Combined functional genome survey of therapeutic targets for clear cell carcinoma of the kidney.

OBJECTIVE: Emerging molecular targeting therapeutics have been incorporated into the management of advanced renal cell carcinoma; however, their efficacy remains limited. The aim of this study was to catalog potential therapeutic target molecules for renal cell carcinoma. METHODS: We first selected genes up-regulated in clear cell renal cell carcinoma relative to surrounding normal kidney tissues in 10 patients (Study Cohort) using high-density exon arrays that detect all potential transcripts predicted in the human genome. The selected genes were subjected to independent validation in another set of 10 patients (Validation Cohort) using real-time reverse transcriptase polymerase chain reaction and functional screening using small interfering RNA in six clear cell renal cell carcinoma cell lines. RESULTS: We identified 164 genes whose expression was significantly elevated in clear cell renal cell carcinoma (P< 0.0001 [Student's t-test] and at least a 3-fold change in transcription signal). We finally extracted 33 genes required for maintaining cell proliferation in at least two clear cell renal cell carcinoma cell lines. The 33 genes included 13 genes known to be associated with the development/progression of renal cell carcinoma, including CAIX and FLT-1, confirming the robustness of the current strategy. CONCLUSIONS: Through a combination of genome-wide expression and functional assays, we identified a set of genes with high potential as targets for drug development. This method is rapid and comprehensive and could be applied to the discovery of diagnostic biomarkers and therapeutic targets for cancers other than clear cell renal cell carcinoma.

Nucleotide excision repair gene polymorphisms may predict acute toxicity in patients treated with chemoradiotherapy for bladder cancer.

AIMS: Platinum-based chemoradiotherapy (CRT) as bladder conservation therapy has shown promising results for muscle-invasive bladder cancer. However, treatment-related toxicity remains a major consideration in therapeutic planning. Some common polymorphisms in genes involved in DNA repair (encoding enzymes that repair DNA damaged by platinum agents and ionizing radiation) are reported to result in modulation of the repair capacity. We investigated associations between functional genetic polymorphisms involved in DNA repair and acute toxicity of CRT to determine the predictive value of these polymorphisms for toxicity. MATERIALS & METHODS: The study group comprised of 101 bladder cancer patients treated with platinum-based CRT, and seven polymorphisms in XPC (Lys939Gln, rs2228001), XPD (Lys751Gln, rs13181), XPG (Asp1104His, rs17655), XRCC1 (Arg399Gln, rs25487), XRCC3 (Thr241Met, rs861539), TP53 (Arg72Pro, rs1042522) and MDM2 (SNP309, T>G, rs2279744) were genotyped. RESULTS: More than two total variant alleles in nucleotide excision repair genes, including XPC, XPD and XPG, were significantly associated with grade 3 or 4 neutropenia (adjusted odds ratio [aOR]: 6.8; 95% CI: 2.0-26; p = 0.0026). There were no significant associations between any genotypes and grade 2 or greater nausea/vomiting or diarrhea. Any grade 3 or 4 hematological toxicity was significantly associated with the Gln/Gln or Lys/Gln + Gln/Gln genotypes of XPC compared with Lys/Lys (aOR: 10; 95% CI: 2.0-65; p = 0.0070 or aOR: 6.3; 95% CI: 1.9-29; p = 0.0069; respectively). CONCLUSION: These results suggest that nucleotide excision repair gene polymorphisms, especially in XPC, might potentially be predictive factors for acute toxicity of CRT for bladder cancer, helping individual patient selection for bladder conservation therapy. However, further studies with larger sample sizes are needed to draw final conclusions.

Genetic alterations at 13q14 may correlate with differences in the biological behavior of prostate cancer between Japanese and Caucasian men.

