TSU-Pr1 Cells Research Articles

CINP is a novel cofactor of KLF5 required for its role in the promotion of cell proliferation, survival and tumor growth.

Krüppel-like factor 5 (KLF5) both suppresses and promotes tumor growth depending on cellular context. The mechanisms underlying tumor promotion could be targetable for therapy. Although a number of transcriptional targets of KLF5 have been identified and implicated in KLF5-mediated tumor growth, how KLF5 regulates these genes remains to be addressed. Here we performed coimmunoprecipitation (co-IP) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the TSU-Pr1 bladder cancer cell line, in which KLF5 is shown to promote tumor growth, to identify KLF5-interacting nuclear proteins that are necessary for KLF5's tumor promoting function. LC-MS/MS revealed 122 potential KLF5 binding proteins in the nuclear proteins precipitated by the KLF5 antibody, and the top nine candidates included AHNAK, TFAM, HSDL2, HNRNPC, CINP, IST1, FBL, PABPC1 and SNRNP40. SRB assays of these nine proteins indicated that silencing CINP had the most potent inhibitory effect on cell growth in KLF5-expressing cells but did not affect parental TSU-Pr1 cells. Further analyses not only confirmed the physical interaction between KLF5 and CINP, also demonstrated that knockdown of CINP attenuated the effects of KLF5 on cell cycle progression, apoptosis and tumorigenesis. Silencing CINP also attenuated the effect of KLF5 on the expression of a number of genes and signaling pathways, including cell cycle regulator Cyclin D1 and apoptosis-related Caspase 7. These results suggest that CINP is a cofactor of KLF5 that is crucial for the promotion of tumor growth, and that the KLF5-CINP interaction could be a novel therapeutic target for inhibiting KLF5-promoted tumor growth.

Andrographolide sensitizes prostate cancer cells to TRAIL-induced apoptosis.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising agent for anticancer therapy. The identification of small molecules that can establish the sensitivity of prostate cancer (PCa) cells to TRAIL-induced apoptosis is crucial for the targeted treatment of PCa. PC3, DU145, JAC-1, TsuPr1, and LNCaP cells were treated with Andrographolide (Andro) and TRAIL, and the apoptosis was measured using the Annexin V/PI double staining method. Real time-polymerase chain reaction (PCR) and Western blot analysis were performed to measure the expression levels of target molecules. RNA interference technique was used to down-regulate the expression of the target protein. We established a nude mouse xenograft model of PCa, which was used to measure the caspase-3 activity in the tumor cells using flow cytometry. In this research study, our results demonstrated that Andro preferentially increased the sensitivity of PCa cells to TRAIL-induced apoptosis at subtoxic concentrations, and the regulation mechanism was related to the up-regulation of DR4. In addition, it also increased the p53 expression and led to the generation of reactive oxygen species (ROS) in the cells. Further research revealed that the DR4 inhibition, p53 expression, and ROS generation can significantly reduce the apoptosis induced by the combination of TRAIL and Andro in PCa cells. In conclusion, Andro increases the sensitivity of PCa cells to TRAIL-induced apoptosis through the generation of ROS and up-regulation of p53 and then promotes PCa cell apoptosis associated with the activation of DR4.

Development of a Detection System for Epigenetic Modifications By Enzyme Fused Zinc Finger Protein

