Expression of WWOX and FHIT is downregulated by exposure to arsenite in human uroepithelial cells

Toxicology Letters 220 (2013) 118–125

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Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet

Expression of WWOX and FHIT is downregulated by exposure to arsenite in human uroepithelial cells Ya-Chun Huang a , Wen-Chun Hung b,c , Wan-Tzu Chen d , Hsin-Su Yu e,f , Chee-Yin Chai a,b,d,∗ a

Department of Pathology, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan National Institute of Cancer Research, National Health Research Institute, Taipei, Taiwan d Department of Pathology, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan e Department of Dermatology, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan f Department of Dermatology, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan b c

h i g h l i g h t s • Low ATR, WWOX and FHIT expressions in urothelial cancer of blackfoot disease areas. • Arsenite decreased ATR, WWOX and FHIT expressions via ERK1/2 activation. • These dysregulations may play a role in arsenite-induced carcinogenesis.

a r t i c l e

i n f o

Article history: Received 4 January 2013 Received in revised form 12 April 2013 Accepted 15 April 2013 Available online 22 April 2013 Keywords: Arsenite ATR WWOX FHIT Urothelial carcinoma Human uroepithelial cells

a b s t r a c t Ecological studies in Taiwan, Chile, Argentina, Bangladesh, and Mexico have confirmed significant dosedependent associations between ingestion of arsenic-contaminated drinking water and the risk of various human malignancies. The FHIT and WWOX genes are active in common fragile sites FRA3B and FRA16D, respectively. Reduced expression of FHIT or WWOX is known to be an early indicator of carcinogeninduced cancers. However, the effect of arsenite on the expressions and molecular mechanisms of these markers is still unclear. The aims of this study were (i) to observe the expression of ATR, WWOX and FHIT proteins in urothelial carcinoma (UC) between endemic and non-endemic areas of blackfoot disease (BFD) by immunohistochemical analyses; (ii) to compare expression of these genes between arsenite-treated SV-HUC-1 human epithelial cells and rat uroepithelial cells; and (iii) to determine the role of DNMT and MEK inhibitors on expressions of WWOX and FHIT in response to arsenite in SV-HUC-1. The experiments revealed that expressions of ATR, WWOX and FHIT in UC significantly differed between BFD areas and non-BFD areas (p = 0.003, 0.009 and 0.021, respectively). In fact, the results for the arsenite-treated groups showed that ATR, WWOX and FHIT are downregulated by arsenite in SV-HUC-1. However, the inhibitors suppressed the effects of arsenite on WWOX and FHIT proteins and mRNA expression. In conclusion, arsenite decreased expressions of ATR, WWOX and FHIT via ERK1/2 activation in SV-HUC-1 cells. These findings confirm that dysregulations of these markers may contribute to arsenite-induced carcinogenesis. © 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Abbreviations: ATR, ataxia telangiectasia and Rad3 related; WWOX, WW domain-containing oxidoreductase; FHIT, fragile histidine triad; BFD, blackfoot disease; ERK1/2, extracellular signal-regulated kinase1/2; CFSs, common fragile sites; DNMT, DNA methyltransferase; MEK, mitogen-activated protein kinase (MAPK) kinase. ∗ Corresponding author at: Department of Pathology, Kaohsiung Medical University Hospital, No. 100, Tzyou 1st Road, Kaohsiung 807, Taiwan. Tel.: +886 7 3208233; fax: +886 7 3136681. E-mail address: [email protected] (C.-Y. Chai). 0378-4274/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.toxlet.2013.04.007

Arsenicals have been documented as a human carcinogen comes from studies using pesticide worker, smelters, and people ingested high levels of arsenic drinking water. There are contain high concentration of arsenic in well water in several different areas of the world, including Bangladesh, India, China, Chile, Mexico, United States, Argentina, and Taiwan (Garelick and Jones, 2008). Arsenic exposure is reported significantly associated with various human internal organ cancers in the blackfoot disease (BFD) endemic areas (Chen et al., 1986; Chen and Wang, 1990; Wu et al., 1989). The BFD is a unique peripheral artery disease associated with chronic arsenic

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Table 1 The WWOX, FHIT and GAPDH oligonucleotide primers for RT-PCR. Primer WWOX F R FHIT F R GAPDH F R

Sequence

Annealing temperature (◦ C)

Product size (bp)

Reference

57

1490

55

599

60

512

Park et al. (2004) Kannangai et al. (2004) Cho et al. (2007)

5 -GAGTTCCTGAGCGAGTGGAC-3 5 -CCCCAGGAATTCCCTGCTT-3 5 -CATCCTGGAAGCTTTGAAGCT-3 5 -TCCTCTGATCTCCAAGAGGC-3 5 -GAGTCAACGGATTTGGTCGT-3 5 -TGTGGTCATGAGTCCTTCCA-3

exposure in several southwestern townships of Taiwan. The residents of the BFD area had significantly dose-response relationship between concentration of arsenic in water and high mortality of bladder cancer (Brown and Beck, 1996; Chiou et al., 2001; Lamm et al., 2003). The fragile histidine triad (FHIT) gene at human chromosome 3p14.2 is a candidate tumor suppressor gene that encompasses the common fragile site FRA3B (Ohta et al., 1996). The FHIT protein, which is encoded by the FHIT gene, is a diadenosine triphosphate (Ap3A) hydrolase belonging to the histidine triad superfamily (HIT) of nucleotide-binding proteins (Barnes et al., 1996) and bears a high structural homology with Ap4A hydrolase form Schizosaccaromyces pombe (Huang et al., 1995; Ohta et al., 1996). Various human cancers, particularly those resulting from environmental carcinogen exposure (Croce et al., 1999), exhibit aberrant mRNA and protein expression and inactivation of the FHIT gene resulting from allelic losses, homozygous deletions and epigenetic modifications (Chang et al., 2002; Tanaka et al., 1998). The human WW domain-containing oxidoreductase (WWOX) gene maps to the second most active common fragile site FRA16D located at chromosome 16q23. The gene encodes a 414 amino acid that forms 46 kDa protein (Bednarek et al., 2000). The WWOX protein contains two WW domains and a short-chain dehydrogenase/reductase domain (SDR) (Bednarek et al., 2000; Chang et al., 2001). The multiple functions of WWOX in physiological and pathological processes include cell growth, differentiation, tumor suppressor and apoptosis. The WWOX gene inactivation and alterations in human tumors may result from genomic, post-translational and epigenetic modification (Iliopoulos et al., 2005; Qin et al., 2006; Ramos et al., 2008). Partial or complete loss of WWOX expression has been reported in human cancers (Maeda et al., 2010; Pluciennik et al., 2006; Watson et al., 2004). Many studies have reported dysregulated ATR, WWOX and FHIT in several tumor types. However, the effects of arsenic on ATR, WWOX and FHIT expression in human uroepithelial cells and the relationships between expressions of these markers and clincopathological parameters of urothelial carcinoma (UC) have never been compared between BFD and non-BFD areas. This study therefore investigated the role of these genes in arsenic-induced carcinogenesis.

