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 diacylglycerol levels generated by hydrolysis of phosphatidylcholine (PC) by PC-hydrolyzing phospholipase C (PC-PLC) is oncogenic, we generated stable transfectants of NIH 3T3 cells expressing the gene encoding PC-PLC from Bacillus cereus. We found that constitutive expression of this gene (plc) led to transformation of NIH 3T3 cells as evidenced by anchorage-independent growth in soft agar, formation of transformed foci in tissue culture, and loss of contact inhibition. The plc transfectants displayed increased intracellular levels of diacylglycerol and phosphocholine. Expression of B. cereus PC-PLC was confirmed by immunoperoxidase and immunofluorescence staining with an affinity-purified anti-PC-PLC antibody. The NIH 3T3 clones expressing plc induced DNA synthesis, progressed through the cell cycle in the absence of added mitogens, and showed significant growth in low-concentration serum. Transfection with an antisense plc expression vector led to a loss of PC-PLC expression accompanied by a complete reversion of the transformed phenotype, suggesting that plc expression was required for maintenance of the transformed state. Taken together, our results show that chronic stimulation of PC hydrolysis by an unregulated PC-PLC enzyme is oncogenic to NIH 3T3 cells.
Johansen
T
T
Department of Biochemistry, University of Tromsø, Norway.
Bjørkøy
G
G
Overvatn
A
A
Diaz-Meco
M T
MT
Traavik
T
T
Moscat
J
J
D016475:3T3 Cells / Q000166:cytology / Q000378:metabolism; D000818:Animals; D001409:Bacillus cereus / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D002453:Cell Cycle; D004247:DNA / Q000096:biosynthesis; D016373:DNA, Antisense / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004075:Diglycerides / Q000378:metabolism; D005822:Genetic Vectors; D006868:Hydrolysis; D051379:Mice; D008969:Molecular Sequence Data; D010641:Phenotype; D010713:Phosphatidylcholines / Q000378:metabolism; D014162:Transfection; D010738:Type C Phospholipases / Q000235:genetics*
D016373:DNA, Antisense; D004269:DNA, Bacterial; D004075:Diglycerides; D010713:Phosphatidylcholines; D004247:DNA; D010738:Type C Phospholipases