This continuation application investigates the role of N-ras in signal transduction, its role in thymic lymphoma development and the analysis of tumor suppressor genes that cooperate with ras in tumor formation. The grounds for that analysis have been laid out during the previous grant period, during which Dr. Pellicer obtained N-ras transgenic lines carrying the overexpressed normal gene (N-rasN) or the oncogene (N- rasT), knockout N-ras mice (KONras), and has identified the frequent involvement of the INK4b locus(p15) in tumors derived from these mouse strains. These experiments permitted the identification of a specific response to PMA+ ionomycin treatment in KONras derived cells, prompting the proposal of a series of experiments to elucidate where in the pathway this signal becomes N-ras specific, and which domain in the molecule is responsible for the specificity. The observation, that there is an increased lymphoma yield when N-rasT mice are crossed with KONras mice compared to mice wild type for N-ras suggests that the normal allele has a blunting effect on the action of the oncogenic allele. To explore the differences between the N-rasT and the wild type, the effects of increasing amounts of normal allele on the effects of oncogenic N-ras will be measured in vivo and in vitro. The biochemical effects of this competition, the affinity of both N-Ras isoforms for their targets and the inhibitory effect of NF1 will also be measured. Results have been obtained indicating that the INK4b locus is involved in the development of thymic lymphomas. To understand the role of this locus in lymphoma development, the impact of ras genes on cell cycle regulators will be investigated in vivo, using the transgenic lines. The recent identification of a new molecule derived from the INK4b locus, p10, opens the way to investigate the role of p10 in lymphoma development versus p15, and the mechanism of action of p10 will be studied and its role in vivo analyzed.
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