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Carcinogenesis Advance Access originally published online on March 6, 2007
Carcinogenesis 2007 28(7):1430-1436; doi:10.1093/carcin/bgm029
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

A modified host-cell reactivation assay to measure repair of alkylating DNA damage for assessing risk of lung adenocarcinoma

Luo Wang, Qingyi Wei*, Qiuling Shi, Zhaosheng Guo, Yawei Qiao and Margaret R. Spitz

Department of Epidemiology, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA

* To whom correspondence should be addressed. Tel: +1 713 792 3020; Fax: +1 713 563 0999; Email: qwei{at}mdanderson.org


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
The nicotine-derived nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induces lung adenocarcinoma through formation of DNA adducts. Our previous research on susceptibility to tobacco-induced carcinogenesis focused on benzo[a]pyrene diol epoxide (BPDE) as the in vitro mutagen for phenotype measurements of DNA repair capacity (DRC) in mammalian cells. Here, we present a modified host-cell reactivation (HCR) assay to measure lymphocytic DRC for alkylating DNA damage as is induced by the tobacco-specific nitrosamine, NNK. We substituted dimethyl sulfate (DMS) to create alkylating damage in pCMVluc plasmid DNA and established the damage-repair dose–response curves in both normal and nucleotide excision repair-deficient lymphoblastoid cell lines and in phytohemagglutinin (PHA)-stimulated primary lymphocytes. We then successfully measured the DRC in PHA-stimulated lymphocytes from 48 patients with lung adenocarcinoma and 45 cancer-free controls and tested our hypothesis that lower DRC for alkylating damage is associated with an increased risk of lung adenocarcinoma. The cases exhibited a lower mean DRC than did the controls. A >3-fold increased risk (odds ratio = 3.21; 95% confidence interval = 1.25–8.21) was found for those with DRC levels below the control median. There was no correlation between the DRC measured with this DMS-HCR assay and that from the parallel BPDE-HCR assay. Interestingly, risk increased to >10-fold for those with sub-optimal DRC measured by both DMS- and BPDE-HCR assays. We conclude that variability in DRC is a risk factor for lung cancer and our results provide proof of principle for a new assay that can assess DRC for NNK-induced DNA damage.

Abbreviations: BPDE, benzo[a]pyrene diol epoxide; CI, confidence interval; DMS, dimethyl sulfate; DRC, DNA repair capacity; FBS, fetal bovine serum; HCR, host-cell reactivation; NER, nucleotide excision repair; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; PHA, phytohemagglutinin


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
The nicotine-derived nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent and selective inducer of lung adenocarcinoma, now the leading histological subtype of lung cancer in the USA. Our research, to date, on susceptibility to tobacco-induced carcinogenesis has focused on benzo[a]pyrene diol epoxide (BPDE) as the in vitro mutagen in phenotype assays of bulky DNA damage and repair (1–3). However, since the 1960s, levels of benzo[a]pyrene in cigarette smoke have decreased as a result of lower average levels of nicotine and tar in cigarettes. In contrast, NNK levels in cigarettes have increased from 0.5% in the mid-1970s to 1.2–1.5% since then (4). The metabolic activation of NNK to DNA reactive species occurs via hydroxylation of the carbons adjacent to the N-nitroso group, producing the metabolites, pyridyloxobutyl- and methyl-diazohydroxide. These metabolites can pyridyloxobutylate or methylate guanine nucleobases in DNA, causing primarily N7- and O6-guanine lesions (5,6). Although the exact repair mechanism for these DNA lesions is unknown, we hypothesized that diminished capacity to repair this alkylating DNA damage would increase the risk of smoking-related cancers, particularly adenocarcinoma of the lung. However, lack of a specific DNA repair capacity (DRC) assay for NNK-induced lesions has been the limiting factor to explore this hypothesis.

A number of studies have demonstrated that DRC for different types of DNA damage can be quantitatively measured by using a host-cell reactivation (HCR) assay in mitogen-stimulated peripheral blood lymphocytes (1,7,8). The unique feature of the HCR assay is that it uses a non-replicating recombinant plasmid containing a reporter gene pre-treated with a specific DNA-damaging agent, such as BPDE used in our previous research on cancers of the lung and head and neck. In this assay, the transcriptional activity of the reporter gene reactivated in the host cell is monitored following a defined incubation period after the transfection (9). The DRC of an individual is defined as the relative ratio (expressed as a percentage) of activity from the damaged reporter gene to that from the undamaged reporter gene, derived from parallel experiments performed with phytohemagglutinin (PHA)-stimulated lymphocytes. Therefore, it is a reflection of intrinsic cellular repair capacity. The HCR assay was originally developed with a recombinant plasmid DNA pCMVcat harboring a chloramphenicol acetyltransferase reporter gene (9), and it has also been successfully modified using a recombinant luciferase reporter plasmid pCMVluc (10).

