The aim of the present study was to elucidate the effects of
ataxia telangiectasia mutated (ATM)
kinase on the regulation of the extrinsic
tumor necrosis factor-related apoptosis-inducing
ligand (
TRAIL) receptor 2/DR5-mediated death pathway in human
melanoma cells. We revealed that total
ATM protein levels were high in some human
melanoma lines compared with normal cells. The basal levels of active form ATM phospho-Ser(1981) were also detectable in many
melanoma lines and could be further up-regulated by gamma-irradiation. Pretreatment of several
melanoma lines just before gamma-irradiation with the inhibitor of ATM
kinase KU-55933 suppressed p53 and
nuclear factor-kappaB (
NF-kappaB) activation but notably increased radiation-induced DR5 surface expression, down-regulated cFLIP (caspase-8 inhibitor) levels, and substantially enhanced exogenous TRAIL-induced apoptosis. Furthermore, gamma-irradiation in the presence of
KU-55933 rendered TRAIL-resistant HHMSX
melanoma cells susceptible to TRAIL-mediated apoptosis. In addition, suppression of ATM expression by the specific
short hairpin RNA also resulted in down-regulation of cFLIP levels, up-regulation of surface DR5 expression, and TRAIL-mediated apoptosis in
melanoma cells. Besides p53 and
NF-kappaB, crucial regulators of DR5 expression,
transcription factor STAT3 is known to negatively regulate DR5 expression. Suppression of Ser(727) and Tyr(705) phosphorylation of STAT3 by
KU-55933 reduced STAT3 transacting activity accompanied by elevation in DR5 expression. Dominant-negative STAT3beta also efficiently up-regulated the DR5 surface expression and down-regulated cFLIP levels in
melanoma cells in culture and in vivo. Taken together, our data show the existence of an ATM-dependent STAT3-mediated antiapoptotic pathway, which on suppression sensitizes human
melanoma cells to TRAIL-mediated apoptosis.