The long-term objective of these studies is to better understand the cell and molecular mechanisms involved in the environmental manipulation of the stage of promotion in multistage hepatocarcinogenesis in the rat. The studies will utilize two model systems developed in our laboratory: 1) acute fasting and refeeding of rats during the stage of promotion and 2) a transgenic model expressing an endogenous promoting agent, transforming growth factor alpha (TGFa). In order to learn more of the mechanisms of the fasting effect, the expression of multiple genes in the fasting as compared with fed animals will be determined utilizing oligonucleotide microarrays. Based on these findings with normal animals fasted and fed we will utilize Laser Capture Microdissection (LCM) to obtain sufficient material from altered hepatic foci (AHF) to prepare cDNA using high fidelity RNA amplification with subsequent determination of the expression of specific genes by quantitative RT-PCR (QPCR) in order to define the principal alterations in gene expression in apoptotic pathways occurring in preneoplastic hepatocytes during the fasting period, especially those related to the c-myc apoptotic pathway. Using this same system, we plan to determine the effect of caloric restriction and chemical inhibitors of tumor promotion on the regrowth of AHF following short-term fasting. Alterations in gene expression will also be studied in normal and preneoplastic hepatocytes following such treatments. Utilizing rats bearing the metallothionein-TGFalpha minigene as a transgene, we propose to study the effects of transgene expression, both basal and induced, by zinc on both induced and spontaneous initiation and promotion in contrast to nontransgenic animals. The effect of short-term fasting in the presence and absence of zinc and phenobarbital (PB) will be investigated to determine the potential role of endogenous promotion by the transgene product. The kinetics of changes in AHF during fasting and refeeding in these animals will also be compared with nontransgemc animals. Genes found to be dramatically altered in nontransgenic animals will be investigated in these studies in the transgenic animals. From these studies we hope to obtain a better understanding of the regulation of the stage of tumor promotion in rat hepatocarcinogenesis by exogenous and endogenous promoting agents as well as the mechanisms of the apoptosis seen in the fasting normal and preneoplastic hepatocyte.
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