Valproic Acid Alters Angiogenic and Trophic Gene Expression in Human Prostate Cancer Models.

BACKGROUND/AIM: Only a minority of men succumb to prostate cancer (PCa). Therapy to prevent progression would change treatment paradigms. We investigated the effect of valproic acid (VPA) on PCa cell proliferation and the effects on both angiogenesis and PCa-specific signaling. MATERIALS AND METHODS: LNCaP cells were treated with VPA for 72 h and proliferation was measured. Cellular RNA extracts were used to measure gene expression with RT-profiler2 arrays. Genes with alterations were validated using real-time polymerase chain reaction and western blot. RESULTS: VPA led to a dose-dependent decrease in proliferation. Expression array data revealed an impact on modulators of angiogenesis. Additionally, several cell-cycle control transcripts were affected. There was a strong correlation between gene and protein expression levels for validated targets. CONCLUSION: VPA decreases cellular proliferation of PCa cells in vitro and also affects gene expression suggestive of anti-angiogenic effect with a concomitant decrease in proliferation-related genes.

p38MAPK activation and DUSP10 expression in meningiomas.

The mitogen activated protein kinase (MAPK) p38MAPK has been implicated in regulation of cell proliferation and apoptosis. However, expression, activation and regulation has not been studied in meningiomas, to our knowledge. p38MAPK is regulated, in part, by dual specificity phosphatases (DUSP) that inactivate signaling by dephosphorylation. DUSP10 is also a likely participant in regulating meningioma proliferation. Five fetal and an adult human leptomeninges and 37 meningioma cultures (MC) were evaluated for DUSP10 as well as phosphorylation of its substrates p38MAPK and p44/42MAPK by western blot and DUSP10 expression by polymerase chain reaction. Platelet derived growth factor-BB (PDGF-BB), transforming growth factor B1 (TGFB1) and cerebrospinal fluid effects on DUSP10 and signaling were also studied in vitro. DUSP10 and phospho-p38MAPK and phospho-p44/42MAPK were detected in all six leptomeninges. DUSP10 was detected in 13 of 17 World Health Organization grade I, 11 of 14 grade II and four of six grade III meningiomas. Phospho-p38MAPK was detected in nine of 17 grade I, two of six grade II, and four of six grade III meningiomas. In the majority of meningiomas DUSP10 expression correlated inversely with phosphorylation of p38MAPK. PDGF-BB increased DUSP10 in MC2 and MC4 and weakly in MC3. TGFB1 increased phosphorylation of p38MAPK and caspase 3 activation. Thus p38MAPK and DUSP10 likely participate in the pathogenesis of meningiomas.

APOBEC3B expression in human leptomeninges and meningiomas.

Nucleic acid-editing enzymes of the apolipoprotein B mRNA-editing enzyme (APOBEC) family have been associated with somatic mutation in cancer. However, the role of APOBEC catalytic subunit 3B (APOBEC3B) editing in the pathogenesis of base substitutions in meningiomas is unknown. In the present study, the expression of APOBEC3B was examined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analyses in five fetal and one adult human leptomeninges and 38 meningiomas. Genomic DNA was sequenced using the Illumina Tru-Seq Cancer Panel. Three meningioma primary cultures were also established and treated with cerebrospinal fluid form patients without neurological disease or platelet-derived growth factor-BB (PDGF-BB), prior to evaluation of APOBEC3B expression. By western blotting, APOBEC3B was revealed to be present in 100% of the fetal leptomeninges, and in 88% of World Health Organization grade I, 100% of grade II and 83% of grade III meningiomas tested, but was not different between grades. RT-qPCR revealed no difference in the mRNA expression of APOBEC3B between grades. Sequencing revealed no elevated levels of the C>T mutations that are characteristic of APOBEC3B editing of genomic DNA. Treatment with cerebrospinal fluid and PDGF-BB had no effect on APOBEC3B protein expression in the leptomeningeal or meningioma cells. These findings suggest that the mutations associated with increased APOBEC3B expression may not be central to the pathogenesis of meningiomas.

Bone morphogenetic protein-4 and 7 and receptors regulate vascular endothelial growth factor and receptors in human fetal leptomeninges.

