cyclin-d1 and Urinary-Bladder-Neoplasms

Treatment of muscle invasive urothelial bladder carcinoma (BCa) remains a major challenge. Comprehensive genomic profiling of tumors and identification of driver mutations may reveal new therapeutic targets. This manuscript discusses relevant molecular drivers of the malignant phenotype and agents with therapeutic potential in BCa. Small molecule pan-FGFR inhibitors have shown encouraging efficacy and safety results especially among patients with activating FGFR mutations or translocations. mTOR inhibitors for patients with TSC1 mutations and concomitant targeting of PI3K and MEK represent strategies to block PI3K/AKT/mTOR pathway. Encouraging preclinical results with ado-trastuzumab emtansine (T-DM1) exemplifies a new potential treatment for HER2-positive BCa along with innovative bispecific antibodies. Inhibitors of cell cycle regulators (aurora kinase, polo-like kinase 1, and cyclin-dependent kinase 4) are being investigated in combination with chemotherapy. Early results of clinical studies with anti-CTLA4 and anti-PDL1 are propelling immune modulating drugs to the forefront of emerging treatments for BCa. Collectively, these novel therapeutic targets and treatment strategies hold promise to improve the outcome of patients afflicted with this malignancy.

Topics: Ado-Trastuzumab Emtansine; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Aurora Kinases; B7-H1 Antigen; Biomarkers, Tumor; Carcinoma, Transitional Cell; Cell Cycle Proteins; Clinical Trials as Topic; CTLA-4 Antigen; Cyclin D1; Cyclin-Dependent Kinase 4; Heat-Shock Proteins; Humans; Immunotherapy; Maytansine; Molecular Targeted Therapy; Mutation; Neoplasm Invasiveness; Phosphatidylinositol 3-Kinases; Polo-Like Kinase 1; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Receptor, ErbB-2; Receptor, Fibroblast Growth Factor, Type 1; Signal Transduction; TOR Serine-Threonine Kinases; Translocation, Genetic; Trastuzumab; Tuberous Sclerosis Complex 1 Protein; Tumor Suppressor Proteins; Urinary Bladder Neoplasms

Published studies on the association between cyclin D1 (CCND1) G870A polymorphism and bladder cancer risk have yielded conflicting results. Thus, a systemic review and meta-analysis of published studies were performed to assess the possible association. All eligible studies of G870A polymorphism and bladder cancer risk were collected from the PubMed and the Cochrane Library. Statistical analyses were performed by Review Manager 5.0 and Stata 11.0. Significant association between G870A polymorphism and bladder cancer susceptibility was found under recessive model in overall population (OR = 1.21, 95% CI 1.01-1.45, P = 0.04). When stratifying for the race, our analysis suggested that CCND1 G870A was associated with bladder cancer risk in Asians when using homogeneous codominant (OR = 1.72, 95% CI 1.34-2.20, P < 0.0001), recessive (OR = 1.46, 95% CI 1.21-1.77, P < 0.0001), dominant (OR = 1.36, 95% CI 1.10-1.69, P = 0.004), and allelic models (OR = 1.30, 95% CI 1.15-1.47, P < 0.0001) to analyze the data. However, no significant associations were found in Caucasians. After stratifying the studies by control source, G870A polymorphism was significantly associated with bladder cancer risk under recessive model (OR = 1.31, 95% CI 1.03-1.67, P = 0.03) in hospital-based case-control studies, but not in population-based case-control studies. This meta-analysis suggested that G870A polymorphism most likely contributes to increased susceptibility to bladder cancer in the overall population, hospital-based case-control studies, and Asians.

Topics: Alleles; Case-Control Studies; Cyclin D1; Genetic Predisposition to Disease; Genotype; Humans; Odds Ratio; Polymorphism, Single Nucleotide; Prognosis; Urinary Bladder Neoplasms

Perturbations in cell cycle and DNA repair genes might affect susceptibility to cancer. The aim of this meta-analysis is to generate large-scale evidence to determine the degree to which common Cyclin D1 (CCND1) G870A (dbSNP: rs603965) and xeroderma pigmentosum group C (XPC) Ala499Val (dbSNP: rs2228000) polymorphisms are associated with susceptibility to bladder cancer. The electronic databases PubMed, Embase, Web of Science, and CNKI were searched for relevant studies (with an upper date limit of July 25, 2013). The principal outcome measure for evaluating the strength of association was crude odds ratios (ORs) along with their corresponding confidence intervals (95%CIs). We found and reviewed nine case-control studies on CCND1 G870A with a total of 6,823 subjects and seven studies on XPC Ala499Val with a total of 7,674 subjects. Our meta-analysis provides evidence that the variant genotype of CCND1 G870A showed a significant association in the occurrence of invasive bladder tumors in former and current smokers. The XPC Ala499Val polymorphism correlated with significant differences between patients and unaffected subjects, but when the groups were stratified by ethnicity, the magnitude of the overall effect was similar only among Caucasian populations. Results from our meta-analysis support the view that the G870A polymorphism may modulate the risk of bladder cancer in conjunction with tobacco smoking and that the Ala499Val polymorphism may contribute to the susceptibility to bladder cancer in Caucasian populations. Our findings, however, warrant larger well-designed studies to investigate the significance of these two polymorphisms as markers of susceptibility to bladder cancer.

Topics: Case-Control Studies; Cyclin D1; DNA-Binding Proteins; Genetic Predisposition to Disease; Humans; Polymorphism, Genetic; Publication Bias; Risk; Urinary Bladder Neoplasms

To evaluate the relationship between cyclin D1 overexpression and bladder cancer prognosis.. A systematic literature search up to July 27, 2013 was carried out in PubMed and Wanfang databases, and the references of retrieved articles were screened. The hazard ratios (HRs) and their 95% CIs were used to combine the data. Heterogeneity and publication bias were also evaluated.. A total of 15 studies containing 2,591 cases were included. We found that increased cyclin D1 levels were significantly correlated with progression-free survival with a pooled HR estimate of 0.54 (95% CI: 0.32-0.92). There was a significant degree of heterogeneity (I² = 93.8%, P <0.001). Moreover, subgroup analysis indicated that elevated cyclin D1 levels were significantly associated with overall survival in muscle-invasive bladder patients (HR: 0.95, 95% CI: 0.91-0.99), without a significant heterogeneity in the data (I² = 0.0%, P = 0.456).. Increased cyclin D1 expression level detected by immunohistochemistry is associated with good progression-free survival for bladder cancer. Because of the limited number of studies, further well-designed prospective studies are warranted to confirm the findings from our study.

Topics: Biomarkers, Tumor; Cyclin D1; Humans; Prognosis; Survival Rate; Urinary Bladder Neoplasms

Cyclin D1 (CCND1) is a key protein in regulation of cell cycle at the G1-to-S transition phase and is essential for regulation of cell proliferation, differentiation, and transcriptional control. We hypothesized that the CCND1 G870A polymorphism is associated with risk of bladder cancer. The CCND1 G870A polymorphism was genotyped in a hospital-based case-control study of 402 bladder cancer cases and 402 control subjects using the polymerase chain reaction-restriction fragment length polymorphism method. Unconditional univariate and multivariate logistic regression analyses were used to evaluate the associations between the CCND1 G870A polymorphism and bladder cancer risk. A significantly increased risk of bladder cancer was associated with the combined variant CCND1 870GA/AA genotypes (adjusted odds ratio, 1.54; 95% confidence interval, 1.08-2.20) compared with the GG genotype, particularly among subgroups of age ≥65 years (1.74; 1.06-2.88), men (1.67; 1.15-2.44), and smokers (1.82; 1.12-2.93). Further, the G870A polymorphism was significantly associated with risk of developing superficial bladder cancer (grade 1). In addition, a meta-analysis of the G870A polymorphism and bladder cancer risk showed that the variant 870GA/AA genotypes were associated with an increased risk of bladder cancer in Asians, but not in Caucasians, which was consistent with the results of our study. The CCND1 G870A polymorphism may be a marker for the development of bladder cancer in Chinese populations. Larger studies are required to validate these findings in diverse populations.

Topics: Aged; Alleles; Asian People; Case-Control Studies; Cyclin D1; Female; Gene Frequency; Genotype; Humans; Male; Middle Aged; Polymorphism, Genetic; Retrospective Studies; Risk; Urinary Bladder Neoplasms

There is much information on the genetic alterations that contribute to the development of bladder cancer. Because it is hypothesised that the genotype of the cancer cell plays a major role in determining phenotype, this genetic information should impact on clinical practice. To date however, this has not happened. Some of the alterations identified in bladder cancer have clear associations with outcome-for example, mutational inactivation of the cell cycle regulator proteins p53 and the retinoblastoma protein (Rb). However, as single markers, these events have insufficient predictive power to be applied in the management of individual patients. The use of panels of markers is a potential solution to this problem. Examples of suitable panels include those genes/proteins with known impact on specific cell cycle checkpoints or with impact on cellular phenotypes, such as immortalisation, invasion, or metastasis. To evaluate such marker panels, large tumour series will be needed-for example, archival samples from completed clinical trials. The use of these valuable resources will require coordination of sample provision. This might involve central collection and distribution of tissue blocks, sections, or tissue arrays and the provision of patient follow up information to laboratories participating in a study. With the availability of microarray technologies, including cDNA and comparative genomic hybridisation arrays, the transcriptome and genome of transitional cell carcinomas of different phenotypes can be compared and will undoubtedly provide a wealth of information with potential diagnostic and prognostic uses. Although these studies can be initiated using small local tissue collections, high quality collection of fresh tissues from new clinical trials will be crucial for proper evaluation of associations with clinical outcome. Funding for molecular pathological studies to date has been poor. To begin to translate molecular information from the laboratory to the clinic and to make maximum use of valuable urological patient resources in the UK, adequate funding and scientific energy are required. Whereas the latter is not in doubt, present funding for this type of translational research is inadequate.

Topics: Carcinoma, Transitional Cell; Cyclin D1; Gene Deletion; Genes, p16; Genes, p53; Genes, Retinoblastoma; Genetic Markers; Humans; Loss of Heterozygosity; Mutation; Prognosis; Proteins; Research; Tumor Suppressor Protein p14ARF; Urinary Bladder Neoplasms

Topics: Apoptosis; Cyclin D1; Gene Expression Regulation, Neoplastic; Genes, bcl-2; Genes, p53; Humans; Nuclear Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-mdm2; Proto-Oncogene Proteins p21(ras); Retinoblastoma; Thrombospondins; Tumor Suppressor Protein p53; Urinary Bladder Neoplasms

This study aimed to investigate the effects of scaffold matrix attachment region binding protein 1 (SMAR1) on the development of bladder cancer (BCa). SMAR1 expression in paired tumor and corresponding adjacent normal tissues from 55 BCa patients was detected by quantitative reverse transcription-polymerase chain reaction. BCa cells were transfected to regulate SMAR1 expression. BCa cells were treated with XAV-939, LiCl and 2-deoxyglucose. The effect of SMAR1 on the viability, proliferation, migration, invasion and Warburg effect of BCa cells was researched by counting kit-8, colony formation assay, Transwell and aerobic glycolysis assays. Western blot was performed to detect protein expression. BCa cell growth in vivo was recorded in nude mice. Immunohistochemical staining was performed for clinical and xenografted tumor tissue specimens. SMAR1 expression was down-regulated in BCa patients, associating with worse prognoses. SMAR1 knockdown enhanced the viability, proliferation, migration, invasion, EMT and Warburg effect of BCa cells. The opposite effect was found in the SMAR1 overexpression BCa cells. XAV-939 treatment reversed the elevation of β-catenin, c-Myc and Cyclin D1 proteins expression and Warburg effect in Bca cells post-SMAR1 knockdown. LiCl treatment abrogated the inhibition of β-catenin, c-Myc and Cyclin D1 proteins expression and Warburg effect proteins due to SMAR1 overexpression in BCa cells. SMAR1 overexpression inhibited the growth of BCa cells in vivo. SMAR1 might suppress the Wnt/β-catenin signaling pathway activity to inhibit the progression of BCa. It might be an effective treatment target for BCa.

Topics: Animals; beta Catenin; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Mice; Mice, Nude; Urinary Bladder Neoplasms; Wnt Signaling Pathway

To date, no reported studies have explored the impacts of microcystin-LR (MC-LR) on bladder tissues, and even the occurrence of bladder cancer. The current study explores the role of MC-LR in the development of bladder cancer through human observation and experimental research. In the population study, the odds ratio of bladder cancer for MC-LR was 6.073 (95 % CI, 2.117-17.422) after adjusting interference confounders. MC-LR is mainly located in the nucleus of epithelial cells in bladder cancer tissues instead of normal tissues. A positive association was observed between MC-LR and advanced tumor stage in serum and tissues. The animal study confirmed that prolonged MC-LR treatment promoted the bladder cancer phenotype accompanied by urinary bladder proliferation. In vitro, we indicated that MC-LR activated the PI3K/AKT/GSK3β/Cyclin D1 and JAK2/STAT3/Bcl2 signaling pathways to induce the growth of SV-HUC-1 cells. Moreover, MC-LR promoted the angiogenesis of SV-HUC-1 cells through PI3K/AKT/mTOR/HIF-1α/VEGF pathway. Our study provided the first evidence that prolonged MC-LR treatment increases the incidence of bladder cancer from human investigations, mice models, and in vitro studies, implying the profound importance of the investigation of MC-LR for public health.

Topics: Animals; Carcinogenesis; Cell Proliferation; Cyclin D1; Environmental Exposure; Glycogen Synthase Kinase 3 beta; Humans; Marine Toxins; Mice; Microcystins; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; TOR Serine-Threonine Kinases; Urinary Bladder; Urinary Bladder Neoplasms; Vascular Endothelial Growth Factor A

Extensive research confirmed that circRNA can play a regulatory role in various stages of tumors by interacting with various molecules. Identifying the differentially expressed circRNA in bladder cancer and exploring its regulatory mechanism on bladder cancer progression are urgent. In this study, we screened out a circRNA-circGLIS3 with a significant upregulation trend in both bladder cancer tissues and cells. Bioinformatics prediction results showed that circGLIS3 may be involved in multiple tumor-related pathways. Function gain and loss experiments verified circGLIS3 can affect the proliferation, migration, and invasion of bladder cancer cells in vitro. Moreover, silencing circGLIS3 inhibited bladder cancer cell growth in vivo. Subsequent research results indicated circGLIS3 regulated the expression of cyclin D1, a cell cycle-related protein, and cell cycle progression. Mechanically, circGLIS3 upregulates the expression of SKP1 by adsorbing miR-1273f and then promotes cyclin D1 expression, ultimately promoting the proliferation of bladder cancer cells. In summary, our study indicates that circGLIS3 plays an oncogene role in the development of bladder cancer and has potential to be a candidate for bladder cancer.

Topics: Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Humans; Male; MicroRNAs; RNA, Circular; S-Phase Kinase-Associated Proteins; Urinary Bladder Neoplasms

Non-schistosomiasis related-squamous cell carcinoma of urinary bladder (NSR-SCCUB) is a rare tumor subtype distinct from urothelial carcinoma (UC). Studies assessing molecular biomarkers in bladder cancer have generally focused on UC, and genomic data of NSR-SCCUB is limited. We aim to provide additional insight into the molecular underpinnings of this rare entity.. NSR-SCCUB patients were identified retrospectively at Princess Margaret Cancer Centre between 2002 and 2017. Demographics, disease characteristics, therapeutic approaches, and outcomes were collected. Tissue samples were interrogated using the Oncomine Comprehensive Assay v3 (ThermoFisher). Kaplan-Meier method was used to estimate the disease-free survival and overall survival (OS).. Overall, 11 patients with NSR-SCCUB were identified between 2002 and 2017 with adequate tissue samples. Median age was 71 years (45-86), predominantly male (63.6%). At time of diagnosis, 9 patients (81.8%) had muscle-invasive disease, 1 (9.1%) had non-muscle invasive, and 1 (9.1%) had advanced disease. Nine (81.8%) patients had radical cystectomy and pelvic lymph nodes dissection. Eight (72.7%) patients had pT3 or pT4 with N0, and 5 (45.5%) were grade 3. Median OS was 12.5 months (95% CI 7.7-17.2 months). Single nucleotide variants or insertion/deletions were identified in TP53, TERT, PIK3CA, PTEN, CREBBP, FBXW7, and FGFR3. Amplifications were found in CCND1, and EGFR.. NSR-SCCUB has potentially actionable genomic alterations with anticancer agents and many of these aberrations are also seen in UC. The recruitment of NSR-SCCUB patients harboring such mutations should be considered in biomarker driven urinary bladder cancer studies.

Topics: Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoma, Squamous Cell; Class I Phosphatidylinositol 3-Kinases; CREB-Binding Protein; Cyclin D1; ErbB Receptors; F-Box-WD Repeat-Containing Protein 7; Female; Humans; Male; Middle Aged; Mutation; PTEN Phosphohydrolase; Receptor, Fibroblast Growth Factor, Type 3; Telomerase; Tumor Suppressor Protein p53; Urinary Bladder Neoplasms

ZFP36L1 is a tandem zinc-finger RNA-binding protein that recognizes conserved adenylate-uridylate-rich elements (ARE) located in 3'untranslated regions (UTR) to mediate mRNA decay. We hypothesized that ZFP36L1 is a negative regulator of a posttranscriptional hub involved in mRNA half-life regulation of cancer-related transcripts. Analysis of

Topics: 3' Untranslated Regions; Animals; Breast Neoplasms; Butyrate Response Factor 1; Carcinogenesis; Cell Cycle; Cell Hypoxia; Cell Line, Tumor; Cyclin D1; E2F1 Transcription Factor; Epigenesis, Genetic; Female; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Mice; Mutation; RNA Processing, Post-Transcriptional; RNA Stability; RNA, Messenger; RNA, Small Interfering; Urinary Bladder Neoplasms; Xenograft Model Antitumor Assays; Zinc Fingers

Downregulation of miR-502-5p has emerged as a critical factor in tumour progression in several cancers. Herein, we elucidated the role of miR-502-5p in bladder cancer.. RT-qPCR was performed to examine the expression of miR-502-5p in bladder cancer. And DNA methylation analysis showed that epigenetic mechanisms may contribute to the downregulation of miR-502-5p. Then, wound-healing assay, transwell assay, colony formation assay, CCK8 assay and flow cytometry analysis were applied to evaluate the function of miR-502-5p in bladder cancer cell lines. Western blot was conducted to measure the protein levels of related genes. Furthermore, dual-luciferase reporter assay, in vivo tumorigenesis assay and immunohistochemical staining were also conducted as needed.. MiR-502-5p is frequently downregulated in BCa. Meanwhile, hypermethylation of CpG islands contributes to the downregulation of miR-502-5p. Functionally, overexpression of miR-502-5p inhibited cell proliferation and migration in vitro and repressed tumour growth in vivo. CCND1, DNMT3B and NOP14 were identified as direct targets of miR-502-5p. Interestingly, DNMT3B and miR-502-5p established a positive feedback loop in the regulation of bladder cancer. In addition, rescue experiments further validated the direct molecular interaction between miR-502-5p and its targets.. Our study proposed and demonstrated that the miR-502-5p-mediated regulatory network is critical in bladder cancer; this network may be useful in the development of more effective therapies against bladder cancer.

Topics: Animals; Cell Line; Cell Line, Tumor; Cell Movement; Cell Proliferation; CpG Islands; Cyclin D1; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; DNA Methyltransferase 3B; Down-Regulation; Gene Expression Regulation, Neoplastic; HEK293 Cells; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Nude; MicroRNAs; Nuclear Proteins; Urinary Bladder Neoplasms

Pleomorphic rhabdomyosarcoma (PRMS) is a rare but highly aggressive soft tissue tumor, accounting for 3% of soft tissue sarcomas. PRMS is the most frequent subtype of RMS in adulthood and it is mainly located in the large muscles of the extremities, particularly the lower limbs and the trunk, more rarely in other locations especially in the bladder. At our knowledge, only six cases of adult pleomorphic rhabdomyosarcoma of the bladder have been reported in the literature. In this study, we report a case of PRMS of bladder with a very poor prognosis. In fact, the patient died a month after surgery. The tumor was characterized by poorly differentiated, medium-sized sometimes rhabdoid cells, mixed with large-sized and pleomorphic elements with evident anisonucleosis, and with large areas of necrosis. We used an extensive immunohistochemical panel to exclude other tumors much more frequently reported at this site. The positivity for myogenic markers such as actin, desmin, myogenin and MyoD1 allowed the correct diagnosis. Furthermore, since preliminary studies highlighted a series of specific molecular alterations in PMRS cell lines, we analyzed a panel of specific mutations and gene rearrangements by RT-PCR and FISH methods. We showed a copy gains of CCND1 and MALT genes in our samples, suggesting an accurate molecular characterization of PRMS to establish a better management of patients and new therapeutic opportunities.

Topics: Biomarkers, Tumor; Cyclin D1; DNA Mutational Analysis; Fatal Outcome; Humans; Male; Middle Aged; Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein; Rhabdomyosarcoma; Urinary Bladder Neoplasms

The aim of the present study was to analyze copy number variations (CNV) of multiple oncogenes and tumor suppressor genes in genomic DNA from primary tumor tissue, lymph node metastasis and cell-free DNA (cfDNA) from serum of 72 urothelial carcinoma of bladder (UCB) patients treated with radical cystectomy (RC), using multiplex ligation-dependent probe amplification (MLPA). We hypothesized that primary tumor and lymph node metastasis show similar CNV profiles, and CNV are more present in lymph node metastasis compared to primary tumor tissue. Samples from 43 (59.7%) patients could be analyzed. In total, 35 (83%), 26 (68%) and 8 (42%) patients had CNV in primary tumor, serum and lymph node metastasis, respectively. MYC, CCND1, ERBB2 and CCNE1 displayed the most frequent amplifications. In particular, CNV in ERBB2 was associated with aggressive tumor characteristics. CNV in both ERBB2 and TOP2A were risk factors for disease recurrence. The current findings show that CNV are present in various oncogenes and tumor suppressor genes in genomic DNA from primary tumor, lymph node metastasis and cfDNA from serum. CNV were more present in genomic DNA from primary tumor tissue compared to cfDNA from serum and genomic DNA from lymph node metastasis. Patients with CNV in ERBB2 and TOP2A are at increased risk for disease recurrence following RC. Further studies are necessary to validate, whether these genes may represent promising candidates for targeted-therapy.

Topics: Aged; Aged, 80 and over; Cyclin D1; Cyclin E; Cystectomy; DNA Copy Number Variations; Female; Humans; Lymph Node Excision; Lymphatic Metastasis; Male; Middle Aged; Multiplex Polymerase Chain Reaction; Oncogene Proteins; Proto-Oncogene Proteins c-myc; Receptor, ErbB-2; Urinary Bladder Neoplasms

Cancer stem cells (CSC) are highly associated with poor prognosis in cancer patients. Our previous studies report that isorhapontigenin (ISO) down-regulates SOX2-mediated cyclin D1 induction and stem-like cell properties in glioma stem-like cells. The present study revealed that ISO could inhibit stem cell-like phenotypes and invasivity of human bladder cancer (BC) by specific attenuation of expression of CD44 but not SOX-2, at both the protein transcription and degradation levels. On one hand, ISO inhibited cd44 mRNA expression through decreases in Sp1 direct binding to its promoter region-binding site, resulting in attenuation of its transcription. On the other hand, ISO also down-regulated USP28 expression, which in turn reduced CD44 protein stability. Further studies showed that ISO treatment induced miR-4295, which specific bound to 3'-UTR activity of usp28 mRNA and inhibited its translation and expression, while miR-4295 induction was mediated by increased Dicer protein to enhance miR-4295 maturation upon ISO treatment. Our results provide the first evidence that ISO has a profound inhibitory effect on human BC stem cell-like phenotypes and invasivity through the mechanisms distinct from those previously noted in glioma stem-like cells.

Topics: 3' Untranslated Regions; Binding Sites; Cell Line, Tumor; Cyclin D1; Down-Regulation; Gene Expression Regulation, Neoplastic; Humans; Hyaluronan Receptors; MicroRNAs; Neoplastic Stem Cells; Promoter Regions, Genetic; RNA, Messenger; SOXB1 Transcription Factors; Stem Cells; Stilbenes; Transcription, Genetic; Ubiquitin Thiolesterase; Urinary Bladder Neoplasms

Long non‑coding RNA (lncRNA) metastasis associated lung adenocarcinoma transcript 1 (MALAT1) has been demonstrated to participate in the development and progression of some common cancer types, including bladder cancer (BC). However, the regulatory mechanism of MALAT1 underlying BC growth and metastasis remains to be fully elucidated. The present study revealed that MALAT1 was significantly upregulated in BC tissues and cell lines compared with the adjacent non‑tumour tissues and the normal urinary tract epithelial cell line SV‑HUC‑1, respectively. The expression levels of MALAT1 were higher in stage III‑IV BC tissues when compared with that in stage I‑II tissues. Furthermore, knockdown of MALAT1 significantly inhibited BC cell proliferation and migration by targeting microRNA (miR)‑34a. The expression levels of miR‑34a were significantly decreased in BC tissues and cell lines compared with that of adjacent non‑tumour tissues and SV‑HUC‑1 cells. In addition, the expression of miR‑34a was inversely correlated with the expression of MALAT1 in BC tissues. The present study revealed that cyclin D1 (CCND1) was identified as a target gene of miR‑34a, and its expression was negatively mediated by miR‑34a in BC cells. Notably, the upregulation of CCND1 impaired the effect of MALAT1 inhibition on BC cell proliferation and migration. In addition, the expression levels of CCND1 were significantly increased in BC tissues and cell lines. In conclusion, the present findings demonstrated that the knockdown of lncRNA MALAT1 inhibits the proliferation and migration of BC cells by modulating the miR‑34a/CCND1 axis, suggesting that the MALAT1/miR‑34a/CCND1 axis may be a potential therapeutic target for BC treatment.

Topics: Adult; Aged; Base Sequence; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Male; MicroRNAs; Middle Aged; RNA, Long Noncoding; Signal Transduction; Up-Regulation; Urinary Bladder Neoplasms

To investigate the expression of several immunohistochemical (IHC) markers and their predictive ability for the recurrence-free and progression-free survival of papillary urothelial bladder cancer (UBC) pTa/pT1 G2 (WHO 1973) compared to classical anatomo-clinical variables using a multidimensional analysis.. A population-based cohort of 213 primary stage UBC (pTa/pT1) G2 (WHO 1973) was evaluated by classic anatomopathological variables and characterized by immunohistochemistry (23 IHC markers, representative of different oncogenic pathways). The most important variables as a predictor of recurrence-free and progression-free survival were selected using multidimensional statistical models, such as random survival forests and least absolute shrinkage and selection operator (. Recurrence and progression-free survival of the previously selected variables were also calculated.. Mean follow-up was 58 ± 33.5 months. Recurrence and progression rates were 54.5% (n = 116) and 17,4% (n = 37), respectively. The most influential variables in the low recurrence-free survival were in order: number of resected tumors, high expression of Ki67 (>10%), Cyclin D1 (>10%), and low cytoplasmic staining of p16INK4a. Regarding low progression-free survival, the most important variables were Ki67 (>15%), multicentric tumor arrangement and Survivin nuclear expression (>20%). Kaplan-Meier and cox-regression model analyses showed that the variables selected by multidimensional models were able to discriminate the clinical outcome.. Ki67 index is the most useful IHC marker, since it can improve the prediction of both recurrence and progression-free survival in papillary UBC pTa/pT1 G2 (WHO 1973). There are other markers, whose utility is specific to recurrence-free survival, such as Cyclin D1 and p16INK4a or in progression-free survival, such as Survivin.

Topics: Aged; Biomarkers, Tumor; Carcinoma, Papillary; Cohort Studies; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Female; Follow-Up Studies; Humans; Ki-67 Antigen; Male; Middle Aged; Neoplasm Grading; Neoplasm Recurrence, Local; Neoplasm Staging; Predictive Value of Tests; Survival Rate; Survivin; Urinary Bladder Neoplasms

The prognosis for patients with bladder cancer (BCa) with lymph node (LN) metastasis is poor, and it is not improved by current treatments. Long noncoding RNAs (lncRNAs) are involved in the pathology of various tumors, including BCa. However, the role of Differentiation antagonizing non-protein coding RNA (DANCR) in BCa LN metastasis remains unclear. In this study, we discover that DANCR was significantly upregulated in BCa tissues and cases with LN metastasis. DANCR expression was positively correlated with LN metastasis status, tumor stage, histological grade, and poor patient prognosis. Functional assays demonstrated that DANCR promoted BCa cell migration, invasion, and proliferation in vitro and enhanced tumor LN metastasis and growth in vivo. Mechanistic investigations revealed that DANCR activated IL-11-STAT3 signaling and increased cyclin D1 and PLAU expression via guiding leucine-rich pentatricopeptide repeat containing (LRPPRC) to stabilize mRNA. Moreover, oncogenesis facilitated by DANCR was attenuated by anti-IL-11 antibody or a STAT3 inhibitor (BP-1-102). In conclusion, our findings indicate that DANCR induces BCa LN metastasis and proliferation via an LRPPRC-mediated mRNA stabilization mechanism. DANCR may serve as a multi-potency target for clinical intervention in LN-metastatic BCa.