The incidence of prostate cancer and the resultant mortality rates in Japanese men are lower compared with the rates for Caucasians; however, the Gleason score at diagnosis is higher in Japanese men compared with Caucasians. Loss of 13q is one of the most common chromosomal alterations in prostate cancer. To elucidate the difference in the rate of loss of 13q between Japanese and Caucasian men, we examined the allelic imbalance (AI) on chromosome 13q in 32 Japanese and 39 German prostate cancer patients with a fluorescent polymerase chain reaction technique using 12 microsatellite markers. Benign and malignant histology was identified by a single pathologist and laser capture microdissection was used to gather cancer cells. Although there were no statistical differences in patient background characteristics, the frequency of AI at 13q14 (D13S1253) and at 13q21 (D13S166) was significantly higher in Japanese patients compared with German patients (p = 0.0128 and p = 0.0078, respectively). The frequency of AI at 13q14 was significantly higher in tumors with high Gleason scores (GS) compared with tumors with low GS (p = 0.0478). The present observations suggest that the frequency of genetic alterations at 13q14 may underlie differences in the biological behavior of prostate cancer between Japanese and Caucasian populations.

Preoperative erythrocyte sedimentation rate is an independent prognostic factor in Japanese patients with localized clear cell renal cell carcinoma.

INTRODUCTION: Few studies have examined the prognostic significance of common laboratory variables in Japanese patients with localized clear cell renal cell carcinoma (CCRCC). We evaluate the prognostic significance of preoperative laboratory variables in Japanese patients with localized CCRCC. PATIENTS AND METHODS: The study included 110 Japanese patients who were pathologically confirmed as nonmetastatic CCRCC (pT1-3 N0M0) after radical nephrectomy at our institution. We assessed the clinical (including laboratory measurements) and pathological findings, with the survival rates after surgery. RESULTS: Tumor stage and erythrocyte sedimentation rate (ESR) were identified as significant independent prognostic factors of progression-free survival in multivariate analysis. As for the prognostic factors for disease-specific survival, tumor stage and ESR had prognostic significance both in univariate and multivariate analyses. When the analysis was limited to pT1, multivariate analysis showed that only ESR was an independent prognostic factor for disease-specific survival. CONCLUSIONS: Preoperative ESR is an independent prognostic factor in Japanese patients with localized CCRCC, especially in patients with pT1.

Risk of concomitant carcinoma in situ determining biopsy candidates among primary non-muscle-invasive bladder cancer patients: retrospective analysis of 173 Japanese cases.

OBJECTIVES: To determine candidates for bladder biopsies among Japanese primary non-muscle-invasive bladder cancer patients according to the risk of concomitant carcinoma in situ (CIS). METHODS: Between January 1992 and August 2006, 173 primary non-muscle-invasive bladder cancer cases underwent transurethral resection of the bladder tumor with bladder biopsies for the detection of CIS. Correlations between biopsy results and preoperative/pathological features were retrospectively analyzed. RESULTS: Positive cytology was statistically associated with the presence of concomitant CIS in multivariate analysis (P < 0.01). Abnormal cystoscopic appearance outside the tumor almost achieved statistical significance in multivariate analysis among preoperative factors (P = 0.06). In our series, one (12.5%) of eight low-risk, 18 (24.7%) of 73 intermediate-risk and 41 (59.4%) of 69 high-risk cases had CIS in normal-looking sites, respectively. In cases with a single papillary tumor and negative cytology, one of 16 (6.3%) had concomitant CIS in their biopsy specimens at the normal-looking sites. CONCLUSIONS: All non-muscle-invasive bladder cancer patients with positive cytology are candidates for additional random biopsies. Targeted biopsies should be performed for all suspicious areas in the bladder mucosa. Random biopsies should be considered in cases with the macroscopic types of cancer for predicting intermediate- and high-risk cancer.

Discrepancies between cytology, cystoscopy and biopsy in bladder cancer detection after Bacille Calmette-Guerin intravesical therapy.

