Rat Bladder Cells Research Articles

The role of urinary pH in o-phenylphenol-induced cytotoxicity and chromosomal damage in the bladders of F344 rats.

o-Phenylphenol (OPP) is a widely used fungicide and antibacterial agent that at high doses has been shown to cause bladder cancer in male F344 rats. The mechanisms underlying OPP-induced bladder carcinogenicity remain unclear but it has been proposed that a non-enzymatic pH-dependent autoxidation of phenylhydroquinone (PHQ), a primary metabolite of OPP, may be a key step in OPP-induced rat bladder carcinogenesis. To investigate this mechanism and to provide insights into the potential human health relevance of OPP-induced cancer, a series of in vitro and in vivo experiments were conducted. In human lymphoblastoid TK-6 cells and rat bladder epithelial NBT-II cells, strong increases in cytotoxicity were seen at a constant concentration of PHQ by increasing the buffer pH as well as by increasing concentrations of PHQ at a constant pH. In in vivo studies, male rats were administered OPP (4,000 and 8,000 ppm) in a diet supplemented with either 1% ammonium chloride or 3% sodium bicarbonate to produce acidic and alkaline urinary pH, respectively. Significant increases in cell proliferation as detected by 5-bromo-2'-deoxyuridine incorporation and micronucleus formation were seen in the bladder cells of OPP-treated rats with neutral or alkaline urinary pH but not in animals with the acidified urine. The results from these in vitro and in vivo studies provide support for the autoxidation hypothesis of bioactivation, and provide additional evidence that urinary pH can significantly influence the genotoxicity and carcinogenicity of this important agent.

Aldehyde dehydrogenase induction in arsenic-exposed rat bladder epithelium

Arsenic is widely distributed in the environment. Many human cancers, including urothelial carcinoma (UC), show a dose-dependent relationship with arsenic exposure in the south-west coast of Taiwan (also known as the blackfoot disease (BFD) areas). However, the molecular mechanisms of arsenic-mediated UC carcinogenesis has not yet been defined. In vivo study, the rat bladder epithelium were exposed with arsenic for 48h. The proteins were extracted from untreated and arsenic-treated rat bladder cells and utilized two-dimensional gel electrophoresis and mass spectrometry. Selected peptides were extracted from the gel and identified by quadrupole-time of flight (Q-TOF) Ultima-Micromass spectra. The significantly difference expression of proteins in arsenic-treated groups as compared with untreated groups was confirmed by immunohistochemistry (IHC) and western blotting. We found that thirteen proteins were down-regulated and nine proteins were up-regulated in arsenic-treated rat bladder cells when compared with untreated groups. The IHC and western blotting results confirmed that aldehyde dehydrogenase (ALDH) protein was up-regulated in arsenic-treated rat bladder epithelium. Expression of ALDH protein was significantly higher in UC patients from BFD areas than those from non-BFD areas using IHC (p=0.018). In conclusion, the ALDH protein expression could be used as molecular markers for arsenic-induced transformation.

Does radio frequency radiation induce micronuclei frequency in exfoliated bladder cells of diabetic rats?

For many years there has been a discussion among both experts and the general public regarding the effects of radio frequency (RF) radiation on the human organism. The purpose of the present study was to evaluate the relationship of micronucleui (MN) frequency and RF radiation in exfoliated bladder cells of non-diabetic and diabetic rats. Three groups were used in the experiment: Group I (n=6): diabetic group without RF exposure; Group II (n=6): diabetic group exposed 2100 MHz RF radiation and Group III (n=6): control animals (non-diabetic group, no RF exposure). RF exposure in the experiment resulted in a whole body average SAR of 0.24 W/kg with an ERMS field of 17.5 V/m in non-thermal levels. Results showed that there was no statistically important differences between non-RF exposed diabetes group and control group; Group I and Group III (p>0.05). There was no statistically important differences between diabetes group and diabetes+RF exposed group (Group I and Group II) (p>0.05). RF exposure did not result in increased MN frequencies in exfoliated bladder cells of diabetic rats with respect to control animals (Group II and Group III), either and this result found no statistically important (p>0.05). This study suggested no possible genotoxic effects of RF radiation among human beings especially with chronic disorders, such as diabetes.

