8-hydroxyguanine and Breast-Neoplasms

To identify possible extrinsic and intrinsic DNA-damaging factors involved in breast cancer etiology, we measured the level of aromatic and lipid peroxidation-related DNA adducts in samples of normal tissue adjacent to breast tumors obtained from 87 breast cancer patients using 32P postlabeling. Twenty-nine cancer-free women who underwent reduction mammoplasty served as controls. Tissue samples from the breast cancer patients contained significantly higher levels of aromatic DNA adducts (mean +/- SEM: 97.4 +/- 23.4 x 109 nucleotides) than did samples obtained from the controls (mean +/- SEM: 23.5 +/- 6.9 x 109 nucleotides). A bulky benzo[a]pyrene (BP)-like adduct was detected in 41% of the cancer patients, but in none of the controls. The level of this adduct was extremely high in some patients (> 1/106). While 88% of the patients with a smoking history had smoking-specific DNA adducts in their breast tissues, the presence of BP-like adduct was not related to smoking history. The cancer patients also had a significantly higher level of lipid peroxidation-related DNA adducts than did controls. The level of these adducts correlated with the presence of the BP-like adduct. To further explore the origin of the BP-like adduct, we examined the level of polycyclic aromatic hydrocarbon (PAH)-DNA adducts and 8-hydroxyguanine (8-OH-G) in tissue sections obtained from 37 breast cancer patients using immunocytochemistry. We found that patients who had the BP-like adduct showed significantly greater immunostaining for PAH adducts than did those without the BP-like adduct (p = 0.04). In addition, we found that adipocytes tended to have greater immunostaining for the PAH adducts than did epithelial cells. On the other hand, epithelial cells tended to have a higher frequency and greater intensity of staining for 8-OH-G than did adipocytes. The detection of PAH adducts, lipid peroxidation-related DNA adducts, and 8-OH-G in normal breast tissues of breast cancer patients suggests that both exogenous and endogenous DNA-damaging factors may be involved in breast cancer. The exogenous source may involve the types of carcinogen exposure other than cigarette smoking, and the endogenous source may involve oxidative stress associated with normal metabolic activities.

Topics: Alcohol Drinking; Breast; Breast Neoplasms; DNA Adducts; DNA Damage; DNA, Neoplasm; Female; Genetic Markers; Genetic Predisposition to Disease; Guanine; Humans; Lipid Peroxidation; Oxidative Stress; Smoking

Chemotherapy is less often prescribed in older individuals due to concerns about post-treatment morbidity and quality of life. We evaluated the physical performance of breast cancer survivors treated with and without adjuvant chemotherapy.. We conducted a case-control study in 56 estrogen receptor positive breast cancer survivors (BCS) on adjuvant aromatase inhibitors 1-2years after definitive surgery. Cases had received adjuvant chemotherapy (n=27; age 70.5±3.6years) versus age-matched controls who had not (n=29; age 70.0±4.3years). Measures of grip strength, physical activity and performance, walking speed, fatigue, and self-reported physical function were collected. Biological correlates of inflammation, frailty and markers of DNA and RNA oxidation were compared.. Grip strength (controls: 21±7.4 vs.. 29.7±5.0kg, p=0.20), physical activity (5403±3204 vs. 6801±9320steps/day, p=0.45), physical performance (short physical performance battery score: 10.1±1.8 vs. 10.4±1.1, p=0.52) and long-distance walking speed (1.2±0.21 vs. 1.3±0.41m/s, p=0.17) were similar between the two groups. Self-reported physical function was marginally lower in cases than controls (controls: 72±24 vs.. 57±34AU, p=0.07). Fatigue disruptiveness was not different between groups (controls: 11.1±13.0 vs.. 15.7±16.2AU, p=0.24). Similarly, the inflammation, oxidation, and frailty markers did not present a significant difference between groups, except for vitamin D levels (p=0.04).. Older women who received chemotherapy reported having slightly lower physical function, but a similar physical performance compared to women who did not. These data suggest that older BCS treated with chemotherapy recover to an extent similar to survivors who only received hormonal therapy.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Activities of Daily Living; Aged; Aromatase Inhibitors; Biomarkers; Breast Neoplasms; Cancer Survivors; Case-Control Studies; Chemotherapy, Adjuvant; Cross-Sectional Studies; Deoxyguanosine; Exercise; Fatigue; Female; Fibrin Fibrinogen Degradation Products; Guanine; Guanosine; Hand Strength; Humans; Insulin-Like Growth Factor Binding Protein 3; Insulin-Like Growth Factor I; Interleukin-6; Oxidation-Reduction; Pyrimidines; Serum Albumin; Tandem Mass Spectrometry; Tumor Necrosis Factor-alpha; Vitamin D; Walking Speed