OBJECTIVES: To evaluate discrepancies in the detection of Bacille Calmette-Guerin (BCG)-resistant bladder cancer by cystoscopy, bladder biopsy and urinary cytology. METHODS: Between January 1992 and August 2006, 127 bladder cancer patients underwent a cycle of eight weekly BCG instillations. Four weeks after the last BCG instillation, urinary cytological analysis and cystoscopy with targeted biopsy in addition to eight-nine selected-site biopsies were performed. RESULTS: Biopsy-proven cancer was found in 11/27 (40.7%), 5/42 (11.9%), and 11/58 (19.0%) of positive, suspicious, and negative cytology cases, respectively. Abnormal and normal cystoscopic findings correlated with a biopsy-proven cancer in 13/53 (24.5%) and 14/74 (18.9%) cases, respectively. The combination of a macroscopic cystoscopic suspicion and a positive cytology missed malignant cases in 15.9% of the cases. In 100 cases without biopsy-proven cancer, the rates of denuded urothelium at biopsy in the cases with positive and non-positive cytology were 7/16 (43.8%) and 16/84 (19.0%), respectively. CONCLUSIONS: According to our study, routine biopsy is recommended in the evaluation of BCG treatment, even if the timing, limitations and disadvantages of the procedure should be taken into account.

Testicular volume, scrotal temperature, and oxidative stress in fertile men with left varicocele.

The testicular volume in fertile men with undetermined varicoceles was comparable with men without varicoceles. Elevation of scrotal temperature without an increase of oxidative stress in fertile men with varicoceles indicates a disturbance of the oxidative stress scavenging system in infertile men with varicoceles.

Development of a peritoneal sclerosis rat model using a continuous-infusion pump.

OBJECTIVE: Encapsulating peritoneal sclerosis (EPS) is a serious complication of continuous ambulatory peritoneal dialysis. Previous studies have created peritoneal sclerosis rat models using daily intraperitoneal injection of chlorhexidine gluconate (CG), but this technique is cumbersome and thickening of the peritoneum makes it difficult to evaluate the injection site. We therefore aimed to make a rat model using a continuous-infusion pump. METHODS: Various concentrations of CG (5%, 8%, 10%, 12%, and 14%) in ethanol were dissolved in saline within the infusion pumps, each of which was placed in the lower abdominal cavity of a male Wister rat. After a peritoneal equilibration test was performed, the rats were sacrificed and the lower anterior parietal and visceral peritoneum was removed. Each excised peritoneum was analyzed by macroscopic and microscopic examinations, including immunohistochemistry for the expression of transforming growth factor-beta 1 (TGF-beta1), vascular endothelial growth factor (VEGF), and alpha-smooth muscle actin (alphaSMA). The results were compared with those of control rats injected with ethanol dissolved in saline within the infusion pump and with no-pump rats. RESULTS: Two of the 5 rats in the 12% CG group and 3 of the 5 rats in the 14% CG group died of ileus within 14 days. All the rats in the 5%, 8%, and 10% CG groups survived to 28 days. Macroscopic examination in the 10% CG group showed bowel dilatation, bowel adhesion, and bloody ascites, similar to those seen in human EPS patients. All rats in each CG group showed the same extent of thickening of the submesothelial compact zone, proliferation of collagen fibers, and presence of numerous cells and neovascularization. Within same CG groups, an equal degree of thickening was observed at all sites of the peritoneum. TGF-beta1, VEGF, and alphaSMA were highly expressed in the peritoneum of the 10% CG group. CONCLUSION: We developed a novel method of creating a peritoneal sclerosis rat model using a continuous-infusion pump. Our technique is simple and highly reproducible, and will be useful in the study of peritoneal sclerosis mechanisms.

Effects of hemodialysis on testicular volume and oxidative stress in humans.

