INHIBITION OF SERUM-INDUCED AND CALCIUM-INDUCED DIFFERENTIATION OF HUMAN KERATINOCYTES BY HPV16 E6 ONCOPROTEIN - ROLE OF P53 INACTIVATION

We have recently shown that human papillomavirus (HPV16) E6 oncoprotei n exhibits two separate biological activities in genital keratinocytes (PHKs). E6 protein by itself is capable of inducing colonies of proli ferating cells resistant to serum and calcium-induced differentiation, whereas both E6 and E7 are required for immortalization of PHK. Using epitope-tagged 56 carboxy-terminal truncation mutants, we mapped the domain between amino acid residues 132 and 141 as being essential for the induction of differentiation resistance (L Sherman and R. Schlegel , J. Virol. 70, 3269-3279, 1996). To determine whether E6 protein's ab ility to alter PHK response to serum and calcium was associated with i ts ability to inactivate p53, we evaluated each of the above E6 mutant s and three 56 natural variants in these respective assays. Our result s demonstrate that the 56 region spanning residues 132-141 is required for p53 degradation and for abrogation of p53 transactivation, sugges ting a possible correlation between 56 biological activity in altering differentiation and loss of p53 function. To evaluate whether selecti ve inactivation of p53 is sufficient for altering the response of PHK to serum and calcium we investigated the capacity of plasmids encoding a dominant mutant human p53 and human MDM-2 to functionally substitut e for 56 in colony formation in PHK. Plasmids were verified for their ability to inactivate wild-type p53 by testing their capacity to abrog ate the p53 transactivation function. The results obtained showed that vectors encoding human MDM-2 and mutant p53, while active in inhibiti on of p53-dependent transactivation and capable of expressing stable p roteins in PHK, failed to induce colonies of proliferating cells resis tant to serum and calcium differentiation. These data argue that p53 i nactivation may not be the sole E6 function required for altering the response of PHK to serum-and calcium-triggered differentiation. (C) 19 97 Academic Press.