Affiliations of authors: Department of Environmental Health Sciences (DOK, MA, JS, GM) and Epidemiology, Mailman School of Public Health (MBT, FFZ, RTS), Columbia University, New York, NY
Correspondence to: Regina M. Santella, Department of Environmental Health Sciences, Rm. 506, Mailman School of Public Health, Columbia University, 701 W. 168th St., New York, NY 10032 (e-mail: rps1{at}columbia.edu)
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ABSTRACT |
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INTRODUCTION |
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In this study, we used sister sets discordant for breast cancer to evaluate the hypothesis that reduced DNA repair capacity in the nucleotide excision repair pathway that removes bulky DNA adducts is associated with cancer risk. A major strength of the family-based design is that it eliminates potential confounding related to population admixture. Sister sets can also reduce potential confounding due to differences in genetic susceptibility as well as behavioral and lifestyle factors, which cluster within families. DNA repair capacity was assayed by measuring the removal of in vitroinduced benzo[a]pyrene diolepoxide (BPDE)DNA adducts in lymphoblastoid cell lines generated from each subjects blood. BPDE is a carcinogenic metabolite of benzo[a]pyrene that is a common environmental pollutant present in cigarette smoke, ambient air, and various foods. Although prior studies have suggested that polycyclic aromatic hydrocarbons may be related to breast cancer risk (9,10), we used BPDE for these studies because of its ability to induce damage that is repaired by the nucleotide excision repair pathway.
Two prior studies have used BPDE as a test mutagen in breast cancer case patients and control subjects. In one study, lymphocytes were treated with BPDE, and DNA repair was assessed using the mutagen sensitivity assay (5). In the other, plasmid DNA was damaged with BPDE, and repair was measured using the host cell reactivation assay (8). In both studies, higher mutagen sensitivity and poorer repair, respectively, were observed in case patients compared with control subjects. We previously reported results on a pilot study of 50 subjects (25 sets of paired sisters) (11) and found poorer repair in case patients. We have now expanded the study to 137 new families, excluding the previous 50 subjects.
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SUBJECTS AND METHODS |
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The study population was selected from families participating in the Metropolitan New York Registry of Breast Cancer Families, one of six sites funded by the National Cancer Institute (NCI) as a part of the Breast Cooperative Family Registry (CFR) (www.cfr.epi.uci.edu) (12). The study was approved by Columbia Universitys Internal Review Board; written informed consent was obtained from all subjects, and strict quality controls and safeguards were used to protect confidentiality. Families were eligible to participate in the New York Registry of Breast Cancer Families if they met one of the following criteria: 1) have a female relative with breast or ovarian cancer diagnosed before age 45 years, 2) have a female relative with both breast and ovarian cancer diagnosed regardless of age at diagnosis, 3) have two or more relatives with breast or ovarian cancer diagnosed after age 45 years, 4) be a male with breast cancer diagnosed at any age, or 5) have a family member with a known BRCA mutation. The present study included all families for whom lymphoblastoid cell lines were available for at least two sisters discordant for breast cancer and consisted of 158 case patients and 154 control subjects from 137 families out of the total of 1250 Registry families. One hundred and seventeen families had one set of sisters and the remaining 20 families had two or three case patients and/or control subjects in the family.
Several questionnaires were administered to each consenting, participating family member on recruitment into the Registry, including a family history instrument that collected information on history of all cancers; an epidemiology questionnaire that collected information on demographics, ethnicity, smoking, alcohol consumption, reproductive history, hormone use, weight, height, and physical activity; and a self-administered food frequency questionnaire. Blood was collected from all participating family members at the time of recruitment. For case patients, blood was collected 5 years after diagnosis, on average.
