1 Department of Cancer Biology and
2 Department of Public Health Sciences, Wake Forest University School of Medicine, Winston-Salem, NC 27157,
3 Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, Livermore, CA 94550 and
4 Lombardi Cancer Center, Georgetown University Medical Center, Washington, DC 20007, USA
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Abstract |
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Abbreviations: APE1, apurinic/apyrimidinic endonuclease; AT, ataxia telangiectasia; BER, base excision repair; CI, confidence interval; FACS, fluorescence-activated cell sorter; FH, family history; HRR, homologous recombination repair; IR, ionizing radiation; Lig III, ligase III; NER, nucleotide excision repair; OR, odds ratio; PARP, poly(ADP-ribose)polymerase; Pol ß, polymerase ß; RFLP, restriction fragment length polymorphism; SNPs, single-nucleotide polymorphisms; XRCC, X-ray repair cross complementing.
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Introduction |
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Hypersensitivity to IR and defective DNA repair were observed in breast cancer patients and healthy women with a positive family history (FH) compared with healthy women without a FH of breast cancer (614). Higher levels of radiation-induced G2 phase delay (i.e. mitotic delay) were present in lymphocytes from ataxia telangiectasia (AT) patients, breast cancer patients and women with a FH of breast cancer (1517). Genetic variability in DNA repair may contribute to IR sensitivity and cancer susceptibility. Several studies have identified many single-nucleotide polymorphisms (SNPs) in genes involved in nucleotide excision repair (NER), BER, double-strand break repair/HRR and cell-cycle checkpoint (1822). These DNA repair genetic variants could be classified as cancer susceptibility genes, particularly if SNPs have functional significance.
In this study, we evaluated the association between IR sensitivity as measured by cell cycle G2 delay and four amino acid substitution variants of three DNA repair genes: XRCC1 (exon 6, codon 194 Arg/Trp and exon 10, codon 399 Arg/Gln), XRCC3 (exon 7, codon 241 Thr/Met) and APE1 (exon 5, codon 148 Asp/Glu). XRCC1 plays an important role in BER and participates as a scaffolding intermediate by interacting with ligase III (Lig III), DNA polymerase ß (pol ß) and poly(ADP-ribose)polymerase (PARP) in the C-terminal, N-terminal and central regions of XRCC1, respectively (2325). XRCC1 mutant cells have increased sensitivity to IR, UV, hydrogen peroxide and mitomycin C (5). The XRCC1 variant allele, Arg194Trp (exon 6), results in a non-conservative substitution in a hydrophobic region of XRCC1, and the SNP of Arg399Gln occurs within the BRCA1 C-terminal domain known to interact with PARP (19). The XRCC1 194Arg and 399Gln alleles were associated with increased risk for oral cavity and pharyngeal cancers (26). The XRCC1 399Gln allele was associated with lung cancer risk, as well as higher levels of DNA adduct and sister chromatid exchange (2729).
XRCC3 is a member of the Rad51 DNA repair gene family; it functions in the HRR pathway for repairing double-strand breaks, which plays important roles in maintaining genome stability (30). XRCC3 mutant cells show moderate hypersensitivity to IR, UV and monofunctional alkylating agents but extreme sensitivity to DNA cross-linking drugs, such as mitomycin C or cisplatin (3033). APE1 (HAP1/Ref-1; EC 4.2.99.18) is the rate-limiting enzyme in the BER pathway (34). It cleaves 5' of DNA abasic sugar residues generated from exogenous factors, such as IR and environmental carcinogens, as well as endogenous agents from normal cellular metabolism (35). Two studies suggested that APE1 allows pol ß and Lig III to enter DNA repair sites and assembles pol ß onto AP sites (36,37). Three variants (L104R, E126D and R237A) of the APE1 gene show reduced endonuclease activity (38).
In contrast to rare, highly penetrant alleles, low-penetrance susceptibility alleles may contribute to a substantial proportion of cancer cases, since some are very common in the general population (39,40). It is clearly important to evaluate genetic variants of DNA repair in human cancer risk, but first, their functional significance must be elucidated. At the present time, one of the most rapidly growing research areas on DNA repair has focused on determining the association between sequence variations with heritable phenotypes and cancer susceptibility. In this study, we investigated the association between prolonged cell cycle delay in response to IR and four amino acid substitution variants of three DNA repair genes.
