Characterization of structural p53 mutants which show selective defects in apoptosis but not cell cycle arrest

Abstract

Suppression of tumor cell growth by p53 results from the activation of both apoptosis and cell cycle arrest, functions which have been shown to be separable activities of p53. We have characterized a series of p53 mutants with amino acid substitutions at residue 175 and show that these mutants fall into one of three classes: class I, which is essentially wild type for apoptotic and cell cycle arrest functions; class II, which retains cell cycle arrest activity but is impaired in the induction of apoptosis; and class III, which is defective in both activities. Several residue 175 mutants which retain cell cycle arrest function have been detected in cancers, and we show that these have lost apoptotic function. Furthermore, several class II mutants have been found to be temperature sensitive for apoptotic activity while showing constitutive cell cycle arrest function. Taken together, these mutants comprise an excellent system with which to investigate the biochemical nature of p53-mediated apoptosis, the function of principal importance in tumor suppression. All of the mutants that showed loss of apoptotic function also showed defects in the activation of promoters from the potential apoptotic targets Bax and the insulin-like growth factor-binding protein 3 gene (IGF-BP3), and a correlation between full apoptotic activity and activation of both of these promoters was also seen with the temperature-sensitive mutants. However, a role for additional apoptotic activities of p53 was suggested by the observation that some mutants retained significant apoptotic function despite being impaired in the activation of Bax- and IGF-BP3-derived promoters. In contrast to the case of transcriptional activation, a perfect correlation between transcriptional repression of the c-fos promoter and the ability to induce apoptosis was seen, although the observation that Bax expression induced a similar repression of transcription from this promoter suggests that this may be a consequence, rather than a cause, of apoptotic death.

FIG. 1

Induction of cell cycle arrest and apoptosis by p53 proteins. (A) Flow cytometry analyses of Saos-2 cells transiently transfected with wild-type p53 and two p53 mutants, 175Pro and 175Asp. Cell cycle arrest function is indicated as the percentage of cells in the G₁ phase of the cell cycle relative to those in the S and G₂/M phases. Apoptotic rates are given as the percentage of cells with sub-G₁ DNA content above that observed in the untransfected population. (B) Representation of the cell cycle arrest and apoptotic functions of a series of mutants with alterations at codon 175 of p53.

FIG. 1

Induction of cell cycle arrest and apoptosis by p53 proteins. (A) Flow cytometry analyses of Saos-2 cells transiently transfected with wild-type p53 and two p53 mutants, 175Pro and 175Asp. Cell cycle arrest function is indicated as the percentage of cells in the G₁ phase of the cell cycle relative to those in the S and G₂/M phases. Apoptotic rates are given as the percentage of cells with sub-G₁ DNA content above that observed in the untransfected population. (B) Representation of the cell cycle arrest and apoptotic functions of a series of mutants with alterations at codon 175 of p53.

FIG. 2

Western blot analyses of lysates from Saos-2 cells which were transfected with p53 constructs as indicated. The nitrocellulose membrane from one polyacrylamide gel was probed with both p53- and p21^Waf1/Cip1-specific monoclonal antibodies. The figure shown is representative of three independent experiments.

FIG. 3

Transcriptional activation by wild-type p53 and the indicated mutants of the p53-responsive promoter plasmids Bax-luc and boxB-luc (IGF-BP3). Assays were carried out following transfection of 5 μg of reporter plasmid and 50 ng of p53 expression plasmid into Saos-2 cells. The values shown are fold trans-activation of the reporters relative to values observed for cells transfected with vector alone.

FIG. 4

Transcriptional repression of the human c-fos promoter. (A) Wild-type p53 and the indicated mutants were cotransfected into Saos-2 cells together with the reporter plasmid fos-luc. The percent luciferase activity caused by each p53 protein was calculated relative to that observed in cells transfected with the empty CMV expression plasmid alone. (B) Comparison of the effects of expression of wild-type p53, the p53 mutants 175Cys, 175Asn, 175Thr, and 175Tyr and Bax on the reporter constructs fos-luc and pJ4Ωβgal. Saos-2 cells were transfected with 5 μg of each reporter construct together with either 5 μg of CMV-driven wild-type or mutant p53 or 1 μg of CMV-driven Bax.

FIG. 5

Induction of apoptosis by wild-type p53 and the indicated mutants at 37°C and 32°C. Saos-2 cells were transfected with 5 μg of p53 expression plasmid, left for 16 h, washed, and then placed at the indicated temperatures for 24 h before being harvested.

FIG. 6

Transcriptional activation of the p53-responsive reporter plasmids Bax-luc and boxB-luc (IGF-BP3) by wild-type p53 and the indicated mutants at 37°C and 32°C. Saos-2 cells were transfected with 5 μg of reporter plasmid and 50 ng of the indicated p53 expression plasmid. Following transfection, the cells were incubated at 37°C for 16 h, washed, and moved to the appropriate temperature for 24 h before being harvested for luciferase and β-galactosidase assays. The values shown represent fold trans-activation relative to cells transfected with the empty CMV expression plasmid alone.