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 carcinomas are poorly understood. CWSV-1 rat hepatocytes, in which p53 protein is inactivated by SV40 large T antigen, respond by inducing p53-independent apoptosis when acutely switched to medium containing low choline (16% apoptotic at 48 h in 5 μM choline) as compared with controls (1% apoptotic at 48 h in 70 μM choline). The rate of apoptosis was inversely correlated with cellular phosphatidylcholine content. Choline deficiency (CD)-induced apoptosis is probably mediated by TGFβ1 and reactive oxygen species, since immunoneutralization of TGFβ1 in the medium or treatment with N-acetyl-cysteine (an antioxidant) or addition of neocuproine (a transition metal chelator) prevented CD-induced apoptosis. CWSV-1 hepatocytes could be gradually adapted to survive in 5 μM choline. CD-adapted cells had increased membrane phosphatidylcholine concentrations (compared with acute CD cells). Adapted cells acquired relative resistance to CD-induced apoptosis (7% of adapted cells compared with 19% of non-adapted cells were apoptotic at 48 h in 5 μM choline). They also became relatively resistant to another p53-independent form of apoptosis (TGFβ1-induced). CD-adapted hepatocytes developed increased capability for anchorage-independent growth and formed tumors when transplanted into nude mice; passage-matched control hepatocytes did not possess these properties. Cell transformation was dependent on exposure to the selective pressure of CD apoptosis, as we observed that when CD apoptosis was inhibited with an antioxidant during adaptation, cells did not become anchorage independent. Acquisition by p53-deficient cells of resistance to p53-independent inducers of apoptosis (CD, TGFβ1 and reactive oxygen species) may leave cells without another important apoptotic defensive barrier and may be responsible for the progression of initiated cells to frank carcinomas.