Download PDF
Geburtshilfe Frauenheilkd 2005; 65(12): 1163-1167
DOI: 10.1055/s-2005-873039
Originalarbeit

Georg Thieme Verlag KG Stuttgart · New York

Expression Profiles of Apoptosis Related Genes and Investigation of HIF-2α Signal Transduction in the Ovarian Cancer Cell Line OvCa-3

HIF-2α Signal Transduction in Ovarian Cancer CellsExpressionsprofile Apoptose-assoziierter Gene und Untersuchung der Signaltransduktion von HIF-2α in der Ovarialkarzinomzelllinie OvCa-3D. Herr1 , C. Keck1 , P. Wiehle1 , B. Phan2 , H. Bettendorf1 , D. Pietrowski1 , 3
  • 1University Medical School Freiburg
  • 2Department of Obstetrics and Gynecology, Da Nang General Hospital, Da Nang City, Vietnam
  • 3Herzzentrum Lahr
Further Information

Publication History

Eingang Manuskript: 27.10.2005

Akzeptiert: 20.11.2005

Publication Date:
18 January 2006 (online)

Also available at
    Permissions and Reprints

Zusammenfassung

Das Tumorsuppressorgen VHL ist ein wichtiger Regulator sowohl von Bcl-2A als auch von HIF-2α. Mit dieser Studie sollte die Frage geklärt werden, ob VHL die HIF-2α-Expression in der Ovarialkarzinom-Zelllinie OvCa-3 über einen Bcl-2A-abhängigen Weg reguliert. Des Weiteren wurde die Expression verschiedener Tumorsuppressorgene, Bcl-2-Familienmitglieder und Hypoxie-abhängiger Transkriptionsfaktoren (HIF) untersucht. Hierzu wurde die Genfunktion von Bcl-2A mittels eines RNA interference assay ausgeschaltet. Die Expression von HIF-2α wurde dann mit einer TaqMan-Realtime-PCR-Analyse quantifiziert. Zusätzlich wurde die Expression verschiedener Tumorsuppressorgene, Bcl-2-Familienmitglieder und Hypoxie-abhängiger Transkriptionsfaktoren (HIF) mittels RT‐PCR nachgewiesen. Es gelang der Nachweis der Expression der Tumorsuppressorgene RB-1, VHL, NF-1, NF-2, Wt-1, p53 und APC, des Hypoxie-induzierten Faktors HIF-2α, und der Bcl-2 Familienmitglieder Bax, Bcl-2A und Bclx in OvCa-3-Zellen. Nach dem Ausschalten der Genfunktion von Bcl-2A ergab die Quantifizierung der HIF-2α-Expression keine signifikante Veränderung. Es ist deshalb davon auszugehen, dass VHL die Expression von HIF-2α in OvCa-3-Zellen nicht über einen Bcl-2A-abhängigen Weg reguliert. Darüber hinaus ist zu vermuten, dass Bcl-2A nicht in die Signaltransduktion von HIF-2α in OvCa-3-Zellen involviert ist.

Abstract

Since the tumor suppressor gene VHL is an important regulator of Bcl-2A as well as of HIF-2α, we wanted to address the question whether VHL regulates the expression of HIF-2α in the ovarian cancer cell line OvCa-3 via Bcl-2A dependent pathways. Furthermore, we investigated expression of different tumor suppressor genes, Bcl-2 family members and hypoxia inducible factors. Gene function of Bcl-2A was knocked down using RNA interference assay. Quantification of HIF-2α expression was performed by TaqMan-Realtime-PCR analysis. In addition, transcript levels of different tumor suppressor genes, Bcl-2 family members and hypoxia inducible factors were determined by RT‐PCR. We demonstrate expression of the tumor suppressor genes RB-1, VHL, NF-1, NF-2, Wt-1, p53, and APC, hypoxia dependent factor HIF-2α, and the Bcl-2 family members Bax, Bcl-2A, and Bclx in OvCa-3 cells. Furthermore, gene function of Bcl-2A was knocked down. After knock-down of Bcl-2A, expression of HIF-2α was quantified using TaqMan-Realtime-PCR-analysis revealing no significant change. We suggest that VHL is not regulating the expression of HIF-2α via Bcl-2A dependent pathways in OvCa-3 cells. Furthermore, we hypothesise that Bcl-2A might not be involved in the signal transduction of HIF-2α in OvCa-3 cells.

