NIH 3T3 CELLS STABLY TRANSFECTED WITH THE GENE ENCODING PHOSPHATIDYLCHOLINE-HYDROLYZING PHOSPHOLIPASE-C FROM BACILLUS-CEREUS ACQUIRE A TRANSFORMED PHENOTYPE

In order to determine whether chronic elevation of intracellular diacy lglycerol levels generated by hydrolysis of phosphatidylcholine (PC) b y PC-hydrolyzing phospholipase C (PC-PLC) is oncogenic, we generated s table transfectants of NIH 3T3 cells expressing the gene encoding PC-P LC from Bacillus cereus. We found that constitutive expression of this gene (plc) led to transformation of NIH 3T3 cells as evidenced by anc horage-independent growth in soft agar, formation of transformed foci in tissue culture, and loss of contact inhibition. The plc transfectan ts displayed increased intracellular levels of diacylglycerol and phos phocholine. Expression of B. cereus PC-PLC was confirmed by immunopero xidase and immunofluorescence staining with an affinity-purified anti- PC-PLC antibody. The NIH 3T3 clones expressing plc induced DNA synthes is, progressed through the cell cycle in the absence of added mitogens , and showed significant growth in low-concentration serum. Transfecti on with an antisense plc expression vector led to a loss of PC-PLC exp ression accompanied by a complete reversion of the transformed phenoty pe, suggesting that plc expression was required for maintenance of the transformed state. Taken together, our results show that chronic stim ulation of PC hydrolysis by an unregulated PC-PLC enzyme is oncogenic to NIH 3T3 cells.