derive from a mutation affecting nonexcision repair processes and should be useful in clarifying the mechanism(s) of postreplication recovery of DNA in mammalian cells.
View details for Web of Science ID A1990CZ34900025
View details for PubMedID 2317821
MOLECULAR-CLONING OF THE BREAKPOINT REGION ON CHROMOSOME-6 IN CUTANEOUS MALIGNANT-MELANOMA - EVIDENCE FOR DELETION IN THE C-MYB LOCUS AND TRANSLOCATION OF A SEGMENT OF CHROMOSOME-12
1989; 4 (10): 1201-1205
Nonrandom involvement of chromosome 6 in cutaneous malignant melanoma have been noted by several investigators. Recently an alteration in the c-myb locus (6q22-23) has been identified by Southern analysis in the WM983A cell line which was derived from a primary melanoma of the vertical growth phase. In the present study, the nature of this rearrangement in the WM983A cell line has been further characterized by molecular cloning and nucleotide sequence analysis of the break-point region in the c-myb locus. The results of this investigation demonstrate that the rearrangement in the 6q22-23 region results in deletion of the 3'-end of the c-myb locus with the concomitant translocation of a portion of chromosome 12 to chromosome 6.
View details for Web of Science ID A1989AU50800006
View details for PubMedID 2677917
ISOLATION AND CHARACTERIZATION OF GLUCOSE-6-PHOSPHATE DEHYDROGENASE-DEFICIENT CHINESE-HAMSTER CELLS DERIVED FROM PURE MUTANT COLONIES
1989; 4 (4): 259-264
Ten pure glucose-6-phosphate dehydrogenase (G6PD)-deficient mutants were isolated from colonies composed entirely of cells which lacked G6PD staining activity. These mutants were analyzed for G6PD enzyme activity and the presence of immunologically cross-reactive proteins using immunoblotting techniques and antiserum directed against bovine G6PD. Four mutants had no detectable enzyme activity and did not contain protein which produces a detectable cross-reaction with G6PD antibody. One mutant had residual enzyme activity and altered electrophoretic mobility but did not have detectable immunological cross-reactivity. These results could be explained by either a DNA deletion or point mutational mechanism. On the other hand, five of the 10 mutants analyzed had characteristics consistent with a point mutation in the G6PD gene. All contain a protein which cross-reacts with the G6PD antibody and have the same subunit molecular weight as the parent cell's G6PD enzyme. Four of the five mutants had residual G6PD enzyme activity. Thus, the mechanism for the formation of pure mutants is not simply DNA deletion but is probably a more complex process involving the transfer of altered genetic information from one DNA strand to the other.
View details for Web of Science ID A1989AG75300003
View details for PubMedID 2674604
AN ASYMMETRIC FIELD INVERSION GEL-ELECTROPHORESIS METHOD FOR THE SEPARATION OF LARGE DNA-MOLECULES
1989; 178 (1): 172-176
An electrophoretic method for separating large DNA molecules which uses periodically inverted electric fields of different magnitude in the two directions is described. Net DNA migration is either in the high field direction or in the low field direction, depending on the relative duration of the pulses. With this approach, molecules of up to 1.6 million base pairs can be separated in parallel lanes after a single run under fixed timing conditions. An inexpensive switching unit is the only device needed in addition to the conventional gel box.
View details for Web of Science ID A1989T871000030
View details for PubMedID 2729570
GENETIC-ANALYSIS OF XR-1 MUTATION IN HAMSTER AND HUMAN HYBRIDS
SOMATIC CELL AND MOLECULAR GENETICS
1989; 15 (1): 71-77
We investigated the dominant/recessive nature of the XR-1 mutant locus in intraspecies Chinese hamster ovary (CHO) hybrids and interspecies hybrids with human cell lines that possess different radioresistances. The XR-1 cell is abnormally sensitive to killing by gamma rays in the G1 phase of the cell cycle, while late-S-phase cells have wild-type resistance. [3H]Thymidine selection was used to eliminate the resistant S-phase population. In both intraspecies and interspecies hybrids, the XR-1 mutation is recessive to the wild-type cell and is not influenced by differences in chromosome ploidy. Analysis of hybrids between human ataxia telangiectasia fibroblasts AT5BI and XR-1 cells revealed that they possess different genetic defects as they complemented each other in three of four hybrids tested. These data suggest that the XR-1 locus is evolutionarily conserved between hamster and human cells.