Topics: Blotting, Western; Cell Movement; Cell Proliferation; Cyclin D1; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation, Neoplastic; HEK293 Cells; Humans; In Vitro Techniques; Interleukin-11; Kaplan-Meier Estimate; RNA, Long Noncoding; Signal Transduction; STAT3 Transcription Factor; Urinary Bladder Neoplasms; Wound Healing

Several studies by our group and others have determined that expression levels of Bcl-2 and/or Bcl-xL, pro-survival molecules which are associated with chemoresistance, are elevated in patients with muscle invasive bladder cancer (MI-BC). The goal of this study was to determine whether combining Obatoclax, a BH3 mimetic which inhibits pro-survival Bcl-2 family members, can improve responses to cisplatin chemotherapy, the standard of care treatment for MI-BC. Three MI-BC cell lines (T24, TCCSuP, 5637) were treated with Obatoclax alone or in combination with cisplatin and/or pre-miR-34a, a molecule which we have previously shown to inhibit MI-BC cell proliferation via decreasing Cdk6 expression. Proliferation, clonogenic, and apoptosis assays confirmed that Obatoclax can decrease cell proliferation and promote apoptosis in a dose-dependent manner. Combination treatment experiments identified Obatoclax + cisplatin as the most effective treatment. Immunoprecipitation and Western analyses indicate that, in addition to being able to inhibit Bcl-2 and Bcl-xL, Obatoclax can also decrease cyclin D1 and Cdk4/6 expression levels. This has not previously been reported. The combined data demonstrate that Obatoclax can inhibit cell proliferation, promote apoptosis, and significantly enhance the effectiveness of cisplatin in MI-BC cells via mechanisms that likely involve the inhibition of both pro-survival molecules and cell cycle regulators.

Topics: bcl-X Protein; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cisplatin; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Dose-Response Relationship, Drug; Drug Synergism; Gene Expression Regulation, Neoplastic; Humans; Indoles; Neoplasm Invasiveness; Proto-Oncogene Proteins c-bcl-2; Pyrroles; Urinary Bladder; Urinary Bladder Neoplasms

Accumulating evidences suggest that circular RNAs play vital roles in human cancers. Previously, we found that circHIPK3 suppressed invasion of bladder cancer cells via sponging miR-558 and downregulating heparanase expression. In this study, we discovered that a circular RNA derived from NR3C1 (circNR3C1) was downregulated in bladder cancer tissues and cell lines according to RNA-Seq data and qRT-PCR analysis. Functionally, we found that overexpression of circNR3C1 could significantly inhibit cell cycle progression and proliferation of bladder cancer cells in vitro, as well as suppress tumor growth in vivo. Mechanistically, we demonstrated that circNR3C1 possessed four targeting sites of miR-27a-3p and could effectively sponge miR-27a-3p to suppress the expression of cyclin D1. Furthermore, we revealed that miR-27a-3p functioned as an oncogene through interacting with 5'UTR of cyclin D1 to enhance its expression, which led to promote cell cycle progression and proliferation in bladder cancer cells. Conclusively, our findings further confirm the hypothesis that circRNAs function as "microRNA sponges", and our data suggest that circNR3C1 and miR-27a-3p would be potential therapeutic targets for bladder cancer treatment.

Topics: 5' Untranslated Regions; Animals; Binding Sites; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Down-Regulation; Gene Expression Regulation, Neoplastic; Humans; Mice, Nude; MicroRNAs; RNA, Circular; Signal Transduction; Tumor Burden; Urinary Bladder Neoplasms

SRY-box 18 (SOX18) is a transcription factor known for its role in regulating cell differentiation and lymphatic and blood vessel development. It has been reported that SOX18 was involved in various diseases, including cancer. This study aimed to explore the significance and biological function of SOX18 in bladder cancer (BCa).. SOX18 expression in BCa and normal tissues was analyzed by immunohistochemistry, and SOX18 expression in BCa cell lines was quantified by western blotting and quantitative real-time PCR. The role of SOX18 on the proliferation, migration and invasion of BCa cells was explored by CCK-8 and transwell invasion assays in vitro. Cell cycle was measured by flow cytometry assays. Western blotting and qRT-PCR were performed to investigate the potential mechanisms by which SOX18 leads to tumor progression.. SOX18 was significantly upregulated in BCa and its expression was associated with clinical features of patients with BCa. Our data demonstrated that SOX18 promoted cell proliferation via accelerating cell cycle and by regulating c-Myc and Cyclin D1, promoted cell invasion via upregulation of MMP-7. Moreover, phosphorylation of c-Met and Akt regulated by SOX18 was identified to be involved in the process of cell migration and invasion, indicating the vital role of SOX18 in the metastasis of BCa.. Our data demonstrated a cancer-promoting effect of SOX18 in BCa, revealed the potential mechanisms of SOX18 in mediating cellular functions, and indicated that SOX18 may serve as a promising progression and prognostic biomarker and a therapeutic target for BCa.

Topics: Aged; Aged, 80 and over; Animals; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Female; G1 Phase; Heterografts; Humans; Male; Matrix Metalloproteinase 7; Mice; Mice, Nude; Middle Aged; Neoplasm Invasiveness; Proto-Oncogene Proteins c-myc; S Phase; SOXF Transcription Factors; Transcriptome; Urinary Bladder Neoplasms

Bladder cancer (BC) is one of the commonest malignancies in the urinary system. Recent evidences have shown that Phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPP) serves as a tumor suppressor in hepatocellular carcinoma and cervical cancer. However, little is known about its function in BC. Here, we aimed to investigate the role of LHPP in BC. We found that LHPP was down-regulated in BC tissues and cells. Knockdown of LHPP promoted the proliferation and growth of BC cells T24 and 5637. Inverse results were observed in SW780 and BIU87 cells with ectopic LHPP expression. LHPP also repressed the glycolysis of BC cells. At the molecular level, LHPP silencing led to enhanced phosphorylation of both AKT and p65, as well as up-regulation of their downstream targets Bcl-2 and Cyclin D1. Inhibition of AKT by MK2206 blunted the increased phosphorylation of p65 caused by LHPP knockdown, suggesting that LHPP silencing activated p65 through AKT. Importantly, p65 inhibitor (caffeic acid phenethyl ester) exhibited larger suppressive effect on the proliferation of LHPP knockdown BC cells as compared with Ctrl cell. Our study demonstrates that LHPP suppresses BC cell growth via inactivating AKT/p65 signaling pathway.

Topics: Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Glucose; Glycolysis; Humans; Inorganic Pyrophosphatase; Lactic Acid; Male; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Transcription Factor RelA; Urinary Bladder Neoplasms

Deregulation of the cell cycle regulating genes is common in urothelial bladder carcinoma (UBC). We aimed to examine the prognostic significance of ki-67, p53, p63 and cyclinD1expression in UBC and to identify optimal cut-off points to help identifying patients at high risk of tumor recurrence. We evaluated the immunohistochemical expression of ki-67, p53, p63 and cyclinD1 in 100 UBCs. The conventional and the classification and regression trees-guided (CART-guided) methods were utilized to determine the independent predictors of tumor recurrence. The p53 and Ki-67 expression didn't associate significantly with tumor recurrence.p63 and cyclinD1 exhibited significant hazard ratios. Using CART, no recurrence was observed when p63 was ≥87.5%. The recurrence incidence increased and the disease free survival (DFS) time shortened as the p63 decreased. CyclinD1 associated significantly with tumor recurrence only if p63 was <35%. Using the CART cut-off values¬, cases were categorized into three groups; (groups I: p63 ≥ 35%, II: p63 < 35% and cyclinD1 < 10% and III: p63 < 35% and cyclinD1 ≥ 10%). Group I patients revealed the least incidence of recurrence at the longest DFS. Group III had the worst prognosis followed by Group II. p63 represents a surrogant biomarker to predict UBC recurrence.CyclinD1 can be used only when p63 is <35%. CART proved helpful with data among which the number of cases with positive outcomes is too small relative to the number of studied predictors. Large cohort studies for ki-67 and p53 are recommended to be performed with standardized criteria as regards patients' characteristics, cut-off values, and follow-up time.

Topics: Adult; Aged; Biomarkers, Tumor; Carcinoma, Transitional Cell; Cyclin D1; Disease Progression; Disease-Free Survival; Female; Humans; Ki-67 Antigen; Male; Membrane Proteins; Middle Aged; Neoplasm Recurrence, Local; Prognosis; Retrospective Studies; Tumor Suppressor Protein p53; Urinary Bladder Neoplasms

The human cyclin D1 gene generates two major isoforms, cyclin D1a and cyclin D1b, by alternative splicing. Although cyclin D1b mRNA is hardly expressed in normal human tissues, it is detected in approximately 60% of human bladder cancer tissues and cell lines. In the present study, to assess the therapeutic ability of cyclin D1b siRNA, we investigated the anti-oncogenic effects of cyclin D1b siRNA on human bladder cancer cell lines, SBT31A and T24, which express cyclin D1b mRNA. Knockdown of cyclin D1b by specific siRNA significantly suppressed cell proliferation, in vitro cell invasiveness and three-dimensional (3D) spheroid formation in these cell lines. Cell cycle analyses revealed that cyclin D1b siRNA inhibited G1-S transition in T24 cells. The increase in the sub-G1 fraction, morphological aberrant nuclei with nuclear fragmentation and caspase-3 activity in SBA31A cells treated with cyclin D1b siRNA showed that cyclin D1b siRNA induced apoptosis. In T24 cells, knockdown of cyclin D1b suppressed the expression of the stem cell marker CD44. Knockdown of cyclin D1b or CD44 suppressed the invasiveness under 3D spheroid culture conditions and expression of N-cadherin. Tumor growth of SBT31A cells in nude mice was significantly inhibited by cyclin D1b siRNA. Taken together, these results indicate that knockdown of cyclin D1b suppresses the malignant phenotypes of human bladder cancer cells via induction of apoptosis and suppression of cancer cell stemness and epithelial-mesenchymal transition. Applying cyclin D1b siRNA will be a novel therapy for cyclin D1b-expressing bladder cancers.

Topics: Animals; Apoptosis; Carcinogenicity Tests; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Female; Gene Knockdown Techniques; Humans; Mice; Mice, Nude; Mice, Transgenic; Neoplasm Invasiveness; Random Allocation; RNA, Small Interfering; RNAi Therapeutics; Transfection; Urinary Bladder Neoplasms; Xenograft Model Antitumor Assays

Bladder cancer has shown great challenge for people's life. Traditional therapeutics against bladder cancer including surgery could not bring much benefit for patients, particularly for the late stage patients. So it is necessary to keep in mind why and how bladder cancer cells survive in our body. In this study, we explored the function and the molecular mechanism of GGN gene in bladder cancer. GGN was shown to be expressed at a high level in bladder cancer tissues compared to the control and was associated with the unsatisfactory survival rate of patients. GGN was also expressed abundantly in bladder cancer cell lines such as T24, 5637 and BIU87. Then GGN was knocked down in 5637 cells and T24 cells at both RNA and protein level. In accordance, aberrant growth and proliferation were demonstrated in bladder cancer cells. The ability of migration and invasion of bladder cancer cells was also inhibited. The in vivo data further proved that the xenograft tumor growth was dramatically suppressed by GGN knockdown. Then we demonstrated that the level of IκB, bax and truncated caspase3 was upregulated after GGN was knocked down in 5637 cells. In contrast, expression level of NFκB, IKK, c-Myc, cyclin D1 and Bcl-2 was reduced. Further, the phosphorylation level of IκB was also downregulated. These data suggest that NFκB/caspase3-mediated apoptosis signaling was regulated by GGN. Conclusively, GGN played a tumor-promoting role in bladder cancer through regulation of NFκB/caspase3-mediated apoptosis signaling. This study provides a new clue for the treatment of patients with bladder cancer.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Humans; Mice; Mice, Nude; NF-kappa B; NF-KappaB Inhibitor alpha; Prognosis; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-myc; RNA, Small Interfering; Signal Transduction; Survival Analysis; Testicular Hormones; Urinary Bladder Neoplasms; Xenograft Model Antitumor Assays

Objective: This study aimed to investigate the expression of cyclin D1 and hnRNP-K in relation to the pathological\ findings in bladder cancer including the type, grade, muscle invasion and bilharzial association. Methods: We studied\ the immunoexpression; as regard the percentage, intensity and score of both cyclin D1 and hnRNP-K in different bladder\ lesions including 10 cases of cystitis; 10 cases of carcinoma insitu (CIS), 20 cases of Squamous cell carcinoma (SCC)\ and 66 cases of urothelial carcinoma (UC). Results: High expression of cyclin D1 was found in UC compared to other\ groups (p<0.001) and in UC with low grade, non-muscle invasive and papillary tumors compared to their counterparts\ (p<0.05, <0.01 and <0.05 respectively), however, bilharzial association does not affect cyclin D1 expression. Higher\ hnRNP-K expression was found in SCC compared to other groups (p <0.001) and in UC with high grade, muscle invasive\ and non-papillary tumors compared to their counterparts (p<0.001each). Bilharzial-associated UC showed higher\ expression of hnRNP-K percent (p<0.05) compared to non-bilharzial cases. Conclusion: This study elucidated a possible\ contribution of cyclin D1 and hnRNP-K expression in the initiation and progression of urinary bladder carcinoma,\ so, both of them can be used in predicting progression of urinary bladder carcinoma and to differentiate between UC\ and SCC in high grade tumors. The possible role of both markers in immunotherapy deserves supplementary studies.

Topics: Adult; Aged; Biomarkers, Tumor; Carcinogenesis; Cyclin D1; Disease Progression; Female; Heterogeneous-Nuclear Ribonucleoprotein K; Humans; Male; Middle Aged; Muscles; Neoplasm Invasiveness; Urinary Bladder; Urinary Bladder Neoplasms

BACKGROUND Bladder cancer caused by exposure to aniline dyes, chronic cystitis, and smoking is detected in approximately 70 000 new cases annually. In the USA alone, it leads to 15 000 deaths every year. In the present study, we investigated the role of 3-((4'-amino-[1,1'-biphenyl]-4-yl)amino)-4-bromo-5-oxo-2,5-dihydrofuran-2-yl acetate (ABDHFA) in the inhibition of bladder cancer cell viability. MATERIAL AND METHODS Viability of cells was examined using MTT assay and distribution of cell cycle was assessed by flow cytometry. Expression of cyclin D1, androgen, prostate-specific antigen (PSA), and miR-449a was analyzed using Western blot and quantitative real-time polymerase chain reaction assays. RESULTS The results demonstrated that ABDHFA treatment inhibited viability of UMUC3 and TCCSUP AR-positive bladder cancer cells. ABDHFA treatment led to break-down of AR in UMUC3 and TCCSUP cells after 48 h in a dose-dependent manner. Up-regulation of miR-449a by lentivirus transfection down-regulated the AR signalling pathway. In UMUC3 and TCCSUP cells, ABDHFA treatment led to inhibition of mRNA and protein expression corresponding to AR. CONCLUSIONS In summary, the present study demonstrates that proliferation of AR-positive bladder carcinoma cells is markedly reduced by ABDHFA treatment through arrest of cell cycle and degradation of AR protein. Thus, ABDHFA, a novel compound, can be used for the treatment of bladder cancer.

Topics: Acetates; Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Furans; Glucosamine; Humans; Kallikreins; MicroRNAs; Prostate-Specific Antigen; Receptors, Androgen; RNA, Messenger; Signal Transduction; Up-Regulation; Urinary Bladder Neoplasms

Although the diverse biological properties of nanoparticles have been studied intensively, research into their mechanism of action is relatively rare. In this study, we investigated the molecular mechanisms of the anticancer activity of heterometallic Au@Pt-nanoseeds (NSs) against bladder cancers.. Mode of action of Au@Pt-NSs was investigated through MTT assay, flow cytometry analysis, Western immunoblots, real-time qPCR, wound-healing migration and invasion assays, zymography, and electrophoretic mobility shift assay (EMSA).. Treatment with Au@Pt-NSs significantly inhibited the proliferation of EJ cells in a dose-dependent manner by inducing G1 phase cell cycle arrest. Among the regulators associated with the G1 cell cycle phase, CDK2, CDK4, cyclin D1, cyclin E, and p21WAF1 were shown to participate in the inhibitory pathways of Au@Pt-NSs. In addition, treatment with Au@Pt-NSs led to upregulation of phospho-p38 MAPK and downregulation of phospho-AKT in EJ cells. Interestingly, Au@Pt-NSs inhibited the migratory and invasive potential of the cells, which was attributed to the suppression of the enzymatic activity of matrix metalloproteinase-9 (MMP-9). Using MMP-9-specific oligonucleotides, we showed that transcription factors such as NF-κB and Sp-1 were responsible for the MMP-9-mediated metastatic potential of EJ cells.. Au@Pt-NSs significantly limited the progression, migration, and invasion of bladder cancer EJ cells. Our data represent a novel insight into developing cisplatin-like chemotherapeutic reagents with fewer side effects and provide useful information on molecular markers to monitor patients under Au@Pt-NSs-based chemotherapy.

Topics: Antineoplastic Agents; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Gold; Humans; Matrix Metalloproteinase 9; Nanostructures; NF-kappa B; p38 Mitogen-Activated Protein Kinases; Platinum; Signal Transduction; Transcription Factors; Urinary Bladder Neoplasms

Topics: 3' Untranslated Regions; Animals; Carcinogenesis; Cell Line; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; HEK293 Cells; Heterogeneous-Nuclear Ribonucleoprotein D; Humans; Mice; Mice, Nude; MicroRNAs; Proto-Oncogene Proteins c-myc; RNA Stability; RNA, Messenger; Transcription Factors; Transcription, Genetic; Tumor Suppressor Proteins; Up-Regulation; Urinary Bladder Neoplasms

There is no curative treatment for advanced bladder cancer. Causing ubiquitinated protein accumulation and endoplasmic reticulum stress is a novel approach to cancer treatment. The HIV protease inhibitor ritonavir has been reported to suppress heat shock protein 90 and increase the amount of unfolded proteins in the cell. If the proteasome functions normally, however, they are rapidly degraded. We postulated that the novel proteasome inhibitor ixazomib combined with ritonavir would kill bladder cancer cells effectively by inhibiting degradation of these unfolded proteins and thereby causing ubiquitinated proteins to accumulate. The combination of ritonavir and ixazomib induced drastic apoptosis and inhibited the growth of bladder cancer cells synergistically. The combination decreased the expression of cyclin D1 and cyclin-dependent kinase 4, and increased the sub-G

Topics: Acetylation; Antineoplastic Agents; Apoptosis; Boron Compounds; Cell Line, Tumor; Cyclin D1; Cyclin-Dependent Kinase 4; Drug Synergism; Endoplasmic Reticulum Stress; Glycine; Humans; Proteasome Inhibitors; Ritonavir; Ubiquitinated Proteins; Ubiquitination; Urinary Bladder Neoplasms

The prognosis of bladder urothelial carcinoma (BLCA) varies greatly even for patients with similar pathological characteristics. We conducted transcriptome sequencing on ten pairs of BLCA samples and adjacent normal tissues to identify differentially expressed genes. Anillin (ANLN) was identified as a transcript that was significantly up-regulated in BLCA samples compared with normal tissues. Prognostic power of candidate gene was studied using qRT-PCR and immunohistochemistry on 40 and 209 patients, respectively. Patients with elevated ANLN expression level was correlated with poorer cancer-specific (median, 22.4 vs. 37.3 months, p = 0.001), progression-free (median, 19.7 vs. 27.9 months, p = 0.001) and recurrence-free survival (median, 17.1 vs. 25.2 months, p = 0.011) compared with low ANLN expression. Public datasets TCGA and NCBI-GEO were analyzed for external validation. Knockdown of ANLN in J82 and 5637 cells using small interfering RNA significantly inhibited cell proliferation, migration, and invasion ability. Moreover, knockdown of ANLN resulted in G2/M phase arrest and decreased expression of cyclin B1 and D1. Microarray analysis suggested that ANLN played a major role in cell migration and was closely associated with several cancer-related signaling pathways. In conclusion, ANLN was identified as a promising prognostic biomarker which could be used to stratify different risks of BLCA.

Topics: Animals; Biomarkers, Tumor; Carcinoma in Situ; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin B1; Cyclin D1; Datasets as Topic; G2 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; High-Throughput Nucleotide Sequencing; Humans; Mice; Microarray Analysis; Microfilament Proteins; Neoplasm Recurrence, Local; Prognosis; RNA, Small Interfering; Signal Transduction; Survival Analysis; Transcriptome; Urinary Bladder Neoplasms; Xenograft Model Antitumor Assays

Mediator complex subunit 19 (Med19), a RNA polymerase II-embedded coactivator, is reported to be involved in bladder cancer (BCa) progression, but its functional contribution to this process is poorly understood. Here, we investigate the effects of Med19 on malignant behaviours of BCa, as well as to elucidate the possible mechanisms. Med19 expression in 15 BCa tissues was significantly higher than adjacent paired normal tissues using real-time PCR and Western blot analysis. Immunohistochemical staining of 167 paraffin-embedded BCa tissues was performed, and the results showed that high Med19 protein level was positively correlated with clinical stages and histopathological grade. Med19 was knocked down in BCa cells using short-hairpin RNA. Functional assays showed that knocking-down of Med19 can suppress cell proliferation and migration in T24, UM-UC3 cells and 5637 in vitro, and inhibited BCa tumour growth in vivo. TOP/FOPflash reporter assay revealed that Med19 knockdown decreased the activity of Wnt/β-catenin pathway, and the target genes of Wnt/β-catenin pathway were down-regulated, including Wnt2, β-catenin, Cyclin-D1 and MMP-9. However, protein levels of Gsk3β and E-cadherin were elevated. Our data suggest that Med19 expression correlates with aggressive characteristics of BCa and Med19 knockdown suppresses the proliferation and migration of BCa cells through down-regulating the Wnt/β-catenin pathway, thereby highlighting Med19 as a potential therapeutic target for BCa treatment.

Topics: Animals; Antigens, CD; beta Catenin; Cadherins; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3 beta; Humans; Male; Matrix Metalloproteinase 9; Mediator Complex; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Grading; Neoplasm Staging; RNA, Small Interfering; Urinary Bladder Neoplasms; Wnt Signaling Pathway; Wnt2 Protein; Xenograft Model Antitumor Assays

Brenner tumors (BT) are rare ovarian tumors encompassing benign, borderline, and malignant variants. While the histopathology of BTs and their clinical course is well described, little is known about the underlying genetic defects. We employed targeted next generation sequencing to analyze the mutational landscape in a cohort of 23 BT cases (17 benign, 2 borderline, and 4 malignant) and 3 ovarian carcinomas with transitional cell histology (TCC). Copy number variations (CNV) were validated by fluorescence in-situ hybridization (FISH) and quantitative PCR-based copy number assays. Additionally, we analyzed the TERT promotor region by conventional Sanger sequencing. We identified 25 different point mutations in 23 of the analyzed genes in BTs and 10 mutations in 8 genes in TCCs. About 57% percent of mutations occurred in genes involved in cell cycle control, DNA repair, and epigenetic regulation processes. All TCC cases harbored TP53 mutations whereas all BTs were negative and none of the mutations observed in BTs were present in TCCs. CNV analysis revealed recurrent MDM2 amplifications in 3 out of 4 of the malignant BT cases with one case harboring a concomitant amplification of CCND1. No mutations were observed in the TERT promoter region in BTs and TCCs, which is mutated in about 50%-75% of urothelial carcinoma and in 16% of ovarian clear-cell carcinomas. In conclusion, our study highlights distinct genetic features of BTs, and detection of the triplet phenotype MDM2 amplification/TP53 wt/TERT wt may aid diagnosis of malignant BT in difficult cases. Moreover, selected genetic lesions may be clinically exploitable in a metastatic setting.

Topics: Adult; Aged; Aged, 80 and over; Brenner Tumor; Carcinoma; Cyclin D1; DNA Copy Number Variations; Female; Humans; Middle Aged; Ovarian Neoplasms; Point Mutation; Promoter Regions, Genetic; Proto-Oncogene Proteins c-mdm2; Telomerase; Tumor Suppressor Protein p53; Urinary Bladder Neoplasms; Urothelium

The leucine zipper-EF-hand containing transmembrane protein 1 (LETM1) is highly expressed in many human malignancies and is correlated with poor prognosis. However, the function of LETM1 in bladder cancer still remains unknown. In the present study, we analyzed the expression levels of LETM1 in bladder cancer tissues and non-cancerous tissues as well as in four bladder cancer cell lines (T24, EJ, 5637 and J82) and a human bladder epithelial immortalized cell line SV-HUC-1. Small interfering RNA (siRNA) was employed to knockdown the expression of LETM1 in the T24 cells. The proliferation of T24 cells was significantly repressed as evaluated by CCK-8 assays. Transwell migration and invasion assays indicated that knockdown of LETM1 suppressed cell migration and invasion significantly. Flow cytometric analysis revealed that cells had accumulated at the S-phase when the expression of LETM1 was suppressed. Moreover, we found that several oncogenic proteins in the Wnt/β-catenin signaling pathway, namely β-catenin, cyclin D1 and c-Myc were significantly decreased by the LETM1 siRNA. Collectively, these results revealed that the knockdown of LETM1 exhibited tumor suppressive effects, possibly by controlling the downstream Wnt/β-catenin signaling pathway.

Topics: Adult; Aged; Apoptosis; beta Catenin; Calcium-Binding Proteins; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Female; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Male; Membrane Proteins; Middle Aged; Neoplasm Invasiveness; Proto-Oncogene Proteins c-myc; RNA, Small Interfering; Urinary Bladder Neoplasms; Wnt Signaling Pathway

To determine the differential protein expression of biomarkers FGFR3, PI3K (subunits PI3Kp110α, PI3KClassIII, PI3Kp85), AKT, p21Waf1/Cip1 and cyclins D1 and D3 in T1 bladder cancer versus healthy tissue and to study their potential role as early recurrence markers.. This is a prospective study that employed a total of 67 tissue samples (55 cases of T1 bladder tumours that underwent transurethral resection and 12 cases of adjacent healthy mucosa). The protein expression levels were assessed using Western blot, and the means and percentages were compared using Student's t-test and the chi-squared test. The survival analysis was conducted using the Kaplan-Meier method and the log-rank test.. Greater protein expression was detected for FGFR3, PI3Kp110α, PI3KClassIII, cyclins D1 and D3 and p21Waf1/Cip1 in the tumour tissue than in the healthy mucosa. However, these differences were not significant for PI3Kp85 and AKT. We observed statistically significant correlations between early recurrence and PI3Kp110α, PI3KClassIII, PI3Kp85 and AKT (P=.003, P=.045, P=.050 and P=.028, respectively), between the tumour type (primary vs. recurrence) and cyclin D3 (P=.001), between the tumour size and FGFR3 (P=.035) and between multifocality and cyclin D1 (P=.039). The survival analysis selected FGFR3 (P=.024), PI3Kp110α (P=.014), PI3KClassIII (P=.042) and AKT (P=.008) as markers of early-recurrence-free survival.. There is an increase in protein expression levels in bladder tumour tissue. The overexpression of FGFR3, PI3Kp110α, PI3KClassIII and AKT is associated with increased early-recurrence-free survival for patients with T1 bladder tumours.

Topics: Aged; Aged, 80 and over; Cyclin D1; Cyclin D2; Cyclin-Dependent Kinase Inhibitor p21; Female; Humans; Male; Middle Aged; Neoplasm Staging; Oncogene Protein v-akt; Phosphatidylinositol 3-Kinases; Prognosis; Prospective Studies; Receptor, Fibroblast Growth Factor, Type 3; Survival Analysis; Urinary Bladder Neoplasms

Transcription factor CCAAT/enhancer-binding protein delta (CEBPD) plays multiple roles in tumor progression. Studies have demonstrated that cisplatin (CDDP) induced CEBPD expression and had led to chemotherapeutic drug resistance. However, the underlying molecular mechanisms of CDDP-regulated CEBPD expression and its relevant roles in CDDP responses remain elusive. MicroRNAs (miRNAs) are small non-coding RNAs that negatively regulate gene expression in a sequence-specific manner. Abnormal miRNAs expression is associated with tumor progression. In current study, a large-scale PCR-based miRNA screening was performed to identify CEBPD-associated miRNAs in urothelial carcinoma cell line NTUB1. Eleven miRNAs were selected with more than twofold changes. MiR-193b-3p, a known tumor suppressor, down-regulated proto-oncogenes Cyclin D1, and ETS1 expression and led to cell cycle arrest, cell invasion, and migration inhibition. The expression of miR-193b-3p was associated with the DNA binding ability of CEBPD in CDDP response. CEBPD knocking-down approach provided a strong evidence of the positive correlation between CEBPD and miR-193b-3p. CDDP-induced CEBPD trans-activated miR-193b-3p expression and it directly targeted the 3'-UTR of Cyclin D1 and ETS1 mRNA, and silenced the protein expression. In addition, miR-193b-3p also inhibited cell migration activity, arrested cell at G1 phase, and sensitized NTUB1 to CDDP treatment. In conclusion, this study indicates that CEBPD exhibits an anti-tumorigenic function through transcriptionally activating miR-193b-3p expression upon CDDP treatment. This study provides a new direction for managing human urothelial carcinoma. J. Cell. Biochem. 118: 1563-1573, 2017. © 2016 Wiley Periodicals, Inc.

