Expression of yeast apurinic/apyrimidinic endonuclease (APN1) protects lung epithelial cells from bleomycin toxicity
Expression of yeast apurinic/apyrimidinic endonuclease (APN1) protects lung epithelial cells from bleomycin toxicity
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DOI：
10.1165/ajrcmb.25.6.4550
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发表时间：
2001-12-01
影响因子：
6.4
通讯作者：
Martin, WJ
中科院分区：
文献类型：
--
作者：
He, YH;Wu, M;Martin, WJ
博莱霉素是一种久负盛名的抗肿瘤药物。它的主要副作用是肺部毒性，限制了它的使用。在本研究中，我们使用了一种DNA修复蛋白，酵母无嘌呤/脱嘧啶核酸内切酶(APN1)来降低博莱霉素对肺细胞的毒性。将携带增强型绿色荧光蛋白(EGFP)和APN1基因的MIEG3逆转录病毒载体导入肺泡上皮细胞系A549。用荧光激活细胞分选仪(FACS)对转导细胞进行分选，并进行克隆。Northern、Western和脱嘌呤/脱嘧啶(AP)核酸内切酶活性分析证实，转导的A549细胞群体和4个表达不同水平的EGFP的克隆均表达了APN1。APN1的表达与EGFP的表达呈正相关。用单细胞凝胶电泳/彗星试验和博莱霉素处理后的克隆存活实验检测APN1对博莱霉素的保护作用。与载体转导的A549细胞相比，表达APN1的A549细胞在20、50和100µg/ml博莱霉素的存在下DNA损伤显著减少；类似地，表达APN1的A549细胞在博莱霉素存在下的存活率也明显高于载体转导的A549细胞。在选定的克隆中，四个表达APN1的克隆中有三个显著提高了细胞存活率。目前的研究表明，酵母DNA修复蛋白APN1可以减少博莱霉素对靶肺细胞的毒性。
Bleomycin is a well-established anti-tumor drug. Its major untoward effect, pulmonary toxicity, has limited its usage. In this study, we used a DNA repair protein, yeast apurinic/apyrimidinic endonuclease (APN1) to reduce the toxicity of bleomycin on lung cells. A549 cells, an alveolar epithelial cell line, were transduced by MIEG3 retroviral vector encoding both enhanced green fluorescent protein (EGFP) and APN1. Transduced cells were sorted by fluorescent-activated cell sorter (FACS) analysis and were cloned. The APN1 expression of transduced A549 cell population and four selected clones expressing different levels of EGFP was confirmed by Northern, Western, and apurinic/apyrimidinic (AP) endonuclease activity analyses. The expression of APN1 was positively correlated with the expression of EGFP. The protective effect of APN1 against bleomycin was determined by single cell gel electrophoresis/Comet assay and by clonogenic survival assay following bleomycin treatment. The A549 population expressing APN1 showed a significant reduction of DNA damage in the presence of 20, 50, and 100 mug/ml bleomycin; similarly, the APN1-expressing A549 population also demonstrated increased survival in the presence of bleomycin compared with the vector-transduced A549 population. In selected clones, three of four APN1-expressing clones resulted in significantly improved cell survival. The current study suggests that the yeast DNA repair protein, APN1, can reduce bleomycin toxicity to target lung cells.