Bone morphogenetic proteins (BMP) 4 and 7 have important roles in neuronal differentiation and cortical development in the murine brain. However BMP4 and BMP7 expression and functions in the developing human brain are unknown. In this study, frozen tissue human fetal leptomeninges, formalin-fixed tissue and primary fetal leptomeningeal cell cultures were studied. By western blot, BMP4, BMP7 and BMPRIa were demonstrated in 15, 17 20, 23 week (wk) human leptomeninges. BMP receptor II was detected at 15 and 17 wks. Immunohistochemically, BMP4 immunoreactivity was also found in 20 to 39 wk human leptomeninges. BMP4 significantly reduced basal DNA synthesis at 22 wks. BMP7 100 and 300 ng/ml stimulated basal DNA synthesis in the 15, 17 and 22 wk leptomeninges. BMP4 and BMP7 increased phosphorylation of SMAD-1, 5, 8 in most cells and had no effect on phosphorylation of p-38MAPK, or p44/42MAPK. BMP4 and BMP7 produced a decrease in VEGF RNA expression in 2 of 4 leptomeninges. BMP4 and BMP7 increased VEGFR1 RNA in 2 or 3 of 4 leptomeningeal cultures respectively. BMP4 produced a decrease in VEGFR2 RNA in 2 of 4 and BMP7 in 3 of 4 while BMP7 reduced VEGFR2 protein in the leptomeninges. The findings show, for the first time, BMP4, BMP7 and receptors are expressed and active in the human fetal leptomeninges. They suggest these BMPs influence vascular development in this tissue by regulating VEGF and its receptors.

GESPA: classifying nsSNPs to predict disease association.

BACKGROUND: Non-synonymous single nucleotide polymorphisms (nsSNPs) are the most common DNA sequence variation associated with disease in humans. Thus determining the clinical significance of each nsSNP is of great importance. Potential detrimental nsSNPs may be identified by genetic association studies or by functional analysis in the laboratory, both of which are expensive and time consuming. Existing computational methods lack accuracy and features to facilitate nsSNP classification for clinical use. We developed the GESPA (GEnomic Single nucleotide Polymorphism Analyzer) program to predict the pathogenicity and disease phenotype of nsSNPs. RESULTS: GESPA is a user-friendly software package for classifying disease association of nsSNPs. It allows flexibility in acceptable input formats and predicts the pathogenicity of a given nsSNP by assessing the conservation of amino acids in orthologs and paralogs and supplementing this information with data from medical literature. The development and testing of GESPA was performed using the humsavar, ClinVar and humvar datasets. Additionally, GESPA also predicts the disease phenotype associated with a nsSNP with high accuracy, a feature unavailable in existing software. GESPA's overall accuracy exceeds existing computational methods for predicting nsSNP pathogenicity. The usability of GESPA is enhanced by fast SQL-based cloud storage and retrieval of data. CONCLUSIONS: GESPA is a novel bioinformatics tool to determine the pathogenicity and phenotypes of nsSNPs. We anticipate that GESPA will become a useful clinical framework for predicting the disease association of nsSNPs. The program, executable jar file, source code, GPL 3.0 license, user guide, and test data with instructions are available at http://sourceforge.net/projects/gespa.

MKP-3 regulates PDGF-BB effects and MAPK activation in meningioma cells.

Autocrine platelet derived growth factor-BB (PDGF-BB) and cerebrospinal fluid, which also contains PDGF, stimulate proliferation of leptomeningeal and meningioma cells, in part, by activation of the Raf-1-MEK-1-MAPK pathway. The negative regulators of this activation are not known. However, PDGF receptors and p44/42 MAPK are regulated, in part, by mitogen activated kinase phosphatase 3 (MKP-3) and Src homology carboxyl terminus protein (SHP-2). Six fetal and one adult human leptomeninges specimens and 22 meningiomas were evaluated for MKP-3, SHP-2, and phospho-SHP-2 as well as activation/phosphorylation of MEK1/2, p44/42 MAPK, Akt and signal transducer and activator of transcription 3 (STAT3) by western blot and MKP3 expression by polymerase chain reaction. PDGF-BB and cerebrospinal fluid effects on these phosphatases and signaling were also studied in vitro. MKP-3 and phospho-p44/42 MAPK were detected in all or six of seven leptomeninges, respectively. MKP-3 was detected in six of eight World Health Organization grade I and II meningiomas. Three of four grade I and five of five grade II with no or low MKP-3 had high levels of phospho-p44/42MAPK. MKP3 was not detected in four of six grade III meningiomas. These had high levels of phospho-p44/42MAPK. SHP2 was found in all leptomeninges and meningiomas while phospho-SHP-2 was found in 11 to 33% of grade I-III meningiomas. Reduced MKP-3 may facilitate PDGF-BB autocrine and paracrine mitogenic effects in a subpopulation of higher grade meningiomas by increasing phospho-p44/42 MAPK.