Schlüsselwörter

Apoptose - HIF-2α - Bcl-2A - OvCa-3-Zellen - Tumorsuppressorgene

Key words

Apoptosis - HIF-2α - Bcl-2A - OvCa-3 cells - tumor suppressor genes

References

  • 1 Folkman J. Angiogenesis and apoptosis.  Semin Cancer Biol. 2003;  13 159-167
    CrossrefPubMedGoogle Scholar
  • 2 Graeber T G, Osmanian C, Jacks T, Housman D E, Koch C J, Lowe S W. et al . Hypoxia-mediated selection of cells with diminished apoptotic potential in solid tumours.  Nature. 1996;  379 88-91
    CrossrefPubMedGoogle Scholar
  • 3 Na X, Wu G, Ryan C K, Schoen S R, di'Santagnese P A, Messing E M. Overproduction of vascular endothelial growth factor related to von Hippel-Lindau tumor suppressor gene mutations and hypoxia-inducible factor-1 alpha expression in renal cell carcinomas.  J Urol. 2003;  170 (2 Pt 1) 588-592
    CrossrefPubMedGoogle Scholar
  • 4 Vogelstein B, Lane D, Levine A J. Surfing the p 53 network.  Nature. 2000;  408 307-310
    CrossrefPubMedGoogle Scholar
  • 5 Huh J E, Kang K S, Chae C, Kim H M, Ahn K S, Kim S H. Roles of p 38 and JNK mitogen-activated protein kinase pathways during cantharidin-induced apoptosis in U937 cells.  Biochem Pharmacol. 2004;  67 1811-1818
    CrossrefPubMedGoogle Scholar
  • 6 Kondo K, Kaelin Jr W G. The von Hippel-Lindau tumor suppressor gene.  Exp Cell Res. 2001;  264 117-125
    CrossrefPubMedGoogle Scholar
  • 7 Kim W Y, Kaelin W G. Role of VHL gene mutation in human cancer.  J Clin Oncol. 2004;  22 4991-5004
    CrossrefPubMedGoogle Scholar
  • 8 Biju M P, Neumann A K, Bensinger S J, Johnson R S, Turka L A, Haase V H. Vhlh gene deletion induces HIF‐1-mediated cell death in thymocytes.  Mol Cell Biol. 2004;  24 9038-9047
    CrossrefPubMedGoogle Scholar
  • 9 Wang G L, Jiang B H, Rue E A, Semenza G L. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension.  Proc Natl Acad Sci USA. 1995;  92 5510-5514
    CrossrefPubMedGoogle Scholar
  • 10 Dachs G U, Stratford I J. The molecular response of mammalian cells to hypoxia and the potential for exploitation in cancer therapy.  Br J Cancer Suppl . 1996;  27 S126-S132
    PubMedGoogle Scholar
  • 11 Jiang B H, Rue E, Wang G L, Roe R, Semenza G L. Dimerization, DNA binding, and transactivation properties of hypoxia-inducible factor 1.  J Biol Chem. 1996;  271 17771-17778
    CrossrefPubMedGoogle Scholar
  • 12 Ratcliffe P J, O'Rourke J F, Maxwell P H, Pugh C W. Oxygen sensing, hypoxia-inducible factor-1 and the regulation of mammalian gene expression.  J Exp Biol. 1998;  201 (Pt 8) 1153-1162
    PubMedGoogle Scholar
  • 13 Semenza G L, Jiang B H, Leung S W, Passantino R, Concordet J P, Maire P. et al . Hypoxia response elements in the aldolase A, enolase 1, and lactate dehydrogenase A gene promoters contain essential binding sites for hypoxia-inducible factor 1.  J Biol Chem. 1996;  271 32529-32537
    CrossrefPubMedGoogle Scholar
  • 14 Zhang H, Akman H O, Smith E L, Zhao J, Murphy-Ullrich J E, Batuman O A. Cellular response to hypoxia involves signaling via Smad proteins.  Blood. 2003;  101 2253-2260
    CrossrefPubMedGoogle Scholar
  • 15 Devarajan P, De Leon M, Talasazan F, Schoenfeld A R, Davidowitz E J, Burk R D. The von Hippel-Lindau gene product inhibits renal cell apoptosis via Bcl-2-dependent pathways.  J Biol Chem. 2001;  276 40599-40605
    CrossrefPubMedGoogle Scholar
  • 16 Reed J C. Bcl-2 family proteins.  Oncogene. 1998;  17 3225-3236
    PubMedGoogle Scholar
  • 17 Goping I S, Gross A, Lavoie J N, Nguyen M, Jemmerson R, Roth K. et al . Regulated targeting of BAX to mitochondria.  J Cell Biol. 1998;  143 207-215
    CrossrefPubMedGoogle Scholar
  • 18 Tsujimoto Y, Shimizu S. VDAC regulation by the Bcl-2 family of proteins.  Cell Death Differ. 2000;  7 1174-1181
