INDUCED EXPRESSION OF THE HUMAN BCL-2 HOMOLOGUE BAK IN S. POMBE LEADS TO GROWTH ARREST AND CELL DEATH - A GENETIC SYSTEM FOR IDENTIFYING APOPTOSIS INHIBITING GENES

Abstract

Non-genotoxic hepatocarcinogenesis may involve suppression of the hepatocyte apoptosis that would normally remove damaged or initiated cells. These protected hepatocytes could then remain as preferential targets for promotion by this class of compounds. Here, we demonstrate clearly that the non-genotoxic liver carcinogens and hepatomitogens cyproterone acetate (CPA) and nafenopin, a peroxisome proliferator, both suppressed the basal level of rat liver apoptosis in vivo to 17 or 77% of controls, respectively. Concomitant with this suppression of apoptosis, BrdU labelling indices and mitotic figures rose confirming a perturbation of both sides of the growth equation between cell death and replication. Withdrawal of CPA or nafenopin resulted in a 100 to 200fold elevation in apoptosis. Tlus was idubited by the readnunstration of either compound. generated upon withdrawal of CPA or nafenopin. Rats were administered BrdU during the hyperplastic phase of compound administration (0-10 days). Livers were examined 5 days after compound withdrawal. With both CPA and nafenopin, apoptotic b d e s and S-phase were predominantly in the periportal region. However, despite this zonal co-localisation , very few (<lo%) of the apoptotic bodies were labelled with BrdU. Overall, our data provide in vivo evidence to support the hypothesis that non-genotoxic hepatocarcinogens such as CPA and the peroxisome proliferators suppress apoptosis. Surprisingly, the majority of the hepatocytes generated during compound-induced hyperplasia were protected from apoptosis during liver regression. These data contribute to our understanding of clonal selection and promotion during non-genotoxic hepatocarcinogenesis.We examined the replicative history of the apoptotic bodies H26EXPRESSION AND ACTIVITY OF p53 DURING LONG TERM QUIESCENCE IN HUMAN DIPLOID FIBROBLASTS Human diploid fibroblasts (HDFs) become quiescent in response to density arrest or serum deprivation in multiple phases. These phases are marked by increasing amounts of time required to enter S-phase in response to stimuli, the longer that they are held quiescent. Previous work has indicated that these phases of quiescence are distinct, with changes in metabolism and gene expression, and not simply a result of a continuous build up of growth inhibitory factors during extended periods of quiescence. We report here that during long term quiescence, p53 protein levels increase without a concomitant rise in RNA levels. This is similar to observations made regarding p53 following DNA damageinduced growth arrest. We also find that the FWA levels of a number of p53 responsive genes including p21 and gadd45 increase with kinetics similar to those seen for p53 protein levels suggesting a direct role for p53 in transactivation of these growth-inhibiton. genes during quiescence. Electrophoretic mobility shiti assays using a p53 consensus sequence show that HDFs contain a distinct p53-specific proteidDNA complex that is not present in growing or in short term (3day) quiescent cells. These results sugges...