Mutagenesis Advance Access originally published online on January 31, 2007
Mutagenesis 2007 22(2):147-153; doi:10.1093/mutage/gel071
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Alkaline unwinding flow cytometry assay to measure nucleotide excision repair
Division of Epidemiology, University of Minnesota, Suite 300, West Bank Office Building, Minneapolis, MN 55454, USA 1Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, MN 55455, USA 2Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA
Nucleotide excision repair (NER), one of the DNA repair pathways, is the primary mechanism for repair of bulky adducts caused by physical and chemical agents, such as UV radiation, cisplatin and 4-nitroquinolones. Variations in DNA repair may be a significant risk factor for several cancers, but its measurement in epidemiological studies has been hindered by the high variability, complexity and laborious nature of currently available assays. An alkaline unwinding flow cytometric assay using UV-C radiation as a DNA-damaging agent was adapted for measurement of NER-mediated breaks. This assay was based on the principle of alkaline unwinding of strand breaks in double-stranded DNA to yield single-stranded DNA with the number of strand breaks being proportional to the amount of DNA damage. This assay measured 50 000 events per sample with several samples being analyzed per specimen, thereby providing very reliable measurements, which can be performed on a large-scale basis. Using area under the curve (AUC) to quantitate amount of NER-mediated breaks, this assay was able to detect increased NER-mediated breaks with increasing doses of UV-C radiation. The assay detected NER-mediated breaks in lymphocytes from normal donors and not in xeroderma pigmentosum lymphoblastoid cell lines indicating specificity for the detection of NER-mediated breaks. The assay measured NER-mediated breaks within G1, S and G2/M phases of the cell cycle; thereby decreasing variability in measurements of NER-mediated breaks due to differences in cell cycle phases. Intraindividual variability for AUC after 120 min of repair was 15% with interindividual variability being 
43% for cells in the G1 phase, indicating substantial between-subject variation and relatively low within-subject variation. Thus, the alkaline unwinding flow cytometry-based assay provides a high-throughput method for the specific measurement of NER-mediated breaks in lymphocytes.
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Introduction |
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Multiple DNA repair strategies have evolved to minimize the deleterious effects of DNA damage induced by numerous endogenous and exogenous agents. The DNA repair pathways, including base excision repair and nucleotide excision repair (NER), operate in concert to repair a wide range of lesions and maintain genomic integrity (1
). Bulky adducts induced by physical and chemical agents such as UV radiation, cisplatin and 4-nitroquinolone 1-oxide contribute to errors in DNA replication and induce mutations that may be carcinogenic or cause cell death (1). One of the DNA repair pathways, the NER pathway, is a common pathway for removal of a wide variety of structurally unrelated bulky DNA lesions that induce DNA helical distortion (1). Deficiency in NER has been demonstrated in rare genetic syndromes such as xeroderma pigmentosum (XP), in which patients have a 1000-fold increased risk of skin cancer (2) and an elevated risk of several other cancers. Numerous studies have also consistently shown an association between low DNA repair and an increased susceptibility to a variety of cancers such as breast cancer, lung cancer and skin cancer (3–5). Thus, variation in NER may be an important factor in determining cancer susceptibility.
