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Micronuclei in EM9 cells expressing polymorphic forms of human XRCC1
Cancer Letters 221 (2005) 91–95 www.elsevier.com/locate/canlet
Micronuclei in EM9 cells expressing polymorphic forms of human XRCC1 Tianli Qua, Eiichi Moriib, Keisuke Obokib, Yuquan Lua, Kanehisa Morimotoa,* a
Department of Social and Environmental Medicine, Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Osaka 565-0871, Japan b Department of Pathology, Osaka University Medical School, 2-2 Yamada-oka, Osaka 565-0871, Japan Received 20 May 2004; received in revised form 27 July 2004; accepted 9 August 2004
Abstract X-ray repair cross-complementing gene 1 (XRCC1) is involved in base excision repair (BER) through interaction with other BER enzymes, and polymorphisms in XRCC1 appear to increase the risk of various cancers. We evaluated how three XRCC1 polymorphisms, Arg194Trp, Arg280His and Arg399Gln, affect the extent of DNA damage and repair using the micronucleus assay. XRCC1 cDNAs containing the wild-type sequence and the three polymorphisms were overexpressed in EM9 cells, which lack the full sequence needed to perform XRCC1 functions. Normal human XRCC1 cDNA corrected the defect in EM9 cells. Only XRCC1 cDNA containing the Arg399Gln polymorphism did not fully correct the DNA repair defect in EM9 cells. These results indicate that the Arg399Gln polymorphism, but not the Arg194Trp or Arg280His polymorphism, influences the ability of XRCC1 to repair DNA. This study may provide a model that can be used to evaluate the functional significance of polymorphisms in DNA repair genes. q 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: XRCC1; EM9 cells; Polymorphism; Cancer susceptibility; DNA repair; Chromosome damage
1. Introduction Among more than 130 human DNA repair genes that mitigate the DNA damage effects of the mutatcarcinogene, X-ray repair cross-complementing gene 1 (XRCC1) was the first mammalian gene shown to play a role in the cellular sensitivity to ionizing radiation (IR) [1]. The human XRCC1 gene is 33 kb in length, and is located on human chromosome * Corresponding author. Tel.: C81 6 6879 3920; fax: C81 6 6879 3923. E-mail address: [email protected] (K. Morimoto).
19q13.2-13.3 [2]. It encodes a 2.2 kb transcript, which corresponds to a putative protein of 633 amino acids (69.5 kDa). The XRCC1 protein forms repair complexes with DNA ligase III [3], poly(ADP-ribose) polymerase (PARP) [4,5], and DNA polymerase b (polyb) [5]. In orchestrating base excision repair (BER), the ternary DNA-XRCC1-polyb complex has been suggested to act as ‘a scaffolding protein’. Three XRCC1 polymorphisms have been reported: Arg194Trp, Arg280His, and Arg399His. Codon 194 and codon 280 reside between the binding domain of polyb and PARP. Codon 399 is located in the vicinity of the PARP binding domain. Studies of Arg194Trp indicate
0304-3835/$ - see front matter q 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2004.08.013
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that this polymorphism may contribute to an increased risk of lung [6], breast [7] and head and neck cancer [8]. The Arg399Gln polymorphism has been linked to an increased risk of lung [9], breast [10], head and neck [8], bladder [11], esophageal [12] cancer. The Arg280His polymorphism has been reported as a lung cancer risk and breast cancer risk [13,14]. Three polymorphic genotype carriers all showed 1.5 odds ratio compared with wildtype genotype carrier for cancer risk. In addition, a gene– environment interaction of XRCC1 polymorphism has been discussed, particularly with regards to lifestyle factors (tobacco smoking and alcohol consumption) combining with the XRCC1 polymorphism to create a higher risk of cancer [15,16]. Some studies even have found no association between XRCC1 polymorphisms and cancer risk [17]. Additional studies will be necessary to establish to what extent XRCC1 polymorphisms are associated with increased cancer risk. EM9 cells, which have a truncated polypeptide lacking two-thirds of the normal hamster XRCC1 sequence, have been used as XRCC1-knockout cells. The frequency of spontaneous sister chromatid exchange (SCE) is 10-fold higher in EM9 cells compared to the parent cell line AA8. This sensitivity is the result of a deficiency in rejoining a DNA singlestrand breaks (SSBs), and is also manifest in sensitivity to SCE induction by agents that induce SSBs, such as alkylating agents (i.e. methyl methanesulfonate and ethyl methanesulfonate) and IR [18]. XRCC1 is thought to be involved in SSB repair. The defective XRCC1 gene function will result in the SSB accumulation or chromosomal DNA instability, which in the following cell cycle, would possibly increase the frequency of micronucleus (MN) because some portions of incompletely repaired of SSB could be possibly converted into double-strand break (DSB) during chromosome replication [19]. We used cytochalasin-B block MN assay to measure DNA damage in this study. This technique is rapid and convenient, permitting the measurement of damage by a wide variety of clastogens. We used bleomycin to induce DNA damage in vitro. Bleomycin is a radiomimetic anticancer drug that produces SSB and DSB in a catalytic way. On average, each bleomycin molecule generates 8–10 DNA breaks (SSB and DSB) [20]; it is admitted that for every six SSB, one DSB is created [21]. The effects of the XRCC1 polymorphisms (Arg194Trp, Arg280His and Arg399Gln) on the
DNA repair function were evaluated. In epidemiological studies, we cannot clearly define which XRCC1 polymorphisms are associated with increased cancer risk. In vitro evidence would be useful for supporting the plausibility of any associations.
