EXPRESSION OF DOMINANT-NEGATIVE CREB REDUCES RESISTANCE TO RADIATION OF HUMAN-MELANOMA CELLS

u.v.-responsive element (URE)-binding proteins were found to include m embers of the AP1 and ATF transcription factor families, To elucidate the functional contribution of URE-bound proteins to the characteristi cs of human melanoma, we have used a dominant negative CREB cDNA which is mutated within the DNA-binding domain and cloned into a mammalian expression vector driven by the RSV promoter (KCREB), As such, KCREB i s still capable of heterodimerizing with its associated proteins, Set, due to its poor binding affinity to DNA it out competes transcription al activity mediated by those proteins, Human melanoma cells (MeWo) we re transfected with KCREB and three clones, designated K1, K2, and K10 which express KCREB transcripts were then selected for further charac terization. When tested for binding activities in gel shift assays, pr oteins prepared from the three clones exhibited a different set of com plexes than the parent MeWo and control MeWo(neo) cells (transfected w ith empty expression vector) under normal growth conditions, and after u.v.-irradiation, Using CAT vector, driven by a tetramer URE construc t, revealed a striking decrease in transcriptional activity in each of the three clones before as well as after u.v.-irradiation, When teste d for radiation resistance MeWo cells were found to exhibit 42% surviv al to a u.v.-dose of 16 J/m(2), whereas, K1, K2 and K10 exhibited only 10.2, 3.9 and 4.2% survival, respectively, Exposure to 2 Gy of X-radi ation led to 62.1% survival of MeWo as compared with 18.5% of K1 and 7 .7% and 6.5% of K2 and K10, respectively, While no significant differe nces were noticed in their growth rate, all three clones exhibited few er, and smaller colonies in soft agar, when compared with parent cells , These findings indicate that through their transcriptional activitie s, CREB and its associated proteins play an important role in the acqu isition of characteristic phenotypes of human melanoma cells including resistance to u.v.-irradiation.