CREB AND ITS ASSOCIATED PROTEINS ACT AS SURVIVAL FACTORS FOR HUMAN-MELANOMA CELLS

cAMP response element-binding protein (CREB) and activating transcript ion factor 1 (ATF-1), members of the CREB/ATF family, have been implic ated in cAMP- and calcium-induced transcriptional activation. We have previously demonstrated that quenching of CREB-associated proteins in metastatic melanoma cells by a dominant-negative CREB (KCREB) that is mutated within its DNA-binding domain decreased their radiation resist ance, and their tumorigenic and metastatic potential in nude mice, As the induction of apoptosis by diverse exogenous signals is dependent o n the elevation of intracellular Ca2+, the purpose of this study was t o determine the role of CREB and its associated proteins in apoptosis using KCREB, We used thapsigargin (Tg), which inhibits endoplasmic ret iculum-dependent Ca2+-ATPase and thereby increases cytosolic Ca2+, to induce apoptosis, MeWo human melanoma cells were transfected with the KCREB expression vector and subsequently analyzed for their susceptibi lity to Tg-induced apoptosis, Here we demonstrate that expression of K CREB in MeWo cells rendered them susceptible to Tg-induced apoptosis, Tg treatment induced phosphorylation of CREB and possibly ATF-1 transc ription factors. Treatment with Tg induced CRE-dependent transcription in parental cells, whereas this activation was reduced in the KCREB-t ransfected cells. In addition, CAT activity driven by the CRE-dependen t promoter was inhibited in parental MeWo cells cotransfected with inc reasing concentrations of KCREB in a dose-dependent manner. We did not observe any changes in Bcl-2 or Bcl-2-related proteins (Bcl-x, Bax, a nd Bad) in control or KCREB-transfected cells before or after treatmen t with Tg, Collectively, these data indicate that CREB and its associa ted proteins act as survival factors for human melanoma cells, and hen ce contribute to the acquisition of the malignant phenotype.