The Role of APEX/Ref-1 (Apurinic/Apyrimidic Endonuclease DNA-Repair Gene) in Renal Cell

The Role of APEX1/Ref-1 (apurinic/apyrimidic endonuclease DNA-repair gene)

in Renal Cell Carcinoma

Wen-Shin Chang1,2,3,*, Chia-Wen Tsai2,3,*, Kuang-Hao Cheng3,*, Chi-Shun Lien3,4, Chi-Ping

Huang4, Wen-Ling Liao3, Meng-Hsuan Lee1,3, Hsi-Chin Wu4, Chao-Hsiang Chang4, Cheng-

Chieh Lin2,3, and Da-Tian Bau1,2,3

1Graduate Institutes of Clinical Medical Science, 2Basic Medical Science, China Medical

University, Taichung, Taiwan.

3Terry Fox Cancer Research Laboratory, China Medical University Hospital, Taichung,

Taiwan.

4Department of Urology, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan,

Republic of China..

Running head: APEX1 IN RENAL CELL CARCINOMA

* These authors contributed equally to this work

*Correspondence to: Da-Tian Bau, Terry Fox Cancer Research Lab, China Medical

University Hospital, 2 Yuh-Der Road, Taichung, 404 Taiwan, Tel: +886 422052121 Ext

1523, Fax: +886 422052121 Ext 1543, E-mail: [email protected]/ [email protected]

1 Abstract

The altered DNA repair capacity plays a key role in genomic stability and renal cancer susceptibility. We aim at evaluation the contribution of the polymorphic variant in apurinic/apyriminidic endonuclease (APEX1) gene to its mRNA level and risk of renal cell carcinoma (RCC). In the hospital-based case-control study, 92 RCC patients and 580 cancer-free controls frequency matched by age and sex were recruited.

Genotyping were performed for APEX1 Asp148Glu (rs1130409) by restriction fragment length polymorphism PCR. At the same time, thirty renal tissue samples with different genotypes were tested to estimate the APEX1 mRNA expression by real-time quantitative reverse transcription method. Compared with the wild-type TT genotype, the TG and GG genotypes of APEX1 Asp148Glu had a risk of RCC of 0.84- and 1.16-fold. We have also examined the in vivo transcriptional (RNA) and translational (protein) levels with renal tissues of various APEX1 Asp148Glu genotypes, revealing that the APEX1 mRNA and protein were of similar levels among people of TT, TG, or GG genotypes. There was no joint gene-environment effect of

APEX1 Asp148Glu genotype and smoking habit on RCC risk. The evidence suggested that the APEX1 Asp148Glu genotype correlates well with its mRNA and protein expression among RCC patients. The genotype of APEX1 Asp148Glu may not be a sensitive marker for prediction of RCC risk in Taiwan.

2 Key words: APEX1/Ref-1, DNA repair, polymorphism, renal cell carcinoma.

3 Introduction

Renal cell carcinoma (RCC) is the most common (> 80%) kidney malignancy, and the frequency of which is keeping increasing in both male and female worldwide

(23, 29). Taiwan is the second-highest prevalence country of end-stage renal disease in the world, and Japan is the first. Although the exact etiology of RCC has not been well identified yet, recent epidemiological investigations have showed that cigarette smoking, hypertension, obesity, occupational exposures, diet, and family history of cancer are associated with RCC (28, 29, 33). Only few exposed individuals develop

RCC in their lifetime, suggesting that genomic susceptibility may play an important role in the development of RCC.

The DNA repair ability of a cell is vital to the integrity of its genome and thus to its normal functioning and that of the organism (34), and mutations or polymorphisms in the DNA repair genes are thought to play a critical role in carcinogenesis (32, 37).

Therefore, genetic variants of DNA repair genes might also involved in the RCC initiation and development.

