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.

Mitochondria play central roles in cellular metabolism and apoptosis and are a major source of reactive oxygen species (ROS). We investigated the role of ROS and mitochondria in radiation-induced apoptosis in multiple myeloma cells. Two distinct levels of ROS were generated following irradiation: a small increase observed early, and a pronounced late increase, associated with depletion of reduced glutathione (GSH) and collapse of mitochondrial membrane potential (Dw m ). Exogenous ROS and caspase-3 induced Dw m drop and cytochrome c release from mitochondria, which could be prevented by molecular (dominant-negative caspase-9) and pharmacologic (zVAD-fmk) caspase inhibitors and overexpression of Bcl-2. Exogenous ROS also induced mitochondrial permeability transition (PT) pore opening and cytochrome c release in isolated mitochondria, which could be blocked by inhibition of PT with cyclosporin A. These results indicate that the late ROS production is associated with increased PT pore opening and decreased Dw m , and GSH, events associated with caspase activation and cytochrome c release.

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Purpose: To investigate the use of MRI alone in post‐implant dosimetry for prostate permanent brachytherapy. Method and Materials: A patient's pelvic MRI was ordered 30 days post implant in addition to the CT scan. The MR images were acquired using all standard pulse sequences. T2 weighted in‐phase images were used to delineate the prostate and out‐of‐phase images to localize the implanted seeds. A post‐implant plan was generated on the MR images. We compared the results of MRI based dosimetry to CT based as well as CT/MRI fused techniques. Results: Phantom studies were performed. A number of dummy seeds were implanted in a tissue‐equivalent phantom and scanned on a Siemens 1.5T MR scanner. The out‐of‐phase MR images manifested significant signal loss artifacts at the seed locations due to susceptibility effects, resulting in enhanced seed visualization. After IRB approval, we acquired the post‐implant CT and MRI for a seed implant patient. The dose coverage based on the CT images was 99.9%. A CT/MRI fusion modified the dose coverage to 90%. After seed localization from the out‐of‐phase MR images, a post‐implant plan was generated on the MR images. The dose coverage was 91.4%. Conclusion: Phantom and patient studies demonstrated the potential to use MRI alone in prostate post‐implant dosimetry. Further investigation will be required to validate this technology. Disclosure: The work was partially supported by a CCF Taussig Cancer Center research grant.