2.2. Western blotting After 24 h pretreatment with 1 ␮M 5-aza-CdR or with 1 ␮M U0126 inhibitors, the cells were treated with various concentrations of arsenite for 48 h. The samples were homogenized with lysis buffer and centrifuged for protein levels analysis. The protein concentration was determined by a Bradford assay (Bio-Rad, Hercules, CA, USA). Samples were subjected to electrophoresis on a SDS-PAGE gel and transferred to a polyvinylidene-difluoride (PVDF) membrane as described previously (Huang et al., 2009). The primary antibody was incubated using ataxia telangiectasia-related (ATR) (Cell Signaling Technology Inc., Beverly, MA, USA), WWOX (Imgenex, San Diego, CA, USA) and FHIT (Neomarkers, Fremont, CA, USA), and secondary antibody conjugated with horseradish peroxidase was added. Protein bands were then detected by enhanced chemiluminescence reagents (Amersham, Buckinghamshire, England). As measured by densimeter, the densitometry data are “fold change” is compared with controls or 4 ␮M arsenic.

2.3. Reverse transcription-polymerase chain reaction Expressions of WWOX and FHIT mRNA were investigated using the One Step reverse transcription-polymerase chain reaction (RT-PCR) kit according to the manufacturer protocol (Qiagen, Hilden, Germany) as described previously (Huang et al., 2011). Table 1 gives the primer sequences, annealing temperatures and predicted sizes of PCR products for WWOX, FHIT and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Cho et al., 2007; Kannangai et al., 2004; Park et al., 2004). After reaction, PCR products were separated on 2% agarose gel, stained with ethidium bromide (EtBr) and visualized under UV light. The GAPDH was used as an internal control. As measured by densimeter, the densitometry data are “fold change” is compared with controls or 4 ␮M arsenic.

2.4. Immunocyto/histochemical staining The immunocytochemical (ICC) or immunohistochemical (IHC) staining procedure was performed as described previously (Huang et al., 2009). Paraffin-embedded tissue samples from BFD and non-BFD areas and rat bladder tissues were then sectioned, deparaffinized, and dehydrated. Antigen retrieval and endogenous peroxidase blocking were then performed at room temperature. After washing with Tris buffer solution (TBS), the sections were incubated with primary antibodies against the rabbit polyclonal antibody used were ATR and WWOX, and the goat polyclonal antibody used was FHIT at room temperature. The DAKO REAL Envision Detection System (DAKO, Denmark) was then performed using biotinylated second antibody and peroxidase-conjugated streptavidin. Finally, sections were incubated in 3 3diaminobenzidine (DAB) and counterstained with hematoxylin. Negative controls were obtained by replacing the primary antibody with non-immune serum. The labeling index (LI), as a percentage of immunostained cells, was determined by counting 1000 cells in the areas of densest immunostaining using light microscope at 400× magnification.

2. Materials and methods 2.5. Case selection 2.1. Cell culture and treatment SV-HUC-1 is SV-40 immortalized human uroepithelial cells. The SV-HUC-1 cell line is derived from transformed normal human urinary tract epithelial cells in vitro after infection with simian virus 40 (SV40) (Christian et al., 1987). The cell line has been tested in toxicological studies previously (Chang et al., 2007; Hirao et al., 1993; Mills et al., 2000; Su et al., 2006). The SV-HUC-1 cells were maintained in Ham’s F-12 medium supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 ␮g/ml streptomycin in 5% CO2 at 37 ◦ C in a humidified incubator. The cells were pretreated with/without 5-aza-deoxycytidine (5-aza-CdR) (BioMol International, LP, USA) or with/without U0126 (Cell Signaling Technology Inc., Beverly, MA, USA) and then stimulated with sodium arsenite (Sigma, St. Louis, MO, USA). The final arsenite concentrations were 1, 4 and 10 ␮M.

The data for the 33 UC patients and the specimens were obtained from the 1993–2000 archives of the Department of Pathology, Kaohsiung Medical University Hospital. The arsenic-contaminated areas (i.e., BFD-endemic areas) analyzed in this study were limited to several southwestern townships of Taiwan (Huang et al., 2011). The residents of the BFD area had regularly used artesian well water with high arsenic content for drinking, cooking, and household use over extended periods. The histological study of all tissue sections was performed by a single experienced pathologist. The clinical profiles of the BFD and non-BFD samples were determined according to the guidelines of the World Health Organization and the TNM system (Eble et al., 2004). This study and all associated tissue acquisitions were approved by the Institutional Review Board of Kaohsiung Medical University Hospital (KMUH-IRB-960470).

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Table 2 Correlation of ATR, WWOX and FHIT proteins in patients with urothelial carcinoma (UC) from blackfoot disease (BFD) areas and non-blackfoot disease (non-BFD) areas. Parameters ATR Low expression High expression WWOX Low expression High expression FHIT Low expression High expression *

BFD (n = 16)

non-BFD (n = 17)

12 (75.0%) 4 (25.0%)

4 (23.5%) 13 (76.5%)

11 (68.8%) 5 (31.2%)

4 (23.5%) 13 (76.5%)

9 (56.3%) 7 (43.7%)

3 (17.6%) 14 (82.4%)

p-Value* 0.003

0.009

0.021

p value was determined by Chi-square test.

