GENETICS OF SENESCENCE IN MELANOCYTES
Adapted and summarized by Prof. DC Bennett from a section of:
Bennett DC and Medrano EE (2002).
Molecular regulation of melanocyte senescence.
Pigment Cell Res., 15, 242-250
Introduction: Cell senescence is the process whereby normal mammalian somatic cells become unable to divide after a finite number of divisions, a process often disrupted in cancer cells. This may have a special significance in melanocytes, as the three genes so far associated with familial melanoma susceptibility – INK4A, CDK4 and ARF, are all implicated in the molecular pathways controlling cell senescence. These pathways are reviewed in the above article, with a discussion of the possible roles of cell senescence in the development and molecular genetics of melanoma and its precursor lesions.
INK4A and ARF: The INK4A and ARF genes are unusual in sharing some coding sequence in different reading frames, within a common locus (INK4A-ARF). Both encode powerful cellular growth inhibitors, which act through key tumour suppressor genes RB1 and p53. INK4A protein, also known as p16INK4A or p16, is an activator of RB1 (by inhibiting kinase CDK4), while ARF is an activator of p53 and its downstream growth inhibitor p21. Some mutations associated with familial melanoma susceptibility affect both INK4A and ARF; others affect INK4A only, and in one case ARF only (see article for literature references).
Genetically engineered inbred mice were used to analyze the effect of an Ink4a-Arf deletion on cell senescence. This deletion abolishes the function of both p16 and Arf. Mouse fibroblasts homozygous for this deletion show very little senescence, and melanocytes of this genotype behaved similarly. However a difference was seen between fibroblasts and melanocytes with only one copy of the deletion (hemizygous, as in most familial melanoma cases). The hemizygous fibroblasts senesced normally, whereas hemizygous melanocytes showed only partial senescence. Another interesting aspect was pigmentation. Normal wild-type mouse melanocytes showed heavy pigmentation and increased cell size and protein content at the time of senescence. These rises did not occur in Ink4a-Arf null melanocytes, and only partially in Ink4a-Arf hemizygous melanocytes. Thus, in mouse melanocytes, both normal copies of the Ink4a-Arf locus are required for normal senescence, and the associated rises in pigmentation and cell size.
To analyze the relative roles of p16 and Arf, wild-type Ink4a and Arf sequences were separately replaced in the Ink4a-Arf-/- melanocytes. p16 expression led to growth arrest, with increased pigmentation and cell size. The cells also expressed acidic -galactosidase, a partial marker of senescence.. Conversely, the cells expressing Arf without p16 had a high rate of cell death, again with apparent high pigmentation but no size increase and no detectable -galactosidase. Moreover, spontaneously immortalized lines of mouse melanocytes and melanoblasts from Ink4a-Arf+/+ mice often expressed Arf protein, but never p16 protein. These findings indicated a key role for the p16/Rb1 pathway in control of mouse melanocyte senescence, with loss of p16 (but not Arf) expression apparently mandatory for immortalization.
Genetic analysis of p16 in melanocyte senescence has also been possible in human melanocytes (work submitted for publication). Two strains of melanocytes from humans with deficiencies in both p16 alleles were cultured. Both showed high rates of apoptosis in a standard melanocyte medium. Addition of either keratinocyte feeder cells or keratinocyte-derived growth factors enabled the cells of both strains to proliferate well and both then achieved a greatly extended lifespan. Thus, p16/RB1 deficiency in melanocytes allows an extended lifespan, but promotes cell death in the absence of keratinocyte products. Blockade of p53 using the HPV16-E6 oncogene showed that the cell death was partially independent of p53. Both strains showed partial upregulation of p53 while growing, with eventual p21 accumulation and senescence. In comparison, normal melanocytes senesced with low p53 and no detectable p21. This with other evidence (see review) supports the view that senescence of human melanocytes is controlled primarily by the p16/RB1 pathway. The p53/p21 pathway can effect only a delayed arrest, when the p16/RB1 pathway is deficient.
p53: Evidence from transgenic mice overexpressing a stable, hyperactive mutant p53, p53m, supported the hypothesis that p53 has little part in normal melanocyte senescence. Although these animals showed accelerated ageing in several tissues, pigmentation in hair and skin did not show appreciably early reduction. Thus, the p53+/m hair bulb melanocytes retained proliferative capacity for a normal time, through repeated hair cycles – further evidence that mouse melanocyte senescence is normally independent of p53.
Extension of melanocyte lifespan by genetic manipulations: One group described the immortalization of two neonatal human melanocyte strains by expression of HPV16 oncogenes E6 and E7, interfering with both p53 and RB. This was interesting but surprising, because no manipulation was performed to restore telomerase activity, which does appear to limit lifespan in cultured melanocytes (see below). It is possible that telomerase became spontaneously reactivated; this was not checked.
Conversely, expression of exogenous hTERT (human telomerase reverse transcriptase) alone, by retroviral transduction, was also followed by marked lifespan extension in human melanocytes, by more than 70 additional doublings. The cells retained normal appearance. The melanocytes did not however reach immortality, because hTERT expression, mediated by a viral promoter, was extinguished at ~ passage 80. These melanocytes showed some intriguing changes in gene expression, including reduced levels of p16 and another growth inhibitor p27Kip-1. It is unknown whether such changes contributed to the extended lifespan, and whether they arose as a result of hTERT expression or spontaneously. The reduction in p16 level is especially interesting.
In summary, most or all the genetic evidence suggests that downregulation of the p16/RB1 pathway may be required, in addition to telomerase activation, for immortalization of human melanocytes. Accordingly, both these changes are predicted to be necessary for the development of a human melanoma, since neoplastic cells are generally immortal.