DNA Damage And Repair In Breast Cancer

乳腺癌中的 DNA 损伤和修复

• 批准号: 7132320
• 负责人: michele k evans
• 金额: --
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7132320
• 关键词: DNA damage; DNA repair; breast neoplasms; carcinogenesis; cell line; free radical oxygen; gas chromatography mass spectrometry; gene mutation; high performance liquid chromatography; mitochondrial DNA; molecular genetics; molecular oncology; neoplastic transformation; oxidative stress; superoxide dismutase

Breast cancer accounts for 15-18% of all deaths among women every year, with about 180,000 new cases being diagnosed every year. Even though the causes of breast cancer remain unknown, several lines of evidence suggest that accumulation of DNA damage coupled with defects in DNA repair play an important role in breast cancer. It has been speculated that DNA base damage may lead to mutations that subsequently can be carcinogenic. Of primary importance are the base lesions caused by reactive oxygen species (ROS). Cellular DNA is exposed to ROS either endogenously by cellular metabolism or through exogenous exposure to environmental mutagens. ROS induce a wide range of DNA lesions. Thymine glycol (Tg) and 8-hydroxyguanine (8-oxoG) are some of the most deleterious oxidative base lesions. Thymine glycol is a toxic lesion that blocks DNA replication and transcription, causing cell death While 8-oxoG is a premutagenic lesion that causes GC to TA transversion mutations. Studies using High Performance Liquid Chromatography and Gas Chromatography-Mass Spectrometry have revealed increased levels of 8-oxoG in invasive ductal breast carcinomas relative to normal breast tissue implicating oxidative DNA damage in the etiology of breast cancer. In order to avoid the harmful effects of DNA damage, organisms have developed elaborate DNA repair mechanisms including nucleotide excision repair (NER) and base excision repair (BER). Although it has been shown that 8-oxoG is repaired via the base excision repair (BER) pathway, the precise repair mechanisms for 8-oxoG or other oxidative DNA base lesions prevailing in breast cancer cells are still unclear. Therefore, it remains to be established unequivocally whether BER of oxidative lesions is altered during breast carcinogenesis. We therefore, hypothesized that the transformation from normal to malignant breast tissue may result from defects in oxidative DNA damage repair, consequently leading to mutations in important genes. Such a defect may occur in the nuclear and/or the mitochondrial genome. Mitochondrial DNA (mtDNA) encodes 13 proteins that are involved in oxidative phosphorylation. Oxidatively induced mutations in the mtDNA can lead to dysfunctional mitochondria, and have been implicated in degenerative diseases, cancer and aging. Therefore, effective oxidative damage repair processes are essential in the maintenance of proper integrity of the mitochondrial genome. We examined the