Carbachol-induced signaling through Thr696-phosphorylation of myosin phosphatase-targeting subunit 1 (MYPT1) in rat bladder smooth muscle cells.

PURPOSE: Lines of evidence suggest that Rho-associated protein kinase (ROCK)-mediated myosin phosphatase-targeting subunit 1 (MYPT1) phosphorylation plays a central role in smooth muscle contraction. However, the physiological significance of MYPT1 phosphorylation at Thr696 catalyzed by ROCK in bladder smooth muscle remains controversial. We attempt to directly observe the quantitative protein expression of Rho A/ROCK and phosphorylation of MYPT1 at Thr696 after carbachol administration in rat bladder smooth muscle cells (RBMSCs). MATERIALS AND METHODS: Primary cultured smooth muscle cells were obtained from rat bladders. The effects of both concentration and time-course induced by the muscarinic agonist carbachol were investigated by assessing the expression of Rho A/ROCK and MYPT1 phosphorylation at Thr696 using Western blot. RESULTS: In the dose-course studies, carbachol showed significant increase in phosphorylation of MYPT1 at Thr696 (p-MYPT1) from concentrations of 15-100 µM based on Western blot results (p < 0.05, ANOVA test). In the time-course studies, treatment of cells with 15 µM of carbachol significantly enhanced the expression of p-MYPT1 from 3 to 15 h (p < 0.05, ANOVA test) and induced the expression of Rho A from 10 to 120 min (p < 0.05, ANOVA test). CONCLUSIONS: Carbachol can induce the expression of ROCK pathway, leading to MYPT1 phosphorylation at Thr696 and thereby sustained RBSMCs contraction.

Urothelium-derived Sonic hedgehog promotes mesenchymal proliferation and induces bladder smooth muscle differentiation.

Induction of smooth muscle differentiation from bladder mesenchyme depends on signals that originate from the urothelium. We hypothesize Sonic hedgehog (Shh) is the urothelial signal that promotes bladder mesenchymal proliferation and induces bladder smooth muscle differentiation. Pregnant FVB mice were euthanized on embryonic day (E) 12.5 and fetal bladders were harvested. Two experimental protocols were utilized: Specimens were sized by serial sectioning. Cell counts were performed after trypsin digestion. Immunohistochemistry was performed to detect smooth muscle-specific protein expression. alpha-Actin expression was quantified using Western blot. All specimens were viable at 72h. BLM cultured without Shh survived but did not grow or undergo smooth muscle differentiation. IB cultured without Shh and BLM cultured with Shh grew and expressed smooth muscle proteins at 72h. IB cultured with Shh were larger and contained more cells than IB cultured without Shh (all p<0.05). Increasing Shh concentration from 48 to 480nM did not change bladder size, cell counts, or the level of alpha-actin expression. Prior to culture, IB did not express alpha-actin. After culture of IB in Shh-deficient media, alpha-actin was detected throughout the mesenchyme except in the submucosal layer. The IB submucosa was thinner after culture with 48nM Shh and smooth muscle completely obliterated the submucosa after culture with 480nM Shh. In fetal mouse bladders, urothelium-derived Shh is necessary for mesenchymal proliferation and smooth muscle differentiation. Shh concentration affects mesenchymal proliferation and patterning of bladder smooth muscle.

Signalling molecules involved in mouse bladder smooth muscle cellular differentiation.

Mouse bladder mesenchyme differentiates into smooth muscle under the influence of urothelium at gestational day 13.5 (E13.5). Sonic hedgehog (Shh) is considered to be the upstream gene arising from the urothelium, which induces smooth muscle in the peripheral bladder mesenchyme. We hypothesize differential gene expression across the full thickness of bladder mesenchyme as a function of proximity to the inducing bladder urothelium and the peripheral location of the smooth muscle. Embryonic bladders from FVB mice were collected at E12.5, 13.5, 15 and 16 and cryosectioned followed by microdissection with a PixCell II laser capture microscope. RNA extraction was performed at the laser captured sites and mRNA expression profiles were measured using SYBR Green quantitative RT-PCR. Smooth muscle a-actin (SMAA) and smooth muscle myosin heavy chain (SM-MHC) were expressed in the E13.5, E15 and E16 bladders in the peripheral layer of mesenchyme, but not in the prospective submucosa. Patched 1 (Ptc1), Gli1 and bone morphogenetic protein (Bmp) 4 expression was consistently elevated in the mesenchymal layer immediately adjacent to the urothelium compared to the peripheral location at E12.5. After E12.5, Ptc1 expression decreased to an undetectable level throughout the bladder mesenchyme. The level of TGF-beta1 was highest in the mesenchymal layer adjacent to the serosa at E13.5. The level of expression of serum response factor (SRF) was also highest at E15 in the peripheral mesenchyme. Genes downstream of Shh are differentially expressed in the prospective submucosa vs. the peripheral bladder mesenchyme as a function gestation age and smooth muscle differentiation.

