DNA Damage And Repair In Breast Cancer

乳腺癌占每年妇女死亡总数的15-18%，每年约有18万新病例被诊断出来。尽管乳腺癌的病因尚不清楚，但一些证据表明，DNA损伤的积累以及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）引起的基础病变。细胞DNA暴露于ROS，要么是内源性的细胞代谢，要么是外源性的环境诱变剂。活性氧诱导广泛的DNA损伤。胸腺嘧啶乙二醇（Tg）和8-羟基鸟嘌呤（8-oxoG）是一些最有害的氧化基病变。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。8-oxoG是一种致突变前病变。为了避免8-oxoG的有害影响，生物体已经发展出修复这种损伤的机制。利用高效液相色谱和气相色谱-质谱技术的研究表明，与正常乳腺组织相比，浸润性导管乳腺癌中8-oxoG水平升高，这可能与乳腺癌病因中的氧化损伤有关。研究表明，8-oxoG是通过碱基切除修复（BER）途径修复的。到目前为止，还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，乳腺癌发生过程中氧化损伤的BER是否发生改变尚不清楚。因此，我们假设正常乳腺组织向恶性乳腺组织的转变可能是由于氧化DNA损伤修复的缺陷，从而导致重要基因的突变。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 （mtDNA）编码13种参与氧化磷酸化的蛋白质。氧化诱导的mtDNA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至关重要。我们检测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切割双寡核苷酸8-oxoG和Tg病变的能力。我们报告了本研究的三个重要发现：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的能力都下降了两倍以上。这种缺陷是8-oxoG所特有的，因为同样的线粒体提取物对Tg的切割与野生型细胞相当。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。我们首次发现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。这种8-oxoG的缺陷修复可能意味着乳腺癌细胞具有高发生率的mtDNA突变。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。我们的研究可能为新的乳腺癌分子干预提供基础。我们进一步提出，其他形式的癌症可能是有缺陷的氧化DNA损伤修复。我们也假设这些细胞的线粒体DNA可能有氧化修复缺陷引起的过度氧化损伤。为了验证这一假设，我们将用LC/GC质谱法分析这些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。我们还将通过使用特定的氧化损伤剂（如美那酮、伽马辐射或过氧化氢）处理细胞来评估氧化DNA损伤的诱导，并通过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brca1基因在氧化损伤修复中的作用。我们正在使用两种细胞系（CRL2336和CRL2337），它们是BRCA-1突变的纯合或杂合。本项目的wt对照为AG10009淋巴母细胞系。初步数据表明，与野生型细胞相比，BRCA-1突变纯合细胞的氧化损伤、8-oxoG、胸腺嘧啶乙二醇和5-羟基胞嘧啶的核修复减少。该突变细胞系的线粒体氧化损伤修复与野生型细胞相当。

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.