To evaluate whether the capacity to repair NNK-induced DNA damage is a susceptibility factor for lung adenocarcinoma, we tested the possibility of inducing adducts in the pCMVluc plasmid by treating with either NNK or its precursor 4-(acetoxy)-NNK. However, we abandoned this approach because the results were irreproducible and the dose–response curves were inconsistent, probably because of the instability of the DNA lesions induced in vitro (wang L., unpublished data). To overcome those technical difficulties, we selected a different approach to assess repair of the alkylating DNA damage induced by NNK. We first generated stable alkylating DNA damage in the pCMVluc plasmid induced by dimethyl sulfate (DMS), a well-known alkylating agent, and then used the treated plasmids in the HCR assay to measure lymphocytic DRC. Here, we outline the methods and report the results with this modified HCR assay and in parallel with the standard BPDE-HCR assay in a proof-of-principle pilot case–control study consisting of 48 patients with lung adenocarcinoma and 45 cancer-free controls.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Cell lines
Normal human B-lymphoblastoid cell lines (GM0131 and GM3798) transformed by Epstein-Barr virus and a XP-A nucleotide excision repair (NER)-deficient homozygote lymphoblastoid cell line (GM2345) were obtained from Coriell Cell Repositories, National Institute of General Medical Sciences (Coriell Institute for Medical Research, Camden, NJ). Lung cancer cell lines (H292, H358 and H1944) and XRCC1-proficient (AA8) or -deficient (EM9) cell lines were purchased from American Type Culture Collection (Manassas, VA). These lymphoblastoid and lung cancer cell lines were grown with 50 U/ml penicillin G sodium and 50 µg/ml streptomycin sulfate in RPMI 1640 medium (Gibco, Invitrogen Corp., Carlsbad, CA) supplemented with 15% fetal bovine serum (FBS) (Sigma–Aldrich Corp., St Louis, MO) for lymphocyte cell lines or 10% for lung cancer cell lines with 2 mmol/l glutamine. AA8 and EM9 cell lines were maintained in {alpha}-minimum essential medium supplemented with 10% FBS without ribonucleosides and deoxyribonucleosides. For use in the HCR assay, cell cultures were grown in RPMI 1640 medium supplemented with 10% FBS and harvested in the logarithmic growth phase (0.6 x 106–1.0 x 106 cells/ml). 4-Amino-1,8-naphthalimide, a specific inhibitor of poly(ADP-ribose) polymerase (11), was purchased from Sigma–Aldrich Corp.

Cryopreserved peripheral blood lymphocytes
An analysis of the association between the BPDE-DRC and lung cancer risk has been conducted on cases and controls enrolled from October 1998 to July 2003 in the Department of Epidemiology at The University of Texas M. D. Anderson Cancer Center (12). Having thawed the cells for the BPDE-DRC assay, cryopreserved viable lymphocytes were still available from some subjects, and we were able to identify 105 samples [54 from patients with histologically confirmed and previously untreated lung adenocarcinoma and 51 from cancer-free controls frequency matched on age (±5 years), sex, ethnicity (non-Hispanic white) and smoking status (ever and never)] to be used for the newly developed DMS-HCR assay. Because some of the cells did not grow well, the DMS-DRC assay was successfully performed for 93 samples (48 cases and 45 controls) that were included in the final analysis.

Pre-stored normal primary lymphocytes had been cryopreserved as described previously (12). Whole blood (~10 ml) had been collected from each study participant, and the lymphocytes had been isolated within 8 h by using Ficoll (Pharmacia Biotech, Piscataway, NJ) gradient centrifugation. The purified lymphocytes had been re-suspended at 10 x 106 cells/ml in an ice-cold freezing medium consisting of 10% dimethyl sulfoxide (Fisher Scientific Co., Pittsburgh, PA), 40% RPMI 1640 and 50% FBS, and 2.0 ml aliquots were stored in a –80°C freezer. For the current study, the samples were thawed in batches of 10 with an equal number of cases and controls for the HCR assay.

Preparation of plasmids with DMS, an alkylating agent
The expression plasmid pCMVluc contains a luciferase reporter gene under the transcriptional control of the promoter/enhancer from the immediate early gene of human cytomegalovirus (9). We induced alkylating DNA damage in this plasmid by using DMS (CAS number 77-78-1; Sigma–Aldrich Corp.). DMS is a strong methylating agent typically used to introduce a methyl group at the N7 site of guanosine, as described in the Maxam–Gilbert chemical sequencing method (13–15). We limited the treatment time up to 10 min when preparing this plasmid containing the N7-methylguanine in the HCR assay and intended to mimic the damage on N7 site of guanine induced by NNK.