INTRODUCTION Epigenetic modifications such as DNA methylation and histone modifications play an important role in gene regulation. Recently, it has been reported that aberrant modifications are related to diseases such as cancer. Therefore, these modifications are regarded as new biomarkers (1). To detect these modifications, usually, quantitative polymerase chain reaction (qPCR) is performed after protein-based precipitation. qPCR is a powerful and simple method to quantify DNA, but this method is not able to distinguish between specific PCR products and nonspecific products. Therefore, to utilize these modifications as a biomarker, more accurate detection method is required. To achieve that, we focused on zinc finger protein (ZF), a well-known DNA binding protein, because it can bind to double stranded DNA with sequence specifically. In addition, to detect DNA methylation level, we have to treat DNA with bisulfite and perform sequencing. Although this method is a standard method to detect the level, it is time consuming and laborious. To detect the level more simply and rapidly, we focused on Methyl CpG-binding domain (MBD). Using MBD, we can separate methylated DNA without bisulfite treatment in shorter time period. Thus we report simple and rapid detection system for epigenetic modifications using ZF and MBD (2,3). METHOD 0. Design of detection system using enzyme fused ZF Our detection method comprises following three steps:1. Methyl CpG-binding domain (MBD) or histone modification antibodies based DNA collection, 2. PCR amplification of the target genomic region, which includes a zinc finger recognition site and 3. Detection of the PCR products by enzyme fused zinc finger protein (Fig.). As reporter enzymes, we chose luciferase and glucose dehydrogenase. We constructed a homogenous detection system combining Bioluminescence Resonance Energy Transfer (BRET) with luciferase fused zinc finger protein (ZF-Luc). We also constructed a simple electrochemical detection system using glucose dehydrogenase fused zinc finger protein (ZF-GDH). 1. Electrochemical detection of DNA methylation using ZF-GDH We detected DNA methylation levels in androgen receptor (AR) promoter regions of human genomic DNA using ZF-GDH. First, we checked genomic DNA copy number dependency. We used highly methylated genomic DNA (Du145) and lowly methylated genomic DNA (LNCaP). These genomic DNA was purified and diluted to certain copy numbers (102 to 106copies), and detected. Next, we tried to detect methylation level in 106copies of genomic DNA. Genomic DNA from LNCaP, Du145, PC3 and TSU-PR1 cells were analyzed and we compared with the results obtained by a conventional method: bisulfite sequencing. 2. Homogeneous detection of histone modifications using ZF-Luc For detection of histone modification, we targeted H3K4me3 at GAPDH promoter region using ZF-Luc. This promoter region is reportedly associated with H3K4me3 in HeLa cells. We then analyzed histone modifications at the AR promoter region. The ARpromoter region is reportedly hypermethylated in Du145 cells and hypomethylated in LNCaP cells. RESULTS AND DISCUSSION 1. As we expected, copy number dependent signal increase was observed only when we detected highly methylated DNA. This result means that we can detect methylated DNA specifically. The methylation level detected by our method was correlated with the result obtained by the conventional method. This shows that the methylation levels of target genomic regions can be correctly determined without bisulfite treatment and sequencing. Importantly we could detect methylation level in 106copies of genomic DNA because it is enough sensitive for a real diagnositics. 2. As the result of histone modifications detection at GAPDH, we detected high luciferase activity from the H3K4me3 sample but not the H3K9me3 sample. Also for ARregion, indeed, we detected high luciferase activity from the s3 sample for Du145 cells and the H3K4me3 sample for LNCaP cells. These results are correlated with reported results and demonstrate that histone modifications were correctly detected by our homogeneous detection method. CONCLUSION We developed a detection system for DNA methylation and histone modification using enzyme fused zinc finger proteins and demonstrated that the DNA methylation levels and histone modification levels can be determined by this system. We believe that DNA methylation levels and histone modification levels can be detected simultaneously by our system and it would be a useful system for the diagnosis of diseases that have a significant impact on human health. REFERENCES 1. Gal-Yam, E. N.; Saito, Y.; Egger, G.; Jones, P. A., (2008), Annu. Rev. Med. 59, 267−280. 2. Hiraoka, D., Yoshida, W., Abe, K., Wakeda, H., Hata, K., & Ikebukuro, K. (2012). Anal. Chem., 84(19) 3. Yoshida, W., Kezuka, A., Abe, K., Wakeda, H., Nakabayashi, K., Hata, K., & Ikebukuro, K. (2013). Anal. Chem., 85(13) Figure 1

Combination of axitinib and dasatinib for anti-cancer activities in two prostate cancer cell lines

<p class="Abstract">Prostate cancer is major cause of cancer related deaths worldwide in men. There are new treatment methods and drugs are being developed with promising results in two of the prostate cancer cell lines (PPC-1 and TSU-Pr1). These two cells were treated with 20 uM of axitinib combined with dasatinib for 6-72 hours. The cell viability assessed by the cytotoxicity assay. Various regulatory genes such as c-KIT, cell cycle and apoptosis and angiogenic factors were also studied. The enzyme activity of apoptosis efector caspase-3 was colorimetrically determined. Axitinib and dasatinib combination lowered the survival rate of PPC-1 cells but enhanced the survival rate of TSU-Pr1 cells. The protein expression levels in apoptosis and angiogenesis factors were also found to be in contrast between the two cell lines. PPC-1 and TSU-Pr1 cells displayed a different response to axitinib with dasatinib, which explains different expression levels of regulators of cell-cycle, apoptosis and angiogenesis.</p><p> </p>