Numerous functional assays have been developed to evaluate DNA repair in cells. Various methods such as the chromatid aberration, P-32 postlabeling, mutagen sensitivity, comet, host cell reactivation assays and ELISA to measure specific DNA adducts have been used to detect DNA damage and repair in human populations. However, the time-consuming and labor-intensive approaches used in these functional assays coupled with high assay variability have limited their widespread use in large epidemiological studies. Also, differences in DNA susceptibility and repair between quiescent and proliferating cells within a single sample contribute to the high variability observed in these assays (6,7). Thus, assays such as the P-32 postlabeling, host cell reactivation and ELISA that cannot measure DNA damage at the single-cell level and measure only average amounts of specific DNA adducts are susceptible to high intra-sample variability, thereby limiting their usefulness in population studies. Hence, single-cell assays, such as comet, the mutagen sensitivity and the chromatid aberration assays, were developed to measure DNA damage within individual cells. The comet and the chromatid aberration assays usually measure DNA damage in a limited number of cells (50–200) leading to substantial analytical variability in the measurement of DNA damage. In addition, it not possible to routinely measure DNA repair in different cell cycle phases using these assays, thus increasing the variability of DNA repair estimates obtained using these assays. The comet assay, where the extent of migration of DNA toward the anode determines the extent of DNA damage in individual cells, is the most commonly used biological assay that is used to estimate DNA repair in human populations (8) and is reported to have an assay variation of 20%, an intra-individual variation of 42% and an interindividual variation of 26% (9). Efforts such as the Fourth International Comet Assay Workshop have tried to standardize protocols for the comet assay in order to improve its reliability. Inspite of these efforts, a major limitation of the comet assay is the high variability involved in measurement of DNA repair (10). Thus, high variability in measurement of DNA damage and repair and low capacity of commonly used DNA repair assays remain major limitations for their routine use in epidemiological studies.
To overcome the problems associated with measuring DNA damage in a limited number of cells, a flow cytometry assay was developed to measure DNA repair in a large number of cells (11). However, limitations in sensitivity of the flow cytometry assay to detect DNA damage limited its routine use (11–13). Recently, the alkaline unwinding flow cytometry assay was modified by Potter et al. (14) to increase sensitivity to detect DNA damage and standardize the analytic methodology. This modified method was used to measure DNA repair due to hydrogen peroxide, gamma radiation and doxorubicin (14). This method allowed for rapid, routine, objective measurement of large numbers of cells (>10 000 cells) within each sample. It also allowed determination of DNA repair in each phase of the cell cycle thereby decreasing sample variability in the measurement of DNA damage. In this paper, we adapted the flow cytometry assay developed by Potter et al. for measurement of NER-mediated breaks. This assay is based on the principle of alkaline unwinding of strand breaks in double-stranded DNA (dsDNA) to single-stranded DNA (ssDNA). Strand breaks can be formed from excision repair sites, double-strand breaks, single-strand breaks, strand breaks associated with alkali labile abasic (apurinic/apyrimidinic) sites and strand discontinuities at transcription forks (15,16). The amount of ssDNA formed after alkali denaturation is proportional to the amount of strand breaks present in the sample (16). The principle of alkaline unwinding in the measurement of DNA damage is also used in other commonly used DNA repair assays such as the comet assay and microgel single-cell assays (8,11–13). A variety of assay parameters were evaluated and optimized to reliably measure NER-mediated breaks. Interindividual and intra-individual variability estimates were obtained by measuring NER-mediated breaks in lymphocytes from 10 normal donors.
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Participant characteristics and blood collection
After obtaining informed consent, blood was collected from six men and four women. These normal donors were between the ages of 18 and 60 years and provided 16 ml of blood in two CPT tubes (Becton, Dickinson and Company, Franklin Lakes, NJ). Lymphocytes were isolated as per manufacturer's recommendations within 20 min of blood collection.
Lymphocyte cultures for the measurement of NER-mediated breaks
Lymphocytes from normal donors were suspended in 3 ml of Hank's Balanced Salt Solution (HBSS). Five hundred microliters of the lymphocyte preparation was seeded into culture flasks containing 7 ml of culture media [1 ml of penicillin–streptomycin (50 U/ml penicillin and 50 µg/ml streptomycin), Invitrogen Corporation, Carlsbad, CA, Cat. No. 15070-063], 100 µl of gentamycin (50 mg/ml, Invitrogen Corporation, Cat. No. 15750-060), 5 ml of L-glutamine (200 mM, Invitrogen Corporation, Cat. No. 25030-081) and 18 ml of fetal bovine serum (FBS) (Invitrogen Corporation, Cat. No. 10082-147) were added to 100 ml of RPMI (50 mg/ml, Invitrogen Corporation, Cat. No. 11875-093) with 5% CO2 at 37°C and cultured for 7 days. Phytohemagglutinin (PHA; 2 µg/ml of culture fluid), a mitogen, was added to stimulate lymphocyte growth.
