Reduced DNA-dependent protein kinase activity is associated with lung cancer
Dennis H. Auckley1,5,
Richard E. Crowell1,
Evelyn R. Heaphy1,
Christine A. Stidley3,
John F. Lechner4,6,
Frank D. Gilliland2,7 and
Steven A. Belinsky4,8
1 Division of Pulmonary, Critical Care and Allergy, University of New Mexico and the New Mexico Veterans Health Care System, Albuquerque, NM 87108,
2 Epidemiology and Cancer Control and
3 Department of Family and Community Medicine, University of New Mexico, Albuquerque, NM 87131 and
4 Lovelace Respiratory Research Institute, PO Box 5890, Albuquerque, NM 87185, USA
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Abstract
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Reduced DNA repair capacity of carcinogen-induced DNA damage is now thought to significantly influence inherent susceptibility to lung cancer. DNA-dependent protein kinase (DNA-PK) is a serine-threonine kinase activated by the presence of double-strand breaks in DNA that appears to play a major role in non-homologous recombination and transcriptional control. The purpose of this study was to determine whether DNA-PK activity varies among individuals and how this affects lung cancer risk. DNA-PK activity in peripheral mononuclear cells from individuals with lung cancer (n = 41) was compared with lung cancer-free controls (n = 41). Interindividual variability was seen within each group, however, significant differences (P = 0.03) in DNA-PK activity between cases and controls were seen when comparing the distribution of enzyme activity among these two groups. The percentages of cases and controls with DNA-PK activity in the ranges 2.55.0 and 7.610.0 units were 39 versus 20% and 7 versus 29%, respectively. The enzyme activity in peripheral mononuclear cells reflected that seen in bronchial epithelial cells, one progenitor cell for lung cancer, supporting the use of peripheral mononuclear cells for larger population-based studies of DNA-PK activity. Its role as a potential modifier for lung cancer risk was supported by the fact that cell growth in bronchial epithelial cells exposed to bleomycin was directly associated with enzyme activity. The results of this study demonstrate that reduced DNA-PK repair activity is associated with risk for lung cancer.
Abbreviations: BECs, bronchial epithelial cells; DNA-PK, DNA-dependent protein kinase; NMVHCS, New Mexico Veterans Health Care System; OR, odds ratio; PMs, peripheral mononuclear cells.
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Introduction
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The major risk factor for lung cancer is clearly cigarette smoking, however, genetic variation in individual response to mutagens and carcinogens may be important for defining inherent susceptibility to this disease (1,2). This conclusion is based on the fact that only a subset of heavily exposed individuals will develop cancer during their lifetime (3). The genes that have been studied most extensively as potential risk modifiers are those involved in carcinogen activation and detoxification (4). However, the rapidly emerging class of genes involved in the recognition and repair of DNA damage may prove to have a critical role in determining inherent susceptibility for lung cancer.
Several studies have demonstrated that decreased capacity to repair DNA damage induced by chemicals or radiation may influence cancer risk. Wei et al. (5) found that individuals with lung cancer are five times more likely than controls to have reduced nucleotide excision repair capacity in peripheral lymphocytes as measured by cellular reactivation of a reporter gene damaged by exposure to benzo[a]pyrene diol epoxide. Exposure of lymphocytes to bleomycin, a radiomimetic agent, also induces chromatid breaks at a higher frequency in cells from lung cancer cases than controls (6). The mechanisms responsible for these differences in DNA repair capacity have not been elucidated, however, a recent study by Shen et al. (7) examining the polymorphic frequency in a series of genes involved in both nucleotide excision repair and double-strand break repair may offer some clues. Nine different amino acid substitution variants were identified in exons of three nucleotide excision repair genes (ERCCI, XPD and XPF), in a gene involved in double-strand break repair/recombination (XRCC3) and in a gene that functions in base excision repair and the repair of radiation-induced damage (XRCCl). For one gene, XRCC1, a Gln399 polymorphism resulting from a guanine
adenine nucleotide substitution was significantly associated with an increase in levels of aflatoxin B1DNA adducts and glycophorin A mutations in erythrocytes (8). Recent studies by our laboratory have linked this functional polymorphism in the XRCC1 gene to an increased risk for adenocarcinoma of the lung (9).
