Fig. 4.

Regulation of degradation of WT p53 and the hot-spot p53 R273H mutant. (A) p53-null HCT116 cells were transfected with pRc/CMV human WT p53 or p53 R273H mutant. After transfection (24 h), cells were cultured for 5 h without (–) or with 300 μM dicoumarol or 60 μM curcumin. Cell extracts were prepared, and the p53 level was determined. In vitro [³⁵S]-labeled WT p53 (B) or p53 R273H mutant (C) was incubated alone (–) or together with (+) recombinant NQO1 in the presence of 1 mM NADH. p53 was immunoprecipitated with mouse anti-human p53 Ab (Pab 1801) (IP: p53). The beads were washed without (–) or with (+)50 μM dicoumarol or 40 μM curcumin before elution and separation on SDS/PAGE. p53 was detected by autoradiography, and NQO1 was detected by immunoblotting with goat anti-NQO1 Ab (IB: NQO1). (D) A31N-ts20 cells were transfected at 32°C with pRc/CMV human WT p53 or p53 R273H mutant without or with pSUPER-NQO1, and 4 h later cells were transferred to 39°C. The level of WT p53 and p53 R273H mutant proteins was determined 24 h after transfection by immunoblotting with anti-human p53 (Pab 1801) Ab.