Cell Culture
Two reference human lymphoblastoid cell lines (GM01989 and GM02485; National Institute of General Medical Sciences Human Genetic Mutant Cell Repository, Coriell Institute for Medical Research, Camden, NJ) and cell lines developed from lymphocytes of sisters in the Breast CFR (as mentioned above) were used. The GM01989 cell line was established from an apparently normal subject, and the GM02485 cell line was from a subject with xeroderma pigmentosum complementation group D. All cell lines from the sisters in the study were developed from frozen lymphocytes stored from less than 1 to 5 years, with some transformations carried out at Coriell Institute and others in our laboratory, both using EpsteinBarr virus isolated from the marmoset line B958 and the same procedure (13). Cells were grown in suspension in 75-cm2 tissue culture flasks (Nalge Nunc International, Rochester, NY) in RPMI-1640 medium supplemented with 1% L-glutamine, antibiotics (Cellgro Mediatech, Herndon, VA), and 10% fetal bovine serum (Fetalclone I, HyClone, Logan, UT) at 37 °C with 5% CO2 for 1 week.
DNA Damage and Repair
The DNA repair assay was carried out essentially as described (11). Lymphoblastoid cell lines from sisters discordant for breast cancer were assayed together with the two reference cell lines (DNA repairproficient line, GM01989, anddeficient line, GM02485). Cells (1.5 x 107) were suspended in 15 mL of serum-free RPMI. Immediately before treatment with (±)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (the anti isomer) (BPDE; NCI Chemical Carcinogen Repository, Midwest Research Institute, Kansas City, MO), 5 mL of the cell suspension was prepared for immunohistochemical analysis by centrifuging at 20 °C for 10 minutes at 1000g, redissolving in 50 µL and dropping approximately 3 µL onto microscope slides, and spreading the cells. The remaining cells (1.0 x 107 in 10 mL) were treated with 5 µM BPDE by adding 10 µL of 5 mM BPDE in tetrahydrofuran and culturing for 30 minutes. The cells were then centrifuged as above and washed with phosphate-buffered saline (PBS). Half of the cells were immediately prepared for immunohistochemical analysis as above, and the remainder were cultured in 5 mL of complete medium with fetal bovine serum for 4 hours to allow the cells to repair the damage before slide preparation. The reference cell lines were assayed with each batch of experimental samples, and both members of each sister set were tested in the same experiment. The laboratory investigator who performed the assays was blinded to casecontrol status.
Immunohistochemical Analysis of BPDE-DNA Damage
The immunohistochemical analysis method performed has been described previously (11,14). Briefly, slides were treated with approximately 100 µL of RNase (0.1 mg/mL in Tris buffer, pH 7.5) for 1 hour at 37 °C, then with 100 µL of proteinase K (10 µg/mL in Tris buffer, pH 7.5) for 10 minutes, and with 100 µL of 4 N HCl for 10 minutes at room temperature followed by neutralization with 100 µL of 50 mM Tris base for 5 minutes at room temperature. Slides were washed twice by immersing in PBS for 5 minutes. Slides were blocked with 100 µL of 10% normal goat serum in Tris buffer, pH 7.5, for 45 minutes at 37 °C. Cells were then incubated with 100 µL of rabbit anti-BPDEDNA polyclonal antiserum #1 (15) at a 1:500 dilution overnight at 4 °C. Next, cells were incubated with 100 µL of secondary fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (ICN Biomedicals, Aurora, OH) at a 1:200 dilution for 45 minutes at 37 °C. Cells were counterstained with 100 µL of 1 µg/mL propidium iodide (Sigma, St. Louis, MO) in PBS for 45 seconds at room temperature. The average nuclear fluorescence intensity for a minimum of 50 randomly selected cells from fields that displayed strong propidium iodide staining (5 cells in each of 10 fields) was determined using a Nikon Eclipse 600 microscope (Nikon, Melville, NY) mounted with a charge-coupled display camera (Hamamatsu C4742-95 [ORCA-100], Bridgewater, NJ) and equipped with image analysis software (MetaView Imaging System, Universal Imaging, West Chester, PA). Fluorescence intensity was collected as an "average gray value," a measure of relative fluorescence intensity.