DNA-dependent protein kinase (DNA-PK) is another nuclear protein involved in the detection and repair of DNA damage (10). This serine-threonine kinase is activated by the presence of double-strand breaks in DNA (11) and phosphorylates several DNA-binding proteins, including DNA ligase IV (10), the p53 tumor suppressor gene product (12) and numerous transcription factors (e.g. Spl, Octl, Fos and RNA polymerase H; ref. 10). Although the exact mechanism of action of DNA-PK has not been fully elucidated, it is thought to participate in non-homologous DNA end joining, cell cycle control and transcriptional regulation (13). DNA-PK activity has been examined in human and rodent cell lines (1417), but enzyme activity has not been characterized in populations with cancer. The fact that tobacco contains carcinogens and mutagens capable of either directly or indirectly inducing a wide spectrum of DNA damage that includes double-strand breaks would necessitate a role for DNA-PK in protecting the cell against heritable chromosome damage.
The purpose of this study was to determine whether DNA-PK activity varies among individuals and how this affects lung cancer risk. DNA-PK activity was compared in peripheral mononuclear cells (PMs) from individuals with lung cancer and cancer-free controls. The relationship of DNA-PK activity observed in PMs to enzyme activity in bronchial epithelial cells (BECs), a progenitor cell for lung cancer, was also established in order to validate the PM as a surrogate cell type for future population-based studies. Finally, a functional outcome of enzyme activity variability was assessed by establishing the relationship between DNA-PK activity and cell growth following exposure to bleomycin.
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Materials and methods
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Subject enrollment
Cases (n = 41) and controls (n = 41) were recruited from the New Mexico Veterans Health Care System (NMVHCS) as part of a hospital-based casecontrol study. This study was approved by the Institutional Review Board and all participants gave written informed consent. Cases were defined as individuals with newly diagnosed (within 2 weeks) non-small cell lung cancer (Stages IIV) seen at the multidisciplinary chest clinic where >80% of NMVHCS patients with lung cancer are evaluated. All cases were approached for participation in the study, however, collection of BECs for research purposes was only performed on patients undergoing bronchoscopy for clinical indications, such as diagnosis or staging of lung cancer. Controls were enrolled from the population of lung cancer-free individuals who were referred for evaluation in the Pulmonary Clinics at NMVHCS for pulmonary diseases other than lung cancer, such as asthma, chronic obstructive pulmonary disease or interstitial lung disease. Patients were approached randomly during clinic visits and asked to participate in the study. All subjects were asked to volunteer for bronchoscopy, however, their participation was not dependent on agreeing to this procedure. Because of the invasiveness of the bronchoscopy most controls did not undergo this procedure, but did complete a questionnaire and donate blood. Exclusion criteria included a history of chemotherapy or radiation therapy, current use of immunosuppressive medications or presence of an infection.
All subjects were interviewed about smoking and occupational histories. Cases and controls were not matched with respect to gender, age or ethnicity during recruitment. Both groups contained current smokers (active smoking within the past year), former smokers (quit >1 year ago and lifetime tobacco use of >1 pack year) and never smokers (lifetime tobacco use <1 pack year). All individuals donated 45 ml of blood for PM isolation within 1 month of enrollment into the study. BECs were obtained during bronchoscopy as previously described (18). BECs were cultured and used in experiments that compared DNA-PK activity between PMs and BECs and for cell growth assays (described below).