Experimental design
The XP lymphoblastoid cell lines were used to determine the assay specificity while assay sensitivity was determined by ability to detect NER-mediated breaks after exposure to different doses of UV-C radiation. The amount of NER-mediated breaks observed using the flow cytometry assay was compared to the amount of 6-4 photoproducts (6-4 PP) and cyclobutane pyrimidine dimers (CPDs) formed immediately after exposure to different doses of UV radiation. The normal lymphocytes were exposed to various doses of UV radiation and allowed to repair the DNA damage for 120 min. The optimal dose of UV radiation was determined by evaluating the number of NER-mediated breaks at a particular UV dose and the ability of the normal lymphocytes to repair DNA damage after 120 min. Subsequently, the variability estimates for different parameters used to estimate NER-mediated breaks were evaluated.
Specificity of the alkaline unwinding flow cytometry assay
Four XP lymphoblastoid cell lines, GM02250F (XPA mutant), GM02248D (XPC mutant), GM022252C (XPB mutant) and GM15698 (XP with unknown mutation) that had 0%, 10–20%, 37% and complete NER, respectively, as previously determined by unscheduled DNA synthesis, were purchased from the Coriell Institute (Camden, NJ). Vials containing frozen XP lymphoblastoid cell lines were rapidly thawed at 37°C and immediately diluted in 10 ml of HBSS. The lymphocytes obtained after centrifugation at 2000 rpm for 10 min at 4°C were washed twice with 10 ml HBSS and suspended in 2 ml HBSS. The XP cell lines were cultured using conditions described above except that no PHA was added to stimulate their growth and the XP lymphoblastoid cell lines were grown for 3 days.
Determination of cell viability
The trypan blue exclusion method was used to determine cell viability prior to the start of the flow cytometry procedure. One hundred microliters of cell suspension was diluted with 300 µl of phosphate-buffered saline (PBS) and subsequently stained with 200 µl of trypan blue. Viability counts were obtained within 15 min of staining the lymphocytes with trypan blue.
ELISA method for quantitation of DNA adducts
Normal lymphocytes from a single normal donor were exposed to two UV doses: 7 and 50 J/m2 after culturing them for 7 days with 2 µg/ml of PHA. An ELISA was performed using monoclonal antibodies for the 6-4 PP (64M-2) and the cyclobutane dimers (TDM-2) as described by Mori et al. (17) with some modifications. A vial of the lyophilized 64M-2 and TDM-2 antibodies, derived from ammonium sulfate precipitation and dialyzed 20 times against 10 mM PBS (generous donation from Dr Mori, Nara, Japan), was made into a stock solution by reconstituting each vial of lyophilized antibody with 100 µl of distilled water and 900 µl of 10 mM PBS. 64M-2 were quantified by adding 100 µl of the 64M-2 antibody (1 : 250 dilution of the stock solution) in 10 mM PBS with 2% FBS to 300 ng of DNA. Biotin-F(ab)'2 and peroxidase-streptavidin were diluted to the same concentrations as described by Mori et al. (17) in 10 mM PBS with 2% FBS. 3,3',5,5'-Tetramethylbenzidine (TMB; Sigma, St Louis, MO), prepared according to manufacturer's recommendation, was used as the substrate and 100 µl was incubated at 37°C for 15 min to determine the amount of 64M-2 formed after 50 J/m2 UV dose while the incubation time after 7 J/m2 was 60 min. CPDs were quantified by adding 100 µl of TDM-2 antibody (1 : 1000 dilution of stock solution) diluted in 10 mM PBS with 10% FBS to 200 ng of DNA. TDM-2 antibody, biotin-F(ab)'2 and peroxidase-streptavidin were diluted to the same concentrations as described earlier in 10 mM PBS with 10% FBS (17). One hundred microliters of TMB substrate was incubated at 37°C for 30 min to determine the amount of CPD adducts formed at both 7 and 50 J/m2. Absorbance at 450 nm was used to estimate the amount of 64M-2 and CPD adducts formed after exposure to different doses of UV radiation. All experiments were performed in quadruplicates.
