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Carcinogenesis Advance Access originally published online on July 6, 2005
Carcinogenesis 2006 27(1):117-122; doi:10.1093/carcin/bgi175
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Carcinogenesis vol.27 no.1 © Oxford University Press 2005; all rights reserved.

The association of DNA-dependent protein kinase activity with chromosomal instability and risk of cancer

Masanori Someya, Koh-ichi Sakata *, Yoshihisa Matsumoto 3, Hiroyuki Yamamoto 1, Manami Monobe 4, Hideyuki Ikeda 2, Koichi Ando 5, Yoshio Hosoi 3, Norio Suzuki 3 and Masato Hareyama

Department of Radiology, 1 First Department of Internal Medicine and 2 Department of Clinical Pathology, Sapporo Medical University, School of Medicine, Hokkaido, Japan, 3 Department of Radiation Research, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, University of Tokyo, Tokyo, Japan, 4 Department of Radiation Biosciences, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Chiba, Japan and 5 Heavy-Ion Radiobiology Research Group, National Institute of Radiological Sciences, Chiba, Japan

* To whom correspondence should be addressed. Tel: +81 11 611 2111 (Ext. 3535); Fax: +81 11 613 9920; Email: sakatako{at}sapmed.ac.jp


   Abstract
Top
 Abstract
Introduction
 Patients and methods
 Results
 Discussion
 References
 
The DNA double-strand breaks (DSBs) repair pathway has been implicated in maintaining genomic integrity via suppression of chromosomal rearrangements. DNA-dependent protein kinase (DNA-PK) has an important role with DNA DSBs repair. In this study, 93 of untreated cancer patients and 41 of cancer-free healthy volunteers were enrolled. Peripheral blood was collected, separated and centrifuged; DNA-PK activity was measured by DNA-pull-down assay. The expressions of DNA-PKcs, Ku70 and Ku86 were examined by RT–PCR assay and western blotting. Chromosomal aberrations were examined by cytogenetic methods. DNA-PK activities of peripheral blood lymphocytes (PBL) in patients with uterine cervix or breast cancer were significantly lower than those in normal volunteers. Age and smoking had no association with DNA-PK activity, whereas DNA-PK activity and the expression of Ku70, Ku86 and DNA-PKcs in RT–PCR were interrelated. A similar tendency was seen in western blot assay but less clear than in RT–PCR. Therefore, the association between DNA-PK activity and expression of DNA-PK in protein level could not be concluded. The frequency of chromosome aberration, such as dicentric chromosomes and excess fragment increased as the DNA-PK activity decreased. In conclusion, DNA-PK activity is associated with chromosomal instability. DNA-PK activity in PBL is associated with risk of breast and uterine cervix cancer. DNA-PK activity in PBL can be used to select individuals for whom an examination should be performed because of their increased susceptibility to breast and uterine cervix cancer.

Abbreviations: DNA-PK, DNA-dependent protein kinase; DSBs, double strand breaks; DNA-PKcs, DNA-PK catalytic subunit; HPV, human papilloma virus; HR, homologous recombination; NHEJ, non-homologous end-joining; PBL, peripheral blood lymphocytes; SNPs, single nucleotide polymorphisms


   Introduction
Top
 Abstract
 Introduction
Patients and methods
 Results
 Discussion
 References
 
One of the hallmarks of malignant transformation is genomic instability, which promotes a wide range of mutations, including chromosome deletions, gene amplifications, translocations and polyploidy (1). The presence of genomic instability in cells is known to play an important role in the multistage carcinogenesis of various organs in both humans (1) and experimental animals (2), and genes involved in the maintenance of genomic stability can be considered as a caretaker-class of tumor suppressor genes.

Cancer suppression genes have been classified into two groups: gatekeepers and caretakers (3). Gatekeepers are genes that control cell proliferation and death, whereas caretakers are DNA repair genes, whose inactivation leads to genetic instability. Abrogation of both caretaker and gatekeeper function markedly increases cancer susceptibility.

