| Budget Amount *help |
¥10,000,000 (Direct Cost: ¥10,000,000)
Fiscal Year 1999: ¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 1998: ¥6,900,000 (Direct Cost: ¥6,900,000)
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| Research Abstract |
Molecular pathways and mechanism of ionizing radiation (IR)-induced apoptosis in p53-expressing, unexpressing, and mutant p53-expressing human cells were studied. The main pathway was p53-dependent. IR activated p53 through phosphorylation at least on Ser-15 and accumulation. Activated p53 up regulated prodeath Bax, but down-regulated prosurvival Bcl-2 without affecting antiapoptotic Bcl-XL expression. After irradiation, cytosolic Bax moved to isolated heavy mitochondria (HMT), but Bcl-2 family proteins were mostly independent of heterodimerization in HMT. Although Bcl-2 binding to VDAC was not significant, Bax bound to VDAC on the outer membrane of HMT was upregulated as a function of time after irradiation, whereas VDAC-bound Bcl-XL was down downregulated. HMT of unirradiated cells had exclusively the Bcl-XL/VDAC channel without Bax/VDAC channel. The cytochrome c (Cytc) release increased as the Bax/VDAC channel increased. Thus, the increasing Bax/VDAC heteromeric channel plays a crucial role for releasing Cytc upon IR apoptosis, while the Bcl-2 or Bcl-XL ion channel and Bcl-XL/VDAC channel prevent it. In addition, we observed hyperpolarization of HMT during the initial 3 h and afterwards, increasing HMT swelling, ΔΨ loss and ROS generation during a late half of 3 to 6 h. Thus, p53-dependent IR apoptosis involves the early Bax/VDAC channel- and the late MT swelling-mediated Cytc release, followed by the activation of the caspase-9/3 cascade. Further, we cloned DNase γ gene which induced apoptotic DNA ladder in a presumably p53-independent pathway.
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