Percent DNA repair capacity was calculated using the average gray value as the measure of fluorescence intensity of nuclear staining. The fluorescence intensity of each subjects untreated cells was subtracted from the fluorescence intensity of her BPDE-treated cells stained immediately after treatment and after 4 hours of repair. DNA repair capacity was calculated from the formula percent DNA repair capacity equals [(I0 IC) (I4 IC)]/(I0 IC) x 100% where I0 is the intensity of treated cells immediately after treatment, I4 is that after 4 hours of repair, and IC is that for untreated control cells. The daily variation in the determination of the percent DNA repair capacity was 8.9 (95% confidence interval [CI] = 5.4 to 12.4, N = 22) for the DNA repairdeficient reference cell line GM02485 and 48.2 (95% CI = 43.7 to 52.8, N = 22) for the repairproficient cell line GM01989.
Statistical Methods
Students t tests and one-way analysis of variance were used to compare mean percent DNA repair capacity between case patients and control subjects. In addition to these univariate analyses, the differences between sister sets were examined using conditional logistic regression (16). In these regression models, the association between the percentage of adducts removed and the participants status was estimated while simultaneously adjusting for potential confounding variables such as age at blood donation, current body mass index (BMI, [weight in kilograms]/[height in meters squared]), and smoking. Odds ratios (ORs) and Ptrend values were calculated using maximum likelihood methods (16). Differences were considered statistically to be significant if P<.05. Both median and quartiles of DNA repair capacity in control subjects were used as cut points for the calculation of odds ratio (see Table 2). Medians were used to categorize the intervals between age at diagnosis and age at interview, BMI, and smoking level.
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RESULTS |
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The association of age, ethnicity, BMI, and smoking status on DNA repair capacity was also investigated (Table 1). Case patients younger than 50 years had lower percent DNA repair capacity than case patients 50 years old or older, although this difference was not statistically significant (difference = 5.7, 95% CI = 3.4 to 15.2, P = .235). For control subjects, in contrast, percent DNA repair capacity was higher in younger than in older subjects, but again, the difference was not statistically significant (P = .164). Comparing case patients and control subjects, statistically significant differences were observed in those younger than 40 years. There were no statistically significant differences in percent DNA repair capacity by ethnicity, BMI, or smoking status in either case patients or control subjects, although differences between case patients and control subjects were observed in non-Caucasians and those with a BMI of 25 or greater.
When percent DNA repair capacity values were dichotomized using the median value in control subjects, values below the median percent DNA repair capacity were associated with an elevated breast cancer risk (crude OR = 2.29, 95% CI = 1.38 to 3.79; Table 2). After adjustment for age at blood donation, BMI, and smoking status, the OR was 2.43 (95% CI = 1.44 to 4.08). Furthermore, categorizing percent DNA repair capacity values into quartiles based on data in control subjects resulted in a statistically significant dose-dependent association between deficient DNA repair capacity and breast cancer risk. Using the quartile with the highest percent DNA repair capacity as the referent group, adjusted ORs increased from 1.23 (95% CI = 0.57 to 2.65) to 2.38 (95% CI = 1.17 to 4.86) and 2.99 (95%CI = 1.45 to 6.17) as percent DNA repair capacity decreased (Ptrend = .002). Similar results were obtained when the analysis was limited to the 117 families with a single set of sisters; ORs increased from 1.46 (95% CI = 0.62 to 3.44) to 1.80 (95% CI = 0.77 to 4.20) and 2.77 (95% CI =1.21 to 6.37) as DNA repair capacity decreased (Ptrend =.029).