View details for Web of Science ID A1989T192900008
View details for PubMedID 2916163
CELL-CYCLE-DEPENDENT REPAIR OF POTENTIALLY LETHAL DAMAGE IN THE XR-1 GAMMA-RAY-SENSITIVE CHINESE-HAMSTER OVARY CELL
1988; 115 (2): 325-333
Repair of potentially lethal damage (PLD) was investigated in a gamma-ray-sensitive Chinese hamster cell mutant, XR-1, and its parent by comparing survival of plateau-phase cells plated immediately after irradiation with cells plated after a delay. Previous work indicated that XR-1 cells are deficient in repair of double-strand DNA breaks and are gamma-ray sensitive in G1 but have near normal sensitivity and repair capacity in late S phase. At irradiation doses from 0 to 1.0 Gy (100 to 10% survival), the delayed- and immediate-plating survival curves of XR-1 cells were identical; however, at doses greater than 1.0 Gy a significant increase in survival was observed when plating was delayed (PLD repair), approaching a 20-fold increase at 8 Gy. Elimination of S-phase cells by [3H]thymidine suicide dramatically increased gamma-ray sensitivity of plateau-phase XR-1 mutant cells and reduced by 600-fold the number of cells capable of PLD repair after a 6-Gy dose. In contrast, elimination of S-phase cells in plateau-phase parental cells did not alter PLD repair. These results suggest that the majority of PLD repair observed in plateau-phase XR-1 cells occurs in S-phase cells while G1 cells perform little PLD repair. In contrast, G1 cells account for the majority of PLD repair in plateau-phase parental cells. Thus, in the XR-1 mutant, a cell's ability to repair PLD seems to depend upon the stage of the cell cycle at which the irradiation is delivered. A possible explanation for these findings is discussed.
View details for Web of Science ID A1988P773800010
View details for PubMedID 3406371
ISOLATION AND CHARACTERIZATION OF BLEOMYCIN-SENSITIVE CHINESE-HAMSTER OVARY CELLS
1987; 47 (6): 1588-1592
Nineteen bleomycin-sensitive Chinese hamster ovary cell mutants have been isolated using a replica plating and photography approach. As judged by the dose which reduces cell survival to 37% of the untreated control, these mutants are from 2.5- to 32-fold more sensitive to a 16-h bleomycin treatment than the parental cell, while for chronic bleomycin exposure, the increase in sensitivity was 5 to 58 times that of the parental cell. Four bleomycin-sensitive mutants had increased sensitivities to killing by gamma-rays (2- to 3-fold), mitomycin C (2-fold), and ethyl methane sulfonate (4- to 5-fold), while six other mutants were resistant to these agents. Nine other bleomycin-sensitive mutants displayed a variable pattern of cross-sensitivities to these agents. Using the technique of alkaline elution, the relative frequency of single-strand DNA breaks introduced by varying concentrations of bleomycin was examined in one mutant and its parent cell. The elution profiles of both cells were similar, suggesting that the bleomycin sensitivity of this mutant is not due to a greater frequency of single-strand breaks introduced by bleomycin.