Alkaline unwinding flow cytometry DNA repair assay
We adapted a flow cytometry assay described earlier for the measurement of NER-mediated breaks (14). The assay methods were similar to those described previously (14) except that acridine orange (AO) staining and flow cytometric measurements were performed at room temperature instead of at 4°C. Briefly, cultured lymphocytes were centrifuged at 2000 rpm at 4°C and the lymphocyte pellet was suspended in 3 ml of culture media. Three hundred microliters of cell suspension was added to 700 µl of fresh culture media and subjected to a dose of 2.3 W/sec of UV-C radiation (Phillips UV light bulb wavelength of 254 nm) for various lengths of time and a final UV dose between 4 and 50 J/m2. The radiation dose was measured by a radiometer (UVX digital radiometer; UVP Inc, Upland, CA). The cells were allowed to repair for various times (0, 15, 30, 45, 60, 75, 90 and 120 min) by incubating the cells at 37°C. Controls for this experiment included lymphocytes that were incubated at 37°C for similar times without exposure to UV-C radiation. The cells were fixed in 40% alcohol in PBS, embedded in 2.25% ultra low-melting agarose gel in PBS and subjected to alkaline denaturation (pH = 13) for 20 min. The alkali was then washed twice with a neutral-buffered solution (pH = 7.4). The agarose gel was then melted by heating it to 65°C in a water bath for 1 min. After melting the gel, a solution containing ice-cold 0.1% Triton X-100, 0.08N HCl and 0.15N NaCl was added to the cells and subsequently stained with AO for 5 min at room temperature. All flow cytometry measurements were performed using a FACS Vantage instrument (BD Bioscience, San Jose, CA). After excitation with a 488-nm argon laser, AO (22.1 uM concentration; Molecular Probes, Eugene, OR, Cat. No. A-1301) produced red fluorescence when bound to ssDNA (monitored at >645 nm) and green fluorescence when bound to dsDNA (monitored at 500–530 nm) (18). To ensure comparability of experiments across days, chicken erythrocyte nuclei (CEN) (Biosure Controls, Grass Valley, CA, Cat. No. 1006) were added to each tube and used as an internal standard to normalize all readings to the CEN fluorescence.
Data analysis
The ratio of ssDNA to dsDNA and sum of ssDNA and dsDNA were calculated using FACS Assistant software. All data analysis was performed using Cell Quest data analysis software. The sum of ssDNA and dsDNA was used to identify cells in different phases of the cell cycle. It has been previously shown that except in cases of extreme DNA damage, the sum of ssDNA and dsDNA can be used to reliably distinguish between cells in different phases of the cell cycle (14). The ssDNA/dsDNA ratio was then calculated to estimate DNA damage for each phase in the cell cycle. The raw fluorescence readings were divided by the corresponding CEN readings to adjust for differences in AO staining intensity. The adjusted fluorescence readings from cells not exposed to UV-C radiation were then subtracted from the adjusted fluorescence readings from cells exposed to UV-C radiation to give an estimate of NER-mediated breaks in lymphocytes after exposure to UV-C radiation.
Statistical analysis
All statistical analyses were performed using Microsoft Excel 2003. Mean and standard deviations (SDs) were calculated for various parameters used to estimate NER-mediated breaks that included the ssDNA/dsDNA ratio at 15 min, the maximum ssDNA/dsDNA ratio, the area under the curve (AUC) (ssDNA/dsDNA ratio over time) after repair for 120 min (AUC120) and 60 min (AUC60), the percentage of cells in different cell cycle phases and the baseline level of DNA damage prior to UV-C radiation in each cell cycle phase. Two sample t-tests were used to test differences between mean levels of all parameters in different cell cycle phases. P values <0.05 were considered statistically significant. Correlation coefficient (r) was used to evaluate the correlation between different parameters. Data for the dose-response experiments were means of three experiments. The average coefficient of variation (CV) and corresponding 95% confidence intervals (CIs) for the intra-individual variability were based on duplicate experiments on 10 normal donors while the average CV and the corresponding 95% CIs (19) for the interindividual variability were based on single experiments done on 10 normal donors.
