Activation of a cAMP pathway and induction of melanogenesis correlate withassociation of p16(INK4) and p27(KIP1) to CDKs, LOSS of E2F-binding activity, and premature senescence of human melanocytes

There is strong evidence that the senescent phenotype, whether induced by t elomere shortening, oxidative damage, or oncogenic stimuli, is an important tumor suppressive mechanism. The melanocyte is a cell of neural crest orig in that produces the pigment melanin and can develop into malignant melanom as. To understand how malignant cells escape senescence, it is first crucia l to define what genes control senescence in the normal cell. Prolonged exp osure to high levels of cAMP results in accumulation of melanin and termina l differentiation of human melanocytes. Here we present evidence that activ ation of a cAMP pathway correlates with multiple cellular changes in these cells: (1) increased expression of the transcription factor microphthalmia; (2) increased melanogenesis; (3) increased association of the cyclin-depen dent kinase inhibitors (CDK-Is) p27(KIP1) and p16(INK4) with CDK2 and CDK4, respectively; (4) failure to phosphorylate the retinoblastoma protein (pRB ); (5) decreased expression of E2F1, E2F2, and E2F4 proteins; (6) loss of E 2F DNA-binding activity; and (7) phenotypic changes characteristic of senes cent cells. Senescent melanocytes have potent E2F inhibitory activity, beca use extracts from these cells completely abolished E2F DNA-binding activity that was present in extracts from the early proliferative phase. We propos e that increased activity of the CDK-Is p27 and p16 and loss of E2F activit y in human melanocytes characterize a senescence program activated by the c AMP pathway. Disruption of cAMP-mediated and melanogenesis-induced senescen ce may cause immortalization of human melanocytes, an early step in the dev elopment of melanomas. (C) 1999 Academic Press.