View details for Web of Science ID A1987G548300020
View details for PubMedID 2434221
DELAYED MUTATION IN CHINESE-HAMSTER CELLS
SOMATIC CELL AND MOLECULAR GENETICS
1987; 13 (1): 57-66
The possibility was examined that mutational events can be delayed for more than one or two cell divisions following treatment of Chinese hamster cells with the DNA alkylating agent ethyl methane sulfonate. If mutations in mammalian cells are delayed, the proportion of mutant cells in colonies grown from single mutagen-treated cells will reflect the cell division at which the mutation is genetically fixed, i.e., a first division mutation yields a 1/2 mutant colony, a fifth division mutation produces a 1/32 mutant colony, etc. In the present study, replating of cells from single colonies grown for six to seven days after mutagen treatment resulted in the discrete ratios of glucose-6-phosphate dehydrogenase (G6PD)-deficient mutant to wild-type colonies expected for a delayed mutational process which produces mutations over at least 8-10 cell generations. Further, when cells from 7- to 10-day colonies, grown from ethyl methane sulfonate (EMS)-treated cells were replated into selective medium containing 6-thioguanine (6TG), the number of 6TG-resistant colonies obtained per flask was distributed over a very wide range, consistent with a mutational delay process. These results could not be explained by differences in the number of cells per colony or plating efficiency in selective medium. Assuming that the relative number of 6TG-resistant colonies per flask reflects the time of mutation, EMS treatment produced two groups of mutational events: one which occurred within the first five cell generations and another uniformly distributed over at least the next eight to nine divisions. These results support the conclusion that EMS induces mutants for at least 10-14 cell generations after treatment and raise the possibility that current methods to assess the mutagenic potential of an agent might lead to significant underestimation. The role of delayed mutation in the phenomenon of "mutation expression time" is also discussed.
View details for Web of Science ID A1987G091300006
View details for PubMedID 3468633
CELL CYCLE-DEPENDENT REPAIR OF DOUBLE-STRAND DNA BREAKS IN A GAMMA-RAY-SENSITIVE CHINESE-HAMSTER CELL
SOMATIC CELL AND MOLECULAR GENETICS
1985; 11 (5): 485-491
A Chinese hamster cell mutant has been isolated which is extremely sensitive to killing by gamma-irradiation in the G1 and early S phases of the cell cycle (LD50 of 20 vs. 250 rads for parent), but which has nearly normal resistance in late S. The mutant cell is able to repair single-stranded DNA breaks introduced by gamma-radiation. However, in comparison to its parental cell, the mutant is deficient in the repair of double-stranded DNA breaks produced by gamma-irradiation during the sensitive G1-early S period, while in the resistant late S period, the repair is nearly the same for both cell types. This correlation between gamma-ray sensitivity and repair strongly suggests that an inability to repair double-strand DNA breaks in G1 is the basis for the hypersensitivity of the mutant to killing by gamma-rays in this phase of the cell cycle. It also provides direct evidence in mammalian cells that the ability to repair double-strand DNA breaks induced by ionizing radiation is an important biochemical function in cell survival and supports the hypothesis that unrepaired double-strand breaks are a major lethal lesion in mammalian cells. A plausible explanation for the appearance of the cell cycle phenotype of the mutant is that in normal cells there are at least two pathways for the repair of double-strand breaks, one of which functions primarily in late S phase, and the other, either throughout the cell cycle or only in the G1 and early S phases.
View details for Web of Science ID A1985ARK8400008
View details for PubMedID 3862244
TIMING OF MUTATION FIXATION EVENTS IN ETHYL METHANE SULFONATE TREATED CHINESE-HAMSTER CELLS
SOMATIC CELL AND MOLECULAR GENETICS
1984; 10 (4): 429-434
Exposure of single Chinese hamster ovary (CHO) cells to the mutagen, ethyl methane sulfonate, produces two types of mutant colonies lacking glucose-6-phosphate dehydrogenase activity: colonies uniformly deficient in enzyme activity, and mosaic colonies containing both mutant and nonmutant cell phenotypes in various relative proportions and sectored patterns (1/8, 1/4, 1/2). We find that the relative size of the mutant sector in these mosaic colonies primarily reflects the cell division at which the mutation was genetically fixed. Thus, the mutation-fixation event occurs before the first cell division in 1/2 sector and pure mutant colonies, between the first and second divisions for 1/4 sectors, and between the second and third divisions for 1/8 sectors. Delay in the formation of mutations could also explain the phenomenon of "mutation expression time" which is observed when drug resistance is used to select for mutants. Colony sectoring offers for the first time in mammalian cells the opportunity to observe an agent's effect on the timing of the mutational process.
