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This version was published on September 1, 2006
Journal of Biomolecular Screening, Vol. 11, No. 6, 678-687 (2006)
DOI: 10.1177/1087057106289234

Development of a Cell-Based Reporter Assay for Screening of Inhibitors of Hypoxia-Inducible Factor 2-Induced Gene Expression

Girma M. Woldemichael

National Cancer Institute, Molecular Targets Development Program, Center for Cancer Research, Frederick, Maryland

James R. Vasselli

National Cancer Institute, Clinical Research Center, Urologic Oncology Branch, Bethesda, Maryland

Roberta S. Gardella

SAIC-Frederick, Inc., Basic Research Program, NCI-Frederick, Frederick, Maryland

Tawnya C. Mckee

National Cancer Institute, Molecular Targets Development Program, Center for Cancer Research, Frederick, Maryland

W. Marston Linehan

National Cancer Institute, Clinical Research Center, Urologic Oncology Branch, Bethesda, Maryland

James B. McMahon

National Cancer Institute, Molecular Targets Development Program, Center for Cancer Research, Frederick, Maryland

Reporter cell lines have been developed for the identification of inhibitors of gene expression enhanced by hypoxia-inducible factor 2, which has been implicated as a transcription factor involved in the tumorigenesis of clear cell renal carcinoma. Stably transformed reporter clones of the human renal clear cell carcinoma cell line 786-O were generated by transfection or retroviral infection. Luciferase reporter expression in the vectors used was driven by either the natural human vascular endothelial growth factor (VEGF) promoter-enhancer or by the VEGF and the human endothelial nitric oxide synthase enhancers modulating minimal human cytomegalovirus promoter. Utility of the generated reporter cell lines was validated by introducing the von Hippel-Lindau protein complex and testing for reporter inducibility by hypoxia. The dynamic range in reporter activity under hypoxic stress was found to be at least 30- to 40-fold, with a signal-to-noise ratio of 60:1. Properties of the cell lines such as tolerance to up to 3% DMSO, signal stability with multiple in vitro passages, and utility in both 96- and 384-well plate formats indicated their suitability for use in a high-throughput screen. In addition, the potential use of these reporter lines in the evaluation of high-throughput screening hits in vivo in various mice models has been demonstrated.

Key Words: HIF-2 • cell-based assay • reporter assay • hypoxia response element • VHL disease

