Masao Deguchi

β-Catenin Mutations Correlate with Over Expression of C-myc and Cyclin D1 Genes in Bladder Cancer

PURPOSE We hypothesized that over expression of c-myc and cyclin D1 genes is transcriptionally activated by beta-catenin mutation independent of gene amplification in bladder cancer. To test this hypothesis we investigated the relationship of beta-catenin mutation to c-myc and cyclin D1 mRNA with special reference to the changes in copy number of the 2 genes. MATERIALS AND METHODS Genomic DNA and total RNA were extracted from 59 bladder cancer specimens and from 31 histologically normal specimens of bladder mucosa. We performed beta-catenin deletion screening by polymerase chain reaction (PCR) using primers spanning exons 3 (including the glycogen synthase kinase-3beta consensus motif), 5 and 6. Mutational changes in beta-catenin in exons 3, 5 and 6 were detected by each PCR-single strand conformational polymorphism analysis followed by direct DNA sequencing. mRNA expression and copy numbers of c-myc and cyclin D1 were determined by semiquantitative reverse transcriptase-PCR and competitive genomic PCR. RESULTS Missense mutations of beta-catenin found in grade 3 bladder cancer were involved in the consensus motif of glycogen synthase kinase-3beta in exon 3. These cancers showed strong intracellular accumulation of beta-catenin and intense expression of c-myc and cyclin D1 mRNA compared with samples lacking the beta-catenin mutation. When grade 3 cancers were compared, expression levels of c-myc and cyclin D1 mRNA were still higher in those with versus without the beta-catenin mutation. In bladder cancers with beta-catenin mutations copy numbers of the c-myc and cyclin D1 genes did not amplify. CONCLUSIONS Bladder cancer harboring a beta-catenin mutation may represent aggressive biological behavior with enhanced proliferating activity. These findings are important for understanding the role of beta-catenin mutation in the pathogenesis of bladder cancer.

Functional Loss of the γ-Catenin Gene through Epigenetic and Genetic Pathways in Human Prostate Cancer

γ-Catenin is a cell adhesion molecule and a candidate mediator of Wnt signal transduction. We hypothesized that impaired regulation of γ-catenin through genetic and epigenetic pathways is associated with the pathogenesis of prostate cancer. To test this hypothesis, cytosine-phosphate-guanine methylation, loss of heterozygosity (LOH), and mutation status of the γ-catenin gene were analyzed in cultured prostate cancer cell lines, 180 localized prostate cancers, 69 benign prostatic hyperplasias, and 11 hormone refractory prostate cancers (HRPC). In prostate cancer cell lines (DuPro, LNCaP, ND-1, and PC3), γ-catenin mRNA transcripts were increased after 5-aza-2′-deoxycytidine treatment. In localized prostate cancer, γ-catenin expression was lower but prevalence of γ-catenin methylation was higher compared with benign prostatic hyperplasia. However, γ-catenin methylation did not correlate with Gleason sum, pT category, or capsular penetration. Among localized prostate cancers with positive γ-catenin methylation, the presence of LOH at chromosome 17q21 was closely related to down-regulation of γ-catenin mRNA expression. The γ-catenin mutations were not found in localized prostate cancers, whereas six mutations were found in five HRPCs within or close to the GSK-3β consensus motif phosphorylation site, among which four HRPCs showed strong nuclear γ-catenin accumulation. In these four HRPCs, Bcl-2 expression was increased, whereas the target of the Wnt signal, c-myc , was only expressed in one HRPC. Therefore, although epigenetic γ-catenin methylation is an early event in the development of prostate cancer, simultaneous events of epigenetic cytosine-phosphate-guanine methylation and genetic LOH may be responsible for functional loss of γ-catenin. The γ-catenin mutation related to Bcl-2 overexpression has a significant effect on the pathogenesis of HRPC. This is the first report to characterize the epigenetic and genetic regulation of γ-catenin in human prostate cancer.

Catechol-O-methyltransferase Gene Polymorphisms in Benign Prostatic Hyperplasia and Sporadic Prostate Cancer