2. Materials and methods EM9 and AA8 cells were obtained from the ATCC (Manassas, Virginia, USA). Bleomycin was purchased from Nihonkagaku (Tokyo, Japan), while Cytochalasin B was from Sigma. Blood was obtained from a healthy donor. XRCC1 cDNA was amplified by RT-PCR, and cloned into pcDNA3.1 (Invitrogen). The XRCC1 polymorphisms were constructed by inducing point mutations into the cDNA, then cloning the cDNAs into pcDNA 3.1. The sequences were verified with a DNA sequencer (Model 3100, Applied Biosystems, Forster City, CA). pcDNA3.1 plasmids containing wild-type XRCC1 cDNA and the XRCC1 cDNAs containing each of the three polymorphisms were transfected into EM9 cells using Lipofectimine2000 reagent (Invitrogen) according to the procedures described by the manufacturer. Clones expressing XRCC1 cDNA were selected with Zeocin (Invitrogen) and CldUrd (Sigma). The adherent cells were harvested, and total RNA was isolated using Trizol reagent (Invitrogen) according to the manufacturer’s instructions and quantified by optical density. One microgram of total RNA was added to a reverse transcriptase (RT) reaction in RT buffer, 0.5 mg oligo d (T) primer, 200 units SuperScript II RT and Rnase H. One microliter of cDNA from the RT was added directly to a 30 ml PCR buffer containing 20 mM Tris–HCl (pH 8.4), 50 mM KCl, 25 mM MgCl2, 10 mM dNTP, 0.25 unit Taq DNA polymerase. Oligonucleotide primers were designed as follows: 5 0 -CAC CTC ATC TGT GCC TTT GC-3 0 and 5 0 -ACC CTC CTC AGC TCA TCC TC-3 0 , a total of 410 bp. Cells were lysed with buffer containing 20 mM HEPEs (pH 7.9), 0.4 M NaCl, 25% glycerol, 1 mM EDTA, 10% protease inhibitor cocktail (Sigma) and 2.5 mM DTT. The cell lysates were frozen and thawed, then centrifuged. The supernatant was subjected to electrophoresis and the separated protein transferred to a nylon membrane. The membranes were blocked and incubated with anti-XRCC1, clone 33-2-5 (Kamiya,
T. Qu et al. / Cancer Letters 221 (2005) 91–95
Fig. 1. XRCC1 gene expression in tranfected cells. (A) RT-PCR of expressed XRCC1 protein. (B) Image density as measured by software (NIH Image 1.62). Densitometry of XRCC1 bands were normalized to the AA8 value for each sample. Relative densitometry was shown for the RT-PCR data in panel A.
Seattle, WA) at 4 8C overnight. The signal specific to the XRCC1 protein was detected after exposure to X-ray film. Cells of 5!105 were plated in six-well plates containing growth mediums supplemented with 3 mg/ml cytochalasin B. Bleomycin was added at concentrations of 0, 3, 10 and 30 mg/ml. The cells were cultured in the dark at 37 8C for 48 h. The cytokinesis block MN assay was based on the technique of French and Morley [22]. The bleomycin treated cell pellets were treated with 0.075 M KCl at room temperature for 15 min. After that, the cells were fixed in fresh Carnoy’s solution (methanol– acetic acid, 3:1) at 4 8C for 30 min, washed with Carnoy’s solution three times. The fixed cells were resuspended in the 1% acetic acid by gentle pipetting and spread onto pre-cleaned 15-mm frosted microscope slides (Matsunami). After drying, the slides were stained with 3% Giemsa (Merck, pH 6.8). The number of binucleated cell per 1000 cells was 250– 300. Micronuclei were counted in 500 binucleated cells under a microscope. The results were expressed as the meanGSD of triplicate determinations from independent experiments. Sample-dependent t-test was used for multiple comparisons.
