Gu Y, Desai T, Gutierrez P L, Lu A L
Department of Biochemistry and Molecular Biology, School of Medicine, University of Maryland, Baltimore, Maryland 21201, USA.
Med Sci Monit. 2001 Sep-Oct;7(5):861-8.
Oxidative stress is a major causative agent of carcinogenesis, aging, and a number of diseases. 8-oxoG is the most stable and deleterious lesion of oxidative DNA damage. The 8-oxoG lesions can be eliminated by human repair systems consisting of three enzymes hMTH1, hOGG1, and hMYH homologous to E. coli MutT, MutM, and MutY proteins, respectively.
Human cells (P1, P2, and P3) resistant to H(2)O(2) were derived from the non-tumorigenic human breast cell line MCF10A by sequential treatment of the cells with H(2)O(2). The protein expression levels of DNA repair enzymes were analyzed by Western blotting. The DNA binding and glycosylase activities of hMYH and hOGG1 were measured in the extracts of the H(2)O(2) resistant cells.
The H(2)O(2) resistant cells displayed tremendously greater anchorage-independent growth capability and higher expression of the anti-apoptotic protein BCL-2 than the parental cells. H(2)O(2) detoxification ability was elevated in P1 and P2 cells, but not in P3 cells, suggesting P3 cells might employ a different defense mechanism from P1 and P2 cells. In P3 cells, both hOGG1 and hMYH glycosylase activities were reduced but their protein levels increased. Two A/8-oxoG binding complexes were detected with cell extracts: the fast-migrating complex (bottom form) was dominated in MCF10A cells, and was greatly reduced in P3 cells. Interesting, the P3 cells showing the least amount of bottom form had the weakest hMYH glycosylase activity.
Our results demonstrated, for the first time, that alteration of base excision repair pathways is correlated to cell resistance to oxidative stress.
氧化应激是致癌、衰老及多种疾病的主要致病因素。8-氧代鸟嘌呤（8-oxoG）是氧化性DNA损伤中最稳定且有害的损伤形式。8-oxoG损伤可被人体修复系统清除，该系统由三种分别与大肠杆菌MutT、MutM和MutY蛋白同源的酶hMTH1、hOGG1和hMYH组成。
通过用H₂O₂对非致瘤性人乳腺细胞系MCF10A进行连续处理，获得对H₂O₂具有抗性的人细胞（P1、P2和P3）。通过蛋白质印迹法分析DNA修复酶的蛋白表达水平。在H₂O₂抗性细胞提取物中测量hMYH和hOGG1的DNA结合及糖基化酶活性。
与亲代细胞相比，H₂O₂抗性细胞表现出极大的非锚定依赖性生长能力以及抗凋亡蛋白BCL-2的更高表达。P1和P2细胞中H₂O₂解毒能力增强，但P3细胞中未增强，这表明P3细胞可能采用了与P1和P2细胞不同的防御机制。在P3细胞中，hOGG1和hMYH糖基化酶活性均降低，但其蛋白水平升高。用细胞提取物检测到两种A/8-oxoG结合复合物：快速迁移复合物（底部形式）在MCF10A细胞中占主导，在P3细胞中大大减少。有趣的是，底部形式含量最少的P3细胞具有最弱的hMYH糖基化酶活性。
我们的结果首次证明，碱基切除修复途径的改变与细胞对氧化应激的抗性相关。
