Document Detail

ER-targeted Bcl-2 and inhibition of ER-associated caspase-12 rescue cultured immortalized cells from ethanol toxicity.
MedLine Citation:
PMID:  20727705     Owner:  NLM     Status:  MEDLINE    
Alcohol abuse, known for promoting apoptosis in the liver and nervous system, is a major public health concern. Despite significant morbidity and mortality resulting from ethanol consumption, the precise cellular mechanism of its toxicity remains unknown. Previous work has shown that wild-type Bcl-2 is protective against ethanol. The present study investigated whether protection from ethanol toxicity involves mitochondrial Bcl-2 or endoplasmic reticulum (ER) Bcl-2, and whether mitochondria-associated or ER-associated caspases are involved in ethanol toxicity. Chinese hamster ovary (CHO695) cells were transiently transfected with cDNA constructs encoding wild-type Bcl-2, mitochondria-targeted Bcl-2, or ER-targeted Bcl-2. MTT assay was used to measure cell viability in response to ethanol. Ethanol treatments of 1 and 2.5 M reduced cell viability at 5, 10, and 24 h. Wild-type Bcl-2, localized both to mitochondria and ER, provided significant rescue for CHO695 cells treated with 1M ethanol for 24 h, but did not rescue toxicity at 2.5 M. ER-targeted Bcl-2, however, provided significant and robust rescue following 24 h of 1 and 2.5 M ethanol. Mitochondria-targeted Bcl-2 offered no protection at any ethanol concentration and generally reduced cell viability. To follow up these experiments, we used a peptide inhibitor approach to investigate which caspases were responsible for ethanol-induced apoptosis. Caspase-9 and caspase-12 are known to be downstream of mitochondria and the ER, respectively. CHO695 cells were treated with a pan-caspase inhibitor, a caspase-9 or caspase-12 inhibitor along with 1.5 M ethanol, followed by MTT cell viability assay. Treatment with the pan-caspase inhibitor provided significant rescue from ethanol, whereas inhibition of caspase-9 did not. Inhibition of ER-associated caspase-12, however, conferred significant protection from ethanol toxicity, similar to the pan inhibitor. These findings are consistent with our transfection data and, taken together, suggest a significant role for the ER in ethanol toxicity.
Andreea G Balan; Barret J Myers; Jansi L Maganti; D Blaine Moore
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2010-08-21
Journal Detail:
Title:  Alcohol (Fayetteville, N.Y.)     Volume:  44     ISSN:  1873-6823     ISO Abbreviation:  Alcohol     Publication Date:  2010 Sep 
Date Detail:
Created Date:  2010-09-17     Completed Date:  2011-01-07     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  8502311     Medline TA:  Alcohol     Country:  United States    
Other Details:
Languages:  eng     Pagination:  553-63     Citation Subset:  IM    
Copyright Information:
Copyright © 2010 Elsevier Inc. All rights reserved.
Department of Biology, Kalamazoo College, Kalamazoo, MI, USA.
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MeSH Terms
CHO Cells
Caspase 12 / antagonists & inhibitors*,  genetics
Cell Line, Transformed
Cell Survival
Endoplasmic Reticulum / chemistry,  enzymology,  physiology*
Enzyme Inhibitors / pharmacology
Ethanol / administration & dosage,  toxicity*
Genes, bcl-2 / genetics
Green Fluorescent Proteins / genetics
Microscopy, Fluorescence
Mitochondria / chemistry,  enzymology,  physiology
Proto-Oncogene Proteins c-bcl-2 / physiology*
Recombinant Fusion Proteins / analysis,  genetics
Grant Support
//Howard Hughes Medical Institute
Reg. No./Substance:
0/Enzyme Inhibitors; 0/Proto-Oncogene Proteins c-bcl-2; 0/Recombinant Fusion Proteins; 147336-22-9/Green Fluorescent Proteins; 64-17-5/Ethanol; EC 3.4.22.-/Caspase 12

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