Topics: 3' Untranslated Regions; Carcinoma, Transitional Cell; CCAAT-Enhancer-Binding Protein-delta; Cell Cycle; Cell Line, Tumor; Cell Movement; Cisplatin; Cyclin D1; Down-Regulation; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; Proto-Oncogene Protein c-ets-1; Urinary Bladder Neoplasms

Long noncoding RNA 19 (H19) has been shown to promote bladder cancer cell proliferation and metastasis. However, little is known about how miR-675, mature product of H19, contributes to bladder cancer cell proliferation. In this study, we first evaluated the expression of miR-675 in bladder cancer tissues by quantitative real-time PCR (qRT-PCR) and defined its biological functions by flow cytometry and Western blotting. We found that miR-675 expression levels were remarkably increased in bladder cancer tissues as compared with adjacent noncancerous tissues or normal bladder tissue from health donors; moreover, enhanced miR-675 expression was also observed in bladder cancer cell lines. Ectopic expression of H19 significantly increased bladder cancer cell proliferation and miR-675 expression in vitro. Furthermore, overexpression of miR-675 promoted bladder cancer cell proliferation, while suppression of miR-675 induced G1 phase cell cycle arrest and promoted cell apoptosis. Western blotting analysis further identified that miR-675 inhibited p53 activation, decreased the ratio of Bax/Bcl-2 and cyclin D1 expression in bladder cancer cells; those effects may result in the abnormal proliferation of bladder cancer cells. In conclusion, abnormal enhanced miR-675 expression increases bladder cancer growth by regulating p53 activation, and thus may be helpful in the development of effective treatment strategies for bladder cancer.

Topics: Actins; Adult; Aged; Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Humans; Male; MicroRNAs; Middle Aged; Real-Time Polymerase Chain Reaction; RNA, Long Noncoding; Tumor Suppressor Protein p53; Urinary Bladder Neoplasms

o-Nitroanisole is an intermediate in the manufacture of azo dyes. In a National Toxicology Program stop-exposure study,o-nitroanisole induced hyperplasia, papillomas, and papillary carcinomas in the urinary bladder of Fischer 344/N rats.o-Nitroanisole was investigated since occupational or environmental exposure to aniline and azo dyes is a risk factor for urinary bladder cancer in humans. The current study describes the morphology of urinary bladder neoplasms seen in rats with respect to those observed in humans. This study also evaluated immunohistochemical expression of the cell cycle-related proteins cyclin D1 and p53 and the differentiation markers cytokeratin 20 and uroplakin III in hyperplastic (n= 11) and neoplastic (n= 6 papillomas,n= 11 carcinomas) lesions of the urinary bladder epithelium from rats treated with o-nitroanisole and in normal (n= 6) urinary bladders from untreated rats. The tumors observed were more similar to the papillary type rather than the muscle-invasive type of urinary bladder cancer in humans. The preneoplastic and neoplastic lesions observed suggest progression from hyperplasia to papilloma to papillary carcinoma. With neoplastic progression (hyperplasia to papilloma to carcinoma), cyclin D1 immunoreactivity progressively increased in intensity, percentage of cells staining, and distribution. Overexpression of p53 was not found. Cytokeratin 20 staining decreased in superficial cells, while uroplakin III staining increased in intermediate and basal cells with progression from hyperplasia to carcinoma. The results are consistent with increased cell cycle dysregulation or proliferation (cyclin D1), decreased differentiation (cytokeratin 20), and abnormal differentiation (uroplakin III) as lesions progress toward malignancy.

Topics: Animals; Anisoles; Biomarkers, Tumor; Carcinoma, Papillary; Cyclin D1; Disease Models, Animal; Female; Humans; Hyperplasia; Immunohistochemistry; Keratin-20; Male; Papilloma; Precancerous Conditions; Rats; Rats, Inbred F344; Tumor Suppressor Protein p53; Urinary Bladder; Urinary Bladder Neoplasms; Uroplakin III

TRIM29 overexpression has been reported in several human malignancies and showed correlation with cancer cell malignancy. The aim of the current study is to examine its clinical significance and biological roles in human bladder cancer tissues and cell lines.. A total of 102 cases of bladder cancer tissues were examined for TRIM29 expression by immunohistochemistry. siRNA and plasmid transfection were performed in 5637 and BIU-87 cell lines. Cell Counting Kit-8, flow cytometry, western blot, and real-time polymerase chain reaction were performed to examine its biological roles and mechanism in bladder cancer cells.. We found that TRIM29 overexpression showed correlation with invading depth (p=0.0087). Knockdown of TRIM29 expression in bladder cancer cell line 5637 inhibited cell growth rate and cell cycle transition while its overexpression in BIU-87 cells accelerated cell proliferation and cell cycle progression. TRIM29 overexpression also inhibited cell apoptosis induced by cisplatin. In addition, we demonstrated that TRIM29 depletion decreased while its overexpression led to upregulated expression of cyclin D1, cyclin E, and Bcl-2. We also showed that TRIM29 knockdown inhibited protein kinase C (PKC) and nuclear factor κB (NF-κB) signaling while its overexpression stimulated the PKC and NF-κB pathways. BAY 11-7082 (NF-κB inhibitor) partly attenuated the effect of TRIM29 on expression of cyclin and Bcl-2. Treatment with PKC inhibitor staurosporine resulted in ameliorated TRIM29 induced activation of NF-κB.. The current study demonstrated that TRIM29 upregulates cyclin and Bcl family proteins level to facilitate malignant cell growth and inhibit drug-induced apoptosis in bladder cancer, possibly through PKC-NF-κB signaling pathways.

Topics: Aged; Apoptosis; Cell Line, Tumor; Cell Proliferation; Cisplatin; Cyclin D1; DNA-Binding Proteins; Female; Flow Cytometry; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Male; Middle Aged; NF-kappa B; Protein Kinase C; Proto-Oncogene Proteins c-bcl-2; RNA, Small Interfering; Signal Transduction; Transcription Factors; Urinary Bladder Neoplasms

Although the precursor protein of NFκB2 (p100) is thought to act as a tumor suppressor in mammalian cells, the molecular mechanism of its anti-tumor activity is far from clear. Here, we are, for the first time, to report that p100 protein expression was dramatically decreased in bladder cancers of N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN)-treated mice and human patients. Knockdown of p100 in cultured human bladder cancer cells promoted anchorage-independent growth accompanied with elevating abundance of cell-cycle-related proteins and accelerated cell-cycle progression. Above effects could be completely reversed by ectopically expression of p100, but not p52. Mechanistically, p100 inhibited Cyclin D1 protein translation by activating the transcription of LARP7 and its hosted miR-302d, which could directly bind to 3'-UTR of cyclin d1 mRNA and inhibited its protein translation. Furthermore, p100 suppressed the expression of PHLPP2 (PH domain and leucine-rich repeat protein phosphatases 2), thus promoting CREB phosphorylation at Ser133 and subsequently leading to miR-302d transcription. Taken together, our studies not only for the first time establish p100 as a key tumor suppressor of bladder cancer growth, but also identify a novel molecular cascade of PHLPP2/CREB/miR-302d that mediates the tumor suppressive function of p100.

Topics: Animals; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; Mice; NF-kappa B p52 Subunit; Phosphoprotein Phosphatases; Protein Biosynthesis; Tumor Suppressor Proteins; Urinary Bladder Neoplasms

Cancers often utilize microRNAs to suppress tumor suppressor genes, thus facilitating their potential for growth and invasion. In the present study, we report the novel findings that miR-892b inhibits proliferation, migration and invasion of bladder cancer cells. The basal expression level of miR‑892b was significantly lower in 3 different bladder cancer cell lines than in normal human urothelial cells. Transfection of miR-892b mimics to bladder cancer cells resulted in dose‑dependent growth arrest. Flow cytometric analysis of the cell cycle showed that miR-892b-transfected bladder cancer cells were subject to arrest in the G1 phase, which was due to the downregulation of cyclin D1 and CDK6 followed by upregulation of p19ARF. In addition, overexpression of miR-892b impeded the migration and invasion of EJ cells. Expression of MMP-9 in EJ cells was blocked by transfection of miR-892b; the effect was regulated, at least in part, by activation of the Sp-1 transcription factor. Overall, we verified that miR-892b regulates the p19ARF/cyclin D1/CDK6 and Sp-1/MMP-9 signaling networks in bladder cancer cells and may provide a treatment option for advanced-stage bladder cancers.

Topics: Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 6; Cyclin-Dependent Kinase Inhibitor p16; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Humans; Matrix Metalloproteinase 9; MicroRNAs; Neoplasm Invasiveness; Signal Transduction; Sp1 Transcription Factor; Urinary Bladder Neoplasms

Simvastatin is currently one of the most common drugs for old patients with hyperlipidemia, hypercholesterolemia and atherosclerotic diseases by reducing cholesterol level and anti-lipid properties. Importantly, simvastatin has also been reported to have anti-tumor effect, but the underlying mechanism is largely unknown. We collected several human bladder samples and performed microarray. Data analysis suggested bladder cancer (BCa) was significantly associated with fatty acid/lipid metabolism via PPAR signalling pathway. We observed simvastatin did not trigger BCa cell apoptosis, but reduced cell proliferation in a dose- and time-dependent manner, accompanied by PPARγ-activation. Moreover, flow cytometry analysis indicated that simvastatin induced cell cycle arrest at G0/G1 phase, suggested by downregulation of CDK4/6 and Cyclin D1. Furthermore, simvastatin suppressed BCa cell metastasis by inhibiting EMT and affecting AKT/GSK3β. More importantly, we found that the cell cycle arrest at G0/G1 phase and the alterations of CDK4/6 and Cyclin D1 triggered by simvastatin could be recovered by PPARγ-antagonist (GW9662), whereas the treatment of PPARα-antagonist (GW6471) shown no significant effects on the BCa cells. Taken together, our study for the first time revealed that simvastatin inhibited bladder cancer cell proliferation and induced cell cycle arrest at G1/G0 phase via PPARγ signalling pathway.

Topics: Anilides; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Enzyme Activation; Epithelial-Mesenchymal Transition; ErbB Receptors; G1 Phase Cell Cycle Checkpoints; Glycogen Synthase Kinase 3 beta; Humans; Neoplasm Invasiveness; Oxazoles; PPAR gamma; Proto-Oncogene Proteins c-akt; Simvastatin; Tyrosine; Urinary Bladder; Urinary Bladder Neoplasms

Dimethylarsinic acid (DMA(V) ), the major urinary metabolite of inorganic arsenic, is a urinary bladder carcinogen and bladder tumor promoter in adult rats. Increased urothelial cellular proliferation has been considered as an earlier phenotype in DMA(V) -induced bladder carcinogenesis. The present study examined the ultrastructural changes of bladder epithelial cells and expressions of proliferation factors, as well as the secretion of inflammatory cytokines in rats exposed to DMA(V) for 10 weeks by transmission electron microscopy (TEM), qRT-PCR, immunohistochemical staining and ELISA methods. The results showed that DMA(V) administered in the drinking water produced urothelial cytotoxicity and ultrastructural changes in rats. PCNA, cyclin D1 and COX-2 mRNA expressions and immunoreactivities were elevated in bladder urothelium. In addition, 200 ppm DMA(V) treatment increased the transforming growth factor-beta 1 (TGF-β1) secretion and decreased tumor necrosis factor-alpha (TNF)-α level in the urine of rats. These data suggest that chronic inflammation, bladder epithelium lesions and proliferation might be the basic process of the chronic toxicity effects in DMA(V) -treated rats.

Topics: Animals; Cacodylic Acid; Cell Proliferation; Cyclin D1; Cyclooxygenase 2; Cytokines; Enzyme-Linked Immunosorbent Assay; Female; Inflammation; Interleukin-6; Male; Microscopy, Electron, Transmission; Proliferating Cell Nuclear Antigen; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Urinary Bladder; Urinary Bladder Neoplasms; Urothelium

Long intergenic noncoding RNAs (lincRNAs) play important roles in regulating various biological processes in cancer, including proliferation and apoptosis. However, the roles of lincRNAs in bladder cancer remain elusive. In this study, we identified a novel lincRNA, which we termed AATBC. We found that AATBC was overexpressed in bladder cancer patient tissues and positively correlated with tumor grade and pT stage. We also found that inhibition of AATBC resulted in cell proliferation arrest through G1 cell cycle mediated by cyclin D1, CDK4, p18 and phosphorylated Rb. In addition, inhibition of AATBC induced cell apoptosis through the intrinsic apoptosis signaling pathway, as evidenced by the activation of caspase-9 and caspase-3. The investigation for the signaling pathway revealed that the apoptosis following AATBC knockdown was mediated by activation of phosphorylated JNK and suppression of NRF2. Furthermore, JNK inhibitor SP600125 could attenuate the apoptotic effect achieved by AATBC knockdown, confirming the involvement of JNK signaling in the induced apoptosis. Moreover, mouse xenograft model revealed that knockdown of AATBC led to suppress tumorigenesis in vivo. Taken together, our study indicated that AATBC might play a critical role in pro-proliferation and anti-apoptosis in bladder cancer by regulating cell cycle, intrinsic apoptosis signaling, JNK signaling and NRF2. AATBC could be a potential therapeutic target and molecular biomarker for bladder cancer.

Topics: Aged; Animals; Apoptosis; Blotting, Western; Cell Line; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Male; Mice, Inbred NOD; Mice, SCID; Middle Aged; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; RNA, Long Noncoding; Tumor Burden; Urinary Bladder Neoplasms; Xenograft Model Antitumor Assays

MicroRNAs (miRNAs) are small, endogenous RNAs that play important gene-regulatory roles by binding to the imperfectly complementary sequences at the 3'-UTR of mRNAs and directing their gene expression. Here, we first discovered that miR-576-3p was down-regulated in human bladder cancer cell lines compared with the non-malignant cell line. To better characterize the role of miR-576-3p in bladder cancer cells, we over-expressed or down-regulated miR-576-3p in bladder cancer cells by transfecting with chemically synthesized mimic or inhibitor. The overexpression of miR-576-3p remarkably inhibited cell proliferation via G1-phase arrest, and decreased both mRNA and protein levels of cyclin D1 which played a key role in G1/S phase transition. The knock-down of miR-576-3p significantly promoted the proliferation of bladder cancer cells by accelerating the progression of cell cycle and increased the expression of cyclin D1. Moreover, the dual-luciferase reporter assays indicated that miR-576-3p could directly target cyclin D1 through binding its 3'-UTR. All the results demonstrated that miR-576-3p might be a novel suppressor of bladder cancer cell proliferation through targeting cyclin D1.

Topics: 3' Untranslated Regions; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Down-Regulation; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; Urinary Bladder Neoplasms

Bladder cancer is the most common malignancy in urinary system and the ninth most common malignancy in the world. MicroRNAs (miRNAs) are small, non-coding RNAs that regulate gene expression by targeted repression of transcription and translation and play essential roles during cancer development. We investigated the expression of miR-135a in bladder cancer and explored its bio-function during bladder cancer progression.. The expression of miR-135a in bladder cancer cells and tissues are performed by using Real-time PCR assay. Cell viability assay (MTT assay), colony formation assay, anchorage-independent growth ability assay and Bromodeoxyuridine labeling and immunofluorescence (BrdUrd) assay are used to examine cell proliferative capacity and tumorigenicity. Flow cytometry analysis is used to determine cell cycle progression. The expressions of p21, p27, CyclinD1, Ki67, PHLPP2 and FOXO1 are measured by Western blotting assay. Luciferase assay is used to confirm whether FOXO1 is the direct target of miR-135a.. miR-135a is upregulated in bladder cancer cells and tissues. Enforced expression of miR-135a promotes bladder cancer cells proliferation, whereas inhibition of miR-135a reverses the function. Furthermore, for the first time we demonstrated PHLPP2 and FOXO1 are direct targets of miR-135a and transcriptionally down-regulated by miR-135a. Suppression of PHLPP2 or FOXO1 by miR-135a, consisted with dysregulation of p21, p27, Cyclin D1 and Ki67, play important roles in bladder cancer progression.. Our study demonstrates that miR-135a promotes cell proliferation in bladder cancer by targeting PHLPP2 and FOXO1, and is performed as an onco-miR.

Topics: Base Sequence; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Forkhead Box Protein O1; Forkhead Transcription Factors; Gene Expression Regulation, Neoplastic; Humans; Ki-67 Antigen; MicroRNAs; Models, Biological; Molecular Sequence Data; Phosphoprotein Phosphatases; Transcription, Genetic; Urinary Bladder Neoplasms

We have previously demonstrated that miR-1180-5p has potent ability to upregulate p21 expression by targeting promoter and inhibit bladder cancer. This prompted us to conjecture that a candidate dsRNA (dsP21-397) with perfect complementarity to the miR-1180-5p target site of p21 promoter may also trigger p21 expression. Transfection of dsP21-397 into T24 and EJ cells significantly activated p21 expression at 72 h and the activation presented in a time-course and dose-dependent manner. Moreover, the p21-activated activities of dsP21-397 and miR-1180-5p are not significantly different. Overexpression of p21 downregulated Cyclin D1, CDK4/6, and Cyclin A2 expression, and thereby induced cell cycle arrest and inhibited proliferation. Moreover, dsP21-397 suppressed bladder cancer largely depended on manipulating p21. In conclusion, our study identifies a pair of miRNA-dsRNA mediating endogenous p21 overexpression.

Topics: Apoptosis; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; Promoter Regions, Genetic; RNA, Double-Stranded; Urinary Bladder Neoplasms

We previously found that two neighboring G-quadruplexes behave as a molecular switch controlling the expression of HRAS (Cogoi, S.; Schekotikhin, A. E.; Xodo, L. E. Nucl. Acids Res. 2014, DOI: 10.1093/nar/gku574). In this study we have designed anthrathiophenediones with two chloroacetamidine-containing side chains (CATDs) as G-quadruplex binders and have examined their anticancer activity in T24 bladder cancer cells bearing mutant HRAS and in T24 xenografts. The designed CATDs (3a-e), bearing alkyl side chains of different length, penetrate T24 cancer cells more than their analogues with guanidine-containing side chains. The lead compounds 3a and 3c inhibit HRAS expression, metabolic activity, and colony formation in T24 cancer cells. They also activate a strong apoptotic response, as indicated by PARP-1, caspases 3/7, and annexin V/propidium iodide assays. Apoptosis occurs under conditions where cyclin D1 is down-regulated and the cell cycle arrested in G2 phase. Finally, compound 3a inhibits the growth of T24 xenografts and increases the median survival time of nude mice.

Topics: Acetamides; Alleles; Animals; Antineoplastic Agents; Apoptosis; Biological Transport; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Drug Design; G-Quadruplexes; G2 Phase Cell Cycle Checkpoints; Genes, ras; Humans; Mice; Substrate Specificity; Survival Analysis; Thiophenes; Urinary Bladder Neoplasms; Xenograft Model Antitumor Assays

MicroRNAs (miRs) serve either as oncogenes or tumor-suppressor genes in tumor progression. MicroRNA-20b (miR‑20b) is known to be involved with the oncomirs of several types of cancers. However, in the present study we describe how miR-20b inhibits the proliferation, migration and invasion of bladder cancer EJ cells. In the present study, miR-20b was downregulated in bladder cancer cell lines, and its overexpression resulted in a significant reduction in the proliferation of EJ cells. In addition, via a bioinformatics approach, we identified cell cycle-regulated genes that are the putative targets of miR-20b. The transfection of miR-20b into EJ cells induced G1 phase cell cycle arrest via the decreased expression of cyclin D1, CDK2 and CDK6 without affecting another G1 phase cell cycle regulator, cyclin E. The cell cycle inhibitor p21WAF1 was upregulated in the miR-20b transfected cells. Moreover, the enforced expression of miR-20b resulted in impaired wound-healing migration and invasion in the EJ cells. Based on our target prediction analysis of miRs, we confirmed that miR-20b overexpression strongly impedes MMP-2 expression via suppressive activation of the Sp-1 binding motif, an important transcription factor present in the MMP-2 promoter. Herein, we report the novel concept that miR-20b exerts a suppressive effect on both cell cycle-modulated proliferation and MMP-2-mediated migration and invasion in bladder cancer EJ cells.

Topics: Cell Cycle Checkpoints; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 6; Gene Expression Regulation, Neoplastic; Humans; Matrix Metalloproteinase 2; MicroRNAs; Neoplasm Invasiveness; Transfection; Urinary Bladder Neoplasms

Bmi1 is a member of the polycomb group family of proteins, and it drives the carcinogenesis of various cancers and governs the self-renewal of multiple types of stem cells. However, its role in the initiation and progression of bladder cancer is not clearly known. The present study aimed to investigate the function of Bmi1 in the development of bladder cancer. Bmi1 expression was detected in human bladder cancer tissues and their adjacent normal tissues (n=10) by immunohistochemistry, qRT-PCR and Western blotting, respectively. Bmi1 small interference RNA (siRNA) was synthesized and transfected into human bladder carcinoma cells (EJ) by lipofectamine 2000. The Bmil expression at mRNA and protein levels was measured in EJ cells transfected with Bmil siRNA (0, 80, 160 nmol/L) by qRT-PCR and Western blotting, respectively. Cell viability and Ki67 expression (a marker of cell proliferation) were determined in Bmi1 siRNA-transfected cells by CCK-8 assay and qRT-PCR, respectively. Cell cycle of transfected cells was flow-cytometrically determined. Immunofluorescence and Western blotting were used to detect the expression levels of cell cycle-associated proteins cyclin D1 and cyclin E in the cells. Pro-apoptotic proteins Bax and caspase 3 and anti-apoptotic protein Bcl-2 were detected by Western blotting as well. Additionally, xenograft tumor models were established by inoculation of EJ cells (infected with Bmil shRNA/pLKO.1 lentivirus or not) into nude mice. The tumor volumes were measured every other day for 14 days. The results showed that the Bmil expression was significantly increased in bladder tumor tissues when compared with that in normal tissues (P<0.05). Perturbation of Bmi1 expression by using siRNA could significantly inhibit the proliferation of EJ cells (P<0.05). Bmi1 siRNA-transfected EJ cells were accumulated in G1 phase and the expression levels of cyclin D1 and cyclin E were down-regulated. Bax and caspase-3 expression levels were significantly increased and Bcl-2 levels decreased after Bmi1 knockdown. Tumor volume was conspicuously reduced in mice injected with EJ cells with Bmi1 knockdown. Our findings indicate that Bmi1 is a potential driver oncogene of bladder cancer and it may become a potential treatment target for human bladder cancer.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Carcinogenesis; Carcinoma; Caspase 3; Cell Line, Tumor; Cyclin D1; Cyclin E; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Humans; Injections, Intralesional; Ki-67 Antigen; Mice; Mice, Nude; Polycomb Repressive Complex 1; Proto-Oncogene Proteins c-bcl-2; RNA, Small Interfering; Signal Transduction; Tumor Burden; Urinary Bladder; Urinary Bladder Neoplasms; Xenograft Model Antitumor Assays

The CCND1 gene encodes the protein CyclinD1, which is an important promoter of the cell cycle and a prognostic and predictive factor in different cancers. CCND1 is amplified to a substantial proportion in various tumors, and this may contribute to CyclinD1 overexpression. In bladder cancer, information about the clinical relevance of CCND1/CyclinD1 alterations is limited. In the present study, amplification status of CCND1 and expression of CyclinD1 were evaluated by fluorescence in situ hybridization and immunohistochemistry on tissue microarrays from 152 lymph node-positive urothelial bladder cancers (one sample each from the center and invasion front of the primary tumors, two samples per corresponding lymph node metastasis) treated by cystectomy and lymphadenectomy. CCND1 amplification status and the percentage of immunostained cancer cells were correlated with histopathological tumor characteristics, cancer-specific survival and response to adjuvant chemotherapy. CCND1 amplification in primary tumors was homogeneous in 15% and heterogeneous in 6% (metastases: 22 and 2%). Median nuclear CyclinD1 expression in amplified samples was similar in all tumor compartments (60-70% immunostained tumor nuclei) and significantly higher than in non-amplified samples (5-20% immunostained tumor nuclei; P<0.05). CCND1 status and CyclinD1 expression were not associated with primary tumor stage or lymph node tumor burden. CCND1 amplification in primary tumors (P=0.001) and metastases (P=0.02) and high nuclear CyclinD1 in metastases (P=0.01) predicted early cancer-related death independently. Subgroup analyses showed that chemotherapy was particularly beneficial in patients with high nuclear CyclinD1 expression in the metastases, whereas expression in primary tumors and CCND1 status did not predict chemotherapeutic response. In conclusion, CCND1 amplification status and CyclinD1 expression are independent risk factors in metastasizing bladder cancer. High nuclear CyclinD1 expression in lymph node metastases predicts favorable response to chemotherapy. This information may help to personalize prognostication and administration of adjuvant chemotherapy.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Agents; Biomarkers, Tumor; Biopsy; Cell Nucleus; Chemotherapy, Adjuvant; Cyclin D1; Cystectomy; Female; Gene Amplification; Humans; Immunohistochemistry; In Situ Hybridization, Fluorescence; Kaplan-Meier Estimate; Lymph Node Excision; Lymph Nodes; Lymphatic Metastasis; Male; Middle Aged; Multivariate Analysis; Neoplasm Staging; Patient Selection; Precision Medicine; Predictive Value of Tests; Proportional Hazards Models; Prospective Studies; Risk Factors; Tissue Array Analysis; Urinary Bladder Neoplasms

Thirty-nine epithelial bladder tumor samples from 37 animals affected with bovine enzootic hematuria (BEH) were selected for immunohistochemical studies. The expression of structural proteins such as uroplakin III (UPIII) and cytokeratin 7 (CK7) and the cell cycle-related proteins cyclin D1 and p53 were evaluated in urothelial papillomas and carcinomas. Loss of UPIII and CK7 expression was seen in both high-grade and high-stage urothelial carcinomas (P < .001 and P < .02). Cyclin D1 expression showed no statistically significant association with grade or stage. In contrast, p53 immunoreactivity was positive in high-grade and high-stage carcinomas (P < .05 and P < .01), confirming its association with the highest malignant behavior of the bladder tumors in BEH.

Topics: Animals; Cattle; Cattle Diseases; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Hematuria; Immunohistochemistry; Keratin-7; Tumor Suppressor Protein p53; Urinary Bladder Neoplasms; Uroplakin III

CRKL encodes an adaptor protein that has been recently reported to be overexpressed in various cancers and associate with the malignant behavior of cancer cells. However, the expression pattern of CRKL protein and its clinical significance in human bladder cancer have not been well characterized to date. In the present study, CRKL expression was analyzed in 82 archived bladder cancer specimens using immunohistochemistry, and the correlations between CRKL expression and clinicopathological parameters were evaluated. We found that CRKL was overexpressed in 31 of 82 (37.8%) bladder cancer specimens. A significant association was observed between CRKL overexpression and tumor status (p = 0.019). To further explore the biological functions of CRKL in bladder cancer, we overexpressed CRKL in BIU-87 and 5637 cell lines. Using CCK8 assay and colony formation assay, we showed that CRKL upregulation increased cell proliferation. In addition, transwell assay showed that CRKL could also facilitate invasion. Further study demonstrated that CRKL upregulation increased cyclin D1 expression and ERK phosphorylation. In conclusion, CRKL is overexpressed in bladder cancer and regulates malignant cell growth and invasion, which makes CRKL a candidate therapeutic target for bladder cancer.

Topics: Adaptor Proteins, Signal Transducing; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Extracellular Signal-Regulated MAP Kinases; Humans; Neoplasm Invasiveness; Nuclear Proteins; Phosphorylation; Urinary Bladder; Urinary Bladder Neoplasms

Caspase recruitment domain and membrane-associated guanylate kinase-like domain protein 3 (CARMA3) was reported as an oncoprotein overexpressed in several cancers. The expression pattern of CARMA3 and its clinical significance in human bladder cancer have not been well characterized. In the present study, CARMA3 expression was analyzed in 90 archived bladder cancer specimens using immunohistochemistry, and the correlation between CARMA3 expression and clinicopathological parameters was evaluated. We found that CARMA3 was overexpressed in 35 of 90 (38.8%) bladder cancer specimens. Significant association was observed between CARMA3 overexpression with tumor status (p = 0.081) and tumor grade (p = 0.027). To further explore the biological functions of CARMA3 in bladder cancer, we depleted CARMA3 in T24 and 5637 cell lines using small interfering RNA (siRNA). Using cell counting kit-8 (CCK8) assay and colony formation assay, we were able to show that CARMA3 depletion inhibited cell proliferation and colony number. Further study demonstrated that CARMA3 depletion decreased an expression of nuclear factor kappa B (NF-κB) targets cyclin D1 and Bcl-2 expression, as well as IκB phosphorylation. Luciferase reporter assay showed that CARMA3 depletion could downregulate NF-κB reporter activity. In conclusion, CARMA3 is overexpressed in bladder cancer and regulates malignant cell growth and NF-κB signaling, which makes CARMA3 a candidate therapeutic target for bladder cancer.