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Dose-response experiments
The amount of NER-mediated breaks was determined in normal lymphocytes for six doses of UV-C radiation: 4, 7, 10, 20, 30 and 50 J/m2 at five time points (0, 15, 30, 60 and 120 min) using three normal donors. NER-mediated breaks, as estimated by the AUC, increased with increasing UV-C dose for G1, S and G2/M phases of the cell cycle (Figure 1) and NER-mediated breaks after 4 J/m2 of UV radiation were significantly greater than the baseline levels of NER-mediated breaks (P < 0.05). The increase in NER-mediated breaks was linear from 4 to 10 J/m2. NER-mediated breaks substantially increased as UV-C dose increased from 20 to 30 J/m2 followed by no increase when the UV-C dose increased to 50 J/m2. NER-mediated breaks were significantly higher in the G1 phase as compared to G2/M and S phases (P < 0.05) at all UV-C doses except at 4 and 50 J/m2 (Figure 1). NER-mediated breaks in cells in G2/M and S phases were not significantly different from each other (P > 0.05) at all UV-C doses (Figure 1). The increase in NER-mediated breaks from 7 to 50 J/m2, as estimated using absorbance readings at 450 nm, was associated with a significant increase in levels of 64M-2 from 0.08 at 7 J/m2 to 0.4 at 50 J/m2 (P < 0.05) immediately after exposure to UV radiation and a significant increase in levels of cyclobutane dimer adducts from 0.12 at 7 J/m2 to 0.5 at 50 J/m2 (P < 0.05) immediately after exposure to UV radiation as determined by an ELISA. The ELISA also showed a 50% decrease in 64M-2 following repair for 120 min following exposure to 7 J/m2 of UV radiation while the corresponding decrease after exposure to 50 J/m2 of UV radiation was
23%. The CPD adducts did not show significant change after repair for 120 min at both UV doses. The maximum ssDNA/dsDNA ratio increased from 4 to 7 J/m2 (data not shown). However, subsequently there was no further increase in maximum ssDNA/dsDNA when the UV dose was increased to 20 J/m2. Though the maximum amount of ssDNA/dsDNA increased at 30 and 50 J/m2 as compared to 7 J/m2 there was not a substantial amount of repair after 120 min at these higher doses. Thus, the maximal amount of ssDNA/dsDNA that was repaired substantially within 120 min appeared to be reached at a dose of 7 J/m2 indicating that 7 J/m2 would be an optimal UV dose to evaluate NER-mediated breaks occurring in 120 min.
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Specificity of the assay for NER
The viability of the XP lymphoblastoid cell lines after exposure to 4, 7 and 10 J/m2 doses of UV-C radiation was >90% for all three doses after 120 min. To ensure comparability between experiments conducted on XP lymphoblastoid cell lines and those conducted on normal lymphocytes obtained from normal donors, 7 J/m2 of UV-C radiation was used to measure NER-mediated breaks in XP lymphoblastoid cell lines. Results from the XP lymphoblastoid cell lines for cells in the G1 phase are shown in Figure 2. The cells in G2/M and S phases of the cell cycle were similar to the results presented for the G1 phase. Little to no increase in ssDNA/dsDNA was seen in the three XP lymphoblastoid cell lines, which were known to have significantly reduced NER (Figure 2). However, in the fourth XP cell line, which had normal NER, an initial increase in ssDNA/dsDNA at 15 min and subsequent decrease in ssDNA/dsDNA to baseline levels at 120 min were noted (Figure 2).