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DISCUSSION |
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In analysis stratified by age, case patients younger than 50 years showed a somewhat lower DNA repair capacity than those older than 50 years and, in control subjects, percent DNA repair capacity decreased with age. As a result, the largest difference in DNA repair capacity between case patients and control subjects was in the youngest age group. The observation in control subjects is in agreement with other studies showing decreased DNA repair capacity with increasing age in the general population (17,18). In addition, Ramos et al. (7) observed that younger breast cancer case patients had a more substantial reduction in capacity to repair UV damage compared with age-matched control subjects than did older case patients and control subjects. In a study of basal cell carcinoma, older patients (aged 40 years and older) also had statistically significantly higher DNA repair capacity compared with their matched control subjects and a higher DNA repair capacity than younger case patients, although this was not statistically significant (19). With regard to ethnicity, the DNA repair capacity differences were similar between Caucasians and non-Caucasians (African Americans, Hispanics, and Asians) within case patients or control subjects although the number of non-Caucasians was small (Table 1). Nor was there an association of smoking with DNA repair capacity in either case patients or control subjectsthe difference in percent DNA repair capacity between smokers and nonsmokers was not statistically significant. Similar results have been observed in a study of upper aerodigestive tract cancer (20).
A unique strength of this study was the family-based design using sisters from the same families as case patients and control subjects. Because each case patients control subject was her sister, any potential confounding related to population admixture was eliminated. Using sisters as control subjects also reduces some of the confounding due to differences in genetic susceptibility as well as behavioral and lifestyle factors that cluster within families. The fact that such large differences in DNA repair capacity were observed in sisters that have other risk factors in common (family history, lifestyle, and behavior) further strengthens these results.
Another strength of our study is that our measurement of DNA repair capacity was more specific than that of many other studies. Most prior studies analyzed mutagen sensitivity, which is a combined measure of DNA damage formation and repair capacity immediately after treatment. Hence, those studies could not distinguish between actual repair processes and other exogenous and endogenous factors modifying the initial level of DNA damage, such as decomposition of the mutagen in aqueous solution or detoxification processes. In contrast, by treating cells with a direct-acting carcinogen, we avoided potential bias due to individual differences in efficiency of metabolism of the parent mutagen, benzo[a]pyrene, into a reactive form. The assay used in the present study allows independent estimation of individual sensitivity to the test mutagen and DNA repair capacity. Individual sensitivity levels were estimated by measuring BPDEDNA adducts in cells harvested immediately after mutagen treatment, but no differences were observed between case patients and control subjects.
This study also has several limitations. Ideally, DNA repair capacity should be measured in the target tissue, mammary epithelial cells. However, most studies use fresh or cryopreserved lymphocytes because these are more readily available than target tissue. In this study, transformed lymphoblastoid cell lines generated from peripheral lymphocytes were used because the cell lines were readily available as a core resource of the Breast CFR. The advantage of using lymphoblastoid cell lines is that they are an unlimited resource and can be thawed for use whenever needed. Our finding that these cell lines allow the detection of differences in DNA repair capacity between case patients and control subjects widely expands the types of repair studies that can be carried out. Indeed, prior studies have successfully used lymphoblastoid cell lines in establishing DNA repair assays (21), and a correlation between DNA repair capacity in primary lymphocytes and lymphoblastoid cell lines has been observed (22).
Another limitation is that blood samples were collected from case patients after diagnosis; 50% were collected more than 5 years after diagnosis. However, we separately analyzed those who provided blood samples within 5 years after diagnosis and those who provided blood more than 5 years after diagnosis and saw no difference in results.
In summary, these data support the hypothesis that deficient DNA repair capacity is associated with susceptibility to breast cancer and may be a valuable in vitro biomarker to identify high-risk subjects, especially in familial breast cancer families. It is unclear at this time whether there are any interventions that could alter DNA repair capacity and what effect such interventions might have on risk.
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NOTES |
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We thank Irina Gurvich for the processing, storage, and inventory of biospecimens for the study.
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Manuscript received June 11, 2004; revised October 28, 2004; accepted November 15, 2004.
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