Cell isolation and lysis
PMs were isolated immediately after blood collection by room temperature Histopaque-1077 centrifugation at 400 g for 30 min. Cells were washed with HEPES-buffered saline (Sigma, St Louis, MO), pelleted by centrifugation and lysed in a low salt (25 mM KCI, 10 mM NaCl, 1 mM MgCl2) buffer by cooling and freezing. Lysed PMs were then stored at 80°C at 5x106 cells/15 µl lysate buffer until assayed. BECs obtained at bronchoscopy were plated on 100 mm fibronectin-coated plates and grown in Bronchial Epithelial Growth Medium (Clonetics, San Diego, CA) as previously described (18). Cells were harvested at 7080% confluence (first or second passage) by trypsinization and resuspended in medium. Lysis of the BECs followed the protocol outlined for PMs. BEC lysates were stored at 80°C at 1x106 cells/15 µl lysate buffer until assayed. Samples were assayed within 2 months of freezing.
Protein extraction
Samples were thawed in the presence of protease inhibitors (0.5 mM phenylmethylsulfonyl fluoride and 2 µg/ml each leupeptin and pepstatin), then subjected to salt extraction (0.5 M NaCl, 10 mM MgCl2, 0.5 mM DTT). Extracts were made by centrifugation at 13 000 g for 2 min at 4°C. The pellet was then re-extracted and the supernatant combined with the first extract. Protein was quantified by the Bio-Rad protein assay using bovine serum albumin as the standard.
DNA-PK assay
Protein extracts (1 mg) were assayed as previously described (19) in the presence of sonicated calf thymus double-strand DNA fragments, 32P-labeled ATP and a synthetic peptide: either PESQEAFADLWKK (PSQ) (Research Genetics, Huntsville, AL), which contains a serine-threonine site, or EPPLSEQFADLWKK (SEQ) (Research Genetics), which does not contain a serine-threonine site. Incubations were for 5 min at 30°C and reactions were stopped by addition of 15% acetic acid with cold ATP. Reactions were then spotted onto filter paper (Whatman P81), washed four times in 15% acetic acid and counted in a scintillation counter (Packard 2500 TR). DNA-PK activity was calculated by subtracting the SEQ peptide activity from the PSQ peptide activity and dividing by the protein concentration to yield activity in pmol phosphate incorporated/min/mg protein (units). Each sample was run in duplicate within each assay and again in duplicate in a second assay. A coefficient of variability of <0.25 units between samples was required as a reproducibility criterion. If this was not achieved by the duplicate assay a third assay was conducted. For all cases and controls the medians for the coefficient of variation of DNA-PK activity were 0.09 (range 0.010.25) and 0.10 (range 0.010.23), respectively. These data confirm the reproducibility of the DNA-PK assay and also indicate that differences do not exist between cases and controls with respect to individual variability. One subject underwent bronchoscopy twice for clinical reasons 18 months apart; BEC collection and blood sampling for PM isolation were performed at both procedures. DNA-PK activity was virtually identical in the duplicate samples with enzyme activities of 6.0 and 6.2 in BECs and 5.6 and 5.5 in PMs, respectively. Thus, the procedures used for collection, processing and analysis of enzyme activity were very reproducible.
Cell killing after bleomycin exposure
Cryopreserved BECs from nine cases and four controls were thawed in the presence of DNase (Biofluids, Rockville, MD) and soybean trypsin inhibitor, washed in medium and replated at 2.5x103 cells/100 mm fibronectin-coated dishes with 7 ml of medium. After incubation for 48 h under normal growth conditions, medium was removed and the cells were exposed for 2 h to bleomycin (ICN, Costa Mesa, CA) in medium at the following concentrations: 0, 5, 10 and 20 µg/ml. Bleomycin was removed, cells were washed with HEPES, fresh medium added and the cells placed back in the incubator under normal growth conditions for 8 days. Assays were conducted with triplicate plates at each exposure concentration. Cells were subsequently fixed in 3:1 methanol:acetic acid, stained with crystal violet and colonies with >32 cells were scored under a white light microscope. The number of populations/nine colonies/dish (27 total colonies) was determined by counting the number of cells in each individual colony (20). The mean and standard deviations were calculated. Reproducibility between triplicate plates was excellent, with the median for the coefficient of variation being 0.11 (range 0.040.16) for all assays. Finally, the percent differences in mean population doublings between control and bleomycin-treated cells were calculated.