One of the DNA repair pathways is the DNA base excision repair (BER) pathway, which repairs the DNA damage induced by mainly oxidation and alkylation and thus protects cells against the cytotoxic effects from both endogenous and exogenous agents (12, 17). The altered bases of purine or pyrimidine are recognized

4 and excised by specific DNA glycosylases in the early repair stage, leaving some abasic sites. Then apurinic/ apyrimidinic endonucleases incise the DNA 5’ to the abasic sites; further repair proceeds to short-patch (when the gap is only one nucleotide) or long-patch (when the gap is two or more nucleotides) subpathways of

BER system (25). The major human apurinic/apyrimidinic endonucleases, APEX1

(also known as APE, APE1, APEX, HAP1, and Ref-1), plays a central role in the BER system, which initiates the repair of apurinic/apyrimidinic sites in altered DNA produced either spontaneously hydrolyzing the 5’-phosphodiester bone of the apurinic/apyrimidinic site or after enzymatic removal of damaged bases. To sum up, the repair activity of APEX1 serves to protect the cell from the apurinic/apyrimidinic sites that can accumulate in DNA via endogenous and exogenous sources. In addition to its apurinic/apyrimidinic endonuclease activity, APEX1 can also act as a 3’- phosphodiesterase, initiating repair of DNA strands breaks with 3’-blocking damage, which are produced either directly by reactive oxygen species or indirectly through the enzymatic removal of damaged bases (13, 21). Moreover, APEX1 also functions as a reduction-oxidation activators of several transcription factors that are identified to be important in carcinogenesis, such as activator protein (Fos/Jun), hypoxia-inducible factor 1, cAMP-responsive element binding protein, and p53 (11, 35). Deficiency in

APEX1 induces embryonic lethality in knockout mice showed that and highlights the

5 importance of its function to the genomic stability and cellular functions (6, 22).

It is been shown that genetic polymorphisms in DNA repair genes have conferred predisposition to many types of cancer, and Hirata et al. have investigated the association between some polymorphisms of DNA repair genes and the risk for

RCC, such as XRCC1, XPC, ERCC1, XRCC3, and XRCC7 (16). However, no study had yet confirmed the association between the polymorphisms of APEX1, which is a central gene in human BER, and the risk of RCC. In recelt year, a few epidemiological studies have investigated the association between the APEX1 polymorphism and the risk for different types of cancers, including bladder (14), lung

(26, 30, 31), and gastric cancer (15). We assumed that APEX1 polymorphism may also contribute to RCC risk. Thus, the present hospital-based matched case-control study is aiming at evaluating the relationship between polymorphisms of APEX1 and the risk for RCC in the high-prevalent Taiwan population. Among the identified single nucleotide polymorphisms in the APEX1 gene, we selected the most common polymorphism in literature, APEX1 Asp148Glu (rs1130409), to investigate the association of the APEX1 genetic polymorphism with RCC risk. Also, we have examined the mRNA expression levels of various APEX1 genotypes in vivo by reversed transcript PCR. To the best of our knowledge, this is the first study to evaluate the association between the APEX1 Asp148Glu polymorphism and RCC

6 susceptibility.

Materials and Methods

Study Population

The hospital-based case-control study recruited 92 RCC patients and 580 cancer- free controls matched by age and gender frequency. All the RCC patients were diagnosed and histopathologically confirmed RCC by Drs. Wu, Chen, Lien and

Chang, without any prior history of other types of cancer. All the age- and gender- matched cancer-free controls were genetically unrelated to the RCC patients and had no individual history of cancer. Extra exclusion criteria of the controls were that if they had symptoms suggestive of RCC, such as hematuria. Each patient donated 3 to

5 ml venous blood after providing a written informed consent. The study was approved by the institutional review board of China Medical University.

Genotyping Protocol

The total genomic DNA of each subject was extracted from the leucocytes of peripheral blood using a QIAamp Blood Mini Kit (Blossom, Taipei, Taiwan) and stored as previously published (2-5, 7-10, 27, 36, 38-40). The primers used for

APEX1 Asp148Glu (rs1130409) were: forward 5’-CCAGCTGAACTTCAGGAGCT-3’, and reverse 5’-CTCGGCCTGCATTAGGTACA-3’. The following cycling conditions

7 were performed: one cycle at 94oC for 5 min; 35 cycles of 94oC for 30 s, 55oC for 30 s, and 72oC for 30 s; and a final extension at 72oC for 10 min. The PCR resultant 350 bp PCR product was mixed with 2 U MnlI. The G form PCR products could be further digested while the T form could not. Two fragments 252 and 98 bp were present if the product was digestible C form. The reaction was incubated for 2 h at 37 oC. Then, 10 l of product was loaded into a 3% agarose gel containing ethidium bromide for electrophoresis.

mRNA APEX1 Expression Pattern

To evaluate the correlation between the APEX1 mRNA expression and APEX1 genotype, 30 surgically removed renal tissue samples adjacent to tumors with different genotypes were subjected to extraction of the total RNA using Trizol

Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol.