Combined therapeutic effects of adenoviral vector-mediated GLIPR1 gene therapy and radiotherapy in prostate and bladder cancer models

ObjectivesThe objectives of this study are to explore the potential benefits of combining AdGlipr1 (or AdGLIPR1) gene therapy with radiotherapy using subcutaneous prostate and bladder cancer models. Materials and methodsCombination adenoviral vector-mediated gene therapy and radiotherapy were applied to 178-2 BMA and TSU-Pr1 cells in vitro and colony formation and apoptosis were analyzed. In addition, combination therapies were administered to mice bearing subcutaneous 178-2 BMA and TSU-Pr1 tumors, and tumor growth suppression and survival extension were compared with the monotherapies (AdGlipr1/AdGLIPR1 and radiotherapy) or control vector Adv/CMV/βgal, as well as single-cycle treatment with 2-cycle treatment. ResultsCombination treatment significantly suppressed colony formation and increased apoptosis in vitro. In vivo, combination therapy produced significant 178-2 BMA and TSU-Pr1 tumor growth suppression and survival extension compared with the monotherapies or the control. Further tumor growth suppression and survival extension were observed after 2 cycles of the combination treatment. ConclusionsCombining AdGlipr1 (AdGLIPR1) with radiotherapy may achieve additive or synergistic tumor control in selected prostate and bladder tumors, and additional therapeutic effects may result with repeated treatment cycles.

Expression of Heat Shock Protein 27 in Prostate Cancer Cell Lines According to the Extent of Malignancy and Doxazosin Treatment

PurposeHeat shock protein 27 (HSP27) is known as the material that plays a role in apoptosis control in tumor and cell protection including the immune response, drug tolerance, and so on. In this study, HSP27 expression according to the prostate cancer malignancy level was evaluated, and HSP27 expression was also analyzed after inducing apoptosis by doxazosin treatment of the prostate cancer cell lines.Materials and MethodsReverse transcription polymerase chain reaction (RT-PCR) and immunofluorescence staining of the HSP27 was implemented by the culture of RWPE-1, LNCaP, androgen-independent human prostate cancer cells (PC-3), and TSU-Pr1 cell lines. Analysis was separately conducted in the control group, control vector group treated by dimethyl sulfoxide, and groups treated with 10 µM or 25 µM doxazosin. The expression of HSP27 in RT-PCR and immunofluorescence staining was observed and evaluated after conversion to numerical values.ResultsIn the RT-PCR results, depending on the cell type, LNCaP, TSU-Pr1 showed the highest HSP27 expression followed by PC-3, LNCaP and RWPE-1 in sequence. After doxazosin treatment, the expression detected by RT-PCR was stronger at a 25-µM doxazosin concentration compared to that at a 10-µM concentration, and the result was similar by immunofluorescence staining.ConclusionsHSP27 expression increased depending on the prostate cancer cell line. This meant that HSP27 expression was related to the prostate cancer malignancy level. Additionally, the higher the treatment concentration in PC-3 was, the higher the HSP27 expression was. This result showed that doxazosin induced apoptosis of prostate cancer.

Multi-target siRNA based on DNMT3A/B homologous conserved region influences cell cycle and apoptosis of human prostate cancer cell line TSU-PR1

Abnormal genome hypermethylation participates in the tumorigenesis and development of prostate cancer. Prostate cancer cells highly express DNA methyltransferase 3 (DMNT3) family genes, essential for maintaining genome methylation. In the present study, multi-target siRNA, based on the homologous region of the DNMT3 family, was designed for the in vitro investigation of its effects on the proliferation, migration, and invasion of TSU-PR1 prostate cancer cells. The consequential cell-cycle derangement, through DNMT3A/B or only DNMT3B silencing, was partially efficient, without affecting apoptosis. DNMT3A silencing had absolutely no effect on changing TSU-PR1 cell biological behavior. Hence, DNMT3B alone apparently plays a key role in maintaining the unfavorable behavior of prostate-cancer cells, thereby implying its potential significance as a promising therapeutic target, with DNMT3A simply in the role of helper.