Incidental computed tomographic bladder wall abnormalities: harbinger or herring?

OBJECTIVE: To evaluate the outcomes of incidental radiographically identified bladder wall abnormalities in the absence of other urologic indications for evaluation. METHODS: All screening cystoscopy evaluations performed at our center over 4 years were identified using surgical logs. We identified patients for whom cystoscopy was performed for a radiographic bladder wall abnormality, defined as diffuse bladder wall thickening, focal bladder wall thickening, or intraluminal bladder mass. Patients with other indications for cystoscopy such as previous bladder cancer, pelvic radiation, or hematuria were excluded. The outcomes including any relevant biopsy or malignant diagnosis were recorded. RESULTS: A total of 2483 cystoscopies were performed in 1418 unique patients, with 34 (2%) performed for radiographic bladder wall abnormalities in the absence of other indications for cystoscopy. Eleven of 34 patients (32.4%) were evaluated for diffuse bladder wall thickening, of which 2 had high-grade carcinoma. Fifteen patients (44.1%) had focal bladder wall thickening, all negative at cystoscopy. Four of the 8 patients (23.5%) evaluated for bladder mass had disease (1 high grade, 3 low grade). CONCLUSION: Although generally nonspecific for malignancy, incidental radiographic finding of bladder wall abnormality led to diagnosis of urothelial carcinoma in >15% of our patients including 3 worrisome tumors. This finding argues for routine cystoscopy in patients with radiographic bladder wall abnormality even in the absence of hematuria.

Repetitive percutaneous epididymal sperm aspirations (PESA's) resulted in asthenospermia and significant inflammation.

INTRODUCTION: In obstructive azoospermia, choosing a sperm retrieval method for intracytoplasmic sperm injection (ICSI) depends on the preference and expertise of both the urologist and the reproductive endocrinologist. Generally, a percutaneous epididymal sperm aspiration (PESA) is attempted first. Not uncommonly, multiple PESA's are necessary. This study utilizes a rat model to provide an understanding of sperm parameter and histological changes resulting from repetitive PESA procedures. MATERIALS AND METHODS: A cohort of 30 male Wistar rats of reproductive age (68-73 days) was divided into three groups of 10 (G1-G3). All three groups underwent a left epididymal head PESA using a 253/8 gauge needle. The untouched right epididymis acted as the control. At 14 day intervals, G2 and G3 underwent a second and third PESA respectively. Fourteen days after the final PESA, both epididymides and a 1 cm segment of both vas deferentia were harvested for sperm and histological evaluations. RESULTS: The percentage of vas specimens with a sperm count ≥ 5 x104/cc was 100%, 22%, and 20% for the G1, G2, G3 PESA samples respectively. Moreover, the percentage of the vas specimens with sperm motility ≥ 10% was 90%, 22%, and 20%, respectively. Epididymal granulomas were not seen in the control side, but formed in 70%, 100%, and 80% of G1, G2, G3 PESA specimens, respectively. CONCLUSIONS: In a rat model, PESA resulted in significant epididymal inflammation and a reduction in both sperm concentration and motility.

CIP2A and PP2A in human leptomeninges, arachnoid granulations and meningiomas.

Previously we have found that mitogens stimulate proliferation of meningioma cells of all grades, in part, by activation of the PI3K-PKB/Akt-PRAS40-mTOR pathway regulated to some degree by the tumor suppressor phosphatase PP2A. PP2A activity is inhibited by the oncoprotein cancerous inhibitor of PP2A (CIP2A), which has not been studied in meningiomas to our knowledge. Six fetal and one adult human leptomeningeal samples and 38 meningiomas were evaluated by western blot. Fifteen adult arachnoid granulations and 58 formalin-fixed meningiomas (36 World Health Organization grade I, 15 grade II and seven grade III) were evaluated by immunohistochemistry. The effects of the mitogens platelet derived growth factor-BB (PDGF-BB) and cerebrospinal fluid on CIP2A were also studied. By western blot, CIP2A and PP2A were found in the five fetal and one adult leptomeninges and all meningiomas. By immunohistochemistry, CIP2A was detected in the arachnoid granulations and all meningiomas. CIP2A tended to be higher in grade III tumors. Three fetal leptomeningeal (two grade I and one grade II) and meningioma cells treated with PDGF-BB and/or human cerebrospinal fluid resulted in a slight increase in CIP2A in the leptomeningeal cells but not meningioma cells. Considered the mechanism of action and seen in other neoplasms, these findings raise the possibility that CIP2A may participate in the biology of meningiomas.