The total RNA was measured by real-time quantitative RT-PCR using FTC-3000 real- time quantitative PCR instrument series (Funglyn Biotech Inc., Canada). GAPDH was used as an internal quantitative control. The primers used for amplification of APEX1 mRNA were 5’-GCCCACTCAAAGTTTCTTAC-3’ as forward one and 5’-

TGTGCCACATTGAGGTCTCC-3’ as reverse one, and the primers for GAPDH were

5’-GAAATCCCATCACCATCTTCCAGG-3’ as forward one and 5’-

8 GAGCCCCAGCCTTCTCCATG-3’ as reverse one. Fold changes were normalized by the levels of GAPDH expression, and each assay was done in at least triplicate.

Western Blotting Analysis

The specimens were homogenized in RIPA lysis buffer (Upstate Inc., Lake

Placid, NY, USA), the homogenates were then centrifuged at 10,000 g for 30 min at

4°C, and the supernatants were used for Western blotting. Samples were denatured by heating at 95°C for 10 min, then separated on a 10% SDS-PAGE gel, and transferred to a nitrocellulose membrane (Bio-Rad). The membrane was blocked with 5% non-fat milk and incubated overnight at 4°C with anti-APEX1 antibody (1:1000; Santa Cruz

Biotechnology), then with the corresponding horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody (Chemicon, Temecula, CA) for 1 h at room temperature. After reaction with ECL solution (Amersham, Arlington Heights, IL,

USA), a bound antibody was visualized using a chemiluminescence imaging system

(Syngene, Cambridge, UK). Finally, the blots were incubated at 56°C for 18 min in stripping buffer (0.0626 M Tris-HCl, pH 6.7, 2% SDS, 0.1 M mercaptoethanol) and re-probed with a monoclonal mouse anti-β-actin antibody (Sigma) as the loading control. The optical density of each specific band was measured using a computer- assisted imaging analysis system (Gene Tools Match software; Syngene).

9 Statistical Analyses

To ensure that the controls used were representative of the general population and to exclude the possibility of genotyping error, the deviation of the genotype frequencies of APEX1 single nucleotide polymorphisms in the control subjects from those expected under the Hardy-Weinberg equilibrium was assessed using the goodness-of-fit test. Pearson’s Chi-square test or Fisher’s exact test (when the expected number in any cell was less than five) was used to compare the distribution of the APEX1 genotypes between cases and controls. The associations between the

APEX1 polymorphisms and RCC risk were estimated by computing odds ratios (ORs) and their 95% confidence intervals (CIs) from unconditional logistic regression analysis with the adjustment for possible confounders. The expression levels of mRNA were examined by unpaired Student’s t-test. Those P < 0.05 were considered statistically significant, and all statistical tests were two-sided.

Results

Basic Comparisons between the Case and Control Groups

The characteristics of the control and case subjects are summarized and compared in Table I. No differences between the case and control group was found on

10 age, gender, smoking or alcohol drinking status, diabetes or family history of cancer

(P>0.05). However, there were more hypertension syndrome in RCC cases than in the controls (66.3% versus 52.1%), and the significance (P = 0.0130) suggested that hypertension may be one of the risky factor for RCC.

Association of APEX1 Genotypes with RCC Risk

The genotypic distributions of the APEX1 Asp148Glu polymorphism in RCC cases and controls are presented in Table II. The ORs after adjusting the confounding factors (age, gender, smoking and alcohol drinking status) for the people carrying TG and GG genotypes were 0.84 (95% CI = 0.52-1.38) and 1.16 (95% CI = 0.58-2.29) respectively, compared to those carrying TT wild-type genotype. The P for trend was not significant (P = 0.5555). In the dominant (TG plus GG versus TT) or recessive

(GG versus TT plus TG) models, the association between APEX1 Asp148Glu polymorphism with the risk for RCC was not statistically significant either. (Table II).

