title: Cigarette smoking extract causes hypermethylation and inactivation of WWOX gene in T-24 human bladder cancer cells.
abstract: Genomic, epigenetic and expression alterations of WW domain containing oxidoreductase (WWOX) have been implicated in multiple tumor types. The current study was designed to examine the expression of WWOX in tumor tissues of human bladder transitional cell carcinoma (BTCC) and the influence of cigarette smoke extract (CSE) on WWOX expression and methylation status in T-24 bladder cancer cells. WWOX protein expression was evaluated by immunohistochemistry staining in a series of tumor samples from 78 patients with BTCC and 26 normal bladder tissues. The expression level and methylation status of WWOX in CSE-treated cells were examined by using quantitative Real-Time RT-PCR and methylation specific PCR, respectively. The expression levels of DNA methyltransferases (DNMTs) 1, 3A and 3B were also detected. We found that WWOX expression was absent or reduced in 79.5% (62/78) of BTCC tissues, but only in 19.2% (5/26) of normal bladder tissues. Loss of WWOX expression was correlated with tumor grade (P=0.019) and cigarette smoking (P=0.031), but was not associated with age, gender, tumor size and tumor number. Hypermethylation of WWOX promoter and exon 1 was specifically induced by CSE with a kinetics concurrent to the suppression of WWOX mRNA in T-24 cells. Furthermore, CSE treatment induced a significant time-dependent increase in the level of DNMT1, but has no effects on DNMT3A and DNMT3B. Taken together, these novel findings suggest that hypermethylation of WWOX induced by cigarette smoking may represent one underlying mechanism for the loss expression of WWOX in bladder cancer.
authors.affiliation: Department of Urology, the First Affiliated Hospital of China Medical University, Shenyang, P. R. China.
authors.forename: W
authors.initials: W
authors.lastname: Yang
authors.forename: S
authors.initials: S
authors.lastname: Cui
authors.forename: J
authors.initials: J
authors.lastname: Ma
authors.forename: Q
authors.initials: Q
authors.lastname: Lu
authors.forename: C
authors.initials: C
authors.lastname: Kong
authors.forename: T
authors.initials: T
authors.lastname: Liu
authors.forename: Z
authors.initials: Z
authors.lastname: Sun
mesh_terms: D015153:Blotting, Western; D002295:Carcinoma, Transitional Cell / Q000235:genetics* / Q000473:pathology; D016022:Case-Control Studies; D019175:DNA Methylation / Q000187:drug effects*; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007124:Immunoenzyme Techniques; D008297:Male; D008875:Middle Aged; D010088:Oxidoreductases / Q000235:genetics*; D011379:Prognosis; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D012907:Smoking / Q000009:adverse effects*; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins / Q000235:genetics*; D001743:Urinary Bladder / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D001749:Urinary Bladder Neoplasms / Q000235:genetics* / Q000473:pathology; D000074584:WW Domain-Containing Oxidoreductase
chemical_list: D012333:RNA, Messenger; D025521:Tumor Suppressor Proteins; D010088:Oxidoreductases; D000074584:WW Domain-Containing Oxidoreductase; C486071:WWOX protein, human