Fabrication of a light-emitting shape memory polymeric web containing indocyanine green.

Dye-containing polymers are highly desired for a number of commercially and medically relevant applications, such as sensors, medical devices, and drug delivery. In particular, dyes that emit light in the NIR region of the electromagnetic spectrum are of great interest due to the window of transparency for mammalian soft tissue in this range. While the incorporation of dyes into polymeric hosts by diffusion is a method that has been widely used, this approach is problematic in that it lacks uniformity and control over the incorporation. Here, we sought to develop NIR-emitting polymeric materials with high fluorescence intensity, addressing the problem of uniformity by incorporating the dye in a polymer host using dissolution in a mutual solvent and subsequent electrospinning into a fibrous web. This web could be prepared as a free-standing film, a coating or, as we will show, a shrink-wrap medical device label. The primary findings of this study were that an optimal concentration of dye in the polymer host exists, that the fluorescence intensity for fibrous webs greatly exceed that of comparable cast films, and that the dye-containing webs feature water-triggered contraction of use for application to medical devices, such as feeding tubes or catheters.

Polymorphism in the SCN9A voltage-gated sodium channel gene associated with interstitial cystitis/bladder pain syndrome.

OBJECTIVE: To determine whether an association exists between interstitial cystitis/bladder pain syndrome (IC/BPS) and a nonsynonymous single nucleotide polymorphism in the SCN9A voltage-gated sodium channel gene previously associated with other chronic pain syndromes. MATERIALS AND METHODS: Germline deoxyribonucleic acid was sampled from archived bladder biopsy specimens from patients with a documented diagnosis of IC/BPS. Deoxyribonucleic acid from hysterectomy specimens was obtained as a control population. The genotype of single nucleotide polymorphism rs6746030 was determined by deoxyribonucleic acid sequencing after polymerase chain reaction amplification. Contingency analysis of genotypes was performed using Pearson's chi-square test and Fisher's exact test. RESULTS: Polymerase chain reaction product was obtained from 26 of 31 control specimens and from 53 of 57 IC/BPS biopsy specimens. Of the 26 control subjects, 3 (11.5%) were genotype AG and 23 were GG. In contrast, AA or AG genotypes were present in 21 of 53 (39.6%) patients with IC/BPS, a statistically significant difference compared with the controls (Pearson's chi-square, P=.036). Similarly, the A allele was at a greater frequency in the IC/BPS group using Fisher's exact test (P=.009). CONCLUSION: These data strongly suggest that pain perception in at least a subset of patients with IC/BPS is influenced by this polymorphism in the SCN9A voltage-gated sodium channel.

Valproic acid decreases urothelial cancer cell proliferation and induces thrombospondin-1 expression.

BACKGROUND: Prevention of bladder cancer recurrence is a central challenge in the management of this highly prevalent disease. The histone deacetylase inhibitor valproic acid (sodium valproate) has anti-angiogenic properties and has been shown to decrease bladder cancer growth in model systems. We have previously shown reduced expression of thrombospondin-1 in a mouse model and in human bladder cancer relative to normal urothelium. We speculated that inhibition of angiogenesis by valproate might be mediated by this anti-angiogenic protein. METHODS: Bladder cancer cell lines UMUC3 and T24 were treated with valproate or another histone deacetylase inhibitor, vorinostat, in culture for a period of three days. Proliferation was assessed by alamar blue reduction. Gene expression was evaluated by reverse transcription of RNA and quantitative PCR. RESULTS: Proliferation assays showed treatment with valproate or vorinostat decreased proliferation in both cell lines. Histone deacetylase inhibition also increased relative expression of thrombospondin-1 up to 8 fold at 5 mM valproate. CONCLUSIONS: Histone deacetylase inhibitors warrant further study for the prevention or treatment of bladder cancer.

