The long term goal is the elucidation of the functional significance of specific carcinogen-protein interactions in liver carcinogenesis. Our recent findings have determined that the principal target of a hepatic carcinogen, N-2-fluorenylacetamide (FAA; 2-acetylaminofluorene), is a liver cytoplasmic 14,000-dalton polypeptide that is specifically associated with mitosis in normal adult rat hepatocytes, and with early and persistent foci of hyperplasia of hepatocytes caused by the two carcinogens, FAA and 3'-methyl-4-dimethyl-aminoazobenzene. A 17,500-dalton polypeptide that is related to the cytoplasmic 14,000-dalton target polypeptide is tightly bound to chromatin in normal adult rat liver. Mitosis in normal adult hepatocytes is associated with a marked elevation in the apparent level of the 14,000-dalton polypeptide in cytoplasm. In addition, feeding of either of the two hepatocarcinogens causes early and persistent polypeptide foci of hepatocytes, in which there appears to be a great overload of the 14,000-dalton polypeptide in cytoplasm, and a near absence of 17,500-dalton polypeptide in most nuclei. Our hypothesis is that the cytoplasmic 14,000-dalton polypeptide target of the hepatic carcinogen has a positive but unknown function in cell multiplication in normal adult hepatocytes, and in liver oncogenesis by these carcinogens. The proposed research will purify the two polypeptides or their fragments in forms suitable for physical, chemical and immunological characterizations. In search of the identities of the two polypeptides and their physiological interrelationships, their amino acid sequences will be determined, evaluated for homology between them, and searched for matches or relatedness to known protein and nucleic acid sequences in large computer databases. The ability of the nuclear 17,500-dalton polypeptide to bind DNA in a specific manner will be investigated. The rates of turnover of the two polypeptides, and their initial rates of accumulation will be examined in livers of rats. The relationships of the early and persistent polypeptide foci of hepatocytes to liver neoplasia caused by the two carcinogens will be investigated. The extent to which the nuclear 17,500-dalton polypeptide may be a direct target of FAA will be directly examined. Finally, histochemical staining methods will quantitatively establish the degrees to which the early and the persistent polypeptide foci overlap with the islands of hyperplastic hepatocytes and with foci of elevated gamma-glutamyl transpeptidase activity during hepatocarcinogenesis in rats. It seems reasonable to speculate that future manipulations of the levels of the two polypeptides in normal and preneoplastic hepatocytes may possibly provide new approaches to the prevention or cessation of the early processes that lead to liver neoplasia in animals and humans.
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