To sum up, these data indicated that individuals carrying variant G allele at APEX1

Asp148Glu may not have a higher risk of RCC.

Association of the APEX1 Asp148Glu Genotype with the Expression Levels of the

APEX1 mRNA and Protein

We have collected 30 surgically removed normal renal tissue samples adjacent to

11 tumors, which were obtained from the RCC patients before any therapy. The frequencies of the TT, TG, and GG genotypes of the APEX1 Asp148Glu were 13, 12, and 5, respectively. The effects of these three genotypes on the transcriptional expression of mRNA levels and translational expression of protein levels were measured and evaluated by real-time quantitative RT-PCR and Western blotting assay, respectively (Fig. 1). There was no statistically difference in their mRNA or protein levels found among samples from the three genotypes (Fig. 1).

12 Discussion In this study, the association of APEX1 genotype and RCC risk was worldwide firstly investigated in Taiwan, where the prevalence of end-stage renal disease was second-highest all over the world (only after Japan). The APEX1 Asp148Glu variants are the most common APE1 polymorphisms that result in single amino acid substitutions identified in the general population (19). In previous literature, the association of APEX1 Asp148Glu genotype in other types of cancer is very few and controversial. The reports of APEX1 Asp148Glu genotype in lung cancer is most common, but the findings are conflicting (18, 20, 24). But there is no report investigating its association with RCC.

In the genotyping results, we found that individuals carrying TG or GG genotypes were not of higher risk of RCC compared with those carrying TT genotype on APEX1 Asp148Glu polymorphic site (Table I). We have further investigated the correlation of APEX1 Asp148Glu genotype on its transcriptional (mRNA) and translational (protein) expression levels. The results showed that the renal tissues from people of TT, TG or GG APEX1 Asp148Glu genotypes were of similar level of APEX1 mRNA and protein levels (Fig. 1). All the data collected from DNA, RNA and protein levels may tell us the following stories. First, APEX1 indeed plays such a critical role in BER that the subtle polymorphism on APEX1 Asp148Glu, which cause the amino- acid substitution variants and may lead to a decrease in the overall DNA repair

13 capacity, was not found among RCC patients. Of course, further works aiming at revealing other potential polymorphic sites of APEX1 gene, such as the promoter

-141T/G (rs1760944), is urgently warranted (26). Second, as mentioned, we have provided evidence that the genetic variants of APEX1 Asp148Glu may not cause a significant difference at the consequent RNA or protein levels (Fig. 1), instead, the functional difference of its enzymatic activity may be found among the groups of different genotypes.

The APEX1 gene consists of five exons and four introns with a 2.21-kb span. It is located at chromosome 14q11.2-q12 and encodes a 317 amino acid protein. It is the essential enzyme in the BER pathway, which is the primary mechanism for the repair of endogenous DNA damage resulting from cellular metabolisms including those resulting from reactive oxygen species, methylation, deamination, and hydroxylation

(17). In addition to its role in DNA repair, APEX1 is also known as a transcriptional coactivator for numerous transcription factors such as AP-1, NF-B, Myb, HIF-1,

HLF, PAX, and p53 that are involved in cancer promotion and progression by regulating the expression of their target downstream genes (1).

We have also evaluated the joint effect of the polymorphism of APEX1 genes stratified by smoking status. We found that APEX1 Asp148Glu genotype was not associated with a higher risk of RCC (Table III), suggesting that APEX1 Asp148Glu

14 genotype only had a minor effect on adding the risk of smokers or non-smokers to

RCC risk.

The present study has some limitations to be improved in the future. First, our sample size is moderate, which may restrict the reliability and feasibility of stratification and interaction analyses. Second, the insufficient clinical and behavioral information, such as occupational exposure, daily diet and physical exercise habits, limited our capacity of performing risky factor analysis. Last, the small sample size of both mRNA and Western blot analysis, especially those tissues from people with the

GG genotype on APEX1 Asp148Glu, should be further validated in both tumor tissues and normal adjacent tissues in our future studies.