Repair of DNA damage is critical for maintaining the genomic integrity of cells. DNA polymerase lambda (POLL/Pol λ) is suggested to function in base excision repair (BER) and nonhomologous end-joining (NHEJ), and is likely to play a role in damage tolerance at the replication fork. Here, using next-generation sequencing, it was discovered that the POLL rs3730477 single-nucleotide polymorphism (SNP) encoding R438W Pol λ was significantly enriched in the germlines of breast cancer patients. Expression of R438W Pol λ in human breast epithelial cells induces cellular transformation and chromosomal aberrations. The role of estrogen was assessed as it is commonly used in hormone replacement therapies and is a known breast cancer risk factor. Interestingly, the combination of estrogen treatment and the expression of the R438W Pol λ SNP drastically accelerated the rate of transformation. Estrogen exposure produces 8-oxoguanine lesions that persist in cells expressing R438W Pol λ compared with wild-type (WT) Pol λ-expressing cells. Unlike WT Pol λ, which performs error-free bypass of 8-oxoguanine lesions, expression of R438W Pol λ leads to an increase in mutagenesis and replicative stress in cells treated with estrogen. Together, these data suggest that individuals who carry the rs3730477 POLL germline variant have an increased risk of estrogen-associated breast cancer.. The Pol λ R438W mutation can serve as a biomarker to predict cancer risk and implicates that treatment with estrogen in individuals with this mutation may further increase their risk of breast cancer. Mol Cancer Res; 14(11); 1068-77. ©2016 AACR.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Transformation, Neoplastic; DNA Damage; DNA Polymerase beta; DNA Repair; Estrogens; Female; Genetic Predisposition to Disease; Germ-Line Mutation; Guanine; High-Throughput Nucleotide Sequencing; Humans; Polymorphism, Single Nucleotide

Recent studies have established that metformin (MET), an oral anti-diabetic drug, possesses antioxidant activity and is effective against different types of cancer in several carcinogen-induced animal models and cell lines. However, whether MET can protect against breast cancer has not been reported before. Therefore, the overall objectives of the present study are to elucidate the potential chemopreventive effect of MET in non-cancerous human breast MCF10A cells and explore the underlying mechanism involved, specifically the role of cytochrome P4501A1 (CYP1A1)/aryl hydrocarbon receptor (AhR) pathway. Transformation of the MCF10A cells into initiated breast cancer cells with DNA adduct formation was conducted using 7,12-dimethylbenz[a]anthracene (DMBA), an AhR ligand. The chemopreventive effect of MET against DMBA-induced breast carcinogenesis was evidenced by the capability of MET to restore the induction of the mRNA levels of basic excision repair genes, 8-oxoguanine DNA glycosylase (OGG1) and apurinic/apyrimidinic endonuclease1 (APE1), and the level of 8-hydroxy-2-deoxyguanosine (8-OHdG). Interestingly, the inhibition of DMBA-induced DNA adduct formation was associated with proportional decrease in CYP1A1 and in. quinone oxidoreductase 1 (NQO1) gene expression. Mechanistically, the involvements of AhR and nuclear factor erythroid 2-related factor-2 (Nrf2) in the MET-mediated inhibition of DMBA-induced CYP1A1 and NQO1 gene expression were evidenced by the ability of MET to inhibit DMBA-induced xenobiotic responsive element and antioxidant responsive element luciferase reporter gene expression which suggests an AhR- and Nrf2-dependent transcriptional control. However, the inability of MET to bind to AhR suggests that MET is not an AhR ligand. In conclusion, the present work shows a strong evidence that MET inhibits the DMBA-mediated carcinogenicity and adduct formation by inhibiting the expression of CYP1A1 through an AhR ligand-independent mechanism.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Anticarcinogenic Agents; Breast Neoplasms; Carcinogenesis; Carcinogens; Cell Line, Tumor; Cytochrome P-450 CYP1A1; DNA Adducts; Female; Guanine; Humans; Metformin; Mice; NAD(P)H Dehydrogenase (Quinone); Receptors, Aryl Hydrocarbon; RNA, Messenger; Signal Transduction