3. Results We constructed expression vectors encoding the wild-type human XRCC1 gene and polymorphic
93
human XRCC1 gene, and introduced it into EM9 cells to isolate stable transformants (ExhX-1). We isolated EM9 cells overexpressing XRCC1 cDNAs containing three polymorphisms: Arg194Trp, Arg280His and Arg399Gln; these cell lines were designated Exm6X-1, Exm9X-1 and Exm10X-1, respectively. We also introduced an empty pcDNA3.1 vector into EM9 cells to isolate stable transformants (EXV) and provide a negative control. We examined the XRCC1 gene expression levels of every cell lines by RT-PCR. The XRCC1 gene was overexpressed in ExhX-1 cells, its amount being approximately 1.7 times that in AA8 (Fig. 1A and B). No XRCC1 band was found in EM9 cells or the negative control EXV cells (Fig. 1A). The quality of XRCC1 in ExhX-1, Exm6X-1, Exm9X-1 and Exm10X-1 cells is comparable (Fig. 1A and B). To further confirm the XRCC1 protein quantity of the cell lines, we carried out Western blot. The XRCC1 gene was overexpressed in ExhX-1 cells, its amount being approximately 2 times that in AA8 (Fig. 2A and B). No XRCC1 band was found in EM9 cells or the negative control EXV cells (Fig. 1A). The quality of XRCC1 in ExhX-1, Exm6X-1, Exm9X-1 and Exm10X-1 cells is comparable (Fig. 2A and B). To assess chromosomal DNA damage (and, indirectly, repair) in ExhX-1 and EM9 cells, we carried out the cytochalasin B-cytokinesis block MN assay. Table 1 shows the MN frequency in cell lines exposed to increasing doses of bleomycin. At a bleomycin
Fig. 2. Western blotting of expressed XRCC1 protein. (A) Western blot of expressed XRCC1 protein. (B) Image density as measured by software (NIH Image 1.62). Densitometry of XRCC1 bands were normalized to the AA8 value for each sample. Relative densitometry was shown for the Western blot data in panel A.
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Table 1 Micronucleus frequency per 1000 cells analyzed on 500 binucleate cells treated with bleomycin
4. Discussion
study, we treated the cell lines with oxidative agent; ExhX-1 cells overexpressing XRCC1 protein were relatively resistant to this agent. It is possible that the overexpression of XRCC1 protein in EM9 cells confers resistance to other exogenous chemical agents and IR. The XRCC1 protein, however, has no enzymatic activity. During BER and SSB repair, the XRCC1 protein interacts with DNA polyb, PARP and DNA ligase III [2]. The XRCC1 protein may also up-regulate the activity of many other DNA repair enzymes. XRCC1 polymorphisms appear to be associated with the risk of acquiring various types of cancer. Three polymorphisms genotype all showed 1.5 odds ratio compared with wild-type genotype for cancer risks. Epidemiology studies alone cannot determine whether polymorphisms like Arg194Gln, Arg280His or Arg399Gln have any functional significance in cancer risk. Data from other studies found no association between Arg399Gln and cancer risk [17]. In this study, we examined the ability of XRCC1 polymorphisms to repair DNA in vitro. Our data suggests that the Arg399Gln polymorphism affects DNA repair. Arg399Gln is located in the BRCT1 domain, which is necessary for accumulation of the protein at SSB via interaction with PARP. This maybe one reason why this polymorphism is most frequently found among the XRCC1 polymorphisms
Micronuclei induced in response to DNA damage may be formed from acentric chromosomal fragments or whole chromosomes that remain separate to the main nucleus after nuclear division. The number of micronuclei within a cell can therefore be indicative of the extent of chromosomal insult [22–24]. The MN assay is not a direct measure of DNA repair, but the magnitude of the responses might be influenced by DNA repair. We initially examined the human XRCC1 gene function in the following cell lines: ExhX-1 cells (EM9 cells overexpressing wild-type XRCC1), EXV cells (an empty vector control for EM9 cells), repairdefective EM9 cells by using the MN assay. The data indicated that bleomycin produced fewer micronuclei in ExhX-1 cells than in EM9 cells, indicating that XRCC1 expression resulted in a higher degree of genetic stability. In the present
Fig. 3. Effect of XRCC1 polymorphisms on micronucleus (MN) induction by bleomycin. Polymorphisms in exon 6 (Arg194Trp), exon 9 (Arg280His) and exon 10 (Arg399Gln) were evaluated. The micronuclei number of 500 binucleated cells per sample was scored. *P!0.001, sample-dependent t-test.