No genotoxic effect in exfoliated bladder cells of rat under the exposure of 1800 and 2100 MHz radio frequency radiation

In this study, we aimed to investigate the effects of 1800 and 2100 MHz Radio Frequency (RF) radiation on the number of micronucleus (MN) in exfoliated bladder cells of rat which shows the genotoxic damage. Exposure period was 30 min/day, 6 days/week for a month and two months exposure periods. Thirty male wistar albino rats were used for five groups: Group I (n = 6): 1800 MHz RF exposed animals for one month, Group II (n = 6): 2100 MHz RF exposed animals for one month, Group III (n = 6): 2100 MHz RF exposed for two months, Group IV (n = 6): control group for one month, Group V (n = 6): control group for two months. Rats of the control groups were housed in their home cages during the entire experimental period without subjecting to any experimental manipulation. 1800 and 2100 MHz RF exposures did not result in any significant MN frequencies in rat bladder cells with respect to the control groups (p > 0.05). There was no statistically significant difference between 2100 MHz RF exposed groups, either. Further studies are needed to demonstrate if there is any genotoxic effect, micronucleus formation in other tissues of rats.

Clonal growth of human melanocytes using cell‐free extracellular matrix

Clonal growth of human melanocytes using cell‐free extracellular matrix

Photodynamic therapy with hexyl aminolevulinate induces carbonylation, posttranslational modifications and changed expression of proteins in cell survival and cell death pathways

Photodynamic therapy (PDT) using blue light and the potent precursor for protoporphyrin IX, hexyl aminolevulinate (HAL), has been shown to induce apoptosis and necrosis in cancer cells, but the mechanism remains obscure. In the present study, we examined protein carbonylation, expression levels and post-translational modifications in rat bladder cells (AY-27) after PDT with HAL. Altered levels of expression and/or post-translational modifications induced by PDT were observed for numerous proteins, including proteins required for cell mobility, energy supply, cell survival and cell death pathways, by using two-dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry (MS). Moreover, 10 carbonylated proteins associated with cytoskeleton, transport, oxidative stress response, protein biosynthesis and stability, and DNA repair were identified using immunoprecipitation, two-dimensional gel electrophoresis and MS. Overall, the results indicate that HAL-mediated PDT triggers a complex cellular response involving several biological pathways. Our findings may account for the elucidation of mechanisms modulated by PDT, paving the way to improve clinic PDT-efficacy.

Photo induced hexylaminolevulinate destruction of rat bladder cells AY-27

Photodynamic therapy (PDT) is of increasing interest as a relevant treatment for human urinary bladder cancer. In the present experiments, the rat bladder transitional carcinoma cell line AY-27 was used as a model to study cell destruction mechanisms induced by PDT. Red LED light (630 nm) PDT with hexylaminolevulinate (HAL) as precursor for the photosensitizer protoporphyrin IX (PpIX) was used in treatment of the cells. Flow cytometry with fluorescent markers annexin V, propidium iodide and YO-PRO-1, as well as MTT assay and confocal microscopy, were used to map cell inactivation after PDT. Dark toxicity of HAL alone was low in these procedures and LD(50) (24 h, MTT assay) was approximately 1.6 J cm(-2) for standard red light (LED) irradiation (36 mW cm(-2)). Measurements done 1 h after HAL-PDT showed a maximum apoptotic level of about 10% at 6 J cm(-2), however the dominating mode of cell death was necrosis. Forward light scattering indicated an increase in cell size at low doses, possibly due to necrosis. Survival curves had a dual-slope shape, a fit to single hit, multi-target approximation gave a parameter estimate of n = 10 and D(0) about 2.6 J cm(-2). Replacing continuous light with fractionated light delivery (45 s light/60 s darkness) did not affect the treatment outcome.

