CP-31398, a putative p53-stabilizing molecule tested in mammalian cells and in yeast for its effects on p53 transcriptional activity

Abstract

Background: CP-31398 is a small molecule that has been reported to stabilize the DNA-binding core domain of the human tumor suppressor protein p53 in vitro. The compound was also reported to function as a potential anti-cancer drug by rescuing the DNA-binding activity and, consequently, the transcription activation function of mutant p53 protein in mammalian tissue culture cells and in mice.

Results: We performed a series of gene expression experiments to test the activity of CP-31398 in yeast and in human cell cultures. With these cell-based assays, we were unable to detect any specific stimulation of mutant p53 activity by this compound. Concentrations of CP-31398 that were reported to be active in the published work were highly toxic to the human H1299 lung carcinoma and Saos-2 cell lines in our experiments.

Conclusion: In our experiments, the small molecule CP-31398 was unable to reactivate mutant p53 protein. The results of our in vivo experiments are in agreement with the recently published biochemical analysis of CP-31398 showing that this molecule does not bind p53 as previously claimed, but intercalates into DNA.

Figure 1

Human p53 protein activates transcription from a reporter construct in Saccharomyces cerevisiae. (A) Schematic representation of our yeast reporter construct integrated into our yeast strain. The black circle represents a single p53 responsive element from the human p21 promoter. (B) β-galactosidase assay to measure activation of the lacZ reporter gene. Wild type p53 and the indicated point mutant variants were transformed into the p53 responsive reporter strain and β-galactosidase activity in solution was determined. The activity of wild type p53 was arbitrarily set to 100%. p53R282W and p53V173A showed about 40% of activation compared to wild type p53. No activation of the reporter gene was detected in yeast cells containing the other point mutant variants. Average and standard deviation were determined from three independent experiments. (C) Growth on selective plates containing 20 mM 3-AT depends on expression of the HIS3 reporter gene and correlates with the activation of the lacZ reporter gene. Control plates consist of standard drop-out plates lacking the corresponding growth marker without 3-AT. Growth under selective conditions was dependent on activation of the p53 dependent reporter gene.

Figure 2

Treatment with the p53 stabilizing compound CP-31398 shows no effect on reporter gene activity in yeast. Yeast cells expressing wild type p53 (lanes 1–5), p53V173A (lanes 6–10) or empty vector (-, lanes 11–15, white bars) were incubated with the concentrations of CP-31398 indicated (0–500 μg/ml) and expression of β-galactosidase was determined. β-galactosidase activity of wild type p53 without CP-31398 treatment was arbitrarily set to 100%. Yeast cells were treated with CP-31398 for 16 hours.

Figure 3

Treatment of H1299 lung carcinoma cells with CP-31398 provokes massive cell death and p53 independent decline of luciferase reporter gene activity. (A) H1299 cells were transfected with expression constructs for wild type p53 (lanes 1–3) and p53V173A (lanes 4–6). All the samples were cotransfected with a p53-responsive luciferase reporter (p21 luciferase, containing a single p53 responsive p53 binding site from the human p21 promoter, termed WWP-luc, see material and methods) and a constitutive reference β-galactosidase construct (CMV-lacZ) for normalization. These cells were subsequently incubated with 0, 10, 15 μg/ml CP-31398 respectively and relative luciferase activities were determined. (B) H1299 cells were transfected with an expression construct for the synthetic activator GAL4-VP16. All samples were cotransfected with a gal4p responsive luciferase reporter (UAS_G luciferase) and a reference β-galactosidase plasmid (CMV-lacZ) for normalization. The control cells were transfected with CMV-lacZ and UAS_G luciferase only. These cells were subsequently incubated with 0, 10 and 15 μg/ml CP-31398 and relative luciferase activities were determined. The cells were treated with CP-31398 for 16 hours.

Figure 4

Treatment of HeLa cervical carcinoma cells with CP-31398 leads to p53 dependent induction of the luciferase reporter. HeLa cells were transfected with a p53 responsive reporter gene (WWP-luc) and a reference β-galactosidase plasmid (CMV-lacZ) for normalization. Control cells were transfected with CMV-lacZ alone. The cells were subsequently incubated with CP-31398 (0–10 μg/ml) and relative luciferase activities were determined. Cells were treated for 16 hours.

Figure 5

Western Blot analysis of HeLa cells treated with CP-31398 and daunorubicin. (A) HeLa cells were treated with increasing concentrations of CP-31398 and protein extracts were subjected to SDS-PAGE and subsequent detection with an anti-p53 antibody (DO-1). (B) HeLa cells were treated with the established p53 inducing agent daunorubicin. Protein extracts were subjected to SDS-PAGE and subsequent detection with an anti-p53 antibody (DO-1). Expression of actin is detected as a loading control in experiments 5A and B.