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Parameters evaluated for their ability to estimate NER in normal donors
A majority of the normal lymphocytes (81 ± 3%) were in the G1 phase of the cell cycle with 13 ± 4% of the normal lymphocytes in the G2/M phase and 4 ± 2% of the cells in the S phase of the cell cycle. Cell viability was >75% for all experiments. Baseline levels of strand breaks were significantly higher among normal lymphocytes in the S phase (3.55 ± 0.68) as compared to normal lymphocytes in the G1 phase (2.73 ± 0.36) or the G2/M phase (2.73 ± 0.30) (P = 0.001). Figure 3 shows the baseline level of strand breaks seen in normal lymphocytes from one normal donor before exposure to UV radiation and the subsequent increase in strand breaks observed after exposure to 7 J/m2 of UV radiation. All parameters were analyzed separately for each cell cycle phase: G1, S and G2/M. Since estimation of NER-mediated breaks using a measurement at one time point is attractive for use in epidemiological studies, the ssDNA/dsDNA ratio at 15 min was evaluated as an end point. This time point was chosen as an end point since a majority of the normal donors (60%) reached the maximum level of ssDNA/dsDNA by 15 min with the remaining cases reaching 70–80% of the peak ssDNA/dsDNA in 15 min. Hence, ssDNA/dsDNA ratio at 15 min was used as a surrogate measure for the peak ssDNA/dsDNA ratio. The interindividual CV for the ssDNA/dsDNA ratio at 15 min, between normal lymphocytes of 10 normal donors, was 31% and the average intraindividual CV (duplicate experiments on same individual) was 19% for cells in the G1 phase (Table I). The peak ssDNA/dsDNA ratio had an average intraindividual CV of 15% and interindividual CV of 30% for cells in the G1 phase (Table I). AUC120 had an average intraindividual CV of 16% and an interindividual variability of 43% indicating a wide range in NER-mediated breaks between normal donors for cells in the G1 phase (Table I). Similarly, the AUC60 had an average intraindividual CV of 13% and an interindividual CV of 34% (Table I). AUC60 represented 66–68% of the AUC120 and had a correlation of 0.96 with AUC120. For the cells in the G1 phase, the correlation between AUC60, AUC120 and peak DNA damage was
0.96 while the correlation between DNA damage at 15 min and AUC120 was 0.86 (Table II).
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Similar analyses in G2/M and S phases of the cell cycle for the normal lymphocytes showed that AUC60 was more variable in G2/M and S phases compared to the G1 phase with the intraindividual CV being 22% in the S phase and 20% in the G2/M phase (Table I). The interindividual CV for AUC60 was 34% in the S phase and 48% in the G2/M phase. NER-mediated breaks, as determined using AUC60, indicated a wide variability in NER-mediated breaks between normal donors and was significantly lower in the G2/M phase (195 ± 94 units) and the S phase (210 ± 72 units) of the cell cycle as compared to the NER-mediated breaks in the G1 phase (329 ± 113 units) of the cell cycle (P < 0.001) (Table III and Figure 4). There was no statistical difference in the mean AUC60 values for the cells in G2/M and S phase of the cell cycle (P = 0.49). The AUC60 had a significant correlation of 0.73 between normal lymphocytes in G1 phase and G2/M phase of the cell cycle, 0.69 between S phase and G1 phase and 0.65 between G2/M phase and S phase.
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Discussion |
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The alkaline unwinding flow cytometry-based assay, using UV-C radiation as a DNA-damaging agent, appears to be a specific and sensitive assay for the measurement of NER-mediated breaks. Results also indicate that AUC can be used to measure NER-mediated breaks and it is feasible to use the assay in molecular epidemiology studies.
