INDUCTION OF APOPTOTIC DNA-DAMAGE AND CELL-DEATH BY ACTIVATION OF THE SPHINGOMYELIN PATHWAY
INDUCTION OF APOPTOTIC DNA-DAMAGE AND CELL-DEATH BY ACTIVATION OF THE SPHINGOMYELIN PATHWAY
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DOI：
10.1073/pnas.91.1.73
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发表时间：
1994-01-04
影响因子：
11.1
通讯作者：
GRANT, S
中科院分区：
文献类型：
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作者：
JARVIS, WD;KOLESNICK, RN;GRANT, S
在人白血病细胞系 HL-60 和 U937 以及鼠纤维肉瘤细胞系 L929/LM 和 WEHI 164/13 中，通过多种生化策略评估了神经酰胺相关信号传导过程在肿瘤坏死因子 a 诱导细胞凋亡中的潜在参与。这些细胞暴露于肿瘤坏死因子α，导致基因组DNA发生核小体间裂解，当通过琼脂糖凝胶电泳解析时，产生具有凋亡特征的寡核小体片段的阶梯模式；暴露于外源性鞘磷脂酶或合成神经酰胺后观察到类似的反应。定量分光荧光光度法表明，这些处理促进了 DNA 的时间和浓度依赖性降解，导致小 DNA 片段（小于或等于 3.0 kb）的形成并最终释放。通过增强荧光碱性解旋分析证明了对大量 DNA 的相应损伤。 DNA 片段化不是由磷脂酶 C 或合成甘油二酯诱导的；事实上，鞘磷脂酶和神经酰胺的作用因同时接触这些药物而大大降低，这表明甘油二酯和神经酰胺介导的信号在细胞凋亡调节中发挥相反的作用。磷脂酶 A2 和花生四烯酸不能促进 DNA 断裂，磷脂酶 D 也是如此。通过碱性和中性洗脱分析对 DNA 链断裂的表征证实，神经酰胺的作用仅限于成熟双链 DNA 的断裂，而不是新生 DNA 的断裂。 DNA 损伤的诱导与细胞凋亡形态的出现和克隆形成性的降低有关。这些结果表明，神经酰胺依赖性信号系统选择性诱导细胞凋亡，并提出神经酰胺激活酶代表参与程序性细胞死亡调节的信号级联中的重要组成部分的可能性。
The potential involvement of ceramide-related signaling processes in the induction of apoptosis by tumor necrosis factor a was assessed by multiple biochemical strategies in the human leukemic cell lines HL-60 and U937 and the murine fibrosarcoma cell lines L929/LM and WEHI 164/13. Exposure of these cells to tumor necrosis factor alpha resulted in internucleosomal cleavage of genomic DNA, yielding laddered patterns of oligonucleosomal fragments characteristic of apoptosis when resolved by agarose gel electrophoresis; similar responses were observed after exposure to exogenous sphingomyelinase or synthetic ceramides. Quantitative spectrofluorophotometry demonstrated that these treatments promoted time- and concentration-dependent degradation of DNA, resulting in the formation of and eventual release of small DNA fragments (less-than-or-equal-to 3.0 kb). Corresponding damage to bulk DNA was demonstrated by enhanced-fluorescence alkaline unwinding analysis. DNA fragmentation was not induced by phospholipase C or synthetic diglyceride; in fact, the effects of sphingomyelinase and ceramide were substantially reduced by coexposure to these agents, suggesting opposing roles for diglyceride- and ceramide-mediated signals in the regulation of apoptosis. Phospholipase A2 and arachidonic acid failed to promote DNA fragmentation, as did phospholipase D. Characterization of DNA strand breaks by alkaline and neutral elution analyses confirmed that ceramide action was restricted to breakage of mature, double-stranded DNA but not of nascent DNA. The induction of DNA damage was associated with appearance of apoptotic morphology and decreased clonogenicity. These results demonstrate that the ceramide-dependent signaling system selectively induces apoptosis and raise the possibility that ceramide-activated enzymes represent important components in a signaling cascade involved in the regulation of programmed cell death.