Brossas Jean-Yves, Tanguy Ronan, Brignole-Baudouin Francoise, Courtois Yves, Torriglia Alicia, Tréton Jacques
INSERM Unité 450, Association Claude Bernard, Paris, France.
Mol Vis. 2004 Jan 27;10:65-73.
To analyse the mechanism of ethanol-induced cell death, and particularly, the activation of the leucocyte elastase inhibitor (LEI) pathway.
Cultured ARPE-19 cells were exposed to 0-13% ethanol for 24 h. Cytotoxicity was estimated by morphologic changes within the nucleus and breakdown of DNA, assessed by agarose gel electrophoresis or flow cytometry cell sorter. Poly(ADP-ribose)polymerase cleavage (PARP) was determined by western blot analysis. Changes in transcription and translation of LEI were assessed by analysis of mRNA levels and expression of protein product (immunohistochemistry), respectively.
We established the ability of ethanol to induce cell death in ARPE-19 cells. After a 24 h incubation with 4% ethanol, 50% of the cells died; all the cells died in the presence of 10% ethanol. After ethanol incubation, we observed nuclear condensation and DNA fragmentation; the amount of fragmentation was proportional to the ethanol level. By flow cytometry analysis and agarose gel electrophoresis, the pattern of DNA cleavage exhibited a sub-G1 peak, suggesting necrotic cell death. However, other observations, i.e. nuclei shrinkage, PARP cleavage and inhibition of cell death by cycloheximide, and activation of a caspase independent LEI/DNase II pathway were observed and are features associated with apoptotic cell death. During ethanol stress, an LEI/L-DNase II intermediate was lost, leading to complete activation L-DNase II (24 kDa). RT-PCR analysis showed an early and specific increase of the LEI mRNA. Cycloheximide inhibited LEI synthesis and protected cells against apoptosis.
Our data indicate that ethanol stress on ARPE-19 cells can induce a pathway which is a form of programmed cell death with characteristics of both apoptosis and necrosis, possibly by triggering conversion of LEI to L-DNase II.
分析乙醇诱导细胞死亡的机制，特别是白细胞弹性蛋白酶抑制剂（LEI）途径的激活机制。
将培养的ARPE - 19细胞暴露于0 - 13%的乙醇中24小时。通过细胞核内的形态变化和DNA断裂来评估细胞毒性，采用琼脂糖凝胶电泳或流式细胞仪细胞分选仪进行检测。通过蛋白质印迹分析确定聚（ADP - 核糖）聚合酶裂解（PARP）情况。分别通过分析mRNA水平和蛋白质产物表达（免疫组织化学）来评估LEI转录和翻译的变化。
我们确定了乙醇诱导ARPE - 19细胞死亡的能力。用4%乙醇孵育24小时后，50%的细胞死亡；在10%乙醇存在的情况下所有细胞均死亡。乙醇孵育后，我们观察到细胞核浓缩和DNA片段化；片段化程度与乙醇浓度成正比。通过流式细胞术分析和琼脂糖凝胶电泳，DNA裂解模式呈现亚G1峰，提示坏死性细胞死亡。然而，还观察到其他现象，即细胞核收缩、PARP裂解以及环己酰亚胺对细胞死亡的抑制，还有半胱天冬酶非依赖性的LEI/DNase II途径的激活，这些都是与凋亡性细胞死亡相关的特征。在乙醇应激期间，一种LEI/L - DNase II中间体丢失，导致L - DNase II（24 kDa）完全激活。逆转录 - 聚合酶链反应（RT - PCR）分析显示LEI mRNA早期特异性增加。环己酰亚胺抑制LEI合成并保护细胞免受凋亡。
我们的数据表明，乙醇对ARPE - 19细胞的应激可诱导一种程序性细胞死亡途径，该途径具有凋亡和坏死的特征，可能是通过触发LEI向L - DNase II的转化实现的。