Bleomycin (mg/ml)
EM9 (meanGSD)
EXV (meanGSD)
ExhX-1 (meanGSD)
0 3 10 30
22G4 64.7G5* 100.7G9* 116G6**
21.3G6.1 66.7G6.1* 96.7G4.2* 120.7G4.6**
20.7G1.2 44.7G5 52.7G7 49.3G7
*Sample-dependent t-test, P!0.05, when compared with the value of ExhX-1. **Sample-dependent t-test, P!0.001, when compared with the value of ExhX-1.
concentration of 30 mg/ml, the MN frequency in EM9 or EXV cells was at least 2-fold greater than the frequency in ExhX-1 cells. We next examined whether or not the polymorphisms affected the induction of DNA damage or the ability of the human XRCC1 protein to repair damaged DNA in vitro. Cells were exposed to 10 mg/ml bleomycin for 48 h at 37 8C. No significant difference in the MN frequency was observed among ExhX-1, Exm6X-1 and Exm9X-1 cells (Fig. 3). In contrast, Exm10X-1 cells had a 1.53 times higher MN frequency than that of ExhX-1 cells (P!0.05, sample-dependent t-test).
T. Qu et al. / Cancer Letters 221 (2005) 91–95
in cancer cells, resulting in the present differences in the MN frequencies in the different DNA polymorphisms of repair abilities. At present study, we have provided an in vitro model for further studying relationships between genetic polymorphisms and their effects on DNA repair.
Acknowledgements We thank Dr Xiangchun Wang, Department of Biochemistry, Osaka University, Japan, for technical assistance.
References [1] L.H. Thompson, K.W. Brookman, N.J. Jones, S.A. Allen, A.V. Carrano, Molecular cloning of the human XRCC1 gene, which corrects defective DNA strand break repair and sister chromatid exchange, Mol. Cell. Biol. 10 (1990) 6160–6171. [2] L.H. Thompson, M.G. West, XRCC1 keeps DNA from getting stranded, Mutat. Res. 459 (2000) 1–18. [3] K.W. Caldecott, C.K. Mckeown, J.D. Tucker, S. Ljungquist, L.H. Thompson, An interaction between the mammalian DNA repair protein XRCC1 and DNA ligase III, Mol. Cell. Biol. 14 (1994) 68–76. [4] K.W. Caldecott, S. Aoufouchi, P. Johnson, S. Shall, XRCC1 polypeptide interacts with DNA polymerase b and possibly poly(ADP-ribose) polymerase, and DNA ligase III is a novel molecular ‘nick-sensor’ in vitro, Nucleic Acids Res. 24 (1996) 4387–4394. [5] M. Masson, C. Niedergang, V. Schreiber, S. Muller, J.M. Murcia, G. Murcia, XRCC1 is specifically associated with poly (ADP-ribose) polymerase and negatively regulate its activity following DNA damage, Mol. Cell. Biol. 18 (1998) 3563–3571. [6] S. Chen, D. Tang, K. Xue, L. Xu, G. Ma, Y. Hsu, S.S. Cho, DNA repair gene XRCC1 and XPD polymorphism and risk of lung cancer in a Chinese population, Carcinogenesis 23 (2002) 1321–1325. [7] T.R. Smith, M.S. Miller, K. Lohman, E.M. Lange, L.D. Case, H.W. Mohrenweiser, J.J. Hu, Polymorphisms of XRCC1 and XRCC3 genes and susceptibility to breast cancer, Cancer Lett. 190 (2003) 183–190. [8] E.M. Sturgis, E.J. Castillo, R. Zhen, S.A. Eicher, Polymorphisms of DNA repair gene XRCC1 in squamous cell carcinoma of the head and neck, Carcinogenesis 20 (1999) 2125–2129. [9] W. Zhou, G. Liu, D.P. Miller, S.W. Thurston, L.L. Xu, J.C. Wain, et al., Polymorphisms in the DNA repair genes XRCC1 and ERCC2, smoking, and lung cancer risk, Cancer Epidemiol. Biomarkers Prev. 12 (2003) 359–369.
