DETECTION OF PHOSPHATIDYLCHOLINE-SPECIFIC PHOSPHOLIPASE-C IN NIH-3T3 FIBROBLASTS AND THEIR H-RAS TRANSFORMANTS - NMR AND IMMUNOCHEMICAL STUDIES

Although evidence supports constitutive activation of phosphatidylchol ine specific phospholipase C (PC-plc) in ras-transformed fibroblasts, no studies have been devoted to measure the basal activity levels of t his enzyme, its molecular characteristics and subcellular localization . This paper reports for the first time measurements of the activity o f different enzymes responsible for PC hydrolysis (PC-plc; phospholipa ses A(2) (pla(2)) and A(1) (pla(1))) in homogenates of murine NIH-3T3 fibroblasts (3T3) and their transformants obtained by human H-ras tran sfection (3T3(ras)). To this end P-31 NMR analyses were carried out on total cell homogenates, incubated in the presence of mixed diheptanoy lphosphatidylcholine: sphingomyelin (DHPC:SM) unilamellar vesicles (SL UV), in which DHPC acts as a suitable substrate for water-soluble lipo lytic enzymes. The basal PC-plc activity levels (0.66 +/- 0.14 and 0.3 8 +/- 0.10 nmol/10(6) cells . hour in 3T3 and 3T3(ras) fibroblasts, re spectively), were substantially higher (over 30-50x) than those report ed in the literature for normal mammalian cells (dog heart myocytes). Moreover the PC-plc activity was about 15-30 times lower than the over all PC deacylation activity in both clones. The use of high titer poly clonal antibodies, raised in a rabbit against bacterial PC-plc, allowe d identification of one cross-reactive mammalian PC-plc component (M(r ) 66 kD) in cell lysates of both 3T3 and 3T3(ras) fibroblasts, and det ection, by indirect immunofluorescence, of its subcellular localizatio n. In control 3T3 fibroblasts (in the late log-phase of growth) the en zyme was exclusively located in the cytosol, while in H-ras transforme d cells it was massively exposed on the external side of the membrane. This new finding strongly suggests that the oncogenic product p21(ras ) is able to induce (or mediate) translocation of PC-plc across the pl asma membrane of ras transformed cells with possible implications not only on cell biochemistry (enhancement of PC-plc activity and conseque nt production of intra- and extracellular PCho and accumulation of neu tral lipids) bur also on cell-cell interaction mechanisms which facili tate tumour invasion and metastasis of oncogene-transformed cells.