CHOLINE DEFICIENCY SELECTS FOR RESISTANCE TO P53-INDEPENDENT APOPTOSIS AND CAUSES TUMORIGENIC TRANSFORMATION OF RAT HEPATOCYTES

The mechanisms which drive initiated cells to progress to form carcino mas are poorly understood, CWSV-1 rat hepatocytes, in which p53 protei n is inactivated by SV40 large T antigen, respond by inducing p53-inde pendent apoptosis when acutely switched to medium containing low choli ne (16% apoptotic at 48 h in 5 mu M choline) as compared with controls (1% apoptotic at 48 h in 70 mu M choline), The rate of apoptosis was inversely correlated with cellular phosphatidylcholine content, Cholin e deficiency (CD)-induced apoptosis is probably mediated by TGF beta 1 and reactive oxygen species, since immunoneutralization of TGF beta 1 in the medium or treatment with N-acetylcysteine (an antioxidant) or addition of neocuproine (a transition metal chelator) prevented CD-ind uced apoptosis. CWSV-1 hepatocytes could be gradually adapted to survi ve in 5 mu M choline. CD-adapted cells had increased membrane phosphat idylcholine concentrations (compared with acute CD cells), Adapted cel ls acquired relative resistance to CD-induced apoptosis (7% of adapted cells compared with 19% of non-adapted cells were apoptotic at 48 h i n 5 mu M choline), They also became relatively resistant to another p5 3-independent form of apoptosis (TGF beta 1-induced), CD-adapted hepat ocytes developed increased capability for anchorage-independent growth and formed tumors when transplanted into nude mice; passage-matched c ontrol hepatocytes did not possess these properties, Cell transformati on was dependent on exposure to the selective pressure of CD apoptosis , as we observed that when CD apoptosis was inhibited with an antioxid ant during adaptation, cells did not become anchorage independent, Acq uisition by p53-deficient cells of resistance to p53-independent induc ers of apoptosis (CD, TGF beta 1 and reactive oxygen species) may leav e cells without another important apoptotic defensive barrier and may be responsible for the progression of initiated cells to frank carcino mas.