Topics: Adult; Aged; CARD Signaling Adaptor Proteins; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Female; Humans; Male; Middle Aged; NF-kappa B; Signal Transduction; Urinary Bladder Neoplasms

Given that the tumor-promoting inflammation has been previously established in squamous cell carcinoma of the bladder but its contribution to development of urothelial carcinoma (UC) still remains elusive, our aim was to study changes in expression and activity of inflammation-mediating NF-κB and STAT3 transcription factors in human urothelial bladder carcinoma as well as expression of their target genes cyclin D1, VEGFA and TGFβ1.. Gene expression of STAT3, NF-κB, TGFβ1, cyclin D1 and VEGFA was measured by quantitative real-time polymerase chain reaction in both tumor and healthy bladder tissue from 36 patients with UC of the bladder. Activation of STAT3 and NF-κB was assessed with immunohistochemistry and immunoblot.. Urothelial bladder carcinoma displayed elevated expression as well as activation of NF-κB (P = 5.38e-10) and STAT3 (P = 0.002) transcription factors. Furthermore, elevated level of expression was observed for cyclin D1, VEGFA and TGFβ1 (P = 9.71e-09, P = 9.71e-09, P = 5.38e-10). Preliminary statistical analysis indicated that the level of upregulation of STAT3 or NF-κB was probably not dependent upon the grade (P = 0.984 and 0.803, respectively) and invasiveness of the tumor (0.399 and 0.949), nor to the gender (0.780 and 0.536) and age (0.660 and 0.816) of the patients.. NF-κB and STAT3 signaling pathways, as main inflammatory mediators, are found to be activated in urothelial bladder carcinoma indicating that chronic inflammatory processes are accompanying development of this tumor type. Future studies will have to determine possible causative role of inflammatory processes in development of urothelial bladder carcinomas.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma; Cohort Studies; Cyclin D1; Female; Humans; Male; Middle Aged; NF-kappa B; Pilot Projects; RNA, Messenger; STAT3 Transcription Factor; Transforming Growth Factor beta1; Urinary Bladder Neoplasms; Urothelium; Vascular Endothelial Growth Factor A

To investigate the changes of gene expression file in transitional cell carcinoma of bladder after hepatocyte cell adhesion molecule(hepaCAM) overexpression.. Affymetrix Human Genome U133 Plus 2.0 Array was used to investigate the changes of gene expression profile between adenovirus-green fluorescent protein(GFP) -hepaCAM group and GFP group in transitional cell carcinoma of bladder EJ cells.Significant Analysis of Microarray(SAM) was used to screen the differentially expressed genes, DAVID software was used to conduct gene ontology analysis and wikiPathway analysis based on the differentially expressed genes. Reverse transcription-polymerase chain reaction and Western blot were applied to verify microarray data.. Compared with the GFP group, a total of 2469 genes were up-regulated or down-regulated by more than 2 times in the GFP-hepaCAM group. Among these genes, 1602 genes were up-regulated and 867 were down-regulated.Most of the differentially expressed genes were involved in the function of cell proliferation and cell cycle regulation. The mRNA expressions of nibrin, liver kinase B1, and cyclin D1 detected by reverse transcription-polymerase chain reaction in three different bladder cancer cell lines were consistent with the microarray data.The protein expressions of nibrin and liver kinase B1 in these three cell lines measured by Western blot were consistent with the mRNA expression.. HepaCAM can alter the gene expression profile of bladder cancer EJ cells. The well-known anti-tumor effect of hepaCAM may be mediated by regulating the gene expression via multiple pathways.

Topics: AMP-Activated Protein Kinase Kinases; Carcinoma, Transitional Cell; Cell Cycle Proteins; Cell Line, Tumor; Cyclin D1; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Humans; Nuclear Proteins; Protein Serine-Threonine Kinases; Proteins; Urinary Bladder Neoplasms

Special AT-rich sequence-binding protein-1 (SATB1) has been recently reported to be overexpressed in various cancers and associate with the malignant behavior of cancer cells. However, the expression and potential roles of SATB1 in bladder cancer remains unclear. In the present study, SATB1 expression was analyzed in 85 archived bladder cancer specimens using immunohistochemistry and the correlations between SATB1 expression and clinicopathological parameters were evaluated. To further explore the biological functions of SATB1 in bladder cancer, siRNA knockdown was performed in 5637 and T24 bladder cancer cell lines. We then carried out CCK8 assay and examined cisplatin-induced apoptosis to address the roles of SATB1 in proliferation and apoptosis. We found that SATB1 was overexpressed in 33 of 85 (38.8 %) bladder cancer specimens. SATB1 overexpression associated with tumor grade (p = 0.002) and tumor stage (p = 0.027). SATB1 depletion in 5637 and T24 cells decreased cell proliferation while upregulating cisplatin-induced apoptosis. Further study demonstrated that SATB1 knockdown decreased cyclin D1 and cyclin E expression and upregulated caspase3 cleavage. In conclusion, SATB1 is overexpressed in bladder cancer and regulates malignant cell growth and apoptosis, which makes SATB1 a therapeutic target candidate for bladder cancer.

Topics: Aged; Antineoplastic Agents; Apoptosis; Case-Control Studies; Caspase 3; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Cisplatin; Cyclin D1; Cyclin E; Female; Gene Expression; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Male; Matrix Attachment Region Binding Proteins; Middle Aged; Urinary Bladder; Urinary Bladder Neoplasms

Isorhapontigenin (ISO) is a new derivative of stilbene compound that was isolated from the Chinese herb Gnetum Cleistostachyum and has been used for treatment of bladder cancers for centuries. In our current studies, we have explored the potential inhibitory effect and molecular mechanisms underlying isorhapontigenin anticancer effects on anchorage-independent growth of human bladder cancer cell lines. We found that isorhapontigenin showed a significant inhibitory effect on human bladder cancer cell growth and was accompanied with related cell cycle G(0)-G(1) arrest as well as downregulation of cyclin D1 expression at the transcriptional level in UMUC3 and RT112 cells. Further studies identified that isorhapontigenin downregulated cyclin D1 gene transcription via inhibition of specific protein 1 (SP1) transactivation. Moreover, ectopic expression of GFP-cyclin D1 rendered UMUC3 cells resistant to induction of cell-cycle G(0)-G(1) arrest and inhibition of cancer cell anchorage-independent growth by isorhapontigenin treatment. Together, our studies show that isorhapontigenin is an active compound that mediates Gnetum Cleistostachyum's induction of cell-cycle G(0)-G(1) arrest and inhibition of cancer cell anchorage-independent growth through downregulating SP1/cyclin D1 axis in bladder cancer cells. Our studies provide a novel insight into understanding the anticancer activity of the Chinese herb Gnetum Cleistostachyum and its isolate isorhapontigenin.

Topics: Animals; Antineoplastic Agents, Phytogenic; Binding Sites; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Disease Models, Animal; Down-Regulation; Gene Expression Regulation, Neoplastic; Humans; Male; Mice; Promoter Regions, Genetic; Sp1 Transcription Factor; Stilbenes; Transcription, Genetic; Urinary Bladder Neoplasms; Xenograft Model Antitumor Assays

Increasing evidence demonstrated that TPX2 was highly expressed and tightly associated with human tumor development and progression. However, its precise role in bladder carcinoma remains to be delineated. In the present study, we revealed the high expression of TPX2 at both mRNA and protein levels in bladder carcinoma tissues and cells, and TPX2 levels in pN1-3 and pT2-4 status were significantly higher than those in pN0 and pTa-T1 status, respectively. Additionally, high TPX2 level was strongly associated with pT status (P = 0.001), higher histological grade (P = 0.001), lymph node metastasis (P = 0.022), and shorter survival time (P = 0.0279). Further investigation showed that TPX2 level in T24 cells was markedly higher than those in 5637, J82 and RT4 cells, in which RT4, a well-differentiated cell line derived from bladder carcinoma with low-grade non-invasive T0, displayed the lowest TPX2 mRNA and protein levels. Besides, TPX2 overexpression promoted proliferation and tumorigenicity, shortened cell cycle in G0/G1 phase, and suppressed cell apoptosis in T24 cells; conversely, TPX2 depletion exhibited opposite effects. Furthermore, TPX2 overexpression evoked the elevation of cyclin D1 and cdk2 levels as well as reduction of p21 level and caspase-3 activity, whereas reversed effects were observed in TPX2-depleted T24 cells. Taken altogether, TPX2 may play a central role in the development and progression of bladder carcinoma, and thus inhibition of TPX2 level may be a novel strategy for therapy of the patients with bladder carcinoma.

Topics: Apoptosis; Blotting, Western; Caspase 3; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 2; Female; G1 Phase; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Lymphatic Metastasis; Male; Microtubule-Associated Proteins; Middle Aged; Neoplasm Staging; Nuclear Proteins; Phenotype; Prognosis; Resting Phase, Cell Cycle; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Survival Analysis; Urinary Bladder Neoplasms

MicroRNA-16 (miR-16) has been demonstrated to regulate proliferation and apoptosis in many types of cancers, but its biological function in bladder cancer remains unknown. Here, we found expression of miR-16 to be downregulated in bladder cancer in comparison with the adjacent normal tissues. Enforced expression of miR- 16 was able to inhibit cell proliferation in TCHu-1 cells, in line with results for miR-16 antisense oligonucleotides (antisense miR-16). At the molecular level, our results further revealed that cyclin D1 expression was negatively regulated by miR-16. Therefore, the data reported here demonstrate that miR-16 is an important regulator in bladder cancer, which will contribute to better understanding of important mis-regulated miRNAs.

Topics: Apoptosis; Blotting, Western; Cell Cycle; Cell Proliferation; Cyclin D1; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Luciferases; MicroRNAs; Oligonucleotides, Antisense; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Cells, Cultured; Urinary Bladder Neoplasms

PIN2/TRF1-interacting telomerase inhibitor1 (PinX1) was recently suggested as a putative tumor suppressor in several types of human cancer, based on its binding to and inhibition of telomerase. Moreover, loss of PinX1 has been detected in many human malignancies. However, the possible involvement of PinX1 and its clinical/prognostic significance in urothelial carcinoma of the bladder (UCB) are unclear.. The PinX1 expression profile was examined by quantitative real-time polymerase chain reaction (qRT-PCR), western blotting, and immunohistochemistry (IHC) in UCB tissues and adjacent normal urothelial bladder epithelial tissues. PinX1 was overexpressed and silenced in UCB cell lines to determine its role in tumorigenesis, development of UCB, and the possible mechanism.. PinX1 expression in UCB was significantly down-regulated at both mRNA and protein level as compared with that in normal urothelial bladder epithelial tissues. PinX1 levels were inversely correlated with tumor multiplicity, advanced N classification, high proliferation index (Ki-67), and poor survival (P < 0.05). Moreover, overexpression of PinX1 in UCB cells significantly inhibited cell proliferation in vitro and in vivo, whereas silencing PinX1 dramatically enhanced cell proliferation. Overexpression of PinX1 resulted in G1/S phase arrest and cell growth/proliferation inhibition, while silencing PinX1 led to acceleration of G1/S transition, and cell growth/proliferation promotion by inhibiting/enhancing telomerase activity and via the p16/cyclin D1 pathway.. These findings suggest that down-regulation of PinX1 play an important role in the tumorigenesis and development of UCB and that the expression of PinX1 as detected by IHC is an independent molecular marker in patients with UCB.

Topics: Animals; Carcinoma, Transitional Cell; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Female; Gene Expression; Gene Expression Regulation, Neoplastic; Humans; Kaplan-Meier Estimate; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Multivariate Analysis; Neoplasm Transplantation; Proportional Hazards Models; Signal Transduction; Telomerase; Telomere Homeostasis; Tumor Suppressor Proteins; Urinary Bladder; Urinary Bladder Neoplasms

Invasive urothelial carcinoma of the bladder (UCB) is characterized by alterations in cell-cycle regulatory pathways. Defects in the expression of cyclin D1, a key cell-cycle regulator, have been implicated in progression of various types of cancer. In the present study, we investigated whether cyclin D1 expression is associated with clinicopathological parameters and whether it has any potential prognostic value in determining risk of UCB recurrence.. Tissue microarrays containing bladder cancer specimens (n=212) and adjacent normal bladder tissues (n=131) were immunostained using an antibody against cyclin D1. The association between cyclin D1 and clinicopathological parameters including stage, lymph node metastasis, and disease-free survival, were evaluated. Cyclin D1 mRNA expression data from human normal bladder (n=14) and cancer specimens (n=28) were extracted from the public Oncomine database.. Cyclin D1 mRNA and protein expression were significantly higher in UCB compared to adjacent non-malignant bladder tissue (for mRNA p=0.003, for protein p=0.001). Cyclin D1 protein expression was significantly higher in non-invasive tumors than in muscle-invasive UCB (p=0.016). Among patients with muscle-invasive UCB, increased cyclin D1 expression in tumor cells significantly correlated with lymph node metastasis (p<0.001), and there was a trend of cyclin D1 together with lymph node positivity to be associated with disease recurrence (p=0.678). Loss of nuclear cyclin D1 expression in tumor cells was likewise significantly associated with the presence of lymph node metastasis (p<0.001).. Altered expression of cyclin D1 is associated with lymph node metastasis and risk of UCB recurrence. Cyclin D1 expression may therefore have clinical value as a prognostic marker and potential therapeutic target.

Topics: Adult; Aged; Aged, 80 and over; Cell Line, Tumor; Cyclin D1; Female; Humans; Male; Middle Aged; RNA, Messenger; Urinary Bladder Neoplasms

Recent studies suggest that metformin, a widely used antidiabetic agent, may reduce cancer risk and improve prognosis of certain malignancies. However, the mechanisms for the anti-cancer effects of metformin remain uncertain. In this study, we investigated the effects of metformin on human bladder cancer cells and the underlying mechanisms. Metformin significantly inhibited the proliferation and colony formation of 5637 and T24 cells in vitro; specifically, metformin induced an apparent cell cycle arrest in G0/G1 phases, accompanied by a strong decrease of cyclin D1, cyclin-dependent kinase 4 (CDK4), E2F1 and an increase of p21waf-1. Further experiments revealed that metformin activated AMP-activated protein kinase (AMPK) and suppressed mammalian target of rapamycin (mTOR), the central regulator of protein synthesis and cell growth. Moreover, daily treatment of metformin led to a substantial inhibition of tumor growth in a xenograft model with concomitant decrease in the expression of proliferating cell nuclear antigen (PCNA), cyclin D1 and p-mTOR. The in vitro and in vivo results demonstrate that metformin efficiently suppresses the proliferation of bladder cancer cells and suggest that metformin may be a potential therapeutic agent for the treatment of bladder cancer.

Topics: AMP-Activated Protein Kinases; Animals; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; G1 Phase Cell Cycle Checkpoints; Humans; Hypoglycemic Agents; Male; Metformin; Mice; Mice, Inbred BALB C; Mice, Nude; Proliferating Cell Nuclear Antigen; Signal Transduction; TOR Serine-Threonine Kinases; Transplantation, Heterologous; Urinary Bladder Neoplasms

Bladder cancer is the fifth most common type of cancer in the USA, with over 70,000 new cases diagnosed each year. Treatment often involves invasive surgical therapies, as chemotherapy alone is often ineffective and associated with high recurrence rates. Identification of estrogen receptor-β (ERβ) in up to 75 % of urinary tumors raises the question of whether this receptor could be targeted to effectively treat bladder cancer. In this study, a panel of five bladder cancer cell lines representing a variety of disease stage and grades were treated with the antiestrogens 4-hydroxytamoxifen, raloxifene, or the pure antagonist ICI 182,780. All cell lines were ERβ positive while only a few expressed estrogen receptor-α (ERα). Notably, all but the TCCSUP cell line were growth inhibited 20-100 % by at least two antiestrogens. Using RT4 cells, we demonstrate that growth inhibition by raloxifene is ER dependent and either ERα or ERβ can mediate this response. Activation of caspase-3 and its effector poly-ADP ribose polymerase (PARP) demonstrate that raloxifene-induced growth inhibition is in part the result of increased apoptosis; this PARP cleavage was ER dependent. Moreover, changes in the expression of cell cycle genes indicate that cell proliferation is also affected. Specifically, raloxifene treatment results in the stabilization of p27 protein, likely via the downregulation of S-phase kinase-associated protein (SKP2). Expression of the negative cell cycle regulator B-cell translocation gene (BTG2) is also increased, while cyclin D1 transcription is reduced. These results indicate that antiestrogens may be useful therapeutics in the treatment of bladder cancer by targeting ER and inhibiting growth via multiple mechanisms.

Topics: Apoptosis; Carcinoma, Transitional Cell; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Down-Regulation; Estradiol; Estrogen Antagonists; Estrogen Receptor alpha; Estrogen Receptor beta; Fulvestrant; Humans; Immediate-Early Proteins; MCF-7 Cells; Poly(ADP-ribose) Polymerases; Raloxifene Hydrochloride; S-Phase Kinase-Associated Proteins; Tamoxifen; Tumor Suppressor Proteins; Urinary Bladder Neoplasms

Transcripts from the four genes encoding cyclin D1, MCM7, TRIM29, and UBE2C have previously been included in gene expression signatures for outcome prediction in stage Ta/T1 urothelial carcinomas. We investigated the prognostic value of the protein expressions in Ta/T1 urothelial carcinomas patients. We used four different tissue microarrays (TMAs) with a total of 859 Ta/T1 urothelial carcinomas from Danish, Swedish, Spanish, and Taiwanese patient cohorts with long-term follow-up. Protein expression was measured by IHC, and antibody specificity was validated by Western blotting. We found the expression of cyclin D1, MCM7, TRIM29, and UBE2C to be significantly associated with progression to muscle-invasive bladder cancer (log-rank test; P < 0.001) in the Danish training cohort (n = 283). Multivariate Cox regression analysis identified cyclin D1 (P = 0.003), TRIM29 (P = 0.001), and UBE2C (P < 0.001) as independent prognostic markers. The prognostic value of the four proteins was validated in a joint validation cohort from Sweden, Spain, and Taiwan (n = 576). Computer-assisted image analysis of the prognostic markers produced results comparable to those obtained by manual scoring. Finally, a four-protein maximum-likelihood classifier was trained on the Danish training cohort and applied to the validation cohort. The four protein markers may help optimize treatment of patients with Ta/T1 bladder cancer. Additional prospective studies are needed for further validation of their clinical relevance.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Cell Cycle Proteins; Cohort Studies; Cyclin D1; Denmark; Disease-Free Survival; DNA-Binding Proteins; Female; Humans; Male; Middle Aged; Minichromosome Maintenance Complex Component 7; Multivariate Analysis; Muscles; Neoplasm Invasiveness; Nuclear Proteins; Proportional Hazards Models; Reproducibility of Results; Risk Factors; Spain; Sweden; Taiwan; Transcription Factors; Ubiquitin-Conjugating Enzymes; Urinary Bladder Neoplasms; Young Adult

A number of histone demethylases have been identified and biochemically characterized, yet their biological functions largely remain uncharacterized, particularly in the context of human diseases such as cancer. In this study, we describe important roles for the histone demethylase KDM3A, also known as JMJD1A, in human carcinogenesis. Expression levels of KDM3A were significantly elevated in human bladder carcinomas compared with nonneoplastic bladder tissues (p < 0.0001), when assessed by real-time PCR. We confirmed that some other cancers including lung cancer also overexpressed KDM3A, using cDNA microarray analysis. Treatment of cancer cell lines with small interfering RNA targeting KDM3A significantly knocked down its expression and resulted in the suppression of proliferation. Importantly, we found that KDM3A activates transcription of the HOXA1 gene through demethylating histone H3 at lysine 9 di-methylation by binding to its promoter region. Indeed, expression levels of KDM3A and HOXA1 in several types of cancer cell lines and bladder cancer samples were statistically correlated. We observed the down-regulation of HOXA1 as well as CCND1 after treatment with KDM3A siRNA, indicating G(1) arrest of cancer cells. Together, our results suggest that elevated expression of KDM3A plays a critical role in the growth of cancer cells, and further studies may reveal a cancer therapeutic potential in KDM3A inhibition.

Topics: Cell Line, Tumor; Cell Proliferation; Cyclin D1; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Histones; Homeodomain Proteins; Humans; Jumonji Domain-Containing Histone Demethylases; Lung Neoplasms; Methylation; Oligonucleotide Array Sequence Analysis; RNA Interference; RNA, Small Interfering; Transcription Factors; Transcription, Genetic; Urinary Bladder Neoplasms

Invasive bladder cancer is a lethal disease for which effective prognostic markers as well as potential therapy targets are still lacking. Nkx2.8 (Nk2 homeobox 8), a novel member of the NK-2 gene family, was reported to play an important role in the development and progression of human cancer. Herein, we reported that Nkx2.8 was markedly reduced in bladder cancer tissues compared with matched adjacent normal urothelial tissues. Nkx2.8 levels were inversely correlated with advanced T classification, N classification, tumor multiplicity, high proliferation index (Ki-67) and poor survival of patients. Furthermore, we found that overexpression of Nkx2.8 in bladder cancer cells significantly inhibited cell proliferation in vitro and in vivo, whereas silencing Nkx2.8 dramatically enhanced cell proliferation. Moreover, we demonstrated that overexpression of Nkx2.8 resulted in G(1)/S phase arrest, accompanied by upregulation of p27(Kip1), downregulation of cyclin D1 and p-FOXO3a and inhibition of MEK/ERK pathway activity. Meanwhile, silencing Nkx2.8 led to acceleration of G(1)/S transition, downregulation of p27(Kip1), upregulation of cyclin D1 and p-FOXO3a and increase of MEK/ERK pathway activity. These findings suggest that Nkx2.8 plays a potential tumor suppressor role in bladder cancer progression and represents a valuable clinical prognostic marker of this disease.

Topics: Aged; Aged, 80 and over; Animals; Biomarkers, Tumor; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Disease Progression; Female; Forkhead Box Protein O3; Forkhead Transcription Factors; Homeodomain Proteins; Humans; Male; MAP Kinase Signaling System; Mice; Mice, Inbred BALB C; Middle Aged; Mitogen-Activated Protein Kinases; Prognosis; S Phase Cell Cycle Checkpoints; Transcription Factors; Tumor Suppressor Proteins; Up-Regulation; Urinary Bladder; Urinary Bladder Neoplasms

Bladder cancer is the most common neoplasm in the urinary system. This study assesses arctigenin anti-tumor activity in human bladder cancer T24 cells in vitro and the underlying molecular events. The flow cytometry analysis was used to detect cell-cycle distribution and apoptosis. Western blotting was used to detect changes in protein expression. The data showed that arctigenin treatment reduced viability of bladder cancer T24 cells in a dose- and time-dependent manner after treatment with arctigenin (10, 20, 40, 80, and 100 μmol/L) for 24 hr and 48 hr. Arctigenin treatment clearly arrested tumor cells in the G1 phase of the cell cycle. Apoptosis was detected by hoechst stain and flow cytometry after Annexin-V-FITC/PI double staining. Early and late apoptotic cells were accounted for 2.32-7.01% and 3.07-7.35%, respectively. At the molecular level, arctigenin treatment decreased cyclin D1 expression, whereas CDK4 and CDK6 expression levels were unaffected. Moreover, arctigenin selectively altered the phosphorylation of members of the MAPK superfamily, decreasing phosphorylation of ERK1/2 and activated phosphorylation of p38 significantly in a dose-dependent manner. These results suggest that arctigenin may inhibit cell viability and induce apoptosis by direct activation of the mitochondrial pathway, and the mitogen-activated protein kinase pathway may play an important role in the anti-tumor effect of arctigenin. The data from the current study demonstrate the usefulness of arctigenin in bladder cancer T24 cells, which should further be evaluated in vivo before translation into clinical trials for the chemoprevention of bladder cancer.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Cell Cycle Checkpoints; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Flow Cytometry; Furans; Humans; Lignans; Mitogen-Activated Protein Kinases; Tumor Cells, Cultured; Urinary Bladder Neoplasms

Cell-cycle proteins are important predictive markers in urothelial carcinoma but may also exhibit exposure-specific heterogeneity.. Tumor tissue from 491 bladder cancer cases enrolled in the Maine and Vermont component of the New England Bladder Cancer Study was assembled as tissue microarrays and examined for aberrant expression of p53, p63, p16, cyclin D1, Rb, and Ki-67. The association between expression and histopathology, demographics, and cigarette smoking was examined using χ(2) tests, multivariable Poisson, and multinomial regression models.. We found that overexpression of p53 and Ki-67 was associated with high-stage/grade tumors [relative risk (RR), 1.26; P(trend) = 0.003; and RR, 3.21; P(trend) < 0.0001, respectively], whereas expression of p63 and p16 was decreased in high-stage/grade tumors (RR, 0.52; P(trend) < 0.0001; and RR, 0.88; P(trend) = 0.04, respectively). No significant aberrations of cell-cycle proteins were identified using various smoking variables and multiple statistical models.. The results of this population-based study of histologically confirmed urothelial carcinomas show significant aberration of cell-cycle proteins p53, p63, p16, and Ki-67, but not Rb or cyclin D1. p53 showed the most significant heterogeneity with respect to tumor stage and grade, especially when stratified for different staining intensities using novel digital image analysis techniques. Our findings do not support that smoking modifies expression of cell-cycle proteins.. Our study shows significant heterogeneity in the expression of key cell-cycle proteins that are associated with disease progression in bladder cancer. Further studies may lead to the identification of biomarkers and their multiplexed interactions as useful prognostic and therapeutic targets.

Topics: Adult; Aged; Cell Cycle; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Female; Humans; Ki-67 Antigen; Maine; Male; Membrane Proteins; Middle Aged; Neoplasm Proteins; Tissue Array Analysis; Tumor Suppressor Protein p53; Urinary Bladder Neoplasms; Vermont

To elucidate the role of replication protein A (RPA) in both superficial (Ta-T1) and muscle-invasive (T2-T4) urothelial carcinomas (UCs), investigating its potential prognostic usefulness.. Paraffin-embedded tissue from 156 patients with bladder UC was immunostained for RPA1 and RPA2.. RPA1 and RPA2 labelling indexes (LIs) decreased with increasing histological grade (both P < 0.001) and T-category in the entire cohort (P = 0.008 and P < 0.001, respectively) and in muscle-invasive carcinomas (P = 0.014 and P = 0.012, respectively). RPA1 expression was positively correlated with RPA2 (Spearman's correlation coefficient ρ = 0.309, P < 0.001). Both RPA1 and RPA2 LIs were positively correlated with cyclin D1 expression (ρ = 0.354, P < 0.001 and ρ = 0.934, P < 0.001). In survival analysis of the entire cohort decreased RPA2 and RPA1 correlated with a lesser probability of survival (P < 0.001 and P = 0.018). In non-muscle-invasive tumours (Ta-T1) only lower RPA2 (P < 0.001) was correlated with shortened survival, whereas in muscle-invasive tumours (T2-T4) decreased RPA2 and RPA1 expression levels were associated with adverse prognosis (P = 0.035 and P = 0.042, respectively). In multivariate survival analysis of the entire cohort and in non-muscle-invasive cases RPA2 expression remained significant, even when adjustment for cyclin D1 expression was applied.. RPA1 and RPA2 overexpression seems to be more important during early T-categories of bladder carcinogenesis. Showing similar kinetics with cyclin D1. RPA2 expression emerges as a valuable marker of favourable prognosis in the entire cohort and in non-muscle-invasive tumours, supplementing the information obtained by standard clinicopathological prognosticators.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Cyclin D1; Female; Humans; Male; Middle Aged; Neoplasm Invasiveness; Prognosis; Replication Protein A; Survival Analysis; Urinary Bladder Neoplasms; Urothelium

The cell cycle regulator cyclin D1 (CCND1) is thought to play a major role in the transition of cell cycle from G1 to S phase. It is known that a common CCND1 G870A genotype is associated with bladder cancer in Japan and China, but not in the Western World. There is neither a report about its role in Taiwan's population, nor its genetic role of CCND1 G870A in another worldwide urothelial cancer, upper tract urothelial cancer (UTUC). Therefore, we aimed at investigating the role of CCND1 G870A in both bladder cancer and UTUC in Taiwanese cohorts. The CCND1 G870A genotypes of 171 (101 bladder cancer and 70 UTUC) patients and 243 control subjects were determined by PCR-RFLP and their correlation with clinical and histopathological data was evaluated. The genotype analysis results showed that CCND1 GG genotype was associated with a lower risk overall in urothelial (P = 0.008, OR = 0.44, 95% CI = 0.24-0.81) and bladder cancer patients (P = 0.008, OR = 0.34, 95% CI = 0.15-0.76) than those of the AA genotype. In addition, patients carrying the AG genotype had a 0.29-fold lower odds ratio of muscle-invasive cancer procession (95% CI = 0.12-0.70) compared with those carrying the AA genotype in bladder cancer patients. Surprisingly, the GG genotype had a 5.88-fold higher odds ratio of muscle-invasive cancer procession (95% CI = 1.08-32.01) compared with those carrying the AA genotype in UTUC. No association between any CCND1 G870A genotype and higher-grade risk was found. Our results suggest that the G allele of the CCND1 G870A polymorphism may be a potential predictive and prognostic biomarker to distinguish between bladder cancer and UTUC in Taiwan.

