Gene expression in normal Urothelium depends on location within the bladder:A possible link to bladder carcinogenesis

Objectives: Clinical studies have shown that more than 70% of primary bladder tumours arise in the area around the ureteric orifice. In this study a genomic approach was taken to explore the molecular mechanisms that may influence this phenomenon.

Methods: RNA was isolated from each individual normal ureteric orifice and the dome biopsy from 33 male patients. Equal amounts of the pooled ureteric orifice and dome mRNAs were labelled with Cy3 and Cy5, respectively before hybridising to the gene chip (UniGEM 2.0, Incyte Genomics Inc., Wilmington, Delaware, USA). Results: Significant changes (more than a twofold difference) in gene expression were observed in 3.1% (312) of the 10,176 gene array: 211 genes upregulated and 101 downregulated. Analysis of Cdc25B, TK1, PKM, and PDGFra with RT-PCR supported the reliability of the microarray result. Seladin-1 was the most upregulated gene in the ureteric orifice: 8.3-fold on the microarray and 11.4-fold by real time PCR.

Conclusions: Overall, this study suggests significant altered gene expression between these two anatomically distinct areas of the normal human bladder. Of particular note is Seladin-1, whose significance in cancer is yet to be clarified. Further studies of the genes discovered by this work will help clarify which of these differences influence primary bladder carcinogenesis. (c) 2006 European Association of Urology. Published by Elsevier B.V. All rights reserved.

Inhibition of Mouse Urinary Bladder Carcinogenesis by A double dagger ai Fruit (Euterpe oleraceae Martius) Intake

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Effects of ginger (Zingiber officinale Roscoe) on DNA damage and development of urothelial tumors in a mouse bladder carcinogenesis model

Extracts of the spice ginger (Zingiber officinale Roscoe) are rich in gingerols and shogaols, which exhibit antioxidant, anti-inflammatory, antifungal, anti mycobacterial, and anticarcinogenic proprieties. The present study evaluated the chemoprotective effects of a ginger extract on the DNA damage and the development of bladder cancer induced by N-butyl-N-(4-hydroxibutyl) nitrosamine (BBN)/N-methyl-N-nitrosourea (MNU) in male Swiss mice. Groups G1-G3 were given 0.05% BBN in drinking water for 18 weeks and four i.p. injections of 30 mg/kg body weight MNU at 1, 3, 10, and 18 weeks. Group G4 and G5 received only the BBN or MNU treatments, respectively, and groups G6 and G7 were not treated with BBN or MNU. Additionally, Groups G2, G3, and G6 were fed diets containing 1, 2, and 2% ginger extract, respectively, while Groups G1, G4, G5, and G7 were fed basal diet. Samples of peripheral blood were collected during the experiment for genotoxicity analysis; blood collected 4 hr after each MNU dose was used for the analysis of DNA damage with the Comet assay (assay performed on leukocytes from all groups), while reficulocytes collected 24 hr after the last MNU treatment of Groups G5-G7 were used for the micronucleus assay. At the end of the experiment, the urinary bladder was removed, fixed, and prepared for histopathological, cell proliferation, and apoptosis evaluations. Ginger by itself was not genotoxic, and it did not alter the DNA damage levels induced by the BBN/MNU treatment during the course of the exposure. The incidence and multiplicity of simple and nodular hyperplasia and transitional cell carcinoma (TCC) were increased by the BBN/MNU treatment, but dietary ginger had no significant effect on these responses. However, in Group G2 (BBN/MNU/2% ginger-treated group), there was an increased incidence of Grade 2 TCC. The results suggest that ginger extract does not inhibit the development of BBN-induced mouse bladder tumors.