UV-C radiation predominantly induces only two types of DNA damage, CPD adducts and 64M-2, which are both repaired almost exclusively by the NER pathway (1). CPD and 6-4 PP do not cause single-strand breaks (20). However, DNA repair intermediates formed as a result of NER, lead to single-strand breaks following alkali denaturation (20). Higher baseline levels of ssDNA/dsDNA among normal lymphocytes and XP lymphoblastoid cell lines in the S phase of the cell cycle as compared to G1 or G2/M phase are reflective of higher ssDNA due to increased number of strand discontinuities at transcription forks (21) and provide evidence for detection of strand breaks using this assay. XP lymphoblastoid cell lines used in our experiments were defective in NER and had mutations in the XPA, XPC and XPB genes. All these genes are involved in DNA damage recognition and processing which occur prior to the incision events and very few single-strand breaks should be produced in these DNA repair-deficient XP cell lines as indicated by our flow cytometry assay. A substantial increase in ssDNA was observed only in the XP lymphoblastoid cell line that demonstrated complete NER, indicating that the NER pathway must be functional in addition to the presence of 6-4 PP or CPD adducts for generation of ssDNA in this assay. The ssDNA formed in this assay is proportional to the number of DNA repair intermediates, formed as a result of excision of DNA adducts and the ssDNA/dsDNA reflects an equilibrium between DNA damage recognition and completion of DNA repair (20). Thus, the initial increase in the ssDNA/dsDNA probably reflects an increase in DNA intermediates due to increase in the rate at which DNA adducts are being recognized and subsequently excised with relatively little completion of DNA repair. However, at later time points there is a decrease in the ssDNA/dsDNA reflecting a decrease in the DNA repair intermediates due to a relative decrease in number of DNA adducts being detected coupled with an increased number of DNA intermediates being completely repaired. Adjustment for the baseline amount of DNA damage present in cells untreated with UV radiation using control cells provided a more accurate estimate of damage due to UV radiation and AUC.
Previous studies have shown that the ratio of CPD adducts to the 6-4 PP formed following UV-C radiation is close to 3 : 1 with increasing amounts of both adducts being formed with increasing UV-C dose (22). Studies evaluating the kinetics of removal of DNA adducts have shown that 80–90% of 6-4 PP formed after UV-C damage are rapidly removed within 3 h (22). The CPD adducts, on the other hand, are repaired more slowly with only 10% of adducts being removed in the same time period (22). This study also showed substantial repair of 6-4 PP in 2 h and almost no repair of the CPD adducts in the same time period when both these adducts were measured in a single individual. Hence, the DNA repair curve in this study, which observed over a 2-h time period, is likely to be more reflective of the repair of 6-4 PP as compared to the CPD adducts.
Measurement of DNA damage at a single time point after exposure to a DNA-damaging agent is a commonly used measure of DNA repair that is used to determine differences in NER between normal donors in epidemiological studies (4,23,24). Our study utilized predominantly proliferating lymphocytes and maximal single-stranded breaks were found between 15 and 30 min. In previous studies, utilizing predominantly resting lymphocytes, the maximum amount of single-stranded breaks was measured at 60–90 min (25,26). Differences in rates of DNA repair between proliferating and non-proliferating lymphocytes may account for the differences in kinetics of DNA repair between this study and previous studies evaluating NER in lymphocytes (27). In this study, which has measurements at multiple time points, it was found that the peak ssDNA/dsDNA ratio was reached at different time points in 10 normal donors reflecting variations in the kinetics of NER between normal donors. Thus, measuring NER-mediated breaks at a single time point will result in a less precise estimation of NER than one considering multiple time points, within the same individuals. Measurement of NER-mediated breaks over a period of time using AUC takes into account the variation between time points and gives a more complete picture of fluctuations in repair following DNA damage. Although not statistically significant, AUC is a more reliable measure of NER as indicated by the CV of the assay, which is lower than the single time-point measures. Thus, AUC may be a more appropriate end point to measure total NER as compared to NER-mediated breaks at a single time point for this assay.
