Cigarette smoking is the single most important epidemiological risk factor for bladder cancer but it is not known whether exposure of urothelial cells to the systemic soluble contents of cigarette smoke is directly causative to bladder cancer and the associated epigenetic changes such as tumor suppressor gene hypermethylation. changes at tumor suppressor gene loci in the chronically CSE-treated cells versus the passage-matched untreated control cells. The RUNX3 tumor suppressor gene promoter was hypomethylated with a significant increase in proportion of the completely unmethylated haplotype after the long-term CSE treatment; whereas RUNX3 promoter hypermethylation was previously reported for bladder cancers of smokers. Hypomethylation induced by the long-term CSE treatment was also observed for the IGF2-H19 locus. The methylation status at the PRSS8/prostasin and 16 additional loci however, was unaffected by the chronic CSE treatment. Transient CSE treatment over 1 buy 51833-76-2 daily regimen resulted in transcriptional down-regulation of RUNX3 and H19, but only the H19 transcription was down-regulated in the chronically CSE-treated urothelial cells. Transcription of a key enzyme in one-carbon metabolism, dihydrofolate reductase (DHFR) was greatly reduced by the long-term CSE treatment, potentially serving as a mechanism for the hypomethylation phenotype via a reduced supply of methyl donor. In conclusion, chronic cyclic CSE treatment of urothelial cells induced hypomethylation rather than hypermethylation at specific loci. Introduction Cigarette smoking is the single most important epidemiological risk factor for bladder cancer as smokers have a four-fold higher risk versus nonsmokers; while about half of bladder cancers among men and women may be associated with or attributed to cigarette smoking [1]. The mainstream cigarette smoke or the cigarette smoke condensate/extract (CSC/CSE) has been shown to cause all sorts of genomic DNA damages in experimental animals as well as in humans, and is regarded as a systemic human mutagen with a direct role in causing eight different cancers including that of the urinary bladder [2]. In recent years, the role of epigenetic/epigenomic modifications in carcinogenesis, at all stages including initiation, promotion, and progression has been recognized. Bladder cancer was the model with which Peter Jones and colleagues expanded the Knudson two-hit hypothesis to include the mechanism of tumor suppressor silencing by epigenetic modifications in the consideration in addition to the mechanism of DNA damages [3]. The molecular pathways that lead to the development of papillary, low-grade, non-invasive urothelial carcinomas versus those to the development of the invasive type are believed to be distinct and mutually exclusive. Aberrant DNA methylation correlates with increased bladder tumor invasion and progression and is buy 51833-76-2 implicated for a causative role [3]. In the non-muscle invasive urothelial carcinomas, DNA methylation at six of twenty cancer-associated genes examined in a 105-patient cohort Cd24a was shown to associate with recurrence, while methylation at one of the genes predicts prolonged disease-free interval [4]. In bladder cancers, promoter DNA hypermethylation in tumor suppressor genes has been associated with cigarette smoking [5]. Promoter methylation in the p16-INK4A gene in bladder cancers was associated buy 51833-76-2 with cigarette smoking, with a greater than doubled risk for smokers versus nonsmokers, and with a greater risk in patients with more recent history of smoking [6]. DNA methylation at the RUNX3 (runt-related transcription factor 3) tumor suppressor gene naturally increases with age and occurs significantly earlier in bladder cancers of buy 51833-76-2 smokers than nonsmokers [7]. The associated effects of cigarette smoking on bladder cancer development appear direct, early, and more aggressive than the effects of other factors. While the epidemiological evidence is strong for a causative role of cigarette smoking in bladder cancer development and in epigenetic changes in bladder cancer specimens, there is no direct proof that exposure of urothelial cells to cigarette smoke causes these changes. A recent study reported that long-term exposure of airway epithelial cells to cigarette smoke condensate/extract resulted in cellular transformation marked by increased soft-agar clonogenicity and induced hypomethylation of repetitive DNA but hypermethylation of tumor suppressor genes; epigenetic changes buy 51833-76-2 that are consistent with pulmonary carcinogenesis [8]. In our study presented here, we exposed urothelial cells to cigarette smoke extract (CSE).