Abstract

Compared to normal melanocytes, melanoma cell lines exhibit overexpression of hyperphosphorylated retinoblastoma tumor suppressor protein (Rb) or a marked decrease in, or lack of, expression of Rb. Hyperphosphorylation of Rb results in increased E2F-mediated transactivation of target genes and cell cycle progression. Using a combination of gene disruption and ectopic expression in growth factor-dependent mouse melanocytes, we studied the roles of E2F1 and the p16INK4A and p21WAF1/CIP1 CKIs (cyclin dependent kinase inhibitors) in the acquisition of TPA (12-O-tetradecanoyl phorbol-13-acetate)-independent growth in culture, a hallmark of melanomas. Surprisingly, melanocytes from p16INK4A- or p21WAF1/CIP1-null mice remained TPA-dependent, and disruption of p21WAF1/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 E2F1E132 mutant conferred TPA-independence upon melanocytes and was associated with sequestration of Rb and constitutive expression of E2F1 target genes, including p21WAF1/CIP1. We conclude that neutralization of Rb by E2F1E132, but not the disruption of p16INK4A or p21WAF1/CIP1, resulted in the accumulation of free E2F and cell cycle progression. Thus, mechanisms other than the loss of p16INK4A or p21WAF1/CIP1 that activate E2F may play an important role in melanomas.