796 EFFECTS OF HERPES SIMPLEX VIRUS VECTOR-MEDIATED INTERLEUKIN-4 GENE THERAPY ON BLADDER PAIN AND OVERACTIVITY

You have accessJournal of UrologyUrodynamics/Incontinence/Female Urology: Basic Research1 Apr 2011796 EFFECTS OF HERPES SIMPLEX VIRUS VECTOR-MEDIATED INTERLEUKIN-4 GENE THERAPY ON BLADDER PAIN AND OVERACTIVITY Tomohiko Oguchi, Hitoshi Yokoyama, Osamu Nishizawa, William F. Goins, James R. Goss, Joseph C. Glorioso, and Naoki Yoshimura Tomohiko OguchiTomohiko Oguchi Pittsburgh, PA More articles by this author , Hitoshi YokoyamaHitoshi Yokoyama Pittsburgh, PA More articles by this author , Osamu NishizawaOsamu Nishizawa Matsumoto, Japan More articles by this author , William F. GoinsWilliam F. Goins Pittsburgh, PA More articles by this author , James R. GossJames R. Goss Pittsburgh, PA More articles by this author , Joseph C. GloriosoJoseph C. Glorioso Pittsburgh, PA More articles by this author , and Naoki YoshimuraNaoki Yoshimura Pittsburgh, PA More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.614AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Bladder inflammation associated with production of inflammatory cytokines has been proposed as a potential pathophysiology of bladder pain syndrome/interstitial cystitis (BPS/IC). On the other hands, interleukin-4 (IL-4) is a prototypical anti-inflammatory cytokine, which can inhibit secretion of inflammatory cytokines such as IL-1β. We examined effects of gene therapy using replication-deficient herpes simplex virus (HSV) vectors expressing IL-4 (S4IL4) on bladder overactivity and pain behavior induced by intravesical application of resiniferatoxin (RTx) in rats. METHODS The vector expressing LacZ, but not IL-4, was used as a control (SHZ). One or 2 weeks prior to study, 20 μl of viral suspension (3.9×109 pfu/ml S4IL4 or 5×108 pfu/ml SHZ) were injected to the bladder wall of the female SD rat. (1) Virus infection was examined by immunohistochemistry. (2) Murine IL-4 in S4IL4 injected rats was quantified by ELISA. (3) Cystometry was performed under urethane anesthesia. After 2 hours saline infusion, 10nM RTx was administered into the bladder to induce bladder overactivity (n=9 each). (4) In a conscious condition, 3μM RTx (0.3 ml, 1 min) was injected to the bladder through a urethral catheter to evaluate nociceptive behaviors such as licking (lower abdominal licking) and freezing (motionless head-turning) were counted and recorded every 5 sec for 15 min (n=7–8). (5) HSV vector-treated rats were sacrificed 6 hours after 3μM RTx application (0.3ml, 1min) to quantify IL-1β in the bladder by ELISA (n=4 each). RESULTS (1) Lac Z-positive staining was identified in the bladder and L6 dorsal root ganglion (DRG) cells of SHZ rats. (2) Murine IL4 was detected in the bladder (5.6 ± 1.6 pg/mg total protein [TP]) and L6 DRG (13.3 ± 3.8 pg/mgTP) in S4IL4 rats. (3) There were no significant difference in intercontraction interval (ICI) between two groups during saline infusion (p=0.15). After RTx administration, SHZ rats showed a significant reduction in ICI (p<0.05) while ICI did not change significantly (p=0.47) in S4IL4 rats. (4) RTx-induced freezing behavior was significantly less in S4IL4 rats by 47% than SHZ rats (30 ± 5 vs. 57 ± 6 times, p<0.05). (5) Bladder IL-1β was significantly reduced in S4IL4 rats compared to SHZ rats (42.1 ± 8.0 vs. 84.4 ± 9.6 pg/mgTP, p<0.05). CONCLUSIONS These results indicate that HSV vector-mediated IL-4 gene therapy reduces bladder overactivity and pain behavior after RTx administration partly due to cytokine inhibition in rats. IL-4 gene therapy could be a new strategy for treating bladder pain and/or urinary frequency in patients with BPS/IC. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e320 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Tomohiko Oguchi Pittsburgh, PA More articles by this author Hitoshi Yokoyama Pittsburgh, PA More articles by this author Osamu Nishizawa Matsumoto, Japan More articles by this author William F. Goins Pittsburgh, PA More articles by this author James R. Goss Pittsburgh, PA More articles by this author Joseph C. Glorioso Pittsburgh, PA More articles by this author Naoki Yoshimura Pittsburgh, PA More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Is There Any Possible Genotoxic Effect in Exfoliated Bladder Cells of Rat Under the Exposure of 1800 MHz GSM-Like Modulated Radio Frequency Radiation (RFR)?

