The long-term objective of the studies outlined in this proposal is to better our understanding of the cell and molecular mechanisms involved in the stages of initiation and promotion in multistage hepatocarcinogenesis in the rat. A series of five experiments will be performed to test the thesis that single glutathione-S transferase, placental form (PGST)-positive (+) hepatocytes comprise a significant part of the initiated cell population. During the stage of promotion altered hepatic foci (AHF) comprise only about 1% of the number of single PGST+ hepatocytes. Reasons for this difference will be sought in determining ploidy characteristics of the single PGST+ hepatocytes as well as the phenotypic distributions of AHF induced by different promoting agents. The operational reversibility of AHF growth during the stage of promotion will be investigated by combining the removal of the -promoting agent after 4 months of administration, together with two sequential periods of short-term fasting in order to obtain the most effective loss of AHF within a relatively short time period. Parameters involved in the acute loss of hepatocytes within AHF by this procedure will be studied by determining the most effective format of the fasting period(s), the effect of the time of tumor promotion prior to the fasting period, the effect of continued administration of the promoting agent during the fasting period, and the effects of omission and addition of the same or other promoting agents or inhibitors of the stage of promotion during the refeeding period on the AHF number and growth. Since apoptosis is reportedly a principal mechanism in the loss of cells from AHF upon removal of the promoting stimulus, as well as in liver on acute fasting, we will investigate both in vivo and in hepatocyte culture in normal hepatocytes and those of AHF levels of the growth factors, TGF-(, HGF, and TGF-(1, as well as TNF-(, and their receptors during and following the period of fasting and promoting agent removal. The effect of fasting on the expression of members of the Bcl-2 and Bax gene families will also be investigated. From these studies we hope to better establish the cell biology of the stage of initiation and develop a better understanding of mechanisms involved in the reversibility of the stage of tumor promotion as induced by a combination of removal of the promoting agent and acute dietary restriction in the form of fasting.
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