Although in this study we did not find any significant association of APEX1 genotype, which is very critical in BER system, we could not ignore the contribution of APEX1 in the etiology of RCC. It is possibly that some other polymorphic site, such as the promoter -141T/G (rs1760944) may related to an alteration in the expression level of mRNA, protein, and consequently its function, which lead to an increased risk of RCC. Also, the personal habits of smoking and alcohol drinking, which may cause lots of oxidative damage related to BER, should be adaptively prohibited by all of the subjects in our society. Only by doing so, the predictive, preventive, personalized and participatory medicine and therapy is actionable and

15 feasible.

In conclusion, the present study has provided evidence at DNA, RNA and protein levels of central dogma of molecular biology, evaluating the contribution of functional

APEX1 Asp148Glu polymorphism to the RCC in Taiwan. The strategy and system is very promising and further functional studies are warranted to reveal the role of DNA repair genes, such as APEX1, in RCC carcinogenesis.

Acknowledgements

This study was supported by research grants from Terry Fox Cancer Research

Foundation and National Science Council (NSC-101-2320-B-039-045). The assistance from Ping-Fang Wang in clinical sample and data collection, and that from

Fang-Jing Li, Huang-Ting Chiang, Yi-Ting Chang, Hong-Xue Ji in genotyping, RT-

PCR and Western blot were highly appreciated by the authors.

Figure legend

Fig. 1 Analysis of APEX1 mRNA and protein expression levels. A, Quantitative RT-

PCR for APEX1 from the renal tissue samples of TT, TG and GG genotypes was performed and GAPDH was used as an internal quantitative control. Fold changes were normalized by the levels of GAPDH expression, and each assay was done in at least triplicate. B, The Western blotting analysis was performed from the renal tissue

16 samples of TT, TG and GG genotypes and quantitatied for comparison. -tubulin was used as an internal standard.

References

1. Ando, K., Hirao, S., Kabe, Y., Ogura, Y., Sato, I., Yamaguchi, Y., Wada, T. and

Handa, H. A new APE1/Ref-1-dependent pathway leading to reduction of NF-

kappaB and AP-1, and activation of their DNA-binding activity. Nucleic Acids

Res. 36:4327-4336, 2008.

2. Bau, D.T., Chang, C.H., Tsai, R.Y., Wang, H.C., Wang, R.F., Tsai, C.W., Yao,

C.H., Chen, Y.S., Shyue, S.K. and Huang, C.Y. Significant association of

caveolin-1 genotypes with bladder cancer susceptibility in Taiwan. Chin. J.

Physiol. 54:153-160, 2011.

3. Bau, D.T., Tsai, M.H., Huang, C.Y., Lee, C.C., Tseng, H.C., Lo, Y.L., Tsai, Y.

and Tsai, F.J. Relationship between polymorphisms of nucleotide excision

repair genes and oral cancer risk in Taiwan: evidence for modification of

smoking habit. Chin. J. Physiol. 50:294-300, 2007.

4. Bau, D.T., Tsai, M.H., Lo, Y.L., Hsu, C.M., Tsai, Y., Lee, C.C. and Tsai, F.J.

Association of p53 and p21(CDKN1A/WAF1/CIP1) polymorphisms with oral

cancer in Taiwan patients. Anticancer Res. 27:1559-1564, 2007.

5. Bau, D.T., Wang, H.C., Liu, C.S., Chang, C.L., Chiang, S.Y., Wang, R.F., Tsai,

17 C.W., Lo, Y.L., Hsiung, C.A., Lin, C.C. and Huang, C.Y. Single-nucleotide

polymorphism of the Exo1 gene: association with gastric cancer susceptibility

and interaction with smoking in Taiwan. Chin. J. Physiol. 52:411-418, 2009.

6. Burdak-Rothkamm, S., Rube, C.E., Nguyen, T.P., Ludwig, D., Feldmann, K.,

Wiegel, T. and Rube, C. Radiosensitivity of tumor cell lines after pretreatment

with the EGFR tyrosine kinase inhibitor ZD1839 (Iressa). Strahlenther. Onkol.

181:197-204, 2005.