2.6. Scoring of IHC antibody staining The immunoreactivities of ATR, WWOX and FHIT were evaluated in terms of percentages of positive cells and reactivity intensities at high magnification as described previously (Huang et al., 2011). The immunoreactivity of ATR, WWOX and FHIT was evaluated for percentage of positive cells and intensity of reactivity at high magnification. The intensity was evaluated as follows: 0, negative (no staining of any nuclear and cytoplasm at high magnification); 1, weak (only visible at high magnification); 2, moderate (easily visible at low magnification); and 3, strong (strongly positive at low magnification). The proportion of tumor cytoplasm showing positive staining was also recorded as follows: 0, staining in <1% of tumor cells; 1, staining in 1–10%; 2, staining in 11–50%; and 3, staining in >50% of tumor cells. The total score was obtained by multiplying the proportion and intensity scores, which ranged from 0 to 9. For further analysis, a cut-off point was established to separate the groups in terms of protein expression into a low expression group (staining patterns with total scores ≤ 4.5) and a high expression group (staining patterns with total scores >4.5). 2.7. Animal experiment Twenty-eight male F344/N (6–8 week-old) rats purchased from the National Laboratory of Animal Breeding and Research Center (Taipei, Taiwan, R.O.C.) were quarantined for 1 week before starting the experiment. The rats were housed in stainless steel wire mesh cages placed in a controlled environment with a 12 h lightdark cycle at 22 ± 3 ◦ C and 60 ± 10% humidity. The rats were then randomly assigned to experimental and control groups, each consisting of seven rats. The animal model used in this study was a modification of the model used in Sessions et al. (2002). For 2 days, rats in the experimental group were given D.D water 4 times daily. The water given 4 times daily contained varying concentrations of sodium arsenite (0, 1, 4 and 10 ␮M, approximately 0.13, 0.52 and 1.3 ppm, respectively). The control group rats were given D.D water alone. After 2 days, the rats were euthanized, and the bladders were removed, immediately frozen in liquid nitrogen, stored at -80◦ C for western blotting and fixed in 10% formalin solution. Expressions of ATR, WWOX and FHIT proteins in the urothelial cells of the F344 rat bladders were then detected by IHC and Western blotting. 2.8. Statistical analysis In human tumor sample experiments, the correlations of clinicopathologic characteristics and expressions of ATR, WWOX and FHIT proteins in UC were compared by Chi-square test between samples from BFD and non-BFD areas. Statistical analysis of LI was performed according to Student’s t-test. The obtained data were then analyzed with SPSS 14.0 statistical software program (Chicago, IL, USA). Results were expressed as mean ± SD. A p value of <0.05 or <0.001 was considered statistically significant.

3. Results 3.1. ATR, WWOX and FHIT protein expression in UC patients from BFD areas and non-BFD areas and correlations with clinicopathologic parameters The immunostaining results for ATR, WWOX and FHIT showed that low expression of these proteins was more common in patients with UC from BFD areas than in UC patients from non-BFD areas: 12 out of 16 (75.0%) versus 4 out of 17 (23.5%); 11 out of 16 (68.8%) versus 4 out of 17 (23.5%) and 9 out of 16 (56.3%) versus 3 out of 17 (17.6%), respectively (Table 2 and Fig. 1). Analysis of the UC patients by Chi-square test further confirmed that residence in a

Fig. 1. Comparison of immunohistochemical staining results for ATR, WWOX and FHIT in urothelial carcinoma (UC) patients from blackfoot disease (BFD) areas and non-blackfoot disease (non-BFD) areas. Low immunoactivity of ATR, WWOX and FHIT was observed more frequently in UC patients from BFD areas than those from non-BFD areas. Original magnification: 200× (scale bar = 10 ␮m).

BFD-endemic area was significantly associated with lower expressions of ATR, WWOX and FHIT proteins (p = 0.003, 0.009 and 0.021, respectively) (Table 2). Table 3 shows the correlations between expressions of ATR, WWOX and FHIT and the clinicopathologic parameters. In this UC subset, Chi-square test revealed statically significant correlations between WWOX, FHIT expression and clinicopathologic parameters. The WWOX and FHIT expressions had statistically significant correlation with tumor sites (p = 0.005 and 0.010, respectively) (Table 3). In contrast, expressions of ATR, WWOX and FHIT expression showed no significant correlations with gender, age, tumor invasiveness, histological grade or recurrence (Table 3).

3.2. Reduction of ATR, WWOX and FHIT expression by arsenite in human uroepithelial cells Expressions of ATR, WWOX and FHIT proteins were analyzed in SV-HUC-1 cells treated with varying concentrations of arsenite for 48 h. The LI of ATR in doses of 0, 1, 4 and 10 ␮M arsenite was 93, 58.67, 32.33 and 77%, respectively; and the LI of WWOX was 92.33, 75.67, 41 and 97%, respectively; and the LI of FHIT was 93, 47, 21.33 and 64.33%, respectively (Table 4). Sodium arsenite treatment decreased the LI in a dose-dependent manner at 1–4 ␮M (Table 4). However, the LI of WWOX and FHIT increased with high concentration of arsenite (10 ␮M) (Table 4). The ICC and Western blot results showed that arsenite reduced ATR, WWOX and FHIT proteins in SV-HUC-1 cells (Figs. 2 and 3I). Moreover, RT-PCR showed that arsenite suppressed mRNA expression in WWOX and FHIT (Fig. 4).

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Table 3 Correlation of WWOX, FHIT and ATR proteins and known clinicopathologic parameters in 33 patients with UC from BFD and non-BFD areas. Parameters

Gender Male Female Age ≤67 >67 Site Urinary bladder Kidney Ureter Tumor invasiveness Absent Present Histological grade Low High Recurrence Absent Present *

ATR expression

WWOX expression

Low (%)

High (%)

11(55.0) 5 (38.5)

9 (45.0) 8 (57.1)

6 (42.9) 10 (52.6)

8 (57.1) 9 (47.4)

7 (77.8) 6 (31.6) 3 (60.0)

2 (22.2) 13 (68.4) 2 (40.0)

7 (63.6) 9 (40.9)

4 (36.4) 13 (59.1)

4 (66.7) 12 (44.4)

2 (33.3) 15 (55.6)

8 (42.1) 8 (57.1)

11 (57.9) 6 (42.9)

p value*

Low (%)

High (%)

0.353

FHIT expression p value*

Low (%)

p value*

High(%)

0.948 9 (45.0) 6 (46.2)

11 (55.0) 7 (53.8)

7 (50.0) 8 (42.1)

7 (50.0) 11 (57.9)

7 (77.8) 4 (21.1) 4 (80.0)

2 (22.2) 15 (78.9) 1 (20.0)

4 (36.4) 11 (50.0)

7 (63.6) 11 (50.0)

1 (16.7) 14 (51.9)

5 (83.3) 13 (48.1)

10 (52.6) 5 (35.7)

9 (47.4) 9 (64.3)

0.579

0.201 9 (45.0) 3 (23.1)

11(55.0) 10(76.9)

6 (42.9) 6 (31.6)

8(57.1) 13(68.4)

7 (77.8) 4 (21.1) 1 (20.0)

2(22.2) 15(78.9) 4(80.0)

4 (36.4) 8 (36.4)

7(63.6) 14(63.6)

2 (33.3) 10 (37.0)

4(66.7) 17(63.0)

8 (42.1) 4 (28.6)

11(57.9) 10(71.4)

0.653

0.063

0.506

0.005

0.218

0.010

0.458

0.325

1.000

0.117

0.393

0.865

0.335

0.424

p value was determined by Chi-square test.