Statistical methods
Summary statistics were obtained on five variables: enzyme activity, smoking history, age, ethnicity and gender for both cases and controls. The DNA-PK activity for each individual used in the statistical analyses was based on the mean of all assays for an individual. Cases and controls were compared using the Wilcoxon rank sum test for continuous variables and Fisher's exact test for categorical variables. Because some of the distributions were skewed and some outliers existed, the Wilcoxon rank sum test was used in place of the two-sample t-test, but always gave similar results. Pearson and Spearman correlation coefficients were used to assess the relationship between DNA-PK activity levels in PMs and BECs. To assess the joint effect of enzyme activity and smoking status, logistic regression models were developed with casecontrol status as the binary outcome variable. Smoking status was either categorized with current smokers and former smokers combined as `ever smokers' or expressed as pack years. Because of the small number of observations, it was not possible to simultaneously control for all variables in the same logistic regression model, therefore, insignificant variables were removed. All statistical computing was done in SAS (SAS Institute, Cary, NC).
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Results
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Reduced DNA-PK activity is associated with lung cancer
DNA-PK activity was assessed in PMs from 41 individuals with lung cancer and 41 individuals without lung cancer in order to determine whether reduced enzyme activity was associated with increased lung cancer risk. Significant inter-individual variability was seen within each group. The range of activity was 0.911.0 units in cases and 2.09.6 units in controls (Figure 1
). The median value for DNA-PK activity was only slightly lower in the group with lung cancer than in the control group: 5.5 units (range 0.910.9) versus 5.7 units (range 2.09.6). The main differences in DNA-PK activity between cases and controls can be seen in the distribution plot depicted in Figure 2
. The percent of cases and controls with DNA-PK activity in the range 2.55.0 and 7.610.0 units was 39 versus 20% and 7 versus 29%, respectively. These discrete distributions of enzyme activity are significantly different between cases and controls (P = 0.03).

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Fig. 1. DNA-PK activity in peripheral mononuclear cells from lung cancer cases and lung cancer-free controls. Each value represents one individual. Enzyme activity was determined from assays conducted at least twice. The median activity (represented by the horizontal lines) in cases (5.5 units) and controls (5.7 units) is depicted.
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Fig. 2. Distribution of DNA-PK activity among lung cancer cases and controls. The variation in DNA-PK activity was divided into five groups and the percent cases and controls with that activity is depicted by the bars.
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The effects of age, ethnicity and smoking status on DNA-PK activity in the study groups were evaluated. There was no difference between the median ages of the two groups (Table I
, P = 0.36) and an association between DNA-PK activity and age was also not apparent for either group. Ethnicity was similar between groups and was not associated with differences in enzyme activity. As expected, smoking status and exposure burden among smokers (pack years of tobacco) were higher in cases than controls (Table I
, P = 0.09 and 0.08, respectively). The effect of enzyme activity adjusted for age, ethnicity and smoking (ever versus never) was assessed through logistic regression models. After adjustment for smoking, the odds ratio (OR) for lung cancer for a 1 unit decrease in DNA-PK activity was 1.24 (95% CI, 0.971.57, P = 0.08). The effects of smoking (OR = 4.9, P = 0.06) and enzyme activity appeared to be independent of each other.
DNA-PK activity in PMs reflects activity in a target cell for lung cancer
DNA-PK activity was initially studied in PMs, because these cells were collected through relatively non-invasive procedures and, thus, were ideal for conducting population-based studies. To determine whether PMs could serve as a surrogate for cells at risk for lung carcinogenesis, DNA-PK activity was assessed in BECs from 10 individuals who underwent bronchoscopy. DNA-PK activity ranged from 3.98 to 8.17 units in the PMs of these individuals, encompassing both high and low enzyme activity categories. The enzyme activity in PMs was strongly correlated with the activity seen in BECs (r = 0.83, P = 0.003) for these individuals who were both cases and controls (Figure 3
). The number of BEC population doublings prior to harvest did not affect the correlation between enzyme activity in PMs and BECs (data not shown), making clonal selectivity unlikely.