7. Chang, C.H., Chang, C.L., Tsai, C.W., Wu, H.C., Chiu, C.F., Wang, R.F., Liu,

C.S., Lin, C.C. and Bau, D.T. Significant association of an XRCC4 single

nucleotide polymorphism with bladder cancer susceptibility in Taiwan.

Anticancer Res. 29:1777-1782, 2009.

8. Chang, C.H., Chiu, C.F., Liang, S.Y., Wu, H.C., Chang, C.L., Tsai, C.W.,

Wang, H.C., Lee, H.Z. and Bau, D.T. Significant association of Ku80 single

nucleotide polymorphisms with bladder cancer susceptibility in Taiwan.

Anticancer Res. 29:1275-1279, 2009.

9. Chang, C.H., Wu, H.C., Ke, H.L., Lin, H.H., Shen, W.C., Sun, S.S., Tsai,

C.W., Tsai, R.Y. and Bau, D.T. The genetic role of Cyclooxygenase 2 in ureter

cancer in Taiwan. Adaptive Medicine 3:99-105, 2011.

10. Hsu, C.F., Chang, C.N., Hsu, P.H., Chang, W.S., Wu, C.N., Lin, S.S., Sun,

S.S., Shih, Y.H, Tsai, C.W. and Bau, D.T. The association of Cox-2 genotypes

18 with pterygium formation in Taiwan. Adaptive Medicine 3:21-26, 2011.

11. Evans, A.R., Limp-Foster, M. and Kelley, M.R. Going APE over ref-1. Mutat.

Res. 461:83-108, 2000.

12. Fleck, O. and Nielsen, O. DNA repair. J. Cell Sci. 117:515-517, 2004.

13. Fortini, P., Pascucci, B., Parlanti, E., D'Errico, M., Simonelli, V. and Dogliotti,

E. The base excision repair: mechanisms and its relevance for cancer

susceptibility. Biochimie 85:1053-1071, 2003.

14. Gangwar, R., Ahirwar, D., Mandhani, A. and Mittal, R.D. Influence of XPD

and APE1 DNA repair gene polymorphism on bladder cancer susceptibility in

north India. Urology 73:675-680, 2009.

15. Gu, D., Wang, M., Wang, S., Zhang, Z. and Chen, J. The DNA repair gene

APE1 T1349G polymorphism and risk of gastric cancer in a Chinese

population. PLoS One 6:e28971, 2011.

16. Hirata, H., Hinoda, Y., Matsuyama, H., Tanaka, Y., Okayama, N., Suehiro, Y.,

Zhao, H., Urakami, S., Kawamoto, K., Kawakami, T., Igawa, M., Naito, K.

and Dahiya, R. Polymorphisms of DNA repair genes are associated with renal

cell carcinoma. Biochem. Biophys. Res. Commun. 342:1058-1062, 2006.

17. Hoeijmakers, J.H. Genome maintenance mechanisms for preventing cancer.

Nature 411:366-374, 2001.

18. Hung, R.J., Brennan, P., Canzian, F., Szeszenia-Dabrowska, N., Zaridze, D.,

19 Lissowska, J., Rudnai, P., Fabianova, E., Mates, D., Foretova, L., Janout, V.,

Bencko, V., Chabrier, A., Borel, S., Hall, J. and Boffetta, P. Large-scale

investigation of base excision repair genetic polymorphisms and lung cancer

risk in a multicenter study. J. Natl. Cancer Inst. 97:567-576, 2005.

19. Hung, R.J., Hall, J., Brennan, P. and Boffetta, P. Genetic polymorphisms in the

base excision repair pathway and cancer risk: a HuGE review. Am. J.

Epidemiol. 162:925-942, 2005.

20. Ito, H., Matsuo, K., Hamajima, N., Mitsudomi, T., Sugiura, T., Saito, T.,

Yasue, T., Lee, K.M., Kang, D., Yoo, K.Y., Sato, S., Ueda, R. and Tajima, K.

Gene-environment interactions between the smoking habit and polymorphisms

in the DNA repair genes, APE1 Asp148Glu and XRCC1 Arg399Gln, in

Japanese lung cancer risk. Carcinogenesis 25:1395-1401, 2004.