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Materials and methods |
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PCRRFLP genotyping assays
Genomic DNA was isolated from peripheral lymphocytes using an ASAP Genomic DNA Isolation kit (Boehringer Mannheim, Indianapolis, IN). PCRRFLP assays were used to determine DNA repair genotypes. Four primer pairs, synthesized by the DNA Synthesis Core Laboratory at Wake Forest University School of Medicine were used to individually amplify each genetic region. The primer pairs used were XRCC1 Arg194Trp, forward, 5'-GCCCCGTCCCAGGTA-3' and reverse, 5'-AGCCCCAAGACCC TTT- CACT-3'; XRCC1 Arg399Gln, forward, 5'-TCTCCCTTGGTCTCCAACCT-3' and reverse, 5'-AGTAGTCTGCTGGCTCTGG-3'; XRCC3 Thr241Met, forward, 5'-GGTCGAGTGACAGTCCAAAC-3' and reverse, 5'-TGCAA- CGGCTGAGGGTCTT-3' and APE1 Asp148Glu, forward, 5'-CTGTTTC- ATTTCTATAGGCTA-3' and reverse, 5'-AGGAACTTGCGAAAGGCTTC-3'. About 50200 ng genomic DNA in a total volume of 20 µl was amplified using a GeneAmp 9700 (Perkin Elmer, Foster City, CA). The reaction mixture consisted of GeneAmp PCR Gold buffer (150 mM TrisHCl, pH 8.0, 500 mM KCl), 2.5 mM MgCl2, 0.2 mM each dNTPs, 0.2 µM each primer, 1 U AmpliTaq Gold polymerase (Perkin Elmer) and nuclease-free water (Promega, Madison, WI). Negative controls lacking DNA templates were set up with all PCR reactions. Each genotype assay was repeated at least two to three times to confirm study results. To avoid contamination, barrier tips were used, and all reaction tubes and tips were UV irradiated for at least 15 min prior to reaction mixture preparation. PCR conditions were 95°C for 2 min, followed by 40 cycles of 94°C for 15 s, 57°C for 45 s, 72°C for 45 s and a final elongation step at 72°C for 5 min.
PCR products were digested with specific restriction enzymes obtained from New England Biolabs (Beverly, MA) that recognized and cut either the wild-type or variant sequence site. PCR products were digested at 37°C for 3 h for XRCC1 (exon 6) and at least 3 h to overnight for other genotypes. The digested products were resolved on 1.5% agarose gels and stained with 0.5 µg/ml ethidium bromide (Gibco BRL, Gaithersburg, MD). The PvuII restricted products of XRCC1 codon 194 Arg/Arg, Arg/Trp and Trp/Trp genotypes had band sizes of 490, 490/294/196 and 294/196 bp, respectively. The MspI restricted products of XRCC1 codon 399 Arg/Arg, Arg/Gln and Gln/Gln genotypes had band sizes of 269/133, 402/269/133 and 402 bp, respectively. The use of NlaIII for XRCC3 (exon 7) created an internal control band size of 140 bp. The digested products for XRCC3 codon 241 Thr/Thr, Thr/Met and Met/Met genotypes had band sizes of 315/140, 315/210/140/105 and 210/140/105 bp, respectively. The BfaI restricted products of APE1 codon 148 Asp/Asp, Asp/Glu and Glu/Glu genotypes had band sizes of 164, 164/144/20 and 144/20 bp, respectively.
Cell cycle G2 delay assay
The quick-thawed lymphocytes with >95% viability were incubated in RPMI medium with 20 µg/ml phytohemagglutinin for 72 h. Using a 137Cs source, the cells were exposed to 3 Gy -irradiation, which gave the best discrimination between AT patients and normal controls (15). Cells with or without (as control) exposure to radiation were harvested 24 h later, and the percentages of cells in the G0/G1, G2/M and S phase of the cell cycle were determined by using a dual laser fluorescence activated cell sorter (FACStar Plus; Becton Dickinson, Mountain View, CA) as described previously (11,12). DNA staining was performed by adding 0.05 ml propidium iodide in PBS (250 µg/ml propidium iodide, 5 mg/ml RNase and 10 mg/ml Triton X-100) to the cell suspension. The mitotic delay index was calculated as (percentage of cells in G2/M with IR exposure percentage of control cells in G2/M) / (percentage of control cells in S phase)x100%. The median of the total study population was used as the cut-off for prolonged cell cycle G2 delay (IR hypersensitive).
Statistical analysis
All the laboratory assays were conducted in a blinded fashion and then unblinded for statistical analysis. ANOVA was used to compare the mitotic delay index among different groups of subjects. A log transformation of the mitotic delay data was used to stabilize variances and increase the normality of the residuals. Analysis of covariance on the log transformed data was used to assess the joint effect of FH and genotypes and their two-way interactions on mitotic delay. Logistic regression was used to evaluate the association between cell cycle G2 delay and genotypes. Odds ratio (OR) and 95% confidence interval (CI) were calculated from the logistic model estimates. All the statistical analyses were carried out using the Statistical Analysis System (SAS Institute, Cary, NC).
