Cycloheximide and actinomycin D delay death and affect bcl‐2, bax, and Ice gene expression in astrocytes under in vitro ischemia

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Abstract
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 (α and β), 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 (α and β) 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 (α and β) 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.