3.3. Effect of MEK and DNMT inhibitors on expressions of WWOX and FHIT in human uroepithelial cells treated with arsenite When the cells were preincubated with 1 ␮M 5-aza-CdR or 1 ␮M U0126 for 24 h, the 4 ␮M of arsenite was added, the LI of WWOX and FHIT was 94.67, 95, 97 and 96%, respectively (Table 4). The ICC, western blot and RT-PCR results showed that arsenite reduced expressions of WWOX and FHIT proteins and mRNA (Figs. 2, 3I and 4). This study further investigated how MEK and DNMT inhibitors affect expression of WWOX and FHIT in SV-HUC1 cells exposed to arsenite. The experiments showed that 24 h pretreatment with U0126 or 5-aza-CdR suppressed the effect of arsenite on expressions of WWOX and FHIT proteins and mRNA (Figs. 2, 3II and 4). That is, U0126 and 5-aza-CdR treatment caused restoration of WWOX and FHIT expressions. 3.4. Expressions of ATR, WWOX and FHIT proteins in rat uroepithelial cells Fig. 5 shows the IHC and Western blotting results for ATR, WWOX and FHIT protein expressions in rat urinary bladder lesions. In the rat bladder, urothelial cells of the arsenite-treated groups showed only low nuclear and cytoplasm immunostaining for ATR, WWOX and FHIT compared to the non-exposed controls (Fig. 5I). Further comparisons of Western blot results for expressions of ATR, WWOX and FHIT proteins between control groups and arsenitetreated groups showed that expressions of ATR, WWOX and FHIT were lower in the arsenite-treated groups than in the control groups (Fig. 5II).

4. Discussion Many studies demonstrated that fragile sites are especially susceptible to carcinogens and to nonrandom alterations in precancerous and cancerous lesions (Huebner et al., 1998; Ishii et al., 2003). The FHIT and WWOX genes active in the common fragile sites FRA3B and FRA16D, respectively. Some studies indicate that reduction of FHIT or WWOX is an early event in carcinogeninduced cancers, including lung, esophageal and liver cancers (Iida et al., 2005; Ji et al., 2006; Menin et al., 2000; Nelson et al., 1998; Pylkkanen et al., 2002; Stein et al., 2002). The frequency of general fragile site expression as well as FRA3B expression is also known to be significantly higher in smokers compared to nonsmokers and lung cancer patients who have stopped smoking (Stein et al., 2002). Smoking and asbestos exposure are also associated with FHIT loss in lung cancer patients (Nelson et al., 1998; Pylkkanen et al., 2002). Pylkkanen et al. reported that reduced FHIT protein expression is common in lung cancer patients with (67%) and without (59%) history of asbestos exposure (Pylkkanen et al., 2002). Aberrant FHIT transcripts or protein loss have been reported in nickel-transformed cell lines (Ji et al., 2006). Levels of FHIT expression are reportedly associated with alcohol use (Menin et al., 2000). Additionally, Iida et al. reported that a 2-week exposure to methyleugenol or o-nitrotoluene substantially reduced FHIT and WWOX proteins in the liver of mice, but no investigation was done in the condition that liver exposured to oxazepam, p-nitrotoluene, eugenol or acetaminophen (Iida et al., 2005). In FHIT-deficient cells, the loss of FHIT contributes to a DNA damage response that accumulates cell mutations, promotes preneoplasia initiation and

Table 4 Effects of sodium arsenite on the ATR, WWOX and FHIT labeling index of immunostaining. Sodium arsenite concentration (␮M)

ATR

WWOX

0 1 4 10 5-aza-CdR + 4 ␮M sodium arsenite U0126 + 4 ␮M sodium arsenite

93.00 ± 7.00 58.67 ± 8.62* , a 32.33 ± 4.93* , a 77.00 ± 3.61* , a N.D. N.D.

92.33 75.67 41.00 97.00 94.67 95.00

N.D.: no determined. a Compared with untreated cells (0 ␮M). b Compared with 4 ␮M arsenite treated cells. * p < 0.05.

± ± ± ± ± ±

FHIT 3.21 4.51* , a 3.21* , a 2.00a 3.06* , b 2.00* , b

93.00 47.00 21.33 64.33 97.00 96.00

± ± ± ± ± ±

2.00 3.00* , a 2.00* , a 12.01* , a 1.73* , b 2.00* , b

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Fig. 2. Immunocytochemical analysis of expressions of ATR, WWOX and FHIT proteins in SV-HUC-1 human uroepithelial cells. The SV-HUC-1 cells were pretreated with/without 5-aza-CdR or U0126 for 24 h before arsenite treatment: (A) 0 ␮M; (B) 4 ␮M; (C) 10 ␮M; (D) 1 ␮M 5-aza-CdR and 4 ␮M arsenite and (E) 1 ␮M U0126 and 4 ␮M arsenite (400×). However, the inhibitors (5-aza-CdR and U0126) suppressed the effect of arsenite on expressions of WWOX and FHIT proteins in SV-HUC-1 cells (scale bar = 10 ␮m).

neoplastic progression (Ishii et al., 2007). These data show that exposure to carcinogens (cigarette, asbestos, alcohol, nickel and other substaqnces) increased chromosomal fragile site expression. The data also suggest that FHIT and WWOX genes are common targets of carcinogen-induced damage and have a central role in the early stage of carcinogenesis. The ATR-dependent DNA damage checkpoint pathway plays an important role in regulating the stability of common fragile sites (Casper et al., 2002). A recent study found that binding requires its kinase activity in response to aphidicolin treatment (Wan et al., 2010). The ATR/ATM-regulated DNA damage response network is activated in early dysplastic stages of lung and skin hyperplasia (Bartkova et al., 2005; Gorgoulis et al., 2005). Mutation of the ATR/ATM checkpoint causes cell proliferation, tumor progression, DNA replication stress, and genomic instability (Pichiorri et al., 2008). Deletions of the short arm of chromosome (3p) have been detected by loss of heterozygosity (LOH) and by cytogenetic studies in several human tumors, including bladder tumors (Li et al., 1996; Voorter et al., 1995). Deletion of 3p observed in bladder tumors (Knowles et al., 1994; Presti et al., 1991) has been associated with tumor progression in bladder cancer and loss of 3p (Habuchi et al., 1993; Presti et al., 1991). Some bladder cancer studies have also reported LOH or abnormal FHIT expression (Baffa et al.,