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[10] J.J. Hu, T.R. Smith, M.S. Miller, K. Lohman, L.D. Case, Genetic regulation of ionizing radiation sensitivity and breast cancer risk, Environ. Mol. Mutagen. 39 (2002) 208–215. [11] M.C. Stern, D.M. Umbach, R.M. Lunn, J.A. Taylor, DNA repair gene XRCC3 codon 241 polymorphism, its interaction with smoking and XRCC1 polymorphisms, and bladder cancer risk, Cancer Epidemiol. Biomarkers Prev. 11 (2002) 939–943. [12] D. Xing, J. Qi, X. Miao, W. Lu, W. Tan, D. Lin, Polymorphisms of DNA repair genes XRCC1 and XPD and their association with risk of esophageal squamous cell carcinoma in a Chinese population, Int. J. Cancer 100 (2001) 600–605. [13] D. Ratnasinghe, S. Yao, J.A. Tangrea, Y.L. Qiao, M.R. Andersen, Polymorphisms of the DNA repair gene XRCC1 and lung cancer risk, Cancer Epidemiol. Biomarkers Prev. 10 (2001) 119–123. [14] N. Moullan, D.G. Cox, S. Angele, P. Romestaining, J.P. Gerard, J. Hall, Polymorphisms in the DNA repair gene XRCC1, breast cancer risk, and response to radiotherapy, Cancer Epidemiol. Biomarkers Prev. 12 (2003) 1168–1174. [15] S.Z. Abdel-Rahman, R.A. El-Zein, The 399Gln polymorphism in the DNA repair gene XRCC1 modulates the genotoxic response induced in human lymphocytes by the tobaccospecific nitrosamine NNK, Cancer Lett. 159 (2000) 63–71. [16] H.H. Nelson, K.T. Kelsey, L.A. Mott, M.R. Karages, The XRCC1 Arg399Gln polymorphism, sunburn, and non-melanoma skin cancer: evidence of gene–environment interaction, Cancer Res. 62 (2002) 152–155. [17] R.R. Misra, D. Ratnasinghe, J.A. Tangrea, J. Virtamo, M.R. Andersen, M. Barrett, et al., Polymorphisms in the DNA repair genes XPD, XRCC1, XRCC3, and APE/ref-1, and the risk of lung cancer among male smokers in Finland, Cancer Lett. 191 (2003) 171–178. [18] L.H. Thompson, L.L. Bachinski, R.L. Stallings, G. Dolf, C.A. Weber, A. Westerveld, M.J. Sicilliano, Complementation of repair gene mutation on the homozygous chromosome 9 in CHO: a third repair gene on human chromosome 19, Genomics 5 (1989) 670–679. [19] A. Kuzminov, Single-strand interruptions in replicating chromosomes cause double-strand breaks, Proc. Natl Acad. Sci. USA 98 (2001) 8241–8246. [20] L.F. Povirk, Y.H. Han, R.J. Steighner, Structure of bleomycininduced DNA double-strand breaks: predominance of blunt ends and single-base 5 0 extensions, Biochemistry 28 (1989) 5808–5814. [21] E.B. Cullinan, L.S. Gawron, Y.M. Rustum, T.A. Beerman, Extrachromosomal chromatin: novel target for bleomycin cleavage in cells and solid tumors, Biochemistry 30 (1991) 3055–3061. [22] M. Frenech, A.A. Morley, Measurement of micronuclei in lymphocytes, Mutat. Res. 147 (1985) 29–36. [23] W.U. Muller, C. Streffer, Biological indicators for radiation damage, Int. J. Radiat. Biol. 59 (1991) 863–873. [24] A. Matsuoka, N. Yamazaki, T. Suzuki, M. Hayashi, T. Sofuni, Evaluation of the micronucleus test using a Chinese hamster cell line as an alternative to the conventional in vitro chromosomal aberration test, Mutat. Res. 272 (1992) 223–236.
Micronuclei in EM9 cells expressing polymorphic forms of human XRCC1 Tianli Qua, Eiichi Moriib, Keisuke Obokib, Yuquan Lua, Kanehisa Morimotoa,* a
Department of Social and Environmental Medicine, Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Osaka 565-0871, Japan b Department of Pathology, Osaka University Medical School, 2-2 Yamada-oka, Osaka 565-0871, Japan Received 20 May 2004; received in revised form 27 July 2004; accepted 9 August 2004
Abstract X-ray repair cross-complementing gene 1 (XRCC1) is involved in base excision repair (BER) through interaction with other BER enzymes, and polymorphisms in XRCC1 appear to increase the risk of various cancers. We evaluated how three XRCC1 polymorphisms, Arg194Trp, Arg280His and Arg399Gln, affect the extent of DNA damage and repair using the micronucleus assay. XRCC1 cDNAs containing the wild-type sequence and the three polymorphisms were overexpressed in EM9 cells, which lack the full sequence needed to perform XRCC1 functions. Normal human XRCC1 cDNA corrected the defect in EM9 cells. Only XRCC1 cDNA containing the Arg399Gln polymorphism did not fully correct the DNA repair defect in EM9 cells. These results indicate that the Arg399Gln polymorphism, but not the Arg194Trp or Arg280His polymorphism, influences the ability of XRCC1 to repair DNA. This study may provide a model that can be used to evaluate the functional significance of polymorphisms in DNA repair genes. q 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: XRCC1; EM9 cells; Polymorphism; Cancer susceptibility; DNA repair; Chromosome damage
1. Introduction Among more than 130 human DNA repair genes that mitigate the DNA damage effects of the mutatcarcinogene, X-ray repair cross-complementing gene 1 (XRCC1) was the first mammalian gene shown to play a role in the cellular sensitivity to ionizing radiation (IR) [1]. The human XRCC1 gene is 33 kb in length, and is located on human chromosome * Corresponding author. Tel.: C81 6 6879 3920; fax: C81 6 6879 3923. E-mail address: [email protected] (K. Morimoto).