Various carcinogenic metabolites, including catechol estrogens, play a role in malignant transformation. An enzyme that is capable of neutralizing the genotoxic effects of these compounds is catechol-O-methyltransferase (COMT). A variant form of this enzyme has been shown to reduce its activity by up to 4-fold; thus, we hypothesize that single nucleotide polymorphisms of the COMT gene can be a risk factor for benign prostatic hyperplasia (BPH) and prostate cancer. To test this hypothesis, the genetic distribution of three different COMT polymorphisms at codon 62 (C→T), codon 72 (G→T), and codon 158 (G→A) were analyzed in 131 normal healthy subjects, 134 BPH, and 178 sporadic prostate cancer samples from a Japanese population. Results of these experiments show that the variant genotype at codon 62 (P = 0.060) and codon 158 (P = 0.047) are risk factors for prostate cancer but not BPH when compared with normal controls. Odds ratio (OR) and 95% confidence interval (95% CI) for cancer were 3.24 and 1.38 to 7.61, respectively, for codon 62 T/T genotype when compared with wild type. At codon 158, the A/A variant for cancer had an OR of 3.00 with a 95% CI of 1.38 to 6.54 compared with wild type. Codons 62 and 158 were in linkage disequilibrium (LD), and when compared with the C-G haplotype, other types (C-A, T-G, T-A) were observed to be associated with prostate cancer (P = 0.040) but not BPH. Codon 72 on the other hand, was not in LD with either codon 62 or 158. The homozygous variant on codon 72 was rare in this Japanese population, and the heterozygous G/T at this codon was not associated with either prostate cancer or BPH. When evaluating the risk of COMT polymorphisms with stage or grade of cancer, no associations were observed for any of the genotypes with the exception of a tendency (P = 0.096) for the variant A allele on codon 158 to be correlated with higher stages (≥T3) of cancer. This is the first report that shows the polymorphisms of COMT to be associated with sporadic prostatic carcinogenesis. These results are important in understanding the role of COMT polymorphisms in the pathogenesis of prostate cancer. (Cancer Epidemiol Biomarkers Prev 2006;15(2):238–44)

Endoscopic management of a traumatic disruption of the bulbous urethra using a thin trocar puncture.

Abstract A case is reported in which complete disruption of the bulbous urethra resulted in a straddled‐type injury, which was managed by endoscopic realignment with a thin trocar needle. The endoscopic urethroplasty consisted of: (i) direct observation of the proximal end of the obliterated urethra by an antegrade flexible cystoscope; (ii) adjustment of the direction of trocar penetration under fluoroscopy and direct vision; (iii) confirmation of the exact trocar position and penetration by antegrade flexible cystoscope; and (iv) placement of the guidewire following the trocar penetration as guidance for urethrotomy. A Foley catheter was left in place for 6 weeks. To date, no further endoscopic revision has been required. Although long‐term follow up and more experience are required, this technique is reported because it appears to be safe, reproducible, simple and minimally invasive.

Methylation of the γ-Catenin Gene is Associated With Poor Prognosis of Renal Cell Carcinoma

Purpose: g-Catenin is a cell adhesion protein, and its functional loss is associated with tumor invasion and metastasis. We hypothesize that (1) promoter CpG methylation regulates the expression and function of the g-catenin gene in renal cell carcinoma (RCC) and (2) methylation of the g-catenin gene is associated with poor prognosis of RCC. To test these hypotheses, we analyzed the CpG methylation status of the g-catenin gene and its correlation with clinical outcome in RCC. Experimental Design: Genomic DNA and total RNA were extracted from three renal cancer cell lines (A498, Caki-1, and Caki-2) and 54 RCC tissue samples with their corresponding normal kidney tissue samples. Expression of g-catenin gene was analyzed by reverse transcription-PCR and immunostaining. Promoter methylation was analyzed by two different methylation-specific PCR (MSP-A and MSP-B), and the results were verified by DNA sequencing. Results: The demethylating agent (5-aza-2V-deoxycytidine) increased levels of mRNA transcript of the g-catenin gene in three renal cancer cell lines. g-Catenin mRNA and protein expression were significantly reduced in RCC samples compared with normal kidney samples, respectively (P < 0.05). MSP-A and MSP-B bands were detected in 45 of 54 (83.3%) and 49 of 54 (90.7%) RCC samples, respectively. In normal kidney, weak products of MSP-A and MSP-B were detected in 5 of 54 (9.3%) and 6 of 54 (11.1%) samples, respectively. Likewise, both MSP-A and MSP-B ratios were significantly higher in RCC samples compared with normal kidney samples, respectively (P < 0.01). Multivariate analysis revealed that the MSP-B ratio was a powerful and independent predictor superior to nuclear grade and Robson stage with respect to survival and disease progression (P = 0.029 and 0.0071, respectively). No mutations in the NH2-terminal region of g-catenin were found in this study. Conclusion: Expression of g-catenin is regulated by promoter CpG methylation, and the balance between methylated and unmethylated RCC cell populations could determine its functional role. Because the conventional nuclear grade and/or staging system have some limitations to predict precise clinical outcome, this is the first report demonstrating that promoter CpG methylation of g-catenin can be an independent and superior predictor for survival and disease progression.