The pCMVluc plasmid stock (500 µg/ml) was diluted with Tris (tris(hydroxymethyl)aminomethane - 10 mM)/EDTA (ethylenediamine-tetraacetic acid - 1 mM) (TE) (pH 7.8) to 0.1 µg/µl, and 10 µg of pCMVluc plasmid was treated for each dose of DMS with a final concentration ranging from 0.0 to 0.125% in phosphate buffer solution (PBS) (pH 7.2). The reaction mixture, protected from light, was incubated at room temperature for 10 min, followed by mixing it with 900 µl of ethanol and stored at –80°C for 1 h. The free DMS was removed after the reaction by washing with 80% ethanol and precipitated with centrifugation at least three times. After being re-suspended to 50 µg/ml in Tris buffer solution (TBS) (25 mM Tris–HCl, 137 mM NaCl, 5 mM KCl, 0.464 mM Na2HPO4, 0.136 mM NaH2PO4, 0.7 mM CaCl2 and 0.5 mM MgCl2, pH 7.3), the solution of modified pCMVluc plasmid substrate was aliquoted into 50 µl per tube and stored at –80°C. For each batch of the plasmid substrates, two aliquots were randomly selected for quality testing.

Quality test of pCMVluc plasmid substrate with alkylating DNA damage
The DNA strand with alkylating DNA damage may be unstable, because the modification can lead to depurination at the sites of guanosine or adenosine in DNA under certain conditions. Consequently, many single-strand breaks can be generated, breaking down the integrity of the double-strand DNA, which will affect the DRC measurements. We incubated the pCMVluc plasmid with alkylating DNA damage at 37°C in phosphate buffer solution (pH 7.2) for up to 4 h but did not observe any changes in plasmid amount and conformation. We further used a hot-piperidine depurination assay (6) to test the extent of alkylating DNA damage in the DMS-treated pCMVluc plasmid. Briefly, we transferred 20 µl of the plasmid solution (1 µg) into a 0.5 ml Eppendorf tube and added 100 µl of freshly prepared 12% piperidine (CAS number 110-89-4; Sigma–Aldrich Corp.) solution. After incubation at 80°C for 10 min, the contents were co-precipitated with glycogen (Roche Molecular Systems, Alameda, CA) by adding three volumes of cold ethanol followed by centrifugation. The product was air-dried, dissolved with TE buffer and separated on a 1.5% agarose gel with Tris-acetate-EDTA (TAE) electrophoresis buffer. The gel was stained in freshly prepared ethidium bromide (EB) solution for 10 min and visualized under an ultraviolet illuminator.

We also used a restriction enzyme digestion assay to test the conformation integrity of the DMS-modified pCMVluc plasmid. Each 0.5 µg plasmid DNA treated with different doses of DMS was digested with restriction enzyme BanI (New England Biolabs, Ipswich, MA) at 37°C for 3 h or longer, separated by electrophoresis and visualized under an ultraviolet illuminator after EB staining. A linear response curve was observed within the range of doses between 0.05 and 0.125% of DMS. The dose of DMS for damaging pCMVluc plasmid to be used in the HCR assay was finally determined by an in vivo transfection assay in lymphoblastoid cell lines. We selected 0.075% DMS as the final optimal dose in the pilot study to achieve a testable DRC between 10 and 20% as previously recommended (9,10). The transfections were performed in duplicate for each dose by following the routine transfection procedure, as described previously (12,16,17).

Plasmid damaged with BPDE
The methods to prepare BPDE-damaged plasmids and to assess repair capacity have been described previously (12). Briefly, BPDE (NCI L0137, 99% purity) powder was completely dissolved in tetrahydrofuran (Sigma Chemical Co., St Louis, MO) and further diluted for plasmid treatment. In our previous studies, we used only one dose of BPDE (>60 µM), a dose that does not cause any detectable changes in plasmid conformation (1,12). The purpose of this analysis was to compare the DRC for BPDE-induced DNA adducts with data for DMS-induced DNA damage measured from the same individuals.

Lymphocyte culture and mitogen stimulation
The primary lymphocytes used for the pilot study had been cryopreserved at –80°C for at least 2 years. As described previously (12), the cells in each frozen vial were thawed quickly at 37°C in a water bath and mixed with 7 ml of thawing medium (50% FBS, 40% RPMI 1640 medium and 10% dextrose) before the last trace of ice disappeared. Cell viability was determined in a hemocytometer microscopically by staining with 0.4% trypan blue (Sigma Chemical Co.). The cells were then centrifuged at 900 r.p.m. for 10 min and re-suspended at 3 x 105 ml–1 in RPMI 1640 medium (supplemented with 20% FBS, 2 mM L-glutamine, 100 U/ml penicillin G and 100 µg/ml streptomycin). The cells were cultured again in RPMI 1640 supplemented with 20% fetal calf serum (Gibco BRL) and 56.25 µg/ml PHA (Murex Diagnostics, Norcross, GA) for 72 h at 37°C and with 5% CO2 before used in transfection for the HCR assay.

Transfection and luciferase signal measurement
The adherent cells were seeded into a 48-well plate 1 day before the transfection with 0.5 x 105–1.0 x 105 cells per each well, and FuGENE 6 Transfection Reagent (Roche Diagnostics Co., Indianapolis, IN) was used for transfection. Briefly, a serum-free transfection mixture was added into the well and incubated for 2 h at 37°C. After removing the transfection mixture, RPMI 1640 serum medium was added and the cells were harvested after 24 h incubation.

