Abstract A15: Single-strand breaks in normal breast DNA are associated with measures of breast cancer risk, deficiencies in repair mechanisms, and diminished protection against oxidative processes

Abstract

Abstract Background: Formation of single-strand breaks (SSBs) in DNA is a constant process, estimated to occur 10,000 times per day in each cell, either from endogenous biosynthetic errors or from endogenous or exogenous disruptive agents. Base excision repair in some cases may be impaired or may be exceeded by the rate of DNA damage, leading to cancer. Methods: 136 healthy women were recruited through the Love-Avon Army of Women and breast clinics at Northwestern and Johns Hopkins Universities. Digital or digitized mammograms were evaluated for percent density using Cumulus software. The population medians (ranges) were for age, 51 (36 to 60); BMI, 28.2 (18.7 to 50.8); life-time Gail estimate, 11.8 (5.6 to 38.3); % breast density, 16.2 (2.4 to 52.5); Masood score, 13 (0 to 18). Breast tissue was obtained by random fine needle aspiration (rFNA). A kit from Norgen Inc. was used to separate DNA, RNA, and protein from the pelleted material. A 200 ng aliquot of DNA was taken for the nick translation assay. Incorporation of free nucleotides at SSBs with dCTP labeled with 3H is catalyzed by Polymerase I from E. coli. A quality control preparation prepared from calf thymus DNA and a reagent blank without DNA was included with each set of 6 samples. Separation of free from incorporated 3H-dCTP was accomplished by gel chromatography on an 80 x 15 mm column. The concordance of interassay values was 0.96. RNA (100 ng), purified from rFNA, was reverse transcribed. qPCR reactions were carried out using the TaqMan OpenArray (Applied Biosystems) for MTUS2, XRCC1, APEX1, SOD2, and NRF1 and were normalized to the average expression of GAPDH and HPRT1. Quantitation of DNA methylation was conducted by QM-MSP from cell lysates assessing the cumulative methylation level of eleven cancer-specific genes (AKR1B1, FZD10, TM6SF1, GAS7C, ALX1, CCND2, RARB, RASSF1A, TWIST1, TMEFF2, and SCGB3A1/HIN1) and a cumulative methylation index (CMI) was calculated. Statistical methods: Demographic and gene expression data were summarized and associations between SSBs and genes of interest were assessed via the Spearman correlation. Results: Among measures of risk, SSBs (pmol/μg DNA) were associated with percent breast density (rho = 0.207, p = 0.016) and with CMI (rho = 0.198, p = 0.021). The association with genes expressed in the rFNA are shown in Table 1. Briefly, MTUS2 is a microtubule-associated tumor suppressor inhibited by PARP: when detected, the MTUS2 mRNA was associated with SSPs; XRCC1 functions as a molecular scaffold that interacts with components of the SSB repair process; APEX1 is an endonuclease involved in base excision repair; and SOD2 and NRF1 act in antioxidant functions. Increases in SSBs were associated with decreased PARP, as reflected in increased MTUS2, decreased XRCC1, and decreased antioxidant factors. Table 1.Association of SSBs with gene products related to DNA repair and antioxidant functionmRNA / N / rho / PMTUS2 / 23 / 0.496 / 0.016XRCC1 / 136 / -0.151 / 0.079APEX1 / 131 / 0.035 / 0.690SOD2 / 136 / -0.207 / 0.016NRF1 / 131 / -0.224 / 0.010 Conclusions: 1) SSBs were associated with two measures of breast cancer risk. 2) SSBs were increased under conditions in which DNA repair processes and protection against reactive oxygen species (ROS) were reduced or unchanged. 3) SSBs were not associated with increased activity of repair genes as should occur if increased DNA damaging agents were the primary cause of increased SSBs. (Supported by the Avon Foundation). Citation Format: Robert Treat Chatterton, Mathavi Sahadevan, Oukseub Lee, Hu Hong, Wang Jun, Irene Helenowski, Saraswati Sukumar, Vered Stearns, Mary J. Fackler, Seema A. Khan. Single-strand breaks in normal breast DNA are associated with measures of breast cancer risk, deficiencies in repair mechanisms, and diminished protection against oxidative processes. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr A15.