Trans-10,cis-12 Conjugated Linoleic Acid Inhibits Insulin-Like Growth Factor-I Receptor Signaling in TSU-Pr1 Human Bladder Cancer Cells

Conjugated linoleic acid (CLA) is a mixture of positional and geometric isomers of linoleic acid; the major isomers are trans-10,cis-12 CLA (t10c12) and cis-9,trans-11 CLA (c9t11). CLA has been demonstrated to exert strong anticarcinogenic effects in a variety of experimental cancer models. We previously observed that CLA (a mixture of isomers) and t10c12 decreased the growth of TSU-Pr1 cells, whereas linoleic acid and c9t11 exerted no effects. In the current study, the mechanisms underlying the t10c12-mediated regulation of the growth of these bladder cancer cells were evaluated. TSU-Pr1 cells were incubated in serum-free medium with various concentrations of t10c12 or c9t11 in the presence or absence of insulin-like growth factor (IGF)-I. The incorporation of [(3)H]thymidine into DNA was decreased, and the number of annexin V-stained cells was increased after t10c12 treatment, whereas c9t11 had no effect on apoptosis or [(3)H]thymidine incorporation. Treatment with exogenous IGF-I alone increased the numbers of viable cells but did not counteract the t10c12-induced growth inhibition of TSU-Pr1 cells. t10c12 effected a dose-dependent reduction in IGF-I receptor (IGF-IR) transcripts and protein levels, whereas c9t11 exerted no effects. Additionally, t10c12 inhibited the IGF-I-induced phosphorylation of IGF-IR, the recruitment of the p85 regulatory subunit of phosphoinositide 3-kinase to IGF-IR, and the phosphorylation of Akt and extracellular signal-regulated kinase (ERK)-1/2. These results indicate that the inhibition of IGF-IR signaling and the activation of Akt and ERK-1/2 contributed to decreased cell proliferation and increased apoptosis in TSU-Pr1 cells treated with t10c12.

WIF1, a Wnt pathway inhibitor, regulates SKP2 and c-myc expression leading to G1 arrest and growth inhibition of human invasive urinary bladder cancer cells

Epigenetic silencing of secreted wingless-type (Wnt) antagonists through hypermethylation is associated with tobacco smoking and with invasive bladder cancer. The secreted Wnt inhibitory factor-1 (WIF1) has shown consistent growth-inhibitory effect on various cancer cell lines. Therefore, we assessed the mechanisms of action of WIF1 by either restoring WIF1 expression in invasive bladder cancer cell lines (T24 and TSU-PR1) or using a recombinant protein containing functional WIF1 domain. Both ectopic expression of WIF1 and treatment with WIF1 domain protein resulted in cell growth inhibition via G(1) arrest. The G(1) arrest induced by WIF1 is associated with down-regulation of SKP2 and c-myc and up-regulation of p21/WAF1 and p27/Kip1. Conversely, reexpression of SKP2 in WIF1-overexpressing TSU-PR1 cells attenuated the WIF1-induced G(1) arrest. Furthermore, inhibition of nuclear Wnt signaling by either dominant-negative LEF1 or short hairpin RNA of TCF4 also reduced SKP2 expression. The human SKP2 gene contains two TCF/LEF1 consensus binding sites within the promoter. Chromatin immunoprecipitation/real-time PCR analysis revealed that both WIF1 and dominant-negative LEF1 expression decreased the in vivo binding of TCF4 and beta-catenin to the SKP2 promoter. Together, our results suggest that mechanisms of WIF1-induced G(1) arrest include (a) SKP2 down-regulation leading to p27/Kip1 accumulation and (b) c-myc down-regulation releasing p21/WAF1 transcription. Additionally, we show that WIF1 inhibits in vivo bladder tumor growth in nude mice. These observations suggest a mechanism for transformation of bladder epithelium on loss of WIF1 function and provide new targets such as SKP2 for intervention in WIF1-deficient bladder cancer.

Krupple-like factor 5 promotes breast cancer proliferation through activating fibroblast growth factor-binding protein transcription.

