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J. Biol. Chem., Vol. 280, Issue 10, 9336-9344, March 11, 2005

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Hypoxia-inducible Factor-1-dependent Overexpression of Myeloid Cell Factor-1 Protects Hypoxic Cells against tert-Butyl Hydroperoxide-induced Apoptosis^*

Jean-Pascal Piret, Emmanuel Minet¶, Jean-Philippe Cosse, Noelle Ninane, Christophe Debacq||, Martine Raes, and Carine Michiels, A Senior Research Associate of FNRS**

From the Laboratory of Biochemistry and Cellular Biology, University of Namur, 61 Rue de Bruxelles, 5000 Namur, Belgium and ^||Molecular and Cellular Biology, FUSAGx, 5030 Gembloux, Belgium

Increased levels of Mcl-1 (myeloid cell factor-1) have been reported in several cancers, suggesting an important role played by Mcl-1 in cancer cell survival. Mcl-1 is an anti-apoptotic protein shown to delay or block apoptosis. In this work, using semiquantitative immunofluorescence, real-time PCR, and RNase protection assay, an increase in Mcl-1 expression was detected in hepatoma HepG2 cells incubated under hypoxia or in the presence of cobalt chloride. Through analysis of the Mcl-1 promoter sequence, a putative HIF-1 (hypoxiainducible factor-1) binding site was identified. A Mcl-1 promoter fragment containing this hypoxia-responsive element was able to bind HIF-1 in vitro. It also induced hypoxia-dependent transcription of a luciferase reporter gene, which was suppressed by anti-HIF-1 short interfering RNA. Finally, overexpression of Mcl-1 protected HepG2 cells against apoptosis induced by tert-butyl hydroperoxide as shown by inhibition of caspase-3 activation and DNA fragmentation. All these data suggest a potential anti-apoptotic role of HIF-1 that could protect cells against apoptosis under hypoxia by overexpression of the Mcl-1 protein.

Received for publication, October 19, 2004 , and in revised form, December 17, 2004.

^* This report presents results of the Belgian Program on Interuniversity Poles of attraction initiated by the Belgian State, Prime Minister's Office, Science Policy Programming. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

A fellow of Fonds pour la Recherche dans l'Industrie et l'Agriculture, Belgium.

^¶ A Senior Research Assistant of Fonds National de la Recherche Scientifique (FNRS), Belgium.

^** To whom correspondence should be addressed. Tel.: 32-81-72-41-31; Fax: 32-81-72-41-35; E-mail: carine.michiels{at}fundp.ac.be.

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