title: ER-targeted Bcl-2 and inhibition of ER-associated caspase-12 rescue cultured immortalized cells from ethanol toxicity.
abstract: 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.
authors: Department of Biology, Kalamazoo College, Kalamazoo, MI, USA.
authors: Andreea G
authors: AG
authors: Balan
authors: Barret J
authors: BJ
authors: Myers
authors: Jansi L
authors: JL
authors: Maganti
authors: D Blaine
authors: DB
authors: Moore
mesh_terms: D000818:Animals; D016466:CHO Cells; D053454:Caspase 12 / Q000235:genetics; D061945:Caspase Inhibitors; D002461:Cell Line, Transformed; D002470:Cell Survival; D006224:Cricetinae; D003412:Cricetulus; D004721:Endoplasmic Reticulum / Q000737:chemistry / Q000201:enzymology / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D000431:Ethanol / Q000008:administration & dosage / Q000633:toxicity*; D019254:Genes, bcl-2 / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000737:chemistry / Q000201:enzymology / Q000502:physiology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics; D014162:Transfection
chemical_list: D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D000431:Ethanol; D053454:Caspase 12