Primary mouse keratinocytes provide an ideal model for the study of multistep carcinogenesis. A unique feature of this system is that a direct connection can be established between in vitro and in vivo studies, by grafting of cultured keratinocytes back onto the animal. Our approach will involve introduction of activated growth-regulatory genes (oncogenes) into primary keratinocytes with evaluation of their possible effects on cells (a) in culture (in response to calcium, TPA defined media) and (b) in vivo, by grafting (ability to form well stratified epidermis, Benign or displastic papillomas, frankly invasive carcinomas). Oncogenes will be introduced into cells by means of a retroviral vector (MD) that we have specifically developed for high expression of foreign genes into primary keratinocytes. Genes that will be introduced into cells include: 1) ras oncogene. Use of a replication-defective MD-ras virus will provide a significant contribution to experiments already underway, concerning the role of ras activation as well as surrounding normal cells in benign versus malignant transformation of primary keratinocytes. 2) Nuclear oncogenes. Preliminary studies with two viruses carrying the Ela and p53 oncogenes will be directly continued. Construction of viruses carrying other nuclear oncogenes (myc and myb) will be undertaken. 3) Growth-factor related genes. Viruses with genes for EGF and EGF-receptor and the neu oncogene will be made and used to test the possible role of these genes during neoplastic evolution of epidermal cells. 4) Oncogenes in various combinations. Oncogene-carrying retroviruses will be constructed that carry different selectable markers. These viruses will be used as an alternative approach to helper virus in testing cooperative effects of various oncogenes in keratinocyte transformation.
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