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人数的15-18％，每年约有18万例新病例。尽管乳腺癌的原因尚不清楚，但有几条证据表明，DNA损伤的积累以及DNA修复中缺陷的积累在乳腺癌中起重要作用。据推测，DNA碱基损伤可能导致突变，后来可能是致癌的。最重要的是由活性氧（ROS）引起的碱病变。细胞DNA通过细胞代谢或外源性暴露于环境诱变剂而暴露于ROS。 ROS诱导多种DNA病变。胸腺甘氨酸（TG）和8-羟基鸟嘌呤（8-oxog）是一些最有害的氧化碱病变。胸腺嘧啶甘油是一种毒性病变，可阻断DNA复制和转录，导致细胞死亡，而8-oxog是一种前食病变，导致GC导致GC进行TA转移突变。使用高性能液相色谱和气相色谱质量质谱法的研究表明，相对于乳腺癌病因中氧化性DNA损伤的正常乳腺组织，浸润性导管性乳腺癌中的8-oxog水平增加。 为了避免DNA损伤的有害作用，生物体已经开发出精心的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，通过碱基切除修复（BER）途径可以修复8-oxog，但仍不清楚乳腺癌细胞中普遍存在的8-oxog或其他氧化DNA碱病变的精确修复机制尚不清楚。因此，在乳腺癌发生过程中是否改变了氧化病变的BER是否会明确确定。因此，我们假设从正常到恶性乳腺组织的转化可能是由于氧化DNA损伤修复的缺陷而导致的，因此导致重要基因突变。这种缺陷可能发生在核和/或线粒体基因组中。 线粒体DNA（mtDNA）编码与氧化磷酸化有关的13种蛋白质。 mtDNA中氧化诱导的突变会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正确完整性至关重要。我们检查了核和线粒体提取物从非肿瘤性乳腺上皮细胞系以及乳腺癌MCF-7和MDA-MB-468细胞系从双核核苷酸引起的8-oxog和TG病变。我们在这项研究中报道了三个重要发现：首先，来自MCF-7和MDA-MB-468乳腺癌细胞系中的线粒体提取物在去除8-oxog方面缺乏。两种乳腺癌细胞系都相对于野生型，其切开8-oxog的能力均显示出二倍以上。该缺陷是对8-oxog的特异性，因为相同的线粒体提取物将TG切口与野生型细胞的切口相当。其次，与线粒体提取物相比，来自两种乳腺癌细胞系的核提取物更快，有效地去除了8-oxog。第三，核提取物被证明比8-oxog更快地去除TG。因此，我们首次表明，来自人类乳腺癌细胞系的线粒体在修复8-oxog中有缺陷。 8-oxog的这种缺陷的修复可能意味着乳腺癌细胞的MTDNA突变发生率很高。这些乳腺癌细胞中mtDNA的遗传状况仍有待分析或其他方式确定。因此，我们得出的结论是，线粒体基因组中8-oxog的修复对于乳腺癌的发展可能至关重要。我们的研究可能为乳腺癌的新分子干预提供基础。我们进一步提出，其他形式的癌症可能以缺陷的氧化DNA损伤修复为特征。我们还假设，这些细胞的线粒体DNA可能因氧化性修复缺陷引起的氧化损伤过多。为了解决这一假设，将通过LC/GC质量分光光度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。我们还将通过用特异性氧化损伤剂（例如Menadione，Gamma辐照或过氧化氢处理细胞）来评估氧化DNA损伤，以通过LC/GC质量分光光度计分析病变形成速率。我们使用两种细胞系HCC 1937，即乳腺癌细胞系和AG11134正常的人类乳腺上皮细胞系。 在我们最近的研究中，我们表明，HCC1937乳腺癌细胞系相对于非恶性乳腺上皮细胞表现出严重减少8-oxog的核修复。液相色谱/质谱分析表明，与非恶性乳腺上皮细胞相比，经过过氧化氢处理的HCC1937细胞积累了更高水平的8-oxoG水平，与这些细胞中效率低下的碱基切除途径一致。此外，相对于正常乳腺上皮细胞，过氧化氢处理的HCC1937细胞表现出降低的克隆生存。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水平与非理性细胞系相当，表明OGG1的正常转录调控。 HCC1937细胞中8-oxog的效率低下并不是由于遗传缺陷引起的，因为编码HOGG1 1A的基因的序列分析显示，没有可能影响蛋白质活性的突变。添加纯化的HOGG1改善了HCC1937无细胞提取物的8-oxog特异性切口活性。有趣的是，HCC1937细胞具有显着升高的超氧化物歧化酶1和2（SOD 1和SOD 2），这与上调抗氧化剂的理论是增强细胞存活的机制。这些发现为HCC1937人类乳腺癌细胞系中8-oxog的修复不足提供了证据，并进一步暗示了OGG1在此缺陷中。