For PHA-stimulated primary lymphocytes from each subject, the cells were divided into four aliquots, each containing ~2 x 106 cells. The diethylaminoethyl–dextran (Pharmacia Biotech) method (18) was used to transfect two aliquots with the untreated pCMVluc and the other two aliquots with the DMS-treated pCMVluc. The 40 h time course for this transfection procedure has been well established, because the highest luciferase signal from this pCMVluc plasmid can be obtained at 40 h after transfection (8,12,16,17).

After harvesting the cells by removing the culture medium and lysing with a lysis buffer (0.5 M potassium phosphate buffer/2.5% Triton X-100, pH 7.8), the luciferase activity signal in arbitrary light intensity units was measured in a MoonlightTM 3010 luminometer (BD Biosciences, San Jose, CA) with mixture of the cell lysis supernatant and luciferase assay substrate (Promega US, Madison, WI) in a 12 x 50 mm tube at room temperature. Background readings for blank cell samples without the plasmids transfected were typically <100 light intensity units, whereas effective readings with undamaged pCMVluc plasmids (the assay controls) were always of 104 light intensity units or above. DRC (%) was calculated as the ratio of the readings from DMS-treated plasmid to that from the untreated plasmids, multiplied by 100.

Statistical analyses
Demographic characteristics, such as age, sex, smoking status and pack-years smoked, were included in our analysis. Those who had smoked fewer than 100 cigarettes in their lifetimes were defined as never-smokers and the others as ever-smokers. Of the ever-smokers, those with pack-years smoked above the median value in the control group were considered as heavy smokers. DRC data were analyzed as a continuous variable. Student’s t-test was used to compare the means of DRC value between the cases and the controls by the selected variables. Pearson’s Rho correlation coefficient (r) was calculated to measure the correlation of the DRC values of the DMS-HCR and BPDE-HCR assays. The chi-squared test was used to test the differences in the distributions of the selected variables. The distribution of the DRC values was also analyzed by performing Kolmogorov–Smirnov goodness-of-fit tests. The median DRC value of the control group was used as the cut-off to dichotomize as either efficient or sub-optimal repair capacity. Odds ratios and their 95% confidence intervals (CIs) were calculated by logistic regression analysis with adjustment for age (in years), gender, smoking status, pack-years smoked and the storage time of the lymphocytes. P values were determined by using two-sided tests, and a P value < 0.05 was considered to be statistically significant for any test or model fitting. All statistical tests were performed with Statistical Analysis System Software (Version 8.0; SAS Institute, Cary, NC).


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Preparation and quality test of DMS-treated pCMVluc plasmids
To demonstrate the extent of alkylating DNA damage in the DMS-treated pCMVluc plasmids under normal physiological conditions, we used a phosphate buffer solution of pH 7.2 instead of pH 8.0, as was used in the piperidine assay (6). There was no apparent alteration in the integrity of the pCMVluc plasmids treated with different doses of DMS (Figure 1A). However, in the hot-piperidine assay, we demonstrated that at the N7 site of guanine in the pCMVluc plasmids, a methyl group was successfully introduced in a dose–response manner (Figure 1B), because plasmid DNA was clearly cleaved at the methylated sites under piperidine treatment (6).


Figure 1
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  		Fig. 1. Preparation and quality validation of pCMVluc plasmid substrate with alkylating DNA damage and in vivo testing in HCR assay. (A) Direct electrophoresis after treatment with different doses of DMS; (B) Piperidine assay; (C) Restriction enzyme BanI digestion assay and in vivo testing HCR assays to measure the DRC for alkylating DNA damage with pCMVluc plasmid substrate in (D) normal and XP-A NER-deficient lymphoblastoid cell lines (E) in XRCC1-proficient (AA8) and deficient (EM9) cell lines. M indicates DNA marker.

 
The alkylating DNA damage in the pCMVluc plasmids may alter plasmid conformation and, therefore, could decrease the transfection efficiency, a factor that would affect reproducibility of the HCR assay. Therefore, we next tested the conformation integrity of the DMS-treated pCMVluc plasmids by using the digestion assay of the restriction enzyme BanI. There are six recognition sites in the sequence of pCMVluc plasmid for BanI, GGYRCC (Y = C or T and R = A or G). The digestion pattern would be changed if plasmid conformation was altered by the modification on this recognition sequence. The resultant fragments were presented as expected, indicating that there were no apparent alterations in the conformation of the DMS-treated plasmids (Figure 1C). Therefore, we concluded that modification by DMS should not affect transfection efficiency, as suggested in our previous study with BPDE (19).