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Results |
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In this study, we evaluated genotype and cell cycle G2 delay data from 135 disease-free women. Their ages ranged from 26 to 82 with a median of 53 years. Sixty-four percent of the women were 50 years or older. A 20 page questionnaire was used by the Biomarker Core to collect information on race, FH, menstrual history, parity and medication. Data on FH were available for 83 women; 38 (46%) of these had at least one first degree relative with breast cancer. Twenty-one (16%) only had mothers with breast cancer, nine (7%) only had sisters and eight (6%) had both. Twenty-six of the women in our sample (31%) had one first degree relative with breast cancer, 11 (13%) had two and one (1%) had three. Many study subjects did not return the questionnaire, probably because the questionnaire was extremely time consuming. Unfortunately, the Institutional Review Board guidelines did not allow us to contact study subjects for missing information. To address the potential bias related to missing FH data, we first ran analyses to compare G2 delay and genotype data between the two groups of subjects, women with and without questionnaire information. There was no significant difference in the distribution of DNA repair genotypes and G2 delay marker between these two groups. Additionally, age was similar for those with and without FH data. Therefore, it is unlikely that missing questionnaire data will introduce serious systematic error or bias in this study. However, missing questionnaire data lowered the sample size and statistical power for those analyses including FH information.
Eighty-five percent of the subjects carried the XRCC1 (exon 6) wild-type/wild-type (WW) genotype; the remaining 15% were wild-type/variant (WV). The distribution of XRCC1 (exon 10) is 44% WW, 46% WV and 10% VV. These percentages are 38% WW, 46% WV and 16% VV for APE1 (exon 5) genotype and 37% WW, 47% WV and 16% VV for XRCC3 (exon 7) genotype. Genotype distributions at each locus were consistent with HardyWeinberg equilibria.
Analysis of variance on the log transformed values of delay index was used to assess the univariate effect of subject characteristics and amino acid substitution variants in DNA repair genes on cellular response to IR. Results are summarized in Tables I and II. In Table I
, there was a slight but non-significant increase of mitotic delay with age (P = 0.57). The correlation between mitotic delay and age considered continuously was also non-significant (P = 0.39). The mean ± SD of the mitotic delay index in women with 0, 1, and 2+ affected first-degree relatives was 32.87 ± 11.12 (n = 45), 30.14 ± 13.09 (n = 26) and 34.27 ± 13.33 (n = 12), respectively. The mitotic delay did not differ significantly among these three groups of women (P = 0.35).
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Multivariable regression models were then used to assess the joint effect of the variables shown in Tables I and II and their two-way interactions on mitotic delay index. Due to the large number of individuals with missing FH information, models involving FH which included only the 83 subjects with these data were initially assessed. Interestingly, it appears there is a significant interaction between FH and APE1 (exon 5) genotype (P = 0.007) and between FH and XRCC1 (exon 10) genotype (P = 0.005) in mitotic delay. Summary statistics for delay index broken down by FH for each genotype are shown in Table III
. In the absence of FH, neither genotype is significantly associated with mitotic delay. For those with one involved relative, both APE1 (exon 5) and XRCC1 (exon 10) are significantly associated with mitotic index, with both WV and VV differing from WW. Data are too sparse to make conclusions regarding differences in genotypes for those with 2+ involved relatives. None of the genegene interactions were statistically significant, even the one between APE1 and XRCC1, although both play critical roles in BER and single-strand break repair.
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Data in Table IV show the distribution of subjects with prolonged G2 delay for combined XRCC1 and APE1 genotypes. Even though this interaction was not significant in the logistic regression model just described, the results in Table IV
suggest that prolonged cell cycle delay was significantly associated with number of variant alleles when APE1 Asp148Glu and XRCC1 Arg399Gln genotypes were evaluated in a four-level model (
2 for linear trend = 10.9; P = 0.001). Odds ratio (OR) = 1.0 for those without variant allele (referent); OR = 0.81 (95% CI 0.272.45) for those with one variant allele; OR = 2.19 (95% CI 0.687.02) and OR = 6.67 (95% CI = 1.3832.26) for those with at least three variant alleles. Future larger studies are warranted to further test this potential combined effect of XRCC1 and APE1 genotypes.
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Discussion |
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Exposure to IR has been implicated in the etiology of cancer (1), and lesions induced by IR are repaired by BER and HRR (24,30). Hypersensitivity to IR could result from somatic mutations of repair genes, polymorphisms of BER genes (e.g. APE1 and XRCC1), and a reduction in protein expression secondary to other cellular dysfunction. Our data suggest that amino acid substitution variants in BER genes are associated with mitotic delay in response to IR. Since inherited hypersensitivity to IR and deficient repair of IR-induced damage may serve as markers for low-penetrant predisposition genes in breast cancer, it is reasonable to conclude that amino acid substitution variants in BER genes may contribute to hereditary IR sensitivity and cancer susceptibility. However, detailed structurefunction relationships between amino acid substitution variants and enzyme activities remain to be elucidated.
