FUNCTIONAL-ANALYSIS OF THE HUMAN CALCYCLIN GENE PROMOTER IN A PANEL OF HUMAN-MELANOMA CELL-LINES

By comparing two subsequent human tumor stages we previously described calcyclin as a new potential melanoma associated neoplastic progressi on marker positively linked with metastasis. In this study the calcycl in expression levels in a representative panel of human melanoma cell lines were correlated with the occurrence of DNase I hypersensitive (D H) regions and potential enhancer elements in a 6 kb genomic fragment spanning the human calcyclin gene. Examination of the chromatin struct ure of the transcription unit revealed no qualitative differences in D H sites within the panel of tested human melanoma cells, but especiall y the sequences around the transcription start site and a 1.5 kb upstr eam region appeared more accessible to the nuclease in frequently (BLM , MV3) as compared to poorly (530, 1F6) metastasizing cells. The genom ic fragments that harbor one or more DH sites were subjected to functi onal analysis by luciferase reporter gene assays. Thus, an enhancer el ement was detected between 361 and 167 bp upstream of the transcriptio n start site. This enhancer displayed equal activating potential (2-3 fold) both in weakly and in frequently metastasizing cells and was app arently recognized by transcription factors present in both types of h uman melanoma cell lines. We. conclude that, in addition to a slight a mplification of the encoding gene, the elevated calcyclin mRNA levels are only reflected in a selectively increased accessibility of the chr omatin structure to DNaseI in metastasizing melanoma cells. (C) 1995 A cademic Press, Inc.