MUTATIONAL ANALYSIS OF HUMAN PAPILLOMAVIRUS TYPE-16 E6 DEMONSTRATES THAT P53 DEGRADATION IS NECESSARY FOR IMMORTALIZATION OF MAMMARY EPITHELIAL-CELLS

We have previously demonstrated that normal human mammary epithelial c ells (MECs) are efficiently immortalized by human papillomavirus type 16 (HPV16) E6. HPV16 E6 binds to and induces p53 degradation in vitro and induces a marked reduction of p53 protein in MECs. Low-risk HPV6 E 6 is defective for p53 binding and degradation in vitro but immortaliz ed MECs at low efficiency. The HPV6 E6-immortalized MECs had markedly reduced levels of p53. To directly investigate whether the ability of MPV16 E6 to stimulate p53 degradation is required for E6-induced immor talization, a series of HPV16 E6 mutants were analyzed for the ability to bind and degrade p53 in vitro, induce a reduction in p53 levels in vivo, and immortalize MECs. We observed that one set of mutants effic iently immortalized MECs, caused a reduction in p53 levels in vivo, an d degraded p53 in vitro. Other mutants immortalized MECs with low effi ciency and either induced p53 degradation at low levels or were unable to induce p53 degradation in vitro; however, all of the immortal clon es displayed low levels of p53. A third class of mutants did not immor talize MECs and failed to induce a reduction in p53 levels in vivo or degrade p53 in vitro. These results demonstrate that a reduction in p5 3 protein levels due to enhanced degradation is essential for MEC immo rtalization by HPV16 E6.