Topics: Asian People; Carcinoma, Transitional Cell; Case-Control Studies; Cyclin D1; Genetic Predisposition to Disease; Genotype; Humans; Neoplasm Invasiveness; Polymorphism, Single Nucleotide; Prognosis; Taiwan; Urinary Bladder Neoplasms

Parthenolide, the principal component of sesquiterpene lactones present in medical plants such as feverfew (Tanacetum parthenium), has been reported to have anti-tumor activity. In this study, we evaluated the therapeutic potential of parthenolide against bladder cancer and its mechanism of action. Treatment of bladder cancer cells with parthenolide resulted in a significant decrease in cell viability. Parthenolide induced apoptosis through the modulation of Bcl-2 family proteins and poly (ADP-ribose) polymerase degradation. Treatment with parthenolide led to G1 phase cell cycle arrest in 5637 cells by modulation of cyclin D1 and phosphorylated cyclin-dependent kinase 2. Parthenolide also inhibited the invasive ability of bladder cancer cells. These findings suggest that parthenolide could be a novel therapeutic agent for treatment of bladder cancer.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Carcinoma; Cell Line, Tumor; Cell Survival; Cyclin D1; Cyclin-Dependent Kinase 2; Diffusion Chambers, Culture; Flow Cytometry; G1 Phase; Humans; Microscopy; Neoplasm Invasiveness; Phosphorylation; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Sesquiterpenes; Signal Transduction; Tanacetum parthenium; Urinary Bladder Neoplasms

Cigarette smoking is the main risk factor for bladder cancer development. Among the mediators of this effect of smoking is nuclear factor-kappa B. Curcumin suppresses cellular transformation by downregulating the activity of nuclear factor-kappa B. Prima-1 is a compound that induces apoptosis in human tumor cells, restoring the function of mutant p53. Our study aimed to evaluate the effects of curcumin and prima-1 in an animal model of bladder cancer.. Tumor implantation was achieved in six- to eight-week-old female C57BL/6 mice by introducing MB49 bladder cancer cells into the bladder. Intravesical treatment with curcumin and Prima-1 was performed on days 2, 6, 10, and 14. On day 15, the animals were sacrificed. Immunohistochemistry was used to determine the expression of cyclin D1, Cox-2, and p21. Cell proliferation was examined using PCNA.. Animals treated with curcumin exhibited a higher degree of necrosis than animals in other groups. Immunohistochemistry showed reduced expression of cyclin D1 in the curcumin-treated group. All of the cells in mice treated with curcumin were p21 positive, suggesting that the p53 pathway is induced by this compound. Prima-1 did not induce any change in tumor size, necrosis, cell proliferation, or the expression of proteins related to the p53 pathway in this animal model.. Curcumin showed activity in this animal bladder cancer model and probably acted via the regulation of nuclear factor-kappa B and p53. Therefore, curcumin is a good choice for the use in clinical trials to treat superficial bladder cancer as an alternative to bacillus Calmette-Guerin. In contrast, Prima-1 does not seem to have an effect on bladder cancer.

Topics: Animals; Antineoplastic Agents; Aza Compounds; Bridged Bicyclo Compounds, Heterocyclic; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Curcumin; Cyclin D1; Cyclooxygenase 2; Disease Models, Animal; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Drug Synergism; Female; Immunohistochemistry; Mice; Mice, Inbred C57BL; Tumor Suppressor Protein p53; Urinary Bladder Neoplasms

Urinary bladder cancer is a heterogeneous disease with tumors ranging from papillary noninvasive (stage Ta) to solid muscle infiltrating tumors (stage T2+). The risk of progression and death for the most frequent diagnosed type, Ta, is low, but the high incidence of recurrences has a significant effect on the patients' quality of life and poses substantial costs for health care systems. Consequently, the purpose of this study was to search for predictive factors of recurrence on the basis of genetic profiling. A clinically well characterized cohort of Ta bladder carcinomas, selected by the presence or absence of recurrences, was evaluated by an integrated analysis of DNA copy number changes and gene expression (clone-based 32K, respectively, U133Plus2.0 arrays). Only a few chromosomal aberrations have previously been defined in superficial bladder cancer. Surprisingly, the profiling of Ta tumors with a high-resolution array showed that DNA copy alterations are relatively common in this tumor type. Furthermore, we observed an overrepresentation of focal amplifications within high-grade and recurrent cases. Known (FGFR3, CCND1, MYC, MDM2) and novel candidate genes were identified within the loci. For example, MYBL2, a nuclear transcription factor involved in cell-cycle progression; YWHAB, an antiapoptotic protein; and SDC4, an important component of focal adhesions represent interesting candidates detected within two amplicons on chromosome 20, for which DNA amplification correlated with transcript up-regulation. The observed overrepresentation of amplicons within high-grade and recurrent cases may be clinically useful for the identification of patients who will benefit from a more aggressive therapy.

Topics: 14-3-3 Proteins; Cell Cycle Proteins; Chromosome Aberrations; Comparative Genomic Hybridization; Cyclin D1; Female; Gene Amplification; Gene Dosage; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genetic Predisposition to Disease; Humans; Male; Neoplasm Recurrence, Local; Neoplasm Staging; Proto-Oncogene Proteins c-mdm2; Proto-Oncogene Proteins c-myc; Receptor, Fibroblast Growth Factor, Type 3; Syndecan-4; Trans-Activators; Urinary Bladder; Urinary Bladder Neoplasms

Previous reports suggested that bladder cancer patients who continue to smoke while receiving chemotherapy have poorer outcomes than their nonsmoking counterparts. Nicotine, the major addictive compound in cigarette smoke, is known to induce chemoresistance in some cancer cells. Chemoresistance has been linked to the activation of Stat3 (signal transducer and activator of transcription). The objective of this study was to identify the role of Stat3 in chemoresistance induced by nicotine in human bladder cancer cell line, T24 cells. Chemoresistant T24 cells were established by persistent nicotine treatment. Apoptosis and cell cycle parameters were analyzed by Annexin V staining, poly(ADP-ribose) polymerase degradation, caspase activity, and propidium iodide staining. Signal transduction mediating the chemoresistance was detected by Western blotting and small interfering RNA (siRNA) transfection. We provide evidence for the first time that nicotine strongly activated Stat3, leading to Cyclin D1 overexpression, cell cycle perturbations, and chemoresistance. Furthermore, nicotine mobilized Stat3 signaling, resulting in the loss of extracellular signal-regulated protein kinase 1/2 (ERK 1/2) activation and reduced chemosensitivity via nicotinic acetylcholine receptors and beta-adrenoceptors. Inhibition of Stat3 by siRNA or a specific inhibitor restored chemosensitivity in T24 cells. Stat3 could be the major target for increasing chemosensitivity in patients who develop chemoresistance during chemotherapy, and avoidance of cigarette smoking or nicotine-based treatments may increase the efficacy of chemotherapy.

Topics: Apoptosis; Carcinoma, Transitional Cell; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Drug Resistance, Neoplasm; Humans; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nicotine; Nicotinic Agonists; Proliferating Cell Nuclear Antigen; Receptors, Adrenergic, beta; Receptors, Nicotinic; RNA, Small Interfering; Signal Transduction; STAT3 Transcription Factor; Transfection; Urinary Bladder Neoplasms

Perturbations in the cell cycle and apoptotic genes have been implicated in human malignancies. Cell cycle (MDM2 and Cyclin D1) and apoptotic (Fas) genes that are differentially expressed in urinary bladder cancer (UBC) were investigated with the susceptibility to UBC in northern India. A total of 212 UBC patients and age- and sex-matched 250 controls were investigated for MDM2 G309T, Cyclin D1 G870A, and Fas A670G polymorphisms by polymerase chain reaction and restriction fragment length polymorphism. MDM2 309GG was at reduced risk for developing UBC when compared with TT genotype (p = 0.027; odds ratio, 0.55). GG genotype was also associated with reduced risk in nonmuscle invasive bladder cancer (NMIBC) and muscle invasive BC (p = 0.006 and p = 0.049). Cyclin D1 AA genotype was associated with high risk in NMIBC (intermediate risk) stage (p = 0.015; odds ratio, 4.55). MDM2-Cyclin D1 interaction revealed overall protective effect. The GG genotype of MDM2 also showed a protective effect and high recurrence-free survival in Bacillus Calmette-Guerin-treated NMIBC patients (log rank p = 0.008). MDM2 GG genotype had protective effect and MDM2T309G polymorphism had profound influence in Bacillus Calmette-Guerin-treated UBC patients in the North Indian population.

Topics: Apoptosis; BCG Vaccine; Cell Cycle; Cyclin D1; Genes; Genotype; Humans; India; Mycobacterium bovis; Polymerase Chain Reaction; Polymorphism, Genetic; Polymorphism, Restriction Fragment Length; Population Groups; Proto-Oncogene Proteins c-mdm2; Racial Groups; Risk Factors; Urinary Bladder; Urinary Bladder Neoplasms

Aneuploidy, centrosome abnormalities and gene amplification are hallmarks of chromosome instability (CIN) in cancer. Yet there are no studies of the in vivo behavior of these phenomena within the same bladder tumor.. Twenty-one paraffin-embedded bladder tumors were analyzed by conventional comparative genome hybridization and fluorescence in situ hybridization (FISH) with a cyclin D1 gene (CCND1)/centromere 11 dual-color probe. Immunofluorescent staining of alpha, beta and gamma tubulin was also performed.. Based on the CIN index, defined as the percentage of cells not displaying the modal number for chromosome 11, tumors were classified as CIN-negative and CIN-positive. Fourteen out of 21 tumors were considered CIN-positive. All T1G3 tumors were included in the CIN-positive group whereas the majority of Ta samples were classified as CIN-negative tumors. Centrosome clustering was observed in six out of 12 CIN-positive tumors analyzed. CCND1 amplification in homogeneously staining regions was present in six out of 14 CIN-positive tumors; three of them also showed amplification of this gene in double minutes.. Complex in vivo behavior of CCND1 amplicon in bladder tumor cells has been demonstrated by accurate FISH analysis on paraffin-embedded tumors. Positive correlation between high heterogeneity, centrosome abnormalities and CCND1 amplification was found in T1G3 bladder carcinomas. This is the first study to provide insights into the coexistence of CCND1 amplification in homogeneously staining regions and double minutes in primary bladder tumors. It is noteworthy that those patients whose tumors showed double minutes had a significantly shorter overall survival rate (p < 0.001).

Topics: Adult; Aged; Biomarkers, Tumor; Centrosome; Chromosomal Instability; Chromosomes, Human, Pair 11; Comparative Genomic Hybridization; Cyclin D1; Female; Fluorescent Antibody Technique; Gene Amplification; Gene Expression Regulation, Neoplastic; Humans; In Situ Hybridization, Fluorescence; Kaplan-Meier Estimate; Male; Micronuclei, Chromosome-Defective; Middle Aged; Mitosis; Neoplasm Staging; Paraffin Embedding; Prognosis; Time Factors; Tubulin; Urinary Bladder Neoplasms

The aim of this study was to assess immunohistochemical expression of p53, pRb, p16, and cyclin D1, alone or in combination, as prognostic indicators and to investigate their correlation with clinocopathologic features of urothelial carcinoma. Immunohistochemical staining for p53, pRb, p16, and cyclin D1 was performed on a tissue microarray from 103 patients with urothelial carcinoma who underwent radical cystectomy. Of the patient samples analyzed, 36 (35%), 61 (59%), 47 (46%) and 30 (29%) had altered expression of p53, pRb, p16, and cyclin D1, respectively. Abnormal expression of p53 and pRb correlated with depth of invasion (P=0.040 and P=0.044, respectively). Cyclin D1 expression was associated with tumor stage and recurrence (P=0.017 and P=0.036, respectively). Altered pRb was significantly correlated with overall survival (P=0.040). According to the expression pattern of pRb and p53, p53/pRb (altered/normal) had worse survival than p53/pRb (normal/altered) (P=0.022). Alteration of all markers had worse survival than all normal (P=0.029). As determined by multivariate analysis, tumor stage, lymph node metastasis and the combined expression of p53 and pRb are independent prognostic factors. In conclusion, immunohistochemical evaluation of cell cycle regulators, especially the p53/pRb combination, might be useful in planning appropriate treatment strategies.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Transitional Cell; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Female; Humans; Immunohistochemistry; Lymphatic Metastasis; Male; Middle Aged; Multivariate Analysis; Neoplasm Staging; Prognosis; Recurrence; Retinoblastoma Protein; Survival Rate; Tumor Suppressor Protein p53; Urinary Bladder Neoplasms

The ras oncogene needs a second factor to induce transformation and tumorigenicity of the cell. In this study, we show that mouse fibroblast 7-4-Stat3C cells overexpressing both Ha-ras(val12) oncogene and active-form Stat3 (Stat3C) showed higher colony formation in soft agar and xenograft tumor growth in BALB/c mice. Further studies show that both serine-727 and tyrosine-705 of Stat3 were phosphorylated while Ha-ras was overexpressed. Interleukin-6 (IL-6)-induced phosphorylation of tyrosine-705 and serine-727, as well as DNA-binding and transcriptional activity of Stat3 were further enhanced by Ha-ras overexpression. In addition, overexpression of Stat3C in 7-4-Stat3C cells prevented the cells from morphological change and apoptosis triggered by the Ha-ras oncogene under serum-depleted conditions. We demonstrate that Ha-ras and Stat3 acting together synergistically induce Stat3 phosphorylation at serine-727 phosphorylation and cyclin D1 expression and further enhance transformation and tumorigenicity of the cell. Ha-ras-induced Stat3 phosphorylation at serine-727 plays a pivotal role in transcriptional activation of cyclin D1 and suppression of cell apoptosis. The effect of Ha-ras on Stat3 phosphorylation at serine-727 was also detected in human bladder (T24) and lung (H460) cancer cells. Stat3 phosphorylation at serine-727 is important in Ras-related tumorigenesis.

Topics: Amino Acid Sequence; Animals; Apoptosis; Cell Line, Tumor; Cell Transformation, Neoplastic; Cyclin D1; Genes, ras; Humans; Interleukin-6; Lung Neoplasms; Mice; Neoplasm Transplantation; Phosphorylation; Serine; STAT3 Transcription Factor; Transcriptional Activation; Up-Regulation; Urinary Bladder Neoplasms

Alternative splicing in the cyclin D1 gene produces cyclin D1b variant which lacks a C-terminal region containing the threonine-286 (T286) phosphorylation site required for nuclear export. We have shown that the expression of the cyclin D1b variant is detected in about 60% of human bladder cancer tissues (15/26) and cell lines (3/5). To examine the role of the cyclin D1b variant in bladder carcinogenesis, we introduced wild-type cyclin D1a, cyclin D1b variant or mutant cyclin D1-T286A cDNAs into a human bladder cancer cell line, SBT991, in which cyclin D1b transcript was not expressed, and compared their oncogenic activities. Ectopic expression of cyclin D1b promoted cell invasiveness and anchorage-independent growth of the cancer cells. On the other hand, cyclin D1-T286A enhanced anchorage-independent growth, but did not promote cell invasiveness. The amount of nuclear-localized cyclin D1b and cyclin D1-T286A was higher than that of nuclear-localized cyclin D1a. In addition, introduction of siRNA specific for cyclin D1b into cells of the T24 bladder cancer cell line, in which cyclin D1b transcript was expressed, significantly suppressed cell invasiveness. Immunoprecipitation analysis revealed that cyclin D1a and cyclin D1-T286A could bind to cyclin-dependent kinase 4 (CDK4) but cyclin D1b has lost its capacity to associate with CDK4. Unlike cyclin D1a and cyclin D1-T286A, expression of cyclin D1b did not enhance phosphorylation of Rb protein in SBT991 cells. These results indicate that cyclin D1b promotes cell invasiveness independent of binding to CDK4 to enhance Rb phosphorylation.

Topics: Aged; Aged, 80 and over; Alternative Splicing; Blotting, Western; Cell Cycle; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; Female; Fluorescent Antibody Technique; Gene Expression Regulation, Neoplastic; Humans; Immunoenzyme Techniques; Immunoprecipitation; Male; Middle Aged; Neoplasm Invasiveness; Phosphorylation; Protein Isoforms; Retinoblastoma Protein; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Subcellular Fractions; Transfection; Urinary Bladder Neoplasms

Celecoxib and other non-steroidal anti-inflammatory drugs (NSAIDs) are being evaluated in the prevention of bladder and other cancers. Here we investigate molecular effects of celecoxib independent of cyclooxygenase (COX)-2 expression levels in urothelial carcinoma of the bladder.. Low-grade RT-4 and high-grade UM-UC-3 bladder cancer cells were treated with 0-50 muM celecoxib. Growth, cell cycle and apoptosis were measured by crystal violet elution and flow cytometry. Western analysis was performed for COX-2, Rb, cyclin B1/D1, and phospho-cyclin B1/D1. COX-2 induction was achieved with phorbol ester.. Celecoxib inhibited growth of RT-4 and UM-UC-3, with G(1) cell cycle arrest and altered cyclin B1/D1 expression in RT-4, whereas Rb up-regulation occurred in UM-UC-3. Apoptosis occurred in both cell lines.. Celecoxib induces G(1) cell cycle arrest in low- and high-grade bladder cancer by different pathways. This heterogeneous molecular response supports combination approaches to prevention and treatment.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Antineoplastic Agents; Apoptosis; Celecoxib; Cell Cycle; Cell Growth Processes; Cell Line, Tumor; Cyclin B1; Cyclin D1; Cyclooxygenase 2; G1 Phase; Humans; Pyrazoles; Retinoblastoma Protein; Sulfonamides; Urinary Bladder Neoplasms

The role of aristolochic acid in the etiology of Balkan endemic nephropathy (BEN) and associated upper urothelial carcinoma (UUC) was recently confirmed. The aim of this study was to determine the marker(s) specific for BEN-associated UUC. A total of 82 patients with UUC (38 from the BEN region and 44 control tumors) were included in the study. The Ki-67 index in BEN tumors correlated with the grade and multifocality (p < 0.05), but in regression analysis, only the grade of BEN tumor. The p53 index was significantly higher in BEN than in control tumors (p < 0.05), as well as the alteration of p53 (p < 0.05). BEN low-stage tumors, tumors without limphovascular invasion (LVI), and tumors of the renal pelvis had a higher p53 index than the control tumors (p < 0.05, 0.01, 0.05, respectively). The Ki-67 index was higher in control tumors with high-stage and solid growth than in BEN UUC (p < 0.050, 0.005). The Ki-67 correlated with the grade, growth, stage, LVI, and multifocality of UUC on the best way, but not with the group. In regression analysis, only multifocality of UUC had predictive influence on Ki-67 activity (p < 0.001). P53 correlated with the grade, growth, and group (p < 0.05). This investigation identifies the p53 pathway as the specific cell cycle marker involved in BEN-associated UUC.

Topics: Adult; Aged; Aged, 80 and over; Aristolochia; Aristolochic Acids; Balkan Nephropathy; Biomarkers, Tumor; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Female; Humans; Kidney Pelvis; Male; Middle Aged; Neoplasm Proteins; Receptor, ErbB-2; Risk Factors; Tumor Suppressor Protein p53; Ureter; Ureteral Neoplasms; Urinary Bladder Neoplasms; Young Adult

The oncogenic isoform of the p63 protein, delta Np63 (delta Np63), plays an important role in the pathogenesis of many epithelial carcinomas, and emerging evidences suggest that delta Np63 is a promising drug target. However, the functions of delta Np63 in transitional cell carcinoma of bladder (TCCB) are poorly defined. In this study, a delta Np63 shRNA expression vector was transfected into TCCB cell line 5637 and cell cycling, cell proliferation and protein expression were assessed by flow cytometry and 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-dimethyl tetrazolium bromide (MTT) assay, and immunohistochemistry, respectively. The delta Np63 shRNA expression vector was also injected into 5637 cell xenograft tumors in nude mice, and tumor size was measured, tumor tissue morphology was assessed by immunohistopathology and transmission electron microscopy. In the in vitro study, delta Np63 shRNA transfection caused successful delta Np63 gene silencing and resulted in significant arrest of cell cycling and cellular proliferation (p<0.05) as well as cyclin D1 expression. In the nude mouse xenograft model, delta Np63 shRNA greatly inhibited tumor growth, induced tumor cell apoptosis (p<0.05) and resulted in cyclin D1 downregulation. Our data suggest that delta Np63 may play an oncogenic role in TCCB progression through promoting cell survival and proliferation. Intratumoral administration of delta Np63-specific shRNA suppressed tumor delta Np63 expression and cellular proliferation while promoted tumor cellular apoptosis, and therefore inhibited tumor growth and improved survival of xenograft-bearing mice, which was not accompanied by significant signs of systemic toxicity.

Topics: Animals; Carcinoma, Transitional Cell; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Disease Progression; Female; Humans; Mice; Mice, Nude; Microscopy, Electron, Transmission; Models, Biological; Neoplasm Transplantation; Trans-Activators; Transcription Factors; Tumor Suppressor Proteins; Urinary Bladder Neoplasms

To investigate the proliferation, apoptosis and mechanisms on T24 cell of transitional cell carcinoma of bladder (TCCB) by crocin.. MTT assay was used to evaluate the proliferation of T24 cells. The changes of cell cycle and cell apoptotic percentage were measured by flow cytometry. T24 cells were inoculated into BALB/c nude mice to establish model of carcinoma of bladder. The mice were randomly divided into control group and experimental group. After treatment with 50 mmol x L(-1) crocin, the inhibited growth of tumor was observed. Electronic microscope was used to observe the morphological changes. The expressions of Bcl-2, Bax, Survivin and Cyclin D1 were detected by immunohistochemistry.. The growth of T24 cells was remarkably inhibited after treatment of crocin. Flow cytometric profiles revealed that crocin led to the increase of the cells in G0/G1 phase, the percentage of cell apoptosis was also increased. Crocin could inhibit the growth of BALB/c xenograft tumor. The morphology changes of cell apoptosis were observed. Bcl-2, Cyclin D1 and survivin expressions determined by immunohistochemical staining were down-regulated after treatment with Bax expression up-regulated.. Crocin exerts both in vitro and in vivo anti-cancer effect on TCCB T24 cell line. The mechanisms may change tumour cell cycle and induce tumour cell apoptosis by down-regulating the expression of Bcl-2, Survivin, Cyclin D1 and up-regulating the expression of Bax.

Topics: Animals; Apoptosis; Carcinoma, Transitional Cell; Carotenoids; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Inhibitor of Apoptosis Proteins; Mice; Mice, Inbred BALB C; Mice, Nude; Microscopy, Electron, Transmission; Microtubule-Associated Proteins; Proto-Oncogene Proteins c-bcl-2; Repressor Proteins; Survivin; Transplantation, Heterologous; Urinary Bladder Neoplasms

It was the aim of this study to assess the expression of selected cell cycle regulation genes in urothelial and sinonasal inverted papillomas (IP).. Archived surgically resected specimens from 18 urothelial and 19 sinonasal IP were studied immunohistochemically for p16, p53, cyclin D1 and Ki67. Staining results were semiquantified and compared between IP and adjacent control mucosa (CM).. p53 expression did not differ between sinonasal and urothelial IP. Although there was a trend of higher p53 expression in IP compared with the adjacent CM in sinonasal and urothelial specimens, this trend failed to be statistically significant. p16 expression was significantly higher in urothelial IP and CM in comparison with their sinonasal counterparts, but did not differ significantly between IP and its adjacent CM either in urothelial or sinonasal specimens. There were no significant differences in the mean scores for cyclin D1 or Ki67.. The changes in p53 expression seen in both types of IP compared with adjacent CM suggest that sinonasal and urothelial IP may share some common ground in terms of their evolution. Although p16 appears not to be directly involved in the development of sinonasal or urothelial IP, the differing recurrence patterns of sinonasal versus urothelial IP may be attributable in part to different p16 expression.

Topics: Adult; Aged; Biomarkers, Tumor; Cell Cycle Proteins; Cyclin D1; Female; Humans; Ki-67 Antigen; Male; Middle Aged; Papilloma, Inverted; Paranasal Sinus Neoplasms; Retrospective Studies; Tumor Suppressor Protein p53; Urinary Bladder Neoplasms; Urothelium

Recent epidemiological studies have indicated that high dietary consumption of fruit and vegetables results in lower risk of bladder cancer. To confirm these findings, we investigated in the current study the effects of dietary administration with beta-cryptoxanthin extracted from Citras unshiu oranges on N-butyl-N-(4-hydroxybutyl)nitrosamine (OH-BBN)-induced urinary bladder carcinogenesis in mice. Male ICR mice were divided into 6 experimental and control groups. Groups 1 through 4 were given OH-BBN (500 ppm) in drinking water for 6 weeks to induced urinary bladder neoplasms. Mice in groups 2, 3 and 4 were fed the diets mixed with 1, 5 and 25 ppm of beta-cryptoxanthin, respectively, starting 1 week after the cessation of OH-BBN exposure, and kept on these diets for 24 weeks until the termination of the study. Group 5 was treated with the diet containing the test compound (25 ppm) alone, and group 6 served as an untreated control. All animals were sacrificed at week 32 for histopathology and immunohistochemistry (cyclin D1). Feeding with beta-cryptoxanthin decreased the incidence and multiplicity of preneoplastic and neoplastic lesions of urinary bladder. Notably, the highest dose (25 ppm) of the test chemical significantly lowered the occurrence of bladder carcinoma, in conjunction with reducing the cyclin D1-positive cell ratio. These findings suggest that beta-cryptoxanthin is able to prevent OH-BBN-induced bladder carcinogenesis in mice.

Topics: Animals; Anticarcinogenic Agents; Butylhydroxybutylnitrosamine; Carcinogens; Cryptoxanthins; Cyclin D1; Male; Mice; Mice, Inbred ICR; Urinary Bladder Neoplasms; Xanthophylls

ErbB receptor tyrosine kinases can transit to nuclei in tumor cells, where they have been shown to regulate gene expression as components of transcriptional complexes. Quantitative analysis of a human bladder cancer tissue microarray identified nuclear epidermal growth factor receptor (EGFR) in tumor cells and also showed an increased frequency of this histologic feature in cancer relative to normal tissues. This observation suggests a potential role for nuclear EGFR in bladder cancer. We confirmed that EGFR could be induced to transit to nuclei in cultured human bladder cancer cells in response to the urothelial cell growth factor and EGFR ligand heparin-binding EGF-like growth factor (HB-EGF). Mass spectrometric analysis of EGFR immune complexes from a transitional carcinoma cell line (TCCSUP) identified the phosphoinositide kinase, PIKfyve, as a potential component of the EGFR trafficking mechanism. RNA silencing indicated that PIKfyve is a mediator of HB-EGF-stimulated EGFR nuclear trafficking, EGFR binding to the cyclin D1 promoter, and cell cycle progression. These results identify a novel mediator of the EGFR transcription function and further suggest that nuclear EGFR and the lipid kinase PIKfyve may play a role in bladder oncogenesis.

Topics: Cell Line, Tumor; Cell Nucleus; Cyclin D1; ErbB Receptors; Humans; Immunohistochemistry; Mass Spectrometry; Phosphatidylinositol 3-Kinases; Promoter Regions, Genetic; Protein Binding; RNA, Small Interfering; Signal Transduction; Transfection; Urinary Bladder Neoplasms

Effective strategies are lacking for the management of urinary bladder cancer for which smoking is a potential risk factor. Herein, we evaluated chemoprevention of urinary bladder cancer by natural chemopreventive agents, silymarin and silibinin, in a preclinical animal (ICR mouse) model of bladder cancer induced by tobacco smoke carcinogen N-butyl-N-(4-hydroxybutyl) nitrosamine (OH-BBN). Mice were fed p.o. with saline or OH-BBN (0.05%, w/v) in drinking water for 6 weeks or with silymarin or silibinin (200 mg/kg body weight for both) starting 1 week before OH-BBN exposure for 51 weeks. Silymarin and silibinin strongly arrested OH-BBN-induced tumor progression at the stage of mucosal dysplasia with a striking reduction in papillary nodular dysplasia as well as invasive carcinoma. Some silymarin- or silibinin-treated mice developed no urothelial lesions in spite of OH-BBN exposure. Immunohistochemical analyses at study conclusion revealed that silymarin and silibinin decreased cell proliferation by 42% (P < 0.001) and 44% (P < 0.001) and increased apoptosis by 4-fold (P < 0.05) and 6-fold (P < 0.05) in OH-BBN-induced urothelium, respectively. Antiproliferative and apoptotic effects of silymarin and silibinin were associated with decreases in (a) cyclin D1 protein level and extracellular signal-regulated kinase-1/2 phosphorylation and in (b) protein levels of survivin and nuclear phospho-p65 (Ser(276) and Ser(536)), respectively. Together, these results suggest that silymarin and silibinin inhibit chemically induced urinary bladder tumor growth and progression possibly by inhibiting cell proliferation and enhancing apoptosis.

Topics: Animals; Anticarcinogenic Agents; Apoptosis; Butylhydroxybutylnitrosamine; Carcinogens; Cell Proliferation; Cyclin D1; Male; Mice; Mice, Inbred ICR; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; NF-kappa B; Phosphorylation; Silybin; Silymarin; Urinary Bladder Neoplasms

KLF5 is a transcription factor that plays important roles in multiple physical and pathological processes, including cell growth, cell cycle regulation, and angiogenesis. To better characterize KLF5 function in bladder carcinogenesis, we established stable TSU-Pr1 cell clones expressing different levels of KLF5. These clones were then characterized for cell growth, cell cycle progression, tumorigenesis, and alteration in gene expression. Overexpression of KLF5 promoted tumorigenesis of the TSU-Pr1 cancer cells in mice. Consistently, KLF5 increased G1 to S phase transition, which was accompanied by the upregulation of cyclin D1, phosphorylation of MAPK and Akt, and reduced protein levels for CDK inhibitors p27 and p15. Microarray analysis combined with expression verification in different cell systems identified a number of additional genes that are potentially regulated by KLF5, including HBP17, ITGA6, and RAIG1. These findings suggest that the KLF5 transcription factor plays an oncogenic role in the TSU-Pr1 bladder cancer cell line through the regulation of a subset of genes.

