The experimental model proposed for this study of the carcinogenesis process are human keratinocytes. These populations can exhibit differentiative function, (keratin) formation and terminal differentiation in vitro on the same substratum as that required to grow the fibroblast upon. We propose to either study the induction stage of carcinogenesis in keratinocytes under conditions where the epithelial morphology of the cells is maintained in vitro and keratin production is repressed, or we can study the carcinogen insult with the cell under conditions where keratin synthesis is operational. We propose to examine the modifying effects compounds such as insulin, PMA or anthralin have on the carcinogenesis process. These compounds when used prior to carcinogen treatment of the fibroblasts have heightened the response to the carcinogen insult. However, keratinocytes have a different program for S phase entry as a function of time following either insulin, PMA or anthralin administration. So it will be of interest to evaluate the carcinogenic response and whether there is a requirement for treatment of keratinocytes with carcinogen in early S or G1/S phase for a heightened response to the insult. Three probes will be used to follow the modifying effect of either PMA, anthralin or insulin on the carcinogenesis process. Calmodulin synthesis and localization in the cell will be followed by indirect immunofluorescent techniques; keratin formation, a marker of cell differentiation, will be followed by indirect immunofluorescent techniques and scheduled DNA synthesis will be followed by 3H-CH3-thymidine incorporation. We will then evaluate the requirement for specific carcinogen-DNA adduct formation and persistence of adducts under conditions where the expression of the carcinogen insult can be modified. Lastly, we will address the assay systems we have developed for measuring a carcinogenic response in vitro. Do human carcinoma cells that exhibit anchorage independent growth, exhibit cellular invasiveness and neoplasia as evaluated in the nude mouse similar to the invitro carcinogen transformations of human keratinocytes?
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