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Sunlight's UVB radiation triggers cell signaling at multiple sites to induce apoptosis. The integration of these signal entry sites is not understood. Here we show that P53 and E2fl constitute a UV-inducible apoptosis switch. At low-UV doses, wild-type cells resemble the OFF state of an siP53-treated cell, whereas at high-UV doses, the apoptosis frequency transitions to the fully ON behavior of an siE2f1-treated cell. The switch's target is Bcl-2: Rapid Bcl-2 down-regulation in response to UVB-induced DNA photoproducts is lost in P53-deficient cells, but, as for apoptosis, is restored when both P53 and its inhibited target E2f1 are absent. P53's down-regulation of Bcl-2 is mediated entirely through E2f1. Bcl-2 is also down-regulated by a separate pathway triggered by DNA photoproducts in the absence of P53 and E2f1. Four UV pathways terminating on Bcl-2 contribute to apoptosis after UVB irradiation. The apoptosis lost in [p53.sup.-/-] is completely restored by siBcl-2, implying that Bcl-2 is a rate-limiting member of this network. These results identify Bcl-2 as an integrator of several UV-induced proapoptotic signals and show that it, in turn, suppresses a direct UV-apoptosis pathway. UV-induced apoptosis requires both UV activation of the direct pathway and a separate UV disinhibition of this pathway through P53-E2f1-Bcl-2. E2fl | P53 | stress response | network | systems biology

Melanin protects the skin against DNA damage induced by direct absorption of sunlight's UV radiation. Yet, irradiating melanin in vitro or in cultured cells also generates active oxygen species such as superoxide, which can indirectly induce oxidative base lesions and DNA strand breaks. This photosensitization is greater for pheomelanin (yellow and red melanin) than for eumelanin (brown and black). The in vivo photosensitizing ability of melanin is unknown. We used congenic mice of black, yellow, and albino coat colors to investigate the induction of DNA lesions and apoptosis after exposure to predominantly UVB (280-320 nm) or UVA (320-400 nm) radiation. Cyclobutane pyrimidine dimers induced by direct UVB absorption were equal in all three strains, as was apoptosis measured as sunburn cells or as keratinocytes containing active caspase-3. However, terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL)-positive cells were [approximately equal to] 3-fold more frequent in black and yellow mice after UVB or UVA irradiation than in albino. In epidermal sheets, TUNEL-positive cells lined the upper portion of the hair follicle, consistent with UV-induced photosensitization by melanin in the hair shaft. Because the concentration of eumelanin in black mice was three times that of pheomelanin in yellow mice, pheomelanin had 3-fold greater specific activity. We conclude that UV-irradiated melanin, particularly pheomelanin, photosensitizes adjacent cells to caspase-3 independent apoptosis, and this occurs at a frequency greater than the apoptosis induced by direct DNA absorption of UV. Melanin-induced apoptosis may contribute to the increased sensitivity of individuals with blonde and red hair to sunburn and skin cancer.

Once mutated, a single cell must expancl into a clone before becoming significant for carcinogenesis. The forces driving clonal expansion and the obstacles that must be overcome are poorly understood. In a genetic mechanism, acquiring a second mutation conferring a proliferative advantage would enable the cell to expand autonomously. If carcinogen exposure instead induced a physiological change, clonal expansion would require the carcinogen's continued presence. To determine which is the case, we studied microscopic clones of keratinocytes mutated in the p53 tumor suppressor gene. Carcinogen exposure was controlled by irradiating mice with 280-320 nm UV radiation (UVB), sunlight's principal carcinogenic component; expansion of mutant clones was observed in epidermal sheets, p53-mutant clones grew only during chronic UVB exposure. Therefore, clonal expansion was not triggered by a proliferative mutation but was instead continually driven by UVB. Unexpectedly, the clone size distribution showed periodicity with maxima at estimated intervals of 16 [+ or -] 6 cells, the size of the epidermal proliferating unit in murine dorsal skin. In the absence of UVB, rare 'imprisoned clones' increased in cell number without increasing in area. We conclude that: stem cell compartments act as physical barriers to clonal expansion of a p53-mutant keratinocyte; a rate-limiting step in clonal expansion is the colonization of an adjacent compartment; and sustained UVB enables the p53-mutant keratinocyte to colonize without incurring an additional mutation.