People are exposed to many carcinogenic and mutagenic chemicals in their everyday lives. These include antineoplastic drugs, Polycyclic aromatic hydrocarbons (PAH)s, aromatic amines, nitrosamines, metals, and electromagnetic radiation. Based on the state of knowledge acquired during the last 50 years of research on possible biological effects of electromagnetic fields (EMF), the majority of the scientific community is convinced that exposure to EMF below the existing security limits does not cause a risk to the health of the general public. However, this position is questioned by others, who are of the opinion that the available research data are contradictory or inconsistent and, therefore, unreliable.In this study, we aimed to investigate if there is any effect of 1800 MHz GSM modulated radio frequency radiation (RFR) on the number of micronucleus in exfoliated bladder cells of rat which will be informative about the genotoxic damage. Exposure period was 20 min/day, 5 days/week during a month. Six female Wistar rats were used for two groups: Group I (n=6): controls; Group II (n=6): 1.8 GHz exposed animals. 1800 MHz RFR did not showed a significant MN frequencies in rat bladder cells when compared with the control group (p>0.05). 1800 MHz RFR-exposed animals did not produce any genotoxic effect when compared with the control group (p>0.05). Kinetic studies are important for any biomarker, especially those in which tissue differentiation and maturation processes will heavily influence the time between induction of damage and collection of damaged cells for micronucleus analysis.

Abstract 5235: Fluorometric imaging system for visualizing submillimeter level tumors using an ultrathin endoscope

Abstract [Background and purpose] Direct observation of pathological lesions by a relatively noninvasive approach is important for understanding pathophysiological mechanism and for assessing the objective response to therapeutic interventions. On the other hand, it is very difficult to perform direct and relatively noninvasive observation of minimal lesions inside a diseased model using a small live animal. However, recent technological developments in imaging, especially significant progress in fluorescent methods and in endoscopic examinations, have enabled detection and tracing of micro-lesions inside small animal models. We have developed a fluorescence imaging technique using high-resolution ultrathin endoscope, which is accessible to hollow organs of small animals. In addition, we have established an orthotopic rat bladder cancer model in which the cancer cells express a fluorescent protein. The aim of this study was to establish a high-resolution imaging method for visualizing the vascular distribution and mucosal morphology of a bladder tumor. [Materials and Methods] GFP-expressing bladder tumor in a rat orthotopic model: Firstly, we established a cell line with stable integration of GFP constructs that were produced by transfection of pTurboGFP into rat bladder cells (AY27). The stable AY27+tGFP populations were purified by repeated selections using Geneticin (G418). AY27-tGFP cells were implanted in the bladders of female Fischer F344 rats, resulting in development of submillimeter-sized bladder tumors. Development of fluorescence ultrathin endoscopy: We developed a fluorescence ultrathin endoscopic system installed with a 180 W Xenon lamp with excitation filters and 3CCD camera with emission filters. The outer diameter of the ultrathin endoscopic fiber is only 0.8 mm, but its number of pixels reaches 15,000, resulting in a spatial resolution of 50μm. [Results] Fluorescence from AY27-tGFP cells implanted in the bladder was clearly visualized by the ultrathin endoscope placed in the bladder by the transurethral approach. Furthermore, when fluorescence-probed tumor-directive micelles were injected (i.v.) into the animals as a model of drug delivery, fluorescence from the probe agents was visualized in accordance with tumor distribution. In both cases, we detected fluorescence from tumors of less than 1 mm in size. [Conclusion] We established an orthotopic GFP-tumor rat model using a GFP-expressing tumor cell line (AY27-tGFP). Submillimeter tumors in this model were visualized by the newly developed ultrathin endoscopic system. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5235.

Identification of interspecies concordance of mechanisms of arsenic-induced bladder cancer

