Analysis of radiation-induced apoptosis using radioresistant mutants

细胞凋亡是一种重要的生理过程，可以被各种物理和化学刺激所激活，如电离辐射和各种化学物质。为了分析辐射诱导细胞凋亡的分子机制，我们从辐射敏感的小鼠胸腺淋巴瘤3SB细胞系中分离到了经甲烷磺酸乙酯(EMS)处理的抗凋亡突变株。经重复X射线照射后，EMS处理的细胞在含有0.25%琼脂的半固体中生长，分离出12个独立的集落。将这些克隆细胞系在液体培养基中培养数周，然后在5GyX射线照射后接种到半固体培养基中，以筛选出更稳定的抗辐射突变株。我们最终获得了5个稳定的细胞系，发现其中一个克隆1B1C4比其他克隆更能抵抗X射线诱导的细胞死亡。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1B1C4细胞中，即使在孵育24小时后，也没有发生这种凋亡。用琼脂糖凝胶电泳法对X射线照射的1B1C4细胞的DNA进行琼脂糖凝胶电泳法观察到类似的辐射抗性。对含有外显子5~9的P53基因片段的PCR-SSCP分析表明，1B1C4和1D5-8两个克隆含有突变型P53基因。PCR直接序列分析显示，1B1C4细胞在P53密码子238的第二位有从C到T的转变。序列特异性DNA结合分析显示，该突变导致了P53蛋白的功能缺陷。这些结果表明，辐射诱导小鼠胸腺淋巴瘤细胞系的凋亡过程可能至少有两条途径，即P53依赖或非依赖途径。

Apoptosis is an important physiological process that can be activated by various physical and chemical stimuli such as ionizing radiation and various chemicals. In order to analyze molecular mechanism of radiation-induced apoptosis, we isolated apoptosis-resistant mutants from a radiosensitive mouse thymic lymphoma 3SB cell line following treatment with ethyl methanesulfonate (EMS). After enrichment by repeated X-irradiation, EMS treated cells were grown in a semi-solid medium containing 0.25% agar, and 12 independent colonies were isolated. These clonal cell lines were cultured in a liquid medium for a few weeks and then inoculated into the semi-solid medium following irradiation with 5 Gy X-rays, to isolate more stable radioresistant mutants. We finally obtained 5 stable cell lines and found that one clone, 1B1C4, is more resistant to X-ray-induced apoptotic cell death than other clones. 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 following irradiation. However, no such apoptosis occurred in 1B1C4 cells, even after incubation for 24 h. Similar radioresistance was observed using agarose gel electrophoresis of DNA from X-irradiated 1B1C4 cells. PCR-SSCP analysis of p53 cDNA fragments containing exons 5 to 9 suggested that two clones, 1B1C4 and 1D5-8 cells, contain a mutant p53 gene. PCR direct sequence analysis revealed that 1B1C4 cells have a transition from C to T in the second position of p53 codon 238. This mutation was found to cause a functional defect in the p53 protein as revealed by the sequence-specific DNA binding assay. These results indicate that the process of radiation-induced apoptosis in the mouse thymic lymphoma cell line may take at least two routs, p53-dependent or -independent pathways.