View details for Web of Science ID A1984TC41700012
View details for PubMedID 6589795
ISOLATION OF CELL CYCLE-DEPENDENT GAMMA-RAY-SENSITIVE CHINESE-HAMSTER OVARY CELL
SOMATIC CELL GENETICS
1983; 9 (2): 165-173
A technique for the isolation of gamma ray-sensitive Chinese hamster ovary (CHO) cell mutants is described, which uses nylon cloth replica plating and photography with dark-field illumination to directly monitor colonies for growth after gamma irradiation. Two gamma ray-sensitive mutants were isolated using this method. One of these cells (XR-1) had a two-slope survival curve: an initial steep slope and then a flattening of the curve at about 10% survival. Subsequently, it was found that this cell is sensitive to gamma irradiation in G1, early S, and late G2 phases of the cell cycle, whereas in the resistant phase (late S phase) its survival approaches that of the parental cells. The D37 in the sensitive G1 period is approximately 30 rads, compared with 300 rads of the parental cell. This mutant cell is also sensitive to killing by the DNA breaking agent, bleomycin, but is relatively insensitive to UV light and ethyl methane sulfonate, suggesting that the defect is specific for agents that produce DNA strand breakage.
View details for Web of Science ID A1983QJ46700002
View details for PubMedID 6836453
The Apoptosis Repressor With a CARD Domain (ARC) is Essential for the Survival of VHL Deficient Renal Cancer Cells
ELSEVIER SCI LTD. 2012: S120-S120
View details for Web of Science ID 000313036501011
Double Knockdown of Prolyl Hydroxylase and Factor-Inhibiting Hypoxia-Inducible Factor With Nonviral Minicircle Gene Therapy Enhances Stem Cell Mobilization and Angiogenesis After Myocardial Infarction
LIPPINCOTT WILLIAMS & WILKINS. 2011: S46-S54
Under normoxic conditions, hypoxia-inducible factor (HIF)-1? is rapidly degraded by 2 hydroxylases: prolyl hydroxylase (PHD) and factor-inhibiting HIF-1 (FIH). Because HIF-1? mediates the cardioprotective response to ischemic injury, its upregulation may be an effective therapeutic option for ischemic heart failure.PHD and FIH were cloned from mouse embryonic stem cells. The best candidate short hairpin (sh) sequences for inhibiting PHD isoenzyme 2 and FIH were inserted into novel, nonviral, minicircle vectors. In vitro studies after cell transfection of mouse C2C12 myoblasts, HL-1 atrial myocytes, and c-kit(+) cardiac progenitor cells demonstrated higher expression of angiogenesis factors in the double-knockdown group compared with the single-knockdown and short hairpin scramble control groups. To confirm in vitro data, shRNA minicircle vectors were injected intramyocardially after left anterior descending coronary artery ligation in adult FVB mice (n=60). Functional studies using MRI, echocardiography, and pressure-volume loops showed greater improvement in cardiac function in the double-knockdown group. To assess mechanisms of this functional recovery, we performed a cell trafficking experiment, which demonstrated significantly greater recruitment of bone marrow cells to the ischemic myocardium in the double-knockdown group. Fluorescence-activated cell sorting showed significantly higher activation of endogenous c-kit(+) cardiac progenitor cells. Immunostaining showed increased neovascularization and decreased apoptosis in areas of injured myocardium. Finally, western blots and laser-capture microdissection analysis confirmed upregulation of HIF-1? protein and angiogenesis genes, respectively.We demonstrated that HIF-1? upregulation by double knockdown of PHD and FIH synergistically increases stem cell mobilization and myocardial angiogenesis, leading to improved cardiac function.
View details for DOI 10.1161/CIRCULATIONAHA.110.014019
View details for Web of Science ID 000294782800006
View details for PubMedID 21911818
Proceedings of the Oxygen Homeostasis/Hypoxia Meeting.
