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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 E2f1 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(-/-) 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.

Ultraviolet (UV) light exposure is a common cause of epithelial-derived skin cancers, and the epidermal response to UV-light has been extensively studied using both mouse models and cultured human keratinocytes (KCs). Elimination of cells with UV-induced DNA damage via apoptosis provides a powerful mechanism to minimize retention or expansion of genetically abnormal cells. This cell editing function has largely been ascribed to the biological role of the p53 tumor suppressor gene, as mutations or deletions involving p53 have been linked to skin cancer development. Rather than introducing mutations, or using cells with complete loss of wild-type p53, we used an siRNA-based approach to knockdown, but not eliminate, p53 levels in primary cultures of human KCs followed by UV-irradiation. Surprisingly, when p53 levels were reduced by 50-80% the apoptosis induced by exposure to UV-light was accelerated and markedly enhanced (two- to three- fold) compared to control siRNA treated KCs. The p53 siRNA treated KCs were characterized by elevated E2F-1 levels accompanied by accelerated elimination of the Mcl-1 and Bcl-x(L) antiapoptotic proteins, as well as enhanced Bax oligomerization. Forced overexpression of either Mcl-1 or Bcl-x(L) reduced the UV-light enhanced apoptotic response in p53 siRNA treated KCs. We conclude that p53 not only can provide proapoptotic signals but also regulates a survival pathway influencing Mcl-1 and Bcl-x(L) levels. This overlooked survival function of p53 may explain previous paradoxical responses noted by investigators using p53 heterozygous and knockout mouse models, and opens up the possibility that not all liaisons within the cell involving p53 necessarily represent fatal attractions.

A key step in cancer development is clonal expansion. The increased number of mutant cells allows a clinical phenotype and increases the probability that one of the cells will be mutated in an additional gene. For skin cancer, observations on p53-mutant keratinocyte clones in epidermal sheets of UVB-irradiated mice reveal that mutant stem cells are normally restrained within their stem cell compartment. Chronic UVB exposure drives clonal expansion by a non-mutational mechanism, and this mechanism allows mutant stem cells to escape from their own stem cell compartment and colonize adjacent compartments. In the absence of escape, cells proliferate without occupying additional territory. The non-mutational clonal expansion mechanism appears to be UVB-induced apoptosis, which deletes DNA-damaged cells in unmutated stem cell compartments but will preferentially spare death-resistant p53-mutant cells. An additional source of apoptotic selection pressure may come from UV-irradiated melanin.

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.

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 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.

The E2f1 transcription factor, which regulates genes required for S-phase entry, also induces apoptosis by transcriptional and post-translational mechanisms. As E2f1 is inducible by DNA damage we investigated its importance in vivo in ultraviolet (UV)-induced apoptosis, a protective mechanism that prevents the epidermis from accumulating UV-induced mutations. Contrary to expectation, E2f1-/- mice demonstrated enhanced keratinocyte apoptosis after UVB exposure, whereas apoptosis was suppressed by epidermis-specific overexpression of human E2F1. Apoptosis induced by -radiation was also repressed by E2f1. E2f1-/-;Trp53-/- double knockout mice exhibited the elevated UVB-induced apoptosis of E2f1-/- alone, rather than the profound apoptosis defect seen in Trp53-/- mice, indicating that Trp53 (p53) lies functionally upstream of E2f1. Transfecting E2F1 into E2f1-/-;Trp53-/- primary fibroblasts suppressed UVB-induced apoptosis and this suppression was relieved by Trp53. The double knockout also reverted the abnormal sex ratio and early-onset tumours of Trp53-/- mice. These results imply that E2f1 functions as a suppressor of an apoptosis pathway that is initiated by DNA photoproducts and perhaps genetic abnormalities; p53 relieves this suppression.

Decitabine, an epigenetic modifier that reactivates genes otherwise suppressed by DNA promoter methylation, is effective for some, but not all cancer patients, especially those with solid tumors. It is commonly recognized that to overcome resistance and improve outcome, treatment should be guided by tumor biology, which includes genotype, epigenotype, and gene expression profile. We therefore took an integrative approach to better understand melanoma cell response to clinically relevant dose of decitabine and identify complementary targets for combined therapy. We employed eight different melanoma cell strains, determined their growth, apoptotic and DNA damage responses to increasing doses of decitabine, and chose a low, clinically relevant drug dose to perform whole-genome differential gene expression, bioinformatic analysis, and protein validation studies. The data ruled out the DNA damage response, demonstrated the involvement of p21(Cip1) in a p53-independent manner, identified the TGFbeta pathway genes CLU and TGFBI as markers of sensitivity to decitabine and revealed an effect on histone modification as part of decitabine-induced gene expression. Mutation analysis and knockdown by siRNA implicated activated beta-catenin/MITF, but not BRAF, NRAS or PTEN mutations as a source for resistance. The importance of protein stability predicted from the results was validated by the synergistic effect of Bortezomib, a proteasome inhibitor, in enhancing the growth arrest of decitabine in otherwise resistant melanoma cells. Our integrative analysis show that improved therapy can be achieved by comprehensive analysis of cancer cells, identified biomarkers for patient's selection and monitoring response, as well as targets for improved combination therapy.