21. Izumi, T., Hazra, T.K., Boldogh, I., Tomkinson, A.E., Park, M.S., Ikeda, S. and

Mitra, S. Requirement for human AP endonuclease 1 for repair of 3'-blocking

damage at DNA single-strand breaks induced by reactive oxygen species.

Carcinogenesis 21:1329-1334, 2000.

22. Jayaraman, L., Murthy, K.G., Zhu, C., Curran, T., Xanthoudakis, S. and

Prives, C. Identification of redox/repair protein Ref-1 as a potent activator of

p53. Genes Dev. 11:558-570, 1997.

23. Jemal, A., Murray, T., Ward, E., Samuels, A., Tiwari, R.C., Ghafoor, A., Feuer,

20 E.J. and Thun, M.J. Cancer statistics, 2005. CA-Cancer J. Clin. 55:10-30,

24. Kiyohara, C., Takayama, K. and Nakanishi, Y. Association of genetic

polymorphisms in the base excision repair pathway with lung cancer risk: a

meta-analysis. Lung Cancer 54:267-283, 2006.

25. Krokan, H.E., Nilsen, H., Skorpen, F., Otterlei, M. and Slupphaug, G. Base

excision repair of DNA in mammalian cells. FEBS Lett. 476:73-77, 2000.

26. Li, Z., Guan, W., Li, M.X., Zhong, Z.Y., Qian, C.Y., Yang, X.Q., Liao, L., Li,

Z.P. and Wang, D. Genetic polymorphism of DNA base-excision repair genes

(APE1, OGG1 and XRCC1) and their correlation with risk of lung cancer in a

Chinese population. Arch. Med. Res. 42:226-234, 2011.

27. Lin, H.H., Ke, H.L., Hsiao, K.H., Tsai, C.W., Wu, W.J., Bau, D.T. and Chang,

L.L. Potential role of CCND1 G870A genotype as a predictor for urothelial

carcinoma susceptibility and muscle-invasiveness in Taiwan. Chin. J. Physiol.

54:196-202, 2011.

28. Lindblad, P. Epidemiology of renal cell carcinoma. Scand. J. Surg. 93:88-96,

29. Lipworth, L., Tarone, R.E. and McLaughlin, J.K. The epidemiology of renal

cell carcinoma. J. Urol. 176:2353-2358, 2006.

30. Lo, Y.L., Jou, Y.S., Hsiao, C.F., Chang, G.C., Tsai, Y.H., Su, W.C., Chen, K.Y.,

21 Chen, Y.M., Huang, M.S., Hu, C.Y., Chen, C.J. and Hsiung, C.A. A

polymorphism in the APE1 gene promoter is associated with lung cancer risk.

Cancer Epidemiol. Biomarkers Prev. 18:223-229, 2009.

31. Lu, J., Zhang, S., Chen, D., Wang, H., Wu, W., Wang, X., Lei, Y., Wang, J.,

Qian, J., Fan, W., Hu, Z., Jin, L., Shen, H., Huang, W., Wei, Q. and Lu, D.

Functional characterization of a promoter polymorphism in APE1/Ref-1 that

contributes to reduced lung cancer susceptibility. Faseb J. 23:3459-3469,

32. Miller, K.L., Karagas, M.R., Kraft, P., Hunter, D.J., Catalano, P.J., Byler, S.H.

and Nelson, H.H. XPA, haplotypes, and risk of basal and squamous cell

carcinoma. Carcinogenesis 27:1670-1675, 2006.

33. Murai, M. and Oya, M. Renal cell carcinoma: etiology, incidence and

epidemiology. Curr. Opin. Urol. 14:229-233, 2004.

34. Sugimura, T., Kumimoto, H., Tohnai, I., Fukui, T., Matsuo, K., Tsurusako, S.,

Mitsudo, K., Ueda, M., Tajima, K. and Ishizaki, K. Gene-environment

interaction involved in oral carcinogenesis: molecular epidemiological study

for metabolic and DNA repair gene polymorphisms. J. Oral Pathol. Med.

35:11-18, 2006.

35. Tell, G., Damante, G., Caldwell, D. and Kelley, M.R. The intracellular

localization of APE1/Ref-1: more than a passive phenomenon? Antioxid.