ability of nuclear and mitochondrial extracts from a non-neoplastic mammary epithelial cell line and breast cancer MCF-7 and MDA-MB-468 cell lines to incise 8-oxoG and Tg lesions from duplex oligonucleotides. We have reported three important findings in this study: first, mitochondrial extracts from both MCF-7 and MDA-MB-468 breast cancer cell lines are deficient in the removal of 8-oxoG. Both breast cancer cell lines exhibited more than two-fold decrease in their ability to incise 8-oxoG relative to the wild type. This defect was specific for 8-oxoG since the incision of Tg by the same mitochondrial extracts was comparable to that of wild type cells. Second, nuclear extracts from both breast cancer cell lines removed 8-oxoG more rapidly and efficiently than mitochondrial extracts. Third, nuclear extracts were shown to remove Tg more rapidly than 8-oxoG. We have therefore shown for the first time that mitochondria from human breast cancer cell lines are defective in the repair of 8-oxoG. This defective repair of 8-oxoG may imply that breast cancer cells have a high incidence of mtDNA mutations. The genetic status of mtDNA from these breast cancer cells remains to be determined through sequence analyses or otherwise. Therefore, we conclude that repair of 8-oxoG in the mitochondrial genome may be crucial in the development of breast cancer. Our studies may provide a basis for novel molecular interventions of breast cancer. We further propose that other forms of cancer may be characterized by defective oxidative DNA damage repair. We have also hypothesized that mitochondrial DNA of these cells may have excessive oxidative damage caused by defective oxidative repair. To address this hypothesis, mitochondrial and genomic DNA from these and other breast cancer cell lines will be analyzed by LC/GC mass spectrophotometry to determine the basal level oxidative damage. We will also assess induction of oxidative DNA damage by treating cells with specific oxidative damaging agents ( e.g. Menadione, gamma irradiation, or hydrogen peroxide), for analysis of rates of lesion formation via LC/GC mass spectrophotometry. We are using two cell lines HCC 1937, a breast cancer cell line and AG11134 a normal human mammary epithelial cell line. In our recent study, we show that HCC1937 breast cancer cell line, exhibits severely diminished nuclear repair of 8-oxoG relative to nonmalignant mammary epithelial cells. Liquid chromatography/mass spectrometry analysis showed that hydrogen peroxide treated HCC1937 cells accumulated higher levels of 8-oxoG compared to the nonmalignant mammary epithelial cells consistent with inefficient base excision repair pathway in these cells. In addition, hydrogen peroxide treated HCC1937 cells exhibited reduced clonogenic survival relative to the normal mammary epithelial cells. Examination of the level of hOGG1 revealed that this enzyme was significantly reduced in HCC1937 cell