In DNA double-strand breaks (DSBs) repair, at least two major repair mechanisms, homologous recombination (HR) and non-homologous end-joining (NHEJ) have been reported (4). In NHEJ pathway, DSBs are directly, or after processing of the DNA ends, rejoined at an appropriate chromosomal end and DNA-dependent protein kinase (DNA-PK) plays an important role in DNA DSBs repair by NHEJ throughout the cell cycle (5). DNA-PK is a serine/threonine kinase, which is composed of DNA-PK catalytic subunit (DNA-PKcs) and heterodimer of Ku70 and Ku86. DNA-PK binds to DSBs in DNA, phosphorylates and activates DNA-binding protein, including XRCC4 and DNA ligase IV, p53 and several transcription factors. Then Ligase IV repairs DNA DSB (6).

This DNA repair pathway has been implicated in maintaining genomic integrity via suppression of chromosomal rearrangements (4,7,8). Consistent with this idea, mice deficient in NHEJ components are characterized by increased sensitivity to agents causing DNA damage, chromosomal instability, immunodeficiency and predisposition to thymic lymphomas (9). Further loss of cell-cycle checkpoints in NHEJ-deficient mice results in a shift from thymomas to pro-B cell lymphomas (7,10–15) or to sarcomas (13,16). These results indicate that DNA-PKcs, Ku70 and Ku86, which compose the DNA-PK complex, are considered to belong to the caretaker class of tumor suppressor genes (17). Therefore, it is very interesting to know how the ability of NHEJ influences the genomic integrity and carcinogenesis in a clinical setting.

Auckley et al. (18) reported that DNA-PK activity in peripheral blood lymphocytes (PBL) from patients with lung cancer was significantly lower than lung cancer-free controls. They also demonstrated a tight correlation between DNA-PK activity in PBL and bronchial epithelial cells (a progenitor cell for lung cancer) that were obtained by bronchoscopy, suggesting that PBL can be used as a surrogate cell type for other kinds of cells.

In this study, we examined the DNA-PK activity in PBL from individuals with various kinds of cancer other than lung cancer. We also investigated the relationship between DNA-PK activity and chromosomal aberrations by cytogenetic methods to elucidate the mechanism of association between DNA-PK activity and cancer risk. We then performed RT–PCR and western blot analysis to find the mechanism of different DNA activity.


   Patients and methods
Top
 Abstract
 Introduction
 Patients and methods
Results
 Discussion
 References
 
Selection eligibility
All subjects were Japanese. A total of 93 untreated cancer patients, who were to receive treatment at Sapporo Medical University and 41 cancer-free normal healthy volunteers were enrolled in this study. Patients with histories of other cancer, those treated with chemotherapy or radiation and those using immunosuppressive medications were excluded. Patients with breast cancer were all sporadic cases.

The study was approved by the appropriate committees for human rights in research in our hospital and written informed consent was obtained from each subject. A summary of the individual's characteristics is shown in Table I.


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  		Table I. Summary of the characteristics of the patients

 
Blood collection and PBL separation
An aliquot of 20 ml of peripheral blood was collected with a sterile heparinized tube from all individuals. PBLs were separated with lymphoprep (Nycomed Pharma AS, Oslo, Norway), centrifuged at 1500 r.p.m. (300x g) for 30 min at 4°C washed twice with Phosphate-Buffer Saline (PBS) and stored at –80°C.

PBL lysis, protein extraction, concentration assay
PBL was thawed with high salt buffer [20 mM HEPES–KOH (pH 7.9), 400 mM KCl, 1 mM EDTA, 1 mM EGTA, 0.02% Tween-20, 10% Glycerol, 1 mM DTT, 1 mM PMSF and 1 g/ml leupeptin, pepstatin and antipain, respectively], and the suspension was lysed by three rounds of freeze–thaw cycle, i.e. repeated freezing in liquid nitrogen bath followed by thawing in water bath at 30°C and clarified by centrifugation at 15 000 r.p.m. (18 000x g) for 7 min at 4°C. Protein concentration was assayed using a BCA protein assay kit (Pierce, Rockford, IL, USA) with bovine serum albumin (BSA) as the standard.

