DNA Damage And Repair In Breast Cancer

乳腺癌中的 DNA 损伤和修复

• 批准号: 6815311
• 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/6815311
• 关键词: DNA damage; DNA repair; brca gene; breast neoplasms; human tissue; mass spectrometry; mitochondria; neoplasm /cancer genetics; oxidative stress

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. 8-oxoG is a premutagenic lesion. In order to avoid the harmful effects of 8-oxoG, organisms have developed mechanisms for repairing this damage. 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 damages in the etiology of breast cancer. It has been shown that 8-oxoG is repaired via the base excision repair (BER) pathway. To date, there are no reports on the removal of 8-oxoG or other oxidative DNA base lesions in breast cancer cells. Therefore, it remains to be established 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 order for the cell to maintain the 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 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. 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 defective in 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. In our most recent work, we have begun to evaluate the role of the BRCA 1 gene in oxidative damage repair. We are using two cell lines (CRL2336 and CRL2337) that are either homozygous or heterozygous for BRCA-1 mutation. The wt control for this project is the AG10009 lymphoblast cell line. Preliminary data suggests that nuclear repair of oxidative lesions, 8-oxoG, thymine glycol and 5-hydroxycytosine is reduced in cells homozygous for the BRCA-1 mutation relative to wild-type cells. Mitochondrial repair of oxidative lesions in this mutant cell line is comparable to that of wild-type cells. Once we have confirmed the repair phenotype of the BRCA1 mutant cell lines, further investigation will be directed to examining whether the specific repair enzymes involved in oxidative lesion repair (e.g. human endonuclease III (hNTH1) for thymine glycol) complexes with BRCA1 and other members of the BASC complex (Brca1-associated genome surveillance complex) as defined Wang et. al.( BRCA1, ATM, NBS1, BLM, MRE-11, RAD50, MSH2, MLH1, MSH6 ). It is possible that the BRCA1 gene may play an important role in oxidative DNA repair in mammary tissue possibly partially explaining one its roles breast tumorigenesis.

乳腺癌占每年妇女死亡总数的15-18%，每年诊断出约18万例新病例。尽管乳腺癌的原因仍然未知，但一些证据表明，DNA损伤的积累加上DNA修复缺陷在乳腺癌中起着重要作用。据推测，DNA碱基损伤可能导致随后可能致癌的突变。最重要的是由活性氧（ROS）引起的基底病变。细胞DNA通过细胞代谢内源性地或通过外源性暴露于环境诱变剂而暴露于ROS。ROS可引起广泛的DNA损伤。胸腺嘧啶乙二醇（Tg）和8-羟基鸟嘌呤（8-oxoG）是一些最有害的氧化性碱基病变。胸腺嘧啶乙二醇是一种毒性损伤，阻断DNA复制和转录，导致细胞死亡。8-oxoG是一种前诱变损伤。为了避免8-oxoG的有害影响，生物体已经开发出修复这种损伤的机制。使用高效液相色谱法和气相色谱-质谱法的研究已经揭示了相对于正常乳腺组织，浸润性导管乳腺癌中8-oxoG的水平增加，这暗示了乳腺癌病因学中的氧化损伤。已显示8-oxoG通过碱基切除修复（BER）途径修复。迄今为止，没有关于去除乳腺癌细胞中8-oxoG或其他氧化DNA碱基病变的报道。因此，氧化损伤的BER是否在乳腺癌发生过程中改变仍有待确定。因此，我们假设从正常乳腺组织到恶性乳腺组织的转化可能是由于氧化DNA损伤修复缺陷，从而导致重要基因突变。这种缺陷可能发生在细胞核和/或线粒体基因组中。线粒体DNA编码13种参与氧化磷酸化的蛋白质。线粒体DNA的氧化诱导突变可导致线粒体功能失调，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细胞维持线粒体基因组的完整性是必不可少的。我们检查了来自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体寡核苷酸切割8-oxoG和Tg病变的能力。我们在这项研究中报告了三个重要的发现：第一，MCF-7和MDA-MB-468乳腺癌细胞系的线粒体提取物都缺乏8-oxoG的去除。相对于野生型，两种乳腺癌细胞系切割8-oxoG的能力均表现出超过两倍的降低。这种缺陷是特定的8-oxoG，因为相同的线粒体提取物的Tg的切口是可比的野生型细胞。第二，从两个乳腺癌细胞系的核提取物去除8-oxoG更迅速，更有效地比线粒体提取物。第三，核提取物被证明比8-oxoG更迅速地去除Tg。我们首次证明了来自人类乳腺癌细胞系的线粒体在8-oxoG的修复中存在缺陷。8-oxoG的这种缺陷性修复可能意味着乳腺癌细胞具有较高的mtDNA突变发生率。来自这些乳腺癌细胞的mtDNA的遗传状态仍有待通过序列分析来确定。因此，我们得出结论，线粒体基因组中8-oxoG的修复可能在乳腺癌的发展中至关重要。我们的研究可能为乳腺癌的新的分子干预提供依据。我们进一步提出，其他形式的癌症可能在氧化DNA损伤修复方面存在缺陷。我们还假设，这些细胞的线粒体DNA可能有过度的氧化损伤引起的缺陷氧化修复。为了解决这一假设，将通过LC/GC质谱法分析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。我们还将评估通过用特定氧化损伤剂（例如甲萘醌、γ辐射或过氧化氢）处理细胞诱导氧化DNA损伤，以通过LC/GC质谱法分析损伤形成率。在我们最近的工作中，我们已经开始评估BRCA 1基因在氧化损伤修复中的作用。我们正在使用两种细胞系（CRL 2336和CRL 2337），它们对于BRCA-1突变是纯合或杂合的。本项目的wt对照是AG 10009淋巴母细胞系。初步数据表明，相对于野生型细胞，BRCA-1突变纯合子细胞中氧化损伤、8-oxoG、胸腺嘧啶乙二醇和5-羟基胞嘧啶的核修复减少。线粒体修复的氧化损伤，在这个突变体细胞系是可比的野生型细胞。 一旦我们证实了BRCA 1突变细胞系的修复表型，进一步的研究将针对检测参与氧化损伤修复的特异性修复酶（例如胸腺嘧啶乙二醇的人核酸内切酶III（hNTH 1））是否与BRCA 1和BASC复合物（Brca 1相关基因组监视复合物）的其他成员复合，如Wang et. al.（BRCA1、ATM、NBS1、BLM、MRE-11、RAD 50、MSH2、MLH1、MSH6）。BRCA 1基因可能在乳腺组织DNA氧化修复中起重要作用，这可能部分解释了其在乳腺肿瘤发生中的作用。