Estrogen effects on fetal penile and urethral development in organotypic mouse genital tubercle culture.

PURPOSE: We developed an organotypic genital tubercle culture system in vitro and used it to investigate the direct effects of the hyperestrogenic state on fetal mouse penile and urethral development. MATERIALS AND METHODS: Genital tubercles were dissected from embryonic day 14.5 C57B/L6 male mouse fetuses and cultured using an air-liquid interface on a microporous membrane support soaked in synthetic medium. Cultures were separated into 4 groups. Groups 1 to 3 were supplied with 10 nM dihydrotestosterone, estradiol and 10 nM dihydrotestosterone plus estradiol, respectively. Group 4 was cultured in hormone-free medium. After 36 to 72-hour culture morphological, histological, proliferation, apoptosis, androgen signaling and activating transcription factor 3 analyses were done. RESULTS: The physiological concentration of 10 nM dihydrotestosterone was essential for genital tubercle growth in vitro. Androgen induced growth and urethral development were significantly suppressed by high dose estrogen. Concurrently we observed increased apoptosis and decreased proliferation in the mesenchyma. Androgen signaling was disrupted and activating transcription factor 3, a factor related to hypospadias genesis, was up-regulated. CONCLUSIONS: High dose estrogen suppressed male genital tubercle development in vitro. The organotypic genital tubercle culture system in vitro consisting of urethral epithelial and mesenchymal cells can recapitulate the hormonal sensitivity of fetal penile and urethral development. This method is potentially useful for studying the effects of various factors, particularly endocrine disruptors.

Differential expression of microRNAs in mouse embryonic bladder.

MicroRNAs (miRNAs) are involved in several biological processes including development, differentiation and proliferation. Analysis of miRNA expression patterns in the process of embryogenesis may have substantial value in determining the mechanism of embryonic bladder development as well as for eventual therapeutic intervention. The miRNA expression profiles are distinct among the cellular types and embryonic stages as demonstrated by microarray technology and validated by quantitative real-time RT-PCR approach. Remarkably, the miRNA expression patterns suggested that unique miRNAs from epithelial and submucosal areas are responsible for mesenchymal cellular differentiation, especially regarding bladder smooth muscle cells. Our data show that miRNA expression patterns are unique in particular cell types of mouse bladder at specific developmental stages, reflecting the apparent lineage and differentiation status within the embryonic bladder. The identification of unique miRNAs expression before and after smooth muscle differentiation in site-specific area of the bladder indicates their roles in embryogenesis and may aid in future clinical intervention.

Genomic variants of ATF3 in patients with hypospadias.

PURPOSE: ATF3, an estrogen responsive gene expressed during genital development, could be implicated in the etiology of hypospadias. ATF3 is up-regulated in the foreskin of patients with hypospadias and is implicated in suppression of the cell cycle, which may interfere with urethral cell growth. We sought to investigate the sequence of ATF3 in patients with hypospadias. MATERIAL AND METHODS: Direct sequencing of coding exons and splice sites of ATF3 was performed in 41 boys with hypospadias and 30 controls. In addition, ATF3 expression in 1 human fetal penis with and 1 without hypospadias was studied by immunohistochemical analysis. RESULTS: A missense variant (L23M) was identified in a boy with anterior hypospadias. This amino acid is highly conserved. Three genomic variants (C53070T, C53632A, Ins53943A) were found in or close to exon 6 in patients with perineal, penoscrotal and anterior hypospadias. This important exon includes splice sites for an alternative transcript (ATF3DeltaZip) that have been implicated in regulation of the function of ATF3. None of these genomic variants was present in controls. Immunochemical analysis revealed that in normal fetuses ATF3 is not expressed in and around the urethra, while in patients with hypospadias ATF3 is over expressed in the urethral plate and subcutaneous tissue, especially around the ectopic orifice of the urethra. CONCLUSIONS: Genomic variants of ATF3 are present in 10% of our patients with hypospadias. We also report an abnormal expression pattern of ATF3 in a hypospadiac fetus. The direct implication of ATF3 in the occurrence of hypospadias remains to be confirmed by functional studies of the genomic variants we describe.