Near infrared fluorescence imaging after intravenous indocyanine green: initial clinical experience with open partial nephrectomy for renal cortical tumors.

OBJECTIVE: To evaluate the safety of near infrared fluorescence (NIRF) of intravenously injected indocyanine green (ICG) during open partial nephrectomy, and to demonstrate the feasibility of this technology to identify the renal vasculature and distinguish renal cortical tumors from normal parenchyma. METHODS: Patients undergoing open partial nephrectomy provided written informed consent for inclusion in this institutional review board-approved study. Perirenal fat was removed to allow visualization of the renal parenchyma and lesions to be excised. The patients received intravenous injections of ICG, and NIRF imaging was performed using the SPY system. Intraoperative NIRF video images were evaluated for differentiation of tumor from normal parenchyma and for renal vasculature identification. RESULTS: A total of 15 patients underwent 16 open partial nephrectomies. The mean cold ischemia time was 26.6 minutes (range 20-33). All 14 malignant lesions were afluorescent or hypofluorescent compared with the surrounding normal renal parenchyma. NIRF imaging of intravenously injected ICG clearly identified the renal hilar vessels and guided selective arterial clamping in 3 patients. No adverse reactions to ICG were noted, and all surgical margins were negative on final pathologic examination. CONCLUSION: The intravenous use of ICG combined with NIRF is safe during open renal surgery. This technology allows the surgeon to distinguish renal cortical tumors from normal tissue and highlights the renal vasculature, with the potential to maximize oncologic control and nephron sparing during open partial nephrectomy. Additional study is needed to determine whether this imaging technique will help improve the outcomes during open partial nephrectomy.

Thrombin expression in prostate: a novel finding.

INTRODUCTION: A variable repertoire of coagulation protein expression is observed in different cancers. We evaluated expression of thrombin in prostate tissue. METHODS: Detection of thrombin was performed using quantitative real-time PCR in fresh tissue and in situ hybridization (ISH) in archival prostate tissue and by immunohistochemistry of prostate tissue microarrays. RESULTS: (Pro)thrombin mRNA expression was detected in cancer tissue and localized to prostatic epithelium and stroma by ISH. Thrombin protein was detected in stroma of benign and malignant epithelium (p <.05). CONCLUSIONS: Prostate tissue is a rich reservoir of thrombin. This may have potential for developing antithrombin-based cancer therapy.

Fibroblast growth factor receptor-3 expression in meningiomas with stimulation of proliferation by the phosphoinositide 3 kinase-Akt pathway.

OBJECT: Fibroblast growth factor receptors (FGFRs)-1, -2, and -3 are expressed in the developing brain and may participate in CNS neoplasia. Fibroblast growth receptor-3 has not been demonstrated in the human CNS or its tumors. Nonetheless, it has been implicated in the pathogenesis of several other forms of neoplasia. METHODS: Twenty-four human meningiomas were evaluated using Western blot analysis for expression of FGFR3, its ligand acidic FGF, and concomitant phosphorylation/activation of p44/42 mitogen-activated protein kinase (MAPK), Akt, and STAT3. Mutations in exons 7 and 10 of the FGFR3 gene were analyzed by polymerase chain reaction in 10 meningiomas. Primary meningioma cells cultured from 10 human meningiomas were also treated with acidic FGF and evaluated for cell proliferation or activation/phosphorylation of p44/42 MAPK, Akt, and STAT3. RESULTS: Immunoblotting demonstrated the presence of FGFR3 in 12 (71%) of 17 primarily fibroblastic and transitional WHO Grade I meningiomas. The FGFR3 was detected in 4 (80%) of 5 WHO Grade II, and 2 of 2 Grade III tumors. Acidic FGF was detected in 3 (18%) of 17 Grade I, 1 (20%) of 5 Grade II, and 1 (50%) of 2 Grade III meningiomas. In WHO Grade I meningiomas, 3 of 6 tumors with no detectable FGFR3 had no detectable p-STAT3. In WHO Grades II and III meningiomas, FGFR3 expression was associated with p-STAT3, p-Akt, and p-p44/42 MAPK expression. No mutations were demonstrated in exons 7 or 10 by polymerase chain reaction in any meningioma. Treatment with acidic FGF, a ligand for FGFR3, stimulated meningioma cell proliferation and activation of Akt and STAT3 in primary meningioma cell cultures. CONCLUSIONS: These findings suggest that FGFR3 and acidic FGF are expressed in adult human leptomeninges as well as WHO Grades I and II meningiomas. Fibroblast growth factor receptor-3 activation stimulates meningioma cell proliferation by activation of the phosphoinositide 3 kinase-Akt-PRAS40-mTOR and STAT3 pathways.

