An understanding of oncogene activation during tumor progression would add to our knowledge of the molecular basis of neoplasia and could aid in terms of diagnosis, prevention and treatment of cancer. The unique mouse skin model of tumor progression in which there are defined stable intermediate stages and lesions will be used in these studies. Using this unique model and molecular biology techniques, genes which are activated by point mutation or over-expression during tumor progression will be identified and their functional role in tumorigenesis will be assessed. Alterations in the expression of known oncogenes will be studied by northern hybridization in tumor promoter dependent and independent papillomas, non-metastasizing and metastasizing squamous cell carcinomas compared to normal epidermis, as well as acutely and chronically tumor promoter treated epidermis. Changes in known cellular oncogene organization in terms of methylation, rearrangement and amplification will be studied in DNA isolated from these various lesions. To identify and clone genes that are responsible for acquisition of the """"""""initiated"""""""" or malignant phenotype by DNA transfection, genomic DNAs from carcinomas, papillomas and """"""""putatively initiated"""""""" cells will be co-transfected with the pSV2 neo plasmid into primary epidermal cells or """"""""putatively initiated"""""""" cells and/or papilloma cells. The method of suppressor rescue for isolation of eukaryotic genes using the supF fragment as a marker will be used to clone the gene(s). To identify which genes are upregulated during skin tumor progression, cDNA libraries, enriched for stage specific mRNAs by selective hybridization techniques, will be prepared and screened by dot hybridization for genes whose expression are stage specific. An attempt will be made to obtain full-length cDNA copies of these upregulated genes in an Okayama-Berg expression vector to determine the functional significance of the upregulated genes by transfection of normal epidermal cells, """"""""putatively initiated"""""""" cells and papilloma cells. If oncogenes either with or without homology to the known retroviral oncogenes are activated in the progression of chemically induced skin tumors in mice, this experimental approach will identify those genes.
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