Takuwa N, Takuwa Y, Rasmussen H
Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06510.
Biochem J. 1987 May 1;243(3):647-53. doi: 10.1042/bj2430647.
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.
用促肿瘤佛波酯12 - O -十四烷酰佛波醇13 - 乙酸酯（TPA）处理瑞士小鼠3T3成纤维细胞，1,2 -二酰基甘油（1,2 - DG）的绝对量会随时间和剂量呈依赖性增加。将TPA（0.1微摩尔）添加到汇合的单层细胞中1分钟内，细胞内1,2 - DG含量显著增加，并在10分钟时达到比对照高近4倍的值。然后1,2 - DG含量下降，但在长达60分钟内仍保持在基础值的两倍。TPA对1,2 - DG含量的影响呈剂量依赖性，6纳摩尔时可获得半数最大效应。对一系列肿瘤促进剂的研究表明，对1,2 - DG形成的刺激作用与其作为肿瘤促进剂的相对效力以及对3T3细胞的促有丝分裂作用密切相关。TPA对磷脂酰肌醇周转率没有任何刺激作用，磷脂酰肌醇周转率是包括肽促有丝分裂剂蛙皮素在内的天然激动剂形成1,2 - DG的主要机制，但能显著抑制蛙皮素诱导的磷脂酰肌醇周转率。然而，同时用蛙皮素和TPA处理的细胞中1,2 - DG的含量甚至比单独用蛙皮素处理的细胞更高。此外，二酰基甘油激酶抑制剂R59022与蛙皮素协同增加1,2 - DG含量，但与TPA无协同作用。对各种磷脂绝对量的测量表明，尽管1,2 - DG显著增加，但TPA不会增加磷脂酸。对磷脂代谢的进一步研究表明，TPA刺激磷脂酰胆碱（PC）的代谢周转，而不改变PC的质量。TPA刺激[3H]胆碱和[32P]Pi掺入PC，并刺激预标记细胞释放[3H]胆碱代谢物。这些发现表明，TPA主要通过增加PC周转和抑制二酰基甘油激酶来增加细胞内1,2 - DG含量。本研究为TPA对瑞士小鼠3T3细胞中1,2 - DG代谢的新作用提供了证据。
