We used EJ capacity as marker of DSB repair capacity in lymphoblastoid cell lines from sisters discordant for breast cancer. Different substrates, either with sticky or blunt ends, were tested with an in vitro
assay to rapidly visualize on agarose gels the products of rejoining activity (dimers, trimers and high molecular weight multimers). The aim was to determine if reduced and/or altered DSB repair is associated with increased breast cancer risk. We used a non-radioactive method with the fluorescent dye SYBR® Green rather than a previously used radioactive probe (31
). Although we believe that SYBR® Green is a less sensitive detection system than 32
P, fluorescent quantification of EJ reactions using 20 ng of substrate allowed the assessment of gel DNA in amounts as little as 0.5 ng (32
The mean repair activity was non-significantly lower in breast cancer cases than controls with both substrates. When using EJ values as a continuous variable, the adjusted ORs indicated an increased risk of 1.07-fold (1.01–1.13) and 1.08-fold (1.01–1.15) for reduced repair capacity with EcoRI- and HincII-linearized pUC18, respectively. Stratifying data into tertiles based on the controls, we observed a 4-fold increased breast cancer risk (OR
4.22, 95% CI
= 1.22–14.6) for those with the poorest repair capacity using the EcoRI substrate. To date, only Bau et al.
) used an in vitro
EJ assay within the epidemiological setting of a case–control study. They provided evidence that lymphoblastoid cells from breast cancer patients had lower capacity to religate a DNA substrate bearing cohesive ends than those from healthy controls (34
). They had also demonstrated previously that whole-cell extracts from BRCA1-deficient breast cell lines compared with BRCA-proficient ones had decreased EJ capacity (21
). Our observations with EcoRI-linearized pUC18 (cohesive ends) are in agreement with their results, although we used a different substrate that was not radioactively labeled.
Bau et al.
) also used a luciferase-containing plasmid with blunt ends due to cutting with HindIII to detect the EJ fidelity in transfected mononuclear cells of 22 breast cancer cases and 20 controls. We instead assayed in vitro
DNA substrates with two different types of termini, confirming the reduced EJ capacity observed with EcoRI-linearized pUC18. Blunt DNA termini are the easiest type to be repaired by NHEJ, usually with an accurate ligation. However, it has also been shown that different types of DNA termini are processed by different enzymatic complexes recruited by the NHEJ machinery, depending upon the type of DSB (35
). There was a correlation between activity measured with both substrates in cases and controls (0.677 and 0.810, P
< 0.0001, respectively). The comparable extent of EJ repair observed with the two different types of substrate supports the hypothesis that decreased repair is the result of a general impairment of DSB repair and not the result of a single NHEJ subpathway. The lack of an increased breast cancer risk in the tertile with the poorest EJ activity, between cases and controls, observed with the HincII substrate might be accounted for a higher day-to-day variability (28.4%) compared with the EcoRI substrate (19.4%).
These same subjects were included in a larger prior study in which we observed increased risk associated with decreased NER capacity with an OR of 2.99 (95% CI
1.45–6.17) for those in the quartile with the poorest repair. NER was also lowest in the youngest cases and oldest controls. Similarly for EJ capacity, we observed that the youngest cases had the lowest DNA repair capacity, although the trend with age was not significant. When looking at the combination of repair capacity in the two pathways, NER and EJ, those with decreased repair in both had an OR of 4.92 (95% CI
1.36–17.8), compared with the ORs of 2.44 (1.46–4.11) for those with NER below the median (25
) and 1.23 (0.46–3.30) for those with EJ below the median. This is in contrast to studies of upper aerodigestive track cancer patients in which the mutagen sensitivity assay was carried out using bleomycin and benzo[a
]pyrene diol epoxide as test mutagens (36
). Subjects with sensitivity to both agents were at a more than additive 19.2-fold increased risk compared with those not sensitive to either agent.
Several other studies have reported on DNA repair capacity as a risk factor for breast cancer development. Using the comet assay, Smith et al.
) found decreased capacity to repair DNA damage induced by ionizing radiation in peripheral lymphocytes from breast cancer cases compared with controls. Using the host cell reactivation assay, Ramos et al.
) showed that younger breast cancer cases less efficiently repaired UV-induced DNA damage than older cases or age-matched control subjects. Studies of non-melanoma skin cancer provide additional evidence on age effects; younger patients affected by basal cell carcinoma had a lower capability to repair DNA compared with their matched control subjects and repair capacity was also lower compared with older cases (37
). In our study, after age stratification, mean values of EJ capacity among controls decreased with age until age >60 years for both substrates (). As DSB repair is expected to become less efficient during normal aging (41
), the lack of a decrease in the repair of DNA in the oldest controls may be due to the small number older than 60 years. In addition to a reduced efficiency of EJ activity, age-related genomic instability might be also be induced by the infidelity of NHEJ (42
). Pre-senescent and senescent fibroblasts have a compromised fidelity of NHEJ and, in particular, older cells do not accurately ligate cohesive ends and tend to generate large deletions at the junctions (43
Similar to Bau et al.
), we used lymphoblastoid lines derived from breast cancer patients and healthy women. This provides outcomes with more biological significance compared with previous studies that used embryonic fibroblasts and breast cancer cell lines to investigate DSB repair (44
). However, in contrast to Bau et al.
), we used a family-based design of at least one affected sister with breast cancer and one unaffected sister. This partially removes potential confounding related to population admixture and reduces confounding by family-level factors. The sister-set design has been recently discussed in terms of its usefulness compared with other study types (25
The present study also has limitations. Because the DNA repair tests are quite time consuming, our sample size is relatively small. Another constraint faced by most DNA repair phenotyping studies is the use of a surrogate tissue; in our case, transformed lymphoblastoid cells that might not reflect the actual EJ capacity of the target tissue, the mammary epithelium. Nevertheless, due to the difficulty of sampling target tissues, most DNA repair phenotyping studies have been performed on lymphocytes (6
). In addition, frozen viable cells are much more readily available than target tissues and, for lymphoblastoid cells, have the advantage of an almost unlimited source of material for molecular studies. Bleomycin sensitivity was found to be similar for mononuclear cells compared with lymphoblastoid cell lines (47
Another potential limitation is that blood samples were collected after diagnosis and our breast cancer cases were prevalent; blood samples were collected on average 5 years after diagnosis. To address these concerns, we stratified the main model by years from breast cancer diagnosis to evaluate the potential impact of disease or treatment on the association between EJ capacity and breast cancer risk. We found that the relationship between EJ capacity and breast cancer risk was similar for women diagnosed within 2 years of blood draw compared with women diagnosed >2 years from blood draw. Without conducting a prospective study, we cannot entirely exclude an effect of treatment or disease. However, a recent study, using the COMET assay, demonstrated no difference in DNA damage of lymphoblastoid cells developed from blood samples collected before or after breast cancer diagnosis (48
). Given the complexities of DNA repair phenotyping assays, there are currently no prospective studies of DNA repair capacity and breast cancer risk.
In summary, the present data add support to the hypothesis that inefficient DNA repair is associated with breast cancer susceptibility. The possibility to non-invasively detect biomarkers indicating compromised DNA repair capacity can greatly impact the determination of breast cancer risk and prognosis in high-risk subjects, especially in breast cancer families.