Background: New methods for detection of bladder cancer are needed because cystoscopy is both invasive and expensive and urine cytology has low sensitivity. We screened proteins as tumor markers for bladder cancer by proteomic analysis of cancerous and healthy tissues and investigated the diagnostic accuracy of one such marker in urine. Methods: Three specimens of bladder cancer and healthy urothelium, respectively, were used for proteome differential display using narrow-pH-range twodimensional electrophoresis. To evaluate the presence of calreticulin (CRT) as detected by Western blotting, we obtained 22 cancerous and 10 noncancerous surgical specimens from transurethral resection or radical cystectomy. To evaluate urinary CRT, we collected 70 and 181 urine samples from patients with and without bladder cancer, respectively. Anti-CRT COOH-terminus antibody was used to detect CRT in tissue and urine. Results: Proteomic analysis revealed increased CRT (55 kDa; pI 4.3) in cancer tissue. Quantitative Western blot analysis showed that CRT was increased in cancer tissue (P ‫؍‬ 0.0003). Urinary CRT had a sensitivity of 73% (95% confidence interval, 62-83%) at a specificity of 86% (80 -91%) for bladder cancer in the samples tested. Conclusions: Proteomic analysis is useful in searching for candidate proteins as biomarkers and led to the

Using proteomic analysis, we previously identified calreticulin (CRT) as a potentially useful urinary marker for bladder cancer. Now, we have also identified γ γ γ γ-synuclein (SNCG) and a soluble isoform of catechol-o-methyltransferase (s-COMT) as novel candidates for tumor markers in bladder cancer, by means of proteomic analysis. In the process of establishing a superior tumor marker system, we investigated the diagnostic value of a combination assay of these three proteins. Voided urine samples were obtained from 112 bladder cancer and 230 control patients. Urinary CRT, SNCG, and s-COMT were measured as a combined marker by quantitative western blot analysis. Relative concentration of each protein was calculated and the diagnostic value of a concomitant examination of these markers was evaluated by receiver operator characteristic analysis. With the best diagnostic cutoff, the overall sensitivity of the combined markers was 76.8% (95% confidence interval, 69-81%) with a specificity of 77.4% (72-80%), while those of a single use of CRT were 71.4% and 77.8%, respectively. When evaluated in relation to tumor characteristics, such as grade, stage, size, and outcome of urinary cytology, the diagnostic capacity of the combined markers was equal to or better than that of CRT in all categories. Concomitant use of CRT, SNCG, and s-COMT had higher sensitivity for detection of bladder cancer than did single use of CRT. Our study suggests that use of this panel of markers will improve the diagnosis of bladder cancer and may allow the development of a protein microarray assay or multi-channel enzyme-linked immunosorbent assay. (Cancer Sci 2004; 95: 955-961) ladder cancer is a common urothelial cancer with an estimated 57,400 and 13,000 diagnosed cases per year in the United States and Japan, respectively. 1, 2) Approximately 70% of bladder cancers are superficial 3) and respond well to endoscopic transurethral resection. However, 50% to 70% of these patients experience tumor recurrence, and 10% to 15% of recurrent tumors progress to muscle invasive disease. 4) Because the propensity for local recurrence extends over the lifetime, patients with superficial bladder cancer must undergo life-long surveillance.Cystoscopy is the mainstay in diagnosing bladder cancer. However, this procedure is unsuitable for screening of large groups because of its invasiveness and expense. In addition, follow-up cystoscopy for bladder cancer patients treated endoscopically represents a considerable part of the workload of any urological unit. Therefore, new tests with high specificity and sensitivity that are easy to perform are needed for both screening and monitoring the response to treatment of bladder cancer. Voided urine cytology (VUC) has been used in both diagnosis and follow-up of superficial bladder cancer since its first description by Papanicolaou and Marshall in 1945, but it has poor sensitivity although high specificity, particularly in well-differentiated cancer.5) To date, several urine-based markers for bladder cancer have ...

