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Purpose: We compared the differential expression of cyclooxygenase-2 in normal bladder tissue, primary bladder transitional cell carcinoma and transitional cell carcinoma metastases to lymph nodes, and determined whether cyclooxygenase-2 expression is associated with molecular alterations commonly found in bladder transitional cell carcinoma and clinical outcomes after radical cystectomy., Materials and Methods: Immunohistochemical staining for cyclooxygenase-2, survivin (Novus Biologicals, Littleton, Colorado), p21, p27, pRB, p53, MIB-1, Bax, Bcl-2, cyclin D(1) (Dakotrade mark), cyclin E (Oncogene, Cambridge, Massachusetts) and caspase-3 (Cell Signaling, Beverley, Massachusetts) was performed on archival bladder specimens from 9 subjects who underwent cystectomy for benign causes, 21 patients who underwent transurethral resection and 157 consecutive patients after radical cystectomy, and on 41 positive lymph nodes., Results: Cyclooxygenase-2 was expressed in none of the 9 normal bladder specimens (0%), 52% of transurethral resection specimens, 62% of cystectomy specimens and 80% of lymph nodes involved with transitional cell carcinoma. Cyclooxygenase-2 expression was associated with higher pathological stage, lymphovascular invasion and metastases to lymph nodes (p=0.001, 0.045 and 0.002, respectively). Cyclooxygenase-2 expression was associated with altered expression of p53 (p=0.039), pRB (p=0.025), cyclin D1 (p=0.034) and caspase-3 (p=0.014). On univariate analysis cyclooxygenase-2 expression was associated with an increased risk of disease recurrence and bladder cancer specific mortality (p=0.0189 and 0.0472, respectively). However, on multivariate analysis only pathological stage and metastases to lymph nodes were associated with disease recurrence (p<0.001 and <0.001) and survival (p<0.001 and 0.015, respectively)., Conclusions: Cyclooxygenase-2 is not expressed in normal bladder urothelium. Cyclooxygenase-2 over expression is associated with pathological and molecular features of biologically aggressive disease, suggesting a role for cyclooxygenase-2 in bladder cancer development and invasion.

Purpose: Nitric oxide (NO) is an antibacterial factor that is produced by the enzyme inducible NO synthase (iNOS). Uroepithelial cells express iNOS in experimental models of urinary tract infection but the stimulatory and regulatory mechanisms are still unclear. We investigated the influence of uropathogenic Escherichia coli strains with different fimbrial expression and the effect of proinflammatory cytokines on the host iNOS response., Materials and Methods: A498 human kidney epithelial cells were stimulated with different uropathogenic E. coli strains, namely the P and type 1-fimbriated clinical isolate AD110, the recombinant P-fimbriated strain E. coli HB101(pPIL110-75) and the recombinant type 1-fimbriated strain E. coli AAEC191A(pPKL4). NO production was determined as nitrite production in cell culture medium. Studies of nuclear factor-kappaB (NF-kappaB) binding to the iNOS promoter and reverse transcriptase-polymerase chain reaction of iNOS mRNA were performed to investigate iNOS gene activation in response to uropathogenic E. coli. The effect of interleukin (IL)-6, IL-8 and transforming growth factor-beta on NO production was also examined., Results: E. coli per se failed to induce NO production and iNOS mRNA in A498 cells. However, in combination with interferon-gamma AD110 and the type 1-fimbriated strain caused a small increase in NO production and iNOS mRNA. AD110 stimulated A498 cells demonstrated weak binding of NF-kappaB to a human iNOS promoter sequence. IL-6, IL-8 and transforming growth factor-a did not affect NO production in A498 cells., Conclusions: Uropathogenic bacteria are weak inducers of human uroepithelial iNOS, which may be related to insufficient binding of NF-kappaB to iNOS promoter. The uroepithelial iNOS response did not appear to be regulated by proinflammatory cytokines.