19q13.2-13.3 [2]. It encodes a 2.2 kb transcript, which corresponds to a putative protein of 633 amino acids (69.5 kDa). The XRCC1 protein forms repair complexes with DNA ligase III [3], poly(ADP-ribose) polymerase (PARP) [4,5], and DNA polymerase b (polyb) [5]. In orchestrating base excision repair (BER), the ternary DNA-XRCC1-polyb complex has been suggested to act as ‘a scaffolding protein’. Three XRCC1 polymorphisms have been reported: Arg194Trp, Arg280His, and Arg399His. Codon 194 and codon 280 reside between the binding domain of polyb and PARP. Codon 399 is located in the vicinity of the PARP binding domain. Studies of Arg194Trp indicate
0304-3835/$ - see front matter q 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2004.08.013
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that this polymorphism may contribute to an increased risk of lung [6], breast [7] and head and neck cancer [8]. The Arg399Gln polymorphism has been linked to an increased risk of lung [9], breast [10], head and neck [8], bladder [11], esophageal [12] cancer. The Arg280His polymorphism has been reported as a lung cancer risk and breast cancer risk [13,14]. Three polymorphic genotype carriers all showed 1.5 odds ratio compared with wildtype genotype carrier for cancer risk. In addition, a gene– environment interaction of XRCC1 polymorphism has been discussed, particularly with regards to lifestyle factors (tobacco smoking and alcohol consumption) combining with the XRCC1 polymorphism to create a higher risk of cancer [15,16]. Some studies even have found no association between XRCC1 polymorphisms and cancer risk [17]. Additional studies will be necessary to establish to what extent XRCC1 polymorphisms are associated with increased cancer risk. EM9 cells, which have a truncated polypeptide lacking two-thirds of the normal hamster XRCC1 sequence, have been used as XRCC1-knockout cells. The frequency of spontaneous sister chromatid exchange (SCE) is 10-fold higher in EM9 cells compared to the parent cell line AA8. This sensitivity is the result of a deficiency in rejoining a DNA singlestrand breaks (SSBs), and is also manifest in sensitivity to SCE induction by agents that induce SSBs, such as alkylating agents (i.e. methyl methanesulfonate and ethyl methanesulfonate) and IR [18]. XRCC1 is thought to be involved in SSB repair. The defective XRCC1 gene function will result in the SSB accumulation or chromosomal DNA instability, which in the following cell cycle, would possibly increase the frequency of micronucleus (MN) because some portions of incompletely repaired of SSB could be possibly converted into double-strand break (DSB) during chromosome replication [19]. We used cytochalasin-B block MN assay to measure DNA damage in this study. This technique is rapid and convenient, permitting the measurement of damage by a wide variety of clastogens. We used bleomycin to induce DNA damage in vitro. Bleomycin is a radiomimetic anticancer drug that produces SSB and DSB in a catalytic way. On average, each bleomycin molecule generates 8–10 DNA breaks (SSB and DSB) [20]; it is admitted that for every six SSB, one DSB is created [21]. The effects of the XRCC1 polymorphisms (Arg194Trp, Arg280His and Arg399Gln) on the
DNA repair function were evaluated. In epidemiological studies, we cannot clearly define which XRCC1 polymorphisms are associated with increased cancer risk. In vitro evidence would be useful for supporting the plausibility of any associations.
