DNA damage is a prerequisite for p53-mediated proteasomal degradation of HIF-1alpha 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-1alpha protein, resulting in considerably lower steady-state levels of HIF-1alpha protein in hypoxic MCF-7 cells but not in Saos-2 cells. Overexpression of HIF-1alpha relieved the MC-induced repression in MCF-7 cells, confirming regulation at the HIF-1alpha level. Similarly, CA9 promoter activity was downregulated by MC in HCT 116 p53(+/+) but not the isogenic p53(-/-) cells. Activated p53 decreased HIF-1alpha protein levels by accelerated proteasome-dependent degradation without affecting significantly HIF-1alpha transcription. In summary, our results demonstrate that the presence of wtp53 under hypoxic conditions has an insignificant effect on the stabilization of HIF-1alpha protein and HIF-1-dependent expression of CAIX. However, upon activation by DNA damage, wt p53 mediates an accelerated degradation of HIF-1alpha protein, resulting in reduced activation of CA9 transcription and, correspondingly, decreased levels of CAIX protein. A model outlining the quantitative relationship between p53, HIF-1alpha, and CAIX is presented.

FIG. 1.

(A) Effect of MC treatment on CAIX expression in wt p53 MCF-7 and p53-null Saos-2 cells. Cells were seeded at 40,000/cm² (1% O₂ and DFO) or 160,000/cm² (density), allowed to attach for 5 h, pretreated with MC for 2 h, and exposed to 1% O₂ or 100 μM DFO for 24 h in the presence of MC. Total protein lysates (40 μg) were tested for CAIX, total p53 (DO1), phospho p53 (S15 and S20), and β-actin by Western blotting. Total protein yield is expressed as percentages of control. (B) p53 transactivation effects in MCF-7 and Saos-2 cells. Cells were cotransfected with the PG13 (containing 13 copies of the p53 response element and the firefly luciferase gene) and pRL-tk (expressing Renilla luciferase) plasmids. After 16 h the transfectants were trypsinized, seeded at 40,000 cells/cm², and treated as in panel A. p53 activity is expressed as the ratio of firefly activity to Renilla activity, and each of the bars represents the mean value (x ± SD) from at least three individual experiments.

FIG. 2.

The effect of MC treatment on CA9 promoter activity in MCF-7 and Saos-2 cells. Cells were cotransfected with the [−46, +14] CA9 reporter construct (containing firefly luciferase gene) and pRL-tk for 16 h, trypsinized, seeded at 40,000/cm², allowed to attach for 5 h, pretreated with MC for 2 h, and exposed to 0.5% O₂ or 100 μM DFO for 24 h in the presence of MC. CA9 promoter activity was expressed as the ratio of firefly activity to Renilla activity and set as 1 in control cells (21% O₂). Activities under various treatments are expressed as the level of induction relative to the control, and each of the bars represents the mean value (x ± SD) from at least three individual experiments.

FIG. 3.

The effect of MC treatment on PR1 and HRE activity in MCF-7 cells. Cells were cotransfected with 3 × PR1-Luc or 3 × HRE-Luc and pRL-tk as in Fig. 2. Promoter activities are expressed as in Fig. 2.

FIG. 4.

(A) The effect of MC treatment on HIF-1α levels in MCF-7 and Saos-2 cells. Cells were seeded at 40,000/cm² and treated as described in Fig. 1A. Total protein lysates (40 μg) were tested for HIF-1α and β-actin by Western blotting. (B) Cotransfected HIF-1α rescues CA9 promoter activation in MC-treated MCF-7 cells. Cells were cotransfected with the [−173, +31] CA9 reporter construct, pRL-tk, and 500 ng of pCEP4 (EV) or pCEP-HIF-1α (HIF-1α) as in Fig. 2. CA9 promoter activity in the presence of EV in control cells (21% O₂) was set as 1, and the rest is expressed as the level of induction as in Fig. 2.

FIG. 5.

(A) RT-PCR analysis of HIF-1α and CA9 transcription in MC-treated MCF-7 cells. Cells were seeded and treated as in Fig. 1A and were harvested 16 h later, and total RNA was isolated, reverse-transcribed, and amplified. NC, negative control. (B) Real-time PCR analysis of HIF-1α transcription in MC-treated MCF-7 cells. The relative HIF-1α transcription for each sample was expressed in arbitrary units as the ratio of HIF-1α and β-actin values. (C) MC-activated p53 accelerates proteasome-dependent degradation of HIF-1α in MCF-7 cells. Cells were pretreated with MC and LLnV for 90 min, exposed to 1% hypoxia for 18 h in the presence of both reagents, and lysed in lysis buffer I. Insoluble pellet was solubilized in lysis buffer II, and both fractions were tested for HIF-1α, phospho-p53 (S15), and β-actin by Western blotting.

FIG. 6.

Effect of MC treatment on CA9 promoter activity in HCT 116 p53^+/+ and p53^−/− cells. Cells were cotransfected with the [−173, +31] CA9 reporter construct and pRL-tk as in Fig. 2. CA9 promoter activity in p53^+/+ cells under normoxic conditions was set as 1, and the rest is expressed as the level of induction as in Fig. 2.

FIG. 7.

Effect of MC treatment on HIF-1 α levels in HCT 116 p53^+/+ and p53^−/− cells. Total protein lysates were prepared as in Fig. 4A and tested for HIF-1α, total p53 (DO1), phospho-p53 (S15), and β-actin by Western blotting.

FIG. 8.

The effect of cotransfected wt and mutant p53 on CA9 promoter activity in HCT 116 p53^−/− cells. Cells were cotransfected with the [−173, +31] CA9 reporter construct, pRL-tk, and 10 ng of pCEP4 (EV) or a construct expressing wt or mutant p53 as in Fig. 2. CA9 promoter activity in the presence of EV under normoxic conditions was set as 1, and the rest is expressed as the level of induction relative to the control.

FIG. 9.

(A) Quantitative relationship between wt p53, HIF-1α, and CAIX under various conditions. The symbols + and − represent relative amounts of protein. (B) Outline of the proposed mechanism responsible for downregulation of HIF-1α protein by activated wt p53. Final steady-state levels of HIF-1 α protein reflect relative efficiency of the proteasome-mediated degradation of HIF-1α under various conditions. Under normoxia, there is no p53 stabilization and activation; HIF-1α becomes hydroxylated, ubiqitylated, and rapidly degraded. Hypoxia inhibits hydroxylation, and therefore large amounts of HIF-1α escape degradation. A small amount of activated p53, induced by strong hypoxia, is unable to interfere significantly with stabilization of HIF-1α and function of HIF-1. Under hypoxic conditions in the presence of DNA-damaging agent high levels of stabilized and activated p53 are induced. This p53 is capable of promoting proteasome-mediated degradation of HIF-1α that results in lower steady-state levels of HIF-1α and lowered HIF-1 activity.