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Intracellular damage caused by anti-cancer drugs and radiation, analysis of regulatory mechanism of apoptosis in topoisomerase mutant cells

抗癌药物和放射线引起的细胞内损伤、拓扑异构酶突变细胞凋亡调控机制分析

基本信息

批准号：
09670826
负责人：
FUNABIKI Tetsunori
金额：
$ 1.09万
依托单位：
Yokohama City University School op Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09670826/
关键词：
topoisomerase II apoptosis drug resistance cell cycle

项目摘要

(1) We established the method of quantification of apoptotic cells, i.e. BrdU was incorporated to fixed cells by TdT, and apoptotic cell were identified by enzyme linked immunoassay with anti-BrdU antibody conjugated with FITC.(2) We quantified the apoptotic human leukemia cells which have wild type topoisomerase II (topo II) and drug-resistant sublines which carries mutation in topo II after the exposure of anti-cancer drugs. The degree of resistance to anti-cancer drug was inversely proportional to the number of apoptotic cells.(3) We found that both of wild type cells and drug resistant cells accumulated in G2-M phase when treated with anti-topo II drugs such as doxorubicin or VP-16. Thus, doxorubicin and VP-16 seem to cause apoptosis of cells at G2-M phase.(4) Recently the expression of topo II was shown to be down regulated before the apoptotic change of the cell. We examined the regulatory mechanism between cell cycle progression and the expression of topo II. The expression of topo II was slightly decreased in both wild type cell and drug resistant cell lines, one with low expression of normal protein and the other with wild type cell equivalent expression of mutant protein. These results suggested the existence of the apoptotic pathway which induced by the suppression of topo II activity.

(1)建立了凋亡细胞的定量方法，即用TdT法将BrdU掺入固定细胞，用FITC偶联抗BrdU抗体酶联免疫法鉴定凋亡细胞。(2)我们定量分析了抗肿瘤药物暴露后具有野生型拓扑异构酶II （topo II）和携带topo II突变的耐药亚群的凋亡人白血病细胞。对抗癌药物的耐药程度与凋亡细胞数量成反比。(3)我们发现，在抗topo II药物如阿霉素或VP-16的作用下，野生型细胞和耐药细胞均在G2-M期积累。因此，阿霉素和VP-16似乎导致G2-M期细胞凋亡。(4)近年来发现topo II的表达在细胞凋亡变化前下调。我们研究了细胞周期进程和topo II表达之间的调控机制。野生型细胞和耐药细胞株中topo II的表达均略有下降，其中一株正常蛋白表达量低，另一株突变蛋白表达量与野生型细胞相当。这些结果提示存在通过抑制topo II活性诱导细胞凋亡的途径。