Measurement of NER-mediated breaks in each cell cycle phase is important because NER-mediated breaks have been shown to be significantly lower in G2/M and S phase of the cell cycle as compared to NER-mediated breaks among lymphocytes in the G1 phase (P < 0.0005). Lower NER-mediated breaks may reflect lower DNA repair among cells in S and G2/M phases as compared to cells in the G1 phase or that UV-induced DNA damage may be lower in S and G2/M phases as compared to the G1 phase. It is not possible to identify the reasons for the lower NER-mediated breaks in S and G2/M phases of the cell cycle based on the results of this study. Since a majority of lymphocytes in peripheral circulation are in the G1 phase (80–90%), overall NER-mediated breaks measured across all cell cycle phases reflect predominantly NER-mediated breaks among lymphocytes in the G1 phase. Consistent with this observation, the correlation between overall NER-mediated breaks and NER-mediated breaks in the G1 phase of the cell cycle was 0.96 in this study. However, since mutations occurring in actively dividing cells are thought to be important in cancer initiation, differences in NER-mediated breaks in the G2/M phase may be an important predictor of overall cancer susceptibility as compared to overall NER-mediated breaks. Similarly, various radiotherapy and chemotherapeutic agents are most effective against actively dividing cells. Hence, NER-mediated breaks in actively dividing cells may be a more relevant biological marker to predict response to cancer treatment or predict development of therapeutic resistance. The moderate correlation observed between different cell cycle phases (r = 0.6–0.7) indicates the importance of measuring NER-mediated breaks in different cycle phases as normal donors may be misclassified using a single overall measure of NER-mediated breaks. Thus, the rapid assessment and measurement of NER-mediated breaks within each cell cycle phase may add valuable information in a variety of studies ranging from that evaluating cancer susceptibility to studies evaluating therapeutic response to various treatment modalities.
Potter et al. (14) found that the sensitivity of an alkali unwinding flow cytometry-based assay was comparable to the comet assay, for the detection of DNA damage due to hydrogen peroxide and gamma radiation. We have shown that NER-mediated breaks were induced by a UV dose as low as 4 J/m2 and in separate experiments (data not shown) have shown that NER-mediated breaks with UV dose as low as 1.5 J/m2 can be detected using the flow cytometry assay indicating that sensitivity of the alkaline unwinding flow cytometry assay is similar to those observed in studies using the comet assay (25). This assay was able to detect variations in NER-mediated breaks in 10 normal donors. Differences in NER of 30–40% have been detected for various polymorphisms in genes involved in NER (24,28). The differences in NER-mediated breaks observed in this assay are comparable to the differences seen in other DNA repair assays evaluating genotype–phenotype relationships indicating that this is a sensitive assay for measurement of NER-mediated breaks.
An important advantage of the alkaline unwinding flow cytometry assay is that NER-mediated breaks can be routinely and rapidly measured in a large number of individual cells per sample. This provides statistical validity and ensures accurate representation of the cells in a particular sample. Both the comet assay and the flow cytometry alkaline unwinding assay can measure DNA repair in each phase of the cell cycle separately. However, the flow cytometry assay decreases the analytic variability in such measurements by vastly increasing the numbers of cells evaluated in each phase of the cell cycle. As expected, the intraindividual variability in NER-mediated breaks in all three cell cycle phases was substantially lower than the intraindividual variability reported in the comet assay (13–22% in the alkaline unwinding flow cytometry assay as compared to 42% in the comet assay) (9). Furthermore, the rapid speed of sample collection and measurement of DNA damage using the flow cytometry assay makes it feasible to measure DNA damage and repair using more complex endpoints, such as AUC, to estimate NER-mediated breaks in large population studies.
In this paper, an alkaline unwinding flow cytometry developed by Potter et al. has been optimized for measurement of NER-mediated breaks and use in epidemiological studies. We have also shown that this assay is a highly reproducible, sensitive and specific assay for measurement of NER-mediated breaks in human normal donors. This assay may be useful in epidemiological studies evaluating the role of DNA repair in cancer susceptibility or in studies evaluating the effectiveness of various cancer treatment modalities.
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Acknowledgments |
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We acknowledge Dr Mori's (Nara Medical University, Nara, Japan) generous donation of CPD and 6-4 photoproduct antibodies for use in the ELISA assays.
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* To whom correspondence should be addressed at Department of Laboratory Medicine and Pathology, University of Minnesota, MMC 609, 420 Delaware Street SE, Minneapolis, MN 55455, USA. Tel: +612 624 5417; Fax: +612 273 6994; Email: gross{at}epi.umn.edu
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Received on April 1, 2006; revised on November 21, 2006; accepted on November 21, 2006.
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