Abstract Abstract #4071 Breast cancer is the second leading cause of cancer death in U.S. women. The estrogen receptor negative basal breast cancer is usually invasive and the prognosis is poor. Kruppel-like Factor 5 (KLF5) is a zinc-finger transcription factor promoting cell proliferation, survival, cell cycle, and angiogenesis. High expression level of KLF5 has been shown to be associated with shorter breast cancer patient survival time. In our previous microarray studies, fibroblast growth factor binding protein (FGF-BP) is suggested to be one of the KLF5's downstream target genes in the TSU-Pr1 bladder cancer cell line. FGF-BP has been appreciated to be overexpressed in breast tumors and promotes tumorigenesis. To test whether KLF5 promotes cell proliferation through inducing FGF-BP gene transcription in breast cells, we first examined the KLF5 and FGF-BP protein levels in a panel of breast cell lines and found that KLF5 and FGF-BP are co-expressed in ER-negative basal breast cell lines. Over-expression of KLF5 by adenovirus dramatically increased the FGF-BP mRNA and protein expression in MCF7 and BT474. In contrast, KLF5 knockdown decreased the FGF-BP mRNA and protein expression in HCC1937, BT20, and SW527. Importantly, we found that FGF-BP, like KLF5, promotes cell proliferation in MCF10A, SW527 and TSU-Pr1 cells by H3-thymidine incorporation assay. Furthermore, we demonstrated that KLF5 activates the FGF-BP promoter through a putative KLF5 binding site by luciferase reporter assays. We further showed that KLF5 binds to the FGF-BP promoter in vitro by oligo pull down assays and in vivo by chromatin immunoprecipetation (CHIP) assays. These findings suggest that KLF5 may promote breast cancer cell proliferation through directly activating the FGF-BP mRNA transcription. This study will yield valuable insights into the mechanism of KLF5 induced cancer pathogenesis, and will result in useful diagnostic and therapeutic targets. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4071.

Transcriptional Effect of DEFB1 Gene 5′ Untranslated Region Polymorphisms

To the Editor: Human β-defensin-1 (hBD-1) has been shown to be a candidate tumor suppressor gene by Sun et al. (1) who reported that single nucleotide polymorphisms (SNP) in the DEFB1 gene are able to modify the transcriptional activity of hBD-1 promoter. In particular, the −44C/G SNP was described to enhance transcription up to 2.3 times more than the wild-type sequence in DU145 or TSU-Pr1 cell lines.Recently, we showed that significant correlation exists between SNPs in the 5′ untranslated region (UTR) of the DEFB1 gene and the risk of being infected with HIV-1 in Italian and Brazilian children (2, 3).With the aim of verifying an eventual transcriptional functional effect of the SNPs in the 5′UTR region of DEFB1 gene, we employed a dual luciferase assay, the same technique used by Sun et al. (1).Starting from the wild-type DEFB1 5′UTR region, we generated three mutated inserts using primers designed to introduce the desired mutation into the wild-type sequence. The four different fragments (wild-type, −52A, −44G, and −20A) have been cloned into pGL3 promoter vector (Promega), and Caco2 cells were transfected for 48 h with 1 μg of the reporter plasmids and 20 ng of control Renilla luciferase expression plasmid (phRG-TK, Promega) using 5 μL of GenePORTER transfection reagent; a dual luciferase assay (Promega) was done according to the manufacturer's instruction. To ensure the reproducibility of the results, all tests have been done in quadruplicate and repeated at a later time to confirm the obtained results.Our results show that the three SNPs do possess a functional activity leading to an impaired production of the gene downstream to the mutated promoter.11More detailed information is available at http://www.bbcm.univ.trieste.it/~bbcm/defb1funct/. Both the −52A and the −20A alleles cause a 25% mean reduction of expression. Although quantitatively relevant, this reduction was not statistically significant. The −44G mutation, contrary to the data presented by Sun et al. (1), drag to a markedly reduced expression of about 53% in our experimental setup.Based on our results (2, 3) and because all the studied SNPs could hamper an effective production of hBD-1 in vivo, we hypothesized that they could be related to reduced response from the innate immune system.Despite the fact that DU145, TSU-Pr1, and Caco2 are all carcinoma-derived cell lines, their inherent differences could lead to variations in dual luciferase assay results: we believe that the functional effect of the DEFB1 5′UTR SNPs is still controversial and deserves a more comprehensive study.