Topics: Animals; Blotting, Northern; Blotting, Western; Cell Line, Tumor; Cell Proliferation; Clone Cells; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; Kruppel-Like Transcription Factors; Male; Mice; Mice, SCID; Mitogen-Activated Protein Kinases; Neoplasm Transplantation; Neoplasms, Experimental; Oligonucleotide Array Sequence Analysis; Proto-Oncogene Proteins c-akt; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; S Phase; Transplantation, Heterologous; Tumor Burden; Urinary Bladder Neoplasms

Aberrant activation of the Wingless-type (Wnt) pathway plays a significant role in the pathogenesis of several human cancers. Wnt inhibitory factor-1 (Wif-1) was identified as one of the secreted antagonists that can bind Wnt protein. We hypothesize that Wif-1 plays an important role in bladder cancer pathogenesis.. To test this hypothesis, epigenetic and genetic pathways involved in the Wif-1 gene modulation and expression of Wnt/beta-catenin-related genes were analyzed in 4 bladder tumor cell lines and 54 bladder tumor and matched normal bladder mucosa.. Wif-1 mRNA expression was significantly enhanced after 5-aza-2'-deoxycytidine treatment in bladder tumor cell lines. Wif-1 promoter methylation level was significantly higher and Wif-1 mRNA expression was significantly lower in bladder tumor samples than in bladder mucosa samples. In the total bladder tumor and bladder mucosa samples, an inverse correlation was found between promoter methylation and Wif-1 mRNA transcript levels. However, loss-of-heterozygosity at chromosome 12q14.3 close to the Wif-1 gene loci was a rare event (3.7%). Nuclear accumulation of beta-catenin was significantly more frequent in bladder tumor than in bladder mucosa and inversely correlated with Wif-1 expression. In addition, known targets of the canonical Wnt/beta-catenin signaling pathway, such as c-myc and cyclin D1, were up-regulated in bladder tumor compared with bladder mucosa, and this up-regulation was associated with reduced Wif-1 expression at both mRNA and protein levels. Furthermore, transfection of Wif-1 small interfering RNA into bladder tumor cells expressing Wif-1 mRNA transcripts had increased levels of c-myc and cyclin D1 and accelerated cell growth.. This is the first report showing that CpG hypermethylation of the Wif-1 promoter is a frequent event in bladder tumor and may contribute to pathogenesis of bladder cancer through aberrant canonical Wnt/beta-catenin signaling pathway. The present study elucidates novel pathways that are involved in the pathogenesis of bladder cancer.

Topics: Adaptor Proteins, Signal Transducing; Adult; Aged; Aged, 80 and over; Antimetabolites, Antineoplastic; Azacitidine; Base Sequence; beta Catenin; Carcinoma, Transitional Cell; Carrier Proteins; Cell Line, Tumor; Cyclin D1; Decitabine; DNA Modification Methylases; Epigenesis, Genetic; Gene Expression Regulation, Neoplastic; Humans; Middle Aged; Molecular Sequence Data; Proto-Oncogene Proteins c-myc; Repressor Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Urinary Bladder Neoplasms; Wnt Proteins

Cruciferous vegetable-derived isothiocyanates (ITCs) display potent cancer chemopreventive activity, but also markedly stimulate oncogenic activator protein 1 (AP-1). AP-1 is well known to promote cell survival and proliferation. We examined the impact of AP-1 activation on antiproliferative activity of ITCs, using bladder cancer cells and phenethyl isothiocyanate (PEITC) as models. AP-1 transactivation induced by PEITC was almost completely suppressed by a dominant-negative c-jun (TAM67). However, suppression of AP-1 transactivation did not affect PEITC-induced apoptosis or cell-cycle arrest. Moreover, we previously showed that in response to ITC treatment c-jun was predominantly stimulated among AP-1 family members largely by c-jun N-terminal kinase (JNK) [Food Chem Toxicol 2005; 43: 1373-1380], but neither JNK inhibition nor forced expression of c-jun altered the antiproliferative activity of PEITC. In addition, cyclin D1, which is considered as an AP-1 target gene and promotes cell proliferation, was markedly elevated in PEITC-treated cells. Unexpectedly, neither TAM67 or JNK inhibition, nor forced c-jun expression had a significant impact on cyclin D1 induction by PEITC, indicating that c-jun/AP-1 does not play an important role in cyclin D1 induction by PEITC. In conclusion, despite the known role of c-jun/AP-1 as a stimulator of cell growth and proliferation, our data show that its activation does not diminish the antiproliferative activity of PEITC and is not responsible for cyclin D1 induction by PEITC.

Topics: Anticarcinogenic Agents; Apoptosis; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Humans; Isothiocyanates; JNK Mitogen-Activated Protein Kinases; Peptide Fragments; Proto-Oncogene Proteins c-jun; Replication Protein C; Signal Transduction; Transcription Factor AP-1; Tumor Cells, Cultured; Urinary Bladder Neoplasms

To retrospectively assess the relationship between immunohistochemical expression of p53, p21, p16, and cyclin D1, with recurrence, progression and survival in superficial bladder cancer.. 163 patients undergoing transurethral resection for superficial bladder cancer between February 1995 and March 2004. Tumor samples were included in a tissue microarray support that was serially sectioned for immunohistochemical staining. Grade and stage associations for each marker were evaluated by the Chi-square test. Assessment of the relationship with recurrence, progression, and survival Kaplan-Meier curves and log-rank test were used.. There were no statistically significant differences in marker expression depending on tumor grade and stage, with the exception of Cyclin D1, that was significantly different depending on tumor stage (p=0.030). p21 expression was related to tumor recurrence (p=0.035), progression (p=0.008) and survival (p=0.034). p16 expression was also related to recurrence (p=0.048) and survival (p=0.047), but not to tumor progression (p=0.116). p53 and Cyclin D1 were not statistically associated with tumor recurrence, progression or survival.. In our experience, only p16 and p21 may be useful in the management of superficial bladder tumors, as they are predictors of recurrence and survival in Ta and T1 patients.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Transitional Cell; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; Female; Humans; Immunohistochemistry; Male; Microarray Analysis; Middle Aged; Retrospective Studies; Tumor Suppressor Protein p53; Urinary Bladder Neoplasms

Proteins controlling cell growth, differentiation, apoptosis, and oncogenic stress are often deregulated in tumor cells. However, whether such deregulations affect tumor behavior remains poorly understood in many tumor types. We recently showed that the urothelium-specific expression of activated H-ras and SV40 T antigen in transgenic mice produced two distinctive types of tumors strongly resembling the human superficial papillary tumors and carcinoma in situ of the bladder, respectively. Here we assessed the expression of a key set of cell cycle regulators in these mouse tumors and in a new transgenic line expressing a cyclin D1 oncogene in the urothelium. We found that urothelia of the wild-type and cyclin D1 transgenic mice exhibited a profile of cell cycle regulators found in quiescent (G(0)) cells, indicating that urothelium overexpressing the cyclin D1 (an 8-fold increase) is reminiscent of normal urothelium and remains slow-cycling. Low-grade superficial papillary tumors induced by activated H-ras had no detectable Rb family proteins (Rb, p107, and p130) and late cell cycle cyclins and kinases (cyclin A, E, and CDK1), but had increased level of p16, p53, and MDM2. These data suggest that the inactivation of the Rb pathway plays an important role in H-ras-induced superficial papillary tumors and that oncogenic H-ras can induce a compensatory activation of alternative tumor suppressor pathways. In contrast, carcinoma in situ of the bladder induced by SV40 T antigen had increased expression of cell cycle regulators mainly active in post-G(1) phases. The fact that phenotypically different bladder tumors exhibit different patterns of cell cycle regulators may explain why these tumors have different propensity to progress to invasive tumors. Our results indicate that the transgenic mouse models can be used not only for studying tumorigenesis but also for evaluating therapeutic strategies that target specific cell cycle regulators.

Topics: Animals; Antigens, Polyomavirus Transforming; Carcinoma, Papillary; Cell Cycle; Cell Cycle Proteins; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Genes, ras; Hyperplasia; Mice; Mice, Transgenic; Proliferating Cell Nuclear Antigen; Retinoblastoma Protein; Tumor Suppressor Proteins; Urinary Bladder Neoplasms; Urothelium

The modifying effects of dietary administration of 1,4-phenylene diisothiocyanate (DITC) on N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN)-induced urinary bladder carcinogenesis during the initiation and post-initiation phases were examined in male ICR mice. Five-week-old animals were divided into 5 groups. Groups 1-3 were given BBN (500 ppm) in drinking water for 6 weeks starting at age 6 week. Mice in Group 2 were given the diet containing 100 ppm DITC for 8 weeks during the initiation phase, starting 1 week before BBN exposure. Animals in Group 3 were fed the experimental diet for 24 weeks during the post-initiation phase starting 1 week after the cessation of BBN exposure. Mice in Group 4 were given only the diet containing the test compound, and those in Group 5 were given the basal diet alone throughout the experiment (32 weeks). The frequency of bladder lesions, neoplasms, dysplasia and hyperplasia, was analyzed histopathologically. The cell-proliferation activity estimated by the 5-bromodeoxyuridine labeling index (BrdU-LI), and cell cycle progression by counting cyclin D1-positive cell ratios were compared among the groups using immunohistochemistry. Administration of DITC in the initiation phase reduced significantly the incidence of urinary bladder carcinoma and dysplasia. The frequencies of any lesions of urinary bladder were not reduced by DITC in post-initiation phase. Dietary exposure of this agent in initiation phase reduced significantly both BrdU-LI and cyclin D1-positive cell ratios in any bladder lesions. Administration of DITC in post-initiation phase also significantly reduced BrdU-LI in bladder neoplasms and hyperplasia and cyclin D1-positive cell ratios in urinary bladder carcinoma as well as dysplasia. These results suggest that dietary DITC could be a preventive agent against BBN-induced bladder carcinogenesis in mice when fed during the initiation phase.

Topics: Animals; Anticarcinogenic Agents; Butylhydroxybutylnitrosamine; Carcinogens; Cyclin D1; Immunohistochemistry; Male; Mice; Mice, Inbred ICR; Thiocyanates; Urinary Bladder Neoplasms

Activation of the epidermal growth factor receptor (EGFR) and downstream signaling pathways, such as phosphatidylinositol-3 kinase/Akt and Ras/mitogen-activated protein kinase (MAPK), have been implicated in causing resistance to EGFR-targeted therapy in solid tumors, including the urogenital tumors. To investigate the mechanism of resistance to EGFR inhibition in bladder cancer, we compared EGFR tyrosine kinase inhibitor (Gefitinib, Iressa, ZD1839) with respect to its inhibitory effects on three kinases situated downstream of EGFR: MAPK, Akt, and glycogen synthase kinase-3beta (GSK-3beta). We found that the resistance to the antiproliferative effects of gefitinib, in vitro as well as in vivo in nude mice models, was associated with uncoupling between EGFR and MAPK inhibition, and that GSK-3beta activation and degradation of its target cyclin D1 were indicators of a high cell sensitivity to gefitinib. Further analysis of one phenotypic sensitive (253J B-V) and resistant (UM-UC13) cell lines revealed that platelet-derived growth factor receptor-beta (PDGFRbeta) activation was responsible for short circuiting the EGFR/MAPK pathway for mitogenic stimuli. However, invasion as well as actin dynamics were efficiently reduced by EGFR inhibition in UM-UC13. Chemical disruption of signaling pathways or of PDGFR kinase activity significantly reduced the inactive pool of cellular GSK-3beta in UM-UC13 cells. In conclusion, our data show that the uncoupling of EGFR with mitogenic pathways can cause resistance to EGFR inhibition in bladder cancer. Although this uncoupling may arise through different mechanisms, we suggest that the resistance of bladder cancer cells to EGFR blockade can be predicted early in the course of treatment by measuring the activation of GSK-3beta and of nuclear cyclin D1.

Topics: Antineoplastic Agents; Carcinoma, Transitional Cell; Cell Growth Processes; Cell Line, Tumor; Cyclin D1; Drug Resistance, Neoplasm; Enzyme Activation; Enzyme Induction; ErbB Receptors; Gefitinib; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Neoplasm Invasiveness; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Quinazolines; Receptors, Platelet-Derived Growth Factor; Urinary Bladder Neoplasms

To determine the prognostic value of a subset of regulators of the transition from G1-to-S phase of cell cycle in stage T1 grade 3 bladder cancers.. Fifty-one such cases were investigated to determine the significance on patient's survival of p53, p21Waf1, p27Kip1, Cyclin D1, Cyclin D3, and ki67-MIB1 (proliferation index). The statistical analysis included Kaplan-Meier methodology with Log-rank test and Cox' proportional hazard analysis.. Tumor size (p=0.0034), and the labeling index of ki67-MIB1 (p=0.0034), p53 (p=0.0332), p27kip1 (p=0.0059) and Cyclin D1 (p=0.0103) were associated to disease-free survival. Progression-free survival was related to tumor size (p<0.0001), ki67 (p=0.0163), p53 (p=0.0041), p27kip1 (p=0.0161), Cyclin D1 (p<0.0001) and Cyclin D3 (p<0.0001). Patient's overall survival was associated to Cyclin D3 (p<0.0001), p53 (p=0.0017), p21Waf1 (p=0.0142), Cyclin D1 (p<0.0001), ki67-MIB1 (p=0.0450), and tumor size (p=0.0296). Down-regulation of p27kip1 and Cyclin D3 over-expression (disease-free), over-expression of p53, Cyclin D1 and Cyclin D3 (progression-free), and over-expression of Cyclin D3 (overall survival) were independent predictors by Cox's multivariate analysis. Down-regulation of p27kip1 (p<0.001, R.R. 0.997, 95%C.I. 0.995-0.999), and over-expression of Cyclin D1 (p<0.001, R.R. 1.009, 95%C.I. 1.004-1.014) and Cyclin D3 (p=0.005, R.R. 1.013, 95%C.I. 1.004-1.022) were the main independent predictors.. Down-regulation of p27kip1 and over-expression of Cyclin D1 and Cyclin D3 might be relevant predictors of survival in stage T1 grade 3 bladder cancers, thus selecting a group of patients at higher risk of malignant behavior.

Topics: Adjuvants, Immunologic; Aged; Aged, 80 and over; BCG Vaccine; Cell Cycle; Cell Cycle Proteins; Cell Division; Cyclin D1; Cyclin D3; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclins; Female; Genes, p53; Humans; Ki-67 Antigen; Male; Middle Aged; Neoplasm Staging; Predictive Value of Tests; Prognosis; Survival Rate; Tumor Suppressor Proteins; Urinary Bladder Neoplasms

Cell cycle proteins are important markers in predicting tumor behavior in urothelial carcinoma of the bladder. The objectives of this study were 1) to determine the expression levels of some of those markers in a series of patients with bladder carcinoma, 2) to define their value in distinguishing T1a (minimally invasive) from T1b (invasive) tumors, and 3) to evaluate their use as predictive factors in the progression of T1a and T1b tumors.. Tumor specimens from 101 patients were included (22 Ta specimens, 34 T1a specimens, 15 T1b specimens, and 30 T2 specimens). A tissue microarray from the 101 paraffin embedded tissue blocks was constructed. Immunohistochemistry for p16, p27, p21, p53, cyclin D1, and Ki-67 were performed. To evaluate T1a and T1b tumor progression, clinical and follow-up data were available for all 49 patients.. Cyclin D1 and p27 were the only markers that showed a significant association with tumor stage and tumor grade (cyclin D1: P = 0.002 and P > 0.00, respectively; p27: P = 0.024 and P = 0.031, respectively). The results indicated that a combination of p21 (odds ratio, 5.7; 95% confidence interval [95% CI], 1.3-24.8 [P = 0.022]) and p16 (odds ratio, 3.7; 95% CI, 0.8-16.5 [P = 0.081]) may have potential use in distinguishing T1b tumors from T1a tumors. Finally, none of the markers examined were found to have predictive value for T1a and T1b tumor progression.. The expression of cyclin D1 and p27 was associated with the most important prognostic factors (tumor stage and grade). The combination of p21 and p16 may have value in distinguishing T1b tumors from T1a tumors, although this finding must be evaluated in much larger series. Finally, none of the markers studied appeared to have predictive value for disease progression in patients with T1a and T1b urothelial bladder tumors.

Topics: Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoma, Transitional Cell; Cell Cycle Proteins; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclins; Disease Progression; Female; Humans; Ki-67 Antigen; Male; Middle Aged; Neoplasm Invasiveness; Predictive Value of Tests; Prognosis; Tumor Suppressor Protein p53; Tumor Suppressor Proteins; Urinary Bladder Neoplasms; Urothelium

Loss of heterozygosity, mutations or deletions of the RB1 gene usually result in loss of pRb expression, which has been regarded as an indicator of loss of pRb function in human tumours. It has previously been shown that in addition to loss of pRb expression, aberrantly high (pRb2+) pRb expression also indicates loss of pRb function in bladder tumours compared with moderate (normal, pRb1+) pRb expression. The aim of this study was to elucidate the mechanism by which pRb is functionally inactivated in bladder tumours expressing aberrantly high levels of pRb. Constitutive phosphorylation was therefore investigated as a mechanism of pRb inactivation in bladder tumours. Of 28 bladder tumours examined, western blotting demonstrated pRb hyperphosphorylation in 5/7 (71%) pRb2+ bladder tumours compared with only 4/11 (36%) pRb1+ tumours (p = 0.002). All cases with undetectable pRb showed moderate to high p16 expression and none showed cyclin D1 expression by immunohistochemistry. All pRb1+ tumours with underphosphorylated pRb showed p16 but not cyclin D1 expression. All pRb2+ tumours with hyperphosphorylated pRb showed loss of p16 expression and/or cyclin D1 overexpression. Thus, elevated pRb expression was associated with pRb hyperphosphorylation, which, in turn, was associated with loss of p16 expression and/or increased cyclin D1 expression. In order to analyse this association in vitro, T24 cells, which express high levels of pRb, were transfected with p16 cDNA. Transfection with p16 cDNA resulted in a marked decrease in pRb phosphorylation, decreased cell proliferation, and a change in expression of pRb from high to moderate phenotype as assessed by immunohistochemistry. This paper gives the biological basis for constitutive alteration of pRb function in human tumours in the presence of an intact, expressed pRb protein; the mechanism of pRb inactivation is through hyperphosphorylation, which results from loss of p16 expression and/or cyclin D1 overexpression. Immunohistochemical expression of pRb appears to be a reliable indicator of pRb function.

Topics: Blotting, Western; Cell Division; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Humans; Image Processing, Computer-Assisted; Immunoenzyme Techniques; Neoplasm Proteins; Phosphorylation; Retinoblastoma Protein; Tumor Cells, Cultured; Urinary Bladder Neoplasms

To evaluate the prognostic value of CCND1 polymorphism in superficial and invasive transitional cell cancer of the bladder.. CCND1 polymorphism of blood DNA from patients with transitional cell cancer of the bladder was evaluated using the polymerase chain reaction-restriction fragment length polymorphism method.. No statistically significant difference was found in the recurrence-free survival of patients with superficial (pTa-T1) transitional cell cancer after transurethral resection among different genotype groups (AA versus GG, P = 0.746; GA versus GG, P = 0.979). In patients with superficial bladder cancer, the occurrence of primary carcinoma in situ was significantly greater in patients with the AA genotype compared with those with the GA or GG genotypes (P = 0.006, chi-square test). No statistically significant difference was found in disease-specific survival after radical cystectomy among the different genotype subgroups (AA versus GG, P = 0.245; GA versus GG, P = 0.649).. Although CCND1 polymorphism is not able to serve as a prognostic marker for bladder cancer, the CCND1 variant A allele may recessively increase the risk of carcinoma in situ incidence in patients with superficial bladder cancer.

Topics: Aged; Alternative Splicing; Antineoplastic Agents, Alkylating; BCG Vaccine; Carcinoma in Situ; Carcinoma, Transitional Cell; Combined Modality Therapy; Cyclin D1; Cystectomy; Disease Progression; Female; Follow-Up Studies; Genetic Predisposition to Disease; Genotype; Humans; Immunotherapy, Active; Incidence; Life Tables; Male; Middle Aged; Mitomycin; Polymorphism, Genetic; Prognosis; Proportional Hazards Models; Proto-Oncogenes; Risk; Salvage Therapy; Survival Analysis; Treatment Outcome; Urinary Bladder Neoplasms

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) has been shown to stimulate the growth of a variety of cells in an autocrine or paracrine manner. Although HB-EGF is widely expressed in tumors compared with normal tissue, its contribution to tumorigenicity is unknown. HB-EGF can be produced as a membrane-anchored form (pro-HB-EGF) and later processed to a soluble form (s-HB-EGF), although a significant amount of pro-HB-EGF remains uncleaved on the cell surface. To understand the roles of two forms of HB-EGF in promoting tumor growth, we have studied the effects of HB-EGF expression in the process of tumorigenesis using in vitro and in vivo systems. We demonstrate here that in EJ human bladder cancer cells containing a tetracycline-regulatable s-HB-EGF or pro-HB-EGF expression system, s-HB-EGF expression increased their transformed phenotypes, including growth rate, colony-forming ability, and activation of cyclin D1 promoter, as well as induction of vascular endothelial growth factor in vitro. Moreover, s-HB-EGF or wild-type HB-EGF induced the expression and activities of the metalloproteases, MMP-9 and MMP-3, leading to enhanced cell migration. In vivo studies also demonstrated that tumor cells expressing s-HB-EGF or wild-type HB-EGF significantly enhanced tumorigenic potential in athymic nude mice and exerted an angiogenic effect, increasing the density and size of tumor blood vessels. However, cells expressing solely pro-HB-EGF did not exhibit any significant tumorigenic potential. These findings establish s-HB-EGF as a potent inducer of tumor growth and angiogenesis and suggest that therapeutic intervention aimed at the inhibition of s-HB-EGF functions may be useful in cancer treatment.

Topics: Animals; Blotting, Northern; Cell Division; Cell Membrane; Cell Movement; Cell Transformation, Neoplastic; Colony-Forming Units Assay; Cyclin D1; Cytosol; Epidermal Growth Factor; Gene Expression Regulation; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Matrix Metalloproteinase 3; Matrix Metalloproteinase 9; Mice; Mice, Nude; Neovascularization, Pathologic; Phenotype; Promoter Regions, Genetic; Tumor Cells, Cultured; Urinary Bladder Neoplasms; Vascular Endothelial Growth Factor A; Wound Healing

We studied 159 cases of superficial (stage Ta or T1) bladder tumors to determine the significance on survival of a subset of regulators of transition from G1 to S phase of the cell cycle (p53, p21Waf1, p27Kip1, cyclin D1, cyclin D3) and tumor proliferation (Ki-67 [MIB-1]). Clinical findings (patient age, sex, tumor size, grade, stage [Ta or T1]) were included in the analysis. Univariate analysis revealed association of tumor size (P = .0353), grade in stage Ta tumors (P = .0074), cyclin D1 expression (P = .0182), and Ki-67 index (P = .0033) with disease-free survival and of tumor size (P = .0005), stage (P = .0494), cyclin D3 expression (P = .0105), and Ki-67 index (P = .0272) with overall survival. Cox multivariate analysis revealed cyclin D1 expression and high proliferation index (disease-free) and tumor size, cyclin D3 expression, and high proliferation index (overall survival) as independent predictors. Results suggest that alterations of the progression from the G1 to S phase of the cell cycle are common in papillary urothelial bladder tumors. High tumor proliferation, expression of cyclins D1 and D3, and tumor size at diagnosis might be relevant predictors of survival in patients with stage Ta and T1 bladder urothelial tumors.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Transitional Cell; Cell Cycle; Cell Cycle Proteins; Cell Division; Cyclin D1; Cyclin D3; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclins; Disease-Free Survival; Female; Humans; Male; Middle Aged; Neoplasm Staging; Prognosis; Risk Factors; Tumor Suppressor Protein p53; Tumor Suppressor Proteins; Urinary Bladder Neoplasms

Specific chromosome aberrations in peripheral blood lymphocytes (PBLs) in chromosomes 9 and 11 may be associated with bladder cancer. To investigate this hypothesis, in this study, we used a whole-chromosome painting technique to detect chromosomal aberrations in PBLs from 100 patients with bladder cancer and 100 matched controls. We also used a locus-specific fluorescence in situ hybridization technique to study 9p21 and cyclin D1 gene (CCND1) aberrations in PBLs of 10 patients and 10 controls and in tumor tissues of 38 additional cases. The chromosome-painting analysis showed that there were more aberrations of chromosomes 9 and 11 in bladder cancer patients than in controls. When categorized by type, the number of deletions of 9p and of translocations of chromosome 11 was significantly higher in patients than in controls (P < 0.05). Stratified analysis showed a larger odds ratio (OR) for bladder cancer in individuals with either a 9p deletion or a chromosome 11 translocation/amplification and an even larger OR in individuals with both aberrations. Using locus-specific analysis, we found that 9p21 aberrations occurred more frequently in bladder cancer patients (12.1 per 1,000 interphase cells) than in controls (6.4 per 1,000 interphase cells, P < 0.05); CCND1 translocation and amplification were observed only in bladder cancer patients. Tumor tissue analysis showed that aberrations of 9p21 (40.0 per 100 interphase cells) and CCND1 (43.8 per 100 interphase cells) were very common. Thus, we concluded that 9p deletions and translocations of chromosome 11, especially at 9p21 and CCND1, are associated with bladder cancer.

Topics: Chromosome Aberrations; Chromosome Painting; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 9; Cyclin D1; Female; Genetic Predisposition to Disease; Humans; In Situ Hybridization, Fluorescence; Lymphocytes; Male; Middle Aged; Risk; Urinary Bladder Neoplasms

Dysregulation of cell cycle control may lead to genomic instability, neoplastic transformation and tumor progression. In terms of the particular roles in regulation of the cell-cycle, p21(WAF1) causes growth arrest through inhibition of cyclin-dependant kinases required for G1/S transition. P16 (INK4A) and p15 (INK4B) are thought to act as tumor suppressors, since their inactivation and/or deletion are observable in various types of malignancies. Cyclin D1 is hypothesized to control cell cycle progression through the G1-S check point. The present study evaluated p21 expression, p16 and p15 gene deletion and cylin D1 expression in bladder carcinoma among Egyptian patients, in relation to different clinicopathological features of the tumors and presence or absence of bilharziasis. Tissue specimens were obtained from 132 patients with bladder carcinoma and 50 normal tissue samples from the same patients served as control. P21 was determined by Western blot (WB) and enzyme immunoassay (EIA), p16 and p15 gene deletions were examined by polymerase chain reaction (PCR) and Cyclin D1 was detected by WB. Levels of p21 were lower in malignant tumors than in normal tissues. Lower expression of p21 was evident in lymph node positive, well differentiated tumors and squamous cell carcinoma (SCC) than in lymph node negative, poorly differentiated tumors and transitional cell carcinoma (TCC). In all normal samples, p15 and p16 genes were detected while cyclin D1 was not detected. P16 and p15 genes were deleted in 38.7% (41/106) and 30.2% (32/106) of bladder tumors respectively. The deletion of both genes was associated with poor differentiation grade and presence of bilharziasis. P16 deletion was also correlated to advancing tumor stage. Cyclin D1 was expressed in 57.5% of bladder tumors (69/120), where its expression was correlated to early stage, well differentiation grade, schistomiasis, and low levels of p21. Cell cycle is dysregulated in bladder carcinoma. This was evident from the increased expression of cyclin D1, the decreased levels of p21 and the deletion of p15 and p16 genes. Moreover, p16 and p15 gene deletion was related to tumor progression and might have a role in bilharzial bladder carcinogenesis. Cyclin D1 over-expression appears to be an early event in bladder cancer and might explain bilharzial associated bladder carcinogenesis.

Topics: Adult; Aged; Aged, 80 and over; Cell Cycle; Cell Cycle Proteins; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p15; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; Female; Gene Deletion; Humans; Male; Middle Aged; Placenta; Pregnancy; Schistosomiasis; Tumor Suppressor Proteins; Urinary Bladder Neoplasms

To observe the effect of curcumin on bladder cancer cell line EJ in vitro.. Cell morphology, MTT, flow cytometer, immunocytochemical method for detecting NF-KB, Cyclin D1 were used to observe the effect of 5,10,20 mg/L curcumin on bladder cancer cell line EJ in vitro.. All concentrations curcumin resulted in the growth suppression significantly [Suppression ratio > or = (27.5 + 3.1)%, P < 0.05]. Above 10 mg/L concentrations curcumin induced apoptosis [Apoptosis ratio > or = (14.6 +/- 1.8)%, P < 0.05] and down-regulated of the expression of NF-kappaB [Expression ratio < or = (35.8 +/- 4.2)%, P < 0.05], Cyclin D1 [Expression ratio < or = (29.7 +/- 3.2)%, P < 0.05]. The cell phase arrest induced by curcumin was G1 phase arrest mainly with significant decrease of S phase.. Curcumin can suppress the growth, induce apoptosis of bladder cancer EJ cell in vitro. Its mechanism is related with down-regulations of the expressions of NF-kappaB and Cyclin D1. Curcumin has great potential for the treatment of bladder cancer.

Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Proliferation; Curcuma; Curcumin; Cyclin D1; Down-Regulation; Drugs, Chinese Herbal; Flow Cytometry; G1 Phase; Humans; NF-kappa B; Urinary Bladder Neoplasms

The neurotrophin (NTR) receptor (p75(NTR)) is a cell-surface glycoprotein that binds to the neurotrophin family of growth factors, of which the prototypic member is nerve growth factor (NGF). This receptor was previously shown to retard cell-cycle progression by inducing accumulation of cells in G(1) with a concomitant reduction of cells in the S phase of the cell cycle. Furthermore, p75(NTR) was shown to be an effective tumor suppressor of bladder cancer cell growth in vivo. In order to investigate the mechanism of p75(NTR)-dependent suppression of cell-cycle progression, we utilized transgenic clones of bladder tumor cells that express p75(NTR) in increasing concentrations to demonstrate an effect of p75(NTR) on the levels of cell-cycle regulatory proteins that modulate proliferation of tumor cells. A rank-order (dose-dependent) increase in p75(NTR) protein expression was associated with a decrease in cell proliferation. This p75(NTR)-dependent suppression of proliferation was rescued with NGF. In the absence of ligand, a dose-dependent increase in p75(NTR) protein expression was associated with reduced expression of cyclin D1, cyclin E, and cyclin-dependent kinase 2 (cdk2) as well as decreased cdk2 activity. There was also a decrease in the expression of hyper-phosphorylated retinoblastoma protein, the transcription factor E2F1, and proliferating cell nuclear antigen, and there was an increase in expression of hypophosphorylated Rb and the cdk inhibitor p16(Ink4a) with increasing p75(NTR) expression. Treatment of tumor cells with NGF ameliorated these p75(NTR)-dependent changes in the levels of cell-cycle regulatory proteins and rescued the tumor cells from p75(NTR)-dependent inhibition of proliferation. Hence, it can be concluded that p75(NTR) inhibits proliferation by altering the expression of cell-cycle regulatory proteins and that NGF ameliorates this effect.

