A tumour promoter, 12-O-tetradecanoylphorbol 13-acetate, increases cellular 1,2-diacylglycerol content through a mechanism other than phosphoinositide hydrolysis in Swiss-mouse 3T3 fibroblasts.
Treatment of Swiss-mouse 3T3 fibroblasts with a tumour-promoting phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), increases the absolute amount of 1,2-diacylglycerol (1,2-DG) in a time- and dose-dependent manner. Within 1 min of TPA (0.1 microM) addition to confluent monolayer cells, cellular 1,2-DG content increases significantly, and reaches a value nearly 4-fold above control at 10 min. The content of 1,2-DG then falls, but still remains at twice the basal value for up to 60 min. The effect of TPA on 1,2-DG content is dose-dependent, with a half-maximal effect obtained with 6 nM. Studies with a series of tumour promoters have revealed that stimulating effect on 1,2-DG formation is closely related to their relative potency as a tumour promoter, as well as their mitogenic effect on 3T3 cells. TPA does not have any stimulatory effect on phosphoinositide turnover, which is a major mechanism of 1,2-DG formation by natural agonists, including a peptide mitogen, bombesin, but significantly inhibits bombesin-induced phosphoinositide turnover. Nevertheless, the content of 1,2-DG is even higher in cells treated with both bombesin and TPA than in cells treated with bombesin alone. In addition, a diacylglycerol kinase inhibitor, R59022, increases 1,2-DG content synergistically with bombesin, but not with TPA. Measurements of the absolute amounts of various phospholipids show that TPA does not increase phosphatidic acid, despite a remarkable increase in 1,2-DG. Further studies on phospholipid metabolism reveal that TPA stimulates metabolic turnover of phosphatidylcholine (PC), without changing the mass of PC. TPA stimulates the incorporation of both [3H]choline and [32P]Pi into PC, and the release of [3H]choline metabolites from prelabelled cells. These findings suggest that TPA increases cellular 1,2-DG content mainly via increased PC turnover and diacylglycerol kinase inhibition. The present study provides evidence for a novel effect of TPA on the metabolism of 1,2-DG in Swiss-mouse 3T3 cells.
Takuwa
N
N
Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06510.
Takuwa
Y
Y
Rasmussen
H
H
D000818:Animals; D001839:Bombesin / Q000494:pharmacology; D002273:Carcinogens / Q000494:pharmacology; D002478:Cells, Cultured; D002794:Choline / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis; D004075:Diglycerides / Q000378:metabolism*; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism*; D005989:Glycerides / Q000378:metabolism*; D051379:Mice; D010716:Phosphatidylinositols / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
C011439:1,2-diacylglycerol; D002273:Carcinogens; D004075:Diglycerides; D005989:Glycerides; D010716:Phosphatidylinositols; D004247:DNA; D002794:Choline; D013755:Tetradecanoylphorbol Acetate; D001839:Bombesin