PURPOSE: Male infertility is a serious problem in patients on hemodialysis. Our understanding is that end stage renal disease or hemodialysis causes poor semen quality but the mechanism leading to impaired spermatogenesis is largely unknown. MATERIALS AND METHODS: Testicular volume in 120 patients on maintenance hemodialysis was compared with that in age matched healthy controls. Volume was correlated with clinical findings. In 10 testicular biopsy specimens from patients on hemodialysis who visited our infertility clinic Western blotting was performed to examine the generation of 4-HNE modified proteins, which are markers of oxidative stress, and the expression of proliferating cell nuclear antigen. Interstitial fibrosis was determined by Masson's trichrome staining. RESULTS: Mean bilateral testicular volume in patients on hemodialysis was significantly smaller than that in healthy controls (31.7 vs 36.4 ml, p <0.01) in a hemodialysis duration dependent manner (r = -0.32, p <0.01). The increase in serum ferritin correlated inversely with testicular volume (r = -0.25, p <0.01). The generation of 4-HNE modified proteins was significantly increased 3.1-fold in patients on hemodialysis, following the 60% decreased expression of proliferating cell nuclear antigen. Quantitative analysis of Masson's trichrome staining revealed increased interstitial fibrosis in patients on hemodialysis compared with that in controls (41.5% vs 14.8%, p <0.01). Serum ferritin, proliferating cell nuclear antigen expression and interstitial fibrosis correlated with the generation of 4-HNE modified proteins (p <0.05). CONCLUSIONS: Testicular volume, which is a parameter of spermatogenesis, is impaired in patients on hemodialysis and oxidative stress is considered to be involved in the process. Serum ferritin is a useful parameter for predicting oxidative stress in the testis.

Clinical significance of lymphovascular invasion in upper urinary tract urothelial cancer.

OBJECTIVES: To clarify the significance of lymphovascular invasion (LVI) in patients with pT3N0M0 upper urinary tract (UUT) urothelial carcinoma (UC) relative to prognosis in terms of disease-specific survival, as LVI, which implies both blood vessel and lymph vessel involvement, is reportedly a poor prognostic factor in patients with UUT-UC. PATIENTS AND METHODS: The clinical records of 90 patients who had surgery for UUT-UC were reviewed retrospectively. The median patient age was 71 years and the median follow-up was 42 months. The prognostic significances of LVI (with vs without), T stage (< 1 vs 2-4), grade (1-2 vs 3), N stage (0 vs 1-2), age (< or = 70 vs > 70 years), gender and tumour location (renal pelvis vs ureter) for survival time were evaluated. RESULTS: LVI of UUT-UC was found in 34 patients (37.8%). There were significantly higher frequencies of LVI with advancing stage and lymph node metastasis. Kaplan-Meier analysis showed that LVI was strongly associated with disease-specific survival in all patients (P < 0.001) and in patients with pT3N0M0 disease (P < 0.001). Univariate analyses showed that LVI, T stage, N stage and tumour grade were significantly related to disease-specific survival in all patients (P < 0.001, < 0.001, 0.003 and 0.007, respectively). Multivariate analysis using Cox proportional hazards model showed that LVI was the only prognostic factor with independent significance for disease-specific survival (P < 0.001). CONCLUSIONS: LVI appears to be an important and independent prognostic factor for UUT-UC in patients treated by nephroureterectomy. Our data suggest that the LVI status might be a predictive marker for disease-specific survival in patients with T3N0M0 UTT-UC.

DNA repair gene polymorphisms may be associated with prognosis of upper urinary tract transitional cell carcinoma.

Upper urinary tract transitional cell carcinoma (UUT-TCC) is quite an uncommon disease, and its prognosis differs among individuals irrespective of tumor stage. DNA repair gene polymorphisms are reported to result in the modulation of the repair capacity and might influence the prognosis of UUT-TCC. We examined the associations between functional polymorphisms in five DNA repair genes, and the prognosis of UUT-TCC in 103 UUT-TCC patients. Variant alleles in xeroderma pigmentosum complementation group C, more than three total variant alleles in all DNA repair genes studied and more than two total variant alleles in three nucleotide excision repair genes were independently associated with improved overall and disease-specific survival of UUT-TCC patients in multivariate analysis (P = .0063 and P = .0005 for xeroderma pigmentosum complementation group C, P = .016 and P = .0016 for all genes, and P = .0053 and P = .018 for nucleotide excision repair genes, respectively). These results suggest that some DNA repair gene polymorphisms may preoperatively be valuable as prognostic factors for UUT-TCC beyond tumor stage and grade, helping to provide optimal treatment strategies for individual patients.

