Effect of p53 expression on ubiquitin-mediated degradation of HIF-1α. (A) Interaction of p53 with HIF-1α. Lysates of p53+/+ or p53−/− HCT116 cells exposed to 1%O2 for 8 hr were immunoprecipitated with either anti-p53 antibody or isotype control antibody (C) and the resultant immune complexes were subjected to immunoblot analysis with anti-HIF-1α monoclonal antibody. (B) Differential ubiquitination of HIF-1α in hypoxic p53+/+ and p53−/− HCT116 cells. Cells were cotransfected with pCMVβgal and pCEP4/HIF-1α with either MT107/His6-Ub or empty vector (MT107), and cultured in 1%O2 for 4 hr in the presence of 50 μm MG132. Aliquots of whole-cell extract (WCE) or His-tagged proteins purified from whole-cell lysates (His-Ub) were subjected to immunoblot analysis with anti-HIF-1β antibody. (C) Effect of p53 expression on HIF-1α protein levels in hypoxic ts20TGR and H38-5 cells. Cells transfected with pCMV–p53 or pCMVβgal were maintained at either 35°C or 39°C for 8 hr and exposed to 1%O2 for an additional 8 hr at their respective temperatures. Whole-cell lysates were subjected to immunoblot analysis with anti-HIF-1α or anti-p53 antibodies. (D) Effect of p53 on complex formation between HIF-1α and Mdm2. Lysates of p53−/− HCT116 cells transfected with either pCMV–p53 or empty vector and transferred to 1%O2 for 6 hr were immunoprecipitated with anti-Mdm2 or isotype control antibody, and the resulting immune complexes were subjected to immunoblot assays using an antibody against HIF-1α. (E) Effect of wild-type p53, p53ΔI, or p53Gln22,Ser23 on expression of HIF-1α in response to hypoxia. p53−/− HCT116 cells transfected with pCMVβgal and either pCMV–p53, pCB6 + p53ΔI, pCMV–p53Gln22,Ser23, or empty vector were exposed to 1%O2 for 8 hr. Whole-cell lysates were subjected to immunoblot analysis with anti-HIF-1α or anti-Mdm2 antibodies. (F) Effect of dominant–negative (RING finger) mutants of Mdm2 on hypoxia-induced expression of HIF-1α. p53+/+ and p53−/− HCT116 cells transfected with vectors encoding human Mdm2 (1–440) (pCHDM1–440), Mdm2 (464Ala) (pCHDM464Ala), or pCMVβgal were exposed to 1%O2 for 8 hr. Whole-cell lysates were subjected to immunoblot analysis with anti-HIF-1α, anti-p53, or anti-Mdm2 antibodies. (G) Effect of dominant–negative (RING finger deletion mutant) Mdm2 on p53-mediated inhibition of HIF-1α expression in ts20TGR and H38-5 cells. Cells cotransfected with pCMV–p53 and either pCHDM1–440 or empty vector were maintained at 39°C for 12 hr and then exposed to 20%or 1%O2 for an additional 8 hr at 39°C. Whole-cell lysates were subjected to anti-HIF-1α immunoblot analysis.