Ye X, Georgoff I, Fleisher S, Coffman F D, Cohen S, Fresa K L
Department of Pathology and Laboratory Medicine, Hahnemann University, Philadelphia, Pennsylvania 19102.
Cell Immunol. 1993 Oct 15;151(2):320-35. doi: 10.1006/cimm.1993.1242.
The epipodophyllotoxins, etoposide (VP-16) and teniposide (VM-26), inhibit topoisomerase II activity by stabilization of the cleavable complex between the enzyme and DNA and formation of protein-bound double-stranded DNA breaks. While it is thought that these agents are cytotoxic by preventing cells from completing the S phase or undergoing mitosis, recent evidence suggests that these agents are also potent inducers of programmed cell death or apoptosis in both normal and malignant cells. We have examined the intracellular pathway leading to epipodophyllotoxin-induced apoptosis in normal mouse thymocytes. Epipodophyllotoxin-induced apoptosis may proceed via a mechanism that is independent of inhibition of topoisomerase activity per se because novobiocin and coumermycin, which inhibit the ATPase subunit of topoisomerase II, were relatively inefficient inducers of apoptosis in these cells, under conditions where strong apoptosis by the epipodophyllotoxins and dexamethasone could be observed. In addition, camptothecin, which inhibits topoisomerase I by stabilization of the cleavable complex between that enzyme and DNA, was also a poor inducer of apoptosis in these cells. Our data suggest that epipodophyllotoxin-induced mouse thymocyte apoptosis, like that induced by dexamethasone, proceeds via a mechanism that involves protein kinase C (PKC) or a similar enzyme. Apoptosis induced by VM-26 or by dexamethasone was inhibited by 1-(5-isoquinolinylsulfonyl)-2- methylpiperazine dihydrochloride (H7), an inhibitor of both PKC and cAMP-dependent protein kinases, but was relatively unaffected by N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA1004), a more specific inhibitor of cAMP-dependent protein kinases. A more specific inhibitor of PKC, sangivamycin, also inhibited both VM-26-induced and dexamethasone-induced apoptosis. Both VM-26- and dexamethasone-induced apoptosis were unaffected by EGTA, a calcium (Ca2+) chelator, under conditions that inhibited apoptosis induced by the Ca2+ ionophore A23187. Moreover, while strong increases in intracellular Ca2+ were observed in thymocytes treated with A23187, we failed to detect increases in intracellular Ca2+ in cells induced to apoptose with either VM-26 or dexamethasone within the first 2 hr of culture. These results suggest that in mouse thymocytes there are at least two intracellular pathways leading to apoptosis: one, utilized by glucocorticoid and the epipodophyllotoxins, that proceeds in the absence of detectable increases in intracellular Ca2+ and possibly requires a novel Ca(2+)-independent PKC-like enzyme and another, utilized by Ca2+ ionophores, that is at least partially dependent on increased intracellular Ca2+.
表鬼臼毒素、依托泊苷（VP - 16）和替尼泊苷（VM - 26）通过稳定酶与DNA之间的可裂解复合物以及形成蛋白质结合的双链DNA断裂来抑制拓扑异构酶II的活性。虽然人们认为这些药物通过阻止细胞完成S期或进行有丝分裂而具有细胞毒性，但最近的证据表明，这些药物在正常细胞和恶性细胞中也是程序性细胞死亡或凋亡的有效诱导剂。我们研究了正常小鼠胸腺细胞中导致表鬼臼毒素诱导凋亡的细胞内途径。表鬼臼毒素诱导的凋亡可能通过一种独立于拓扑异构酶活性抑制本身的机制进行，因为抑制拓扑异构酶II的ATP酶亚基的新生霉素和香豆霉素在这些细胞中是相对低效的凋亡诱导剂，而在这些条件下可以观察到表鬼臼毒素和地塞米松强烈诱导凋亡。此外，通过稳定该酶与DNA之间的可裂解复合物来抑制拓扑异构酶I的喜树碱在这些细胞中也是一种较差的凋亡诱导剂。我们的数据表明，表鬼臼毒素诱导小鼠胸腺细胞凋亡，与地塞米松诱导的凋亡一样，通过一种涉及蛋白激酶C（PKC）或类似酶的机制进行。VM - 26或地塞米松诱导的凋亡受到1 -（5 - 异喹啉磺酰基）- 2 - 甲基哌嗪二盐酸盐（H7）的抑制，H7是PKC和cAMP依赖性蛋白激酶的抑制剂，但相对不受N -（2 - 胍基乙基）- 5 - 异喹啉磺酰胺（HA1004）的影响，HA1004是cAMP依赖性蛋白激酶的更特异性抑制剂。PKC的更特异性抑制剂桑吉瓦霉素也抑制VM - 26诱导和地塞米松诱导的凋亡。在抑制Ca2 +离子载体A23187诱导的凋亡的条件下，VM - 26和地塞米松诱导的凋亡均不受EGTA（一种钙（Ca2 +）螯合剂）的影响。此外，虽然在用A23187处理的胸腺细胞中观察到细胞内Ca2 +强烈增加，但我们未能在培养的前2小时内检测到用VM - 26或地塞米松诱导凋亡的细胞中细胞内Ca2 +的增加。这些结果表明，在小鼠胸腺细胞中至少有两条导致凋亡的细胞内途径：一条是糖皮质激素和表鬼臼毒素利用的途径，在细胞内Ca2 +没有可检测到的增加的情况下进行，可能需要一种新的不依赖Ca（2 +）的PKC样酶；另一条是Ca2 +离子载体利用的途径，至少部分依赖于细胞内Ca2 +的增加。
