Stabilization of p53 by CP-31398 inhibits ubiquitination without altering phosphorylation at serine 15 or 20 or MDM2 binding

Abstract

CP-31398, a styrylquinazoline, emerged from a high throughput screen for therapeutic agents that restore a wild-type-associated epitope (monoclonal antibody 1620) on the DNA-binding domain of the p53 protein. We found that CP-31398 can not only restore p53 function in mutant p53-expressing cells but also significantly increase the protein level and promote the activity of wild-type p53 in multiple human cell lines, including ATM-null cells. Cells treated with CP-31398 undergo either cell cycle arrest or apoptosis. Further investigation showed that CP-31398 blocks the ubiquitination and degradation of p53 but not in human papillomavirus E6-expressing cells. Of note, CP-31398 does not block the physical association between p53 and MDM2 in vivo. Moreover, unlike the DNA-damaging agent adriamycin, which induces strong phosphorylation of p53 on serines 15 and 20, CP-31398 exposure leads to no measurable phosphorylation on these sites. We found that CP-31398 could also stabilize exogenous p53 in p53 mutant, wild-type, and p53-null human cells, even in MDM2-null p53(-/-) mouse embryonic fibroblasts. Our results suggest a model wherein CP-31398-mediated stabilization of p53 may result from reduced ubiquitination, leading to high levels of transcriptionally active p53. Further understanding of this mechanism may lead to novel strategies for p53 stabilization and tumor suppression in cancers, even those with absent ARF or high MDM2 expression.

FIG. 1.

CP-31398 stabilizes wild-type p53. (A and B) H460 cells, as well as ATM-positive 2184 and ATM-null 3189 lymphoblasts, were treated with CP-31398 at a final concentration of 15 μg/ml. Adriamycin (ADR) was used as a DNA damage control at a concentration of 0.2 μg/ml. Cells were collected at the indicated time points and subjected to SDS-PAGE. p53 and MDM2 were detected by anti-p53 (Ab-2; Calbiochem) and anti-MDM2 (Ab-1; Calbiochem) antibodies, respectively. Actin was probed as a loading control (data not shown). (C) Northern blot of the total RNA from CP-31398 (CP)-treated H460 cells with the probes of MDM2 and p53, respectively. Lane C, control.

FIG. 2.

CP-31398 does not phosphorylate p53 at ser15 or ser20. (A) Adriamycin (ADR) or CP-31398 was added to H460 cell culture at various concentrations and incubated for 4 h. Cell lysates were subjected to SDS-PAGE and probed with anti-p53 (Ab-2) and anti-phospho-p53 antibodies against ser15, ser20, ser46, and ser392, respectively. (B) H460 cells were treated with ADR at a concentration of 0.2 μg/ml or CP-31398 at a concentration of 15 μg/ml. Cells were lysed at different time points after treatment and subjected to SDS-PAGE and immunoblotting with anti-phospho-ser20 of p53.

FIG. 3.

CP-31398 does not prevent the binding of MDM2 to p53. (A) H460 cells were treated with adriamycin (ADR) or CP-31398 (CP) for 4 h. p53 was immunoprecipitated (IP) with anti-p53 (FL-393; Santa Cruz Biotech). The immunoprecipitated complexes were subjected to SDS-PAGE and probed with anti-p53 (Ab-2; Calbiochem) and anti-MDM2 (Ab-1; Calbiochem) antibodies. (B) H460 cells were infected with Ad/His-p53 and treated with adriamycin or CP-31398. p53 was precipitated with Ni-nitrilotriacetic acid resin prior to SDS-PAGE. MDM2 and p53 were probed with the same antibodies as in panel A. WB, Western blotting; C, control.

FIG. 4.

CP-31398 inhibits the ubiquitination of p53. (A) H460 and PA-1 cells were treated with CP-31398 (CP) at a concentration of 15 μg/ml for 1 h, and then ALLN was added at a concentration of 50 μM. Four hours later, cells were lysed for Western blotting with DO-1 against p53. (B) Dose-dependent inhibition of p53 ubiquitination by CP-31398. (C) Same H460 cell lysates as in panel A, probed with an anti-ubiquitin antibody (Santa Cruz). (D) Immunoblots against pRB, smad2/3, and cyclin E in H460 cells after treatment with adriamycin (ADR) or CP-31398. Lanes C, control.

FIG. 5.

CP-31398 (CP) does not block p53 degradation by HPV E6. H460 and AdH460 Eb-infected cells were treated with CP-31398 at a concentration of 15 μg/ml for 1 h, and then ALLN was added at a concentration of 50 μM. Four hours later, cells were lysed for Western blotting with DO-1 against p53.

FIG. 6.

CP-31398 stabilizes exogenous p53 by inhibiting its ubiquitination. (A) H460 cells were infected with Ad/p53 at multiplicities of infection (MOI) of 2.5, 5, and 10 for 12 h. CP-31398 (CP) was then added to the medium at a concentration of 15 μg/ml and incubated for 8 h. Cells were lysed for SDS-PAGE. p53 was probed with Ab-2. Also shown are p53^−/− HCT116 cells and p53^−/− MDM2^−/− MEF cells infected with Ad/p53 (MOI-5) and treated with CP-31398. C, control. (B) H460 cells were infected with Ad/p53 at a MOI of 5 (second panel). Twelve hours after the infection, CP-31398 was added and incubated for 1 h and then ALLN was added at a concentration of 50 μg/ml. Four hours later, cells were lysed for Western blotting by DO-1 against p53. Also shown are results performed in HCT116/p53^−/− cells (third panel) infected and treated as described for the H460 cells in the second panel.

FIG. 7.

CP-31398 differentially induces p53 target gene expression and enhances sensitivity to TRAIL. (A) Cells were treated with CP-31398 (CP) or adriamycin (ADR) for 8 h and probed with antibodies as indicated. C, control. (B) HCT116/p53^+/+ and HCT116/p53^−/− cells were treated with CP-31398 or adriamycin for 4 h, then TRAIL was added at a final concentration of 20 ng/ml, and cells were incubated for another 4 h. Cells were collected for active caspase 3 staining, and positive cells were counted by fluorescence-activated cell sorter analysis.

FIG. 8.

CP-31398 promotes p53 nuclear accumulation. H460 cells were infected with Ad/GFP-p53. Twelve hours after the infection, CP-31398 was added at a concentration of 15 μg/ml and incubated for 6 h. Cells were photographed under a phase-contrast laser microscope.