3-dimensional neuroanatomy of the human fetal pelvis: anatomical support for partial urogenital mobilization in the treatment of urogenital sinus.

PURPOSE: Retrospective reviews suggest that the functional outcomes of surgery of the urogenital sinus have often been unsatisfactory and to our knowledge the long-term results of newer surgical techniques have yet to be evaluated. A precise understanding of pelvic fetal neuroanatomy is germane for optimizing surgical correction of the urogenital sinus. MATERIALS AND METHODS: The pelves of 10 human female fetuses were serially sectioned. Masson's trichrome staining and immunochemistry for the neuronal marker S100 (Dako Corp., Carpinteria, California) along with anatomical computer reconstruction allowed 3-dimensional analysis of the nerves in relation to the pelvic structures as an animated motion picture. RESULTS: Two types of neuronal structures were identified. 1) A dense perivisceral foil of branching nerves closely surrounded the pelvic organs. The localization of most nerves was on the external faces of the viscera with a limited fraction in the rectovaginal and urethrovaginal septa. This innervation was from the anterior cephalad periurethral area to the posterior caudal perirectal area. 2) A significant amount of nerves surrounded the cephalad urethra on its anterior and posterior faces. CONCLUSIONS: Based on these anatomical data during surgical repair of a urogenital sinus we would advocate minimal mobilization of the lateral faces of the vagina, avoiding dissection of the proximal urethra above the pubic bone and electing a vaginal flap in severe cases.

Mutations of CXorf6 are associated with a range of severities of hypospadias.

OBJECTIVE: Mutations in chromosome X open reading frame 6 (CXorf6), a recently described candidate gene involved in the development of male genitalia, have been found in patients with complex 46,XY disorders of sexual development (46,XY DSD) including micropenis, bifid scrotum, and penoscrotal hypospadias. The objective of this work was to identify genomic variants of CXorf6 in patients with isolated hypospadias, severe or non-severe. DESIGN AND METHODS: Forty-one patients with glandular to perineal hypospadias and thirty controls were studied. Direct sequencing for coding exons 3-6 of CXorf6 and their flanking splice sites was performed on DNA extracted from foreskin collected from surgery. Secondary and tertiary structures of the protein were predicted using NNpredict and Protein Homology/analogY Recognition Engine engines. RESULTS: Four mutations (9.7% of cases) were identified. One missense mutation (1295T>C, V432A) and two deletions (325delG, predicted to cause a stop codon L121X) occurred in patients with penoscrotal and proximal hypospadias. One patient with subcoronal hypospadias had CAG-repeat amplification in the second polyglutamine domain of CXorf6. Secondary structure prediction indicated that this insertion occurred in a helix element of the protein. The tertiary structure prediction showed an alteration of the shape of the protein and crowding between domains. CONCLUSION: CXorf6 mutations are associated with isolated hypospadias of varying severity. However, the pathophysiology of these mutations and the function of the CXorf6 gene product remain to be investigated.

Urothelium patterns bladder smooth muscle location.

Smooth muscle differentiation is induced in the embryonic bladder by the centrally located urothelium in the undifferentiated mesenchyme in the periphery adjacent to the serosa. We hypothesize that under the appropriate signal the entire undifferentiated bladder mesenchyme is capable of smooth muscle differentiation and that the urothelium patterns fibromuscular development. Embryonic bladders of wild-type and Green Fluorescent Protein mice were separated into urothelial and mesenchymal components before smooth muscle differentiation (E12.5-E13). The urothelial layer green fluorescent protein was recombined and grafted with the mesenchyme (wild-type) in an orthotopic position, heterotopic position and ectopic position. In all cases, a zone of smooth muscle inhibition was observed adjacent to the epithelium whether the urothelium was in an orthotopic or heterotypic position. Bladder mesenchyme and bladder epithelium grafted alone did not grow. In conclusion, the full thickness of bladder mesenchyme is capable of smooth muscle differentiation dependent on the location of urothelium. These experiments support the hypothesis that urothelium secretes a diffusible factor that at high concentrations inhibits smooth muscle and at low concentrations induces smooth muscle, thus patterning mesenchymal cell differentiation across the full thickness of the fibromuscular bladder wall.

