Induction of apoptosis via Fas system and enhancement of the induction in bile duct carcinoma cells

Fas系统诱导胆管癌细胞凋亡并增强其诱导作用

• 批准号: 11671276
• 负责人: MARUNO Kaname
• 金额: $ 2.05万
• 依托单位: Teikyo University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11671276/
• 关键词: apoptosis; Fas; human bile duct carcinoma cell; anti-Fas monoclonal antibody; IFN-γ; caspase; IL-2

Results1. 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 … More .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.Conclusions1. 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

结果1.流式细胞仪检测胆管癌细胞Fas表达：HuCCT 1细胞Fas表达率为21.7%，HuH 28细胞Fas表达率为30.9%.用诱导凋亡的抗Fas单克隆抗体（mAb）CH-11处理的BDC细胞的存活率：CH-11在0.01至1.0 μg/ml的浓度范围内剂量依赖性地减少HuCCT 1和HuH 28细胞计数，在第3天分别高达16.4%和71.7%。CH-11或IFN-γ处理的BDC细胞中DNA片段化：在CH-11或IFN-γ处理的HuCCT 1和HuH 28细胞中，与阳性对照细胞对应的峰消失. CH-11或IFN-γ处理后的BDC细胞形态学变化：细胞皱缩、染色质浓缩、核碎裂等形态学变化为典型的凋亡特征.不诱导凋亡的抗Fas mAb 4 B4对CH-11诱导凋亡的抑制作用：4 B4抑制细胞计数减少41 ...更多信息 .7%。IFN-γ处理后的BDC细胞中Fas表达增强：在HuCCT 1细胞中，Fas表达增强了17.6%。IFN-γ对HuH 28细胞Fas表达无影响.用IFN-γ预处理对用CH-11处理的BDC细胞的活力的影响：用IFN-γ预处理在第3天使HuCCT 1和HuH 28细胞计数的减少分别增加41.2%和8.2%。用IL-2预处理对用CH-11处理的BDC细胞的活力的影响：在HuCCT 1细胞中，单独的IL-2在第3天不影响细胞数量。用IL-2预处理也不影响细胞计数的减少。在HuH 28细胞中，单独的IL-2在第3天使细胞计数减少11.2%。用IL-2预处理也不影响细胞计数的减少。9.用INF-γ处理的BDC细胞的存活率：在0.1至100 U/ml的浓度范围内，INF-γ剂量依赖性地减少HuCCT 1和HuH 28细胞计数，在第3天分别高达45.1%和87.8%。用CH-11或INF-γ处理的BDC细胞中的半胱天冬酶。在HuCCT 1细胞中未检测到任何半胱天冬酶。CH-11处理HuH 28细胞8 h后，检测到Caspase-3的表达。Fas在人BDC细胞中表达.抗Fas单克隆抗体和IFN-γ诱导人胆管癌细胞凋亡. 4 B4可抑制CH-11诱导人胆管癌细胞凋亡. IFN-γ可增强人BDC细胞中Fas的表达并诱导细胞凋亡。5. Caspase-3在抗Fas mAb处理的BDC细胞中被激活。少