ADH1, an N-cadherin inhibitor, evaluated in preclinical models of angiogenesis and androgen-independent prostate cancer

The conversion from E-cadherin to N-cadherin has been observed in several human cancer types, including prostate cancer, with more homogenous expression of N-cadherin detected in high-grade prostate tumors. N-cadherin, in vitro, has been shown to promote cell mobility, migration and invasion of several cancer cell lines, indicating the possibility of N-cadherin as a molecular target of cancer therapy. Herein, we examined the potential of an N-cadherin inhibitor, ADH1, in reducing tumor angiogenesis ex vivo and delaying tumor progression in vivo. Our data demonstrate that ADH1, at the dosages evaluated, does not display either antiangiogenic activity in a rat aortic ring assay or antitumor potential in a PC3 subcutaneous xenograft tumor model. We detected cytotoxic activity in human umbilical vein endothelial cells, PC3, and Tsu-Pr1 cells, when ADH1 exposure was evaluated at 500 micromol/l or above.

Human β-Defensin-1, a Potential Chromosome 8p Tumor Suppressor: Control of Transcription and Induction of Apoptosis in Renal Cell Carcinoma

Human beta-defensin-1 (hBD-1) is a candidate tumor suppressor gene located on chromosome 8p23. Previously, we showed that cancer-specific loss of hBD-1 was found in 90% of renal clear cell carcinomas and in 82% of prostate cancers. To investigate the possible mechanisms of decreased gene expression and determine the function of hBD-1 protein in urological cancers, we sequenced hBD-1 gene coding regions in prostatic and renal cancer samples. We then analyzed the frequency distribution of promoter polymorphisms and determined the effect of these base changes on transcriptional activity of the hBD-1 promoter. A polymorphism at -688 bases upstream of the ATG start codon affects hBD-1 promoter activity, leading to a rate of reporter gene transcription that is 40% to 50% lower than the wild-type sequence when tested in either DU145 or TSU-Pr1 cell lines. In addition, a polymorphism at -44 bases was shown to enhance transcription up to 2.3 times more than the wild-type sequence in the same cell lines. In addition, three novel hBD-1 promoter mutations were found in renal and prostate cancer clinical samples. An iso-5-aza-2'-deoxycytidine treatment was effective in transcription up-regulation in DU145, suggesting a possible upstream methylation-dependent effect. Synthetic hBD-1 peptide inhibited bladder cancer cell TSU-Pr1 proliferation. Overexpression of the hBD-1 gene in renal cancer cells SW156 resulted in caspase-3-mediated apoptosis. These data support the hypothesis that hBD-1 is a potential tumor suppressor gene for urological cancers. Promoter point mutations may be responsible for cancer-specific loss of hDB-1 expression.

Characterization of the TSU-PR1 cell line by chromosome painting and flow cytometry

TSU-PR1 was originally reported as a prostatic carcinoma cell line derived from a lymph node metastasis. Recently, however, this cell line was reported to be derived from T24 bladder carcinoma cells, and thus further definition of its origin is needed. Conventional cytogenetic study of TSU-PR1 showed aneuploidy, ranging from 65 to 86 chromosome with a modal number of 80, and with 10 marker chromosomes, thus conventional cytogenetics cannot be used to determine which chromosomes or regions of chromosomes are critical in cancer development and progression of this cell line. The present study was conducted to characterize genetic changes of the cell line using comparative genomic hybridization (CGH), fluorescence in situ hybridization (FISH), and flow cytometry. CGH results showed that green-to-red fluorescence ratios were within the range of 0.85–1.15, except for a few chromosomes, which reflected near tetraploidy in TSU-PR1. Flow cytometric analysis of TSU-PR1 revealed a DNA index of 3.46 n, which is close to the 3.48 n calculated from a modal number of 80. The copy numbers of chromosomes 4, 6, 7, 17, and 20 determined by the DNA index and the CGH analyses were 2.85 ± 0.09, 3.22 ± 0.77, 3.01 ± 0.26, 4.05 ± 0.44, and 4.99 ± 0.48, respectively. These numbers are also in accordance with the chromosome copy numbers determined with FISH: 2.98 ± 0.23, 2.91 ± 0.44, 2.74 ± 0.44, 3.93 ± 0.38, and 5.05 ± 0.78 for chromosomes 4, 6, 7, 17, and 20, respectively ( P > 0.05).

