Cycloheximide and actinomycin D delay death and affect bcl-2, bax, and Ice gene expression in astrocytes under in vitro ischemia.
An in vitro ischemia model was established and the effect of the metabolic inhibitors cycloheximide (CHX) and actinomycin D (ActD) on apoptosis in astrocytes under ischemia studied. CHX decreased by 75% the number of cells dying after 6 hr of ischemia compared with control cultures. TdT-mediated dUTP nick end labelling (TUNEL) staining of comparable cultures was reduced by 40%. ActD decreased cell death by 60% compared with controls. The number of TUNEL-positive cells was reduced by 38%. The nuclear shrinkage in TUNEL-positive astrocytes in control cultures did not occur in ActD-treated astrocytes, indicating that nuclear shrinkage and DNA fragmentation during apoptosis are two unrelated processes. Expression of bcl-2 (alpha and beta), bax, and Ice in astrocytes under similar ischemic conditions, as measured by quantitative reverse transcription-polymerase chain reaction, indicated that ischemia down-regulated bcl-2 (alpha and beta) and bax. Ice was initially down-regulated from 0 to 4 hr, before returning to control levels after 8 hr of ischemia. ActD decreased the expression of these genes. CHX reduced the expression of bcl-2 (alpha and beta) but increased bax and Ice expression. It is hypothesized that the balance of proapoptotic (Bad, Bax) and antiapoptotic (Bcl-2, Bcl-Xl) proteins determines apoptosis. The data suggest that the ratio of Bcl-2/Bad in astrocytes following ActD and CHX treatment does not decrease as much in untreated cells during ischemia. Our data indicate that it is the ratio of Bcl-2 family members that plays a critical role in determining ischemia-induced apoptosis. It is also important to note that ischemia-induced apoptosis involves the regulation of RNA and protein synthesis.
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D001253:Astrocytes / Q000187:drug effects* / Q000378:metabolism; D002545:Brain Ischemia / Q000378:metabolism* / Q000503:physiopathology; D020170:Caspase 1 / Q000235:genetics*; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D003513:Cycloheximide / Q000494:pharmacology; D003609:Dactinomycin / Q000494:pharmacology; D015536:Down-Regulation / Q000187:drug effects / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects / Q000235:genetics; D019254:Genes, bcl-2 / Q000187:drug effects / Q000235:genetics; D051379:Mice; D008813:Mice, Inbred ICR; D008954:Models, Biological; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics*; D012333:RNA, Messenger / Q000187:drug effects / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects / Q000235:genetics; D051028:bcl-2-Associated X Protein
C494827:Bax protein, mouse; D011500:Protein Synthesis Inhibitors; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D051028:bcl-2-Associated X Protein; D003609:Dactinomycin; D003513:Cycloheximide; D020170:Caspase 1