Tumor-infiltrating lymphocytes derived from human renal cell carcinoma: clonal analysis of its characteristics.

AIM: To assess the characteristics of activated tumor-infiltrating lymphocytes (TIL), we report the isolation, growth response, and functional analysis of a CD4(-) CD8(+) TIL-clone derived from human renal cell carcinoma (RCC). METHODS: Bulk TILs were expanded from a human RCC and the lymphocytes were separated into a CD8(+) enriched population. Subsequently, using the limiting dilution technique, a TIL clone was established and its growth response, phenotype and cytotoxic activity were analyzed. RESULTS: A clone, T16-13, by day 94 numbering 1 x 10(7) cells, was harvested and characterized as a CD4(-) CD8(+) clone. On day 144, the cytotoxic activity of this clone against the autologous tumor was relatively high (2.3 +/- 0.7 LU(30)/10(6) cells). Meanwhile, against allogeneic renal tumors, there was no cytotoxic activity (-0.1 LU(30)/10(6) cells). CONCLUSIONS: A TIL clone possessing modest autologous tumor-specific cytotoxicity can be isolated from human RCC. The characteristics analysis of various TIL clones may provide a better understanding of an RCC tumor microenvironment and may help to establish new modalities for the treatment of patients with metastatic kidney cancer.

Involvement of vascular endothelial growth factor on spermatogenesis in testis with varicocele.

The expression and the role of vascular endothelial growth factor in human testes with varicocele were examined. Vascular endothelial growth factor expression was increased in testes with varicocele, and was inversely correlated with total motile sperm count and testicular volume, indicating that excessive vascular endothelial growth factor has harmful effects on spermatogenesis in testes with varicocele.

Evaluation of hematuria and proteinuria positivity in relation to ageing in 6,651 apparently healthy men and women.

We examined the positivity of hematuria and proteinuria in relation to ageing in 6,651 apparently healthy persons (2,556 women and 4,095 men) who underwent multiphasic health screening in our Medical Checkup Center. Commercially available dipsticks were used. The time from urine collection to dipstick analysis was within 60 minutes. The mean age of women was 48.2 years (range 10 to 82) and that of men was 49.9 years (range 7 to 89). Approximately 30.1, 1.5, and 0.7% of the women had hematuria, proteinuria, and hematoproteinuria, respectively; and 11.4, 4.0, 1.5% of the men had the corresponding urine abnormalities, respectively. Hematuria was 2.6 times more common in women than in men, and proteinuria was 2.7 times more common in men than in women. The positivity of hematuria increased linearly with age in women (Rs = 0.943, P = 0.0350). On the other hand, the positivity of proteinuria or hematoproteinuria was not correlated with age (P = 0.8386 and P = 0.0639, respectively). In men, the positivity of hematuria or hematoproteinuria was not correlated with age (P = 0.0845 and P = 0.0845, respectively). However, the positivity of proteinuria in those more than 30-year age group increased linearly with age (Rs = 1.000, P = 0.0455). The true meaning of such gender- and/or age-related differences in urinary abnormalities remains to be determined.

Gain of 5p15.33 is associated with progression of bladder cancer.

OBJECTIVE: To search for a biological marker to distinguish low-risk from high-risk bladder cancer indicating disease progression. METHODS: The whole genome-wide copy numbers were screened in 18 patients with bladder cancer using array comparative genomic hybridization (CGH) consisting of 4,030 bacterial artificial chromosome clones. RESULTS: Gain of 5p15.33, including TPPP (tubulin polymerization-promoting protein)and ZDHHC11 (zinc finger DHHC domain-containing protein 11) genes, was detected in 5 of 9 (55.6%) high-grade bladder cancers and no (0%; n = 9) low-grade bladder cancer. To confirm the preliminary data, 5p15.33 gain was studied by fluorescence in situhybridization (FISH) in 100 patients, and the results were compared with biological characteristics. In FISH analysis, gain of 5p15.33 was significantly correlated with higher histological grade (p < 0.0001) and advanced pathological stage (p = 0.0284). Tumors with a gain of 5p15.33 had a significantly higher progression-free survival rate than those without (p = 0.0006, log-rank test). Multivariate analysis revealed that gain of 5p15.33 was a predictor for disease progression in bladder cancer (hazard ratio: 1.887, 95% confidence interval: 1.215-2.968, p = 0.0050). CONCLUSION: These data suggest that gain of 5p15.33 (TPPP and ZDHHC11) may become a potential biomarker identifying high-risk patients with disease progression in bladder cancer.