Absence of promoting potential of bracken fern (Pteridium aquilinum) in rat urinary bladder carcinogenesis induced by N-butyl-N-(4-hydroxybutyl)-nitrosamine and uracil

Conventional studies on bracken fern (Pteridium aquilinum; PA) carcinogenicity have used high dietary concentrations (around 30%) and long-term exposure (up to 52-70 weeks) without consideration of the multistep character of the chemical carcinogenesis process. The present study evaluated specifically the promoting potential of 3-5% dietary crude PA in the rat urinary bladder mucosa in a 32-week-long initiation-promotion assay for chemical carcinogenesis. Initiation of urothelial carcinogenesis was accomplished with N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN). Uracil (U) was provided through the diet in order to expand the population of initiated cells. Seven groups (C) of male Wistar rats were submitted to the following treatments: G1 = BBN (n = 8); G2 = U (n = 10); G3 = BBN-U (n = 9); G4 = BBN-PA-U-PA (n = 16); G5 = PA (n = 8); G6 = BBN-PA (n = 10); G7 = PA-U-PA (n = 12). At the end of the experiment rats presenting epithelial papillary or nodular hyperplasia (PNH), papillomas (PAP), or simultaneous PNH plus PAP numbered, respectively G1: 2-0-1; G2: 0-0-0; G3: 3-0-2; G4: 4-3-2; G5: 1-0-1; G6: 8-0-0; and G7: 0-0-0, with no significant differences in the incidence of lesions among the groups. More frequent and more severe lesions occurred in BBN-initiated animals, predominantly in those also exposed to uracil (G3 and G4). Low-dose crude bracken fern in the diet does not promote rat urinary bladder carcinogenesis after a 32-week period of exposure, even when the initiated urothelial cell population has been expanded through a mechanical stimulus. (C) 1995 Wiley-Liss, Inc.

Diuron-induced rat urinary bladder carcinogenesis: Mode of action and human relevance evaluations using the International Programme on Chemical Safety framework

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Potential effects of the herbicide Diuron on mammary and urinary bladder two-stage carcinogenesis in a female Swiss mouse model

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Occupational and non-occupational risk factors in bladder cancer patients in an industrialized area located in former East-Germany [Berufliche und außerberufliche Risikofaktoren für das Harnblasenkarzinom in einem ehemaligen Industriegebiet der neuen Bundesländer]

Purpose: Several occupational carcinogens are metabolized by polymorphic enzymes. The distribution of the polymorphic enzymes N-acetyltransferase 2 (NAT2; substrates: aromatic amines), glutathione S-transferase M1 (GSTM1; substrates: e.g., reactive metabolites of polycyclic aromatic hydrocarbons), and glutathione S-transferase T1 (GSTT1; substrates: small molecules with 1-2 carbon atoms) were investigated. Material and Methods: At the urological department in Lutherstadt Wittenberg, 136 patients with a histologically proven transitional cell cancer of the urinary bladder were investigated for all occupations performed for more than 6 months. Several occupational and non-occupational risk factors were asked. The genotypes of NAT2, GSTM1, and GSTT1 were determined from leucocyte DNA by PCR. Results: Compared to the general population in Middle Europe, the percentage of GSTT1 negative persons (22.1 %) was ordinary; the percentage of slow acetylators (59.6%) was in the upper normal range, while the percentage of GSTM1 negative persons (58.8%) was elevated in the entire group. Shifts in the distribution of the genotypes were observed in subgroups who had been exposed to asbestos (6/6 GSTM1 negative, 5/6 slow acetylators), rubber manufacturing (8/10 GSTM1 negative), and chlorinated solvents (9/15 GSTM1 negative). Conclusions: The overrepresentation of GSTM1 negative bladder cancer patients also in this industrialized area and more pronounced in several occupationally exposed subgroups points to an impact of the GSTM1 negative genotype in bladder carcinogenesis. [Article in German]

Phenotypic impact of deregulated expression of class I histone deacetylases in urothelial cell carcinoma of the bladder