View details for PubMedID 15126380
Regulation of insulin-like growth factor-binding protein 1 by hypoxia and 3 ',5 '-cyclic adenosine monophosphate is additive in HepG2 cells
ENDOCRINE SOC. 2000: 3821-3827
Insulin-like growth factor-binding protein 1 (IGFBP-1) is important in regulating minute-to-minute IGF bioavailability in the circulation and is primarily an inhibitor of IGF action systemically and in most cellular systems. Understanding regulation of IGFBP-1 is, thus, important in understanding regulation of IGF actions. The IGFBP-1 promoter contains a cAMP response element, and cAMP stimulates IGFBP-1 gene expression at the transcriptional level. Recently, we have found three consensus sequences for the hypoxia response element in intron 1 of the IGFBP-1 gene. Herein, we have investigated the effects of hypoxia and a cAMP analog, 8-bromoadenosine-3',5'-cyclic monophosphate (8-Br-cAMP), on IGFBP-1 expression in HepG2 cells, a model system for IGFBP-1 gene regulation. HepG2 cells were exposed to normoxia (20% pO2) or hypoxia (2% pO2) for 24 h in the absence or presence of 8-Br-cAMP (0.1, 0.5, and 1 mM). Western ligand blotting revealed IGFBP-1 as the predominant IGFBP in HepG2-conditioned media, which increased in a dose-dependent manner after incubation with 8-Br-cAMP in normoxia and hypoxia (3-fold and 7-fold at 1 mM, respectively). Under hypoxic, compared with normoxic, conditions, IGFBP-1 protein and messenger RNA (mRNA) levels increased approximately 10-fold and 20-fold, respectively. In normoxia, 8-Br-cAMP stimulated IGFBP-1 protein and mRNA levels in a dose-dependent manner (7-fold and 10-fold at 1 mM). Hypoxia and 8-Br-cAMP showed additive stimulatory effects on IGFBP-1 protein and mRNA levels (35-fold and 50-fold at 1 mM) that were time and dose dependent. Primary transcripts of IGFBP-1 mRNA were increased concordantly with IGFBP-1 mRNA. The half-life of the IGFBP-1 mRNA was markedly increased (approximately 6-fold) by hypoxia, and cAMP minimally enhanced this effect. These results demonstrate that hypoxia and compounds that increase intracellular cAMP additively regulate IGFBP-1 gene expression by transcriptional and posttranscriptional mechanisms. Regulation of IGFBP-1 mRNA and protein by cAMP and hypoxia may be important for understanding the physiologic and pathophysiologic roles of IGFBP-1.
View details for Web of Science ID 000089820800059
View details for PubMedID 11061545
CHEMOTHERAPEUTIC TUMOR TARGETING USING CLOSTRIDIAL SPORES
ELSEVIER SCIENCE BV. 1995: 357-364
The toxicity associated with conventional cancer chemotherapy is primarily due to a lack of specificity for tumour cells. In contrast, intravenously injected clostridial spores exhibit a remarkable specificity for tumours. This is because, following their administration, clostridial spores become exclusively localised to, and germinate in, the hypoxic/necrotic tissue of tumours. This unique property could be exploited to deliver therapeutic agents to tumours. In particular, genetic engineering could be used to endow a suitable clostridial host with the capacity to produce an enzyme within the tumour which can metabolise a systemically introduced, non-toxic prodrug into a toxic metabolite. The feasibility of this strategy (clostridial-directed enzyme prodrug therapy, CDEPT) has been demonstrated by cloning the Escherichia coli B gene encoding nitroreductase (an enzyme which converts the prodrug CB1954 to a highly toxic bifunctional alkylating agent) into a clostridial expression vector and introducing the resultant plasmid into Clostridium beijerinckii (formerly C. acetobutylicum) NCIMB 8052. The gene was efficiently expressed, with recombinant nitroreductase representing 8% of the cell soluble protein. Following the intravenous injection of the recombinant spores into mice, tumour lysates have been shown, by Western blots, to contain the E. coli-derived enzyme.
