Tolomeo M, Dusonchet L, Meli M, Grimaudo S, D'Alessandro N, Papoff G, Ruberti G, Rausa L
Chair of Hematology, University of Palermo, Italy.
Cell Death Differ. 1998 Sep;5(9):735-42. doi: 10.1038/sj.cdd.4400406.
Many anticancer drugs are able to induce apoptosis in tumor cells but the mechanisms underlying this phenomenon are poorly understood. Some authors reported that the p53 tumor suppressor gene may be responsible for drug-induced apoptosis; however, chemotherapy-induced apoptosis can also be observed in p53 negative cells. Recently, doxorubicin (DXR) was reported to induce CD95L expression to mediate apoptosis through the CD95/CD95L system. Thus, an impairment of such a system may be involved in drug resistance. We evaluated the in vitro antitumor activity of several cytotoxic drugs on two human p53-negative T-cell lymphoma cell lines, the HUT78-B1 CD95L-resistant cell line and the HUT78 parental CD95L-sensitive cell line. We demostrated by Western blotting assay that DXR and etoposide (VP-16) were able to induce CD95L expression after 4 h of treatment. In contrast, they were unable to induce the expression of p53. DXR, at concentrations ranging from 0.001 - 1 microg/ml, and VP16, at concentrations ranging from 0.05 - 1 microg/ml, were equally cytotoxic and induced apoptosis in both cell lines as assessed by fluorescence microscopy and flow cytometry analyses. Although we observed a slightly reduced percentage of apoptotic cells in HUT78B1 when compared with the parental HUT78 cells after few hours of drug exposure, this difference was no longer evident at 48 or 72 h. Similarly, the exposure of HUT78 cells to a CD95-blocking antibody partially reduced early apoptosis (24 h) without affecting the long-term effects of the drugs including cytotoxicity. Furthermore, as observed with DXR and VP-16, both the CD95L-sensitive and the CD95L-resistant cell lines resulted equally sensitive to the cytotoxic effects of a number of different cytotoxic drugs (vincristine, camptothecin, 5-fluorouracil and methotrexate). The treatment with the Caspase-3 tetrapeptide aldehyde inhibitor, Ac-DEVD-CHO, did not affect the DXR-induced apoptosis whereas it only modestly inhibited apoptosis and cytotoxicity of VP-16, while Z-VAD.FMK, a Caspase inhibitor that prevents the processing of Caspase-3 to its active form, was able to block DXR-induced apoptosis at 24 h but not at 48 h. Thus, our results do not confirm a crucial role for the CD95/CD95L system in drug-induced apoptosis and suggest the involvement of alternative p53-independent pathways at least in this experimental model system.
许多抗癌药物能够诱导肿瘤细胞凋亡，但这种现象背后的机制却知之甚少。一些作者报道，p53肿瘤抑制基因可能是药物诱导凋亡的原因；然而，在p53阴性细胞中也能观察到化疗诱导的凋亡。最近，有报道称阿霉素（DXR）可诱导CD95L表达，通过CD95/CD95L系统介导凋亡。因此，该系统的损伤可能与耐药性有关。我们评估了几种细胞毒性药物对两种人p53阴性T细胞淋巴瘤细胞系（HUT78 - B1 CD95L耐药细胞系和HUT78亲本CD95L敏感细胞系）的体外抗肿瘤活性。我们通过蛋白质印迹分析证明，DXR和依托泊苷（VP - 16）在处理4小时后能够诱导CD95L表达。相比之下，它们不能诱导p53的表达。浓度范围为0.001 - 1微克/毫升的DXR和浓度范围为0.05 - 1微克/毫升的VP16具有同等的细胞毒性，并通过荧光显微镜和流式细胞术分析评估，在两种细胞系中均诱导凋亡。尽管在药物暴露数小时后，我们观察到与亲本HUT78细胞相比，HUT78B1中凋亡细胞的百分比略有降低，但在48或72小时时这种差异不再明显。同样，将HUT78细胞暴露于CD95阻断抗体可部分降低早期凋亡（24小时），而不影响包括细胞毒性在内的药物长期作用。此外，正如用DXR和VP - 16观察到的那样，CD95L敏感和CD95L耐药细胞系对多种不同细胞毒性药物（长春新碱、喜树碱、5 - 氟尿嘧啶和甲氨蝶呤）的细胞毒性作用同样敏感。用半胱天冬酶 - 3四肽醛抑制剂Ac - DEVD - CHO处理并不影响DXR诱导的凋亡，而它仅适度抑制VP - 16的凋亡和细胞毒性，而Z - VAD.FMK是一种半胱天冬酶抑制剂，可阻止半胱天冬酶 - 3加工成其活性形式，它能够在24小时时阻断DXR诱导的凋亡，但在48小时时则不能。因此，我们的结果并未证实CD95/CD95L系统在药物诱导凋亡中起关键作用，并表明至少在这个实验模型系统中存在替代的不依赖p53的途径。