The assay procedure for DNA-PK activity has been described in our earlier publication (19). The PBL cell lysates were diluted to three protein concentrations (0.5, 0.25 and 0.125 mg/ml) with high salt buffer. An aliquot of 5 µl of the diluted lysate was mixed with 15 µl of 1.33x kinase assay buffer (contents of 1x kinase assay buffer: 20 mM HEPES–NaOH (pH 7.2), 5 mM MgCl2, 150 mM KCl, 50 µM [{gamma}-32P]ATP, 1 mM DTT, 0.5 mM NaF and 0.5 mM ß-sodium glycerophosphate), 0.25 µg/µl synthetic peptide hp53-S15 (sequence: EPPLSQEAFADLWKK; synthesized in Sawady Biotechnology, Tokyo, Japan) and with or without 20 ng/µl sonicated salmon sperm DNA. This reaction mixture was incubated at 37°C for 10 min. The reaction was stopped by the addition of 20 µl of 30% acetic acid and absorbed onto a phosphocellulose filter disk (2.3 cm in diameter, Whatman, Maidstone, UK). The filter disks were washed in 15% acetic acid and in 99% ethanol and the remaining radioactivity was counted in a liquid scintillation counter. The net phosphorylation of hp53-S15 was calculated as phosphate incorporation in reaction with DNA minus that in reaction without DNA, divided by the specific radioactivity of ATP. In this study, DNA-PK activity was expressed as amount of ATP (unit is ‘pmol’) used for DNA-dependent phosphorylation of hp53-S15.

Semiquantitative RT–PCR
We selected eight individuals for RT–PCR assay who had various activities of DNA-PK. Total RNA isolated from PBLs of each individual as DNA-PK assay using Triazol reagent (Life Technologies, Rockville, MD) was used for mRNA extraction using a MagExtractor mRNA isolation kit (Toyobo, Tokyo, Japan) according to the manufacturer's protocol. The mRNA samples were used to synthesize the first strand cDNA (Life Technologies). PCR was performed using primers specific for DNA-PKcs, Ku70 and Ku86, and GAPDH genes in duplex PCR reactions. GAPDH served as an internal control of the reaction. Results were analyzed using a multiimage analyzer (Bio-Rad, Hercules, CA).

Western blot analysis
The expressions of DNA-PKcs, Ku70, Ku86 and GAPDH, as an internal control, were examined with western blotting by using the same subjects as RT–PCR. For the detection of DNA-PKcs, mouse monoclonal antibody Ab4 (Neomarkers, Fremont, CA) was used. Rabbit polyclonal antibodies against Ku70 and Ku86, have been previously generated by our group (20). Monoclonal antibodies against GAPDH (MAB374) were purchased from Chemicon International (Temecula, CA). PBL lysates were diluted to 2, 0.5 and 0.0625 mg protein/ml for DNA-PKcs, Ku70 and Ku86 and GAPDH, respectively, and 10 µl of aliquot was analyzed. Other details of western blotting have been described elsewhere (21). The signals were detected using X-ray film (Hyperfilm MP; Amersham) or lumino-image analyzing system LAS1000-Mini (Fuji, Tokyo, Japan).

Spontaneous chromosomal aberration of PBL
Chromosomal aberration of PBL was observed by Giemsa staining in 18 normal volunteers and 23 cancer patients. The procedure has been described by Durante et al. (22). Briefly, 2 ml of peripheral blood was collected with a sterile heparinized tube, and 10 ml RPMI-1640 medium (Sigma Aldrich, Taurtkirchen, Germany), supplemented with 20% fetal calf serum (FCS) and 1% PHA (Murex, Dartford, UK) was added. The medium and cells were incubated at 37°C. After 24 h, 40 ng/ml of colcemid (GIBCO BRL, Grand Island, NY) was added and incubated again for 23 h. Finally, 50 nM of calyculin A (Wako Chemicals, Osaka, Japan) was added and incubated again for 1 h. The cells were then swollen in 0.075 M KCl at 37°C for 20 min, fixed with fixative solution (methanol:acetic acid = 3:1), and stored at –20°C for 24 h. The cells in fixative were spread on humid slides and air-dried. Then the slides were stained with 4% Giemsa solution (Merck, Darmstadt, Germany) for 20 min. The slides were observed with a light microscope under 10 x 100 with oil immersion.