Proteome analysis of bladder cancer with narrow-range pH 2-DE has identified a novel protein on chromosome 7 encoded by ORF 24 (C7orf24) as one of the highly expressed proteins in cancer cells. C7orf24 is currently registered in the protein database as a hypothetical protein with unknown function. The homologs of C7orf24 in other animals have also been registered as putative protein genes. Western blot analysis using a mAb against C7orf24 confirmed its higher expression in bladder cancer compared with normal tissue. Several other cancer cell lines were also found to express C7orf24. However, the introduction of C7orf24 into Rat-1 or NIH3T3 cells did not cause malignant transformation. A stable transfectant of NIH3T3 cells with recombinant retrovirus vector was produced for a growth rate assay, and a higher growth rate was observed in C7orf24-expressing cells compared with the controls. Six kinds of small interfering RNAs (siRNAs) were then produced, and C7orf24-siRNA#5 showed a strong knockdown effect on protein expression and significant antiproliferative effects on cancer cell lines were demonstrated by the MTT assay. Therefore, C7orf24 may have an important role in cancer cell proliferation, and may be an appropriate therapeutic target molecule against cancer.

Although the clinical implications of the over expression of uroplakin III and III-delta4 in nonulcerative interstitial cystitis bladders remains to be clarified, from the diagnostic viewpoint uroplakin III-delta4 is a potential marker for identifying nonulcerative interstitial cystitis.

Hypermethylation of tumor-suppressor genes has been implicated in the pathogenesis of human cancers. Although a growing number of genes showing hypermethylation is being reported in human cancer, methylation profiles of tumor-related genes in testicular neoplasms have not been well elucidated. This study was designed to show the methylation profiles of multiple CpG islands in testicular germ cell tumors (TGCTs) in comparison with those in testicular malignant lymphomas. We studied the methylation status of E-cadherin, CDKN2B, CDKN2A, BRCA1, RB1, VHL, RASSF1A, RARB, and GSTP1 by use of TGCT tissues and testicular malignant lymphoma tissues (25 primary TGCT tissues and three primary testicular lymphoma tissues). Methylation was not observed in E-cadherin, CDKN2B, CDKN2A, BRCA1, RB1, VHL, RASSF1A, RARB, and GSTP1 in any of the TGCT tissues. In contrast, all three (100%) of the testicular lymphoma tissues demonstrated hypermethylation of E-cadherin, RASSF1A, and RARB, but not CDKN2B, CDKN2A, BRCA1, RB1, VHL, and GSTP1. These data demonstrate that a distinctive epigenetic phenotype underlies the TGCTs and testicular lymphomas at the CpG sites of E-cadherin, RASSF1A, and RARB; a distinctive epigenetic phenotype was not observed among seminomatous TGCTs and non-seminomatous TGCTs at the CpG sites examined.

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Abbreviations & Acronyms 2D-DIGE = two-dimensional difference gel electrophoresis 2DE = two-dimensional electrophoresis A-FABP = adipocyte-fatty acid binding protein Apo-A1 = apolipoprotein A1 EGFR = epidermal growth factor receptor ELISA = enzyme-linked immunosorbent assay ESI = electrospray ionization GGCT = gammaglutamylcyclotransferase iTRAQ = isobaric tags for relative and absolute quantification LC = liquid chromatography MALDI = matrix-assisted laser desorption/ionization MS = mass spectrometry MS/MS = tandem mass spectrometry m/z = mass-to-charge ratio SELDI = surface-enhanced laser desorption/ionization SILAC = stable isotope labeling by amino acid in cell culture Abstract: In all creatures including humans, the molecules that function in accordance with the genetic code are mainly proteins. After completing the sequencing of the human genome, rapid progress has been made in proteome analysis. The primary structures of almost all proteins were determined by the human genome sequence. However, the whole picture of proteins cannot be elucidated because of alternative splicing and posttranslational modifications. Therefore, genomic as well as systematic and comprehensive information of proteins is required. Modern methods of proteomics have dramatically improved the quality and speed of protein analysis. Developments in both bioinformatics and mass spectrometry have contributed to the technical improvement, making it possible to identify proteins in a short time with high accuracy even from a very small sample. In the field of cancer research, many studies of useful diagnostic and prognostic biomarkers using these proteomic technologies have been reported, and target molecules for treatment have been explored. The aim of the present review was to summarize the basic technologies of proteomics and recent research in the field of urothelial cancer obtained using proteomic methods.

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