DNA Damage And Repair In Breast Cancer

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Abstract

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 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. 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 AG10097 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. NTH1, the human homolog of endonuclease III for thymine glycol repair) complexes with BRCA1 and other members of the BASC complex (BRCA1-associated genome surveillance complex) as defined in Wang et. al., (2000) Genes Dev. (14) 927-939 ( BRCA1, ATM, NBS1, BLM, MRE-11, RAD50, MSH2, MLH1, MSH6 ). It is possible that the BRCA1 gene may play an important role in modulating oxidative DNA repair in mammary tissue possibly partially explaining one of its roles in breast tumorigenesis.