Chromosomal aberrations are typically subdivided into chromatid breaks and gaps versus chromosome breaks and gaps, triradials, quadriradials and dicentrics. Chromosome break and gaps not accompanying a dicentric chromosome were defined as excess fragments in this analysis. We analyzed 200 cells in metaphase from each individual and counted the number of dicentric chromosomes and excess fragment. We excluded triradials and quadriradials from this analysis because these were not observed.

Statistical methods
The two-sample unpaired t-test was used to compare DNA-PK activities of normal volunteers and cancer patients. Spearman's rank correlation test was used to compare DNA-PK activity and expression of DNA-PKcs, Ku86 and Ku70 in RT–PCR and western blotting. Multiple regression analysis was used to clarify significant variables which correlate with frequency of chromosome aberration. All statistical computing was done with StatView version 4.58 (Abacus Concepts, Berkeley, CA).


   Results
Top
 Abstract
 Introduction
 Patients and methods
 Results
Discussion
 References
 
We performed the following two experiments to confirm the reliability of our method to measure the DNA-PK activity of PBL. First, DNA-PK activity of each sample was measured in three protein concentrations (0.5, 0.25 and 0.125 mg/ml) and the linear correlation between DNA-PK activity and protein concentration was confirmed (data not shown). Next, DNA-PK activity of 134 samples was measured twice to examine reproducibility of DNA-PK activity assay. The correlation coefficient of two measurements was 0.647 and P-value was <0.0001, indicating that the result of the first assay gave good agreement with the second assay. The sample of the same person was simultaneously run for all DNA-PK assays as an internal control. DNA-PK activity values of the sample used as an internal control were 17.6 ± 0.9 pmol in 20 measurements.

Figure 1A demonstrated the DNA-PK activity of PBL in various kinds of cancer patients and normal volunteers. DNA-PK activities of PBL in normal volunteers were 14.2 ± 4.0 pmol (mean ± SD). In uterine cervix cancer patients, in particular, DNA-PK activities were 9.6 ± 4.2 pmol and significantly lower than those in normal volunteers (P < 0.0001). In breast cancer patients, DNA-PK activities were 11.6 ± 4.6 pmol and also significantly lower than those in normal volunteers (P = 0.009). However, patients with head and neck cancer had DNA-PK activity of 13.0 ± 5.1 pmol, esophageal cancer 11.7 ± 2.7 pmol and malignant lymphoma 18.3 ± 2.4 pmol. There were no significant differences among normal volunteers, head and neck cancer, esophageal cancer and malignant lymphoma. Figure 1B demonstrated DNA-PK activity of cancer patients separated into three categories: normal, breast and cervical, and all other cancer types. DNA-PK activities of patients with breast cancer or uterine cervix cancer were 10.9 ± 4.5 pmol and those with other cancer types were 13.5 ± 4.9 pmol. There was the significant difference in DNA-PK activity between normal volunteers and breast or uterine cervix cancer patients (P = 0.0002).



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  		Fig. 1. (A) DNA-PK activity in the peripheral lymphocytes of normal healthy volunteers and various kinds of cancer patients. (B) DNA-PK activity of cancer patients separated into three categories: normal, breast and cervical, and all other cancer types. The horizontal bar indicates the mean value.

 
No relationship was found between age and DNA-PK activity (Figure 2A). In addition, DNA-PK activities of non-smoker were 13.7 ± 3.5 pmol and those of smoker were 14.8 ± 4.6 pmol. There was no relationship between smoking and DNA-PK activity (P = 0.412) (Figure 2B).



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  		Fig. 2. (A) The relationship between age and DNA-PK activity in normal healthy volunteers. (B) The relationship between smoking and DNA-PK activity in normal healthy volunteers. The horizontal bar indicates the mean value.

 
We performed semiquantitative RT–PCR analysis for DNA-PKcs, Ku86 and Ku70 in eight selected subjects, i.e. four cancer patients and four non-cancer volunteers, exhibiting various levels of DNA-PK activity (Figure 3), to clarify the basis underlying such a variation in DNA-PK activity. The expression levels of all three genes correlated well with DNA-PK activities, indicating that the kinase activity might have been largely determined by transcriptional regulation. In addition, the expression levels of all three genes were related to one another, suggesting a possibility of coordinated regulation of these three genes.



