This study will focus on promoter-independent expression of an oncofetal mRNA-transport protein with a molecular weight 65 kDa (p65) which has been functionally characterized by its ability to enhance the transport of mRNA to the exterior of isolated cell nuclei in hepatocarcinogenesis. The overall objective of this study is to elucidate the mechanisms of expression of p65 in rat liver during the course of chemically induced hepatocarcinogenesis. Specifically, (i) Rat p65 will be microsequenced and protein sequence homology searches will be carried out in order to determine if the p65 structure is unique or the same or related to that of other known proteins and whether p65 has some other functions in vivo in addition to the putative stimulation of the mRNA transport. Alternatively, the rat p65 gene will be cloned, sequenced and characterized and its regulation during rat hepatocarcinogenesis will be elucidated using a cDNA probe. (ii) Immunohistochemical techniques will be employed to identify the cellular site(s) of the p65 synthesis at early time-points following the administration of N-nitrosodiethylamine and determine whether minifoci of putative initiated hepatocytes are the main site of p65 synthesis. The p65 production and DNA synthesis will be monitored at specific times after partial hepatectomy, with and without subsequent carcinogen treatment. (iii) Using the two-stage model for hepatocarcino-genesis, the effect of the choline-deficient diet on p65 expression in oval cells and or altered hepatic foci during promotion will be investigated by means of immunohisto-chemistry. The p65 production will be correlated with the level of DNA synthesis and mitotic activity of altered hepatic foci. Cell type specificity of the p65 production in carcinogen induced preneoplastic lesions and malignant tumors will also be investigated using anti-p65 antibodies and recently developed monoclonal antibodies to different preneoplastic liver cell populations induced by chemical carcinogens in rats. (iv) The effects of withdrawal and subsequent re-administration of the choline-deficient diet on p65 expression in developing altered hepatic foci will be correlated with the persistence of reversibility. We will correlate the p65 expression in promoter-independent foci with the expression of some protooncogenes. It will also be established if the use of N- diethylnitrosamine, a genotoxic carcinogen (initiator) is prerequisite for the induction of p65 in the choline-deficiency model.
