Transcriptional regulation of the alpha 4 integrin subunit gene in the metastatic spread of uveal melanoma

Recently, expression of the alpha 4 integrin subunit has been shown to be i nversely correlated with the invasive potential of B16 mouse epidermal mela noma. The purpose of this study was to establish whether expression of the human alpha 4 integrin subunit gene might be similarly regulated in human u veal melanoma which has varying degrees of invasiveness, and whether such m odifications are determined by alterations in the transcriptional activity directed by the alpha 4 gene promoter. Two metastatic variants (MH5 and MH1 0) derived from a human uveal melanoma (SP6.5) were used. Expression studie s were performed by transiently transfecting each of these cell lines with recombinant plasmids bearing various lengths of the alpha 4 promoter fused to the CAT reporter gene, and were further validated by Northern blot analy ses of the alpha 4 transcript. Both transient transfection and mRNA analyse s provided evidence that the transcriptional activity directed by the alpha 4 promoter sequences extending up to position -76 and -120 was indeed inve rsely correlated to the potential of uveal melanoma to yield metastasis. Ex periments in electrophoretic mobility shift assay (EMSA) demonstrated that binding of the nuclear proteins that likely account for transcription of th e alpha 4 gene to alpha 4.1 (namely Bp1, Bp2, Bp4, and Bp5) was dramaticall y reduced in uveal melanoma, but not in normal uveal melanocytes. These res ults highlight the fundamental function the alpha 4 integrin subunit may pl ay in the ability of tumor cells to evade the primary tumor and form metast asis.