Exposure to arsenic causes cancer by inducing a variety of responses that affect the expression of genes associated with numerous biological pathways leading to altered cell growth and proliferation, signaling, apoptosis and oxidative stress response. Affymetrix GeneChip ® arrays were used to detect gene expression changes following dimethylarsinic acid (DMA) exposure to human bladder cells (UROtsa) or rat bladder cells (MYP3) and rat bladder epithelium in vivo at comparable doses. Using different experimental models coupled with transcriptional profiling allowed investigation of the correlation of mechanisms of DMA-induced toxicity between in vitro and in vivo treatment and across species. Our observations suggest that DMA-induced gene expression in UROtsa cells is distinct from that observed in the MYP3 cells. Principal component analysis shows a more distinct separation by treatment and dose in MYP3 cells as compared to UROtsa cells. However, at the level of pathways and biological networks, DMA affects both common and unique processes in the bladder transitional cells of human and rats. Twelve pathways were found common between human in vitro, rat in vitro and rat in vivo systems. These included signaling pathways involved in adhesion, cellular growth and differentiation. Fifty-five genes found to be commonly expressed between rat in vivo and rat in vitro systems were involved in diverse functions such as cell cycle regulation, lipid metabolism and protein degradation. Many of the genes, processes and pathways have previously been associated with arsenic-induced toxicity. Our finding reiterates and also identifies new biological processes that might provide more information regarding the mechanisms of DMA-induced toxicity. The results of our analysis further suggest that gene expression profiles can address pertinent issues of relevance to risk assessment, namely interspecies extrapolation of mechanistic information as well as comparison of in vitro to in vivo response.

Micronuclei and cell proliferation as early biological markers of ortho-phenylphenol-induced changes in the bladder of male F344 rats

ortho-Phenylphenol (OPP) and its sodium salt, sodium ortho-phenylphenate (SOPP), are widely used fungicides and antibacterial agents known to cause tumors in the bladders of male F344 rats. Previous studies in our laboratory have shown that micronuclei and cell proliferation were induced in the bladders of treated rats by a high dose of OPP. In our present studies, we investigated the relationship in dose response between these two biomarkers and previously reported tumor formation in the bladders of male F344 rats. Significant non-linear increases in micronuclei (MN) and BrdU-labeling were seen in the bladder cells of rats treated with the 8000 and 12,500 ppm doses of OPP and at 20,000 ppm SOPP. CREST anti-kinetochore staining showed that the micronuclei originated from both chromosomal loss and breakage. In addition, increases in MN were detected in the bladder but not in the bone marrow, underscoring the value of assessing genotoxicity in the target organ. In summary, these studies clearly show that at high doses, OPP and SOPP are genotoxic to the rat bladder. These results also indicate that micronucleus formation and cell proliferation can detect early OPP-induced changes in the rat bladder and may be useful as biomarkers for bladder carcinogens.

Detection of micronuclei, cell proliferation and hyperdiploidy in bladder epithelial cells of rats treated with o-phenylphenol.

o-Phenylphenol (OPP), a widely used fungicide and antibacterial agent, has been considered to be among the top 10 home and garden pesticides used in the USA. Earlier studies have consistently shown that the sodium salt of OPP (SOPP) causes bladder cancer in male Fischer 344 (F344) rats, whereas OPP has produced variable results. This difference has been attributed to the presence of the sodium salt. To determine cellular and genetic alterations in the rat bladder and the influence of the sodium salt, F344 rats were administered 2% OPP, 2% NaCl and 2% NaCl + 2% OPP in their diet for 14 days. Twenty-four hours before being killed the animals were administered 5-bromo-2'-deoxyuridine (BrdU) by i.p. injection. Bladder cells were isolated, stained with DAPI and scored for the presence of micronuclei and incorporation of BrdU into replicating cells. To determine changes in chromosome number, we used fluorescence in situ hybridization (FISH) with a DNA probe for rat chromosome 4. Significant increases in the frequency of micronuclei and BrdU incorporation were seen in bladder cells of rats from all treatment groups. In contrast, the frequency of hyperdiploidy/polyploidy in treated animals was not increased over that seen in controls. A high control frequency of cells with three or more hybridization signals was seen, probably due to the presence of polyploid cells in the bladder. The presence of polyploid cells combined with cytotoxicity and compensatory cell proliferation makes it difficult to determine whether OPP is capable of inducing aneuploidy in the rat urothelium. In summary, these studies show that OPP can cause cellular and chromosomal alterations in rat bladder cells in the absence of the sodium salt. These results also indicate that at high concentrations the sodium salt can enhance chromosomal damage in the rat urothelium.

DNA damage induced by 3,3'-dimethoxybenzidine in liver and urinary bladder cells of rats and humans.