Animal models of diabetic uropathy.

PURPOSE: Diabetes mellitus is a group of debilitating and costly diseases with multiple serious complications. Lower urinary tract complications or diabetic uropathy are among the most common complications of diabetes mellitus, surpassing widely recognized complications such as neuropathy and nephropathy. Diabetic uropathy develops in individuals with types 1 and 2 diabetes, and little is known about the natural history of these common and troublesome complications. Animal models have the potential to reveal mechanisms and aid in the development of treatment strategies. MATERIALS AND METHODS: We present a review of available animal models of diabetes mellitus relative to their use in the study of diabetic uropathy. RESULTS: Large and small animal models of diabetes mellitus are available. While large animals such as dogs and swine may closely mirror the human disease in size and phenotype, the time between diabetic complication onset and development, and associated husbandry expenditures can make acquiring data on statistically valid sample sizes prohibitively expensive. In contrast, small animal models (rats and mice) have much lower expenditures for a larger number of animals and compressed observation time due to a shorter life span. Also, mice are readily manipulated genetically to facilitate the isolation of the effect of single genes (transgenic and knockout mice). Type 1 diabetes mellitus can be induced chemically with streptozotocin, which is selectively toxic to pancreatic beta cells. Type 2 diabetes mellitus models have been developed by selective breeding for hyperglycemia with or without associated obesity. Diabetic uropathy has been noted in several well characterized, predictable animal models of diabetes mellitus. CONCLUSIONS: Diabetic uropathy, including diabetic bladder dysfunction, has been more frequently studied in small animals with type I diabetes. The recent availability of transgenic models provides a new opportunity for further studies of diabetic uropathy in mouse models of types I and II diabetes mellitus.

Inherited pelvic organ prolapse in the mouse: preliminary evaluation of a new murine model.

The objective of this study was to report the initial anatomic, radiographic, and genetic evaluations of a novel form of spontaneous pelvic organ prolapse (S-POP) in mice. We observed S-POP in a colony of UPII-SV40T transgenic mice developed for studies on bladder cancer. We utilized magnetic resonance imaging and necropsy to characterize this finding. We have established a breeding colony to identify inheritance patterns and for future studies. Selective breeding isolated the S-POP phenotype from the transgene. In contrast to other animal models, the S-POP mouse does not require an obligatory antecedent event to manifest pelvic organ prolapse. Necropsy and imaging demonstrate significant displacement of the pelvic organs distal to the pelvic floor in both sexes. The appearance of the POP is similar to that seen in the human female phenotype. Preliminary breeding studies indicate an autosomal dominant inheritance pattern. This mouse may be an effective animal model for the study of POP in humans.

Decreased bladder cancer growth in parous mice.

OBJECTIVES: To investigate the association between parity and exophytic bladder cancer growth in the UPII-SV40T transgenic mouse model of bladder cancer. METHODS: The UPII-SV40T transgenic mice express the simian virus 40 large T antigen specifically in the urothelium (driven by uroplakin II promoter) and reliably develop bladder cancer. UPII-SV40T transgenic female mice were either never bred (nulliparous; n = 6) or placed in breeding pairs and allowed full-term pregnancies and lactation. Multiparous animals (n = 5) had between 2 and 4 litters. UPII-SV40T transgenic male mice were sham-operated (intact; n = 9) or castrated (n = 8) at 24 weeks of age. Noninvasive, contrast-enhanced, flat panel detector-based, cone beam computed tomographic imaging of animals at 32 weeks of age permitted quantification of bladder cancer volumes. RESULTS: Multiparous animals had significantly smaller bladder cancers than their nulliparous female (P < .001) and intact male (P = .007) counterparts but not different from castrated males. Bladder cancer volume in nulliparous females was significantly larger than castrated males (P < .001) but not different from intact males. CONCLUSIONS: These results suggest that pregnancy, parity, lactation, or a combination of these may play a protective role in bladder cancer by inhibiting tumor growth. This could be an important model system for studying the effects of pregnancy/lactation hormones on bladder cancer, which could lead to identification of additional risk factors of bladder cancer.