Wild-type p53 is a tumor suppressor gene which can activate or repress transcription, as well as induce apoptosis. The human p53 proline-rich domain localized between amino acids 64 and 92 has been reported to be necessary for efficient growth suppression. This study shows that this property mainly results from impaired apoptotic activity. Although deletion of the proline-rich domain does not affect transactivation of several promoters, such as WAF1, MDM2 and BAX, it does alter transcriptional repression, reactive oxygen species production and sequence-specific transactivation of the PIG3 gene, and these are activities which affect apoptosis. Whereas gel retardation assays revealed that this domain did not alter in vitro the specific binding to the p53-responsive element of PIG3, this domain plays a critical role in transactivation from a synthetic promoter containing this element. To explain this discrepancy, evidence is given for a prolinerich domain-mediated cellular activation of p53 DNA binding.

The wild-type protein product of the p53 tumor suppressor gene can activate transcription of genes which are involved in mediating either growth arrest, e.g. WAF1 or apoptotis, e.g. BAX and PIG3. Additionally, p53 can repress a variety of promoters, which, in turn, may be responsible for the functional activities exhibited by p53. This study shows that the Q22, S23 double mutation, which is known to inactivate a p53 transactivation subdomain located within the initial 40 residues of the protein, while abrogating transactivation from the WAF1 promoter, only attenuates apoptosis triggering, transactivation from other p53-responsive promoters and repression of promoters by p53. The Q53, S54 double mutation, which inactivates another p53 transactivation subdomain situated between amino acids 43 and 73 results in attenuation of all of the aforementioned p53 activities. In contrast to the Q22, S23 double mutation, this latter mutation set does not alter mdm-2-mediated inhibition and degradation of p53. Finally, mutation of all four residues results in complete abrogation of every p53 activity mentioned above.

Patients with tienilic acid hepatitis exhibit autoantibodies that recognize unalkylated cytochrome P450 2C9 in humans but recognize 2C11 in rats. Our aim was to determine whether the immune reaction is also directed against neoantigens. Rats were treated with tienilic acid and hepatocytes were isolated. Immunoprecipitation, immunoblotting, and flow cytometry experiments were performed with an anti-tienilic acid or an anti-cytochrome P450 2C11 antibody. Cytochrome P450 2C11 was the main microsomal or plasma membrane protein that was alkylated by tienilic acid. Inhibitors of vesicular transport decreased flow cytometric recognition of both unalkylated and tienilic acid-alkylated cytochrome P450 2C11 on the plasma membrane of cultured hepatocytes. Tienilic acid hepatitis sera that were preadsorbed on microsomes from untreated rats (to remove autoantibodies), poorly recognized untreated hepatocytes in flow cytometry experiments, but better recognized tienilic acid-treated hepatocytes. This recognition was decreased by adsorption with tienilic acid or by preexposure to the anti-tienilic acid or the anti-cytochrome P450 2C11 antibody. We conclude that cytochrome P450 2C11 is alkylated by tienilic acid and follows a vesicular route to the plasma membrane. Tienilic acid hepatitis sera contain antibodies against this tienilic acid adduct, in addition to the previously described anticytochrome P450 autoantibodies. ( J. Clin. Invest. 1996. 98: 1471-1480.)

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The clinical potential of the p53 tumor suppressor gene is being evaluated currently for gene therapy of cancer. We have built a variant of wild-type p53, chimeric tumor suppressor 1 (CTS1), in which we have replaced the domains that mediate its inactivation. CTS1 presents some very interesting properties: (a) enhanced transcriptional activity; (b) resistance to the inactivation by oncogenic forms of p53; (c) resistance to the inactivation by MDM2; (d) lower sensitivity to E6-induced degradation; (e) ability to suppress cell growth; and (f ) faster induction of apoptosis. Thus, CTS1 is an improved tumor suppressor and an alternative for the treatment of wild-type p53-resistant human tumors by gene therapy.

Kin17 is a mammalian nuclear protein sharing a slight sequence homology with the bacterial RecA protein. Kin17 has a zinc-finger motif and binds efficiently to curved DNA, a genomic topology associated with illegitimate recombination junctions. We investigated the relationship between the level of Kin17 protein and genomic alteration due to either impaired wild-type p53 functions or exposure to gamma rays. We used BP cells, a rodent epithelial cell system. The cell lines used were syngeneic and harbored wild-type or mutant p53 alleles and exhibited different sensitivities to gamma irradiation. In radioresistant cells (wild-type p53 genotype), the level of Kin17 protein peaked 30 min after a low dose of radiation (2 Gy), whereas maximum accumulation of p53 protein was observed 3 h postirradiation. Radiosensitive cells carrying the same mutation in both alleles of the p53 gene showed elevated basal levels of both Kin17 and p53 proteins and failed to accumulate Kin17 and p53 proteins after exposure to ionizing radiation. These cells exhibited enhanced cell death by apoptosis after gamma irradiation. Our results indicate that Kin17 protein accumulated immediately after DNA damage in cells carrying a wild-type p53 genotype, and that levels of constitutive Kin17 protein increased in highly proliferating tumorigenic cells when wild-type p53 functions were abrogated.

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