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Fig. 3. Comparison of DNA-PK activity in peripheral mononuclear cells and bronchial epithelial cell pairs from 10 subjects. Enzyme activity was determined in bronchial epithelial cells obtained at bronchoscopy and compared with values in peripheral mononuclear cells from the same subjects. A correlation coefficient of 0.83 (P = 0.003) was observed between the two cell sources.
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Effect of bleomycin exposure on BEC cell survival correlates with DNA-PK activity
A role for reduced DNA-PK activity as a modifier for lung cancer risk would be supported by evidence from a functional assay in which a difference in sensitivity to repair of double-strand breaks in DNA was demonstrated. Cell growth was determined in cultured BECs with differing enzyme activities following exposure to bleomycin, a glycopeptide antibiotic that induces a broad spectrum of DNA damage (21), including double-strand breaks. BEC lines from 13 subjects, with DNA-PK activity ranging from 2.0 to 7.5 units, were exposed to bleomycin. There was no relationship between cell growth and derivation of the BECs (i.e. from a case or control subject). However, cell growth and DNA-PK activity were highly associated following exposure to 5 µg bleomycin (Figure 4
, Spearman correlation coefficient = 0.59, P = 0.03). This association was not as strong nor significant following exposure to the two higher doses of bleomycin, most likely because of a marked increase in cell killing at these doses. This may reflect the inherent fragility of the mortal BECs.

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Fig. 4. Relationship between DNA-PK activity and sensitivity to cell killing by bleomycin. Bronchial epithelial cells were exposed to 5 µg/ml bleomycin for 2 h and colony growth determined 8 days after exposure. Data are expressed as proliferation index versus DNA-PK activity in BECs.
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Discussion
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The results of this study suggest that reduced DNA-PK repair activity is associated with an increased risk for lung cancer. This conclusion is supported by the fact that challenge of cells with bleomycin leads to a reduction in cell growth rates that correlates directly with DNA-PK activity. The tight correlation between DNA-PK activity in PMs and BECs, a progenitor cell for lung cancer, validates the use of PMs as a surrogate cell type for future population-based studies.
DNA-PK activity varied by a factor of 10 in the study groups. The apparent difference in enzyme activity in some persons was not explained by aging or smoking history. The most likely explanation for this variation in enzyme activity is the presence of polymorphism(s) within the DNA-PK complex. A variant form of Ku80 has been identified in B lymphocytes resulting in the ablation of DNA-PK activity (22). Although our study used PMs that contain B lymphocytes, other studies have shown that the percentage of this circulating lymphocyte does not differ between the lung cancer and control groups (23,24). Moreover, DNA-PK activity was tightly correlated between lymphocytes and BECs. While polymorphisms can lead to complete loss of enzyme activity, they are more frequently associated with a modulation of activity. For example, a base pair substitution polymorphism identified in the NAD(P)H:quinone oxidoreductase gene results in a change in amino acid sequence and a reduction in enzyme activity (25). Polymorphisms in genes can also produce a spectrum of enzymatic activity stemming from whether the person is a homozygous or heterozygous carrier of the susceptible allele (26). Finally, persons homozygous for a Gln399 polymorphism that occurs in the poly(ADP) ribose polymerase binding domain of the XRCC1 gene are at increased risk for adenocarcinoma of the lung (9). Alternatively, variation in DNA-PK activity could be mediated by alterations in DNA-PK protein levels or transcriptional regulation.
