New Assay Methods for Monitoring Radiation Effects.

监测辐射效应的新测定方法。

• 批准号: 09557068
• 负责人: SUZUKI Norio
• 金额: $ 1.54万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09557068/
• 关键词: Indicator of sensitivity; X ray induced protein p41; anti-p41 antibody; MOLT-4; cell death; MUC1 mucin

A newly appearing p41 protein after X-irradiation of MOLT4 cells was characterized to access the mechanism(s) and feasibility as a marker of radiation effect. Amount of p41 was radiation dose and time dependent. Newly prepared polyclonal antibody against p41 also stained p42 on western blotting analysis of various tumor cell lines. Amino acid analysis of p41 and p42 showed that p41 was a cleavage product of p42. P42 was generated in vitro by protein synthesis and used as a substrate to identify a particular caspase (unpublished data) activated after X-irradiation of MOLT4 cells.Expression of MUC1 mucin of HT-29 cells after X-irradiation of 1, 3, 6, 10 Gy were analyzed for several days during cell death. Glycosylated MUC1 mucins (500 kDa and 390 kDa) and underglycosylated MUC1 mucins (350 kDa and 240 kDa) were found increased in dose-dependent manner reaching maximum level in 4 days. Transcriptional activity of MUC1 gene was analyzed by CAT assay containing the 5'-flanking sequence of MUC1 gene. Induction of CAT activity in HT-29 cells was found elevated from day 2 to 4 of X-irradiation. These findings with increased mRNA determined by RT-PCR indicate that transcriptional activity of MUC1 gene in HT-29 cells was upregulated by X-irradiation.Further characterization is required prior to practical use of these markers to monitor radiation effect, although the present results indicate such possibility.

对MOLT4细胞x射线照射后新出现的p41蛋白进行了表征，探讨了其作为辐射效应标志物的机制和可行性。p41的量与辐射剂量和时间有关。新制备的抗p41多克隆抗体也对多种肿瘤细胞系的p42进行了western blotting分析。p41和p42的氨基酸分析表明p41是p42的裂解产物。P42是通过蛋白质合成在体外生成的，并作为底物用于鉴定x照射MOLT4细胞后激活的特定caspase（未发表的数据）。观察1、3、6、10 Gy x射线照射HT-29细胞死亡后数天MUC1粘蛋白的表达情况。糖基化MUC1粘蛋白（500 kDa和390 kDa）和低糖基化MUC1粘蛋白（350 kDa和240 kDa）呈剂量依赖性增加，在4天达到最大水平。采用CAT法分析MUC1基因5′侧序列的转录活性。在x射线照射的第2天至第4天，HT-29细胞的CAT活性升高。这些结果和RT-PCR检测到的mRNA升高表明，x辐射上调了HT-29细胞MUC1基因的转录活性。在实际使用这些标记物来监测辐射效应之前，需要进一步的表征，尽管目前的结果表明了这种可能性。

项目成果

Hosoi, Y., Miyachi, H., Matsumoto, Y., Ikehata, H., Komura, J., Ishii, K., H.J.Zhao, Yoshida, M., TakaiY., Yamada, S., Suzuki, N., and Ono, T.: "A phosphatidylionsitol 3-kinase inhibitor, wortmannin, induces radioresistant DNA synthesis, and sensitizes ce

Hosoi, Y.、Miyachi, H.、Matsumoto, Y.、Ikehata, H.、Komura, J.、Ishii, K.、H.J.Zhao、Yoshida, M.、TakaiY.、Yamada, S.、Suzuki, N.

Hosoi,Y.et al.: "A phosphatidylionsitol 3-kinase inhibitor,wortmannin,induces radioresistant DNA synthesis,and sensitizes cells to bleomycin and ionizing radiation." Int.J.Cancer. 78. 642-647 (1998)

Hosoi, Y. 等人：“一种磷脂酰肌醇 3-激酶抑制剂，渥曼青霉素，可诱导抗辐射 DNA 合成，并使细胞对博来霉素和电离辐射敏感。”

鈴木紀夫: "放射線感受性について" 癌の臨床. (印刷中). (1999)

Norio Suzuki：“关于辐射敏感性”临床癌症（出版中）。

Matsumoto,Y.et al.: "A possible mechanism for hyperthermic radiosensitization mediated through hyperthermic lability of Ku subunits in DNA-dependent protein kinase." Biochemical and Biophysical Research Communications. 234. 568-572 (1997)

Matsumoto,Y.et al.：“通过 DNA 依赖性蛋白激酶中 Ku 亚基的高温不稳定性介导的高温放射增敏的可能机制。”

Matsumoto, Y., Umeda, N., Suzuki, N., Sakai, K., and Hirano, K.: "A gel-electrophoretic analysis for improved sensitivity and specificity of DNA-dependent protein kinase activity." Radiat.Res.(in press). (1999)

Matsumoto, Y.、Umeda, N.、Suzuki, N.、Sakai, K. 和 Hirano, K.：“凝胶电泳分析可提高 DNA 依赖性蛋白激酶活性的灵敏度和特异性。”