Repair of DMS-modified pCMVluc plasmids in cell lines and PHA-stimulated primary lymphocytes
Dose dependence of DRC for the DMS-treated pCMVluc plasmids was tested in a normal human B-lymphoblastoid cell line (GM3798) and one XP-A homozygote lymphoblastoid cell line (GM2345) that is NER deficient (Figure 1D). The response curve was linear for DMS doses ranging from 0.05 to 0.125%. The DRC in the XP-A-deficient cells was slightly lower than that in the normal human B-lymphoblastoid cells, but the difference was not statistically significant, suggesting that NER is not a major pathway involved in the repair of alkylating DNA damage. The DRC in the XRCC1-deficient cell line (EM9) was decreased ~17% compared with that in the XRCC1-proficient cell line (AA8) (Figure 1E), indicating that base excision repair pathway through XRCC1 may play a role in repair of alkylating DNA damage. However, there was no apparent difference in AA8 and EM9 cell lines when a specific inhibitor of poly(ADP-ribose) polymerase, 4-amino-1,8-naphthalimide, was used (data not shown).

A similar dose dependency for DRC of alkylating DNA damage was also apparent in the lung adenocarcinoma cell lines (data not shown), as well as in all cell lines tested, suggesting a linear relationship between the arbitrary luciferase signals of the plasmids and DMS concentrations. Table I shows one example of the luciferase signals from the DMS-treated pCMVluc plasmids decreasing with increasing DMS dosage in a dose-dependent relationship. To estimate the reproducibility in measuring the DRC values by this modified assay, we further assayed the two normal lymphoblastoid cell lines used in Table I with plasmids treated by 10 x 10–2% of DMS, with repeated measurements 3 days and 2 weeks later, and we obtained similar DRC results (19.1 ± 1.0 in GM0131 and 16.5 ± 0.4 in GM3798, coefficient of variation% = 5.2 and 2.4, respectively) (data not shown), which were very similar to those (19.8 and 16.1, respectively) in Table I. We later tested the DRC for alkylating DNA damage in PHA-stimulated primary lymphocytes from eight subjects either with lung cancer or cancer-free individuals, and their DRC values ranged between 3.7 and 19.8% (mean ± SD = 10.7 ± 4.6%), a 5-fold variation, similar to the DRC values previously observed in the BPDE-HCR assay (12); we then continued testing for DMS-DRC in the remaining samples. For most of the data used in this study as shown in Figure 2, there was an ~5-fold range of variation in the measurements of BPDE-DRC assay, compared with only an ~3-fold range of variation in the measurements of DMS-DRC assay.


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  		Table I. Luciferase signal and dose–effect relationship of DMS-DRC in two normal lymphoblastoid cell lines (GM00131 and GM03798) and one XP-A-deficient cell line (GM02345)

 


Figure 2
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  		Fig. 2. Correlation of the DRC levels for DNA damage induced by DMS and by BPDE. *Pearson’s correlation test.

 
Case–control analysis
We used questionnaire-derived data collected previously (12) to assess the potential effects of any covariates in this pilot case–control analysis. Demographic and selected characteristics of the study population are shown in Table II. All participants were non-Hispanic whites. All cases (21 men and 27 women) presented with lung adenocarcinoma and were well matched on age, sex and smoking status (ever or never) (Table II). The optimal dose of 0.075% DMS tested in lymphoblastoid cell lines was used as the final dose in the pilot study. The test for normality showed that Kolmogorov–Smirnov values were 0.102 (P > 0.150) for the 45 controls and 0.076 (P > 0.150) for the 48 cases. Therefore, the DMS-DRC data were normally distributed for both controls and cases.


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  		Table II. Distribution of selected variables in the patients with lung adenocarcinoma and matched controls

 
The mean DRC for DMS-induced alkylating DNA damage was 11.4% for the cases and 12.6% for the controls (Table III). A relative 14.1% reduction was observed in younger individuals (≤61 years), but this difference was not statistically significant (P = 0.176). The case–control difference was borderline significant in women (P = 0.099). However, we observed a statistically significant lower DRC between cases and controls in heavy smokers (P = 0.049; Table III). We also dichotomized the DRC level by the median value in the control group (Table IV). It was notable that 70.8% of the cases (34/48) exhibited DRC below the control median, and this difference was associated with an adjusted odds ratio of 3.21 (95% CI = 1.25–8.21) for risk of lung cancer (Table IV), 3.94 (95% CI = 1.11–13.9) in younger subjects (≤61 years), 4.41 (95% CI = 1.21–16.1) in women, 3.03 (95% CI = 1.17–7.82) in ever-smokers and 4.38 (95% CI = 1.27–15.1) in heavy smokers. However, we did not find any evidence of interactions between these variables and sub-optimal DRC (data not shown) because of a limited power in such a pilot study.


