IMMORTALIZATION & TRANSFORMATION OF HUMAN MELANOCYTES

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The hypothesis to be experimentally explored in this project states that some of the multiple steps in the malignant transformation of human melanocytes can involve the activation of specific oncogenes or cellular genes that encode products involved in the growth factor signal transduction pathway in melanocytes. The specific aims to approach this hypothesis include: 1) to identify important biological, biochemical, and molecular markers on cultured cells that represent melanocytes undergoing transformation 2) to transfect oncogenes (v-jun and v-fos) and cellular genes (basic fibroblast growth factor (bFGF) and protein kinase C) into melanocytes from normal human foreskin and from dysplastic nevi and look for the phenotypic alterations defined in the first specific aim 3) to determine the molecular mechanism for the overexpression of the bFGF gene that has been observed in malignant melanoma cell sin comparison to normal melanocytes. In the first specific aim the cell surface antigen to be studied in the different melanocyte cell lines include HLA-DR, GD3, HMB45, HLAI, S100 and ADA. The growth related markers include phorbol ester dependence for growth, growth factor and serum dependence, anchorage- independent growth and rate of growth. The biochemical markers to be studied include protein kinase C (PKC) activity as well as PKC and FGF protein expression. In the second specific aim both plasmid and defective retroviral expression vectors will be used to express the genes of interest in the normal melanocytes or melanocytes from dysplastic nevi. Electroporation will be used to transfect the plasmid DNA into the melanocytes. In the third specific aim the molecular basis for the overexpression of bFGF in malignant melanocytes in comparison to normal melanocytes will be pursued. Two hypotheses related to the overexpression of bFGF will be tested: 1) mutation(s) in the 5' flanking regulatory region of the bFGF gene has occurred in malignant melanoma cells, 2) there are enhanced levels of a transacting factor(s) in malignant melanoma cells that stimulate transcription by binding to cis-regulatory sequences of the bFGF gene. By defining the molecular differences between melanocytes that are at different stages of progression to malignant melanoma new strategies for intervention by chemoprevention can be designed or existing chemoprevention protocols may be monitored for effectiveness by studying alterations in the expression of the critical oncogenes or other cellular genes.