Distinct stages of cytochrome c release from mitochondria: evidence for a feedback amplification loop linking caspase activation to mitochondrial dysfunction in genotoxic stress induced apoptosis

Abstract

Cytochrome c (cyto c) release from mitochondria is a critical event in apoptosis. By investigating the ordering of molecular events during genotoxic stress-induced apoptosis, we found that ionizing radiation (IR) and etoposide induced the release of cyto c from mitochondria in two distinct stages. The early release of low levels of cyto c into the cytosol preceded the activation of caspase 9 and 3, but had no effect on ATP levels or mitochrondrial transmembrane potential (Dc m ). In contrast, the late stage cyto c release resulted in a drastic loss of mitochondrial cyto c and was associated with reduction of ATP levels and Dc m . Moreover, caspases contributed to the late cyto c release since the caspase inhibitor zVAD prevented only the late but not the early-stage cyto c release. Recombinant caspase 3 induced cyto c release from isolated mitochondria in the absence of cytosolic factors. Bcl-2 but not Bid was cleaved during apoptosis after caspase activation. This suggests that Bcl-2 cleavage might contribute to the late cyto c release, which results in mitochondrial dysfunction manifested by the decrease of ATP and Dc m . zVAD prevented the reduction of ATP, Dc m , and nuclear condensation when added up to 8 h after IR, at the time the caspases were highly activated but when the majority of cyto c was still maintained in the mitochondria. These findings link the feedback loop control of caspase-induced cyto c release with mitochondrial dysfunction manifested by ATP and Dc m decline. Cell Death and Differentiation (2000) 7, 227 ± 233.

Cyclin E induction by genotoxic stress leads to apoptosis of hematopoietic cells

“…Cyclin E protein levels and Cdk2 kinase activity are induced by ionizing radiation in hematopoietic cells Treatment with ionizing radiation e ciently kills hematopoietic cells, including multiple myeloma (Chen et al, 2000), and T cell lymphoma cell lines (Gong et al, 1999). To determine the role of cell cycle regulators in apoptosis, we examined the levels and activity of several cyclins and their interacting partner Cdks during radiation-induced apoptosis.…”

“…The levels of ectopic cyclin E were 2 ± 3-fold higher as compared to parental IM-9 cells (Figure 4a), but comparable to that of IM-9 cells following irradiation ( Figure 1a). We next examined the IM-9 transfectants by ®rst examining caspase activation, a crucial biochemical event required for apoptosis induced by many agents including girradiation (Chen et al, 2000;Gong et al, 1999). The DEVD-pNA cleavage, representing caspase 3-like activity in cells undergoing apoptosis, started to increase signi®cantly by 4 h following g-irradiation of IM-9 cells.…”

“…Genotoxic agents, such as ionizing radiation, induce apoptosis in many cell types, most potently in those of hematopoietic origin (Chen et al, 2000;Gong et al, 1999). Apoptosis is frequently associated with proliferating cells, implying the existence of molecules in late G1 and S phase whose activities facilitate execution of the apoptotic process.…”

“…Two key early cellular events associated with apoptosis are the exposure of the phosphatidylserine on the plasma membrane and activation of the ICE/CED-3 family of cysteine proteases, named caspases. Caspase activation is a critical regulator of the execution phase of apoptosis induced by g-irradiation, a prototypic genotoxic agent (Chen et al, 2000;Gong et al, 1999). Caspases are synthesized as inactive zymogens which, after proteolytic cleavage, generate active enzymes which then further cleave other caspases, most important of which is caspase 3, as well as critical cellular proteins (Gong et al, 1999).…”

Regulation of CD20 expression by radiation-induced changes in intracellular redox status

“…Exposure of cells to radiation is known to induce intracellular oxidative stress [26]. To determine the role of intracellular redox changes or oxidative stress on the expression of CD20, cells were treated with two oxidative agents, H 2 O 2 and BSO; BSO increases intracellular oxidative stress by depleting glutathione.…”