S-phase induction and transformation of quiescent NIH 3T3 cells by microinjection of phospholipase C.

Abstract

Two inositol phospholipid-specific phospholipase C (PLC) isozymes (PLC-I and -II) have been purified from bovine brain. When PLC-I or PLC-II was microinjected (100-700 .mu.g/ml) into quiescent NIH 3T3 cells, a time- and dose-dependent induction of DNA synthesis occurred, as demonstrated by [3H]thymidine incorporation into nuclear DNA. In addition, .apprxeq. 8 hr after PLC injection, NIH 3T3 fibroblasts appeared spindle-shaped, refractile, and highly vacuolated, displaying a morphology similar to transformed cells. The morphologic transformation was apparent for 26-30 hr after which the injected cells reverted back to a normal phenotype. Microinjected PLC at a high concentration (1 mg/ml) was cytotoxic, dissolving the cytoplasmic membrane and leaving behind cellular ghosts. PLC is a key regulatory enzyme involved in cellular membrane signal transduction. Introduction of exogenous PLC Into NIH 3T3 cells by microinjection induced a growth and oncogenic potential, as demonstated by the ability of microinjected PLC (.apprxeq. 10,000 molecules per cell) to override the cellular Go block, inducing DNA synthesis and morphologic transformation of growth-arrested fibroblast cells.