ABROGATION OF WILD-TYPE P53-MEDIATED TRANSACTIVATION IS INSUFFICIENT FOR MUTANT P53-INDUCED IMMORTALIZATION OF NORMAL HUMAN MAMMARY EPITHELIAL-CELLS

The p53 protein has become a subject of intense interest since the dis covery that about 50% of human cancers carry p53 mutations. Mutations in the p53 gene are the most frequent genetic lesions in breast cancer , suggesting a critical role for p53 protein in normal mammary epithel ial cell (MEC) growth control, We previously demonstrated that abrogat ion of the p53 function by a cancer-derived p53 mutant, del239, was su fficient to induce immortalization of normal MECs, To further extend t hese findings and to examine the mechanism of mutant p53-induced immor talization of MECs, we tested the immortalizing ability of four select ed p53 mutants (R175H, R248W, R249S, and R273H), which involve residue s that cluster close to N239 in the three-dimensional structure and wh ich are critical for the DNA-binding function of p53, Interestingly, t wo of these mutants (R175H and R249S) reproducibly immortalized 76N no rmal MECs, whereas the other two mutants (R248W and R273H) induced an extension of life span but not immortalization, These results further substantiate that selective ablation of p53 function with dominant-neg ative mutants is sufficient for immortalization of MECs. To determine whether abrogation of the transactivation function of endogenous p53 w as important for the differential immortalizing ability of p53 mutants , ne measured the effects of mutant p53 on the endogenous wild-type p5 3-mediated transactivation of a chloramphenicol acetyltransferase repo rter linked to a consensus p53 binding DNA sequence in transiently tra nsfected 76N MECs. All of the mutants, regardless of their immortalizi ng phenotype, abrogated the endogenous wild-type p53-mediated transact ivation to a similar extent, Thus, abrogation of transactivation funct ion is not sufficient for mutant p53-induced immortalization of normal MECs. The p53-immortalized MECs showed substantial telomerase activit y; however, induction of telomerase activity occurred at late passages and was undetectable in mutant p53-expressing cells prior to immortal ization. We suggest that mechanisms other than abrogation of transacti vation and induction of telomerase activity determine the differential MEC-immortalizing behavior of various p53 mutants.