| Budget Amount *help |
¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 1999: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1998: ¥2,700,000 (Direct Cost: ¥2,700,000)
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| Research Abstract |
Molecular cloning of rat Pax4 cDNA from a rat insulinoma cell line, RINm5F, library by PCR-based cloning strategy revealed four isoforms of the protein. Analysis of tissue disrtibution using Northern blotting and RT-PCR showed specific expression of Pax4 mRNA in pancreatic islets and RIN cells. RT-PCR confirmed that the mRNAs of four isoforms are expressed in RIN cells. These Pax4 variants may regulate the transcriptional activity of Pax4 during the development of pancreatic islets. Cytokines induce apoptosis in pancreatic beta-cells, but the exact mechanisms and sequence of events are not clear. We investigated a role for tumor necrosis factor-α (TNF-α) in the apoptosis of beta-cells. Using the ribonuclease protein assay and the reverse transcriptase-polymerase chain reaction method, we confirmed that tumor necrosis factor receptor 1 (TNFR1), TNFR1-associated death domain protein (TRADD), Fas receptor-associated intracellular protein with death domain (FADD) and FADD-like ICE(FLICE) were expressed in the pancreatic beta-cell line, MIN6 cells. Fluorescent microscopic examination using Hoechst 33342 dye demonstrated that TNF-α induced time- and dose-dependent apoptotic nuclear changes in these beta-cells. In situ end-labeling of DNA analysis and agarose gels electrophresis revealed that 10 nM TNF-αgenerated characteristic apoptotic DNA fragments. In addition, C2- and natural ceramides dispersed in a solvent mixture of ethanol and dodecane induced characteristic features of apoptosis in MIN6 cells, mimicking TNF-induced DNA damage. We also determined endosomal ceramide production after TNF-α (10 nM) treatment in MIN6 cells using diacyblycerol kinase assay. These results suggest that TNF-α can cause apoptosis in pancreatic beta-cells through TNFR1-linked apoptotic factors, TRADD, FADD and FLICE, and that TNF-induced ceramide production may be involved in the pathways.
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