Gonadotropin-Releasing Hormone Induces Actin Cytoskeleton Remodeling and Affects Cell Migration in a Cell-Type-Specific Manner in TSU-Pr1 and DU145 Cells

GnRH was first identified as the hypothalamic decapeptide that promotes gonadotropin release from pituitary gonadotropes. Thereafter, direct stimulatory and inhibitory effects of GnRH on cell proliferation were demonstrated in a number of types of primary cultured cells and established cell lines. Recently, the effects of GnRH on cell attachment, cytoskeleton remodeling, and cell migration have also been reported. Thus, the effects of GnRH on various cell activities are of great interest among researchers who study the actions of GnRH. In this study, we demonstrated that GnRH induces actin cytoskeleton remodeling and affects cell migration using two human prostatic carcinoma cell lines, TSU-Pr1 and DU145. In TSU-Pr1, GnRH-I and -II induced the filopodia formation of the cells and promoted cell migration, whereas in DU145, GnRH-I and -II induced the formation of the cells with stress fiber and inhibited cell migration. In our previous studies, we reported the stimulatory and inhibitory effects of GnRH on the cell proliferation of TSU-Pr1 and DU145 cells. This study provides the first evidence for the effects of GnRH on actin cytoskeleton remodeling and cell migration of cells in which cell proliferation was affected by GnRH at the same time. Moreover, we also demonstrated that the same human GnRH receptor subtype, human type I GnRH receptor, is essential for the effects of GnRH-I and -II on actin cytoskeleton remodeling and cell migration in both TSU-Pr1 and DU145 cells using the technique of gene knock-down by RNA interference.

KLF5 promotes cell proliferation and tumorigenesis through gene regulationin the TSU-Pr1 human bladder cancer cell line

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.

Analysis of gene expression during staurosporine-induced neuronal differentiation of human prostate cancer cells

Staurosporine induces neuronal differentiation and suppresses malignancy of human prostate cancer TSU-Pr1 cells. To investigate the mechanism underlying neuronal differentiation and suppression of malignancy, we used cDNA microarrays to examine gene expression profiles in TSU-Pr1 cells treated with staurosporine. mRNAs isolated from untreated and staurosporine-treated TSU-Pr1 cells were hybridised to microarrays consisting of approximately 9100 genes. Changes in gene expression were verified by Northern blot analysis. Staurosporine-responsive genes were involved in a variety of cellular functions including growth regulation, differentiation, replication, DNA repair, G2/M transition and inhibition of apoptosis. Interestingly, expression of genes associated with cell proliferation and malignancy, such as Cyr61 and CTGF, was reduced. Expression of CD73/NT5E, which is involved in neuronal differentiation, was increased. In the present study, we identified various staurosporine-responsive genes in TSU-Pr1 cells. Further studies of the roles of these genes may clarify the mechanisms underlying neuronal differentiation and inhibition of malignancy by staurosporine and identify better approaches for the prevention and treatment of prostate cancer.

Efficacy of selective estrogen receptor modulators (SERMs) in a murine xenograft model bearing human bladder cancer

4582 We have demonstrated through Western Blot and Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) that estrogen receptor (ER)-beta is expressed in RT-4, 5637, T-24, TSU-PR1 and TCC-SUP human bladder cancer cell lines, while ER-alpha is expressed at very low levels. Raloxifene, a SERM, induced apoptosis and decreased the viability of RT-4, T-24, and 5637 cell-lines in-vitro. Using tissue microarray analysis ER-beta was expressed in 133 (63.3%) of 210 bladder tumors, while ER-alpha was expressed in only 2 tumors. The expression of ER-beta was associated with higher stage and grade. We constructed a murine xenograft model bearing human bladder cancer to evaluate the efficacy of SERMs in an animal model. A total of 107 5637 human transitional cell carcinoma cells (suspended in 0.1 ml of a 1:1 mixture of RPMI with 10% FBS and matrigel) were injected into one site at a mammary fat pad of 6–8 week old female athymic BALB/c nu/nu mice. Only mice that developed measurable subcutaneous tumor (at least 3 mm in one dimension) within 2 weeks were chosen for further study. Raloxifene 100 mg (Sigma-Aldrich) powder was suspended in 9 ml of double-distilled water. One milliliter of 90% CMC and 10% PEG400/Tween80 was added to the raloxifene solution for a final concentration of 10 mg/ml. Five cohorts of mice consisting of 6–8 mice per cohort were administered no therapy, placebo (solvent only without raloxifene), raloxifene 0.01 mg/day, raloxifene 0.1 mg/day and raloxifene 1 mg/day for 5 days a week by oral gavage for 8 weeks. All of the doses of raloxifene significantly inhibited the growth of tumor (p<0.05). In a separate experiment, 107 5637 cells were subcutaneously injected into the flanks of 6–8 week old female athymic nu/nu mice. Four cohorts of mice with measurable tumors with 6–8 mice per cohort were implanted with subcutaneous 60-day time-release pellets (Innovative Research of America, Sarasota, FL) delivering placebo, tamoxifen 0.008 mg/day, tamoxifen 0.125 mg/day and tamoxifen 1.25 mg/day, respectively. Significant inhibition of tumor was observed with all of the doses of tamoxifen (p< 0.01). These data provide the rationale for evaluating SERMs as a targeted therapuetic for patients with bladder cancer. No significant financial relationships to disclose.