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  		Table III. DRC for alkylating DNA damage in lung adenocarcinoma patients and controls stratified on selected variables

 


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  		Table IV. Odds ratios for DRC stratified by selected variables

 
We compared these DMS-DRC data with the BPDE-DRC data from the same subjects of this pilot study. The mean DRC for BPDE-induced DNA damage was non-statistically significantly lower in the cases (8.1 ± 2.8) than in the controls (8.9 ± 2.4) (P = 0.115) (Table II). When dichotomized by the control median value, 72.9% of the cases (35/48) exhibited BPDE-DRC below the control median value (P = 0.017), a pattern similar to that for the DMS-DRC (70.8%), but there was no correlation observed between the measurements of two DRC assays, either in the cases or in the controls (Figure 2). The Pearson’s Rho correlation coefficients were 0.030 for all 93 individuals (P = 0.458), 0.146 for the 48 cases (P = 0.322) and –0.037 for the 45 controls (P = 0.812).

We further analyzed the joint effects on lung cancer risk of exhibiting sub-optimal DRC for both DMS- and BPDE-induced damage. As shown in Table V, the risk was 3.78 for those having lower BPDE-DRC, 6.05 for those with lower DMS-DRC and the joint risk was 10.2 (95% CI = 1.94–53.4) for those who had both lower BPDE-DRC and DMS-DRC. However, we did not find any evidence of interactions between the two DRC measurements (data not shown).


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  		Table V. Joint effects of DRC for BPDE- and DMS-induced damage and lung adenocarcinoma risk

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
In this report, we have summarized the development of a modified HCR assay with a pCMVluc plasmid that measures DRC for DNA-alkylating damage induced by DMS. The purpose of using this DMS-modified plasmid substrate in the HCR assay was to mimic the damage induced by NNK. The long-term goal of developing this assay is to facilitate molecular epidemiological studies of cancer risk by enabling researchers to measure the DRC of DNA damage induced by NNK in the etiology of lung adenocarcinoma. Although we found that the DMS-DRC was different in the XRCC1-deficient cell line compared with that in the XRCC1-proficient cell line (but not in NER-deficient lines), the exact repair mechanism remains unclear and requires further investigation.

NNK is the most potent carcinogen of the tobacco-specific N-nitrosamines and preferentially induces adenoma and adenocarcinoma in animals and the human lung (20,21). The metabolites of NNK can either pyridyloxobutylate or methylate guanine in DNA with primarily N7- and O6-guanine lesions (5). Therefore, the association between NNK exposure and susceptibility to lung adenocarcinoma can be evaluated by the variability in the repair rates of alkylating damage. Our results demonstrated that the pCMVluc plasmid with alkylating damage induced by DMS is a suitable substrate for the HCR assay system, because the induced damage is relatively stable under physiologic conditions (pH 7.2 and 37°C) and the alkylating damage does not alter the conformation of the plasmid DNA. We were able to demonstrate clear dose-dependent relationships in all cell lines tested, including a normal human B-lymphoblastoid cell line, an XP-A-deficient homozygote lymphoblastoid cell line, lung adenocarcinoma cell lines and PHA-stimulated primary lymphocytes. The results in this pilot study support our hypothesis that patients with lung adenocarcinoma exhibit a lower capacity to repair alkylating DNA damage than healthy matched controls. We previously demonstrated in a large case–control study that sub-optimal DRC measured by the HCR assay with BPDE-damaged pCMVluc plasmids was significantly associated with risk of lung cancer (12) and NER was a major pathway to repair BPDE-induced DNA damage (9). In this pilot study, however, we found that there was no correlation between the DRC for the DMS-induced damage and that for the BPDE-induced damage, suggesting that NER may not be involved in repairing the alkylating DNA damage. However, measuring these two distinct repair pathways simultaneously can enhance the risk assessment for lung adenocarcinoma.

DNA adduct formation is a key step in carcinogenesis. If the adducts persist, miscoding during DNA replication may lead to accumulation of permanent mutations during carcinogenesis. There is a strong correlation in mice studies between lung tumor formation and the level of alkylating DNA adducts, such as O6-methylguanine adducts (22,23). DNA repair is, therefore, a critical mechanism to maintain the integrity of the genome, and human individuals display a wide range of capability for repairing DNA damage (12,17). The sub-optimal repair phenotype has been associated with susceptibility to a variety of epithelial cancers, and the HCR assay is to date the best method to measure an individual’s intrinsic DRC as demonstrated in several cancer sites including the lung, skin, head and neck, prostate and breast (2,6,12,17,24–36). The HCR assay has the flexibility of being adapted to monitor the repair response to selected etiological agents, and our DMS-DRC assay is one such example of modification of the assay to measure repair capacity of alkylating DNA damage.

The limitation of this work is obvious. First of all, we did not quantify the absolute amount of N7 adducts in the pCMVluc plasmids induced by different doses of DMS. Secondly, we could not explore the detailed repair mechanism involved in DMS-induced DNA damage but could only indirectly conclude that NER appeared not to be involved in the repair for alkylating DNA damage, and we suggest that XRCC1-related base excision repair could play a role in the repair of alkylating DNA damage, since the DRC in the XRCC1-deficient cell line was decreased by 17%. However, it is probable that multiple mechanisms may be responsible for repairing the alkylating DNA damage, and this should be further investigated. Thirdly, this small pilot study did not have enough statistical power to detect any possible interaction that may be suggested by the data. Finally, the pilot data presented could not provide evidence of a cause–effect relationship between the risk and sub-optimal DRC, because of the retrospective nature of this study.