Effects of 4-hydroxy-2-nonenal, a marker of oxidative stress, on spermatogenesis and expression of p53 protein in male infertility.

PURPOSE: Oxidative stress is involved in male infertility. However, little is known about how it impairs spermatogenesis. We investigated the presence of oxidative stress in human testes by studying the generation of 4-hydroxy-2-nonenal modified proteins and expressions of proliferating cell nuclear antigen and p53. MATERIALS AND METHODS: A total of 47 testicular biopsies from patients with varicocele, obstructive azoospermia and idiopathic infertility were included. Localization and generation of 4-hydroxy-2-nonenal modified proteins were determined by immunohistochemistry and Western blotting. The expressions of proliferating cell nuclear antigen and p53 were assessed by Western blotting. The interaction between 4-hydroxy-2-nonenal modified proteins and p53 was examined by immunoprecipitation. Data were compared to clinicopathological parameters. RESULTS: 4-Hydroxy-2-nonenal modified proteins were strongly positive in spermatogonia, primary spermatocytes and Sertoli's cells, and generation was inversely correlated with expression of proliferating cell nuclear antigen. The expression of p53 was increased in testes with varicocele (p <0.01) and obstructive azoospermia (p <0.05), and there was a positive or inverse correlation with 4-hydroxy-2-nonenal modified proteins and proliferating cell nuclear antigen. Immunoprecipitated p53 was detected by anti-4-hydroxy-2-nonenal modified protein antibody. CONCLUSIONS: 4-Hydroxy-2-nonenal impairs the proliferation of germ cells through the up-regulation of p53 protein, especially in testes with varicocele. Modification by 4-hydroxy-2-nonenal might alter normal function and stabilization of p53 protein.

[Does alpha1-blocker provide additional improvement of quality-of-life in patients with overactive bladder?--a multi-center prospective randomized trial between anti-cholinergic (propiverine chloride) with and without alpha1-blocker (urapidil)].

AIM: Storage/filling symptoms caused by overactive bladder (OAB) are bothersome to patients. The aim of this study is to clarify if alpha1-blocker provides additional benefit in combination with anticholinergic treatment in patients with OAB. METHODS: In total, 100 patients (men/women: 43/57, mean age: 71.3 years) who had frequency (more than eight times a day) and urgency (more than three times a week) were prospectively randomized, and allocated to two groups (monotherapy group [n = 52]: propiverine alone or combination group [n = 48]: propiverine plus urapidil). The primary end point was to compare the improvement of storage symptoms (numbers of frequency, urgency, disappearance of urge incontinence) as well as patients' quality of life (QOL) assessed by King's Health Questionnaires (KHQ) at baseline, 2 weeks, and 6 weeks after the start of treatment in both groups. The second end point was to evaluate the safety of these agents. RESULTS: Statistically significant improvements in terms of urgency and frequency were observed in both groups at two-weeks after the start of treatment as compared with baseline (p < 0.01 and < 0.05, respectively), while no inter-group difference was observed between the two groups. Significant improvement of QOL was observed after six weeks treatment in overall mean score, general health perception, incontinence impact, sleep/energy domains in both groups as compared with baseline. No significant difference was observed in terms of toxic events between the two groups. CONCLUSIONS: Although both groups showed identical improvement of storage symptoms and tolerability, no additional benefit of alpha1-blocker was observed.

Evaluation of lower urinary tract symptoms and how bothersome it was with or without urinary incontinence in apparently healthy persons of both sexes.