Deregulated expression of histone deacetylases (HDACs) has been implicated in tumorigenesis. Herein, we investigated class I HDACs expression in bladder urothelial cell carcinoma (BUCC), its prognostic value and biological significance. Significantly increased transcript levels of all HDACs were found in BUCC compared to 20 normal mucosas, and these were higher in lower grade and stage tumors. Increased HDAC3 levels were associated with improved patient survival. SiRNA experiments showed decrease cell viability and motility, and increased apoptosis. We concluded that class I HDACs play an important role in bladder carcinogenesis through deregulation of proliferation, migration and apoptosis, constituting putative therapeutic targets

DNA methylation patterns in bladder cancer and washing cell sediments: a perspective for tumor recurrence detection

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Transcriptional Profile of Diuron-Induced Toxicity on the Urinary Bladder of Male Wistar Rats to Inform Mode of Action

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Frequent p53 mutations and occasional loss of chromosome 4 in invasive bladder carcinoma induced by N-butyl-N-(4-hydroxybutil)nitrosamine in B6D2F1 mice

B6D2F1 mice (45/group) were treated with N-butyl-N-(4- hydroxybutyl)nitrosamine (BBN) or uracil as follows: Group 1 received 0.05% BBN in drinking water for the entire experiment, Group 2 received 5 mg of BBN by gastric gavage in 0.1 mL of 20% ethanol twice per week for 10 wk, Group 3 received a 2.5% uracil-containing diet for the entire experiment, and Group 4 was controls (received 0.1 mL of 20% ethanol by gavage twice per week for 10 wk). The surviving mice in Group 1 were killed after week 26 and those in the other groups after week 30. By week 15, three of 11 Group 1 and one of 15 Group 2 mice had bladder carcinoma. By 26 and 30 wk, respectively, invasive carcinomas were observed in 33 of 34 and six of 21 mice in Groups 1 and 2 and renal pelvic carcinomas in 11 of 34 and three of 21 mice in Groups 1 and 2. Four of 19 uracil-treated mice had bladder nodular hyperplasia. By polymerase chain reaction-single-strand conformation polymorphism and sequence analyses, 16 of 20 and two of five bladder carcinomas from Groups 1 and 2, respectively, showed mutations in the p53 gene. Ha-ras mutation was present in one case. Loss of heterozygosity analysis with simple-sequence length polymorphism markers for chromosome 4 showed that 10 of 21, two of 15, and nine of 13 mice in Groups 1-3, respectively, had heterozygous or homozygous deletions. B6D2F1 mice are therefore susceptible to the urothelial carcinogenic effects of BBN and develop frequent p53 mutations and chromosome 4 deletions. Chromosome 4 deletions were also seen with uracil.

Comparison of uroplakin expression during urothelial carcinogenesis induced by N-butyl-N-(4-hydroxybutyl)nitrosamine in rats and mice

The expression of uroplakins, the tissue-specific and differentiation- dependent membrane proteins of the urothelium, was analyzed immunohistochemically in N butyl-N-(4-hydroxybutyl)nitrosamine (BBN)-treated rats and mice during bladder carcinogenesis. Male Fischer 344 rats were treated with 0.05% BBN in the drinking water for 10 wk and were cuthanatized at week 20 of the experiment. BBN was administered to male B6D2F1 mice; it was either provided at a rate of 0.05% in the drinking water (for 26 wk) or 5 mg BBN was administered by intragastric gavage twice weekly for 10 wk, followed by 20 wk without treatment. In rats, BBN-induced, noninvasive, low grade, papillary, transitional cell carcinoma (TCC) showed decreased uroplakin-staining of cells lining the lumen but showed increased expression in some nonluminal cells. In mice, nonpapillary, high-grade dysplasia, carcinoma in situ, and invasive carcinoma were induced. There was a marked decrease in the number of uroplakin-positive cells lining the lumen and in nonluminal cells. This occurred in normal-appearing urothelium in BBN-treated mice and in dysplasic urothelium, in carcinoma in situ, and in invasive TCC. The percentage of uroplakin-positive nonluminal cells was higher in control mice than in rats, but it was lower in the mouse than in the rat after BBN treatment. Uroplakin expression was disorderly and focal in BBN-treated urothelium in both species. These results indicate that BBN treatment changed the expression of uroplakins during bladder carcinogenesis, with differences in rats and mice being related to degree of tumor differentiation.

