Deregulated E2F transcriptional activity in autonomously growing melanoma cells

Inactivation of the retinoblastoma tumor suppressor protein (pRb) has been implicated in melanoma cells, but the molecular basis for this phenotype ha s not yet been elucidated, and the status of additional family members (p10 7 and p130, together termed pocket proteins) or the consequences on downstr eam targets such as E2F transcription factors are not known. Because cell c ycle progression is dependent on the transcriptional activity of E2F family members (E2F1-E2F6), most of them regulated by suppressive association wit h pocket proteins, we characterized E2F-pocket protein DNA binding activity in normal versus malignant human melanocytes. By gel shift analysis, we sh ow that in mitogen-dependent normal melanocytes, external growth factors ti ghtly controlled the levels of growth-promoting free E2F DNA binding activi ty, composed largely of E2F2 and E2F4, and the growth-suppressive E2F4-p130 complexes. In contrast, in melanoma cells, free E2F DNA binding activity ( E2F2 and E2F4, to a lesser extent E2F1, E2F3, and occasionally E2F5), was c onstitutively maintained at high levels independently of external melanocyt e mitogens. E2F1 was the only family member more abundant in the melanoma c ells compared with normal melanocytes, and the approximately fivefold incre ase in DNA binding activity could be accounted for mostly by a similar incr ease in the levels of the dimerization partner DP1. The continuous high exp ression of cyclin D1, A2, and E, the persistent cyclin-dependent kinase 4 ( CDK4) and CDK2 activities, and the presence of hyperphosphorylated forms of pRb, p107, and p130, suggest that melanoma cells acquired the capacity for autonomous growth through inactivation of all three pocket proteins and re lease of E2F activity, otherwise tightly regulated in normal melanocytes by external growth factors.