Alcohol induces apoptosis in TM3 mouse Leydig cells via bax-dependent caspase-3 activation.
To investigate whether ethanol induces apoptosis in Leydig cells, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, terminal deoxynuclotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay, 4,6-diamidino-2-phenylindole (DAPI) staining, DNA fragmentation assay, caspase-3 enzyme assay, reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis were performed on TM3 mouse Leydig cells. Through morphological and biochemical analyses, it was demonstrated that TM3 cells treated with ethanol at concentrations of 50 and 100 mM exhibit classical apoptotic features. In addition, it was shown that ethanol induces increases in levels of bax and caspase-3 and a decrease in bcl-2 expression. Based on the results, alcohol appears to activate specific intracellular death-related pathways leading to bax-dependant caspase-3 activation and the induction of apoptosis in Leydig cells.
Jang
Mi-Hyeon
MH
Department of Physiology, College of Medicine, Kyung Hee University, #1 Hoigi-dong, Dongdaemoon-gu, Seoul, 130-701, South Korea.
Shin
Min-Chul
MC
Shin
Hye-Sook
HS
Kim
Khae-Hawn
KH
Park
Heung-Jae
HJ
Kim
Ee-Hwa
EH
Kim
Chang-Ju
CJ
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects; D002492:Central Nervous System Depressants / Q000494:pharmacology*; D053938:DNA Fragmentation / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D000431:Ethanol / Q000494:pharmacology*; D005456:Fluorescent Dyes; D019254:Genes, bcl-2 / Q000235:genetics; D020287:In Situ Nick-End Labeling; D007211:Indoles; D007985:Leydig Cells / Q000187:drug effects* / Q000201:enzymology; D008297:Male; D051379:Mice; D011518:Proto-Oncogene Proteins / Q000502:physiology*; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013778:Tetrazolium Salts / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology; D051028:bcl-2-Associated X Protein
C494827:Bax protein, mouse; D002492:Central Nervous System Depressants; D005456:Fluorescent Dyes; D007211:Indoles; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D013778:Tetrazolium Salts; D013844:Thiazoles; D051028:bcl-2-Associated X Protein; D000431:Ethanol; C007293:DAPI; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases; C022616:thiazolyl blue