HUMAN-PAPILLOMAVIRUS ONCOPROTEINS ALTER DIFFERENTIATION-DEPENDENT CELL-CYCLE EXIT ON SUSPENSION IN SEMISOLID MEDIUM

The suspension of keratinocytes containing episomal forms of the human papillomavirus (HPV)-31 genome in semisolid medium results in the ind uction of Viral late functions. In this study, the suspension in semis olid medium was used to analyze how HPV deregulates the process of cel l cycle exit during differentiation. In cells that contain the entire HPV-31 genome, induction of late protein synthesis was found to be lin ked with the expression of cyclin A Consistent with analyses in organo typic rafts, the expression of the high-risk E7 oncoprotein alone was sufficient to retain cyclin A expression during suspension-induced dif ferentiation. The cyclin-dependent kinase inhibitors (CKIs) p27 and p5 7 were found to be up-regulated in normal keratinocytes, as well as in the lines that express the HPV oncoproteins. The up-regulation of the se CKIs is coincident with the inhibition of cyclin/cdk activity in no rmal keratinocytes. Cells expressing E7 were found to retain significa nt cdk2-associated kinase activity, although it was partially inhibite d, coincident with CKI induction. When the phosphorylation state of Rb was examined during differentiation, cells expressing E7 retained pho sphorylated forms of Rb, whereas Rb in normal keratinocytes was hypoph osphorylated. As previously reported, E7-expressing cells were found t o contain less Rb protein than normal keratinocytes. Interestingly, th e Rb levels decreased during normal keratinocyte differentiation, and this differentiation-dependent reduction in Rb levels was enhanced by EG and E7 expression. This study identified proteins that may be criti cal for cell cycle regulation during normal epithelial differentiation and demonstrated that HPV oncoproteins alter their activities, (C) 19 98 Academic Press.