Exposure to the sun's UV radiation appears to be the most important environmental factor involved in the development of skin cancer. UVA is the major portion of UV radiation in sunlight and is considered to be a human carcinogen. In this study, we have investigated the delayed and sustained activation of ERK MAPK by UVA exposure. In parallel, a delayed Ras activation with a similar time course was observed after UVA exposure. The activated Ras was found to be localized in endomembranes such as the Golgi apparatus instead of plasma membranes. Expression of dominant negative Ras (N17Ras) abolished ERK activation by UVA. The presence of AG1478, an epidermal growth factor (EGF) receptor (EGFR) kinase inhibitor, had no effect on ERK or Ras activation, indicating that EGFR kinase activity is not involved in ERK activation by UVA. In contrast, protein kinase C (PKC) depletion by chronic 12-O-tetradecanoylphorbol-13-acetate treatment nearly abolished UVA-induced ERK and Ras activation. The presence of the Ca 2؉ -dependent-PKC inhibitor Gö 6976 had a similar effect. These findings suggest that ERK activation by UVA is mediated by PKC in a Ras-dependent pathway. In addition, a gradual increase in intracellular calcium level after UVA exposure was detected by flow cytometry. The presence of the PLC inhibitor U73122 or the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N, N,N-tetraacetic acid tetrakis (acetoxymethyl ester) (BAPTA-AM) blocked both ERK and Ras activation, suggesting that both PLC and calcium are required for ERK activation. Our findings demonstrated that, different from UVC and UVB, UVA-induced delayed and sustained ERK activation is EGFR kinase activity-independent, but PLC/calcium/PKC-mediated. The delayed and sustained ERK activation provides a survival signal to human HaCaT keratinocytes, which may serve as an important mechanism for cell transformation and potential skin carcinogenesis in vivo caused by UVA exposure.

Inorganic arsenic is a human carcinogen that targets the skin. Carcinogenesis is a multistep process in which acquired apoptotic resistance is a common event and prior work in non-skin cells shows acquired resistance to apoptosis occurs with chronic arsenite exposure. In the present study, when HaCaT cells, an immortalized, non-tumorigenic human keratinocyte cell line, were continuously exposed to low-level inorganic arsenite (as sodium arsenite; 100 nM) for 28 weeks, the cells acquired a generalized resistance to apoptosis. This included resistance to apoptosis induced by acute high concentrations of arsenite, ultraviolet A (UVA) irradiation, and several chemotherapeutic compounds (cisplatin, etoposide and doxorubicin). These arsenite-tolerant (As-TL) cells showed similar levels of UVA-induced reactive oxygen species (ROS) and oxidative DNA damage when compared to passage match control cells. Because cellular apoptosis is dependent on the balance between proapoptotic and survival pathways, the roles of protein kinase B (PKB), a key antiapoptotic molecule, in this acquired apoptotic resistance were investigated. Stimulation of apoptosis markedly decreased nuclear phosphorylated PKB (P-PKB) levels in control cells, but As-TL cells showed greatly increased stability of nuclear P-PKB. Pretreatment of the As-TL cells with LY294002 or Wortmannin, which specifically inhibit PKB phosphorylation, completely blocked apoptotic resistance in As-TL cells, indicating acquired apoptotic resistance is associated with increased stability of nuclear P-PKB. Because arsenic and UV irradiation are co-carcinogenic in mouse skin, resistance to UV-induced apoptosis in As-TL cells may allow UV-damaged cells to escape normal cell population controls and initiate the carcinogenic cascade. The observation that As-TL cells show no lessening of UV-induced genotoxicity supports this possibility. ' 2005 Wiley-Liss, Inc.