Apoptosis in keratinocytes is required for epidermal turnover, stratum corneum formation, and removal of ultraviolet-damaged premalignant cells. Its role in melanocyte homeostasis and transformation, on the other hand, has not been de¢ned, although apoptosis resistance is a commonly recognized feature of melanoma. We examined the expression of apoptosis regulators in melanocytes, keratinocytes, melanoma, and HaCat cells. Melanocytic cells expressed relatively high levels of Bcl-2, Bcl-X L , Mcl-1, C-IAP-1, C-IAP-2, XIAP, Livin, and Apaf-1. The only apoptotic regulator that was di¡erentially expressed in melanoma cells and not melanocytes was Survivin, whereas Bax was expressed in melanocytes but not in most melanoma lines. Keratinocytic cells, on the other hand, expressed high levels of FLIP and were relatively de¢cient in Bcl-2 family proteins. Levels of p53 were highest in HaCat cells and some of the melanoma lines, and barely detectable in melanocytes and keratinocytes. Next, susceptibility of these cells types to apoptosis induced by ultraviolet B, the tyrosine analog 4-tert-butylphenol, and cytotoxic drugs was examined. Melanocytes were relatively resistant to ultraviolet B, whereas keratinocytes were unresponsive to 4-tert-butylphenol. Melanocytes and keratinocytes were generally less susceptible than melanoma lines and HaCat cells to etoposide, cisplatin, and staurosporine. Induction of apoptosis in these cell types was generally associated with decreased levels of Mcl-1, XIAP, and Livin, and increased levels of p53, whereas levels of other apoptotic regulators were unaltered. These results provide insights into the potential roles of apoptosis in the function and transformation of epidermal melanocytes and keratinocytes.

The mechanism by which a single mutant cell clonally expands is usually assumed to involve an additional mutation in a cell cycle regulatory gene. An alternative mechanism for driving clonal expansion is apoptosis, which might create vacant stem cell compartments that can be repopulated by mutant cells. This model predicts that in a mouse with reduced apoptotic capacity (i) more mutated cells will appear initially but (ii) these cells will expand into clones more slowly than in wild-type animals. To test this hypothesis for ultraviolet B (UVB)-induced skin carcinogenesis, we examined UVB-induced p53 mutant clones and tumors in a transgenic (Tg) mouse (K14-Survivin) with skin-specific expression of the apoptosis inhibitor Survivin. To limit the effects of Survivin on apoptosis, without affecting epidermal proliferation or differentiation, we used Survivin expression levels and UVB doses that resulted in a 2-fold reduction in keratinocyte apoptosis. After 5 weeks of chronic UVB irradiation, newly created p53 mutant keratinocyte clones (indicative of initial mutation frequency) were 1.4-fold more frequent in K14-Survivin mice (P = 4 x 10(-6)). As predicted, this effect was reversed for clones growing by clonal expansion, which were rarer in Tg skin by 1.7-fold (P = 0.047). At 10 weeks large expanding Tg clones were rarer by a magnitude approaching the apoptosis differential (approximately 2-fold, P = 4 x 10(-5)). Survivin expression also retarded clonal expansion at later stages of tumor development. By 20 weeks 95% of animals carried tumors (primarily papillomas), which were 1.6-fold rarer in apoptosis-defective Tg mice (P = 0.03). In contrast, the rate of tumors attaining large size (> or =3 mm, P = 0.048) and converting to carcinoma was increased approximately 2-fold in Tg mice. Thus, Survivin-regulated apoptosis appears to suppress two stages that involve new mutations, initiation and malignant conversion, yet drives clonal expansion of existing p53 mutant cells.

No abstract