2000; Louhelainen et al., 2001; Vecchione et al., 2004; Wada et al., 2001; Xiao et al., 2002). In SW780 FHIT-negative cells, transduction with adenoviral-FHIT inhibited cell growth, increased apoptotic cell population, and suppressed s.c. tumor growth in nude mice (Vecchione et al., 2004). Moreover, these findings suggest that the inactivation of FHIT has an important role in the development of bladder cancer. However, abnormal WWOX expression has been demonstrated at the genomic, mRNA and protein levels in various human tumors, including prostate and bladder tumors (Iliopoulos et al., 2005; Qin et al., 2006; Ramos et al., 2008). An analysis of seven bladder TCCs by Iliopoulos et al. showed that WWOX expression was highly reduced in three, moderately reduced in three, and strong in one (Iliopoulos et al., 2005). Ramos et al. found that progressive loss of WWOX expression may be a predictor of progressive disease in bladder cancer (Ramos et al., 2008). Hypermethylation of the WWOX and FHIT genes has been noted in various human cancers, including bladder and prostate cancer (Gutierrez et al., 2004; Iliopoulos et al., 2005; Maruyama et al., 2001). Wang et al. reported significantly lower WWOX mRNA and protein expression in breast cancer tissues with methylated WWOX CpG islands compared to those without methylation (p < 0.001 and p = 0.008, respectively) (Wang et al., 2009). Overexpression of exogenous WWOX or restoration of endogenous WWOX protein expression caused by infection with Ad-WWOX or treatment

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Fig. 4. Role of inhibitors on expressions of WWOX and FHIT mRNA in response to arsenite in SV-HUC-1 cells. The SV-HUC-1 cells were pretreated with 5-aza-CdR or U0126 for 24 h. After arsenite addition, mRNA levels of WWOX and FHIT were analyzed by RT-PCR. Equal loading was confirmed by GAPDH. * compared with 4 ␮M arsenite treated cells.

Fig. 3. Western blot results showing the effect of inhibitors (5-aza-CdR or U0126) on expressions of ATR, WWOX and FHIT in response to sodium arsenite stimulation in SV-HUC-1 cells. (I) Effect of arsenite on expressions of ATR, WWOX and FHIT in SV-HUC-1 cells. Cells were treated with different concentrations of arsenite for 48 h. Arsenite treatment decreased expressions of ATR, WWOX and FHIT proteins. (II) In cells incubated with 1 ␮M 5-aza-CdR or 1 ␮M U0126 for 24 h, arsenite treatment (4 ␮M) for 48 h suppressed the effect of arsenite on expressions of ATR, WWOX and FHIT. Equal loading was confirmed by ␤-actin. * compared with 4 ␮M arsenite treated cells.

with 5-aza-CdR induces in vitro apoptosis in breast cancer cells and suppression of xenograft tumor growth (Iliopoulos et al., 2007). Nakayama et al. reported that hypermethylation reduces WWOX expression in intraductal papillary mucinous neoplasms of the pancreas (Nakayama et al., 2009). They also reported a 16% promoter methylation frequency in FHIT in bladder cancers. Methylation of FHIT is also associated with poor survival (Maruyama et al., 2001). For example, Gutiérrez et al. reported a 40% methylation frequency in squamous cell carcinoma of the bladder (Gutierrez et al., 2004). Maruyama et al. reported a significantly higher rate of FHIT gene methylation in prostate cancer tissues (15%) compared to nonmalignant tissues (Maruyama et al., 2002). Additionally, the

11–29% methylation of FHIT and WWOX CpGs is sufficient for gene silencing in bladder cancer (Iliopoulos et al., 2005). Cantor et al. reported that epigenetic modulation of endogenous tumor suppressor gene expression in lung cancer xenografts inhibits tumorigenicity, including WWOX and FHIT (Cantor et al., 2007). Reports that EKR activation correlates with DNA methylation are direct evidence of the important role of ERK in regulating DNA methylation in cells (Chang et al., 2006; Chen et al., 2010; Deng et al., 1998; Oelke and Richardson, 2004). Decreased T cell signaling in the ERK pathway may also contribute to the development of lupus by affecting DNA methylation and gene expression (Oelke and Richardson, 2004). Chen et al. further showed that, through epigenetic modification, suppression of the ERK and JNK signaling pathways contributes to gene overexpression in “senescent” CD4+ CD28− T cells (Chen et al., 2010). The ICC and western blot analyses in the present study showed that 48 h arsenite treatment decreased WWOX and FHIT protein expressions. However, pretreatment with 5-aza-CdR or U0126 suppressed the effect of arsenite on WWOX and FHIT protein expression, which suggests that arsenite-induced expression of WWOX and FHIT decreased protein levels through ERK activation and epigenetic alteration in SV-HUC-1 cells. This study provides the first evidence that frequence of ATR, WWOX and FHIT low expressions in UC patients from BFD areas was higher than the UC patients from non-BFD areas (p = 0.003, 0.009 and 0.021, respectively). We further showed that arsenite reduces ATR, WWOX and FHIT expression in SV-HUC-1 human uroepithelial

Fig. 5. Comparison of results for immuohistochemical and Western blot analyses of expressions of ATR, WWOX and FHIT proteins in bladders of control groups and arsenitetreated groups. The (I) ATR, WWOX and FHIT immunoreactivities and (II) protein levels were lower in the bladder urothelium cells of rats in the arsenite-treated groups compared with the control groups. Lane 1: control (incubated without arsenite); lane 2: exposure to 0.13 ppm arsenite; lane 3: 0.52 ppm arsenite; and lane 4: 1.3 ppm arsnite, as analyzed by Western blotting. Original magnification: 400× (scale bar = 10 ␮m).

