NQO1 stabilizes p53 through a distinct pathway

Abstract

Wild-type p53 is a tumor-suppressor gene that encodes a short-lived protein that, upon accumulation, induces growth arrest or apoptosis. Accumulation of p53 occurs mainly by posttranslational events that inhibit its proteosomal degradation. We have reported previously that inhibition of NAD(P)H: quinone oxidoreductase 1 (NQO1) activity by dicoumarol induces degradation of p53, indicating that NQO1 plays a role in p53 stabilization. We now have found that wild-type NQO1, but not the inactive polymorphic NQO1, can stabilize endogenous as well as transfected wild-type p53. NQO1-mediated p53 stabilization was especially prominent under induction of oxidative stress. NQO1 also partially inhibited p53 degradation mediated by the human papilloma virus E6 protein, but not when mediated by Mdm-2. Inhibitors of heat shock protein 90 (hsp90), radicicol and geldanamycin, induced degradation of p53 and suppressed p53-induced apoptosis in normal thymocytes and myeloid leukemic cells. Differences in the effectiveness of dicoumarol and hsp90 inhibitors to induce p53 degradation and suppress apoptosis in these cell types indicate that NQO1 and hsp90 stabilize p53 through different mechanisms. Our results indicate that NQO1 has a distinct role in the regulation of p53 stability, especially in response to oxidative stress. The present data on the genetic and pharmacologic regulation of the level of p53 have clinical implications for tumor development and therapy.

Figure 1

NQO1 activity-dependent stabilization of p53 protein. (A) p53 null HCT116 cells were transfected with 150 ng of pRc/CMV human wild-type p53 with either pSG5 empty vector, 2 μg of pSG5 wild-type HA NQO1, or 3 μg of polymorphic HA C609T NQO1. (B) HCT116 (−) and HCT116 stably expressing HA NQO1 (+) cells were cultured without any treatments (N. T.), γ-irradiated (γ-IR) at 6 Gy, or treated with 100 μM H₂O₂. Cell extracts were prepared from untreated cells and from cells cultured for a half-hour and 4 h post-γ-irradiation and 6 h after the addition of H₂O₂. Protein extraction and immunoblot analysis were carried out as described in Materials and Methods by using PAb 1801 monoclonal anti-p53 antibody. The blots then were stripped and reprobed with monoclonal anti-Ha for the detection of HA NQO1 and with anti-actin antibody as a control for equal protein loading in each lane.

Figure 2

NQO1 partially antagonizes papillomavirus E6 but not Mdm-2-mediated degradation of p53. (A) p53 null HCT116 cells were transfected with 150 ng of pRc/CMV human wild-type p53 without and with 500 ng of pRc/CMV-E6, 2 μg of pSG5 wild-type HA NQO1, or 3 μg of polymorphic HA C609T NQO1. (B) p53 null HCT116 cells were transfected with 150 ng of pRc/CMV human wild-type p53 without or with 300 ng of pCOC-mdm2 X2, 1.5 μg of pCGN-HA-LT, or 2 μg of pSG5 wild-type HA NQO1. (C) p53 null HCT116 cells were transfected with 150 ng of pRc/CMV human wild-type p53 without or with 800 ng of pRc/CMV-E6 or 1.5 μg of pCGN-HA-LT. Immunoblot analysis was carried out by using PAb 1801 monoclonal anti-p53 antibody. The blots then were stripped and reprobed with monoclonal anti-Mdm-2 and anti-HA for the detection of HA NQO1 and HA LT, and anti-actin antibody as a control for equal protein loading in each lane.

Figure 3

LT protects p53 protein from degradation induced by dicoumarol. p53 null HCT116 cells were transfected with 150 ng of pRc/CMV human wild-type p53 without or with 300 ng of pCOC-mdm2 X2 or 1.5 μg of pCGN-HA-LT. Transfected cells (24 h posttransfection) then were cultured for 5 h without or with 300 μM dicoumarol.

Figure 4

Induction of wild-type and mutant p53 degradation by hsp90 inhibitors. (A) M1-t-p53 myeloid leukemic cells were cultured for 6 h at 32°C or 37°C without or with different concentrations of radicicol. (B) Cells were cultured at 32°C for 6 h or 2 h without (−) or with (+) 1 μM radicicol. Cells also were preincubated for 4 h at 32°C and then cultured with 1 μM radicicol or 1 μM geldanamycin (geldan.) for 2 h (2 h*). Immunoblot analysis was carried out by using PAb 240 monoclonal anti-p53 antibody and hamster anti-mouse Bcl-2.

Figure 5

Suppression of wild-type p53-mediated apoptosis by hsp90 inhibitors. M1-t-p53 cells were cultured at 32°C without or with different concentrations of radicicol or geldanamycin. Cell viability was determined 23 h after culture at 32°C.

Figure 6

Degradation of p53 in 7-M12 myeloid leukemic cells and normal thymocytes by dicoumarol and radicicol. (A) 7-M12 cells either were not treated (none), γ-irradiated at 0.4 Gy, or treated with 2.1 μM doxorubicin (Dox.). Cells were cultured for 2 h without (−) or with 100 μM dicoumarol (Dic.) or 5 μM radicicol. (B) Normal thymocytes either were not treated (none) or γ-irradiated at 0.4 Gy and cultured without (−) or with 200 μM dicoumarol (Dic.), 5 μM radicicol, or 5 μM geldanamycin (Geldan.). Cell extracts were prepared after 2 h, and apoptosis was determined after 5 h. Immunoblot analysis was carried out by using PAb 240 monoclonal anti-p53 antibody and rabbit anti-IκB.

Figure 7

Model of the role of NQO1 in p53 accumulation. The model assumes that the level of p53, depicted as a triangle, is oppositely regulated by Mdm-2 and by NQO1. These pathways function independently, with a possible cross-talk between them mediated by production of ROS after γ-irradiation (IR). ROS increases the level of NQO1, which, in turn, reduces ROS by its oxidoreductase activity in a negative feedback loop. ↓, pathway; ⊥, inhibition of a pathway.