We evaluated the effect of urinary incontinence on the degree of being bothersome in apparently healthy males and females by a questionnaire survery. From March to May, 2003 apparently healthy subjects underwent multiphasic health screening after informed of the nature of this study and were asked to fill out the questionnaires of International Prostate Symptom Score (IPSS) with IPSS QOL index (IPSS-QI) and the short form version of the Urogenital Distress Inventory (UDI-6). The data were subjected to analytical studies. Of the 388 participants who responded completely to both questionnaires, 172 (44.3%) had urinary incontinence; 143 were women (36.9%) and 29 men (7.5%). The mean age of the women was 46.0 years (range 18.0 to 76.0) and that of men was 47.5 years (range 22.0 to 76.0). Compared with continent participants, women and men with mixed urinary incontinence had a significantly higher IPSS severity (P = 0.0002 and P = 0.0014, respectively). In terms of contribution on QOL impairment, the women and men with mixed urinary incontinence considered it significantly more bothersome compared with continent participants (P = 0.0004 and P = 0.0003, respectively). These data showed that urinary incontinence was relatively common among apparently healthy women, but not men, and type of incontinence had a different impact on the degree of being bothersome in both sexes.

Nitric oxide produced in the testis is involved in dilatation of the internal spermatic vein that compromises spermatogenesis in infertile men with varicocele.

OBJECTIVE: To determine the concentration of nitric oxide (NO) and the location and change in the expression of NO synthase (NOS) isoforms in the testes of subfertile men with varicocele, and to compare the NO concentration or NOS expression with clinical variables, to determine the role of NO on the pathophysiology of varicocele. PATIENTS AND METHODS: In all, 27 men who had a left varicocelectomy and five with 'normal' spermatogenesis (controls) who had scrotal surgery for other reasons were enrolled. Intratesticular fluid was taken from the men and the NO concentration determined colorimetrically. The expression and location of NOS isoforms were examined by Western blotting and immunohistochemistry, using testicular biopsy specimens, and NADPH-diaphorase (NADPH-d) staining used to identify NO-producing cells. The relationship between the NO concentration and the expression of NOS isoforms or clinicopathological variables was investigated. RESULTS: In testes with grade 2 and 3 varicoceles there were significant increases in the concentration of NO or the expression of inducible NOS. There was no change in the expressions of endothelial NOS, which is located in vascular endothelial cells, while NADPH-d activity was mainly located in these cells. The concentration of NO was significantly correlated with the maximum and total vein diameter (both P<0.01). In patients aged>35 years, the concentration of NO significantly correlated with a deterioration in total motile sperm count (P<0.05). CONCLUSIONS: Increased production of NO in the testis is involved in the enlargement of varicocele and indirectly deteriorates spermatogenesis.

Haploinsufficiency of 8p22 may influence cancer-specific survival in prostate cancer.

Although Knudson's two-hit hypothesis with functional loss of a tumor suppressor gene has been widely accepted, accumulating evidence suggests that several genes are regulated by the quantity of their product in a dose-dependent manner (gene dosage effect). The study was designed to identify the influence of gene dosage effect of 8p22 on patient prognosis. With a median age of 71 years, 40 patients with prostate cancer (11 organ-confined, 13 capsular penetrating, and 16 nodal and/or distant metastatic) were followed for a median of 68.5 months. A fluorescence in situ hybridization (FISH) technique was applied using a region-specific cosmid probe combined with centromeric probe. Allelic losses of 8p22, 8p21.3, 8p21.1 approximately 2, and 8p12 were found in 23, 22, 14, and 9 patients, respectively. A Cox proportional hazard model revealed that decreased fraction (i.e., the fraction of nuclei with a lesser number of cosmid signals than of centromeric probe signals) of 8p22 proved to be the sole independent prognostic factor predicting cancer-specific death, as well as disease progression--but allelic loss of 8p22 was not predictive. Cytogenetic estimation of 8p22 by FISH can yield quantitative evaluation of relevant gene dosage, which may become a useful biomolecular marker predicting poor patient prognosis.