line compared to the nonmalignant mammary epithelial cell line. The hOGG1 1a mRNA level in HCC1937 cell line was comparable to the nonmalignant cell line suggesting normal transcriptional regulation of the OGG1. The inefficient repair of 8-oxoG in HCC1937 cells was not due to a genetic defect since sequence analysis of the gene encoding the hOGG1 1a revealed no mutations that could affect the activity of the protein. Addition of purified hOGG1 improved the 8-oxoG specific incision activity of the HCC1937 cell-free extracts. Interestingly, the HCC1937 cells had significantly elevated levels of superoxide dismutase 1 and 2 (Sod 1 and Sod 2), consistent with the theory of upregulation of antioxidants as a mechanism for enhancing cellular survival. These findings provide evidence for deficient repair of 8-oxoG in HCC1937 human breast cancer cell line and further implicate OGG1 in this defect.

乳腺癌每年占女性死亡总数的 15-18%，每年诊断出约 180,000 例新病例。尽管乳腺癌的病因尚不清楚，但多项证据表明，DNA 损伤的累积以及 DNA 修复缺陷在乳腺癌中发挥着重要作用。据推测，DNA 碱基损伤可能导致突变，随后可能致癌。最重要的是活性氧（ROS）引起的基础损伤。细胞 DNA 通过细胞代谢内源性或通过外源性环境诱变剂暴露于 ROS。 ROS 诱导广泛的 DNA 损伤。胸腺嘧啶乙二醇 (Tg) 和 8-羟基鸟嘌呤 (8-oxoG) 是最有害的氧化碱损伤。胸腺嘧啶乙二醇是一种毒性病变，可阻断 DNA 复制和转录，导致细胞死亡，而 8-oxoG 是一种诱变前病变，可导致 GC 至 TA 颠换突变。使用高效液相色谱法和气相色谱-质谱法的研究表明，相对于正常乳腺组织，浸润性导管癌中 8-oxoG 水平升高，这表明氧化性 DNA 损伤与乳腺癌的病因有关。 为了避免DNA损伤的有害影响，生物体开发了复杂的DNA修复机制，包括核苷酸切除修复（NER）和碱基切除修复（BER）。尽管已经表明8-oxoG通过碱基切除修复（BER）途径进行修复，但乳腺癌细胞中普遍存在的8-oxoG或其他氧化DNA碱基损伤的精确修复机制仍不清楚。因此，氧化损伤的 BER 在乳腺癌发生过程中是否发生改变仍有待明确确定。因此，我们推测正常乳腺组织向恶性乳腺组织的转变可能是由于氧化DNA损伤修复缺陷造成的，从而导致重要基因的突变。这种缺陷可能发生在核和/或线粒体基因组中。 线粒体 DNA (mtDNA) 编码 13 种参与氧化磷酸化的蛋白质。线粒体 DNA 中氧化诱导的突变可导致线粒体功能障碍，并与退行性疾病、癌症和衰老有关。因此，有效的氧化损伤修复过程对于维持线粒体基因组的适当完整性至关重要。我们检查了非肿瘤性乳腺上皮细胞系和乳腺癌 MCF-7 和 MDA-MB-468 细胞系的核和线粒体提取物从双链寡核苷酸切割 8-oxoG 和 Tg 病变的能力。我们在这项研究中报告了三个重要发现：首先，MCF-7 和 MDA-MB-468 乳腺癌细胞系的线粒体提取物缺乏 8-oxoG 的去除。与野生型相比，两种乳腺癌细胞系切割 8-oxoG 的能力均下降了两倍以上。该缺陷是 8-oxoG 特有的，因为相同线粒体提取物对 Tg 的切割与野生型细胞的切割相当。其次，两种乳腺癌细胞系的核提取物比线粒体提取物更快、更有效地去除 8-oxoG。第三，核提取物比 8-oxoG 更快地去除 Tg。因此，我们首次证明来自人类乳腺癌细胞系的线粒体在 8-oxoG 修复方面存在缺陷。 8-oxoG 的这种缺陷修复可能意味着乳腺癌细胞 mtDNA 突变的发生率很高。这些乳腺癌细胞的线粒体 DNA 的遗传状态仍有待通过序列分析或其他方式确定。因此，我们得出结论，线粒体基因组中 8-oxoG 的修复可能对乳腺癌的发生至关重要。我们的研究可能为乳腺癌的新型分子干预提供基础。我们进一步提出，其他形式的癌症可能以氧化 DNA 损伤修复缺陷为特征。我们还假设这些细胞的线粒体 DNA 可能因氧化修复缺陷而导致过度氧化损伤。为了解决这一假设，将通过 LC/GC 质谱分析法分析来自这些和其他乳腺癌细胞系的线粒体和基因组 DNA，以确定基础水平的氧化损伤。我们还将通过用特定的氧化损伤剂（例如甲萘醌、伽马射线照射或过氧化氢）处理细胞来评估氧化 DNA 损伤的诱导，通过 LC/GC 质谱分析损伤形成的速率。我们使用两种细胞系 HCC 1937（乳腺癌细胞系）和 AG11134（正常人乳腺上皮细胞系）。 在我们最近的研究中，我们表明，相对于非恶性乳腺上皮细胞，HCC1937 乳腺癌细胞系的 8-oxoG 核修复严重减弱。液相色谱/质谱分析表明，与非恶性乳腺上皮细胞相比，过氧化氢处理的 HCC1937 细胞积累了更高水平的 8-oxoG，这与这些细胞中低效的碱基切除修复途径一致。此外，与正常乳腺上皮细胞相比，过氧化氢处理的 HCC1937 细胞表现出克隆形成存活率降低。对 hOGG1 水平的检查表明，与非恶性乳腺上皮细胞系相比，HCC1937 细胞系中该酶显着降低。 HCC1937 细胞系中的 hOGG1 1a mRNA 水平与非恶性细胞系相当，表明 OGG1 的转录调节正常。 HCC1937 细胞中 8-oxoG 的低效修复并非由于遗传缺陷所致，因为对 hOGG1 1a 编码基因的序列分析显示没有可能影响该蛋白质活性的突变。添加纯化的 hOGG1 改善了 HCC1937 无细胞提取物的 8-oxoG 特异性切割活性。有趣的是，HCC1937 细胞的超氧化物歧化酶 1 和 2（Sod 1 和 Sod 2）水平显着升高，这与抗氧化剂上调作为增强细胞存活机制的理论一致。这些发现为 HCC1937 人乳腺癌细胞系中 8-oxoG 修复缺陷提供了证据，并进一步表明 OGG1 与该缺陷有关。