L-DNase II associated with active process during ethanol induced cell death in ARPE-19.
OBJECTIVE
To analyse the mechanism of ethanol-induced cell death, and particularly, the activation of the leucocyte elastase inhibitor (LEI) pathway.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Brossas
Jean-Yves
JY
INSERM Unité 450, Association Claude Bernard, Paris, France.
Tanguy
Ronan
R
Brignole-Baudouin
Francoise
F
Courtois
Yves
Y
Torriglia
Alicia
A
Tréton
Jacques
J
D015153:Blotting, Western; D016923:Cell Death / Q000187:drug effects; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D053938:DNA Fragmentation / Q000187:drug effects; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D000431:Ethanol / Q000494:pharmacology*; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D010857:Pigment Epithelium of Eye / Q000187:drug effects* / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015843:Serpins / Q000235:genetics / Q000378:metabolism*
D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D015843:Serpins; D000431:Ethanol; D003513:Cycloheximide; D011065:Poly(ADP-ribose) Polymerases; D004706:Endodeoxyribonucleases; C019329:deoxyribonuclease II