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Fas-Mediated Apoptosis Is Dependent on Wild-Type p53 Status in Human Cancer Cells Expressing a Temperature-Sensitive p53 Mutant Alanine-143¹

Experimental Therapeutics Program, Division of Medical Oncology, Herbert Irving Comprehensive Cancer Center, College of Physicians and Surgeons of Columbia University, New York, New York 10032

The p53 mutant 143Ala is a human temperature-sensitive mutant with two conformational states.To definitively determine whether the Fas signal transduction pathway and the function of the pathway are dependent on p53 status, we have established stable transfectants of p53 mutant 143Ala in two human cancer cell lines: H1299 (lung cancer line) and PC-3 (prostate cancer line), the native state of which contains null p53 status and can grow at 37°C and 32.5°C. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and cell cycle analysis showed inhibition of the growth of cells overexpressing p53 mutant 143Ala in the wild-type p53 form at 32.5°C because of induction of G0/G1 arrest. Transfected cells had increased protein expression of p21, Fas, and MDM2 at the wild-type p53 conformation at 32.5°C, but not in the mutant p53 form at 37°C. However, there was no change in protein expression of FADD, FAP-1, Bcl-2, or Bax at 32.5 or 37°C. Assays for apoptosis demonstrated that anti-Fas antibody CH-11 and FasL induced apoptosis only in cells that overexpress p53 mutant 143Ala at 32.5°C with the wild-type p53 form. Both caspase-3 and caspase-8 activities were increased by anti-Fas antibody CH-11 only in cells at 32.5°C with wild-type p53. Our results demonstrated that Fas-mediated apoptosis in H1299 and PC-3 cells expressing p53 mutant 143Ala occurred only with the wild-type p53 phenotype. These results support the hypothesis that Fas-mediated apoptosis is dependent, at least partially, on the presence of a functional wild-type p53 state. This model may be a useful tool for dissecting the specific interactions between wild-type p53 and the Fas signal transduction pathway in human cancer cells.

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