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(29) DIFFERENTIAL RELEASE OF CYTOCHROME C AND ADENYLATE KINASE IN RESPONSE TO VARIOUS APOPTOTIC TRIGGERS A.N. Murphy, S. Wiley and A.
Andreyev
Activation of caspases by cytochrome c release from mitochondria is a critical event in many forms of apoptotic cell death. However, the mechanism by which cytochrome c is released remains a subject of debate. It is possible that cytochrome c release is a consequence of either the opening of a specific channel or a relatively nonspecific rupture of the outer mitochondrial membrane due to swelling associated with the mitochondrial permeability transition pore. To address this, we have engineered SH-SY5Y cells that stably overexpress the intermembrane space protein adenylate kinase tagged with a carboxy-terminal hemagglutinin epitope (AK-HA). Because it is considerably larger than cytochrome c, AK-HA was used as a marker of nonspecific rupturing or the opening of a large relatively nonspecific channel. After treatment with various apoptotic signals, subcellular localization of cytochrome c and AK-HA were assessed by western blotting. We have found differential release of cytochrome c and AK-HA in these cells. In response to the apoptotic triggers etoposide or thapsigargin, both cytochrome c and AK-HA were released from the mitochondria; whereas, treatment with staurosporine and actinomycin D resulted solely in release of cytochrome c. We observed that only a small fraction of the total pool of cytochrome c translocated from the mitochondria to the cytoplasm. The amount of cytochrome c released was sufficient to stimulate caspase activation but resulted in either no or only mild respiratory inhibition. Thus far, our data suggest that there may be multiple mechanisms for cytochrome c release from the mitochondria. Sponsored in part by NIH NS34154 to ANM.
For further information contact...Carmen Mannella: carmen@wadsworth.org |
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