Melanin accumulation accelerates melanocyte senescence by a mechanism involving p16INK4a/CDK4/pRB and E2F1

Abstract

Cellular and molecular evidence suggests that senescence is a powerful tumor-suppressor mechanism that prevents most higher eukaryotic cells from dividing indefinitely in vivo. Recent work has demonstrated that alpha-melanocyte stimulating hormone (alpha-MSH) or cholera toxin (CT) can activate a cAMP pathway that elicits proliferative arrest and senescence in normal human pigmented melanocytes. In these cells, senescence is associated with increased binding of p16INK4a to CDK4 and loss of E2F-binding activity. Because senescence may provide defense against malignant transformation of melanocytes, and because pigmentation is a strong defense against melanoma, we examined the ability of melanocytes derived from light and dark skin to respond to CT. Here we demonstrate that in melanocytes derived from dark-skinned individuals, CT-induced melanogenesis is associated with accumulation of the tumor suppressor p16INK4a, underphosphorylated retinoblastoma protein (pRb), downregulation of cyclin E, decreased expression of E2F1, and loss of E2F-regulated S-phase gene expression. In contrast to other senescent cell types, melanocytes have reduced or absent levels of the cyclin-dependent kinase inhibitors p27Kip1 and p21Waf-1. Importantly, melanocytes derived from light-skinned individuals accumulated smaller amounts of melanin than did those from dark-skinned individuals under the same conditions, and they continued to proliferate for several more division cycles. This delayed senescence may result from reduced association of p16 with CDK4, reduced levels of underphosphorylated pRb, and steady levels of cyclin E and E2F1. Because cyclin E-CDK2 inhibition is required for p16-mediated growth suppression, upregulation of p16 and downregulation of cyclin E appear essential for maintenance of terminal growth and senescence. Given the rising incidence of melanoma, identification of major growth regulatory proteins involved in senescence should shed light on the biology of this genetically mysterious tumor.