A critical role for DNA-PK in cancer susceptibility is most likely due to its role in facilitating non-homologous recombination, the major mechanism in mammalian cells for repair of double-strand breaks (14). Several studies have linked this process and the genes that regulate it to enhanced genomic instability, mutation frequency, radiosensitivity and increased rates of cancer (2729). For example, cell lines deficient in non-homologous repair or inhibited from performing this process are more susceptible to developing chromosomal aberrations when exposed to DNA-damaging agents than normal cells (27,28). The importance of non-homologous repair is exemplified by the autosomal recessive lethal human disease ataxia telangiectasia. The protein product of the gene (ATM) appears to either participate directly in non-homologous recombination or modulate the pathway (30). Mutations in the ATM gene result in an error-prone recombination of double-strand breaks and a 100-fold increase in susceptibility for leukemia and lymphoma (31). The kinase domain of the ATM gene shares significant homology with the kinase domain of the DNA-PK catalytic subunit (25). Thus, reduced DNA-PK activity could profoundly affect the ability to repair double-strand breaks, resulting in enhanced cell killing and the perpetuation of chromosome damage in the surviving cells.
This conclusion is supported by the current study, which directly assessed the effect of bleomycin, a radiomimetic agent, on cell growth as a function of DNA-PK activity, and by Wu et al. (32), who examined chromosomal lesions in cells surviving exposure to bleomycin. The effect of bleomycin on cell growth following a relatively low dose (5 µg/ml) was directly associated with DNA-PK activity in the exposed cells, while no relationship was observed when the dose of bleomycin was doubled. This suggests that the DNA-PK complex, comprised of the catalytic subunit of DNA-PK and the Ku70 and Ku80 subunits, exhibits a high affinity but low capacity for facilitating the repair of DNA damage. Wu et al. (31) showed that following exposure to bleomycin the proportion of chromosome 5 aberrations is significantly higher in surviving PMs from lung cancer cases than controls. Thus, sensitivity to bleomycin, which has been correlated with lung cancer risk (6), appears linked to lower DNA-PK activity and the survival of cells harboring chromosomal aberrations.
This study has identified the DNA-PK complex as another pathway that may affect the inherent susceptibility to lung cancer. The contribution of double-strand break repair as a risk modifier for lung cancer may be clearly elucidated through future casecontrol studies of DNA-PK activity in former uranium miners. The radon progeny liberated during the mining process decay into a series of short-lived radioisotopes, some of which emit
-particles that are highly effective in damaging DNA in the form of double-strand breaks. These exposures, in conjunction with smoking, have resulted in a markedly increased excess risk of lung cancer in this population (33). Thus, studying this population will help to unravel the role that repair of one form of DNA lesion has in the susceptibility to lung cancer.
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Notes
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5 Present address: MetroHealth Medical Center, Cleveland, OH 44109, USA 
6 Present address: Bayer Diagnostics, Berkeley, CA 94702, USA 
7 Present address: University of Southern California, Los Angeles, CA 91105, USA 
8 To whom correspondence should be addressed Email sbelinsk{at}LRRI.org 
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Acknowledgments
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The authors acknowledge Dr Susan Lees-Miller (University of Calgary, Alberta, Canada) for her advice regarding DNA-PK and the DNA-PK assay and Bill McCurdy and Benita Brennan at the Clinical Research Center, University of New Mexico, for technical support and assistance. The authors thank Elma Perez, Dawn Griffith, Donna Klinge, Marcie Grimes, and Sally Winters at Lovelace Respiratory Research Institute. D.H.A. was supported by a Clinical Research Center Grant, NCRR-GCRC MOIRR.0097, during 19961997 and an American Lung Association Research Training Fellowship during 19971998. The work was also supported by a National Cancer Institute Grant, CA 70190 (D.H.A., R.E.C., E.R.H., C.A.S., F.D.G. and S.A.B.) under US DOE cooperative agreement no. DE-FC04-96AL-76406, with the New Mexico Veterans Health Care System, Albuquerque, NM (R.E.C., E.R.H. and D.H.A.) and by NIOSH SERCA K01 CHO 0142 (F.D.G.).
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Received September 27, 2000;
revised January 12, 2001;
accepted January 18, 2001.