p14ARF基因功能性恢复对人肺癌细胞放射敏感性的影响

Increased radiosensitivity of lung cancer cell lines related with the functionally replaced p14ARF gene

目的 了解专职调控细胞周期的p14ARF蛋白功能恢复或增强能否影响体外肺癌细胞的放射敏感程度。方法 选择了 4株具有不同基因背景的肺癌细胞系 ,为它们引入人野生型p14ARF基因。用RT PCR、Western蛋白印迹和免疫组化检测外源性p14ARF的信使RNA及蛋白表达 ,并筛选出阳性克隆。以母细胞系和转染空载体的阴性细胞为对照 ,分析转染p14ARF的细胞在周期分布、放射存活分数及放射诱导的凋亡比例等指标的变化。结果 外源性p14ARF的引入使 3株p5 3野生型肺癌细胞阻滞于G1期或G1、G2 M期。其中H46 0 p14ARF和A5 49 p14ARF细胞的S期细胞比例显著下降 ,分别为 2 7.9%± 2 .4%、16 .5 %± 0 .3% ,剂量 存活曲线下移 ,4Gy照射后较对照细胞出现G2 期延迟。A5 49 p14ARF细胞照射后凋亡比例增高。结论 野生型p14ARF的恢复能提高肺肿瘤细胞的放射敏感性。p14ARF引起的细胞周期再分布可能是放射增敏的主要原因 ,其引发的p5 3活性增强以及放射诱导的凋亡比例增高也为这种放射增敏提供了部分解释。

Objective To assess the possibility that whether restored or enforced function of p14ARF could influence the radiosensitivity of lung cancer cells in vitro. Methods Human lung cancer cell lines with various endogenous backgrounds in INK4a, p53 and Rb genes were used as the recipients of the wild type p14ARF gene. The expression of p14ARF mRNA and protein was detected with RT PCR, immunohistochemistry and Western immunoblot after G418 selection. Clones expressing both p14ARF mRNA and protein were identified and selected for further experiments. By comparing with the parental and negative control cells prepared with empty vector, the effects of exogenously transfected p14ARF on cell cycle distribution, cell survival fraction and radiation induced proportion of apoptosis were analyzed. Results The cell cycles of three wild type p53 cell lines were arrested in G 1 phase or G 1 and G 2 M phases. A significant decline in the proportion of S phase was observed in H460 p14ARF and A549 p14ARF cells, with decreased survival fraction and prolonged G 2 delay after irradiation. We also observed in A549 p14ARF cells an increased percentage of apoptosis when radiated. Conclusion The exogenously transfected wild type p14ARF could increase the radiosensitivity of some lung cancer cells. It appears that cell cycle redistribution of cells after acquiring p14ARF may be the main explanation for the enhanced sensitivity. The increased apoptosis proportion of A549 p14ARF cells in response to radiation indicates a fortified p53 function and might partly contribute to the increased sensitization.