View details for Web of Science ID A1995TA64700015
View details for PubMedID 7576773
Reduced p53 response to DNA damage in human prostate epithelial cells
10TH ICRR SOCIETY (INT CONGRESS RADIATION RES). 1995: 552-555
View details for Web of Science ID A1995BH47W00115
ACTIVATORS OF PROTEIN-KINASE-C SELECTIVELY MEDIATE CELLULAR CYTOTOXICITY TO HYPOXIC CELLS AND NOT AEROBIC CELLS
PERGAMON-ELSEVIER SCIENCE LTD. 1994: 259-265
By understanding the signal transduction pathways through which a cell responds to changes in environmental oxygen levels, we may be able to therapeutically exploit this response by manipulating these pathways.The human adenocarcinoma cell line A549 was exposed to varying durations of hypoxia alone and then plated for survival, or treated with PKC activating agents for 1 h before plating for survival. Western blots were used to determine the kinetics of PKC epsilon and phospholipase C induction.The level of hypoxic killing was directly related to the time of exposure and inversely related to the level of oxygen in the environment. Exposure of the cells to protein kinase C (PKC) activators for 1 h after chronic hypoxic exposure increased cell killing by at least an additional three logs beyond that found for hypoxia alone. Treatment of cells with an inactive phorbol ester 4 alpha-phorbol-12,13-didecanoate (PDA) resulted in no increase in hypoxic cell killing, even at the highest concentrations of PDA which produced no detectable toxicity under normal aerobic conditions. Using inhibitors of phospholipases A2 and C, we were able to completely inhibit the additional hypoxic cell killing induced by TPA, but not the uninduced hypoxic cell killing.These studies suggest that accumulation of phospholipid breakdown products may be responsible for TPA induced cell killing, and that hypoxic cells differ from aerobic cells in their ability to tolerate these products.
View details for Web of Science ID A1994NN51200007
View details for PubMedID 8195017
THE REGULATION OF GRP78 AND MESSENGER-RNA LEVELS BY HYPOXIA IS MODULATED BY PROTEIN-KINASE-C ACTIVATORS AND INHIBITORS
RADIATION RESEARCH SOC. 1994: S60-S63
In this study, we have shown that steady-state levels of glucose-regulated 78 kDa (GRP78) protein and messenger RNA increase during a 5-h exposure to 0.02% oxygen. This increase in GRP78 protein and mRNA induced by hypoxia can be abolished by a 1-h pretreatment of cells before hypoxia with the protein kinase C (PKC) inhibitors staurosporine and H7 at concentrations at which the drugs themselves do not cause cytotoxicity. Although all studies using protein kinase inhibitors must be interpreted with caution, staurosporine and H7 have been shown to be potent inhibitors of PKC activity, suggesting a role for PKC in mediating the transcriptional regulation of GRP78 by hypoxia. Further support for PKC in regulating GRP78 gene expression by hypoxia stems from gel-mobility shift studies in mixtures of nuclear extracts from aerobic or hypoxic cells with a 36 bp region of the GRP78 promoter (-170 to -135). Binding of this factor could be inhibited by pretreating cells with the PKC inhibitor staurosporine before hypoxia or activated by treating cells with the PKC-activating phorbol ester TPA. These data suggest that activation of this hypoxia-responsive factor is sensitive to oxygen levels and seems to be mediated through a PKC signal transduction pathway.
