Oxidative DNA damage and repair in a cell lineage model of human proliferative breast disease (PBD).
Oxidative DNA damage and repair in a cell lineage model of human proliferative breast disease (PBD).
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人类增殖性乳腺疾病 (PBD) 细胞谱系模型中的氧化 DNA 损伤和修复。
DOI：
10.1093/toxsci/kfg154
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发表时间：
2003
期刊：
影响因子：
--
通讯作者：
Novak,RaymondF
中科院分区：
文献类型：
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作者：
Starcevic,SusanL;Diotte,NicoleM;Zukowski,KimL;Cameron,MarkJ;Novak,RaymondF
DNA 的氧化损伤被认为在突变、衰老和癌症中发挥着重要作用。使用一系列 Ras 蛋白逐渐升高的人乳腺上皮细胞系（MCF-10A、MCF-10AT 和 MCF-10ATG3B）检查对氧化 DNA 损伤的敏感性和 DNA 修复效率。在 DNA 修复抑制剂羟基脲 (HU) 和胞嘧啶阿糖苷 (Ara-C) 不存在和存在的情况下，用 H2O2 处理乳腺上皮细胞。使用碱性单细胞凝胶电泳 (Comet) 测定法通过平均橄榄尾矩 (μm) 评估 DNA 链断裂。在未经处理的细胞中，MCF-10A、MCF-10AT 和 MCF-10ATG3B 细胞的平均橄榄尾矩值分别为 4.3 ± 0.7、8.3 ± 1.1 和 7.1 ± 0.6 μm。 5 分钟的 H2O2 处理会产生浓度依赖性 DNA 损伤，其中 MCF-10A 细胞最敏感，致瘤性 MCF-10ATG3B 细胞最不敏感。与未处理的细胞相比，100 μM H2O2 处理导致 MCF-10A、MCF-10AT 和 MCF-10ATG3B 细胞的平均橄榄尾矩值分别增加约 17、6 和 4.5 倍。 HCC1937 肿瘤细胞系的反应方式与用 H2O2 处理的 MCF-10ATG3B 细胞相当，HU/Ara-C 预处理导致所有三种细胞系的橄榄尾矩值增加约 1.5 倍。测定抗氧化酶的蛋白质水平，包括过氧化氢酶、铜/锌超氧化物歧化酶 (Cu/Zn SOD) 和锰 SOD (MnSOD)，以检查增强对 H2O2 介导的 DNA 损伤的抵抗力的潜在机制。这些酶的水平逐渐增加，在 MCF-10ATG3B 细胞中表达最高。细胞抵抗力的增加也与 p53 蛋白水平的显着下降同时发生。这些结果表明，在该细胞谱系中，对 H2O2 氧化性 DNA 损伤的敏感性随着致瘤性的增加而降低（即 MCF-10A 与 MCF-10ATG3B），并表明 DNA 修复、Ras 和 p53 表达改变或涉及抗氧化酶升高的补偿机制参与介导这些效应。
Oxidative damage to DNA is thought to play a significant role in mutagenesis, aging, and cancer. Sensitivity to oxidative DNA damage and DNA repair efficiency were examined using a series of human breast epithelial cell lines—MCF-10A, MCF-10AT, and MCF-10ATG3B—with progressively elevated Ras protein. Breast epithelial cells were treated with H2O2, in the absence and presence of the DNA-repair inhibitors hydroxyurea (HU) and cytosine arabinoside (Ara-C). DNA strand breaks were assessed by the mean olive tail moment (μm) using the alkaline single-cell gel electrophoresis (Comet) assay. In untreated cells, the mean olive tail moment values were 4.3 ± 0.7, 8.3 ± 1.1, and 7.1 ± 0.6 μm in the MCF-10A, MCF-10AT, and MCF-10ATG3B cells, respectively. Five min H2O2treatment produced concentration-dependent DNA damage, with the MCF-10A cells most susceptible and the tumorigenic MCF-10ATG3B cells the least susceptible. Treatment with 100 μM H2O2resulted in ~17-, 6-, and 4.5-fold increases in mean olive tail moment values in the MCF-10A, MCF-10AT, and MCF-10ATG3B cells, respectively, compared to untreated cells. The HCC1937 tumor cell line responded in a manner comparable to the MCF-10ATG3B cells treated with H2O2, HU/Ara-C pre-treatment resulted in a ~1.5-fold increase in olive tail moment values in all three cell lines. Protein levels of antioxidant enzymes, including catalase, copper/zinc superoxide dismutase (Cu/Zn SOD), and manganese SOD (MnSOD) were determined in order to examine a potential mechanism for increased resistance to H2O2-mediated DNA damage. Levels of these enzymes increased progressively, with highest expression in MCF-10ATG3B cells. Increased cellular resistance also coincided with marked decreases in p53 protein levels. These results demonstrate that, in this cell lineage, sensitivity to oxidative DNA damage by H2O2decreases with tumorigenicity (i.e., MCF-10A vs. MCF-10ATG3B), and show that DNA repair, altered Ras, and p53 expression, or compensatory mechanisms involving elevated antioxidant enzymes are involved in mediating these effects.