Penile and scrotal Paget's disease: 130 Chinese patients with long-term follow-up.

OBJECTIVES: To report a retrospective series of 130 Chinese patients with penoscrotal extramammary Paget's diseases (EMPD), with a long-term follow-up, and thus improve the diagnosis and therapy of this disease. PATIENTS AND METHODS: The history, clinical presentation, pathology, treatment, and prognosis of 130 patients were analysed. All cases were confirmed by skin biopsy, and then the patients had local wide resection to remove the involved skin and subcutaneous tissue. The large defective wound was reconstructed using a split-thickness skin graft or local flap. RESULTS: Forty-five patients were evaluated by frozen-section biopsy of the margins during surgery, five of whom had positive margins and then had an extended resection immediately. Most of these patients had local skin or adjacent scrotal flaps to cover their skin defects. Of the 130 patients, 81 had a mean (range) follow-up of 3.2 (0.5-10) years after surgery. Five of nine patients with positive margins and three (4%) of 72 with negative margins had tumour recurrence. Five patients died from metastatic disease. CONCLUSIONS: Penoscrotal EMPD needs be differentiated from other chronic dermatitis. A 3 cm surgical margin should be sufficient and frozen-section pathological examinations are necessary for some complicated conditions. Skin grafts or local flaps are good for large skin defects.

Estradiol upregulates activating transcription factor 3, a candidate gene in the etiology of hypospadias.

Hypospadias is a penile developmental abnormality that may partly result from in utero exposure to estrogenic compounds. Expression of activating transcription factor 3 (ATF3) is elevated in human foreskin tissue from hypospadic patients, and in utero exposure to ethinyl estradiol (17-EE) causes ATF3 upregulation in a hypospadias mouse model. We investigated the effects of in vitro exposure to EE on ATF3 expression and promoter activity in human foreskin fibroblasts using immunocytochemistry, quantitative polymerase chain reaction (PCR), western blot, and the luciferase activity assay. Immunocytochemistry showed peak positive staining at 2 hours after 0.5 to 3 hours of EE treatment (0.1 microM). Western blot showed significantly increased ATF3 protein expression (P = 0.006) after EE exposure. ATF3 mRNA, as evaluated using reverse transcriptase PCR and TaqMan real-time PCR, also increased (P = 0.146). In addition, the luciferase activity assay showed that ATF3 promoter activity was significantly enhanced after 1 hour of EE exposure (P < 0.0001). Thus, EE upregulates ATF3 expression in fibroblasts in vitro, consistent with our previous results with human tissue and in vivo mouse models. ATF3 is involved in the TGF-beta epithelial-mesenchymal signaling pathway, and its involvement in hypospadias suggests that ATF3 plays a role in development of this anomaly as a result of exposure to estrogenic compounds. Its potential involvement in other manifestations of developmental endocrine disruption is worth investigating.

In utero exposure to benzophenone-2 causes hypospadias through an estrogen receptor dependent mechanism.

PURPOSE: Additives such as benzophenone-2 are commonly used in cosmetic products and food container plastics to filter out ultraviolet light. In pregnant women exposure may result in transplacental transfer of benzophenone-2 to fetuses. Benzophenone-2 is estrogenic in vitro and in the rat uterotropic assay. Estradiol causes hypospadias in mice and estrogen-like compounds are also postulated to cause hypospadias. We determined whether hypospadias would develop in male mice exposed to benzophenone-2 in utero and whether this outcome depended on estrogen receptor pathways. MATERIALS AND METHODS: Timed pregnant C57BL/6 mice were administered benzophenone-2 (6.25 mg) or control vehicle by oral gavage from gestational days 12 through 17 and they were sacrificed on day 18. Fetuses were weighed and sexed, anogenital distance was measured and genital tubercles were harvested for paraffin sections or quantitative reverse transcriptase-polymerase chain reaction analysis of genes purportedly involved in genital tubercle development. RESULTS: Eight of 57 benzophenone-2 treated male fetuses (14%) whose genital tubercles were examined histologically had hypospadias (p = 0.0064). Co-administration of benzophenone-2 with the estrogen receptor antagonist EM-800 resulted in normal genital tubercles, ie no hypospadias, in 26 of 26 mice. Likewise no EM-800 or control treated male genital tubercles showed hypospadias. Benzophenone-2 treated male mice had no changes in body mass adjusted anogenital distance relative to controls. Reverse transcriptase-polymerase chain reaction revealed that genital tubercles of benzophenone-2 treated male mice expressed higher levels of estrogen receptor-beta relative to male controls (p = 0.04). CONCLUSIONS: These findings suggest that benzophenone-2 may cause hypospadias via signaling through the estrogen receptor. Further study of human benzophenone-2 exposure and its effects is needed to support this hypothesis.