FANCJ mutations are genetically linked to the Fanconi anemia complementation group J and predispose individuals to breast cancer. Understanding the role of FANCJ in DNA metabolism and how FANCJ dysfunction leads to tumorigenesis requires mechanistic studies of FANCJ helicase and its protein partners. In this work, we have examined the ability of FANCJ to unwind DNA molecules with specific base damage that can be mutagenic or lethal. FANCJ was inhibited by a single thymine glycol, but not 8-oxoguanine, in either the translocating or nontranslocating strands of the helicase substrate. In contrast, the human RecQ helicases (BLM, RECQ1, and WRN) display strand-specific inhibition of unwinding by the thymine glycol damage, whereas other DNA helicases (DinG, DnaB, and UvrD) are not significantly inhibited by thymine glycol in either strand. In the presence of replication protein A (RPA), but not Escherichia coli single-stranded DNA-binding protein, FANCJ efficiently unwound the DNA substrate harboring the thymine glycol damage in the nontranslocating strand; however, inhibition of FANCJ helicase activity by the translocating strand thymine glycol was not relieved. Strand-specific stimulation of human RECQ1 helicase activity was also observed, and RPA bound with high affinity to single-stranded DNA containing a single thymine glycol. Based on the biochemical studies, we propose a model for the specific functional interaction between RPA and FANCJ on the thymine glycol substrates. These studies are relevant to the roles of RPA, FANCJ, and other DNA helicases in the metabolism of damaged DNA that can interfere with basic cellular processes of DNA metabolism.

Topics: Basic-Leucine Zipper Transcription Factors; Breast Neoplasms; DNA; DNA Adducts; DNA Damage; DNA Helicases; Enzyme Activation; Fanconi Anemia; Fanconi Anemia Complementation Group Proteins; Female; Guanine; Humans; Oxidative Stress; Replication Protein A; Substrate Specificity; Thymine

Breast cancer is the second leading cause of cancer deaths in women in the United States. Although the causes of this disease are incompletely understood, oxidative DNA damage is presumed to play a critical role in breast carcinogenesis. A common oxidatively induced DNA lesion is 8-hydroxyguanine (8-OH-Gua), which has been implicated in carcinogenesis. The aim of this study was to investigate the ability of HCC1937 and MCF-7 breast cancer cell lines to repair 8-OH-Gua relative to a nonmalignant human mammary epithelial cell line, AG11134.. We used oligonucleotide incision assay to analyze the ability of the two breast cancer cell lines to incise 8-OH-Gua relative to the control cell line. Liquid chromatography/mass spectrometry (LC/MS) was used to measure the levels of 8-OH-Gua as its nucleoside, 8-OH-dG in the cell lines after exposure to H2O2 followed by 30 min repair period. Protein expression levels were determined by Western blot analysis, while the hOGG1 mRNA levels were analyzed by RT-PCR. Complementation of hOGG1 activity in HCC1937 cells was assessed by addition of the purified protein in the incision assay, and in vivo by transfection of pFlagCMV-4-hOGG1. Clonogenic survival assay was used to determine sensitivity after H2O2-mediated oxidative stress.. We show that the HCC1937 breast cancer cells have diminished ability to incise 8-OH-Gua and they accumulate higher levels of 8-OH-dG in the nuclear genome after H2O2 treatment despite a 30 min repair period when compared to the nonmalignant mammary cells. The defective incision of 8-OH-Gua was consistent with expression of undetectable amounts of hOGG1 in HCC1937 cells. The reduced incision activity was significantly stimulated by addition of purified hOGG1. Furthermore, transfection of pFlagCMV-4-hOGG1 in HCC1937 cells resulted in enhanced incision of 8-OH-Gua. HCC1937 cells are more sensitive to high levels of H2O2 and have up-regulated SOD1 and SOD2.. This study provides evidence for inefficient repair of 8-OH-Gua in HCC1937 breast cancer cell line and directly implicates hOGG1 in this defect.