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cells. However, inhibition of DNMT or MEK suppresses the effect of arsenic on SV-HUC-1 cells in relation to ATR, WWOX and FHIT. The results of in vivo studies showed lower expressions of ATR, WWOX and FHIT in the arsenite groups than in the control groups. In conclusion, the experimental results indicate that arsenite may dysregulate ATR, WWOX and FHIT by activating ERK1/2 and by deactivating epigenetic activity in human uroepithelial cells. This study confirmed the critical role of ATR and chromosomal fragile site genes in arsenite-induced carcinogenesis. Acknowledgements This research was supported by grant from the National Science Council of Taiwan (NSC 97-2320-B-037-020) and Kaohsiung Medical University Research Foundation (KMUER-002-4). References Baffa, R., Gomella, L.G., Vecchione, A., Bassi, P., Mimori, K., Sedor, J., Calviello, C.M., Gardiman, M., Minimo, C., Strup, S.E., et al., 2000. Loss of FHIT expression in transitional cell carcinoma of the urinary bladder. American Journal of Pathology 156, 419–424. Barnes, L.D., Garrison, P.N., Siprashvili, Z., Guranowski, A., Robinson, A.K., Ingram, S.W., Croce, C.M., Ohta, M., Huebner, K., 1996. FHIT, a putative tumor suppressor in humans, is a dinucleoside 5 ,5 -P1,P3-triphosphate hydrolase. Biochemistry 35, 11529–11535. Bartkova, J., Horejsi, Z., Koed, K., Kramer, A., Tort, F., Zieger, K., Guldberg, P., Sehested, M., Nesland, J.M., Lukas, C., et al., 2005. DNA damage response as a candidate anti-cancer barrier in early human tumorigenesis. Nature 434, 864–870. Bednarek, A.K., Laflin, K.J., Daniel, R.L., Liao, Q., Hawkins, K.A., Aldaz, C.M., 2000. WWOX, a novel WW domain-containing protein mapping to human chromosome 16q23.3-24.1, a region frequently affected in breast cancer. Cancer Research 60, 2140–2145. Brown, K.G., Beck, B.D., 1996. Arsenic and bladder cancer mortality. Epidemiology 7, 557–558. Cantor, J.P., Iliopoulos, D., Rao, A.S., Druck, T., Semba, S., Han, S.Y., McCorkell, K.A., Lakshman, T.V., Collins, J.E., Wachsberger, P., et al., 2007. Epigenetic modulation of endogenous tumor suppressor expression in lung cancer xenografts suppresses tumorigenicity. International Journal of Cancer 120, 24–31. Casper, A.M., Nghiem, P., Arlt, M.F., Glover, T.W., 2002. ATR regulates fragile site stability. Cell 111, 779–789. Chang, H.C., Cho, C.Y., Hung, W.C., 2006. Silencing of the metastasis suppressor RECK by RAS oncogene is mediated by DNA methyltransferase 3b-induced promoter methylation. Cancer Research 66, 8413–8420. Chang, H.R., Lian, J.D., Lo, C.W., Huang, H.P., Wang, C.J., 2007. Aristolochic acidinduced cell cycle G1 arrest in human urothelium SV-HUC-1 cells. Food and Chemical Toxicology 45, 396–402. Chang, K.W., Kao, S.Y., Tzeng, R.J., Liu, C.J., Cheng, A.J., Yang, S.C., Wong, Y.K., Lin, S.C., 2002. Multiple molecular alterations of FHIT in betel-associated oral carcinoma. Journal of Pathology 196, 300–306. Chang, N.S., Pratt, N., Heath, J., Schultz, L., Sleve, D., Carey, G.B., Zevotek, N., 2001. Hyaluronidase induction of a WW domain-containing oxidoreductase that enhances tumor necrosis factor cytotoxicity. Journal of Biological Chemistry 276, 3361–3370. Chen, C.J., Chuang, Y.C., You, S.L., Lin, T.M., Wu, H.Y., 1986. A retrospective study on malignant neoplasms of bladder, lung and liver in blackfoot disease endemic area in Taiwan. British Journal of Cancer 53, 399–405. Chen, C.J., Wang, C.J., 1990. Ecological correlation between arsenic level in well water and age-adjusted mortality from malignant neoplasms. Cancer Research 50, 5470–5474. Chen, Y., Gorelik, G.J., Strickland, F.M., Richardson, B.C., 2010. Decreased ERK and JNK signaling contribute to gene overexpression in “senescent” CD4+ CD28-T cells through epigenetic mechanisms. Journal of Leukocyte Biology 87, 137–145. Chiou, H.Y., Chiou, S.T., Hsu, Y.H., Chou, Y.L., Tseng, C.H., Wei, M.L., Chen, C.J., 2001. Incidence of transitional cell carcinoma and arsenic in drinking water: a follow-up study of 8,102 residents in an arseniasis-endemic area in northeastern Taiwan. American Journal of Epidemiology 153, 411–418. Cho, C.Y., Wang, J.H., Chang, H.C., Chang, C.K., Hung, W.C., 2007. Epigenetic inactivation of the metastasis suppressor RECK enhances invasion of human colon cancer cells. Journal of Cellular Physiology 213, 65–69. Christian, B.J., Loretz, L.J., Oberley, T.D., Reznikoff, C.A., 1987. Characterization of human uroepithelial cells immortalized in vitro by simian virus 40. Cancer Research 47, 6066–6073. Croce, C.M., Sozzi, G., Huebner, K., 1999. Role of FHIT in human cancer. Journal of Clinical Oncology 17, 1618–1624. Deng, C., Yang, J., Scott, J., Hanash, S., Richardson, B.C., 1998. Role of the ras-MAPK signaling pathway in the DNA methyltransferase response to DNA hypomethylation. Biological Chemistry 379, 1113–1120. Eble, J.N., Sauter, G., Epstein, J.I., Sesterhenn, I.A., 2004. Pathology and Genetics of Tumours of the Urinary System and Male Genital Organs. TARC, Lyon.

