Isolation and characterization of apoptosis-resistant mutants from a radiosensitive mouse lymphoma cell line
Isolation and characterization of apoptosis-resistant mutants from a radiosensitive mouse lymphoma cell line
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DOI：
10.2307/3579680
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发表时间：
1998-01-01
影响因子：
3.4
通讯作者：
Suzuki, F
中科院分区：
文献类型：
--
作者：
Kawai, H;Kitamura, Y;Suzuki, F
为了分析与辐射诱导细胞凋亡相关的特异性基因，从辐射敏感的小鼠胸腺淋巴瘤3SB细胞系中分离出12个抗凋亡克隆。对12个克隆细胞系中的5个进行了重新克隆，并检测了它们对X射线诱导的细胞凋亡的敏感性。细胞存活试验表明，所有五个次级细胞系对X射线的抵抗力都是3SB细胞的两到三倍。3SB细胞在5GyX射线照射后8h内，红素B染色的细胞比例迅速增加。然而，在这些次级突变的细胞中没有发生细胞凋亡。与X射线不同，经20J/m(2)紫外线照射后，突变细胞的凋亡率增加，24 h时达到60%~100%，说明X射线和紫外线诱导细胞凋亡的能力不同。用琼脂糖凝胶电泳法对所有X辐射次生系的细胞DNA进行琼脂糖凝胶电泳法也观察到了类似的辐射抗性。Western印迹分析和序列特异性DNA结合分析表明，1B1C4细胞存在P53蛋白的功能缺陷，而其余4株细胞经X射线照射后均显示野生型P53蛋白。我们的结果表明，胸腺淋巴瘤细胞中存在单独的辐射特异性的P53依赖和独立的凋亡。因此，这些耐凋亡细胞系提供了一个有用的工具来鉴定导致X射线特异性细胞凋亡的信号通路中涉及的基因。(C)1998年，由辐射研究学会提供。
To analyze specific genes related to radiation-induced apoptosis, 12 apoptosis-resistant clones were isolated from cells of the radiosensitive mouse thymic lymphoma 3SB line after treatment with ethyl methanesulfonate. Five of 12 clonal cell lines were recloned and were examined for their susceptibility to X-ray-induced apoptosis. A cell survival assay showed that all five secondary cell lines were two to three times more resistant to X rays than 3SB cells. When 3SB cells were exposed to 5 Gy of X rays, the fraction of cells stained with erythrosin B increased quickly within 8 h of incubation after irradiation. However, no apoptosis occurred in these secondary mutant cells. In particular, the percentage of cells undergoing apoptosis in one clone, 1B1C4, was low even after incubation for 48 h. In contrast to X rays, after exposure to 20 J/m(2) UV radiation, the proportion of apoptotic cells in these mutant cells increased and reached about 60 to 100% at 24 h, indicating a difference in the ability of X rays and UV radiation to induce apoptosis. A similar radioresistance was observed using agarose gel electrophoresis of DNA from cells of all X-irradiated secondary lines. Western blot analysis and a sequence-specific DNA-binding assay demonstrated that 1B1C4 cells had a functional defect in p53 protein, but the other four cell lines displayed wild-type p53 after X irradiation. Our results suggest the existence of separate radiation-specific p53-dependent and independent apoptosis in thymic lymphoma cells. Thus these apoptosis-resistant cell lines provide a useful tool to identify the genes involved in the signaling pathways leading to X-ray-specific apoptosis. (C) 1998 by Radiation Research Society.