Depletion of intracellular NAD+ and ATP levels during ricin-induced apoptosis through the specific ribosomal inactivation results in the cytolysis of U937 cells
Depletion of intracellular NAD+ and ATP levels during ricin-induced apoptosis through the specific ribosomal inactivation results in the cytolysis of U937 cells
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DOI：
10.1093/oxfordjournals.jbchem.a022775
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发表时间：
2000-09-01
影响因子：
2.7
通讯作者：
Muramatsu, T
中科院分区：
文献类型：
--
作者：
Komatsu, N;Nakagawa, M;Muramatsu, T
我们前期的研究表明，蓖麻毒素诱导U937细胞凋亡，表现为DNA断裂、细胞核形态改变和caspase样活性增加。在本研究中，我们发现蓖麻毒素处理的U937细胞内NAD（+）和ATP水平下降，并且这种下降伴随着蓖麻毒素介导的蛋白质合成抑制。PARP抑制剂，3-氨基苯甲酰胺（3-ABA），防止NAD（+）和ATP水平的消耗，并同时保护U937细胞免受蓖麻毒素处理后的裂解。因此，3-ABA的保护作用是由于对PARP的抑制，而不是由于其其他药理副作用。此外，暴露于蓖麻毒素3 h后，PARP酶活性逐渐升高，达到最大水平，而未处理细胞的酶活性没有明显变化。然而，3- aba对蓖麻毒素介导的DNA断裂没有影响。在。此外，免疫印迹分析显示，加入蓖麻毒素后12 h以上出现了显著的PARP裂解，而DNA断裂在孵育后6 h内达到最大水平。因此，在蓖麻毒素诱导细胞凋亡的情况下，PARP切割似乎不是与细胞凋亡发生相关的早期凋亡事件。我们的研究结果表明，蓖麻毒素可能触发多种凋亡信号通路。可能，通过PARP激活和NAD（+）耗竭导致细胞裂解的途径独立于导致DNA断裂的途径，而半胱天冬酶可能深刻参与了DNA断裂。其他蛋白质合成抑制剂，包括白喉毒素和环己亚胺，在诱导DNA断裂和细胞溶解方面效果较差，即使在显著抑制蛋白质合成的浓度下也是如此。因此，蓖麻毒素使核糖体失活的特定作用机制可能是导致蓖麻毒素诱导的细胞凋亡事件的原因。
Our previous studies demonstrated that ricin induces the apoptotic death of U937 cells as evidenced by DNA fragmentation, nuclear morphological changes, and increases in caspase-like activities. In this study, we have found that intracellular NAD(+) and ATP levels decrease in ricin-treated U937 cells and that this decrease is followed by the ricin-mediated protein synthesis inhibition. The PARP inhibitor, 3-aminobenzamide (3-ABA), prevents the depletion in NAD(+) and ATP levels and concomitantly protects U937 cells from the lysis that follows ricin treatment. Hence, the protective action of 3-ABA is due to the inhibition of PARP and does not result from its other pharmacological side effects. Moreover, the enzymatic activity of PARP gradually increases and reaches a maximum level after ricin exposure for 3 h, whereas no significant change in activity was observed in untreated cells, However, 3-ABA has no effect on ricin-mediated DNA fragmentation. In. addition, immunoblot analysis revealed that significant PARP cleavage occurred more than 12 h after ricin addition, while DNA fragmentation reached a maximum level within 6 h of incubation. Thus, in the case of ricin-induced apoptosis, it appears that PARP cleavage is not an early apoptotic event associated with the onset of apoptosis, Our results suggest that multiple apoptotic signaling pathways may be triggered by ricin-treatment. Probably, the pathway leading to cell lysis via PARP activation and NAD(+) depletion is independent of the pathway leading to DNA fragmentation in which caspases may be profoundly involved. Other protein synthesis inhibitors, including diphtheria toxin and cycloheximide, were less effective in terms of inducing DNA fragmentation and cytolysis, even at concentrations that cause significant inhibition of protein synthesis, Thus, a specific ricin action mechanism through which ribosomes are inactivated may be responsible for the apoptotic events induced by ricin.