Purpose: Increased prostaglandin production correlates positively with cancer risk and cyclooxygenase (COX)-2, the inducible rate limiting enzyme for prostaglandin synthesis, is elevated in bladder cancer cases. Urinary prostaglandin levels and COX-2 expression in urine particulates may increase in urogenital cancer, including bladder cancer, and with infectious and inflammatory processes, including urinary tract infections and that resulting from bacillus Calmette-Guerin (BCG) treatment for bladder cancer., Materials and Methods: Urinary prostaglandin E2 levels were measured in patients with a urinary tract infection before and after treatment, urogenital cancer (including bladder cancer), bladder cancer in remission and bladder cancer with BCG treatment. COX-1 and COX-2 mRNA and protein were assessed in the human ureter, in normal human bladder muscle and urothelium, and in urine particulates from patients with urinary tract infections, bladder cancer and bladder cancer with BCG treatment., Results: COX-1 protein, and mRNA and COX-2 mRNA were expressed in the ureter, and bladder muscle and urothelium. Urinary prostaglandin E2 levels and COX-2 protein expression in urine particulates were elevated in patients with urinary tract infections and with bladder cancer compared with age matched controls. Successful treatment for urinary tract infections and bladder cancer lowers urinary prostaglandin E2 levels. Urinary prostaglandin E2 and COX-2 protein levels are elevated during BCG treatment., Conclusions: Increased urinary prostaglandin E2 production and COX-2 protein expression correlate with urogenital cancer, urinary tract infections and inflammatory processes, such as those induced by BCG. Patients in whom urinary tract infection was treated with antibiotics or in whom bladder cancer is in remission have reduced urinary prostaglandin E2 compared with those who have active disease.

Purpose: Nitric oxide is a free radical gas synthesized from L-arginine by a family of isoenzymes called nitric oxide synthase that has an important role in smooth muscle relaxation. L-arginine, the substrate for nitric oxide synthase, may be beneficial under pathophysiological conditions in the bladder, as in interstitial cystitis. We determined the localization of nitric oxide synthase and the target enzyme of NO, soluble guanylyl cyclase, in the human bladder., Materials and Methods: Benign bladder tissues were obtained from 18 patients with localized superficial bladder tumors undergoing transurethral bladder resection. Histochemical nicotinamide adenine dinucleotide phosphate-diaphorase staining, nitric oxide synthase immunohistochemical testing and soluble guanylyl cyclase immunoreactivity studies were performed in all benign tissue specimens., Results: A different pattern of nitric oxide synthase expression was confirmed by nicotinamide adenine dinucleotide phosphate-diaphorase staining and immunohistochemical testing for endothelial and neuronal nitric oxide synthase. In addition to endothelial nitric oxide synthase expression, detrusor smooth muscle was recognized as an important location of endothelial nitric oxide synthase, while the urothelium had only small endothelial nitric oxide synthase positive cell clusters. Neuronal nitric oxide synthase expression was only found in nitrinergic fibers of the submucosal surface and between muscle cells. Detrusor and vascular smooth muscle as well as interstitial cells, nerve fibers and transitional epithelium were recognized as targets of nitric oxide, as shown by soluble guanylyl cyclase expression., Conclusions: The distribution of constitutive nitric oxide synthase isoforms and soluble guanylyl cyclase provides evidence of nitric oxide-cyclic guanosine monophosphate mediated regulation of detrusor smooth muscle relaxation, neurotransmission and blood flow. Furthermore, the urothelium may also be a target of nitric oxide.

Purpose: To determine the changes in expression of bikunin in renal epithelial cells on exposure to oxalate and calcium oxalate crystals., Materials and Methods: This study used reverse transcription polymerase chain reaction (RT-PCR) to examine bikunin mRNA expression levels in MDCK cells exposed to oxalate or calcium oxalate crystals. Poly(A)+ RNA was isolated directly from renal epithelial cells, then converted to cDNA with random primers and reverse transcriptase. To quantify the expression level ofbikunin mRNA, we developed a competitive DNA template for competitive PCR analysis. The PCR products were resolved by electrophoresis on 1.3% agarose gel and visualized with ethidium bromide. In this system, we could quantify the exact number of bikunin mRNA transcripts. Bikunin mRNA from rat liver was expressed as a positive control., Results: Bikunin mRNAs and competitive templates from renal epithelial cells were expressed in all samples as 434 bp and 312 bp bands, respectively. Bikunin expression was significantly increased in oxalate exposed cells. Cells exposed to calcium oxalate monohydrate crystals or latex beads showed no significant change in expression of bikunin. Western blotting analysis also showed increased expression of bikunin and inter-alpha-inhibitor-related proteins in the culture medium of oxalate exposed cells., Conclusions: These findings suggest that renal epithelial cells express bikunin gene and have the capability to produce bikunin when stimulated by certain agents such as oxalate. This increased expression and production of bikunin may represent a protective response of renal epithelial cells to nephrotoxic challenges of oxalate.