| Budget Amount *help |
¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 2000: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1999: ¥1,700,000 (Direct Cost: ¥1,700,000)
|
|---|
| Research Abstract |
Ionizing radiation (IR) induced apoptosis is a major causative mechanism underlying cell death by radiation therapy, and is dependent on the function of tumor suppressor p53 protein. We have analyzed the molecular mechanisms of IR-apoptosis in p53 (+) and p53 (-) cells, and found that (1) the final effector molecule of the IR-apoptosis is a well-known CAD (Caspase activated DNase) as well as unknown, Ca^<2+> and Mg^<2+> activated DNase. We also determined that (2) activated p53 induced Bax and Bcl-2 translocation in the miotchondrial (MT) outer menbrane, followed by cytochrome C (CytC) release.
With regards to apoptotic DNase (1), treatments of Caspase 3 inhibitors blocked about 50% of DNA ladder formation, while remaining half of the activity was completely suppressed by the addition of Ca^<2+>/Mg^<2+> chelator. We isolated one of the candidate DNase genes, human DNase γ, and analyzed its possible involvement in IR-apoptosis. However, human DNase γ gene and protein were scarcely expressed in the typical IR-apoptotic cells, indicating that another DNase or unknown regulation of the DNase γ protein would be responsible for the Ca^<2+>/Mg^<2+> dependent DNase activity.
With regards to MT regulation by IR (2), activation of p53 by IR was attributed to Ser 15 phosphorylation in its polypeptide, and then the activated form induced cytosolic Bax translocation to MT outermenbrane. We have found that Bcl-2 was sequestered from MT, and instead Bax traslocation accumulated by a VDAC binding from, indicating that VDAC pore opening was regulated by Bcl-2 negatively but by Bax positively. Our results suggestes that VDAC opening induces CytC release, followed by Caspase activation, which is the major mechanism of IR-apoptosis.
|