    CrossrefPubMedGoogle Scholar
  • 19 Kluck R M, Bossy-Wetzel E, Green D R, Newmeyer D D. The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis.  Science. 1997;  275 1132-1136
    CrossrefPubMedGoogle Scholar
  • 20 Vander Heiden M G, Chandel N S, Williamson E K, Schumacker P T, Thompson C B. Bcl-xL regulates the membrane potential and volume homeostasis of mitochondria.  Cell. 1997;  91 627-637
    CrossrefPubMedGoogle Scholar
  • 21 Yang J, Liu X, Bhalla K, Kim C N, Ibrado A M, Cai J. et al . Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked.  Science. 1997;  275 1129-1132
    CrossrefPubMedGoogle Scholar
  • 22 Shimizu S, Eguchi Y, Kamiike W, Waguri S, Uchiyama Y, Matsuda H. et al . Bcl-2 blocks loss of mitochondrial membrane potential while ICE inhibitors act at a different step during inhibition of death induced by respiratory chain inhibitors.  Oncogene. 1996;  13 21-29
    PubMedGoogle Scholar
  • 23 Marzo I, Brenner C, Zamzami N, Jurgensmeier J M, Susin S A, Vieira H L. et al . Bax and adenine nucleotide translocator cooperate in the mitochondrial control of apoptosis.  Science. 1998;  281 2027-2031
    CrossrefPubMedGoogle Scholar
  • 24 Pastorino J G, Chen S T, Tafani M, Snyder J W, Farber J L. The overexpression of Bax produces cell death upon induction of the mitochondrial permeability transition.  J Biol Chem. 1998;  273 7770-7775
    CrossrefPubMedGoogle Scholar
  • 25 Xiang J, Chao D T, Korsmeyer S J. BAX-induced cell death may not require interleukin 1 beta-converting enzyme-like proteases.  Proc Natl Acad Sci USA. 1996;  93 14559-14563
    CrossrefPubMedGoogle Scholar
  • 26 Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.  Anal Biochem. 1987;  162 156-159
    PubMedGoogle Scholar
  • 27 Greijer A E, van der Wall E. The role of hypoxia inducible factor 1 (HIF‐1) in hypoxia induced apoptosis.  J Clin Pathol. 2004;  57 1009-1014
    CrossrefPubMedGoogle Scholar
  • 28 Herr D, Keck C, Tempfer C, Pietrowski D. Chorionic gonadotropin regulates the transcript level of VHL, p 53, and HIF‐2 alpha in human granulosa lutein cells.  Mol Reprod Dev. 2004;  69 397-401
    CrossrefPubMedGoogle Scholar
  • 29 Haimovich B, Tanaka J C. Magainin-induced cytotoxicity in eukaryotic cells: kinetics, dose-response and channel characteristics.  Biochim Biophys Acta. 1995;  1240 149-158
    PubMedGoogle Scholar
  • 30 Esteller M. Relevance of DNA methylation in the management of cancer.  Lancet Oncol. 2003;  4 351-358
    CrossrefPubMedGoogle Scholar
  • 31 Tweddle D A, Pearson A D, Haber M, Norris M D, Xue C, Flemming C. et al . The p 53 pathway and its inactivation in neuroblastoma.  Cancer Lett. 2003;  197 93-98
    CrossrefPubMedGoogle Scholar
  • 32 Pugh C W, Ratcliffe P J. The von Hippel-Lindau tumor suppressor, hypoxia-inducible factor-1 (HIF‐1) degradation, and cancer pathogenesis.  Semin Cancer Biol. 2003;  13 83-89
    CrossrefPubMedGoogle Scholar
  • 33 Sowter H M, Ratcliffe P J, Watson P, Greenberg A H, Harris A L. HIF‐1-dependent regulation of hypoxic induction of the cell death factors BNIP3 and NIX in human tumors.  Cancer Res. 2001;  61 6669-6673
    PubMedGoogle Scholar
  • 34 Ohh M, Park C W, Ivan M, Hoffman M A, Kim T Y, Huang L E. 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
    CrossrefPubMedGoogle Scholar
  • 35 Blancher C, Harris A L. The molecular basis of the hypoxia response pathway: tumour hypoxia as a therapy target.  Cancer Metastasis Rev. 1998;  17 187-194
    CrossrefPubMedGoogle Scholar

Dr. Detlef Pietrowski

Herzzentrum Lahr

Hohbergweg 2

77933 Lahr

Email: detlef.pietrowski@heart-lahr.com

      >