This new DMS-DRC assay needs further evaluation of assay conditions that could affect intra-individual variation, such as storage time of the lymphocytes and altered variation in cellular response to the stimulation by PHA. Overall, as shown in Figure 2, there was an ~5-fold range of variation in the measurements of BPDE-DRC assay, but there was only an ~3-fold range of variation in the measurements of the DMS-DRC assay, suggesting that the new assay may produce more precise measurements with less assay variation, although larger studies are needed to further validate this finding. We selected 0.075% of DMS as the optimal dose as determined by in vivo testing in lymphoblastoid cell lines, but this dose also needs to be evaluated carefully in future studies because incomplete treatment or over treatment of the plasmids by DMS may also contribute to the assay variation.

In summary, we have demonstrated the ability of a modified HCR assay to measure the DRC for alkylating DNA damage. Our preliminary data support the use of this assay in future case–control studies evaluating the role of alkylating DNA damage in carcinogenesis. The results of our pilot case–control study on lung adenocarcinoma provide the rationale for future etiologic research on NNK-induced lung adenocarcinoma. The preliminary data support our hypothesis that sub-optimal DRC for alkylating DNA damage induced by NNK may increase the risk of tobacco-induced lung cancer. A larger study is underway to further validate the use of this assay, but additional studies exploring the underlying repair mechanisms involved in the repair of alkylating DNA damage are also needed.


   Acknowledgments
 
We thank Stephen S.Hecht of the University of Minnesota for his constructive suggestions, Li-E.Wang and Zhensheng Liu for technical support, Susan Honn for subject recruitment, Jianzhong He for technical assistance and Joanne Sider for manuscript preparation. Grant support: National Institutes of Health grants CA55769 and CA70907 (M.R.S.); ES11740 and CA100264 (Q.W.), CA16672 and Lung SPORE (The University of Texas M. D. Anderson Cancer Center) and FAMRI grant of the Flight Attendant Medical Research Institute (M.R.S.).