Garelick, H., Jones, H., 2008. Arsenic pollution and remediation: an international perspective. Special foreword. Reviews of Environmental Contamination and Toxicology 197, v–vi. Gorgoulis, V.G., Vassiliou, L.V., Karakaidos, P., Zacharatos, P., Kotsinas, A., Liloglou, T., Venere, M., Ditullio Jr., R.A., Kastrinakis, N.G., Levy, B., et al., 2005. Activation of the DNA damage checkpoint and genomic instability in human precancerous lesions. Nature 434, 907–913. Gutierrez, M.I., Siraj, A.K., Khaled, H., Koon, N., El-Rifai, W., Bhatia, K., 2004. CpG island methylation in Schistosoma- and non-Schistosoma-associated bladder cancer. Modern Pathology 17, 1268–1274. Habuchi, T., Ogawa, O., Kakehi, Y., Ogura, K., Koshiba, M., Hamazaki, S., Takahashi, R., Sugiyama, T., Yoshida, O., 1993. Accumulated allelic losses in the development of invasive urothelial cancer. International Journal of Cancer 53, 579–584. Hirao, Y., Kitagawa, H., Yoshie, T., Futami, T., Momose, H., Ozono, S., Okajima, E., 1993. The effects of rhodamine 123 on the cell growth of the cultured cells derived from urogenital carcinoma, Hinyokika Kiyo. Acta Urologica Japonica 39, 1233–1240. Huang, Y., Garrison, P.N., Barnes, L.D., 1995. Cloning of the Schizosaccharomyces pombe gene encoding diadenosine 5 ,5 -P1,P4-tetraphosphate (Ap4A) asymmetrical hydrolase: sequence similarity with the histidine triad (HIT) protein family. Biochemical Journal 312 (Pt 3), 925–932. Huang, Y.C., Hung, W.C., Chen, W.T., Yu, H.S., Chai, C.Y., 2009. Sodium arsenite-induced DAPK promoter hypermethylation and autophagy via ERK1/2 phosphorylation in human uroepithelial cells. Chemico-Biological Interactions 181, 254–262. Huang, Y.C., Hung, W.C., Chen, W.T., Yu, H.S., Chai, C.Y., 2011. Effects of DNMT and MEK inhibitors on the expression of RECK, MMP-9, -2, uPA and VEGF in response to arsenite stimulation in human uroepithelial cells. Toxicology Letters 201, 62–71. Huebner, K., Druck, T., Siprashvili, Z., Croce, C.M., Kovatich, A., McCue, P.A., 1998. The role of deletions at the FRA3B/FHIT locus in carcinogenesis. Recent Results in Cancer Research 154, 200–215. Iida, M., Anna, C.H., Holliday, W.M., Collins, J.B., Cunningham, M.L., Sills, R.C., Devereux, T.R., 2005. Unique patterns of gene expression changes in liver after treatment of mice for 2 weeks with different known carcinogens and noncarcinogens. Carcinogenesis 26, 689–699. Iliopoulos, D., Guler, G., Han, S.Y., Johnston, D., Druck, T., McCorkell, K.A., Palazzo, J., McCue, P.A., Baffa, R., Huebner, K., 2005. Fragile genes as biomarkers: epigenetic control of WWOX and FHIT in lung, breast and bladder cancer. Oncogene 24, 1625–1633. Iliopoulos, D., Fabbri, M., Druck, T., Qin, H.R., Han, S.Y., Huebner, K., 2007. Inhibition of breast cancer cell growth in vitro and in vivo: effect of restoration of Wwox expression. Clinical Cancer Research 13, 268–274. Ishii, H., Ozawa, K., Furukawa, Y., 2003. Alteration of the fragile histidine triad gene early in carcinogenesis: an update. Journal of Experimental Therapeutics and Oncology 3, 291–296. Ishii, H., Wang, Y., Huebner, K., 2007. A FHIT-ing role in the DNA damage checkpoint response. Cell Cycle 6, 1044–1048. Ji, W.D., Chen, J.K., Lu, J.C., Wu, Z.L., Yi, F., Feng, S.M., 2006. Alterations of FHIT gene and P16 gene in nickel transformed human bronchial epithelial cells. Biomedical and Environmental Sciences 19, 277–284. Kannangai, R., Sahin, F., Adegbola, O., Ashfaq, R., Su, G.H., Torbenson, M., 2004. FHIT mRNA and protein expression in hepatocellular carcinoma. Modern Pathology 17, 653–659. Knowles, M.A., Elder, P.A., Williamson, M., Cairns, J.P., Shaw, M.E., Law, M.G., 1994. Allelotype of human bladder cancer. Cancer Research 54, 531–538. Lamm, S.H., Byrd, D.M., Kruse, M.B., Feinleib, M., Lai, S.H., 2003. Bladder cancer and arsenic exposure: differences in the two populations enrolled in a study in southwest Taiwan. Biomedical and Environmental Sciences 16, 355–368. Li, M., Zhang, Z.F., Reuter, V.E., Cordon-Cardo, C., 1996. Chromosome 3 allelic losses and microsatellite alterations in transitional cell carcinoma of the urinary bladder. American Journal of Pathology 149, 229–235. Louhelainen, J., Wijkstrom, H., Hemminki, K., 2001. Multiple regions with allelic loss at chromosome 3 in superficial multifocal bladder tumors. International Journal of Oncology 18, 203–210. Maeda, N., Semba, S., Nakayama, S., Yanagihara, K., Yokozaki, H., 2010. Loss of WW domain-containing oxidoreductase expression in the progression and development of gastric carcinoma: clinical and histopathologic correlations. Virchows Archiv 457, 423–432. Maruyama, R., Toyooka, S., Toyooka, K.O., Harada, K., Virmani, A.K., ZochbauerMuller, S., Farinas, A.J., Vakar-Lopez, F., Minna, J.D., Sagalowsky, A., et al., 2001. Aberrant promoter methylation profile of bladder cancer and its relationship to clinicopathological features. Cancer Research 61, 8659–8663. Maruyama, R., Toyooka, S., Toyooka, K.O., Virmani, A.K., Zochbauer-Muller, S., Farinas, A.J., Minna, J.D., McConnell, J., Frenkel, E.P., Gazdar, A.F., 2002. Aberrant promoter methylation profile of prostate cancers and its relationship to clinicopathological features. Clinical Cancer Research 8, 514–519. Menin, C., Santacatterina, M., Zambon, A., Montagna, M., Parenti, A., Ruol, A., D’Andrea, E., 2000. Anomalous transcripts and allelic deletions of the FHIT gene in human esophageal cancer. Cancer Genetics and Cytogenetics 119, 56–61. Mills, M., Meysick, K.C., O’Brien, A.D., 2000. Cytotoxic necrotizing factor type 1 of uropathogenic Escherichia coli kills cultured human uroepithelial 5637 cells by an apoptotic mechanism. Infection and Immunity 68, 5869–5880. Nakayama, S., Semba, S., Maeda, N., Matsushita, M., Kuroda, Y., Yokozaki, H., 2009. Hypermethylation-mediated reduction of WWOX expression in intraductal