Topics: CDC2-CDC28 Kinases; Cell Cycle; Cell Cycle Proteins; Cell Division; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinases; DNA-Binding Proteins; E2F Transcription Factors; E2F1 Transcription Factor; Epithelial Cells; Genes, Tumor Suppressor; Nerve Growth Factor; Proliferating Cell Nuclear Antigen; Protein Serine-Threonine Kinases; Receptor, Nerve Growth Factor; Receptors, Nerve Growth Factor; Retinoblastoma Protein; Transcription Factors; Tumor Cells, Cultured; Urinary Bladder Neoplasms

Expression of cyclin D1 is believed to lead to progression through the G1-S cell cycle checkpoint, and both experimental and pathological evidence suggest that over-expression of this protein may increase the risk of several cancers, including transition cell carcinoma of the bladder. Two major transcripts have been described for CCND1, the gene encoding cyclin D1. CCND1 870A-->G, a common single nucleotide polymorphism in the splice donor region of exon 4, may modulate expression of these transcripts, with the A variant resulting in an increased pool of the isozyme encoded by transcript form b. A statistically significant 1.8-fold increased risk for bladder cancer among individuals possessing the A/A genotype was recently reported in a hospital-based case-control study conducted among native Japanese. We conducted a population-based case-control study of incidence of bladder cancer among non-Hispanic whites in Los Angeles County to examine the relationship between CCND1 870A-->G genotypes and bladder cancer risk. No association between the A/A genotype and risk was observed (odds ratio = 0.90, 95% confidence interval 0.60-1.33). The null association was not appreciably modified by bladder cancer risk factors, including lifetime smoking history, or by histopathologic classification.

Topics: Adult; Case-Control Studies; Cyclin D1; Female; Genetic Predisposition to Disease; Humans; Male; Middle Aged; Polymerase Chain Reaction; Polymorphism, Single Nucleotide; Risk Factors; Urinary Bladder Neoplasms

Overexpression of the G1 cyclins, D1 and E, and/or downregulation of p27(Kip1) allow uncontrolled tumour cell proliferation. This study investigated the relation between these three cell cycle proteins and tumour proliferation in bladder cancer.. Nuclear expression of cyclin D1, cyclin E, and p27(Kip1) was determined immunohistochemically in 52 primary transitional cell carcinomas, and the Ki-67 proliferation marker was also assessed. For each protein, the percentage of positive tumour cell nuclei was determined and analysed as a continuous variable.. Advancing tumour grade and pathological stage were accompanied by increasing proliferation indices, but decreasing p27(Kip1) and cyclin D1 expression, with no significant change in cyclin E expression. Overall, cyclin D1 and E expression did not correlate with proliferation. However, in cyclin D1 overexpressing tumours (> or = 5% nuclei positive), the level of cyclin D1 expression positively correlated with proliferation. The correlation between cyclin E expression and proliferation changed from positive to negative with increasing levels of cyclin E expression, accompanied by a coordinate increase in p27(Kip1) expression. Overall, there was an inverse association between p27(Kip1) expression and proliferation. However, a subset of tumours displayed high proliferation indices despite high p27(Kip1) expression. The G1 cyclin index (sum of the level of expression of cyclins D1 and E) correlated positively with proliferation in superficial but not muscle invasive tumours. This correlation was stronger when the G1 cyclin index was adjusted for p27(Kip1) expression.. These findings support a role for these proteins in the proliferation, differentiation, and progression of bladder transitional cell carcinomas.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoma, Transitional Cell; Cell Cycle Proteins; Cell Division; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase Inhibitor p27; Female; Humans; Immunohistochemistry; Ki-67 Antigen; Male; Middle Aged; Neoplasm Staging; Statistics, Nonparametric; Tumor Suppressor Proteins; Urinary Bladder Neoplasms

Gene amplification is a common mechanism for oncogene overexpression. High-level amplifications at 11q13 have been repeatedly found in bladder cancer by comparative genomic hybridization (CGH) and other techniques. Putative candidate oncogenes located in this region are CCND1 (PRAD1, bcl-1), EMS1, FGF3 (Int-2), and FGF4 (hst1, hstf1). To evaluate the involvement of these genes in bladder cancer, a tissue microarray (TMA) containing 2317 samples was screened by fluorescence in situ hybridization (FISH). The frequency of gains and amplifications of all genes increased significantly from stage pTa to pT1-4 and from low to high grade. In addition, amplification was associated with patient survival and progression of pT1 tumours. Among 123 tumours with amplifications, 68.3% showed amplification of all four genes; 19.5% amplification of CCND1, FGF4, and FGF3; and 0.8% co-amplification of FGF4, FGF3, and EMS1. Amplification of CCND1 alone was found in 9% of the tumours, while EMS1 alone was amplified in 1.6% and FGF4 in 0.8%. Overall, the amplification frequency decreased with increasing genomic distance from CCND1, suggesting that, among the genes examined, CCND1 is the major target gene in the 11q13 amplicon in bladder cancer.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Carcinoma, Small Cell; Carcinoma, Squamous Cell; Carcinoma, Transitional Cell; Chromosomes, Human, Pair 11; Cortactin; Cyclin D1; DNA, Neoplasm; Female; Fibroblast Growth Factor 3; Fibroblast Growth Factor 4; Fibroblast Growth Factors; Gene Amplification; Genes, bcl-1; Humans; In Situ Hybridization, Fluorescence; Male; Microfilament Proteins; Middle Aged; Neoplasm Staging; Oligonucleotide Array Sequence Analysis; Oncogenes; Phenotype; Prognosis; Proto-Oncogene Proteins; Urinary Bladder Neoplasms

Detrusor muscle invasive transitional cell carcinoma is associated with poor prognosis and is responsible for the majority of bladder cancer related deaths. Amplifications of c-myc and CCND1 are associated with detrusor-muscle-invasive transitional cell carcinoma, however, their precise role in driving disease progression is unclear. Fluorescence in situ hybridisation on archival tissue from 16 patients with primary diagnosis of > or = pT2 transitional cell carcinoma and 15 cases with primary pTa/pT1 disease subsequently progressing to detrusor-muscle-invasion was performed, in the latter group both pre and post muscle invasive events were studied. No patients presenting with >/=pT2 had amplification of c-myc, two out of 16 (12.5%) had CCND1 amplification. Of patients who developed > or = pT2, two out of 15 (13.3%) had amplification of c-myc, both in > or = pT2, five out of 15 (33.3%) had CCND1 amplification, two in pTa/pT1 tumours, three in > or = pT2 transitional cell carcinomas. In total, two out of 31 (6.5%) of patients' > or = pT2 TCCs were amplified for c-myc and six out of 31 (19%) were amplified for CCND1. Eighty-seven per cent (40 out of 46) of tumours were polysomic for chromosome 8 and 80% (37 out of 46) were polysomic for chromosome 11 and this reflected the high copy numbers of c-myc and CCND1 observed. In almost all cases an increase in c-myc/CCND1 copy number occurred prior to invasion and persisted in advanced disease. Amplification of CCND1 or alterations in c-myc/CCND1 early in bladder cancer may have clinical relevance in promoting and predicting progression to detrusor-muscle-invasive transitional cell carcinoma.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Transitional Cell; Chromosome Aberrations; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 8; Cyclin D1; Disease Progression; DNA, Neoplasm; Female; Gene Amplification; Genes, myc; Humans; In Situ Hybridization, Fluorescence; Male; Middle Aged; Neoplasm Invasiveness; Neoplasm Recurrence, Local; Urinary Bladder Neoplasms

Gene p16 encodes a cyclin-dependent kinase inhibitor which functions to regulate cyclin D1, cell cycle progression and malignancies. The relationship between p16 and cyclin D1 is thought to alter bladder cancer formation and tumor progression. We aimed to investigate the expression of p16 and cyclin D1 genes in order to evaluate their clinical significance in bladder cancer.. Tissue samples from 67 patients with transitional cell carcinoma were examined with an immunohistochemical stain for the expression of p16 and cyclin D1 genes. The expression rate was compared to 12 normal urinary bladder mucosa samples obtained from transurethral surgery from noncancer patients.. The results revealed significant differences between normal bladder mucosa (100%) and cancer tissue (40.3%) for the positive staining of p16 protein (p < 0.001), while positive staining for the cyclin D1 protein in the patient group (67.2%) was significantly higher (p = 0.003) than that in the control group (16.7%). With the progression of tumor grade and clinical staging the positive rate of p16 gene expression increased, whereas, that of cyclin D1 decreased. Expression of the p16 gene in the non-invasive group was greater than that in the invasive group and a lower expression rate of the cyclin D1 gene in the non-invasive group compared to the invasive group.. The results revealed that expression of the p16 gene is inversely proportional to the expression of the cyclin D1 gene. Therefore, abnormal expression of the p16 and cyclin D1 genes play important roles in tumorigenesis and tumor progression.

Topics: Adult; Aged; Biomarkers, Tumor; Biopsy, Needle; Carcinoma, Transitional Cell; Case-Control Studies; Confidence Intervals; Culture Techniques; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Genes, p16; Humans; Immunohistochemistry; Male; Middle Aged; Neoplasm Staging; Probability; Prognosis; Reference Values; Sensitivity and Specificity; Urinary Bladder Neoplasms

We hypothesized that over expression of c-myc and cyclin D1 genes is transcriptionally activated by beta-catenin mutation independent of gene amplification in bladder cancer. To test this hypothesis we investigated the relationship of beta-catenin mutation to c-myc and cyclin D1 mRNA with special reference to the changes in copy number of the 2 genes.. Genomic DNA and total RNA were extracted from 59 bladder cancer specimens and from 31 histologically normal specimens of bladder mucosa. We performed beta-catenin deletion screening by polymerase chain reaction (PCR) using primers spanning exons 3 (including the glycogen synthase kinase-3beta consensus motif), 5 and 6. Mutational changes in beta-catenin in exons 3, 5 and 6 were detected by each PCR-single strand conformational polymorphism analysis followed by direct DNA sequencing. mRNA expression and copy numbers of c-myc and cyclin D1 were determined by semiquantitative reverse transcriptase-PCR and competitive genomic PCR.. Missense mutations of beta-catenin found in grade 3 bladder cancer were involved in the consensus motif of glycogen synthase kinase-3beta in exon 3. These cancers showed strong intracellular accumulation of beta-catenin and intense expression of c-myc and cyclin D1 mRNA compared with samples lacking the beta-catenin mutation. When grade 3 cancers were compared, expression levels of c-myc and cyclin D1 mRNA were still higher in those with versus without the beta-catenin mutation. In bladder cancers with beta-catenin mutations copy numbers of the c-myc and cyclin D1 genes did not amplify.. Bladder cancer harboring a beta-catenin mutation may represent aggressive biological behavior with enhanced proliferating activity. These findings are important for understanding the role of beta-catenin mutation in the pathogenesis of bladder cancer.

Topics: beta Catenin; Carcinoma, Transitional Cell; Cyclin D1; Cytoskeletal Proteins; DNA Mutational Analysis; Gene Expression Regulation, Neoplastic; Humans; Mutation, Missense; Neoplasm Staging; Proto-Oncogene Proteins c-myc; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Trans-Activators; Urinary Bladder; Urinary Bladder Neoplasms

Studies on solid cancer(such as breast cancer, hepatocellular cancer, pancreatic cancer) indicated that the abnormal expression of nuclear transcription factor Kappa B (NF-kappa B) regulates angiogenesis and cyclin-related genes. This study was designed to investigate the expression differences of NF-kappa B and its regulated genes between human primary transitional cell carcinoma(TCCs) of bladder and non-cancer bladder mucosa and its clinical significance.. Forty-three frozen sections including 30 bladder cancer and 13 non-cancer bladder mucosa were subjected to immunohistochemistry and nucleus staining for determining levels of NF-kappa B family and I kappa B alpha; Five paired cancer and non-cancer specimens were subjected to Western blot for analysis p65, an important subtype of NF-kappa B; Thirteen paired specimens were subjected to RT-PCR for determination mRNA levels of p50, p52, p65, c-Rel, RelB, I kappa B alpha, CyclinD1, IL-8.. Expressions of p50, p52, p65, c-Rel, RelB, I kappa B alpha, CyclinD1, IL-8 mRNAs in bladder cancer were higher than that in non-cancer bladder mucosa (P < 0.01, P < 0.05, P < 0.01, P < 0.01, P < 0.05, P < 0.05, P < 0.05 and P < 0.05, respectively). Nucelus stainings of p50, p52, p65, c-Rel, RelB were also stronger in bladder cancer(P < 0.01, P < 0.01, P < 0.01, P < 0.01 and P < 0.01, respectively). Moreover, p65 was expressed more in cancer tissue than that in non-cancer mucosa evidenced by Western blot. In bladder cancer, the ranking score differences of p52, p65, c-Rel protein between lymphatic metastasis group and non-lymphatic metastasis group were statistically significant (P < 0.01, P < 0.01, P < 0.05, respectively).. Compared to noncancer bladder mucosa, expressions of NF-kappa B family and its regulated genes in bladder cancer are markedly higher. NF-kappa B may be related to lymphatic metastasis.

Topics: Adult; Aged; Aged, 80 and over; Blotting, Western; Cyclin D1; Female; Humans; I-kappa B Proteins; Immunohistochemistry; Interleukin-8; Male; Middle Aged; NF-kappa B; NF-kappa B p50 Subunit; NF-KappaB Inhibitor alpha; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-rel; Reverse Transcriptase Polymerase Chain Reaction; Transcription Factor RelA; Transcription Factor RelB; Transcription Factors; Urinary Bladder Neoplasms

The modifying effects of dietary administration of a flavonoid antioxidant, silymarin, a mixture of three flavonoids isolated from milk thistle seeds, on N-butyl-N-(4-hydroxybutyl)nitrosamine (OH-BBN)-induced urinary bladder carcinogenesis were examined in male ICR mice. Animals were divided into 5 groups, and groups 1 to 3 were given OH-BBN (500 ppm) in drinking water for 6 weeks. Mice in group 2 were fed a diet containing 1000 ppm silymarin for 8 weeks during the initiation phase starting 1 week before OH-BBN exposure, and mice in group 3 were fed the diet for 24 weeks during the postinitiation phase. Animals in group 4 were given only the test compound, and those in group 5 were given the basal diet alone throughout the experiment. Animals were sacrificed at the end of week 32. The frequency of bladder lesions, cell proliferation and cell cycle progression activity estimated in terms of the 5-bromodeoxyuridine (BrdU) labeling index or cyclin D1-positive cell ratio were compared among the groups. Administration of silymarin in the initiation or postinitiation phase significantly decreased the incidences of bladder neoplasms and preneoplastic lesions. Dietary exposure to this agent significantly reduced the labeling index for BrdU and the cyclin D1-positive cell ratio in various bladder lesions. These findings suggest that silymarin is effective in preventing OH-BBN-induced bladder carcinogenesis in mice.

Topics: Administration, Oral; Animals; Anticarcinogenic Agents; Antioxidants; Bromodeoxyuridine; Butylhydroxybutylnitrosamine; Carcinogens; Carcinoma, Transitional Cell; Cell Cycle; Cyclin D1; Male; Mice; Mice, Inbred ICR; Papilloma; Precancerous Conditions; Silymarin; Urinary Bladder Neoplasms

Cyclin D1 contributes to regulate G1 progression by forming a complex with different cyclin-dependent kinases. It has oncogenic properties and is frequently overexpressed in several human tumor types. In our study, expression of cyclin D1 and Ki67, a proliferation marker, was evaluated by immunohistochemistry in human papillary superficial (pTa-pT1) bladder cancers and was correlated with p27(Kip1), p21(Waf1) and c-erbB-2 expression, with p53 gene status and protein expression, ploidy and cancer progression. Cyclin D1 expression was neither associated with tumor stage nor with tumor grade but high cyclin D1 expression (> or =25% positive nuclei) was significantly associated with p53 gene mutation (p = 0.012), low p21(Waf1) (p = 0.015) and high p27(Kip1) (p = 0.016) protein expression. Ki67 expression was not associated with tumor stage but a high proliferation index (> or =10% positive nuclei) was significantly associated with high tumor grade (p = 0.001) and with DNA aneuploidy (p = 0.005). There was no significant difference in proliferative activity between high and low cyclin D1 expressor tumors. Patients whose tumors showed high expression of cyclin D1 displayed a significantly longer disease-free survival (p < 0.001 by log-rank test). Increased Ki67 expression was significantly associated with shorter disease-free survival (p = 0.003). Both cyclin D1 (p = 0.027; RR = 1.898) and Ki67 (p = 0.047; RR = 1.932) protein expressions were independent predictors of reduced disease-free survival on a multivariate analysis that also included p27(Kip1) expression and tumor stage. The simultaneous presence of low cyclin D1, low p27(Kip1) and high Ki67 expression defined a "high-risk" group of patients who displayed a significantly increased risk of recurrence (p < 0.0001). These results suggest that evaluation of cell cycle-associated markers can help to identify high-risk patients and may affect the management of patients with papillary superficial bladder cancer.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoma, Papillary; Cell Cycle Proteins; Cell Division; Cohort Studies; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Disease Progression; Disease-Free Survival; Female; Follow-Up Studies; Humans; Immunohistochemistry; Ki-67 Antigen; Male; Middle Aged; Ploidies; Predictive Value of Tests; Prognosis; Receptor, ErbB-2; Risk Assessment; Tumor Suppressor Proteins; Urinary Bladder Neoplasms

Cyclin D1 is believed to play an important role in the genesis and/or progression of transitional cell cancer (TCC) of the urinary bladder. Cyclin D1 gene (CCND1) mRNA is alternatively spliced to produce two transcripts, and the splicing pattern may be modulated by a G to A single nucleotide polymorphism within the splice donor site of exon 4. This study was conducted to explore the association between the polymorphism and the susceptibility to and disease status of TCC of the bladder in 222 cases and 317 native Japanese controls. The relationship between the CCND1 polymorphism and the mRNA splicing pattern in TCC cells was evaluated by semi-quantitative reverse-transcription PCR. The CCND1 A allele was more frequently observed in the TCC group than the control group (P = 0.032) with a significant difference in the genotype frequency between the two groups (P = 0.029). The AA genotype was associated with a significantly higher risk of TCC compared with the AG+GG genotypes (adjusted odds ratio (aOR) = 1.76, 95% confidence interval (CI) = 1.09-2.84, P = 0.022). This association was observed more significantly in nonsmoking cases (aOR = 2.53; 95% CI = 1.28-4.51, P = 0.008). Looking at tumor grade, the presence of the A allele was associated with higher grade (= grade 3) tumors with a gene dosage effect (aOR = 1.77, CI = 1.16-2.69, P = 0.008). In tumor stage, although not significant, the AA + AG genotypes tended to be more frequently observed in cases with T1-4 tumors than those with Ta tumors (aOR = 1.94, 95% CI = 0.98-3.82, P = 0.057). The genotype seemed to influence the two alternatively spliced forms of the CCND1 mRNA because the ratio of the CCND1 transcript-b/transcript-a was significantly higher in cases with the AA genotype compared with those with the AG + GG genotypes. These data suggest that the CCND1 variant A allele may be associated with an increased risk of TCC of the bladder, especially in men without a history of smoking, and it may also have an effect on its disease status.

Topics: Age Factors; Aged; Alleles; Alternative Splicing; Carcinoma, Transitional Cell; Case-Control Studies; Cyclin D1; Female; Gene Dosage; Genetic Predisposition to Disease; Genotype; Humans; Male; Middle Aged; Odds Ratio; Polymerase Chain Reaction; Polymorphism, Genetic; Reverse Transcriptase Polymerase Chain Reaction; Risk; RNA, Messenger; Sex Factors; Smoking; Urinary Bladder Neoplasms

Normal cell proliferation is closely regulated by proteins called cyclins. One of these, cyclin D1, in combination with its corresponding cyclin-dependent kinase (cdk), is essential for G(1)/S phase transition. Cyclin/cdk complexes are generally inhibited by cyclin-dependent kinase inhibitors(ckis), some of which are induced by wild-type p53. The aims of this study were: to investigate levels of cyclin D1 expression in transitional cell carcinoma (TCC) of the bladder; to correlate these results with data concerning the expression of p53, waf1, pRb and Ki67; and to determine whether cyclin D1 expression could predict clinical outcome. Paraffin-sections from 150 newly diagnosed bladder tumours (Ta/T1 = 97; T2-T4 = 53) were stained for cyclin D1 using immunohistochemistry and a cyclin D1 index assigned. These results were correlated with data relating to the expression of p53 and waf1 by the same tumours. A representative subset of 54 tumours (Ta/T1 = 28; T2-T4 = 26) was also stained for Ki67 and 55 were stained for pRb. The clinical course of each patient was recorded and multivariate analyses of risk factors for tumour recurrence, stage progression and overall survival were performed. Positive staining for cyclin D1 was found in 83% of tumours. The staining pattern varied between tumours with nuclear, cytoplasmic or a combination of the two evident in different tumours. 89% of Ta/T1 and 74% of T2-T4 tumours showed nuclear staining with or without cytoplasmic staining. The median value for cyclin D1 staining was significantly higher in Ta/T1 tumours (41%) compared with T2-T4 tumours (8%, P< 0.005) with 26% of muscle-invasive tumours demonstrating absent staining. In addition, the median value for cyclin D1 staining was significantly higher in G1/G2 tumours (43%) compared with G3 tumours (14%, P< 0.005). There was a significant positive correlation between expression of cyclin D1 and waf1 expression (P< 0.0001) as well as pRb expression but not between cyclin D1 expression and expression of p53. Ki67 expression was significantly associated with increasing tumour stage (P< 0.005) and histological grade (P< 0.05) but did not correlate with cyclin D1 expression. A cyclin D1 index > or = 8% was associated with significantly better survival in those patients with muscle-invasive disease (T2-T4). In addition, there was a significantly higher progression rate for those patients with Ta/T1 disease whose tumours demonstrated cytoplasmic cyclin D1 staining. These resu

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Transitional Cell; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Disease Progression; Female; Humans; Immunohistochemistry; Ki-67 Antigen; Male; Middle Aged; Neoplasm Recurrence, Local; Neoplasm Staging; Predictive Value of Tests; Retinoblastoma Protein; Survival Analysis; Tumor Suppressor Protein p53; Urinary Bladder Neoplasms

To determine whether the apoptotic index, the Ki67 index, and the expression of the p53, cyclin D1, and retinoblastoma genes correlate with local control, overall survival, and time to distant metastases in invasive bladder cancer treated with external beam radiation.. Paraffin-embedded pretreatment biopsies from 83 patients with invasive transitional cell carcinoma of the bladder were scored morphologically for apoptosis and immunohistochemically for Ki67, p53, cyclin D1, and retinoblastoma gene expression. Survival analysis methods were used to assess overall survival, local control, and freedom from distant metastases. A multiple proportional hazard (PH) regression analysis was performed to study the prognostic value of the abovementioned biologic parameters (all divided into two categories, except Ki67) in addition to classical prognostic factors such as T stage, histologic grade, multifocality of the tumor, and completeness of transurethral resection. All patients were treated with external beam radiation as sole treatment. Median follow-up for the 19 patients still living was 7.5 years.. Apoptotic index varied from 0% to 3.4% with a mean of 0.8% and a median of 0.6%. Ki67 index varied from 0% to 60% with a mean of 14% and a median of 12%. P53 protein was detectable in 61% of the tumors. Overexpression of cyclin D1 was observed in 39% of the tumors and loss of retinoblastoma protein in 23% of the tumors. High Ki67 index was found to be significantly associated with p53 expression (p = 0.04) and cyclin D1 overexpression (p = 0.023). Cyclin D1 overexpression was found more often in Rb-positive tumors than in Rb-negative tumors (p = 0.006). Other associations between the markers are less clear. Biologic markers were not correlated with T stage or grade. In the PH analysis local control was found to be significantly better for tumors with wild-type p53 (p = 0.028). Also, tumors with an apoptotic index above the median value (0.6%) had a significantly better local control rate (p = 0.035). Ki67 index (p = 0.35), retinoblastoma gene expression (p = 0.30) and cyclin D1 overexpression (p = 0.61) were not found to have an additional predictive value regarding local tumor control. None of the tested biologic parameters were found to be associated with overall survival. Time to distant metastases was significantly shorter for tumors with high Ki67 index (p = 0.01) and tumors with an apoptotic index less than median (p = 0.009).. The results of our study provide evidence for a prognostic value of p53 expression and apoptotic index with respect to the radiation response in bladder cancer in addition to more conventional prognosticators. The value of these parameters as a predictive assay for radiation response warrants confirmation in larger and prospective studies.

Topics: Adult; Aged; Aged, 80 and over; Analysis of Variance; Apoptosis; Carcinoma, Transitional Cell; Cyclin D1; Female; Humans; Ki-67 Antigen; Male; Middle Aged; Neoplasm Proteins; Neoplasm Staging; Retinoblastoma Protein; Time Factors; Tumor Suppressor Protein p53; Urinary Bladder Neoplasms

Second nonocular malignancies develop with increased incidence in patients with hereditary retinoblastoma. Osteosarcoma is by far the most common type with an incidence of up to 50%, followed by soft tissue sarcomas. Visceral leiomyosarcoma is extremely rare and only 2 cases have been reported in the past 2 decades, one in the liver and another one in the urinary bladder, both of which developed after cyclophosphamide therapy. Here we report a case of vesical leiomyosarcoma that was diagnosed in a 49-year-old woman 47 years after the diagnosis of a hereditary retinoblastoma. The patient's retinoblastoma was treated with unilateral enucleation without adjuvant radiation or chemotherapy. We believe that this is the first report of vesical leiomyosarcoma occurring in a patient with retinoblastoma without a prior history of radiation or chemotherapy. This report is significant not only because of the rarity of vesical leiomyosarcoma as a second nonocular tumor in retinoblastoma patients, but also because of the infrequency of vesical leiomyosarcoma in general. We also investigated the potential molecular pathogenesis of the leiomyosarcoma.

Topics: Actins; Adult; Cyclin D1; Cystectomy; Desmin; Eye Neoplasms; Female; Hematuria; Humans; Hysterectomy; Leiomyosarcoma; Neoplasms, Second Primary; Ovariectomy; Retinoblastoma; Retinoblastoma Protein; Survivors; Tomography, X-Ray Computed; Tumor Suppressor Protein p53; Urinary Bladder Neoplasms

Alteration in cell cycle regulators is considered to play an important role in carcinogenesis. In order to cast light on changes in reversible hyperplastic and irreversible tumorigenic lesions in the rat urinary bladder, expression of p27(Kip1), cyclin D1 and cyclin E proteins was sequentially compared. In the first study, 3% uracil was fed for 4 weeks to cause urinary calculi and consequent hyperplasia and papillomatosis, both regressing after withdrawal of the insult. Compared with normal bladder epithelium, in papillomatosis at week 4, the BrdU index and immunohistochemical positivities for cyclin D1 and cyclin E were significantly elevated, whereas values for p27(Kip1) tended to be reduced. One week after withdrawal of uracil, the BrdU index and positivities for cyclin D1 and cyclin E were decreased to below the control levels, while positivity for p27(Kip1) was dramatically increased, with a strong staining intensity. In a second study, rats were initiated with a bladder carcinogen, N-butyl-N-(4-hydroxybutyl)nitrosamine for 4 weeks, then fed 3% uracil for 8 weeks. During this latter period, expression of cyclin D1, cyclin E and p27(Kip1) in hyperplastic urothelium were comparable with those in the first study. One week after withdrawal of uracil, most urothelial lesions regressed, showing high p27(Kip1) and low cyclin D1 and cyclin E staining. Two weeks after uracil withdrawal, transitional cell carcinomas, with a low p27(Kip1) and high cyclin D1 and cyclin E staining pattern, could be easily distinguished from surrounding regressing epithelium. These data indicate that during regression of papillomatosis after cessation of a proliferative stimulus, expression of p27(Kip1)is elevated, accompanied by a lowering of cyclin D1 and cyclin E. In irreversible tumorous bladder lesions, on the other hand, persistent low expression of p27(Kip1) and elevated cyclin D1 and cyclin E are characteristic.

