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，而8-oxoG是一种致突变前病变，可导致GC到TA的翻转突变。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，浸润性导管性乳腺癌中8-oxoG水平升高，这可能与乳腺癌的病因中DNA氧化损伤有关。

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.