RELEASE OF CELL-CYCLE CONSTRAINTS IN MOUSE MELANOCYTES BY OVEREXPRESSED MUTANT E2F1(E132), BUT NOT BY DELETION OF P16(INK4A) OR P21(WAF CIP1)/

Compared to normal melanocytes, melanoma cell lines exhibit overexpres sion of hyperphosphorylated retinoblastoma tumor suppressor protein (R b) or a marked decrease in, or lack of, expression of Rb. Hyperphospho rylation of Rb results in increased E2F-mediated transactivation of ta rget genes and cell cycle progression. Using a combination of gene dis ruption and ectopic expression in growth factor-dependent mouse melano cytes, we studied the roles of E2F1 and the p16(INK4A) and p21(WAF1/CI P1) CKIs (cyclin dependent kinase inhibitors) in the acquisition of TP A (12-O-tetradecanoyl phorbol-13-acetate)-independent growth in cultur e, a hallmark of melanomas. Surprisingly, melanocytes from p16(INK4A)- or p21(WAF1/CIP1)-null mice remained TPA-dependent, and disruption of p21(WAF1/CIP1) accelerated cell death in the absence of this mitogen. Disruption of E2F1 had the most profound effect on melanocyte growth, resulting in a fourfold decrease in growth rate in the presence of TPA . Furthermore, enforced overexpression of the DNA-binding-defective E2 F1(E132) mutant conferred TPA-independence upon melanocytes and was as sociated with sequestration of Rb and constitutive expression of E2F1 target genes, including p21(WAF1/CIP1). We conclude that neutralizatio n of Rb by E2F1(E132), but not the disruption of pl6(INK4A) or p21(WAF 1/CIP1), resulted in the accumulation of free E2F and cell cycle progr ession. Thus, mechanisms other than the loss of p16(INK4A) or p21(WAF1 /CIP1) that activate E2F may play an important role in melanomas.