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  		Fig. 3. (A) The semiquantitative RT–PCR analyses of DNA-PKcs, Ku70/86 and GAPDH, as a loading control, in eight individuals who had various activities of DNA-PK (shown below the blots). N-40, N-20, N-14 and N-17 are normal healthy volunteers, whereas C-56 is a patient with breast cancer, and C-52, C-54 and C-26 were patients with uterine cervix cancer. (B–D) The relationship between the DNA-PK activity and the expression levels of (B) DNA-PKcs, (C) Ku86 and (D) Ku70. The expression levels were normalized to that of GAPDH.

 
We also examined western blotting analysis for DNA-PKcs, Ku86 and Ku70 by using the same subjects (Figure 4). Although there was the similar tendency as Figure 3, there are several exceptions, most notably C-52. The association of the expression with the kinase activity in western blotting analysis was less clear than RT–PCR assay.



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  		Fig. 4. (A) The western blotting analyses of DNA-PKcs, Ku70/86 in the same samples as in Figure 3. GAPDH was loaded as a control. (B–D) The expression levels of DNA-PKcs (B), Ku86 (C) and Ku70 (D) were normalized to that of GAPDH.

 
We performed chromosome analysis to examine a possible link between DNA-PK activity and genomic instability. Since the mice lacking either one of DNA-PK subunits exhibit many types of chromosome aberrations in association with tumorigenesis, we performed chromosome analysis of PBL.

In the present analysis from 41 individuals, 10 persons had one dicentric chromosome and the others had no dicentric chromosome. Dicentric chromosomes were observed when their DNA-PK activity was <16.3 pmol (Figure 5A). Excess fragment was seen much higher frequency than dicentric chromosome. An inverse correlation was found between DNA-PK activity and excess fragment (Figure 5B). We further performed multiple regression analysis (Table II). In this analysis, only DNA-PK activity showed significant correlation with the excess fragment frequency.



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  		Fig. 5. The relationship between chromosomal aberration and DNA-PK activity. The numbers of dicentric chromosomes (A) and excess fragments (B) in 200 mitotic metaphase cells were counted and expressed as the number per 100 cells. Open circle, normal healthy volunteers; closed circle, cancer patients. The line in (B) is the regression curve.

 

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  		Table II. Variables that affect yield of excess fragment by multiple regression analysis

 
A variant form of Ku86 has been identified in B lymphocytes, resulting in the ablation of DNA-PK activity (23). Although our study used PBL that contain B lymphocytes, other studies showed that the percentage of this circulating lymphocyte did not differ between the lung cancer and control groups (24,25). We also measured the percentage of this circulating lymphocyte and found that it did not differ among individuals (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
References
 
DNA-PK and chromosomal instability
Reduced DNA-PK activity can profoundly affect the ability to repair double-strand breaks, resulting in the perpetuation of chromosome damage. Unrepaired DNA ends might contribute to the development of translocations by acting as transposable elements (26,27). We demonstrated that there is a relationship between DNA-PK activity and chromosome aberration in PBL (Figure 5).

Recently, in vitro studies that support our results have been reported. Karanjawala et al. (8) examined primary dermal fibroblasts from mice [wild-type, Ku86(+/–), Ku86(–/–), and DNA ligase IV(+/–)] for chromosome breaks. Fibroblasts from Ku86(+/–) or DNA ligase IV(+/–) mice have elevated frequencies of chromosome breaks compared with those from wild-type mice. Fibroblasts from Ku86(–/–) mice have even higher levels of chromosome breaks. The Ku86(+/–) heterozygous fibroblasts have a chromosome break frequency that is intermediate between the wild-type and Ku86(–/–) cells. The DNAligase IV(+/–) cells also have a markedly increased frequency of chromosome breaks relative to that of wild-type cells. This suggests that heterozygous levels of Ku86 and DNA ligase IV in murine fibroblasts have a lower NHEJ ability and are not sufficient to repair many of the spontaneously occurring chromosomal breaks.

