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 progression marker positively linked with metastasis. In this study the calcyclin expression levels in a representative panel of human melanoma cell lines were correlated with the occurrence of DNase I hypersensitive (DH) regions and potential enhancer elements in a 6 kb genomic fragment spanning the human calcyclin gene. Examination of the chromatin structure of the transcription unit revealed no qualitative differences in DH sites within the panel of tested human melanoma cells, but especially the sequences around the transcription start site and a 1.5 kb upstream region appeared more accessible to the nuclease in frequently (BLM, MV3) as compared to poorly (530, 1F6) metastasizing cells. The genomic fragments that harbor one or more DH sites were subjected to functional analysis by luciferase reporter gene assays. Thus, an enhancer element was detected between 361 and 167 bp upstream of the transcription start site. This enhancer displayed equal activating potential (2-3 fold) both in weakly and in frequently metastasizing cells and was apparently recognized by transcription factors present in both types of human melanoma cells lines. We conclude that, in addition to a slight amplification of the encoding gene, the elevated calcyclin mRNA levels are only reflected in a selectively increased accessibility of the chromatin structure to DNaseI in metastasizing melanoma cells.