Upregulated MT1-MMP/TIMP-2 axis in the TSU-Pr1-B1/B2 model of metastatic progression in transitional cell carcinoma of the bladder

Muscle invasive transitional cell carcinoma (TCC) of the bladder is associated with a high frequency of metastasis, resulting in poor prognosis for patients presenting with this disease. Models that capture and demonstrate step-wise enhancement of elements of the human metastatic cascade on a similar genetic background are useful research tools. We have utilized the transitional cell carcinoma cell line TSU-Pr1 to develop an in vivo experimental model of bladder TCC metastasis. TSU-Pr1 cells were inoculated into the left cardiac ventricle of SCID mice and the development of bone metastases was monitored using high resolution X-ray. Tumor tissue from a single bone lesion was excised and cultured in vitro to generate the TSU-Pr1-B1 subline. This cycle was repeated with the TSU-Pr1-B1 cells to generate the successive subline TSU-Pr1-B2. DNA profiling and karyotype analysis confirmed the genetic relationship of these three cell lines. In vitro, the growth rate of these cell lines was not significantly different. However, following intracardiac inoculation TSU-Pr1, TSU-Pr1-B1 and TSU-Pr1-B2 exhibited increasing metastatic potential with a concomitant decrease in time to the onset of radiologically detectable metastatic bone lesions. Significant elevations in the levels of mRNA expression of the matrix metalloproteases (MMPs) membrane type 1-MMP (MT1-MMP), MT2-MMP and MMP-9, and their inhibitor, tissue inhibitor of metalloprotease-2 (TIMP-2), across the progressively metastatic cell lines, were detected by quantitative PCR. Given the role of MT1-MMP and TIMP-2 in MMP-2 activation, and the upregulation of MMP-9, these data suggest an important role for matrix remodeling, particularly basement membrane, in this progression. The TSU-Pr1-B1/B2 model holds promise for further identification of important molecules.

Restoration of Bone Morphogenetic Protein Receptor Type II Expression Leads to a Decreased Rate of Tumor Growth in Bladder Transitional Cell Carcinoma Cell Line TSU-Pr1

Bone morphogenetic proteins (BMPs), potential regulators of cellular growth and metastasis that signal through an interaction with plasma membrane receptors, have been suggested to be important regulators of malignant cells. The present study was carried out to evaluate the potential role of BMP receptor (BMP-R) types IA, IB, and II in bladder transitional cell carcinoma (TCC) cells. Initially, we investigated the expression of these BMP-Rs in 30 archival tissues of human bladder TCC using immunohistochemistry; 10 benign bladder specimens were used for comparison. The results demonstrated that the expression of BMP-Rs is localized preferentially to the transitional epithelium and that there was a significant association between loss of BMP-RII expression and tumor grade. To find a cell line that can serve as a model system for clinical observation, we subsequently examined sensitivity to BMP-4 and expression of BMP-RII, BMP-RIA, and BMP-RIB in three human bladder cancer cell lines, TCC-Sup, RT4, and TSU-Pr1. Of the three cell lines, TSU-Pr1 exhibited a decreased level of BMP-RII expression and was resistant to the growth-inhibitory effect of BMP-4. Overexpression of BMP-RII in TSU-Pr1 cells not only restored BMP-4 responsiveness but also significantly decreased tumorigenicity in vivo. Taken together, these results demonstrate that human bladder TCC tissues have a frequent loss of BMP-RII expression and that overexpression of BMP-RII leads to restoration of BMP signaling and decreased tumor growth in the human bladder TCC cell line TSU-Pr1.