Urothelial sonic hedgehog signaling plays an important role in bladder smooth muscle formation.

During bladder development, primitive mesenchyme differentiates into smooth muscle (SM) under the influence of urothelium. The gene(s) responsible for this process have not been elucidated. We propose that the Sonic hedgehog (Shh) signaling pathway is critical in bladder SM formation. Herein, we examine the role of the Shh-signaling pathway during SM differentiation in the embryonic mouse bladder. Genes in the Shh pathway and SM expression in mouse embryonic (E) bladders (E12.5, 13.5, and 14.5) were examined by immunohistochemistry (IHC), in situ hybridization, and reverse transcription polymerase chain reaction (RT-PCR). To examine the effects of disrupting Shh signaling, bladder tissues were isolated at E12.5 and E14.5, that is, before and after bladder SM induction. The embryonic bladders were cultured on membranes floating on medium with and without 10 muM of cyclopamine, an Shh inhibitor. After 3 days, SM expression was examined by assessing the following: SM alpha-actin (SMAA), SM gamma-actin (SMGA), SM-myosin heavy chain (SM-MHC), Patched, GLI1, bone morphogenic protein 4 (BMP4), and proliferating cell nuclear antigen (PCNA) by IHC and RT-PCR. SM-related genes and proteins were not expressed in E12.5 mouse embryonic bladder before SM differentiation, but were expressed by E13.5 when SM differentiation was initiated. Shh was expressed in the urothelium in E12.5 bladders. Shh-related gene expression at E12.5 was significantly higher than at E14.5. In cyclopamine-exposed cultures of E12.5 tissue, SMAA, SMGA, GLI1, and BMP4 gene expression was significantly decreased compared with controls, but PCNA gene expression did not change. In cyclopamine-exposed E14.5 cultures, SMGA and SM-MHC gene expression did not change compared with controls. Using an in vitro embryonic bladder culture model, we were able to define the kinetics of SM- and Shh-related gene expression. Cyclopamine inhibited detrusor SM actin induction, but did not inhibit SM-MHC induction. SMAA and SMGA genes appear to be induced by Shh-signaling pathways, but the SM-MHC gene is not. Based on Shh expression by urothelium and the effects of Shh inhibition on bladder SM induction, we hypothesize that urothelial-derived Shh orchestrates induction of SM in the fetal mouse bladder.

Ontogeny of androgen receptor and disruption of its mRNA expression by exogenous estrogens during morphogenesis of the genital tubercle.

PURPOSE: The ontogeny of androgen receptor expression in male and female mouse genital tubercles, and the effects of in utero ethinyl estradiol exposure on androgen receptor mRNA expression in the hypospadias model were studied. MATERIALS AND METHODS: Androgen receptor mRNA expression was measured in mouse genital tubercles from fetuses and pups collected on gestational days 12, 14, 16 and 18, and from newborns using immunohistochemistry and real-time quantitative polymerase chain reaction. Pregnant dams were exposed to ethinyl estradiol or corn oil as controls from gestational days 12 to 17. Genital tubercles of gestational day 19 fetuses were then examined by further quantitative polymerase chain reaction analysis after identification of the seam area using a dissecting microscope to diagnose hypospadias in males. RESULTS: Androgen receptor protein was detected in genital tubercles by gestational day 14. Androgen receptor mRNA expression increased gradually in each sex during normal development. However, female genital tubercles expressed a higher level of androgen receptor mRNA throughout development compared to male genital tubercles (p <0.0001). In utero ethinyl estradiol exposure led to a 5.4 and 4.5-fold increase in androgen receptor mRNA in the genital tubercles of female and male embryos (p = 0.004 and 0.001, respectively). Hypospadiac male genital tubercles showed increased androgen receptor mRNA expression compared to control males (p = 0.006). Levels in hypospadiac males did not differ from those in control females but they were less than those in ethinyl estradiol treated females (p >0.05 and 0.01, respectively). CONCLUSIONS: Androgen receptor protein is expressed abundantly in male and female genital tubercles. Androgen receptor mRNA levels are higher in female than in male genital tubercles through development and they increase in response to in utero ethinyl estradiol exposure with ethinyl estradiol treated females having the highest levels of expression, followed by ethinyl estradiol treated hypospadiac males. We infer that higher estrogen in genital tubercles results in a physiological response of increased androgen receptor mRNA expression. We found no direct association between changes in androgen receptor mRNA expression and the presence or absence of hypospadias in males, suggesting that alterations in the expression of proteins other than or in addition to androgen receptor result in anomalous urethral development. This finding supports the idea that the etiology of hypospadias is multifactorial in origin.