View details for Web of Science ID A1994NF37900016
View details for PubMedID 8146329
TUMOR HYPOXIA - THE PICTURE HAS CHANGED IN THE 1990S
TAYLOR & FRANCIS LTD. 1994: 95-102
Since the 1950s, the presence of hypoxic cells in human tumours has been widely regarded as a problem, and a variety of strategies have been developed and tested, both in experimental and clinical studies, to overcome this perceived problem. One of these strategies was the development of bioreductive cytotoxins--drugs which in themselves were relatively innocuous, but when metabolized under hypoxic conditions, became highly cytotoxic, thereby preferentially killing the hypoxic cells. Modelling studies and experimental data with newly developed hypoxic cytotoxins, such as SR 4233 (tirapazamine) and RSU 1069, have led to the realization not only that it is better to kill hypoxic cells in tumours than to radiosensitize or oxygenate them, but also that with these bioreductive cytotoxins hypoxic cells in tumours can be an advantage in cancer therapy. However, to realize the advantage of adding the drug with each radiation dose, the tumour must undergo a process analogous to reoxygenation, which we have termed 'rehypoxiation', by which hypoxic cells are regenerated after each dose of the hypoxic cytotoxin. In addition, we also discuss the fact that hypoxia is a cellular stress which activates many new genes. The activation of these genes will be a major focus for research in coming years and will undoubtedly lead to new approaches in cancer detection and treatment. In summary, the 1990s are bringing a fundamental change in our perception of tumour hypoxia, from a position of being a problem to that of being a solution in cancer treatment.
View details for Web of Science ID A1994NE75800013
View details for PubMedID 7905916
WHAT ROLE DO GLUTATHIONE S-TRANSFERASES PLAY IN THE CELLULAR-RESPONSE TO IONIZING-RADIATION
ELSEVIER SCIENCE INC. 1992: 759-763
The glutathione S-transferases (GST's) are cytosolic dimeric proteins that are composed of three family members, alpha, pi, and mu, and a fourth microsomal member. These four family members are primarily involved in cellular detoxification of xenobiotics and hydroperoxides. Recently, a strong correlation has been found between the overexpression of GST's and resistance to chemotherapeutic drugs. In comparison to chemotherapy, little is known about the role of GST's in the cellular response to ionizing radiation. To determine which GST's may be involved in this response, we have identified Chinese hamster ovary cell lines that possess different levels of alpha and pi GST isozyme activity. The survival of these cell lines to ionizing radiation was similar to that of wild-type Chinese hamster ovary-KI cells from which they were derived. Although differences in GST levels did not affect ionizing radiation sensitivity per se, we found that ionizing radiation decreased the amount of cytosolic pi GST without affecting alpha GST levels. Taken together, these data suggest that GST's are involved in the cellular response against oxidative stress generated by ionizing radiation.
View details for Web of Science ID A1992HJ37700028
View details for PubMedID 1544849
CHARACTERIZATION OF A CHO CELL-LINE RESISTANT TO KILLING BY THE HYPOXIC CELL CYTOTOXIN SR-4233
ELSEVIER SCIENCE INC. 1992: 681-684
One approach to understanding the mechanism of selective hypoxic cell killing by the benzotriazine-di-N-oxide, SR 4233, is to characterize cell lines that exhibit increased resistance to killing by this drug. The Chinese Hamster Ovary cell line BL-10 was originally isolated on the basis of its hypersensitivity to killing by bleomycin. It is 2.7-fold more resistant to hypoxic cell killing by SR 4233 than wild-type CHO on comparison of D0's. However, both BL-10 and CHO possess the same sensitivity to killing by SR 4233 under aerobic conditions. We have excluded the explanation that differential metabolism of SR 4233 is responsible for its increased survival as both BL-10 and CHO produce the two-electron product SR 4317 at the same rate (3 nmoles/hr/10(6) cells). Analysis of free radical production, DNA double-strand break induction, and glutathione (GSH) levels suggested that the resistance of BL-10 to killing by SR 4233 might result from increased intracellular radical scavenger pathways. Using buthionine sulfoximine (BSO) to decrease cellular GSH levels, we found a marked increase in the sensitivity of BL-10 cells to SR 4233 killing under hypoxia, but a much smaller increase in the sensitivity of CHO cells. Taken together, these data imply that the high GSH levels in BL-10 cells is responsible for its resistance to SR 4233 cytotoxicity.
View details for Web of Science ID A1992HJ37700011
View details for PubMedID 1544836