Published in Cancer Detection and Prevention 2000; 24(Supplement 1).

Role of MAPK-p38 in radiation resistance of human melanoma

Z Ronai PhD, VN Ivanov PhD, A Bhoumik MSc

Ruttenberg Cancer Center, Mount Sinai School of Medicine, New York, NY,

AIMS to reveal mechanisms underlying the resistance of human melanoma cells to radiation and chemotherapy. METHODS human melanoma derived cell cultures were analyzed for expression and activities of stress kinases including p38, JNK, IKK and their respective substrates ATF2, c-Jun and NFkB. The role of these components in melanoma sensitivity to radiation and chemical induced apoptosis was monitored via routine FACS analysis. RESULTS Our recent findings demonstrate that changes in the expression and respective activities of TRAF2/GCK, upstream regulators of MEKK1 JNK/p38 and IKK signaling, occur during melanoma development and regulate its sensitivity to UV-induced apoptosis. The finding that stress kinases, which are regulated by TRAF2, play an important role in the acquisition of melanoma resistance to irradiation prompted us to further elucidate the possible modulation of TRAF2 on melanoma sensitization to apoptosis induced by various treatments. Inhibition of TRAF2, by a form of TRAF2, which lacks the ring finger domain, increases activities of p38, ATF2 and correspondingly the level of TNF expression, thus by increasing the relative fraction of cells that undergoes apoptosis. Such changes takes place in melanoma cells that express low levels of Fas (or NF-kB) and sensitizes melanoma cells to treatment by TNF, anisomycin or actinomycin D. Among the mechanisms underlying the role of stress kinases in apoptosis of human melanoma cells is the suppression of Fas expression by p38 signaling. ASK1/p38 downregulates the expression of a Fas via NF-kB/SP1 site on the Fas promoter. Deletion or mutation of NF-kB/SP1 within the Fas promoter abrogates p38 effect. ASK1/p38 silences the Fas promoter by inhibition of IkBa phosphorylation - thereby limiting NF-kB activity. Forced expression of a dominant negative form of p38 (p38-ASP) or treatment with p38 pharmacological inhibitor, SB203580, increases NF-kB activity, Fas expression and the levels of UVC-induced apoptosis in late stage melanoma cells. Inhibition of p38 activity also restored NF-kB activity and Fas expression in early-phase melanoma cells, suggesting that p38 elicited suppression of Fas expression is not restricted to late phase melanoma. Identifying p38-mediated down-regulation of Fas expression illustrates a novel regulatory pathway by which ASK1/MKK6/p38 alters the degree and nature of the UV-induced apoptosis of human melanoma cells. CONCLUSIONS our studies identify specific changes in expression and activities of stress kinases during melanoma development. The contribution of stress kinases to the resistance of melanoma cells to apoptosis in response to radiation or chemical treatments is mediated via their regulation of NF-kB, Fas, and TNF. Our studies also demonstrate that it is possible to use the selective kinases as target to sensitize melanoma to chemical treatments.

KEY WORDS: melanoma, radiation resistance, p38, NFkB, Fas, .

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Paper presented at the International Symposium on Impact of Biotechnology on Cancer Diagnostic & Prognostic Indicators; Geneva, Switzerland; October 28 - 31, 2000; in the section on anticancer strategies.