Topics: Base Sequence; Breast Neoplasms; Carcinoma, Ductal; Cell Extracts; Cell Line, Tumor; Cell Nucleus; DNA Primers; DNA Repair; Female; Guanine; Humans; Hydrogen Peroxide; Mitochondria; Oligodeoxyribonucleotides; Reverse Transcriptase Polymerase Chain Reaction; RNA, Neoplasm; Transfection

Breast cancer is one of the major causes of mortality among women in the United States. Although the causes of breast cancer remain unclear, it has been speculated that DNA base damage may lead to mutations that subsequently can be carcinogenic. Recently, defective oxidative DNA damage repair has been implicated in breast tumorigenesis. The major oxidative DNA lesion, 8-hydroxyguanine (8-oxoG), is increased in breast cancer, suggesting that this lesion may play a crucial role in the etiology of breast cancer. However, it is not known whether the repair of 8-oxoG or other oxidative base lesions is altered during breast carcinogenesis. We examined the ability of nuclear and mitochondrial extracts of two human breast cancer cell lines, MCF-7 and MDA-MB-468, to repair 8-oxoG lesion. We report that mitochondrial extracts from the two breast cancer cell lines are defective in the base excision repair of 8-oxoG relative to two noncancer cell lines. We also show that the incision activity of 8-oxoG was significantly lower in mitochondrial than in nuclear extracts in the breast cancer cell lines. The defective mitochondrial repair activity was not attributable to lower levels of human 8-hydroxyguanine DNA glycosylase, the base excision repair enzyme known to incise 8-oxoG in DNA. The repair of thymine glycol, another major oxidative DNA base lesion that blocks transcription and causes cell death, was similar in cancer and noncancer cells. Furthermore, nuclear extracts incised thymine glycol with a much higher efficiency than 8-oxoG. These data provide evidence for defective repair of 8-oxoG in mitochondria of MCF-7 and MDA-MB-468 breast cancer cell lines. These results may implicate 8-oxoG repair mechanisms in mitochondria of certain breast cancers.

Topics: Blotting, Western; Breast Neoplasms; Cell Nucleus; DNA Repair; DNA-Formamidopyrimidine Glycosylase; Electron Transport Complex IV; Female; Guanine; Humans; Mitochondria; N-Glycosyl Hydrolases; Reverse Transcriptase Polymerase Chain Reaction; Tumor Cells, Cultured

The breast and ovarian cancer susceptibility genes, BRCA1 and BRCA2, are likely to participate in DNA lesion processing. Oxidative lesions, such as 8-oxoguanine, occur in DNA after endogenous or exogenous oxidative stress. We show that deficiency for either BRCA1 or BRCA2 in human cancer cells leads to a block of the RNA polymerase II transcription machinery at the 8-oxoguanine site and impairs the transcription-coupled repair of the lesion, leading to a high mutation rate. Expression of wild-type BRCA1 from a recombinant adenovirus fully complements the repair defect in BRCA1-deficient cells. These results represent the first demonstration of the essential contribution of BRCA1 and BRCA2 gene products in the repair of the 8-oxoguanine oxidative damage specifically located on the transcribed strand in human cells. This suggests that cells from individuals predisposed to breast and/or ovarian cancer may undergo a high rate of mutations because of the deficiency of this damage repair pathway after oxidative stress.

Topics: Adenoviridae; BRCA1 Protein; BRCA2 Protein; Breast Neoplasms; Cell Line, Transformed; DNA Damage; DNA Repair; Female; Fibroblasts; Genes, BRCA1; Genetic Vectors; Germ-Line Mutation; Guanine; Humans; Neoplasm Proteins; Oxidative Stress; Pancreatic Neoplasms; Recombinant Proteins; RNA Polymerase II; Transcription Factors; Transcription, Genetic; Transfection

DNA of invasive ductal carcinomas from five women was analyzed for structural alterations in the purine nucleotides using gas chromatography-mass spectrometry with selected ion monitoring. The results were compared to those for a normal DNA control. The carcinoma DNA showed dramatically higher concentrations of the base modifications 8-hydroxy-guanine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, and 8-hydroxyadenine. For example, the concentration of total identified base modifications represented a more than 9-fold increase over the control value. Base modifications of this type, which arise from radical-induced hydroxylation and cleavage reactions of the purine ring, likely play a major role in initiation and probably contribute to the further transformation of neoplastic cells in cancer of the female breast.

Topics: Adenine; Breast Neoplasms; Carcinoma, Intraductal, Noninfiltrating; Cell Transformation, Neoplastic; Chromatography, Gas; DNA; Female; Guanine; Humans; Hydroxylation; Mass Spectrometry; Pyrimidines