Release of cell cycle constraints in mouse melanocytes by overexpressed mutant E2F1E132, but not by deletion of p16INK4A or p21WAF1/CIP1

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 p16 INK4A and p21 WAF1/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 p16-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 eect on melanocyte growth, resulting in a fourfold decrease in growth rate in the presence of TPA. Furthermore, enforced overexpression of the DNA-binding-defective E2F1 E132 mutant conferred TPAindependence upon melanocytes and was associated with sequestration of Rb and constitutive expression of E2F1 target genes, including p21 WAF1/CIP1. We conclude that neutralization of Rb by E2F1 E132 , but not the disruption of p16 INK4A or p21, resulted in the accumulation of free E2F and cell cycle progression. Thus, mechanisms other than the loss of p16 INK4A or p21 WAF1/CIP1 that activate E2F may play an important role in melanomas.

The Regulation of Normal Melanocyte Proliferation

“…5). This mutant indirectly stimulated endogenous E2F activity, as well as the expression of E2F-responsive genes by sequestering pRb (147). Taken together, these data .…”

“…A positive correlation was observed between the number of functional E2F1 alleles and growth rate in TPA-supplemented medium (147) (Fig. 4B).…”

“…4A). Furthermore, although the CDK inhibitor p21 WAF1/CIP1 was originally identified as the gene product up-regulated in a melanoma cell line by agents that induced differentiation (146), p21 WAF1/CIP1 levels are actually increased in normal human and mouse melanocytes in response to growth factors (147,148).…”

“…We recently examined the role of various components of this pathway in normal melanocytes (147 INK4A ) caused accelerated death in growth factor-deprived medium, suggesting a positive role in melanocyte survival for these, presumably, CKIs (Fig. 4A).…”

Macrophage Migration Inhibitory Factor Deficiency Is Associated with Altered Cell Growth and Reduced Susceptibility to Ras-mediated Transformation

“…Several attempts to express a DNA binding-defective E2F1 E132 mutant in MIF Ϫ/Ϫ fibroblasts resulted in rapid cell death. The activity of this mutant has been associated with sequestration of Rb and constitutive expression of E2F target genes (43). By contrast, the E2F1 E132 mutant stimulated growth in wild-type fibroblasts (data not shown).…”