22 Redox Signal. 7:367-384, 2005.

36. Tseng, H.C., Tsai, M.H., Chiu, C.F., Wang, C.H., Chang, N.W., Huang, C.Y.,

Tsai, C.W., Liang, S.Y., Wang, C.L. and Bau, D.T. Association of XRCC4

codon 247 polymorphism with oral cancer susceptibility in Taiwan.

Anticancer Res. 28:1687-1691, 2008.

37. Vogelstein, B., Alberts, B. and Shine, K. Genetics. Please don't call it cloning!

Science 295:1237, 2002.

38. Wang, C.H., Wu, K.H., Yang, Y.L., Peng, C.T., Tsai, F.J., Lin, D.T., Chiu, C.F.,

Lin, C.C. and Bau, D.T. Association between Ataxia Telangiectasia Mutated

gene polymorphisms and childhood leukemia in Taiwan. Chin. J. Physiol.

54:413-418, 2011.

39. Wang, H.C., Liu, C.S., Wang, C.H., Tsai, R.Y., Tsai, C.W., Wang, R.F., Chang,

C.H., Chen, Y.S., Chiu, C.F., Bau, D.T. and Huang, C.Y. Significant

association of XPD Asp312Asn polymorphism with breast cancer in

Taiwanese patients. Chin. J. Physiol. 53:130-135, 2011.

40. Yang, M.D., Hsu, Y.M., Kuo, Y.S., Chen, H.S., Chang, C.L., Wu, C.N., Chang,

C.H., Liao, Y.M., Wang, H.C., Wang, M.F. and Bau, D.T. Significant

association of Ku80 single nucleotide polymorphisms with colorectal cancer

susceptibility in Central Taiwan. Anticancer Res. 29:2239-2242, 2009.

23 Table I. Distributions of selected characteristics among RCC cases and control subjects

Characteristics Cases (n = 92) Controls (n = 580) P-value N % N % Age (yr) (mean  SD) 58.8  11.7 58.3  11.5 0.8971 < 60 47 51.1% 307 52.9% 0.8223 > 60 45 48.9% 273 47.1% Gender Male 59 64.1% 371 64.0% 1.0000 Female 33 35.9% 209 36.0% Smoking status Smokers 41 44.6% 220 37.9% 0.2499 Non-smokers 51 55.4% 360 62.1% Alcohol drinking status Drinkers 37 40.2% 209 36.0% 0.4848 Non-drinkers 55 59.8% 371 64.0% Hypertension Yes 61 66.3% 302 52.1% 0.0130* No 31 33.7% 278 47.9% Diabetes Yes 21 104 0.2523 No 71 476 Family cancer history Yes 6 6.5% 17 2.9% 0.1125 No 86 93.5% 563 97.1%

24 Table II. Distributions of genotypic frequencies and their association with risk of RCC

RCC Cases (%) Controls (%) Adjusted ORa (95% CI) P-value APEX1 Asp148Glu (rs1130409) TT 40 (43.5) 238 (41.0) 1.00 (ref) TG 37 (40.2) 265 (45.7) 0.84 (0.52-1.38) 0.4647 GG 15 (16.3) 77 (13.3) 1.16 (0.58-2.29) 0.7353 P for trend 0.5555 (TG+GG) vs TT 0.88 (0.54-1.43) 0.7326 GG vs (TT+TG) 1.27 (0.72-2.28) 0.4171 a Adjusted by age, gender, smoking and alcohol drinking status

25 Table III. Association between APEX1 genotype and risk of RCC stratified by personal smoking habits

APEX1 genotype Never smokers Smokers Cases/controls aOR (95% CI) a Cases/controls aOR (95% CI) a Asp148Glu (rs1130409) TT 23/145 1.00 (ref) 17/93 1.00 (ref) TG 20/167 0.74 (0.41-1.48) 17/98 0.94 (0.52-1.98) GG 8/48 1.06 (0.48-2.54) 7/29 1.31 (0.48-3.28) P for trend 0.6248 0.7948 Abbreviations: aOR, adjusted odds ratio. a Data were adjusted for age, gender and alcohol drinking status.

26 Figure 1