3,3'-Dimethoxybenzidine (DMB), a congener of benzidine used in the dye industry and previously found to be carcinogenic in rats, was evaluated for its genotoxic activity in primary cultures of rat and human hepatocytes and of cells from human urinary bladder mucosa, as well as in liver and bladder mucosa of intact rats. A similar modest dose-dependent frequency of DNA fragmentation was revealed by the alkaline elution technique in metabolically competent primary cultures of both rat and human hepatocytes exposed for 20 h to subtoxic DMB concentrations ranging from 56 to 180 microM. Replicating rat hepatocytes displayed a modest increase in the frequency of micronucleated cells after a 48-h exposure to 100 and 180 microM concentrations. In primary cultures of human urinary bladder mucosa cells exposed for 20 h to 100 and 180 microM DMB, the Comet assay revealed a clear-cut increase of DNA fragmentation. In rats given one-half LD50 of DMB as a single oral dose, the GSH level was reduced in both the liver and urinary bladder mucosa, whereas DNA fragmentation was detected only in the bladder mucosa. Taken as a whole, these results suggest that DMB should be considered a potentially genotoxic chemical in both rats and humans; the selective effect on the rat urinary bladder might be the consequence of pharmacokinetic behavior.

Neu Is not involved in N-[4-(5-nitro-2-furyl)-2-thiazolyl]-formamide-induced bladder carcinoma or 2-amino-4-(5-nitro-2-furyl)thiazole transformation of rat bladder epithelial cells

Enhanced c- erbB-2/ neu expression has been linked with a poor prognosis in human bladder cancer. Previous reports have shown that a point mutation at nucleotide T 2012 in the coding region of the transmembrane domain of the rat gene is sufficient to confer transformation potential on this gene. We examined the comparative levels of p185 neu as well as the sequence around the hotspot (T 2012) of the neu gene of rat bladder cells transformed by 2-amino-4-(5-nitro-2-furyl)thiazole (ANFT) or established in culture from N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT)-induced rat bladder tumors. We concluded that increased p185 neu expression did not correlate significantly with tumorigenicity. No alterations in nucleotide sequences of the neu gene were observed in either in vitro model.

Prohibitin gene is overexpressed but not mutated in rat bladder carcinomas and cell lines.

Prohibitin was isolated as a candidate antiproliferating gene in rat liver cells, and it has been suggested as a tumor suppressor gene in human breast cancer. We investigated the steady state level of prohibitin mRNA in rat bladder cell lines and in rat bladder carcinoma induced by N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT) and sodium saccharin, as well as the protein level in rat bladder epithelial cells. We also examined the prohibitin gene for mutations using the polymerase chain reaction (PCR) single strand conformation polymorphism (SSCP) and direct sequencing methods. All rat bladder tumors investigated and several cell lines had unexpectedly increased steady state levels of prohibitin mRNA compared with that of normal rat bladder or liver. The prohibitin protein was easily detected by Western blotting in all cell lines regardless of their malignant status or growth rate. However, PCR-SSCP and direct sequencing analysis showed no mutations in the prohibitin gene. These results These results indicate that prohibitin overexpression, but not mutations, may be involved in the early stage of rat bladder carcinogenesis.

Neu is not involved in N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide-induced bladder carcinoma or 2-amino-4-(5-nitro-2-furyl)thiazole transformation of rat bladder epithelial cells

Enhanced c- erbB-2/ neu expression has been linked with a poor prognosis in human bladder cancer. Previous reports have shown that a point mutation at nucleotide T 2012 in the coding region of the transmembrane domain of the rat gene is sufficient to confer transformation potential on this gene. We examined the comparative levels of p185 neu as well as the sequence around the hotspot (T 2012) of the neu gene of rat bladder cells transformed by 2-amino-4-(5-nitro-2-furyl)thiazole (ANFT) or established in culture from N-[-4-(-5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT)-induced rat bladder tumors. We concluded that increased p185 neu expression did not correlate significantly with tumorigenicity. No alterations in nucleotide sequences of the neu gene were observed in either in vitro model.

Mutations and expression of the p53 gene in rat bladder carcinomas and cell lines.