Y.-C. Huang et al. / Toxicology Letters 220 (2013) 118–125 papillary mucinous neoplasms of the pancreas. British Journal of Cancer 100, 1438–1443. Nelson, H.H., Wiencke, J.K., Gunn, L., Wain, J.C., Christiani, D.C., Kelsey, K.T., 1998. Chromosome 3p14 alterations in lung cancer: evidence that FHIT exon deletion is a target of tobacco carcinogens and asbestos. Cancer Research 58, 1804–1807. Oelke, K., Richardson, B., 2004. Decreased T cell ERK pathway signaling may contribute to the development of lupus through effects on DNA methylation and gene expression. International Reviews of Immunology 23, 315–331. Ohta, M., Inoue, H., Cotticelli, M.G., Kastury, K., Baffa, R., Palazzo, J., Siprashvili, Z., Mori, M., McCue, P., Druck, T., et al., 1996. The FHIT gene, spanning the chromosome 3p14.2 fragile site and renal carcinoma-associated t(3;8) breakpoint, is abnormal in digestive tract cancers. Cell 84, 587–597. Park, S.W., Ludes-Meyers, J., Zimonjic, D.B., Durkin, M.E., Popescu, N.C., Aldaz, C.M., 2004. Frequent downregulation and loss of WWOX gene expression in human hepatocellular carcinoma. British Journal of Cancer 91, 753–759. Pichiorri, F., Ishii, H., Okumura, H., Trapasso, F., Wang, Y., Huebner, K., 2008. Molecular parameters of genome instability: roles of fragile genes at common fragile sites. Journal of Cellular Biochemistry 104, 1525–1533. Pluciennik, E., Kusinska, R., Potemski, P., Kubiak, R., Kordek, R., Bednarek, A.K., 2006. WWOX – the FRA16D cancer gene: expression correlation with breast cancer progression and prognosis. European Journal of Surgical Oncology 32, 153–157. Presti Jr., J.C., Reuter, V.E., Galan, T., Fair, W.R., Cordon-Cardo, C., 1991. Molecular genetic alterations in superficial and locally advanced human bladder cancer. Cancer Research 51, 5405–5409. Pylkkanen, L., Wolff, H., Stjernvall, T., Tuominen, P., Sioris, T., Karjalainen, A., Anttila, S., Husgafvel-Pursiainen, K., 2002. Reduced FHIT protein expression and loss of heterozygosity at FHIT gene in tumours from smoking and asbestos-exposed lung cancer patients. International Journal of Oncology 20, 285–290. Qin, H.R., Iliopoulos, D., Semba, S., Fabbri, M., Druck, T., Volinia, S., Croce, C.M., Morrison, C.D., Klein, R.D., Huebner, K., 2006. A role for the WWOX gene in prostate cancer. Cancer Research 66, 6477–6481. Ramos, D., Abba, M., Lopez-Guerrero, J.A., Rubio, J., Solsona, E., Almenar, S., LlombartBosch, A., Aldaz, C.M., 2008. Low levels of WWOX protein immunoexpression correlate with tumour grade and a less favourable outcome in patients with urinary bladder tumours. Histopathology 52, 831–839. Sessions, A., Eichel, L., Kassahun, M., Messing, E.M., Schwarz, E., Wood, R.W., 2002. Continuous bladder infusion methods for studying voiding function in the ambulatory mouse. Urology 60, 707–713.

125

Stein, C.K., Glover, T.W., Palmer, J.L., Glisson, B.S., 2002. Direct correlation between FRA3B expression and cigarette smoking. Genes, Chromosomes and Cancer 34, 333–340. Su, P.F., Hu, Y.J., Ho, I.C., Cheng, Y.M., Lee, T.C., 2006. Distinct gene expression profiles in immortalized human urothelial cells exposed to inorganic arsenite and its methylated trivalent metabolites. Environmental Health Perspectives 114, 394–403. Tanaka, H., Shimada, Y., Harada, H., Shinoda, M., Hatooka, S., Imamura, M., Ishizaki, K., 1998. Methylation of the 5 CpG island of the FHIT gene is closely associated with transcriptional inactivation in esophageal squamous cell carcinomas. Cancer Research 58, 3429–3434. Vecchione, A., Sevignani, C., Giarnieri, E., Zanesi, N., Ishii, H., Cesari, R., Fong, L.Y., Gomella, L.G., Croce, C.M., Baffa, R., 2004. Inactivation of the FHIT gene favors bladder cancer development. Clinical Cancer Research 10, 7607–7612. Voorter, C., Joos, S., Bringuier, P.P., Vallinga, M., Poddighe, P., Schalken, J., du Manoir, S., Ramaekers, F., Lichter, P., Hopman, A., 1995. Detection of chromosomal imbalances in transitional cell carcinoma of the bladder by comparative genomic hybridization. American Journal of Pathology 146, 1341–1354. Wada, T., Louhelainen, J., Hemminki, K., Adolfsson, J., Wijkstrom, H., Norming, U., Borgstrom, E., Hansson, J., Steineck, G., 2001. The prevalence of loss of heterozygosity in chromosome 3, including FHIT, in bladder cancer, using the fluorescent multiplex polymerase chain reaction. BJU International 87, 876–881. Wan, C., Kulkarni, A., Wang, Y.H., 2010. ATR preferentially interacts with common fragile site FRA3B and the binding requires its kinase activity in response to aphidicolin treatment. Mutation Research 686, 39–46. Wang, X., Chao, L., Jin, G., Ma, G., Zang, Y., Sun, J., 2009. Association between CpG island methylation of the WWOX gene and its expression in breast cancers. Tumour Biology 30, 8–14. Watson, J.E., Doggett, N.A., Albertson, D.G., Andaya, A., Chinnaiyan, A., van Dekken, H., Ginzinger, D., Haqq, C., James, K., Kamkar, S., et al., 2004. Integration of highresolution array comparative genomic hybridization analysis of chromosome 16q with expression array data refines common regions of loss at 16q23-qter and identifies underlying candidate tumor suppressor genes in prostate cancer. Oncogene 23, 3487–3494. Wu, M.M., Kuo, T.L., Hwang, Y.H., Chen, C.J., 1989. Dose–response relation between arsenic concentration in well water and mortality from cancers and vascular diseases. American Journal of Epidemiology 130, 1123–1132. Xiao, Z., Zhang, J., Zheng, S., Li, C., He, Z., Cheng, S., Gao, Y., 2002. Loss of heterozygosity of tumor suppressor genes at chromosome 3p in transitional cell carcinoma of urinary bladder. Zhonghua Yi Xue Za Zhi 82, 1375–1377.