DNA Damage Is a Prerequisite for p53-Mediated Proteasomal Degradation of HIF-1α in Hypoxic Cells and Downregulation of the Hypoxia Marker Carbonic Anhydrase IX

Abstract

We investigated the relationship between the tumor suppressor p53 and the hypoxia-inducible factor-1 (HIF-1)-dependent expression of the hypoxia marker, carbonic anhydrase IX (CAIX). MCF-7 (wt p53) and Saos-2 (p53-null) cells displayed similar induction of CAIX expression and CA9 promoter activity under hypoxic conditions. Activation of p53 by the DNA damaging agent mitomycin C (MC) was accompanied by a potent repression of CAIX expression and the CA9 promoter in MCF-7 but not in Saos-2 cells. The activated p53 mediated increased proteasomal degradation of HIF-1α protein, resulting in considerably lower steady-state levels of HIF-1α protein in hypoxic MCF-7 cells but not in Saos-2 cells. Overexpression of HIF-1α relieved the MC-induced repression in MCF-7 cells, confirming regulation at the HIF-1α level. Similarly, CA9 promoter activity was downregulated by MC in HCT 116 p53^+/+ but not the isogenic p53^−/− cells. Activated p53 decreased HIF-1α protein levels by accelerated proteasome-dependent degradation without affecting significantly HIF-1α transcription. In summary, our results demonstrate that the presence of wtp53 under hypoxic conditions has an insignificant effect on the stabilization of HIF-1α protein and HIF-1-dependent expression of CAIX. However, upon activation by DNA damage, wt p53 mediates an accelerated degradation of HIF-1α protein, resulting in reduced activation of CA9 transcription and, correspondingly, decreased levels of CAIX protein. A model outlining the quantitative relationship between p53, HIF-1α, and CAIX is presented.

This study was supported by a grant from the California Cancer Research Program (00-00789V-20240) and The Avon Foundation. M.I.L. was supported by funds from the National Cancer Institute under contract N01-CO-56 000.

We thank J. Pastorek and S. Pastorekova for the CAIX antibody. We also thank B. Vogelstein for the PG13-Luc construct and for the human colon cancer cell lines HCT 116 p53^+/+ and HCT 116 p53^−/−. The L22Q/W23S mutant p53 cDNA was a kind gift from J. K. Nyborg, and the pCEP-HIF-1α construct was kindly provided by D. Theodorescu. We also acknowledge K. Flick for assistance with real-time PCR.