Activating transcription factor 3 is estrogen-responsive in utero and upregulated during sexual differentiation.

BACKGROUND/AIMS: Synthetic estrogens induce hypospadias, an anomaly of genital tubercle/urethral development. Activating transcription factor 3 (ATF3), which is estrogen-responsive in vitro, is upregulated in hypospadiac human tissue. We used a mouse model of steroid-dependent genital tubercle development to elucidate the ontogeny of ATF3 expression and the developmental response of ATF3 in vivo to estrogen exposure. METHODS: We used quantitative RT-PCR to assess ontogenic expression of ATF3 and its response to estrogen treatment in utero. Immunohistochemistry was used to localize the protein. RESULTS: Quantitative RT-PCR showed that ATF3 mRNA is upregulated in all estrogen-exposed fetal genital tubercles compared to controls (p = 0.024), including specifically in males exposed in utero (p = 0.049). Additionally, its expression increases significantly during the period of sexual differentiation in both sexes and significantly correlates with female development (p = 0.004), a phenomenon that appears to be attributable to higher levels at birth in females. The protein localizes in the nucleus, as expected. CONCLUSIONS: ATF3 is estrogen-responsive in vivo. The response of ATF3 to estrogenic stimulation in utero at an earlier stage may contribute to urethral abnormalities observed in estrogen-exposed male fetuses, although it is likely not the only gene involved, which supports the general understanding that hypospadias is subject to multifactorial influences. ATF3 may therefore be an appropriate gene for further investigations in an endocrine context.

Randomized study of single instillation of epirubicin for superficial bladder carcinoma: long-term clinical outcomes.

PURPOSE: Intravesical instillation of epirubicin (EPI) is one of the most effective adjuvant therapies for nonmuscle-invasive bladder cancer postoperation. We evaluated the long-term efficacy of single dose intravesical epirubicin for superficial bladder carcinoma recurrence. METHODS: Between June 1997 and May 1998, a total of 47 patients with resectable superficial bladder carcinoma (Ta-1, Grade 1-2), primary or recurrent with no recurrence during last one year, were enrolled in this study. All patients were randomized into 3 study groups: Group A-single epirubicin (80 mg/40 mL of normal saline) was administered into the bladder within 6 hours postoperation; Group B-40 mg Epirubicin consecutively; Group C-40 mg mitomycin C, consecutively. In Group B and C, instillation were given every week for 6- 8 weeks and then every one month for 10 months. Patients were followed up at 3, 6, 9, 12, 18, 24, 36, 48, and 60 months of treatment. The analyzed background factors were the therapeutic method, tumor recurrence, and side effects. RESULTS: Of the 47 patients, 43 (91.5 percent) were eligible and were followed up for 5 years postoperation. The disease free intervals of the three groups were found to have no significant differences (F = 10.28, p > 0.05). The recurrence rates were 35.7 percent (5/14), 33.3 percent (5/15), and 40 percent (6/15), respectively (chi(2)= 0.83, p > 0.05). Side effects of group A (13.6 percent) was lower than that of Group B or C (53.3 percent and 46.7 percent, respectively) significantly (chi(2) test, p < 0.01). CONCLUSIONS: These data indicate that single dose of epirubicin instillation postoperation can reduce the recurrence of superficial bladder carcinoma and has low side effects.

Activating transcription factor 3 is up-regulated in patients with hypospadias.