2. Materials and methods EM9 and AA8 cells were obtained from the ATCC (Manassas, Virginia, USA). Bleomycin was purchased from Nihonkagaku (Tokyo, Japan), while Cytochalasin B was from Sigma. Blood was obtained from a healthy donor. XRCC1 cDNA was amplified by RT-PCR, and cloned into pcDNA3.1 (Invitrogen). The XRCC1 polymorphisms were constructed by inducing point mutations into the cDNA, then cloning the cDNAs into pcDNA 3.1. The sequences were verified with a DNA sequencer (Model 3100, Applied Biosystems, Forster City, CA). pcDNA3.1 plasmids containing wild-type XRCC1 cDNA and the XRCC1 cDNAs containing each of the three polymorphisms were transfected into EM9 cells using Lipofectimine2000 reagent (Invitrogen) according to the procedures described by the manufacturer. Clones expressing XRCC1 cDNA were selected with Zeocin (Invitrogen) and CldUrd (Sigma). The adherent cells were harvested, and total RNA was isolated using Trizol reagent (Invitrogen) according to the manufacturer’s instructions and quantified by optical density. One microgram of total RNA was added to a reverse transcriptase (RT) reaction in RT buffer, 0.5 mg oligo d (T) primer, 200 units SuperScript II RT and Rnase H. One microliter of cDNA from the RT was added directly to a 30 ml PCR buffer containing 20 mM Tris–HCl (pH 8.4), 50 mM KCl, 25 mM MgCl2, 10 mM dNTP, 0.25 unit Taq DNA polymerase. Oligonucleotide primers were designed as follows: 5 0 -CAC CTC ATC TGT GCC TTT GC-3 0 and 5 0 -ACC CTC CTC AGC TCA TCC TC-3 0 , a total of 410 bp. Cells were lysed with buffer containing 20 mM HEPEs (pH 7.9), 0.4 M NaCl, 25% glycerol, 1 mM EDTA, 10% protease inhibitor cocktail (Sigma) and 2.5 mM DTT. The cell lysates were frozen and thawed, then centrifuged. The supernatant was subjected to electrophoresis and the separated protein transferred to a nylon membrane. The membranes were blocked and incubated with anti-XRCC1, clone 33-2-5 (Kamiya,
T. Qu et al. / Cancer Letters 221 (2005) 91–95
Fig. 1. XRCC1 gene expression in tranfected cells. (A) RT-PCR of expressed XRCC1 protein. (B) Image density as measured by software (NIH Image 1.62). Densitometry of XRCC1 bands were normalized to the AA8 value for each sample. Relative densitometry was shown for the RT-PCR data in panel A.
Seattle, WA) at 4 8C overnight. The signal specific to the XRCC1 protein was detected after exposure to X-ray film. Cells of 5!105 were plated in six-well plates containing growth mediums supplemented with 3 mg/ml cytochalasin B. Bleomycin was added at concentrations of 0, 3, 10 and 30 mg/ml. The cells were cultured in the dark at 37 8C for 48 h. The cytokinesis block MN assay was based on the technique of French and Morley [22]. The bleomycin treated cell pellets were treated with 0.075 M KCl at room temperature for 15 min. After that, the cells were fixed in fresh Carnoy’s solution (methanol– acetic acid, 3:1) at 4 8C for 30 min, washed with Carnoy’s solution three times. The fixed cells were resuspended in the 1% acetic acid by gentle pipetting and spread onto pre-cleaned 15-mm frosted microscope slides (Matsunami). After drying, the slides were stained with 3% Giemsa (Merck, pH 6.8). The number of binucleated cell per 1000 cells was 250– 300. Micronuclei were counted in 500 binucleated cells under a microscope. The results were expressed as the meanGSD of triplicate determinations from independent experiments. Sample-dependent t-test was used for multiple comparisons.
3. Results We constructed expression vectors encoding the wild-type human XRCC1 gene and polymorphic
93
human XRCC1 gene, and introduced it into EM9 cells to isolate stable transformants (ExhX-1). We isolated EM9 cells overexpressing XRCC1 cDNAs containing three polymorphisms: Arg194Trp, Arg280His and Arg399Gln; these cell lines were designated Exm6X-1, Exm9X-1 and Exm10X-1, respectively. We also introduced an empty pcDNA3.1 vector into EM9 cells to isolate stable transformants (EXV) and provide a negative control. We examined the XRCC1 gene expression levels of every cell lines by RT-PCR. The XRCC1 gene was overexpressed in ExhX-1 cells, its amount being approximately 1.7 times that in AA8 (Fig. 1A and B). No XRCC1 band was found in EM9 cells or the negative control EXV cells (Fig. 1A). The quality of XRCC1 in ExhX-1, Exm6X-1, Exm9X-1 and Exm10X-1 cells is comparable (Fig. 1A and B). To further confirm the XRCC1 protein quantity of the cell lines, we carried out Western blot. The XRCC1 gene was overexpressed in ExhX-1 cells, its amount being approximately 2 times that in AA8 (Fig. 2A and B). No XRCC1 band was found in EM9 cells or the negative control EXV cells (Fig. 1A). The quality of XRCC1 in ExhX-1, Exm6X-1, Exm9X-1 and Exm10X-1 cells is comparable (Fig. 2A and B). To assess chromosomal DNA damage (and, indirectly, repair) in ExhX-1 and EM9 cells, we carried out the cytochalasin B-cytokinesis block MN assay. Table 1 shows the MN frequency in cell lines exposed to increasing doses of bleomycin. At a bleomycin
Fig. 2. Western blotting of expressed XRCC1 protein. (A) Western blot of expressed XRCC1 protein. (B) Image density as measured by software (NIH Image 1.62). Densitometry of XRCC1 bands were normalized to the AA8 value for each sample. Relative densitometry was shown for the Western blot data in panel A.