Abnormalities of the p53 gene are frequently observed in human tumors, including urinary bladder carcinoma, suggesting that p53 plays an important role in human carcinogenesis. However, its role in rat bladder carcinogenesis is unclear. We investigated p53 gene mutations and expression in rat urinary bladder carcinogenesis in vivo and in vitro. Fifteen urothelial cell lines, including six untransformed (nontumorigenic) ones, six transformed (tumorigenic) in vitro, and three derived from tumors induced in vivo, were examined for p53 expression by immunochemical analysis and for p53 mutations; in addition, 81 rat bladders were analyzed immunohistochemically for p53 expression, and 23 rat bladder tumors were analyzed for p53 mutations. Four cell lines had mutations in the p53 gene. Two of these were missense point mutations, and the other two were splicing mutations. On the other hand, no mutations were found in the bladder tumors induced in rats. By immunoprecipitation with PAb240, which is supposed to be specific for mutant p53, we detected mutations in three of the cell lines; PAb240 did not react with wild-type p53. However, in all cell lines and in growing populations of primary cultured bladder urothelial cells, p53 expression was detected immunohistochemically or by western blotting using PAb240 or PAb 421 monoclonal antibodies. In a high percentage of transitional cell carcinomas, wild-type p53 expression was detected by immunohistochemical analysis with PAb240. These results suggest that p53 gene mutations may not occur frequently in rat bladder carcinogenesis in vivo but may occur in vitro and that p53 overexpression detected immunohistochemically is common and may be related to cell proliferation rather than to the presence of mutations in rat bladder carcinogenesis.

In vitro and in vivo accelertration of the neoplastic phenotype of a low‐tumorigenicity rat bladder carcinoma cell line by transfected transforming growth factor‐α

We conducted an experiment to determine whether expression of transforming growth factor-alpha (TGF-alpha) enhances tumorigenicity in a low-tumorigenicity rat bladder carcinoma cell line and whether it is sufficient to induce a tumorigenic phenotype in a nontumorigenic rat bladder cell line. D44c cells (which are nontumorigenic) were derived from a minute nodule from a bladder treated with N-methyl-N-nitrosourea (MNU); G1-200 cl-17 cells (which have low tumorigenicity) were isolated from D44c cells exposed to MNU in vitro. Neither cell line expressed TGF-alpha mRNA. The cells were cotransfected with pSV2neo and pSR alpha-rTGF-alpha. The latter plasmid contains the rat TGF-alpha cDNA under the transcriptional control of the SR alpha promoter. In the low-tumorigenicity G1-200 cl-17 cells, the expression of TGF-alpha mRNA and the subsequent synthesis of TGF-alpha protein activated epidermal growth factor receptors (EGFRs) and markedly enhanced tumorigenicity in nude mice (i.e., shortened the latency period before tumor appearance, accelerated the rate of growth, and increased the size of the tumors) as well as anchorage-independent growth in vitro. In nontumorigenic D44c cells, however, transfected TGF-alpha did not induce either anchorage-independent growth or tumorigenicity in nude mice, in spite of overexpression of EGFR mRNA and the constitutive expression of c-jun and junB mRNA. These results suggest that the increased signal transduction mediated by TGF-alpha enhanced tumorigenicity in a cell that was already tumorigenic but was not sufficient to induce tumorigenicity in a nontumorigenic cell.

Effect of epidermal growth factor and transforming growth factor β1 on growth and invasive potentials of newly established rat bladder carcinoma cell lines

We established 5 rat bladder cell lines (MYU3L, MYU4, MYU6s, MYKU1L and MYP3). EGF stimulated DNA synthesis of all the cells in monolayer culture, regardless of the number of EGF receptors. In soft agar, only MYU3L formed colonies, and EGF enhanced their growth. However, EGF did not induce the other cells to grow in soft agar. In contrast, TGF-beta 1 inhibited the growth of the cells, but a tumorigenic cell and the cells which were established from large in vivo tumors were more resistant than the others to TGF-beta 1. We tested the effect of growth factors on the invasive potential of MYP3 cells (non-tumorigenic), MYU3L cells (tumorigenic/highly invasive but not metastatic) from newly established cell lines, and another metastatic cell line, LMC19. MYP3 expressed only a trace amount of 92-kDa gelatinase (MMP-9), whereas MYU3L expressed interstitial collagenase (MMP-1) and MMP-9, and LMC19 expressed 72-kDa gelatinase (MMP-2) and MMP-9. The release of MMP-2 in LMC19 was stimulated by TGF-beta 1, but EGF had no effect on the release of any MMPs in either type of cells. These observations suggest that EGF acted as a mitogen on all the cells tested, but did not enhance the malignant phenotype. Further, the loss of responsiveness to the suppressive effect of TGF-beta 1 may be an important step toward a malignant phenotype. Some of malignant tumors may utilize TGF-beta 1 for enhancing their invasive and metastatic potential.