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Wei Q, et al. Reduced DNA repair capacity in lung cancer patients. Cancer Res. (1996) 56:4103–4107.[Abstract/Free Full Text]
  2. Wei Q, et al. Benzo(a)pyrene diol epoxide-induced chromosomal aberrations and risk of lung cancer. Cancer Res. (1996) 56:3975–3979.[Abstract/Free Full Text]
  3. Li D, et al. In vitro induction of benzo(a)pyrene diol epoxide-DNA adducts in peripheral lymphocytes as a susceptibility marker for human lung cancer. Cancer Res. (1996) 56:3638–3641.[Abstract/Free Full Text]
  4. Wynder EL, et al. The changing epidemiology of smoking and lung cancer histology. Environ. Health Perspect. (1995) 103(suppl. 8):143–148.
  5. Hecht SS. DNA adduct formation from tobacco-specific N-nitrosamines. Mutat. Res. (1999) 424:127–142.[Web of Science][Medline]
  6. Ziegel R, et al. K-ras gene sequence effects on the formation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-DNA adducts. Chem. Res. Toxicol. (2003) 16:541–550.[CrossRef][Web of Science][Medline]
  7. Wei Q, et al. DNA repair and aging in basal cell carcinoma: a molecular epidemiology study. Proc. Natl Acad. Sci. USA (1993) 90:1614–1618.[Abstract/Free Full Text]
  8. Ahn B, et al. Repair of mitomycin C cross-linked DNA in mammalian cells measured by a host cell reactivation assay. Mol. Cells (2004) 18:249–255.[Web of Science][Medline]
  9. Athas WF, et al. Development and field-test validation of an assay for DNA repair in circulating human lymphocytes. Cancer Res. (1991) 51:5786–5793.[Abstract/Free Full Text]
  10. Qiao Y, et al. Rapid assessment of repair of ultraviolet DNA damage with a modified host-cell reactivation assay using a luciferase reporter gene and correlation with polymorphisms of DNA repair genes in normal human lymphocytes. Mutat. Res. (2002) 509:165–174.[Web of Science][Medline]
  11. Schlicker A, et al. 4-Amino-1,8-naphthalimide: a novel inhibitor of poly(ADP-ribose) polymerase and radiation sensitizer. Int. J. Radiat. Biol. (1999) 75:91–100.[CrossRef][Web of Science][Medline]
  12. Wei Q, et al. Repair of tobacco carcinogen-induced DNA adducts and lung cancer risk: a molecular epidemiologic study. J. Natl Cancer Inst. (2000) 92:1764–1772.[Abstract/Free Full Text]
  13. Maxam AM, et al. A new method for sequencing DNA. Proc. Natl Acad. Sci. USA (1977) 74:560–564.[Abstract/Free Full Text]
  14. Kumar R, et al. Separation of 7-methyl- and 7-(2-hydroxyethyl)-guanine adducts in human DNA samples using a combination of TLC and HPLC. Carcinogenesis (1996) 17:485–492.[Abstract/Free Full Text]
  15. Sambrook J, et al. Molecular Cloning: A Laboratory Manual (1989) NY: Cold Spring Harbor Laboratory Press.
  16. Qiao Y, et al. Modulation of repair of ultraviolet damage in the host-cell reactivation assay by polymorphic XPC and XPD/ERCC2 genotypes. Carcinogenesis (2002) 23:295–299.[Abstract/Free Full Text]
  17. Wei Q, et al. Repair of UV light-induced DNA damage and risk of cutaneous malignant melanoma. J. Natl Cancer Inst. (2003) 95:308–315.[Abstract/Free Full Text]
  18. Emmert S, et al. Relationship of neurologic degeneration to genotype in three xeroderma pigmentosum group G patients. J. Invest. Dermatol. (2002) 118:972–982.[CrossRef][Web of Science][Medline]
  19. Cheng L, et al. Fluorescence in situ hybridization method for measuring transfection efficiency. Biotechniques (1996) 21:486–491.[Web of Science][Medline]
  20. Hoffmann D, et al. Tobacco specific N-nitrosamines: occurrence and bioassays. IARC Sci. Publ. (1982) 41:309–318.[Medline]
  21. Hoffmann D, et al. Tobacco-specific N-nitrosamines and Areca-derived N-nitrosamines: chemistry, biochemistry, carcinogenicity, and relevance to humans. J. Toxicol. Environ. Health (1994) 41:1–52.[Web of Science][Medline]
  22. Peterson LA, et al. O6-methylguanine is a critical determinant of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone tumorigenesis in A/J mouse lung. Cancer Res. (1991) 51:5557–5564.[Abstract/Free Full Text]
  23. Hecht SS, et al. Tobacco-specific nitrosamine adducts: studies in laboratory animals and humans. Environ. Health Perspect. (1993) 99:57–63.[Web of Science][Medline]
  24. Kraemer KH, et al. Xeroderma pigmentosum. Clin. Dermatol. (1985) 3:33–69.[CrossRef][Medline]
  25. Wei Q, et al. The role of DNA repair capacity in susceptibility to lung cancer: a review. Cancer Metastasis Rev. (1997) 16:295–307.[CrossRef][Web of Science][Medline]
  26. Spitz MR, et al. Genetic susceptibility to lung cancer: the role of DNA damage and repair. Cancer Epidemiol. Biomarkers Prev. (2003) 12:689–698.[Free Full Text]
  27. Shi Q, et al. Reduced DNA repair of benzo[a]pyrene diol epoxide-induced adducts and common XPD polymorphisms in breast cancer patients. Carcinogenesis (2004) 25:1695–1700.[Abstract/Free Full Text]
  28. Hall J, et al. DNA repair capacity as a risk factor for non-melanocytic skin cancer—a molecular epidemiological study. Int. J. Cancer (1994) 58:179–184.[Web of Science][Medline]
  29. Zang EA, et al. Differences in lung cancer risk between men and women: examination of the evidence. J. Natl Cancer Inst. (1996) 88:183–192.[Abstract/Free Full Text]
  30. D'Errico M, et al. Factors that influence the DNA repair capacity of normal and skin cancer-affected individuals. Cancer Epidemiol. Biomarkers Prev. (1999) 8:553–559.[Abstract/Free Full Text]
  31. Dybdahl M, et al. Low DNA repair is a risk factor in skin carcinogenesis: a study of basal cell carcinoma in psoriasis patients. Mutat. Res. (1999) 433:15–22.[Web of Science][Medline]
  32. Landi MT, et al. DNA repair, dysplastic nevi, and sunlight sensitivity in the development of cutaneous malignant melanoma. J. Natl Cancer Inst. (2002) 94:94–101.[Abstract/Free Full Text]
  33. Matta JL, et al. DNA repair and nonmelanoma skin cancer in Puerto Rican populations. J. Am. Acad. Dermatol. (2003) 49:433–439.[CrossRef][Web of Science][Medline]
  34. Cheng L, et al. Reduced DNA repair capacity in head and neck cancer patients. Cancer Epidemiol. Biomarkers Prev. (1998) 7:465–468.[Abstract/Free Full Text]
  35. Hu JJ, et al. Deficient nucleotide excision repair capacity enhances human prostate cancer risk. Cancer Res. (2004) 64:1197–1201.[Abstract/Free Full Text]
  36. Ramos JM, et al. DNA repair and breast carcinoma susceptibility in women. Cancer (2004) 100:1352–1357.[CrossRef][Web of Science][Medline]
Received November 15, 2006; revised January 10, 2007; accepted February 5, 2007.


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