Hochegger K, Partik G, Schörkhuber M, Marian B
Institute of Tumor Biology/Cancer Research, University of Vienna, Vienna, Austria.
Int J Cancer. 1999 Nov 26;83(5):650-6. doi: 10.1002/(sici)1097-0215(19991126)83:5<650::aid-ijc14>3.0.co;2-7.
Protein-kinase-C signalling has been blocked in colorectal tumor cells by kinase inhibitors, by TPA down-regulation or by exposure to anti-sense oligonucleotides. This resulted in growth inhibition in all cell lines used. The kinase inhibitors H7 and calphostin induced apoptosis, demonstrated by the appearance of cells with characteristically condensed chromatin and the induction of stand-breaks in the DNA. A cell-death-inducing concentration of 15 microgram/ml H7 down-regulated the bcl-2 levels after 9 hr, while bak levels were not affected. Gö6976,-an inhibitor of Ca(++)-dependent PKC iso-enzymes, was not active in growth inhibition or induction of apoptosis. Analysis of DNA synthesis in inhibitor-treated cultures indicated that H7 caused strong inhibition in all cell lines, while the more specific inhibitor calphostin was effective only in VACO235 adenoma cells. When down-regulation by TPA or anti-sense oligonucleotides was used to block PKC, effects on cell numbers were smaller and delayed. However, induction of apoptosis was significantly increased in SW480 carcinoma cells 4 days after exposure to anti-epsilon and anti-zeta oligonucleotides in SW480 and T84 carcinoma cells. Apoptosis was preceeded by loss of PKC protein and of bcl-2 from day 1 after addition of the oligonucleotides. In VACO235 adenoma cells, no induction of apoptosis could be observed when anti-epsilon and anti-zeta oligonucleotides were used. On the other hand, the adenoma cells were more responsive to anti-alpha and anti-beta oligonucleotides, which strongly inhibited DNA-synthesis 3 days after addition to the culture medium. Our results indicate that the Ca(++)-dependent PKCs alpha and beta are involved in proliferation signals, while the Ca(++)-independent PKCs epsilon and zeta are involved in survival pathways of colorectal tumor cells.
蛋白激酶C信号传导已通过激酶抑制剂、佛波酯（TPA）下调或暴露于反义寡核苷酸而在结肠直肠肿瘤细胞中被阻断。这导致了所用所有细胞系的生长抑制。激酶抑制剂H7和钙泊三醇诱导了细胞凋亡，表现为出现具有特征性浓缩染色质的细胞以及DNA链断裂的诱导。细胞死亡诱导浓度为15微克/毫升的H7在9小时后下调了bcl-2水平，而bak水平未受影响。Gö6976，一种钙（++）依赖性蛋白激酶C同工酶的抑制剂，在生长抑制或细胞凋亡诱导方面没有活性。对抑制剂处理的培养物中DNA合成的分析表明，H7在所有细胞系中都引起了强烈抑制，而更具特异性的抑制剂钙泊三醇仅在VACO235腺瘤细胞中有效。当使用TPA或反义寡核苷酸下调来阻断蛋白激酶C时，对细胞数量的影响较小且延迟。然而，在SW480和T84癌细胞中，暴露于抗ε和抗ζ寡核苷酸4天后，SW480癌细胞中的细胞凋亡显著增加。在添加寡核苷酸后第1天，细胞凋亡之前先出现蛋白激酶C蛋白和bcl-2的丢失。在VACO235腺瘤细胞中，当使用抗ε和抗ζ寡核苷酸时未观察到细胞凋亡诱导。另一方面，腺瘤细胞对抗α和抗β寡核苷酸更敏感，在添加到培养基中3天后强烈抑制DNA合成。我们的结果表明，钙（++）依赖性蛋白激酶Cα和β参与增殖信号，而钙（++）非依赖性蛋白激酶Cε和ζ参与结肠直肠肿瘤细胞的存活途径。