Hypospadias is a congenital anomaly of the genitalia characterized by abnormalities of the urethra and foreskin, with the urethral meatus located in an abnormal position anywhere from the distal ventral penile shaft to the perineum. Because the incidence of hypospadias is approximately 1/200-1/300 live male births, it is one of the most common congenital malformations, but its etiology is largely uncharacterized. Genomic analysis of hypospadic tissue indicated a potential role for activating transcription factor 3 (ATF3) in the development of this anomaly. ATF3 may be involved in homeostasis, wound healing, cell adhesion, or apoptosis, and normally it is expressed at a steady-state in quiescent cells. Additionally, it has been shown to be an estrogen-responsive gene, and the etiology of hypospadias may be related to in utero exposure to estrogenic or anti-androgenic compounds. We examined the expression of ATF3 in tissues from 28 children with hypospadias compared with 20 normal penile skin tissue samples from elective circumcision. Eighty-six percent of the hypospadias samples were immunohistochemically positive, compared with 13% of normal tissue samples. Seventy-five percent of hypospadias samples were positive from in situ hybridization, compared with 1% of circumcision samples. Our results indicate that ATF3 is up-regulated in the penile skin tissues of boys with hypospadias, suggesting a role for this transcription factor in the development of this abnormality. Because the etiology of hypospadias may include exposure to estrogenic compounds, the responsiveness of ATF3 to estrogen is also discussed.

Identification of potential biomarkers of Peyronie's disease.

AIM: To identify proteins that are differentially expressed in cells derived from normal and diseased tunica albuginea (TA) as related to Peyronie's disease (PD). METHODS: Cells with characteristics of fibroblasts were isolated from two tissue sources. Those from the plaque of patients with PD were designated as PT cells, and those from the normally-appearing TA of the same patients were designated as NT cells. Messenger RNAs of these cells were analyzed by real-time polymerase chain reaction (RT-PCR) for the expression of monocyte chemoattractant protein 1 (MCP-1). Crude protein lysates were analyzed by surface-enhanced laser desorption/ionization mass spectrometry (SELDI-MS) with IMAC30-Cu, CM10, and H50 chips. Each lysate was then separated into six fractions, which were further analyzed by SELDI-MS. RESULTS: RT- PCR analysis showed that PT cells expressed higher levels of MCP-1 than their counterpart NT cells. SELDI-MS analysis showed that the crude protein lysates of all four cell strains produced similar and reproducible protein profiles on IMAC30-Cu and CM10 chips. Additional SELDI-MS analyses with the fractionated lysates detected three proteins of 11.6 kDa, 14.5 kDa, 22.6 kDa that were upregulated in PT cells and two proteins of 6.3 kDa and 46.9 kDa that were downregulated in PT cells. CONCLUSION: MCP-1, which is often involved in tissue fibrosis, was expressed at higher levels in PT than that in NT cells. Five potential biomarkers for PD were identified by SELDI-MS analysis.

[Single instillation of epirubicin for the prophylaxis of recurrent primary superficial bladder carcinoma].

OBJECTIVE: To determine the feasibility of single dose intravesical epirubicin in the prevention of recurrent superficial bladder carcinoma. METHODS: We compared the effect of intravesical epirubicin or mitomycin C on tumor recurrence and disease free interval and their side effects after treatment of superficial bladder tumor. 47 postoperative patients with stages Ta to T1 primary superficial bladder carcinoma of grades 1 or 2 were randomized into groups A: single 80 mg epirubicin; B: 40 mg consecutive epirubicin; C: 40 mg consecutive mitomycin C. Patients were followed up for clinical, analytical, and cystoscopic evaluations every 3 months. RESULTS: The disease free intervals of the three groups were found no significant differences (F = 3.25, P > 0.05). The recurrence rate was 6.25% (1/16), 13.3% (2/15), 12.5% (2/16) (chi(2) = 0.496, P > 0.05) in groups A, B, and C at 1 year, and 33.3% (5/15), 26.7% (4/15), 25% (4/16) (chi(2) = 0.290, P > 0.05) at 3 years after operation, respectively. Side effects of group A (13.3%) were lower than those of group B (46.7%) or C (43.8%) (chi(2) = 14.56, P < 0.01). CONCLUSIONS: Single dose of epirubicin given intravesically immediately after tumor resection is effective in preventing tumor recurrence.