Internucleosomal DNA cleavage precedes diphtheria toxin-induced cytolysis. Evidence that cell lysis is not a simple consequence of translation inhibition.
Internucleosomal DNA cleavage precedes diphtheria toxin-induced cytolysis. Evidence that cell lysis is not a simple consequence of translation inhibition.
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发表时间：
1989-09
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通讯作者：
Michael;Chang;John BramhallQII;Scott Graves;Benjamin Bonavida;Bernadine J. WisnieskiS
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作者：
Michael;Chang;John BramhallQII;Scott Graves;Benjamin Bonavida;Bernadine J. WisnieskiS
白喉毒素（DTx）是一种非常有效的蛋白质合成抑制剂。细胞死亡被普遍认为是翻译抑制的直接作用。使用人U937细胞，我们发现DTx中毒导致细胞溶解；事实上，在7小时内可以检测到51Cr和75se标记的蛋白质的释放。然而，当人U937细胞暴露于环己亚胺、氨基酸缺乏培养基或代谢性毒物中20-50小时内，即使蛋白质合成被迅速抑制到DTx所观察到的水平，也很少或没有观察到细胞裂解。同样，对人类K562细胞的研究显示，尽管翻译活性严重降低，但在50小时内对DTx的细胞溶解作用有完全的抗性。这些观察结果表明，抑制蛋白质合成本身并不足以引起细胞裂解。对DTx诱导的细胞溶解的表征表明，DTx有一个很长的滞后期（6-7小时），短时间暴露于低ph的NH4Cl中可以大大缩短这一滞后期，代谢性毒物阻断了DTx的细胞溶解作用，这表明溶解活性需要毒素的内吞摄取。令人惊讶的是，DTx还诱导了广泛的核小体间细胞DNA降解，这是细胞凋亡或程序性细胞死亡的特征。dna断裂发生在细胞裂解之前，在dtx处理的K562细胞和其他蛋白质合成抑制剂处理的U937细胞中没有发生dna断裂。从这些观察结果中，我们得出结论，DTx介导的细胞溶解不是翻译抑制的简单结果，核体间DNA断裂是DTx细胞溶解途径中一个新发现的相对较早的步骤。
Diphtheria toxin (DTx) is an extremely potent inhibitor of protein synthesis. Cell death has been generally accepted as a straightforward effect of translation inhibition. Using human U937 cells, we found that DTx intoxication leads to cytolysis; indeed, release of 51Cr- and 75Se-labeled proteins could be detected within 7 h. However, little or no cell lysis was observed over a 20-50-h period when human U937 cells were exposed to cycloheximide, amino acid-deficient medium, or metabolic poisons even though protein synthesis was rapidly inhibited to levels observed with DTx. Likewise, investigations with human K562 cells revealed full resistance to the cytolytic action of DTx over a 50-h period despite a severe reduction in translation activity. These observations establish that inhibition of protein synthesis per se is not sufficient to provoke cell lysis. A characterization of DTx-induced cytolysis revealed a long lag period (6-7 h) which could be shortened considerably by a short exposure to low pH. NH4Cl and metabolic poisons blocked the cytolytic action of DTx, indicating that endocytic uptake of toxin is required for lytic activity. Surprisingly, DTx also induced extensive internucleosomal degradation of cellular DNA, a characteristic feature of apoptosis or programmed cell death. DNA-fragmentation preceded cell lysis and did not occur in DTx-treated K562 cells or in U937 cells that were treated with the other protein synthesis inhibitors. From these observations, we conclude that DTx-mediated cytolysis is not a simple consequence of translation inhibition and that internucleosomal DNA fragmentation is a newly identified and relatively early step in the cytolytic pathway of DTx.