| Budget Amount *help |
¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 2000: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1999: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Research Abstract |
Results
1. Fas expression in bile duct carcinoma (BDC) cells, analyzed by flow cytometry : Fas was expressed at 21.7% in HuCCT1 cells and at 30.9% in HuH28 cells, respectively.
2. Viability of BDC cells treated with anti-Fas monoclonal antibody (mAb), CH-11 which induces apoptosis : CH-11 dose-dependently reduced HuCCT1 and HuH28 cell counts at the concentration range from 0.01 to 1.0 μg/ml by up to 16.4% and 71.7% on day 3, respectively.
3. DNA fragmentation in BDC cells treated with CH-11 or IFN-γ : In HuCCT1 and HuH28 cells treated with CH-11 or IFN-γ, the peak corresponding to that of positive control cells was observed.
4. Morphological changes of BDC cells treated with CH-11 or IFN-γ : The shrinkage of cells, chromatin condensation and nuclear fragmentation were observed as morphological changes characteristic of apoptosis.
5. Inhibitory effect of anti-Fas mAb 4B4, which does not induce apoptosis, on the induction of apotosis by CH-11 : 4B4 inhibited the reduction of cell counts by 41
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.7% in HuH28 cells.
6. Enhancement of Fas expression in BDC cells treated with IFN-γ : In HuCCT1 cells, the Fas expression was enhanced by 17.6%. In HuH28 cells, however, IFN-γ did not affect the Fas expression.
7. Effect of pretreatment with IFN-γ on the viability of BDC cells treated with CH-11 : Pretreatment with IFN-γ increased the reduction of HuCCT1 and HuH28 cell counts by 41.2% and 8.2% on day 3, respectively.
8. Effect of pretreatment with IL-2 on the viability of BDC cells treated with CH-11 : In HuCCT1 cells, IL-2 alone did not affect the cell number on day 3. Pretreatment with IL-2 also did not affect the reduction of cell counts. In HuH28 cells, IL-2 alone reduced the cell counts by 11.2% on day 3. Pretreatment with IL-2 also did not affect the reduction of cell counts.
9.Viability of BDC cells treated with INF-γ : INF-γ dose-dependently reduced HuCCT1 and HuH28 cell counts at the concentration range from 0.1 to 100 U/ml by up to 45.1% and 87.8% on day 3, respectively.
10. Caspases in BDC cells treated with CH-11 or INF-γ. Any caspase was not detected in HuCCT1 cells. Caspase-3 was detedeted in HuH28 cells treated with CH-11 for 8 hr.
Conclusions
1. Fas was expressed in human BDC cells.
2. Apoptosis was induced by anti-Fas mAb and IFN-γ in human BDC cells.
3. Induction of apoptosis by CH-11 was inhibited by 4B4 in human BDC cells.
4. The expression of Fas and the induction of apoptosis was enhanced by IFN-γ in human BDC cells.
5. Caspase-3 was activated in BDC cells treated with anti-Fas mAb. Less
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