L-DNase II associated with active process during ethanol inducedcell death in ARPE-19

[摘要] Purpose: To analyse the mechanism of ethanol-induced cell death, andparticularly, the activation of the leucocyte elastase inhibitor (LEI)pathway.Methods: Cultured ARPE-19 cells were exposed to 0-13% ethanol for 24h. Cytotoxicity was estimated by morphologic changes within the nucleusand breakdown of DNA, assessed by agarose gel electrophoresis or flowcytometry cell sorter. Poly(ADP-ribose)polymerase cleavage (PARP) wasdetermined by western blot analysis.Changes in transcription andtranslation of LEI were assessed by analysis of mRNA levels andexpression of protein product (immunohistochemistry), respectively.Results: We established the ability of ethanol to induce cell deathin ARPE-19 cells. After a 24 h incubation with 4% ethanol, 50% of thecells died; all the cells died in the presence of 10% ethanol. Afterethanol incubation, we observed nuclear condensation and DNAfragmentation; the amount of fragmentation was proportional to theethanol level. By flow cytometry analysis and agarose gelelectrophoresis, the pattern of DNA cleavage exhibited a sub-G1 peak,suggesting necrotic cell death. However, other observations, i.e. nucleishrinkage, PARP cleavage and inhibition of cell death by cycloheximide,and activation of a caspase independant LEI/DNase II pathway wereobserved and are features associated with apoptotic cell death. Duringethanol stress, an LEI/L-DNase II intermediate was lost, leading tocomplete activation L-DNase II (24 kDa). RT-PCR analysis showed an earlyand specific increase of the LEI mRNA. Cycloheximide inhibited LEIsynthesis and protected cells against apoptosis.Conclusions: Our data indicate that ethanol stress on ARPE-19 cellscan induce a pathway which is a form of programmed cell death withcharacteristics of both apoptosis and necrosis, possibly by triggeringconversion of LEI to L-DNase II.

[学科分类] 生物化学/生物物理