Isolation and Partial Characterization of Potential Melanoma Suppressor Genes Using a Novel Subtractive Melanocyte Library

Abstract

Background: There is increasing evidence that a tumour suppressor plays a role in the pathogenesis of cutaneous melanoma. Objective: Our objective was to isolate a melanoma-tumour suppressor gene. Methods: We constructed a novel subtractive library enriched for cDNAs expressed preferentially in normal melanocytes. Candidate genes were isolated using differential hybridization and were characterized further by Northern blot analysis. Results: Initially, 238 plaques were isolated, of which 57 contained insert cDNA. Ten of the cDNA clones demonstrated expression in normal melanocytes and were not present in at least one of four melanoma cell lines. Three of the clones showed no expression in melanocytes, but did hybridize with at least one of the melanoma lines. The remaining 44 clones were not expressed in either melanocyte or melanoma lines. Partial DNA sequence analysis of four selected clones revealed a cDNA representing the Ret Fused Gene (RFG) and two others highly homologous to tyrosinase-related protein (TRP1). Ret Fused Gene, a gene originally isolated from thyroid gland tumours, has been mapped to chromosome 10, whereas the TRP1 has been mapped to chromosome 9p. Both these genetic loci are known to be altered in melanoma. Conclusion: The method used is a powerful tool for the identification of genes important in the pathogenesis of skin diseases and is applicable to the study of a wide range of neoplastic and nonneoplastic conditions.