Possible link between DNA-PK and carcinogenesis
In the present study, we first demonstrated a significant difference in DNA-PK activity between cancer-free volunteers and patients with various kinds of cancer. When the cancer patients were sorted by the primary sites, the tendency of low DNA-PK activity was evident in the groups of uterine cervix and breast cancer. However, DNA-PK activity of head and neck, esophageal tumor or malignant lymphoma patients did not differ significantly from that of the cancer-free group. Therefore, the association of DNA-PK activity in PBL with the cancer risk might be tissue dependent. Protein products of the familial breast cancer susceptibility genes, BRCA1 and BRCA2 are involved in HR, which is an alternative mechanism of DNA DSB repair to NHEJ. DNA-PK activities in our results were obtained with sporadic cases of the breast cancer. The molecular and genetic determinants of most sporadic breast cancer remain unknown. It is highly probable that breast cancer can result from a variety of sporadic gene mutations that lead to abnormalities in multiple independent pathways (28). Our results indicate that DNA-PK could work as a breast cancer susceptibility gene, especially in sporadic cases.

The high-risk human papilloma virus (HPV) infection is the main etiologic factor for cervical cancer (29). The E6 and E7 products of HPV interfere with the p53 and pRB functions and deregulate the cell cycle. HPV DNA exists in an extrachromosomal form in benign and premalignant lesions. However, HPV DNA is integrated into the host's chromosomes in cervical cancer (30). Integration occurs in the E1/E2 region, disrupting the E2 viral genome (31). E2 represses the promoter from which the E6 and E7 genes are transcribed (32). Thus, after HPV DNA integration with disruption of the E2 gene, the expression of the E6 and E7 genes are accelerated, leading to the accumulation of DNA damage and the development of cancer cell. Since the integration of HPV genomes can be mediated by NHEJ (33), DNA-PK might influence HPV integration which leads to carcinogenesis.

Why is there such a variation in DNA-PK activity among individuals?
We found that DNA-PK activity in PBL varied by a factor of 10 among the individual subjects. The difference in DNA-PK activity in individuals was not explained by aging or smoking history (Figure 2). DNA-PK activity can be influenced by the abundance of DNA-PKcs, Ku86 and Ku70, and also by the activity of each molecule.

We demonstrated the correlation between DNA-PK activity and the expression of these subunits, at mRNA, indicating that the kinase activity might be determined by the transcription of these genes. As the promoter regions of genes of DNA-PKcs, Ku86 and Ku70 have binding sites for many transcription factors, the transcriptional regulation may be one possibility for a variation in DNA-PK activity among individuals.

Another explanation for this variation in enzyme activity is the presence of polymorphism(s) within the DNA-PK complex. Fu et al. (34) have genotyped 30 single nucleotide polymorphisms (SNPs) in all five NHEJ genes (Ku70, Ku86, DNA-PKcs, Ligase IV and XRCC4) in 254 primary breast cancer patients and 379 healthy controls. They found that two SNPs in Ku70 and XRCC4 were associated with breast cancer risk although both SNPs were in introns and probably do not affect protein function. The research for SNPs of DNA-PK which influence the DNA-PK activity is under investigation in our laboratory.

When the results of RT–PCR analysis are compared with those of western blotting analysis, changes at the protein level of Ku70, Ku86 and DNA-PKcs were smaller than changes at the mRNA levels. Yaneba et al. (35) studied the expression of these proteins in PBL during cell proliferation stimulated by phytohemagglutinin. They found that the change of the Ku protein level was significantly less than the change in the mRNA level observed. Our results agreed with their results. This discrepancy of changes between mRNA and protein levels could be partially explained by the high stability of the Ku protein.

In conclusion, our data suggest that DNA-PK activity is associated with chromosomal instability and risk of cancer. This indicates that DNA-PK acts as a caretaker in cancer susceptibility genes whose inactivation leads to genetic instabilities resulting in increased mutation of all genes, including gatekeepers. Therefore, DNA-PK activity in PBL can be used to select individuals for whom an examination should be performed because of their increased susceptiblity to cancer.


   Acknowledgments
 
Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 

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Received April 26, 2005; revised June 5, 2005; accepted June 28, 2005.


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