Topics: Animals; Bromodeoxyuridine; Butylhydroxybutylnitrosamine; Cell Cycle Proteins; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase Inhibitor p27; Male; Microtubule-Associated Proteins; Papilloma; Rats; Rats, Inbred F344; Tumor Suppressor Proteins; Uracil; Urinary Bladder Calculi; Urinary Bladder Neoplasms

Overexpression of ornithine decarboxylase (ODC) has been shown to be characteristic of tumor development and progression in humans and experimental animals. Therefore, we have examined the effects of 1, 3-diaminopropane dihydrochloride (DAP), a potent inhibitor of ODC, on rat two-stage urinary bladder carcinogenesis initiated with N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN). In experiment 1 (36 weeks), 6-week-old F344 male rats were administered 0.05% BBN in drinking water for 4 weeks and then divided into four groups. Animals of groups 1 and 2 received basal diet and drinking water supplemented with or without DAP (2 g/l). Groups 3 and 4 were given diet containing 5% sodium L-ascorbate (NaAsA), a typical urinary bladder tumor promoter, and drinking water with or without DAP. Administration of DAP to group 1 significantly reduced tumor size, multiplicity and incidence, particularly of papillomas, when compared with group 2 values. DAP together with NaAsA (group 3) also decreased tumor size relative to the group 4 case. To determine the effects of DAP on the early stages of bladder carcinogenesis and its mechanisms, a similar protocol was conducted (experiment 2) with death after 20 weeks. DAP treatment caused complete inhibition (0% incidence) of papillary and/or nodular hyperplasia in group 1 but was without influence in group 3, as compared with the respective controls. Moreover, the ODC activity, bromodeoxyuridine labeling indices and mRNA expression levels of cyclin D1 in the urinary bladder mucosa, determined by northern blotting, were markedly lower in group 1 than in group 2, but values were comparable for both groups administered NaAsA. Assessment of mRNA expression levels of the angiogenic vascular endothelial growth factor suggested no involvement in the inhibitory effects of DAP on urinary bladder carcinogenesis. The results indicate that inhibition of ODC could reduce urinary bladder carcinogenesis in rats, particularly in the early stages, through antiproliferative mechanisms.

Topics: Acetyltransferases; Animals; Anticarcinogenic Agents; Apoptosis; Ascorbic Acid; Butylhydroxybutylnitrosamine; Carcinogens; Carcinoma; Cocarcinogenesis; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases; Diamines; Endothelial Growth Factors; Hydrogen-Ion Concentration; Hyperplasia; Lymphokines; Male; Ornithine Decarboxylase; Ornithine Decarboxylase Inhibitors; Papilloma; Polyamines; Proto-Oncogene Proteins; Rats; Rats, Inbred F344; RNA, Messenger; Urinary Bladder; Urinary Bladder Neoplasms; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Effects of a genotoxic bladder carcinogen, N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) and a non-genotoxic bladder promoter, sodium L-ascorbate (Na-AsA), on protein expression, cell proliferation and apoptosis of the bladder epithelium with or without the influence of testicular castration were investigated. Male F344 rats were divided into six groups (groups 1-6). BBN was given with 0.05% drinking water to groups 1 and 4 for 8 weeks, groups 2 and 5 received diet with 5% Na-AsA. Then the animals were treated without any chemicals. Groups 3 and 6 were non-treated controls. Testicular castration was carried out 2 weeks before commencement of chemical treatment on groups 4-6. The total observation period was 18 weeks. Overexpression of cyclin D1 was induced by BBN but not Na-AsA and the degree of overexpression was higher in the order simple hyperplasia, papillary or nodular hyperplasia, papilloma and carcinoma. Metallothionein (MT) was also overexpressed in bladder epithelium treated with BBN but not Na-AsA, but was decreased in papillomas and never found in a carcinoma. Cyclin D1-positive cells were essentially MT-negative. Therefore, it is speculated that MT protects genes from insult by genotoxic carcinogens and its lack is associated with tumor development. Apoptotic cell death occurred during treatment with BBN and Na-AsA and after their withdrawal. Chromatin condensation of many G0/G(1) cells was particularly marked on flow cytometry analysis 1 week after cessation of treatment, this being considered as an early apoptotic change. Although testicular castration had no influence on the above events, it resulted in decreased tumor formation as compared with the case of similarly treated intact animals. Our data demonstrate that overexpression of MT and cyclin D1 is specific for treatment with a genotoxic carcinogen, and suggest that MT overexpression may play an important suppressive role in the early stages of rat urinary bladder carcinogenesis.

Topics: Animals; Apoptosis; Ascorbic Acid; Butylhydroxybutylnitrosamine; Cyclin D1; DNA, Neoplasm; Flow Cytometry; Male; Metallothionein; Microscopy, Electron; Rats; Rats, Inbred F344; Urinary Bladder Neoplasms

Immunoreactivity of p21WAF1/CIP1 and cyclin D1 proteins was assessed in a cohort of 207 patients with superficial (pTa-pT1) bladder cancer followed up for a mean of 4.9 years. The results of the immunostainings were compared with T category, WHO grade, tumor cell proliferation rate (MIB-1 score), the expressions of p53 and bcl-2 as well as survival. Sixty-eight percent and 75% of the tumors were p21WAF1/CIP1 positive (> or = 5% of cells positive) and cyclin D1 positive (> or = 10% of cells positive), respectively. The p21WAF1/CIP1 expression was related to cyclin D1 immunolabelling (P < 0.001) but not to the other variables studied. The expression of cyclin D1 was inversely associated with T category (P = 0.001), WHO grade (P = 0.006), MIB-1 score (P = 0.014), p53 expression (P = 0.001), and bcl-2 (P = 0.011) immunoreactivity. In univariate analysis, T category (P = 0.0001), WHO grade (P < 0.0001), MIB-1 score (P < 0.0001), bcl-2 (P = 0.0092), p53 (P = 0.0016) and p21WAF1/CIP1 (P = 0.009) expressions were significant prognostic factors with regard to tumor progression, whereas cyclin D1 was without any prognostic significance (P = 0.1). Out of 123 p21 positive tumors 21 progressed, whereas only 2 out of 58 p21 negative tumors progressed. In multivariate analysis, the MIB-1 score was the only independent predictor of cancer-specific survival (P = 0.03), whereas tumor grade (P = 0.002) and cyclin D1 expression (P = 0.04) were independent predictors of tumor recurrence. Only the WHO grade (P = 0.04) retained its prognostic value indicating the risk of progression. We suggest that in superficial bladder cancer p21WAF1/CIP1 and cyclin D1 immunohistochemistry provide no additional prognostic information compared with already established prognostic factors for predicting the risk of progressive disease.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Transitional Cell; Cell Division; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Disease Progression; Female; Humans; Male; Middle Aged; Neoplasm Recurrence, Local; Neoplasm Staging; Prognosis; Randomized Controlled Trials as Topic; Urinary Bladder Neoplasms

Cyclin D1 is essential for G1 progression through the cell cycle phase. It is a possible proto-oncogene whose aberrant expression may be responsible for the occurrence of some types of human neoplasms. The objective of the present study was to demonstrate immunohistochemically cyclin D1 expression in bladder cancer tissues and establish any relationship with the histologic findings and the clinical course.. Tissue from 102 patients with bladder cancers and bladder tissue from five normal subjects were used for an immunohistochemical study of cyclin D1 using the avidin-biotin complex method.. Nuclear staining of cyclin D1 was found in 79 (77%) out of the 102 cases of bladder cancer. The five cases of normal epithelium had no immunostaining for cyclin D1. All grade 1 tumors were positive for cyclin D1. With the advance of tumor grade the incidence of cyclin D1 decreased. All pTa tumors stained positively for cyclin D1, whereas the positive staining rates of invasive tumors were 47% in pT1, 73% in pT2, 31% in pT3 and 0% in pT4 tumors. Although a univariate analysis revealed patients with lesions positive to cyclin D1 had more favorable survival rates than those with negative findings, a multivariate analysis showed that positivity for cyclin D1 is not an independent prognostic factor. No relationship was discovered between positivity for cyclin D1 and tumor recurrence in patients with superficial bladder cancers.. These findings suggest that cyclin D1 demonstrated immunohistochemically could be used as an inverse indicator for the level of invasiveness of bladder cancer, but not as an independent prognostic factor.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoma, Transitional Cell; Cyclin D1; Female; Humans; Immunoenzyme Techniques; Male; Middle Aged; Neoplasm Invasiveness; Prognosis; Proto-Oncogene Mas; Survival Rate; Urinary Bladder Neoplasms

During the 11-year period subsequent to the Chernobyl accident, the incidence of urinary bladder cancer in Ukraine has increased from 26.2 to 36.1 per 100,000 population. Cesium-137 (137Cs) accounts for 80-90% of the incorporated radioactivity in this population, which has been exposed to long-term, low-dose ionizing radiation, and 80% of the more labile pool of cesium is excreted via the urine. The present study was performed to evaluate the histopathological features and the immunohistochemical status of p53, p21WAF1/Cip1, cyclin D1 and PCNA (proliferating cell nuclear antigen) in urinary bladder mucosa of 55 males (49-92 years old) with benign prostatic hyperplasia who underwent surgery in Kiev, Ukraine, in 1995 and 1996. Group I (28 patients) inhabiting radiocontaminated areas of the country, group II (17 patients) from Kiev city with less radiocontamination and a control group III (10 patients) living in so-called "clean" areas of Ukraine were compared. In groups I and II, an increase in multiple areas of moderate or severe dysplasia or carcinoma in situ was seen in 42 (93%) of 45 cases. In addition, two small transitional cell carcinomas were found in one patient in each of groups I and II. Nuclear accumulation of p53, PCNA, cyclin D1, and to a lesser extent p21WAF1/Cip1, was significantly increased in both groups I and II as compared with the control group III, indicating possible transformation events or enhancement of repair activities, that may precede the defect in the regulatory pathway itself, at least in the G1 phase of the cell cycle. Our results suggest that early malignant transformation is taking place in the bladder urothelium of people in the radiocontaminated areas of Ukraine and that this could possibly lead sometime in the future to an increased incidence of urinary bladder cancer.

Topics: Aged; Aged, 80 and over; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Humans; Immunohistochemistry; Male; Middle Aged; Nuclear Reactors; Power Plants; Proliferating Cell Nuclear Antigen; Radioactive Hazard Release; Tumor Suppressor Protein p53; Ukraine; Urinary Bladder; Urinary Bladder Neoplasms

The biological behaviour of urinary bladder neoplasms cannot be adequately predicted by histological criteria alone. Cyclin D1 is a cell-cycle regulating protein known to be overexpressed in a proportion of bladder carcinomas. To evaluate the prognostic significance of cyclin D1 expression and its relationship with tumour phenotype, 392 bladder carcinomas were analysed by immunohistochemistry. Clinical follow-up information was available in 337 patients with superficial bladder tumours (stages pTa/pT1). Cyclin D1 positivity was seen in 176 of 392 carcinomas. Cyclin D1 overexpression was strongly linked to papillary tumour growth, low stage, and low histological grade (p<0.005 each). Multivariate analysis showed that papillary tumour growth was the only parameter which was independently linked to cyclin D1 positivity. There was no significant difference in proliferative activity (Ki67 labelling index) between cyclin D1-negative and -positive tumours. Cyclin D1 positivity was not linked to the risk of recurrence or tumour progression, either in pTa or in pT1 carcinomas. It is concluded that cyclin D1 positivity distinguishes a large subgroup of papillary bladder tumours, but there is no evidence of prognostic significance for increased cyclin D1 expression.

Topics: Analysis of Variance; Biomarkers, Tumor; Carcinoma; Cell Division; Chi-Square Distribution; Cyclin D1; Humans; Immunohistochemistry; Neoplasm Staging; Prognosis; Urinary Bladder Neoplasms

The present study examined the expression and role of the thiazolidinedione (TZD)-activated transcription factor, peroxisome proliferator-activated receptor gamma (PPARgamma), in human bladder cancers. In situ hybridization shows that PPARgamma mRNA is highly expressed in all human transitional epithelial cell cancers (TCCa's) studied (n=11). PPARgamma was also expressed in five TCCa cell lines as determined by RNase protection assays and immunoblot. Retinoid X receptor alpha (RXRalpha), a 9-cis-retinoic acid stimulated (9-cis-RA) heterodimeric partner of PPARgamma, was also co-expressed in all TCCa tissues and cell lines. Treatment of the T24 bladder cancer cells with the TZD PPARgamma agonist troglitazone, dramatically inhibited 3H-thymidine incorporation and induced cell death. Addition of the RXRalpha ligands, 9-cis-RA or LG100268, sensitized T24 bladder cancer cells to the lethal effect of troglitazone and two other PPAR- activators, ciglitazone and 15-deoxy-delta(12,14)-PGJ2 (15dPGJ(2)). Troglitazone treatment increased expression of two cyclin-dependent kinase inhibitors, p21(WAF1/CIP1) and p16(INK4), and reduced cyclin D1 expression, consistent with G1 arrest. Troglitazone also induced an endogenous PPARgamma target gene in T24 cells, adipocyte-type fatty acid binding protein (A-FABP), the expression of which correlates with bladder cancer differentiation. In situ hybridization shows that A-FABP expression is localized to normal uroepithelial cells as well as some TCCa's. Taken together, these results demonstrate that PPARgamma is expressed in human TCCa where it may play a role in regulating TCCa differentiation and survival, thereby providing a potential target for therapy of uroepithelial cancers.

Topics: Alitretinoin; Antineoplastic Agents; Apoptosis; Carcinoma, Transitional Cell; Carrier Proteins; Cell Death; Chromans; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; DNA; DNA, Complementary; Dose-Response Relationship, Drug; Fatty Acid-Binding Protein 7; Fatty Acid-Binding Proteins; G1 Phase; Humans; Immunoblotting; In Situ Hybridization; Ligands; Luciferases; Myelin P2 Protein; Neoplasm Proteins; Nicotinic Acids; Receptors, Cytoplasmic and Nuclear; Receptors, Retinoic Acid; Retinoid X Receptors; Ribonucleases; Tetrahydronaphthalenes; Thiazoles; Thiazolidinediones; Transcription Factors; Transcriptional Activation; Transfection; Tretinoin; Troglitazone; Tumor Cells, Cultured; Tumor Suppressor Proteins; Urinary Bladder Neoplasms

We attempted to clarify the significance of cyclin D1 in the development and progression of transitional cell carcinoma of the bladder in humans.. Immunohistochemical staining of archival tissue specimens of transitional cell bladder carcinoma obtained from 163 patients was performed by the labeled streptavidin-biotin-peroxidase method.. Cyclin D1 protein overexpression was observed in 51 of the 163 specimens (31.3%). Cyclin D1 protein overexpression was showed a highly significant inverse correlation with the histological grade of malignancy (p < 0.01). Grade 3 tumors showed a highly significant low incidence of cyclin D1 protein overexpression as compared with grade 2 tumors (p < 0.01). There was no significant correlation between the overexpression of cyclin D1 protein and tumor stage or the Ki-67 labeling index.. Cyclin D1 in transitional cell bladder carcinoma was closely related to tumor differentiation but not to tumor progression. Transitional cell carcinoma of the bladder may utilize another pathway for proliferation that is independent of cyclin D1.

Topics: Carcinoma, Transitional Cell; Cell Division; Cyclin D1; Disease Progression; Gene Expression Regulation, Neoplastic; Humans; Ki-67 Antigen; Urinary Bladder Neoplasms

Growth of cancer cells is characterized by accelerated passage through the cell cycle, which is often caused by deregulation of the G1-->S transition. In this study the expression of G1-->S transition regulatory molecules was analyzed in 32 transitional cell carcinoma specimens and fifteen normal tissues obtained by cystectomy or nephroureterectomy of mainly locally advanced tumors, as well as six bladder cancer cell lines. Expression of mRNAs for cyclins D1 and D2 and cyclin-dependent kinases (CDK) 2 and 4 was investigated by quantitative reverse transcription-polymerase chain reaction. Overexpression of cyclin D1 compared to normal mucosa was observed in 3 tumors (9.4%), but in neither of the cell lines. All tumors with overexpression were moderately differentiated (G2) pT1 or pT2 tumors, and thus among the less advanced specimens. Cyclin D2 was not expressed in normal bladder mucosa or in tumors. The expression of CDK4 mRNA varied within the same range in mucosa, tumors, and cell lines. CDK2 mRNA expression varied more strongly and was diminished in individual tumors and in four cell lines. It is concluded that cyclin D1 overexpression can play an important role in the early stage of urothelial tumorigenesis, driving cell proliferation. Ectopic expression of cyclin D2 or amplification of CDK4 does not occur at a significant frequency in urothelial carcinomas. Different expression patterns of cyclin D1 and CDK2 indicate heterogeneity in the mechanisms of G1-->S transition deregulation in individual bladder tumors which may elicit differences in their biological and clinical behavior.

Topics: Aged; Aged, 80 and over; Carcinoma, Transitional Cell; CDC2-CDC28 Kinases; Cyclin D1; Cyclin D2; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases; Cyclins; Female; G1 Phase; Humans; Male; Middle Aged; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; RNA, Messenger; S Phase; Tumor Cells, Cultured; Urinary Bladder Neoplasms

Genetic alterations leading to neoplastic transformation of the urothelium are likely to involve the activation of oncogenes and loss of functional tumor suppressor genes. Cyclin D1 has been implicated as a putative protooncogene whereas mutations of the p53 gene occur frequently in invasive transitional cell carcinomas (TCCs) of the urinary bladder. In this study, cyclin D1 overexpression and nuclear accumulation of p53 were evaluated and the results correlated with histopathologic features.. TCCs of the urinary bladder from 161 surgical procedures were evaluated for cyclin D1 overexpression and nuclear accumulation of p53. Results were correlated with tumor grade, T classification, and papillary status. Topologic distributions of cyclin D1, p53, and proliferating cellular nuclear antigen (PCNA) were evaluated. Northern blot analysis was performed on selected specimens.. Overexpression of cyclin D1 was observed in 47% (24 of 51) of Grade 1 TCCs and 20% (13 of 65) of Grade 2 TCCs but in no Grade 3 TCCs. Approximately 34% (14 of 41) of Ta classified TCCs and 21% (13 of 63) of T1 classified TCCs were immunoreactive for cyclin D1 whereas none of the TCCs beyond T1 was immunoreactive. Overexpression of cyclin D1 was observed only in papillary type TCCs. Results of Northern blot analysis for cyclin D1 were comparable to those of immunohistochemistry.. The observed significant relation between cyclin D1 overexpression and tumor grade/T classification suggests that cyclin D1 may be a useful biologic marker for biopsied materials or urine cytology specimens. The prognostic significance of cyclin D1 overexpression in TCCs remains to be determined.

Topics: Adult; Aged; Aged, 80 and over; Blotting, Northern; Carcinoma, Transitional Cell; Cell Nucleus; Cyclin D1; Cyclins; Female; Humans; Immunohistochemistry; Male; Middle Aged; Neoplasm Proteins; Oncogene Proteins; Proliferating Cell Nuclear Antigen; Tumor Suppressor Protein p53; Urinary Bladder; Urinary Bladder Neoplasms

Cyclin D1 is a cell cycle regulator essential for G1 phase progression and is frequently overexpressed in several human tumour types as a consequence of gene amplification or chromosomal rearrangements. We analysed the expression of cyclin D1 in 75 patients with transitional cell carcinoma (TCC) to investigate the possible relationship between its expression and clinical outcome as well as histopathological findings using the immunohistochemical method. We observed strong staining (++, > 50% positive cells) for cyclin D1 in 19 cases (25.3%) and weak staining (+, 5-50% positive cells) in 19 cases (25.3%). Overexpression of cyclin D1 was not associated with tumour invasion. No significant association was found between overexpression of cyclin D1 and tumour grade (P > 0.05). We assessed the differences of disease-free interval in superficial tumours and actuarial survival probability in invasive tumours according to the status of cyclin D1 expression. Tumours with (++) staining for cyclin D1 recurred much more rapidly than (-) and/or (+) staining tumours (P < 0.01 for - vs ++; P < 0.05 for + vs ++). However, overexpression of cyclin D1 was not associated with a shortened overall survival of patients with invasive tumours (P < 0.1). These results suggest that genetic alteration of cyclin D1 appears to be an early event in the tumorigenesis of bladder TCC and is associated with early recurrence in superficial tumours.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoma, Transitional Cell; Cyclin D1; Cyclins; Disease-Free Survival; Gene Expression; Humans; Immunohistochemistry; Middle Aged; Neoplasm Recurrence, Local; Oncogene Proteins; Retrospective Studies; Time Factors; Urinary Bladder Neoplasms

Apoptosis is a morphologically and biochemically distinct form of cell death which determines specific patterns of tissue size and shape and balances cell proliferation. In the present study, the sequence of cellular proliferative alterations in urinary bladder epithelium associated with uracil-induced reversible urinary calculi was investigated in male F344 rats. Group 1 consisted of 45 rats, 6 weeks old at commencement of the experiment, which were given a diet containing 3% uracil for 8 weeks and were then returned to basal diet until week 20. Five rats were killed at each of weeks 2, 4, 8, 9, 10, 11, 12, 14 and 20. Group 2 consisted of 15 rats which were given basal diet for 20 weeks. Five rats were killed at each of weeks 0, 8 and 20. Microscopic, reversible papillomatosis, which showed papillary projections of epithelial proliferation, was seen in the urinary bladder of all rats in group 1 through week 8. No epithelial lesions were apparent in any of rats in group 2. Anti-Le(y)(BM-1/JIMRO)-positive areas of the urinary bladder epithelia were immunohistochemically seen in all rats of group 1 at weeks 2-12. At week 9 the percentage of anti-Le(y)-positive areas reached a maximum. Nick-end labeling stained nuclei of cells in the urinary bladder epithelium were observed in all rats of group 1 at weeks 4-14. At week 10 the labeling index was at a maximum. Proliferating cell nuclear antigen (PCNA)- and cyclin D1-positive cells of the urinary bladder epithelium were observed in group 1 at weeks 2, 4 and 8, however, at week 9 there were no PCNA- and cyclin D1-positive cells. In urinary bladder papillomatosis the simultaneous existence of apoptotic cells and proliferating cells was shown by double staining with anti-Le(y) (BM-1/JIMRO) and for PCNA. At week 10 apoptosis, stained by BM-1 and nick-end labeling, occurred extensively in regressing urinary bladder papillomatosis. Agarose gel electrophoresis of DNA in regressing papillomatosis at week 9 showed DNA fragmentation. Thus, these results indicate that apoptosis occurs in the process of papilloma regression following withdrawal of uracil treatment.

Topics: Animals; Apoptosis; Cyclin D1; Cyclins; DNA Fragmentation; Immunohistochemistry; Lewis Blood Group Antigens; Male; Oncogene Proteins; Papilloma; Proliferating Cell Nuclear Antigen; Rats; Rats, Inbred F344; Time Factors; Uracil; Urinary Bladder Calculi; Urinary Bladder Neoplasms

Overexpression of cyclin D1 has been implicated in the malignant transformation of a variety of human cancers, including urinary bladder carcinomas. However, few reports have addressed the significance of cyclin D1 overexpression in chemical carcinogenesis in rodents. In the present study, we evaluated the oncogenic potential of cyclin D1 in experimental rat urinary bladder carcinogenesis and its relationships to the oncogenes cyclin E, K-ras, and H-ras as well as tumor suppressor genes p53 and p21WAF1/Cip1. In addition, proliferation status of preneoplastic lesions and tumors was assessed by proliferating cell nuclear antigen immunohistochemistry. Fisher 344 rats were initiated with 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine in the drinking water for 4 weeks and then administered 5% sodium L-ascorbate in diet. Animals were sacrificed at weeks 4, 8, 12, 18, and 24. Preneoplastic lesions such as papillary or nodular hyperplasia and neoplastic lesions of the urinary bladder were observed during carcinogenesis. By immunohistochemical examination, overexpression of cyclin D1 protein was observed in 17% of papillary or nodular hyperplasias, 66% of papillomas, and 69% of transitional cell carcinomas, whereas nuclear accumulation of p53 was observed in none of the preneoplastic lesions and in fewer than 2% of transitional cell carcinomas. Overexpression of cyclin D1 in preneoplastic lesions and tumors was not dependent on the size of the tumors or their proliferation status. Quantitation of mRNA in tumors by multiplex reverse transcription-PCR showed that average mRNA expression of cyclin D1 and cyclin E was increased, whereas average p21WAF1/Cip1 mRNA expression was decreased. More than 2-fold overexpression of cyclin D1 mRNA was observed in 50 and 60% of tumors at weeks 18 and 24, respectively. Localization of cyclin D1 mRNA expression was demonstrated by in situ hybridization, and the results were comparable to immunohistochemistry findings. None of the 25 tumors we examined by PCR-single-strand conformational polymorphism analysis harbored p53 mutations, H-ras mutations, or K-ras mutations. Thus, during the promotion phase of two-stage bladder carcinogenesis, overexpression of cyclin D1 in tumor cells may provide yet another mechanism by which tumors can gain a growth advantage. In contrast, tumors with mutated p53 may not have a growth advantage. Our results suggest that overexpression of cyclin D1 plays a critical role during urinary bladder carcinogenesi

Topics: Animals; Ascorbic Acid; Butylhydroxybutylnitrosamine; Carcinogens; Carcinoma, Transitional Cell; Cell Nucleus; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Hyperplasia; Male; Neoplasm Proteins; Papilloma; Precancerous Conditions; Proliferating Cell Nuclear Antigen; Rats; Rats, Inbred F344; RNA, Messenger; Tumor Suppressor Protein p53; Urinary Bladder; Urinary Bladder Neoplasms

The present study was conducted to analyze the alterations affecting cyclins D1, E, and A in bilharzial bladder cancer and to assess their potential clinical significance. A total of 125 cases were examined. Histopathological subtypes included 68 squamous cell carcinomas, 55 transitional cell carcinomas, and 2 adenocarcinomas. Immunohistochemical analyses were performed using a panel of well-characterized antibodies. The results were correlated with proliferative index, as assessed by Ki67 antigen expression. The cyclin D1-positive phenotype, defined as the identification of positive immunoreactivity in the nuclei of >/=20% of tumor cells, was found in 33 of 107 (31%) evaluable cases. A significant association was observed between the cyclin D1-positive phenotype and deep muscle invasion (P = 0.02), high tumor grade (P = 0.02), and Ki67 high proliferative index (P = 0.03). The cyclin E-positive phenotype, defined as per cyclin D1, was found in 79 of 106 (75%) evaluable cases. The cyclin A-positive phenotype, defined using the above criteria, was identified in 60 of 108 (56%) evaluable cases. No statistically significant association was found between cyclins E or A and clinicopathological parameters or proliferative index. However, there was a strong association between the expression of cyclin D1 and the coexpression of cyclins A and/or E (P = 0.05). Ki67 proliferative index was considered high when >/=20% of tumor cells displayed positive nuclear staining, a phenotype that was observed in 99 of 115 (86%) cases. These data support the hypothesis that cyclin D1 activation determines the evolution of a particular subset of aggressive bladder tumors. In addition, cyclins E and A seem to follow an unscheduled pattern of expression, based on the high frequency of identifying a positive phenotype for these cyclins and the lack of correlation between their expression and Ki67 high proliferative index. It may be postulated that the expression of G1 cyclin genes is deregulated in bilharzial bladder cancer, and that cyclin D1 acts as an oncogenic event in these neoplasms. Moreover, the moderate number of tumors displaying the cyclin D1-positive phenotype (31%) versus the high frequency observed for both cyclins E (75%) and A (56%), suggests a short G1 disbalanced by a long S phase and a rapid transversal of the cell cycle, as evidenced by a high Ki67 index observed in 86% of these cases. This imbalance in the cell cycle, together with alterations reported on the p5

Topics: Adenocarcinoma; Biomarkers, Tumor; Carcinoma, Squamous Cell; Carcinoma, Transitional Cell; Cyclin A; Cyclin D1; Cyclin E; Female; Humans; Ki-67 Antigen; Lymphatic Metastasis; Male; Middle Aged; Mitotic Index; Neoplasm Staging; Schistosomiasis; Urinary Bladder Neoplasms

11q13 amplifications have been found in several cancers, including bladder tumours. However, the biological significance of this genetic alteration is not yet fully understood. To get more insight into the role of 11q13 amplification in bladder tumour development, we have studied the level of amplification and expression of 4 (protoonco)genes lying within the amplicon; cyclin D1, FGF3, FGF4 and EMS1 DNA amplification was found in 5/46 tumours. There was no correlation between amplification and clinico-pathological data. No expression of FGF3 and FGF4 was detected whereas both cyclin D1 and EMS1 were expressed at higher level in tumours with amplifications. Thus cyclin D1 and EMS1, but not FGF3 and FGF4, are likely to play a pathogenic role in the 11q13 amplification in bladder cancer. However, amplification is not the unique way of activation of these genes. Indeed, in situ hybridisation and Northern blot analysis have shown that most bladder tumours have a fair to high expression of cyclin D1 and EMS1 in contrast to normal urothelium with a moderate expression. Interestingly, a trend towards higher expression occurs in superficial versus invasive tumours (8.8 +/- 2.0 versus 1.9 +/- 0.4; P approximately equal to 13% for cyclin D1 and 4.5 +/- 1.4 versus 2.0 +/- 0.4; P approximately equal to 8% for EMS1). Moreover, the 9 tumours with low expression are all highly malignant, leading to the hypothesis that the tumours developing through a cyclin D1/EMS1 independent pathway are more aggressive.

Topics: Carcinoma, Squamous Cell; Chromosomes, Human, Pair 11; Cortactin; Cyclin D1; Cyclins; Fibroblast Growth Factor 4; Fibroblast Growth Factors; Gene Amplification; Gene Expression; Humans; Microfilament Proteins; Neoplasm Proteins; Neoplasm Staging; Oncogene Proteins; Proto-Oncogene Proteins; RNA, Messenger; Transcription, Genetic; Urinary Bladder Neoplasms