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Table 1 Micronucleus frequency per 1000 cells analyzed on 500 binucleate cells treated with bleomycin
4. Discussion
study, we treated the cell lines with oxidative agent; ExhX-1 cells overexpressing XRCC1 protein were relatively resistant to this agent. It is possible that the overexpression of XRCC1 protein in EM9 cells confers resistance to other exogenous chemical agents and IR. The XRCC1 protein, however, has no enzymatic activity. During BER and SSB repair, the XRCC1 protein interacts with DNA polyb, PARP and DNA ligase III [2]. The XRCC1 protein may also up-regulate the activity of many other DNA repair enzymes. XRCC1 polymorphisms appear to be associated with the risk of acquiring various types of cancer. Three polymorphisms genotype all showed 1.5 odds ratio compared with wild-type genotype for cancer risks. Epidemiology studies alone cannot determine whether polymorphisms like Arg194Gln, Arg280His or Arg399Gln have any functional significance in cancer risk. Data from other studies found no association between Arg399Gln and cancer risk [17]. In this study, we examined the ability of XRCC1 polymorphisms to repair DNA in vitro. Our data suggests that the Arg399Gln polymorphism affects DNA repair. Arg399Gln is located in the BRCT1 domain, which is necessary for accumulation of the protein at SSB via interaction with PARP. This maybe one reason why this polymorphism is most frequently found among the XRCC1 polymorphisms
Micronuclei induced in response to DNA damage may be formed from acentric chromosomal fragments or whole chromosomes that remain separate to the main nucleus after nuclear division. The number of micronuclei within a cell can therefore be indicative of the extent of chromosomal insult [22–24]. The MN assay is not a direct measure of DNA repair, but the magnitude of the responses might be influenced by DNA repair. We initially examined the human XRCC1 gene function in the following cell lines: ExhX-1 cells (EM9 cells overexpressing wild-type XRCC1), EXV cells (an empty vector control for EM9 cells), repairdefective EM9 cells by using the MN assay. The data indicated that bleomycin produced fewer micronuclei in ExhX-1 cells than in EM9 cells, indicating that XRCC1 expression resulted in a higher degree of genetic stability. In the present
Fig. 3. Effect of XRCC1 polymorphisms on micronucleus (MN) induction by bleomycin. Polymorphisms in exon 6 (Arg194Trp), exon 9 (Arg280His) and exon 10 (Arg399Gln) were evaluated. The micronuclei number of 500 binucleated cells per sample was scored. *P!0.001, sample-dependent t-test.
Bleomycin (mg/ml)
EM9 (meanGSD)
EXV (meanGSD)
ExhX-1 (meanGSD)
0 3 10 30
22G4 64.7G5* 100.7G9* 116G6**
21.3G6.1 66.7G6.1* 96.7G4.2* 120.7G4.6**
20.7G1.2 44.7G5 52.7G7 49.3G7
*Sample-dependent t-test, P!0.05, when compared with the value of ExhX-1. **Sample-dependent t-test, P!0.001, when compared with the value of ExhX-1.
concentration of 30 mg/ml, the MN frequency in EM9 or EXV cells was at least 2-fold greater than the frequency in ExhX-1 cells. We next examined whether or not the polymorphisms affected the induction of DNA damage or the ability of the human XRCC1 protein to repair damaged DNA in vitro. Cells were exposed to 10 mg/ml bleomycin for 48 h at 37 8C. No significant difference in the MN frequency was observed among ExhX-1, Exm6X-1 and Exm9X-1 cells (Fig. 3). In contrast, Exm10X-1 cells had a 1.53 times higher MN frequency than that of ExhX-1 cells (P!0.05, sample-dependent t-test).
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in cancer cells, resulting in the present differences in the MN frequencies in the different DNA polymorphisms of repair abilities. At present study, we have provided an in vitro model for further studying relationships between genetic polymorphisms and their effects on DNA repair.
Acknowledgements We thank Dr Xiangchun Wang, Department of Biochemistry, Osaka University, Japan, for technical assistance.
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