ANCHORAGE-INDEPENDENT TRANSCRIPTION OF THE CYCLIN-A GENE INDUCED BY THE E7 ONCOPROTEIN OF HUMAN-PAPILLOMAVIRUS TYPE-16

To develop an experimental model for E7-mediated anchorage-independent growth, we studied the ability of M-expressing NIH 3T3 subclones to e nter 8 phase when they were cultured in suspension. We Pound that expr ession of E7 prevents the inhibition of cyclin E-associated kinase and also triggers activation of cyclin A gene expression in suspension ce lls. A point mutation in the amino terminus of E7 prevented W-driven r escue of cyclin E-associated kinase activity in suspension cells; howe ver, cells with this mutation retained some ability to activate cyclin A gene expression and promote S-phase entry, Activation of cyclin A g ene expression by E7 was correlated with an increased binding of free E2F to a regulatory element in the cyclin A promoter which mediates bo th repression of cyclin A upon loss of adhesion and its reactivation b y E7, Surprisingly, expression of E7 led to a nuclear accumulation of one species of free E2F, namely, an E2F-4-DP-1 heterodimer, that is ex clusively cytoplasmic in the absence of E7. Taken together, the data r eported here indicate that several different E7-dependent changes of c ellular-growth-regulating pathways can cooperate to allow adhesion-ind ependent entry into S phase.