References

  • Krek W: VHL takes HIF’s breath away. Nat Cell Biol 2000;2: E121-E123.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  • Ohh M, Park CW, Ivan N, Hoffman MA, Kim TY, Huang LE, et al: Ubiquitination of hypoxia-inducible factor requires direct binding to the beta-domain of the von Hippel-Lindau protein. Nat Cell Biol 2000;2: 423-427.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  • Hu CJ, Wang LY, Chodosh LA, Keith B, Simon MC: Differential roles of hypoxia-inducible factor 1{alpha} (HIF-1{alpha}) and HIF-2{alpha} in hypoxic gene regulation. Mol Cell Biol 2003;23: 9361-9374.[Abstract/Free Full Text]
  • Talks KL, Turley H, Gatter KC, Maxwell PH, Pugh CW, Ratcliffe PJ, et al: The expression and distribution of the hypoxia-inducible factors HIF-1{alpha} and HIF-2{alpha} in normal human tissues, cancers, and tumor-associated macrophages. Am J Pathol 2000;157: 411-421.[Abstract/Free Full Text]
  • Wiesener MS, Jurgensen JS, Rosenberger C, Scholze C, Horstrup JH, Warnecke C, et al: Widespread, hypoxia-inducible expression of HIF-2{alpha} in distinct cell populations of different organs. FASEB J 2002;16: U348-U369.
  • Raval RR, Lau KW, Tran MGB, Sowter HM, Mandriota SJ, Li JL, et al: Contrasting properties of hypoxia-inducible factor 1 (HIF-1) and HIF-2 in von Hippel-Lindau-associated renal cell carcinoma. Mol Cell Biol 2005;25: 5675-5686.[Abstract/Free Full Text]
  • Krieg M, Haas R, Brauch H, Acker T, Flamme I, Plate KH: Up-regulation of hypoxia-inducible factors HIF-1{alpha} and HIF- 2{alpha} under normoxic conditions in renal carcinoma cells by von Hippel-Lindau tumor suppressor gene loss of function. Oncogene 2000;19: 5435-5443.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  • Iliopoulos O, Kibel A, Gray S, Kaelin WG: Tumor suppression by the human Von Hippel-Lindau gene-product. Nat Med 1995;1: 822-826.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  • Kondo K, Klco J, Nakamura E, Lechpammer M, Kaelin WG: Inhibition of HIF is necessary for tumor suppression by the von Hippel-Lindau protein. Cancer Cell 2002;1: 237-246.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  • Covello KL, Simon MC, Keith B: Targeted replacement of hypoxiainducible factor-1{alpha} by a hypoxia-inducible factor-2{alpha} knock-in allele promotes tumor growth. Cancer Res 2005;65: 2277-2286.[Abstract/Free Full Text]
  • Zimmer M, Doucette D, Siddiqui N, Iliopoulos O: Inhibition of hypoxiainducible factor is sufficient for growth suppression of VHL-/- tumors. Mol Cancer Res 2004;2: 89-95.[Abstract/Free Full Text]
  • Rapisarda A, Uranchimeg B, Scudiero DA, Selby M, Sausville EA, Shoemaker RH, et al: Identification of small molecule inhibitors of hypoxia-inducible factor 1 transcriptional activation pathway. Cancer Res 2002;62: 4316-4324.[Abstract/Free Full Text]
  • Isaacs JS, Jung YJ, Mimnaugh EG, Martinez A, Cuttitta F, Neckers LM: Hsp90 regulates a von Hippel Lindau-independent hypoxia-inducible factor-1{alpha}-degradative pathway. J Biol Chem 2002;277: 29936-29944.[Abstract/Free Full Text]
  • Chau NM, Rogers P, Aherne W, Carroll V, Collins I, McDonald E, et al: Identification of novel small molecule inhibitors of hypoxia-inducible factor-1 that differentially block hypoxia-inducible factor-1 activity and hypoxia-inducible factor-1{alpha} induction in response to hypoxic stress and growth factors. Cancer Res 2005;65: 4918-4928.[Abstract/Free Full Text]
  • Chang H, Shyu KG, Lee CC, Tsai SC, Wang BW, Lee YH, et al: GL331 inhibits HIF-1{alpha} expression in a lung cancer model. Biochem Biophys Res Commun 2003;302: 95-100.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  • Lee YM, Kim SH, Kim HS, Son MJ, Nakajima H, Kwon HJ, et al: Inhibition of hypoxia-induced angiogenesis by FK228, a specific histone deacetylase inhibitor, via suppression of HIF-1{alpha} activity. Biochem Biophys Res Commun 2003;300: 241-246.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  • Nagasawa H, Mikamo N, Nakajima S, Matsumoto H, Uto Y, Hori H: Antiangiogenic hypoxic cytotoxin TX-402 inhibits hypoxia-inducible factor 1 signaling pathway. Anticancer Res 2003;23: 4427-4434.[ISI][Medline] [Order article via Infotrieve]
  • Kung AL, Zabludoff SD, France DS, Freedman SJ, Tanner EA, Vieira A, et al: Small molecule blockade of transcriptional coactivation of the hypoxia-inducible factor pathway. Cancer Cell 2004;6: 33-43.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  • Hasebe Y, Egawa K, Yamazaki Y, Kunimoto S, Hirai Y, Ida Y, et al: Specific inhibition of hypoxia-inducible factor (HIF)-1{alpha} activation and of vascular endothelial growth factor (VEGF) production by flavonoids. Biol Pharm Bull 2003;26: 1379-1383.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  • Palayoor ST, Tofilon PJ, Coleman CN: Ibuprofen-mediated reduction of hypoxia-inducible factors HIF-1{alpha} and HIF-2{alpha} in prostate cancer cells. Clin Cancer Res 2003;9: 3150-3157.[Abstract/Free Full Text]
  • Liu YX, Cox SR, Morita T, Kourembanas S: Hypoxia regulates vascular endothelial growth-factor gene-expression in endothelial-cells: identification of A 5'-enhancer. Circ Res 1995;77: 638-643.[Abstract/Free Full Text]
  • Coulet F, Nadaud S, Agrapart M, Soubrier F: Identification of hypoxia-response element in the human endothelial nitric-oxide synthase gene promoter. J Biol Chem 2003;278: 46230-46240.[Abstract/Free Full Text]
  • Duan DR, Humphrey JS, Chen DYT, Weng Y, Sukegawa J, Lee S, et al: Characterization of the VHL tumor suppressor gene product: localization, complex formation, and the effect of natural inactivating mutations. Proc Nat Acad Sci U S A 1995;92: 6459-6463.[Abstract/Free Full Text]
  • Alleman WG, Tabios RL, Chandramouli GVR, Aprelikova ON, Torres-Cabala C, Mendoza A, et al: The in vitro and in vivo effects of re-expressing methylated von Hippel-Lindau tumor suppressor gene in clear cell renal carcinoma with 5-Aza-2'-deoxycytidine. Clin Cancer Res 2004;10: 7011-7021.[Abstract/Free Full Text]
  • Eli Lilly and Company, National Institutes of Health Chemical Genomics Center: Two-day plate uniformity and signal variability assessment [Online]. Retrieved from http://www.ncgc.nih.gov/guidance/section2.html%23twoday_plate
  • Institute of Laboratory Animal Resources Commission on Life Sciences, National Research Council: Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No. 86-23). Washington, DC: National Academy Press, 1985.
  • Zhang JH, Chung TDY, Oldenburg KR: A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen 1999;4: 67-73.[Abstract]
  • Jost LM, Kirkwood JM, Whiteside TL: Improved short-term and long-term XTT-based colorimetric cellular cytotoxicity assay for melanoma and other tumor-cells. J Immunol Meth 1992;147: 153-165.[ISI][Medline] [Order article via Infotrieve]
  • Hiroshi H, Shinae KK, Masahiro H: Optical imaging of tumor hypoxia and evaluation of efficacy of a hypoxia-targeting drug in living animals. Mol Imaging 2005;4: 182-193.[Medline] [Order article via Infotrieve]

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[Abstract] [PDF]

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