The multifunctional mammalian APE1 is responsible for the repair of AP sites in DNA. This enzyme also functions as a redox factor facilitating the DNA-binding capability of AP-1, and numerous other transcription factors (41). Abasic sites represent ubiquitous DNA lesions that arise spontaneously or are induced by DNA-damaging agents. They block DNA replication and are considered cytotoxic and mutagenic. The major mammalian APE1 plays a central role in BER in two distinct ways (42). First, APE1 initiates repair of AP sites in DNA produced either spontaneously or after removal of uracil and alkylated bases in DNA by monofunctional DNA glycosylases. Second, APE1 can act as a 3'-phosphoesterase to initiate repair of strand breaks, either directly induced by reactive oxygen species or indirectly through the AP lyase reaction of DNA damage-specific glycosylases. Furthermore, although APE1 plays an important role in the repair of DNA strand breaks, other genes, such as pol ß, are rate-limiting factors for uracil repair (42); hence, it will be necessary to evaluate other BER genes as well.
In the current study, we report an association between SNPs of two DNA repair genes and IR sensitivity. Since the SNPs presented in this paper are amino acid substitution variants, structural changes of APE1 and XRCC1 proteins may have functional significance. First, the APE1 148 Glu variant allele has a small but non-significant effect on endonuclease activity (94% of wild-type) and DNA binding activity (Kd, 20.3 ± 3.4 versus 25.8 ± 12.2 nM in wild-type) (38). Although the small differences in binding and lower endonuclease activity may be within experimental error, it is possible that the lower Kd of the variant implies a higher affinity between APE1 protein and damaged DNA after catalysis and as such turns over less effectively. In addition, isolated protein was studied, effects of APE1 interaction with other BER components not studied (38). These other proteins may also affect APE1 turnover. Therefore, future studies are needed to investigate whether APE1 148 Glu allele may alter the ability of the APE1 protein to communicate with other BER proteins and thereby influence BER efficiency. Second, the genetic variant of XRCC1 Arg399Gln occurs within the BRCA1 C-terminal domain, which interacts with PARP (19). Considering the important roles of BRCA1 and PARP in DNA repair, the XRCC1 variant may have functional significance. Future detailed analyses of protein structures and functions are needed to further evaluate the mechanisms involved.
Using a cytogenetic assay, cellular radiosensitivity in family members of radiosensitive (breast cancer patients) and non-sensitive individuals was studied (12). The results from their segregation analysis suggested that radiosensitivity may be heritable, with a single major gene accounting for 82% of the variance among family members. The addition of a second, rarer gene to the model resulted in a better fit of the data. These results further suggest that cancer-predisposing genes are probably common, low-penetrance alleles, such as amino acid substitution variants in DNA repair genes. The results from our study support their proposed model that multiple, low-penetrance, common genes (e.g. APE1 and XRCC1) contribute to IR sensitivity.
To the best of our knowledge, the relationship between APE1 Asp148Glu genotype and cancer susceptibility has not been investigated previously. In an ongoing study, we are evaluating whether this genotype is associated with breast cancer (43). The genetic variant of XRCC1 Arg399Gln has been evaluated in cancer of the head and neck, lung and breast (26,27,43). In this study, we demonstrate that the XRCC1 Arg399Gln genotype may influence cellular response to IR, particularly in women with positive FH. Our data are consistent with the previous findings that the XRCC1 399Gln allele was associated with higher levels of DNA adducts and sister chromatid exchange (28,29).
Ionizing radiation induces many types of damage to DNA, requiring multiple repair pathways (e.g. BER and HRR) to restore genomic integrity. Most of the repair pathways are extremely complex, and many genes are involved in different repair pathways. With limited questionnaire data and sample size, this manuscript focuses on the genotypephenotype relationship. It is conceivable that many other genetic and non-genetic factors (i.e. other SNPs, smoking, alcohol consumption, menopausal status, medication, diet and environmental exposures) may also influence DNA damage/repair, but the study of those relationships is beyond the scope of this paper. At the present time, we are conducting a larger study to test these other factors. The results from this study suggest that amino acid substitution variants of XRCC1 and APE1 may alter the functions of these proteins, resulting in hypersensitivity to IR. Future larger studies are warranted to further test the functional significance of these genotypes.
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Notes |
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Acknowledgments |
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References |
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