Chromosomal imbalances in successive moments of human bladder urothelial carcinoma

Objective: To understand developmental characteristics of urinary bladder carcinomas (UBC) by evaluating genomic alterations and p53 protein expression in primary tumors, their recurrences, and in the morphologically normal urothelium of UBC patients. Methods: Tumors and their respective recurrences, six low-grade and five high-grade cases, provided 19 samples that were submitted to laser microdissection capture followed by high resolution comparative genomic hybridization (HR-CGH). HR-CGH profiles went through two different analyses-all tumors combined or classified according to their respective histologic grades. In a supplementary analysis, 124 primary urothelial tumors, their recurrences, and normal urothelium biopsied during the period between tumor surgical resection and recurrence, were submitted to immunohistochemical analyses of the p53 protein. During the follow-up of at least 21 patients, urinary bladder washes citologically negative for neoplastic cells were submitted to fluorescence in situ hybridization (FISH) to detect copy number alterations in centromeres 7, 17, and 9p21 region. Results and Conclusions: HR-CGH indicated high frequencies (80%) of gains in 11p12 and losses in 16p12, in line with suggestions that these chromosome regions contain genes critical for urinary bladder carcinogenesis. Within a same patient, tumors and their respective recurrences showed common genomic losses and gains, which implies that the genomic profile acquired by primary tumors was relatively stable. There were exclusive genomic alterations in low and in high grade tumors. Genes mapped in these regions should be investigated on their involvement in the urinary bladder carcinogenesis. Successive tumors from same patient did not present similar levels of protein p53 expression; however, when cases were grouped according to tumor histologic grades, p53 expression was directly proportional to tumor grades. Biopsies taken during the follow-up of patients with history of previously resected UBC revealed that 5/15 patients with no histologic alterations had more than 25% of urothelial cells expressing the p53 protein, suggesting that the apparently normal urothelium was genomically unstable. No numerical alterations of the chromosomes 7, 17, and 9p21 region were found by FISH during the periods free-of-neoplasia. Our data are informative for further studies to better understand urinary bladder urothelial carcinogenesis. © 2013 Elsevier Inc.

Genetic predictors of bladder cancer with an emphasis on methylation-related genes

Although tobacco exposure remains the prevailing risk factor for bladder cancer (BC), only a small percentage of exposed individuals develop cancer, suggesting that tobacco-related carcinogenesis is modulated by genetic susceptibility and possibly by DNA methylation-related events. Methylation patterns established by DNA methyltransferases (DNMTs) are influenced by dietary folate and genetic polymorphisms in the methylene-tetrahydrofolate reductase gene (MTHFR). Therefore, we hypothesized that DNA methylation-related genes, such as DNMT3B and MTHFR, might modulate BC risk. ^ In a study of 514 Caucasian BC cases and 498 healthy Caucasian controls examining the DNMT3B C46359T polymorphism, CC genotype was found to be a risk factor in women (Odds Ratio (OR) = 1.79), but not in men. This risk was further increased among women who were never smokers, consumed low dietary folate, and had adverse variants of MTHFR. In addition, higher DNMT3B expression among smokers was a risk factor (OR = 4.27) and correlated with genetic variants of the DNMT3B C46359T polymorphism, providing salient evidence for the risk associated with the CC variant. This suggests that the DNMT3B CC variant may confer a predisposition toward aberrant de novo methylation of CpG islands in critical tumor suppressor genes. ^ The convergence of alterations in DNMT3B, associated with promoter methylation, and reduced dietary folate consumption, accompanying global hypomethylation and genetic instability, may act synergistically to promote bladder carcinogenesis, especially in women. The results of this study unveiled new gender-specific paradigms of BC risk for women and demonstrated that this risk can be modified by folate consumption as well as polymorphisms in the folate pathway. ^