Autophagy is a survival mechanism known to be activated by starvation and able to turn into cell death of either apoptotic or autophagyc type. We have previously shown that 20 mg/Kg of diethylnitrosamine (DENA), unable per se to start liver carcinogenesis, generated initiated hepatocytes in rats refed after prolonged starvation leading to foci, nodules and eventually cancer when promoted by the Solt and Farber regimen. At the time of DENA administration, depression of glutathion-S-transferase activity enhanced the DENA-induced DNA damaged hepatocytes that were stimulated to divide by starvation-induced cell loss resulting in initiated cells. Here we demonstrated that 1 day-starvation reduced the expression of the phosphorilated form of the major inhibitory signal that shuts off autophagy during nutrient abundance, the mammalian target of rapamycin (m-Tor), that virtually disappeared 4 days after starvation. On the contrary, starvation enhanced the expression of the membrane-bound form of microtubule associated protein 1 light chain 3, namely LC3-II, an autophagosomal marker, as shown by western blotting analysis. Paradoxically, the expression of Beclin-1, gene essential for the nucleation of the autophagosomal vesicles, slightly decreased after starvation. However, 1 day-starvation induced the traslocation of Beclin-1 from nucleus to cytoplasm as indicated by immunohistochemical analysis. Autophagic activity progressively reduced with long-term starvation to preserve cellular constituents essential for survival and prolongation of starvation made autophagy to switch into cell death when the cells became unable to survive without energy and nutrients. Activation of the cell death programm resulted by down regulation of the antiapoptotic protein, Bcl2 and up regulation of the proapoptotic bax which in turn activated the release of cytochrome c into the cytoplasm and the DNA fragmentation as shown by the increased % of TUNEL-positive fragmented nuclei in a caspase-independent manner. Starvation-induced caspase-independent cell death resulted in cell loss able to stimulate cell replication of DENA-damaged hepatocytes during refeeding, leading to inhitiate cells by a per se non carcinogenic dose of DENA.

Work supported by: MIUR, Comunità Novarese.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA