transforming-growth-factor-alpha and Urinary-Bladder-Neoplasms

Topics: Adjuvants, Immunologic; Administration, Intravesical; Administration, Oral; Antineoplastic Agents; Antineoplastic Agents, Phytogenic; Bacterial Vaccines; Carcinoma, Transitional Cell; Clinical Trials, Phase I as Topic; Clinical Trials, Phase II as Topic; Cytosine; Exotoxins; Heart Diseases; Hemocyanins; Humans; Immunologic Factors; Immunotherapy; Interferons; Interleukins; Randomized Controlled Trials as Topic; Recombinant Proteins; Transforming Growth Factor alpha; Treatment Outcome; Tumor Necrosis Factor-alpha; Urinary Bladder Neoplasms

Recently, expectations have been raised that molecular biological studies of human tumours may be of value in helping to predict future clinical behaviour, in terms of therapeutic response and long-term survival. The epidermal growth factor receptor (EGFr) is a cell surface receptor for EGF and transforming growth factor-alpha which is overexpressed by a number of human tumours. This article principally reviews previous investigations of the role of the epidermal growth factor receptor in bladder cancer and examines methods of detection, the correlation between EGFr status and known prognostic indicators and the value of assessing EGFr status in predicting clinical outcome in patients with bladder cancer. Recent studies of the c-erbB-2 proto-oncogene in bladder cancer and of cell cycling using Ki-67 are included.

Topics: Biomarkers, Tumor; Epidermal Growth Factor; ErbB Receptors; Humans; Neoplasm Metastasis; Neoplasm Proteins; Neoplasm Recurrence, Local; Prognosis; Proto-Oncogene Mas; Proto-Oncogene Proteins; Receptor, ErbB-2; Transforming Growth Factor alpha; Urinary Bladder Neoplasms

Transforming growth factor alpha-Pseudomonas exotoxin-40 (TP40) is a hybrid fusion protein that selectively binds to cancer cells that express the epidermal growth factor receptor. TP40 is then internalized and kills these cells by virtue of its Pseudomonas exotoxin-derived domains. We studied the safety and short-term antitumor activity of intravesical TP40 in 43 patients with refractory superficial bladder cancer. These patients had resected Ta/T1 disease (n = 19), visible Ta or T1 lesions (n = 11), or carcinoma in situ (n = 13). Patients were treated with increasing dose levels of TP40 at 0.15, 0.3, 0.6, 1.2, 2.4, 4.8, or 9.6 mg/week for 6 weeks and evaluated by comparing pretreatment and posttreatment cystoscopic examinations, cytology, and histopathology. All TP40 doses were well tolerated. No evidence of antitumor activity was seen in any of the patients with Ta or T1 lesions. However, 8 of 9 patients with evaluable carcinoma in situ were judged by histopathology of multiple biopsy specimens to exhibit clinical improvement following TP40 therapy. In most of these responsive patients, cystoscopic examination supported the histopathological findings, although cytology of urine and bladder washings persistently demonstrated malignant cells. Therefore, TP40 appears to be a well-tolerated biological agent that may prove to have utility in treating carcinoma in situ of the bladder.

Topics: Aged; Antineoplastic Agents; Carcinoma in Situ; Dose-Response Relationship, Drug; Exotoxins; Female; Humans; Male; Middle Aged; Neoplasm Staging; Transforming Growth Factor alpha; Urinary Bladder Neoplasms

Topics: Arsenites; Cell Line, Tumor; China; Epidermal Growth Factor; ErbB Receptors; Humans; Receptor, ErbB-2; Transforming Growth Factor alpha; Urinary Bladder; Urinary Bladder Neoplasms; Urothelium

Being best-studied superficial bladder cancer (SBC) chemopreventives, retinoids' negative studies and toxicity were stumbling. With proper understanding of retinoid metabolism, we aimed at investigating combined ketoconazole (a strong inhibitor of retinoic acid-catabolizing cytochrome P450s) all-trans retinoic acid (Keto-atRA) SBC treatment. VEGF and TGFalpha levels are end-point pathogenetic biomarkers involved in early SBC.. Keto-atRA treatment significantly improved survival time and decreased recurrence rate compared to control disease group, with tolerable and reversible side-effects. Treatment normalized induced levels of VEGF and TGFalpha with a positive correlation between these cytokines.. Seven days after TURT visible tumor(s), combined atRA 1 mg/kg for five days a week + Keto 200 mg twice daily for five days a week for three months were given to 16 patients with SBC stages Ta and T1 with various grades. Three months follow up/20 months used white light cystoscopy and urinary cytology. Recurrence rate and survival time were compared to a retrospective group of 25 patients of comparable age, stage and grade with TURT as sole treatment for SBC. VEGF and TGFalpha were measured in urine and serum of 12 normal subjects and treated patients. Samples were collected just before TURT, one week after TURT, at the end of one month and at the end of three months of treatment.. The combination and schedule used for Keto-atRA therapy effectively reduced recurrence rate and increased survival time of SBC patients probably through reduction of VEGF and TGFalpha as major mitogenic/angiogenic factors; possibly by eliminating malignant cells that produce them.

Topics: Aged; Biomarkers; Cytochrome P-450 Enzyme System; Drug Therapy, Combination; Humans; Ketoconazole; Middle Aged; Neoplasm Recurrence, Local; Transforming Growth Factor alpha; Tretinoin; Urinary Bladder Neoplasms; Vascular Endothelial Growth Factor A

Increased activity of the EGF system exerts a cell survival function in the presence of cytotoxic agents. The aim of our investigation was to identify the ligands and receptors from the EGF system, that are induced by the chemotherapeutic DNA damaging agent VP16 in bladder cancer cell lines. By use of real-time RT-PCR assays for all four receptors and six ligands from the EGF system we demonstrate that in HCV29 bladder cancer cells, amphiregulin, HB-EGF, and epiregulin mRNA levels are elevated (more than 100, 5, and 4 fold, respectively) by VP16. The remaining ligands (EGF, TGFalpha and betacellulin) are uninduced. The same was found for T24A bladder cancer cells, except that TGFalpha also was induced. The four receptors were reduced by VP16 in both cell lines. This demonstrates that the induction of the EGF system is mediated by an increased expression of a subset of the ligands, whereas the four receptors are reduced. For amphiregulin and HER1 we investigated with ELISA assays if the effects of VP16 also were observed at the protein level. We found that VP16 increase the amount of amphiregulin peptide both in the cell membrane and the culture medium. Similarly, the reduced EGF receptor mRNA expression correlated with reduced HER1 protein. Several investigations have shown that labile protein factors can be involved in the regulation of stress inducible growth factors and cytokines. We investigated if a labile protein regulates the expression of the subset of ligands that were induced with VP16. Blocking of protein neosynthesis with cycloheximide resulted in induced mRNA expression of exactly the same subset of ligands as observed with VP16 treatment of both HCV29 and T24A cells. This suggests that a labile protein factor regulates either the transcription or degradation of these mRNA's, and it can be speculated that VP16 also operate by inhibiting the activity of this factor. This is further stressed by the observation that combined treatment with cycloheximide and VP16 show no additive effect. In conclusion, we show that a subset of ligands from the EGF system is upregulated by VP16, whereas none of the four receptors are induced. This might represent a physiological response aimed at rescuing the cells.

Topics: Amphiregulin; Antineoplastic Agents, Phytogenic; Betacellulin; Cell Line, Tumor; Cell Membrane; Cycloheximide; Dose-Response Relationship, Drug; EGF Family of Proteins; Epidermal Growth Factor; Epiregulin; ErbB Receptors; Etoposide; Gene Expression Regulation, Neoplastic; Glycoproteins; Humans; Intercellular Signaling Peptides and Proteins; Ligands; Protein Synthesis Inhibitors; RNA, Messenger; Transforming Growth Factor alpha; Urinary Bladder Neoplasms

Cell proliferation is stimulated by growth factors and inhibited by p15 and p16 gene products. We compared cell regulators, TGF-alpha, p15, and p16, in schistosomal and non-schistosomal bladder cancer to explore possible differences in their alterations between the two subtypes and their correlations with proliferation pattern [synthetic phase fraction (SPF)], DNA ploidy, and clinicopathological factors.. Tumor tissue samples were obtained from 120 patients. Expressions of p15 and p16 genes were investigated by the polymerase chain reaction, while TGF-alpha protein expression was measured by an enzyme immunoassay (EIA) method.. Deletion of both p15 and p16 was observed in 62 and 46 bladder tumors, respectively. TGF-alpha was overexpressed in 64 bladder tumors. A highly significant association was observed between the two deleted genes and TGF-alpha positivity. Of the entire group, p15 and p16 alteration and positive TGF-alpha (> or =cutoff value) were significantly expressed in schistosomal bladder cancer (68.1%, 60.9%, and 65.2%), and squamous cell carcinoma type (SCC) (69.1%, 64.7% and 72.1%) compared to those with non-schistosomal bladder cancer (29.4%, 7.8%, and 37.3%) or transitional cell carcinoma (TCC) (28.8%, 3.8%, and 28.8), respectively. A significant association between p15 and p16 deletion and TGF-alpha positivity with high SPF, aneuploid DNA pattern, late stages, and high histological grades was also documented.. Alteration of p15 and p16 genes and overexpression of TGF-alpha appears to be an event in bladder cancer that occurs more frequently in schistosomal bladder cancer and SCC, and may play an important role in their development. These observations may provide insight into treatment guided by molecular changes.

Topics: Carcinoma, Transitional Cell; Cell Cycle Proteins; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p15; Cyclin-Dependent Kinase Inhibitor p16; DNA Primers; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Humans; Immunoenzyme Techniques; Ploidies; ROC Curve; Transforming Growth Factor alpha; Tumor Suppressor Proteins; Urinary Bladder Neoplasms

To study inhibitory effect of recombinant transforming growth factor alpha-Pseudomonas exotoxin fusion protein (TP40) on proliferation of the human bladder cancer T24 cells.. Expression of epidermal growth factor receptor (EGFR) in cultured T24 cells was analyzed with Western blot assay. Human bladder cancer T24 cells were exposed to TP40 at 5 - 1 000 microg/L. Methyl thiazolyl tetrazolium assay was applied to evaluate the cell proliferation by measuring the absorbance (A) at 570 nm with a microplate reader. Tritium labeled thymine deoxyriboside ([(3)H]-TdR) uptake was measured to observe DNA synthesis. Competition assays were performed by the EGF at 1 - 7 500 microg/L.. Expression of EGFR was high in human bladder cancer T24 cells. Cell growth was suppressed by 10%, 19%, 27%, 41%, 47%, 53% and 61% after 96 h treatment with TP40 at 5, 50, 100, 250, 500, 750 and 1 000 microg/L, respectively. [(3)H]-TdR incorporation was 80%, 69%, 48% and 51% after 24 h, 48 h, 72 h, 96 h treatment with TP40 at 750 microg/L, respectively. When the concentration was 1 - 7 500 microg/L, EGF could block the inhibitory effect of TP40 to some extent.. Human bladder cancer T24 cells express EGFR at a high level. TP40 could inhibit the growth of T24 cells effectively in a dose- and time-dependent manner. The cytotoxic effects of TP40 were specifically mediated by EGFR.

Topics: Cell Proliferation; ErbB Receptors; Exotoxins; Humans; Recombinant Fusion Proteins; Transforming Growth Factor alpha; Tumor Cells, Cultured; Urinary Bladder Neoplasms

Members of the epidermal growth factor (EGF) family have been suggested as prognostic markers in patients with bladder cancer. Thus far, there has been no consensus on their usefulness. We report an analysis of six ligands and two receptors of which a subset correlate to tumor stage and survival. Biopsies from bladder cancer tumors were obtained from 73 patients followed for a median of 28 months. The mRNA content for six ligands [EGF, transforming growth factor alpha (TGF-alpha), amphiregulin (AR), betacellulin (betaCL), heparin-binding EGF-like growth factor (HB-EGF), epiregulin (EPI)] and two receptors [EGF receptor I Human EGF Receptor (HER1) and 2 (HER2)] was examined by a newly developed quantitative reverse transcription-PCR method. Five ligands and two receptors (HER1 and HER2) were present in median concentrations of (10(-21) mol/microg RNA) 0.39 (AR), 11 (betaCL), 2.4 (EPI), 40 (HB-EGF), 1.4 (TGF-alpha), 75 (HER1), and 39,000 (HER2). EGF was barely detectable. A significantly higher expression of EPI (P < 0.001), HB-EGF (P < 0.001), and TGF-alpha (P < 0.05) were observed in T2-T4 tumors as compared with Ta tumors. Especially the expression of EPI mRNA correlated strongly to survival (P < 0.0005), but increased expression of TGF-alpha (P < 0.005), AR, and HB-EGF (P < 0.02) was also associated with a reduced life span. For the first time, mRNA expression of six ligands and two receptors of the EGF family have been examined in bladder cancer tumors. Our data emphasize that members of the EGF family, especially EPI, may be potential bladder tumor markers.

Topics: Adult; Aged; Aged, 80 and over; Amphiregulin; Betacellulin; Biomarkers, Tumor; EGF Family of Proteins; Epidermal Growth Factor; Epiregulin; ErbB Receptors; Female; Glycoproteins; Growth Substances; Humans; Immunohistochemistry; Intercellular Signaling Peptides and Proteins; Ligands; Male; Middle Aged; Prognosis; Prospective Studies; Receptor, ErbB-2; RNA, Messenger; Transforming Growth Factor alpha; Urinary Bladder Neoplasms

We analysed the expression of epidermal growth factor receptor (EGFr) and transforming growth factor alpha (TGF-alpha) in human bladder tumours. Tumour biopsies were obtained from 54 patients with primary bladder cancer (18 stage T1 and 36 stage T2-4). The protein and mRNA expression of EGFr and TGF-alpha were quantified by ELISA and competitive RT-PCR, respectively. The EGFr protein level was significantly increased in T2-4 tumours (0.44 x 10(-11); 0.0-27.5 x 10(-11) mol/g) compared with T1 tumours (0.0; 0.0-2.0 x 10(-11) mol/g) (median; range; 2p<0.01). The EGFr protein and mRNA level correlated (Spearman r=0.45, 2p<0.005, n=40). Co-expression of TGF-alpha protein and EGFr protein was significantly associated with muscle invasive tumours (T2-4) (chi-squared=7.9, df=3, p<0.05) and the TGF-alpha protein level correlated significantly with EGFr protein expression (Spearman r=0.56, 2p<0.0001, n=54). While tumour stage correlated with survival, no correlation was observed between survival and the expression of EGFr and/or TGF-alpha. In conclusion, human bladder tumours express both EGFr and TGF-alpha. The expression of EGFr and TGF-alpha are closely correlated, and the expression of EGFr and co-expression of EGFr and TGF-alpha correlate with tumour stage.

Topics: Enzyme-Linked Immunosorbent Assay; ErbB Receptors; Gene Expression; Humans; Neoplasm Invasiveness; Neoplasm Staging; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Survival Rate; Transforming Growth Factor alpha; Urinary Bladder Neoplasms

To investigate the correlation of epidermal growth factor receptor (EGFR) expression and its ligands EGF and transforming growth factor-alpha (TGF-alpha) with disease outcome in a cohort of patients with superficial bladder cancer.. Tumor samples of 21 patients with transitional cell carcinoma of the bladder were analyzed by immunohistochemistry for expression of EGFR, EGF, and TGF-alpha. Disease-related events were recorded during a routine clinical follow-up and analyzed for possible correlation with the expression status of the above-mentioned proteins.. All Stage pT1 transitional cell carcinomas expressed EGFR, and 10 of 21 (48%) tumors showed focal areas of strong EGF and/or TGF-alpha expression. Of these, 80% with EGF positivity (8 of 10) had recurrences, whereas only 9% of patients without EGF staining (1 of 11) did so. The same pattern was observed with TGF-alpha. A strong association was confirmed between EGF/TGF-alpha positivity and tumor recurrence (P <0.005). We also found that EGF and TGF-alpha were expressed in stroma and/or around the vessels of tumor tissue in 48% and 38% of the tumors, respectively. No association was found between the recurrence rate/vascular invasion and the stromal/vascular wall expression of the growth factors.. Expression of EGF and TGF-alpha is correlated with tumor recurrence. Also, there is the ability of vessel walls to express EGF and TGF-alpha in superficial bladder cancer. Further clarification of the impact of this expression on angioinvasion of tumor cells may be helpful in understanding the nature of local invasion and metastasis.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Transitional Cell; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Male; Middle Aged; Prognosis; Transforming Growth Factor alpha; Urinary Bladder Neoplasms

Elevated expression of transforming growth factor-alpha (TGF-alpha) gene has been previously reported in some types of human neoplasms, but its role in the pathogenesis of bladder cancer has still not been investigated. In the present study, we analysed 28 samples of early stage bladder tumours for the presence of TGF-alpha mRNA using reverse transcription-polymerase chain reaction (RT-PCR). We detected TGF-alpha mRNA in 71% (20/28) of these samples. When we related the expression levels of TGF-alpha with local relapses of patients during a follow-up of 2 years, we found that a high TGF-alpha expression level in bladder cancer was significantly associated with local relapses in patients with early stage tumours. The appearance of early relapses in tumours with high TGF-alpha expression levels may suggest the existence of an additional marker in the prediction of local relapses in patients with superficial disease.

Topics: Adult; Aged; Female; Humans; Male; Middle Aged; Neoplasm Recurrence, Local; Polymerase Chain Reaction; Prognosis; RNA, Messenger; RNA, Neoplasm; Transforming Growth Factor alpha; Urinary Bladder Neoplasms

To investigate the patterns of expression of transforming growth factor alpha (TGF-alpha) and epidermal growth factor receptor (EGFR) in squamous metaplasia and squamous cell carcinomas of the urinary bladder with and without schistosomiasis.. Immunohistochemical study of the expression of TGF-alpha and EGFR in squamous metaplasias (n = 12) and various grades of squamous cell carcinomas (n = 21) of the bladder with and without schistosomiasis.. Focal cytoplasmic and membranous positivity for EGFR and TGF-alpha was seen in all cases of squamous metaplasia. The markers were diffusely coexpressed in a concordant pattern in areas of hyperplastic keratinising squamous metaplasia. A similar pattern of positivity was seen in verrucous carcinomas (n = 2) and well differentiated squamous carcinomas (n = 6). Progressive loss of differentiation was associated with increasing loss of EGFR staining while TGF-alpha staining was retained. Squamous cell carcinoma in situ (n = 2) showed focal positivity for TGF-alpha and EGFR. There were no differences in staining patterns between cases with and without schistosomiasis.. The coexpression of TGF-alpha and EGFR by well differentiated squamous cell carcinomas and hyperplastic keratinising squamous metaplasia is consistent with the active regulatory role exerted by this autocrine loop. There is regional absence of expression of EGFR but not of TGF-alpha in squamous cell carcinomas of lesser differentiation, suggesting heterogeneity of such control in these tumours. The focal expression of the two markers in squamous cell carcinomas in situ indicates a possible second pathway of oncogenesis for less differentiated tumours. These observations may have important implications for the effectiveness of putative growth factor based treatments.

Topics: Adult; Aged; Carcinoma, Squamous Cell; ErbB Receptors; Female; Humans; Male; Metaplasia; Middle Aged; Neoplasm Proteins; Precancerous Conditions; Schistosomiasis haematobia; Transforming Growth Factor alpha; Urinary Bladder; Urinary Bladder Neoplasms

Determination of the risk of invasive bladder tumors progressing is still imprecise due to the heterogeneous biological behavior of this neoplasm. The goals of this study were to evaluate the patterns of expression of the epidermal growth factor (EGF) system in invasive bladder cancer and to assess its prognostic value.. This immunohistochemical study was performed using fresh frozen tumor samples and a panel of monoclonal antibodies on a series of 43 invasive bladder cancers treated by cystectomy.. EGF was detected in 45% of the tumors and did not correlate with survival from bladder cancer. Transforming growth factor alpha (TGF alpha) was expressed by 60% of the tumors and correlated strongly with death from bladder cancer. Epidermal growth factor receptor (EGF-R) expression was seen in 86% of cases and had no prognostic significance. c-erbB2 was expressed in 50% of cases and was inversely related to a poor prognosis. When EGF and TGF alpha were both expressed, there was little or no expression of c-erbB2.. The accumulation of several growth factors and the relevant receptor are necessary for the progression of invasive bladder cancers. They could be used as indicators of tumor aggressiveness.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Transitional Cell; Disease Progression; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Immunohistochemistry; Male; Middle Aged; Prognosis; Receptor, ErbB-2; Transforming Growth Factor alpha; Urinary Bladder Neoplasms

Studies on epidermal-growth-factor-like-, fibroblast- and transforming growth factors suggested their implication in tumorigenesis involving effects on tumour-cell proliferation and migration. In human transitional-cell carcinomas (TCC), enhanced expression of TGF alpha and EGF receptors correlated with an aggressive phenotype. However, little is known about functions of these growth factors in invasive TCCs. In this study, we performed protein- and RNA-expression studies on a set of growth factors and their receptors on the newly established invasive human TCC cell line designated 1207. The data were correlated with functional proliferation and migration studies. Similar expression patterns of many cellular markers, growth factors and their receptors were noted both in the original TCC tissue and in its derivative cell line, indicating the relevance of this cell line to the investigation of growth factor functions on TCC cells. The proliferation induction by EGF, TGF alpha, amphiregulin, heregulin alpha, FGF-1 and FGF-7 correlated with the presence of EGF receptors, c-erbB4 and FGFR2 (IIIb), respectively. Amphiregulin and heregulin alpha induced the most proliferation. In conformity with the low expression of TGF beta receptors I and II, TGF beta1, barely inhibited proliferation, while TGF alpha induced invasion of 1207 cells into Matrigel. These data support the notion that notably EGF-like proteins mediate TCC growth and invasion through autocrine pathways which can be reinforced by loss of TGF beta1 regulation.

Topics: Animals; Biomarkers; Carcinoma, Transitional Cell; Cell Division; Epidermal Growth Factor; ErbB Receptors; Female; Fibroblast Growth Factors; Growth Substances; Humans; Immunohistochemistry; Mice; Mice, Nude; Neoplasm Invasiveness; Neoplasm Transplantation; Receptors, Fibroblast Growth Factor; Receptors, Growth Factor; Receptors, Transforming Growth Factor beta; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured; Urinary Bladder Neoplasms

Human bladder carcinomas often express high levels of the epidermal growth factor (EGF) receptor. In three human bladder carcinoma cell lines (OBR, T24, and 647V), we show that two EGF receptor ligands, namely EGF and transforming growth factor alpha, enhanced the apoptosis due to serum starvation on cells cultured as monolayers. Conversely, EGF and transforming growth factor alpha prevented apoptosis when the same serum-starved cells were cultured as three-dimensional spheroids. Both stimulation and inhibition of apoptosis by EGF were associated with p21 WAF1/CIP1 overexpression. In 647V spheroids, EGF protection against radiation-induced apoptosis was negated by genistein and tyrphostin AG1478, suggesting that blockade of the EGF signal transduction in patients with bladder cancer may improve the radiotherapy efficacy.

Topics: Apoptosis; Culture Media, Serum-Free; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Epidermal Growth Factor; ErbB Receptors; Genistein; Humans; Isoflavones; Neoplasm Proteins; Nitriles; Protein-Tyrosine Kinases; Quinazolines; Spheroids, Cellular; Time Factors; Transforming Growth Factor alpha; Tumor Cells, Cultured; Tyrphostins; Up-Regulation; Urinary Bladder Neoplasms

We isolated, in vitro, spontaneous variants of the rat bladder tumor NBT-II cell line with a distinctive morphology. Of five sublines obtained, three (NBT-L1, L2a and L2b) exhibited an elongated shape and moderate to high invasive activity in vitro. The other two sublines (NBT-T1 and T2) formed tight colonies and exhibited very low or negligible invasive activity. The contents of mRNAs coding for E-cadherin and cadherin-associated molecules (alpha-catenin and beta-catenin) were not correlated with the invasive activity of the cells. However, the expression level of the E-cadherin protein, but not those of catenins, was lower in invasive cells (NBT-L1, L2a and L2b) than in noninvasive cells (NBT-T1 and T2). Analysis of mRNAs coding for several growth factors and their receptors showed that the transforming growth factor alpha mRNA content in invasive cells was higher than that in noninvasive cells, and that the content of epidermal growth factor receptor mRNA was low in NBT-T2. Although NBT-II is known to acquire a fibroblastic appearance and cell motility in response to several growth factors, the conditioned media of the invasive sublines hardly affected the morphology or motility of noninvasive cells. These results indicate that the decreased E-cadherin expression is closely associated with the transition from the noninvasive to the invasive phenotype of the bladder tumor cells, and that a post-transcriptional process is important in the control of E-cadherin expression in the cells. These sublines may be useful as models for studies on the progression of bladder tumors.

Topics: alpha Catenin; Animals; beta Catenin; Cadherins; Cell Movement; Clone Cells; Culture Media, Conditioned; Cytoskeletal Proteins; Genetic Variation; Growth Substances; Neoplasm Invasiveness; Rats; Receptors, Growth Factor; RNA, Messenger; Trans-Activators; Transcription, Genetic; Transforming Growth Factor alpha; Tumor Cells, Cultured; Urinary Bladder Neoplasms

We have examined levels of epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha) in neoplastic and non-neoplastic bladder tissue using a standard radioimmunoassay technique. Tumour samples had much higher TGF-alpha levels compared with EGF and TGF-alpha levels in malignant tissue were significantly higher than in benign bladder samples. There was, in addition, a difference in mean EGF levels from 'normal' bladder samples from non-tumour bearing areas of bladder in patients with bladder cancer compared with 'normal' bladder tissue obtained at the time of organ retrieval surgery. Levels of EGF and TGF-alpha did not correlate with levels of EGF receptor (EGFR) as determined by a radioligand binding method but levels of TGF-alpha > 10 ng gm-1 of tumour tissue did correlate with EGFR positivity defined using immunohistochemistry. These data suggest that TGF-alpha is the likely ligand for EGFR in bladder tumours.

Topics: Epidermal Growth Factor; ErbB Receptors; Humans; Radioimmunoassay; Radioligand Assay; Transforming Growth Factor alpha; Urinary Bladder Neoplasms

We tested the role of epidermal growth factor (EGF) in the development of low-grade superficial bladder tumors by using a heterotopically transplanted rat urinary bladder system. Weekly EGF administration (250 ng/0.5 ml of phosphate-buffered 2.1% NaCl solution) for 28 weeks into heterotopically transplanted rat urinary bladders initiated with a low dose of N-methyl-N-nitrosourea resulted in a significant increase in the incidence (17 of 25 versus 6 of 30 rats; P < 0.001) and the mean number of tumors per bladder (1.08 versus 0.20; P < 0.001) as compared with those for a vehicle-only group. Changing to vehicle without EGF for the last 8 weeks resulted in tumors in 8 of 24 rats (P = 0.02 versus the EGF group), comparable to the rate for controls. Switching from vehicle to EGF for the last 8 weeks resulted in tumors in 15 of 24 rats, comparable to the rate in the 28-week EGF group. When tumors were divided into two groups according to size (>4.2 mm3 and

Topics: Animals; Carcinoma, Transitional Cell; Epidermal Growth Factor; ErbB Receptors; Gene Expression; In Situ Hybridization; Male; Membrane Glycoproteins; Methylnitrosourea; Neovascularization, Pathologic; Rats; Rats, Inbred F344; RNA, Messenger; Thrombospondins; Transforming Growth Factor alpha; Urinary Bladder Neoplasms

To investigate the roles of growth factors in bladder cancer, changes in the expression of messenger RNAs (mRNAs) for several growth factors and their receptors were examined during rat bladder carcinogenesis induced with N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN). Northern blot analysis showed that the contents of mRNAs for transforming growth factor-alpha (TGF-alpha) and c-met/hepatocyte growth factor (HGF) receptor increased with BBN treatment. Epidermal growth factor (EGF) receptor mRNA was hardly affected by the treatment; while mRNA for fibroblast growth factor (FGF) receptor 1 and transforming growth factor-beta (TGF-beta) type II receptor decreased with BBN treatment. A rat bladder tumor cell line, NBT-II, expressed both TGF-alpha and c-met mRNAs, and HGF showed apparent scattering and growth-stimulating effects on the cells. These results indicate the possibility that TGF-alpha produced by a bladder cancer, in addition to urinary EGF, plays a role in the development of bladder cancer, and that enhanced cell motility due to activation of the c-met/HGF receptor participates in the invasion and metastasis of the cancer cells.

Topics: Animals; Butylhydroxybutylnitrosamine; Carcinoma, Transitional Cell; Cell Division; Cell Movement; Gene Expression; Growth Substances; Hepatocyte Growth Factor; Male; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-met; Rats; Rats, Sprague-Dawley; Receptor Protein-Tyrosine Kinases; Receptors, Growth Factor; RNA, Messenger; Transforming Growth Factor alpha; Tumor Cells, Cultured; Urinary Bladder Neoplasms

Hyperplasia of transitional cell epithelium adjacent to human transitional cell carcinomas (TCC) is a common finding in pathology. This hyperplasia may be a precancerous aberration. Alternatively, it has been suggested that the hyperplasia is due to paracrine action of tumour-derived growth factors. In this study we tested the latter hypothesis using the mouse tumorigenic TCC cell line NUC-1. Transplantation of NUC-1 tumour cells into the urinary bladder submucosa of syngeneic mice in vivo induced hyperplasia of normal adjacent urothelium in all tested mice. Implantation of normal mouse bladder mucosa did not induce urothelial hyperplasia. In vitro, conditioned medium of NUC-1 cells induced the proliferation of the mouse urothelial cell line g/G, which closely resembles normal urothelial cells. This induction was inhibited by transforming growth factor beta 1 (TGF beta 1). Similarly, TGF beta 1 inhibited the fibroblast growth factor-1 (FGF-1) and FGF-2 induced proliferation of g/G cells. Chemico-physical examination, bioassays with conditioned media, and RNA analysis of NUC-1 cells revealed that these cells secreted a growth factor with FGF-like properties. These results indicate that epithelial hyperplasia surrounding carcinomas is not necessarily a precancerous aberration, but may result from direct paracrine action of tumour-derived growth factors.

Topics: Animals; Carcinoma, Transitional Cell; Cell Division; Cell Line; Culture Media, Conditioned; Dithiothreitol; DNA Replication; Epidermal Growth Factor; Epithelium; Fibroblast Growth Factor 1; Fibroblast Growth Factor 2; Granulocyte-Macrophage Colony-Stimulating Factor; Growth Substances; Humans; Hyperplasia; Mice; Mucous Membrane; Neoplasm Transplantation; Thymidine; Transforming Growth Factor alpha; Transforming Growth Factor beta; Transplantation, Isogeneic; Tumor Cells, Cultured; Urinary Bladder; Urinary Bladder Neoplasms

The effect of two growth factors ellicited in response to surgical woundings (transforming growth factor alpha [TGF alpha] and beta 1) on the regulation of cellular plasminogen activator activity (PAA) in human transitional carcinoma cell lines, with differing baseline PAA (253J--high; 647V--low), was studied. mRNA transcript levels of PA regulatory proteins in both cell lines were responsive to TGF alpha and TGF beta 1. However, both the magnitude and nature of the response differed markedly between the two lines. TGF alpha increased uPA transcript levels in both cell lines in a dose-dependent fashion. In the case of uPA receptor, exogenous TGF alpha concentrations which increased receptor levels over fivefold in the 253J line had no effect on this transcript in the 647V line. This differential responsiveness was even more pronounced for TGF beta 1. TGF beta 1 appeared to increase uPA transcript in the 253J line in a dose dependent manner while decreasing transcript levels in the 647V line. uPAr mRNA in 253J cells increased over a 19-fold range in response to TGF beta 1 while this same transcript was decreased 14-fold in 647V cells. The most pronounced effect of TGF beta 1 was seen on PAI1 transcript levels in the 253J line. This transcript increased in a concentration dependent fashion from non-detectable levels. These findings demonstrate that growth factors ellicited by surgical wounding may alter the biology of neoplastic cells. Both the growth factor milieu, and intrinsic cellular regulatory mechanism, appear important in determing net PAA in transitional carcinoma cell lines.

Topics: Carcinoma, Transitional Cell; Dose-Response Relationship, Drug; Humans; Plasminogen Activators; RNA, Messenger; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured; Urinary Bladder Neoplasms

A protein formed by fusion of transforming growth factor alpha with Pseudomonas exotoxin (TGF alpha-PE40) has been shown to have the ability to kill or inhibit the growth of several carcinoma cell lines. This study was designed to evaluate the in vitro cytotoxic effects of TGF alpha-PE40 on rat and human bladder carcinoma cell lines with different biological potential, and normal rat urothelial cells. The rat cell lines used were D44c, LMC19, and MYU3L, which were established in our laboratory. Human cell lines used were RT4, T24, and 253J. As a normal control, we used the first-passage culture of normal rat bladder urothelium (RU-P1). We examined the number and affinity of epidermal growth factor receptors (EGFR) in these cells, the ability of TGF alpha-PE40 to bind EGFR, and the cytotoxic effect of TGF alpha-PE40 and PE40. Rat cell lines, D44c, LMC19, and MYU3L (EGFR = 4.9 x 10(3)-11.4 x 10(3)/cell) had ED50 values (the concentration of TGF alpha-PE40 needed to reduce the viable cell population by 50%) of 180 pM, 540 pM and 6000 pM respectively; for cI (the concentration required to achieve complete inhibition of growth under continuous serum stimulation) TGF alpha-PE40 concentrations of 10(4) pM, 10(4) pM and higher than 10(4) pM respectively were required. Human cell lines, RT4, T24, and 253J (EGFR = 32 x 10(3)-126 x 10(3)/cell) had ED50 values of 20 pM, 66 pM, and 330 pM respectively and T24 showed cI values of 10(3) pM. RU-P1 (EGFR = 92.6 x 10(3)/cell) had the highest ED50 value of 8000 pM. These data indicate that the susceptibility to TGF alpha-PE40 does not always depend on the number of EGFR, that cells having a relatively small number of EGFR respond well to TGF alpha-PE40, and that normal urothelial cells are more resistant to TGF alpha-PE40 than are cancer cells. The differential effect of TGF alpha-PE40 on normal and neoplastic cells provides a rational basis for its use in vivo to control tumor growth.

Topics: Administration, Intravesical; ADP Ribose Transferases; Animals; Bacterial Toxins; ErbB Receptors; Exotoxins; Humans; Pseudomonas aeruginosa; Pseudomonas aeruginosa Exotoxin A; Rats; Recombinant Fusion Proteins; Transforming Growth Factor alpha; Tumor Cells, Cultured; Urinary Bladder; Urinary Bladder Neoplasms; Virulence Factors

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.

Topics: Animals; Base Sequence; DNA Primers; ErbB Receptors; Gene Expression Regulation, Neoplastic; Genes, fos; Genes, jun; Genes, myc; Mice; Mice, Nude; Molecular Sequence Data; Neoplasm Transplantation; Rats; RNA, Messenger; Transfection; Transforming Growth Factor alpha; Tumor Cells, Cultured; Urinary Bladder Neoplasms

A series of rat monoclonal antibodies (MAbs) has been generated against the extracellular domain of the receptor for EGF which block the binding of EGF and TGF alpha to the receptor and inhibit the growth in vitro of a range of carcinoma cell lines that over-express the receptor for EGF. Some of these antibodies were able also to induce the complete regression of xenografts of EGFR-over-expressing tumours when treatment was started, either at the time of tumour inoculation or later when the tumours were established. The most effective of these antibodies was ICR62, which was also able to activate host immune effector functions. We conclude that antibodies which block growth-factor-ligand interaction can have a profound influence on the proliferative capacity of tumour cells in vivo and may have useful clinical application.

Topics: Animals; Antibodies, Monoclonal; Binding Sites; Breast Neoplasms; Carcinoma; Cell Division; Cell Line; Epidermal Growth Factor; ErbB Receptors; Female; Fibroblasts; Head and Neck Neoplasms; Humans; Immunotherapy; Lung Neoplasms; Mice; Mice, Nude; Ovarian Neoplasms; Rats; Rats, Inbred Strains; Recombinant Proteins; Transforming Growth Factor alpha; Transplantation, Heterologous; Tumor Cells, Cultured; Urinary Bladder Neoplasms; Vulvar Neoplasms

TP-40 is a hybrid fusion protein produced by recombinant technology and consists of a molecule of transforming growth factor-alpha (TGF-alpha) fused to the Pseudomonas exotoxin PE-40. A panel of human and murine bladder cancer cell lines was found to be universally sensitive in vitro to TP-40 in a clonogenic assay. All lines expressed receptor for epidermal growth factor (EGF), though none demonstrated gene amplification for the EGF receptor. The sensitivity to TP-40 may be blocked by preexposure to EGF. Six human bladder tumors taken directly from patients were all sensitive in vitro to TP-40; these included well-differentiated tumors. TP-40 may prove to be effective as an intravesical agent in bladder cancer via selective targeting to cells that express EGF receptors, as do the majority of human bladder cancers.

Topics: ADP Ribose Transferases; Animals; Bacterial Toxins; Drug Screening Assays, Antitumor; ErbB Receptors; Exotoxins; Humans; Immunotoxins; In Vitro Techniques; Mice; Pseudomonas aeruginosa; Pseudomonas aeruginosa Exotoxin A; Recombinant Fusion Proteins; Transforming Growth Factor alpha; Tumor Cells, Cultured; Tumor Stem Cell Assay; Urinary Bladder Neoplasms; Virulence Factors

The epidermal growth factor receptor (EGFR) is overexpressed on the superficial layers of malignant urothelium and is suspected of playing a role in tumor progression. TP40 is a chimeric protein composed of transforming growth factor-alpha (TGF alpha) fused to a modified form of Pseudomonas exotoxin A (PE) that is selectively cytotoxic to EGFR-bearing cells and is currently undergoing clinical study for the intravesical therapy of bladder cancer. We constructed a recombinant toxin PE35/TGF alpha-KDEL as an improved agent for the local therapy of EGFR-bearing bladder cancer. PE35/TGF alpha-KDEL does not require intracellular proteolysis to generate a carboxyl-terminal fragment capable of reaching the target cell cytosol and contains a modified carboxyl-terminal sequence KDEL, that increases toxin activity. These features make PE35/TGF alpha-KDEL from 10- to 700-fold more potent than TP40 on four human bladder cancer cell lines. PE35/TGF alpha-KDEL may be a useful agent for treatment of EGFR-bearing cancers.

Topics: ADP Ribose Transferases; Bacterial Toxins; Drug Design; Drug Screening Assays, Antitumor; ErbB Receptors; Exotoxins; Humans; Immunotoxins; In Vitro Techniques; Pseudomonas aeruginosa; Pseudomonas aeruginosa Exotoxin A; Recombinant Fusion Proteins; Recombinant Proteins; Transforming Growth Factor alpha; Urinary Bladder Neoplasms; Virulence Factors

Topics: Animals; Carcinoma; Fibroblast Growth Factor 1; Fibroblasts; In Vitro Techniques; Neoplasm Invasiveness; Neoplasm Metastasis; Rats; Transforming Growth Factor alpha; Tumor Cells, Cultured; Urinary Bladder Neoplasms

The response to growth factor stimulation was evaluated in clonally derived rat bladder carcinoma cell lines, ranging from nontumorigenic to tumorigenic and metastatic, in athymic nude mice. In the nontumorigenic cell line D44c, epidermal growth factor (EGF)/transforming growth factor (TGF) alpha weakly stimulated anchorage-dependent, but not -independent, growth. In tumorigenic/nonmetastatic cells (G1-200 Cl-17), EGF/TGF-alpha stimulated markedly anchorage-independent, but marginally anchorage-dependent growth, whereas TGF-beta 1 inhibited anchorage-independent growth and DNA synthesis. In the highly tumorigenic/metastatic cell line LMC19, EGF/TGF-alpha stimulated anchorage-dependent growth weakly and anchorage-independent growth strongly. In these cells, TGF-beta 1 did not inhibit anchorage-independent growth and DNA synthesis but increased the size of colonies irrespective of the presence of EGF, and some cells were scattered around colonies in soft agar. None of the cell lines showed evidence of TGF-alpha-specific mRNA transcription. Expression of TGF-beta 1 mRNA increased in parallel to the biological aggressiveness of the cell lines. Highly tumorigenic and metastatic cells also demonstrated gelatinase activity involving 72 kilodalton and 92 kilodalton types. Our data suggest that the growth-stimulatory effect of EGF/TGF-alpha in soft agar may be limited to cells that are already tumorigenic and that EGF/TGF-alpha is not effective in making nontumorigenic cells become tumorigenic (or in making nontumorigenic cells grow in soft agar).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Carcinoma, Transitional Cell; Cell Division; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Neoplastic; Genes, p53; Genes, ras; Lung Neoplasms; Male; Mice; Mice, Nude; Neoplasm Proteins; Neoplasm Transplantation; Proto-Oncogene Proteins; Rats; RNA, Messenger; RNA, Neoplasm; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured; Urinary Bladder Neoplasms

Rodent fibroblastic cells transformed by ras oncogenes can grow in serum-free (S-) medium. We have studied clonal lines of mouse NIH3T3 fibroblasts transfected with the EJ-H-ras oncogene, and observed that practically all become independent of exogenous growth and attachment factors shortly after transfection. Moreover, all the clones tested soon form anchorage-independent (AI) colonies in S- medium, and most give rise to spheroids able to grow in suspension. The cell-conditioned S- medium of the transformed (TR) cells stimulates autocrinally the AI and anchored growth of these cells, in the absence of serum, and it contains growth factors related to TGF-alpha (or EGF), PDGF and bFGF, and other uncharacterized factors. Some of these factors are not found, or are found only in very small amounts, in the S- medium of non-transformed NIH3T3 cells, which also stimulates the growth of the TR cells, in the absence of serum. In addition, the TR cells contain 4-6 times more cell-associated bFGF than the non-transformed cells and release more latent TGF-beta activatable by acid treatments. However, no active TGF-beta is secreted by either cell type. Activated TGF-beta and pure TGF-beta 1 stimulate the growth of the anchored TR and NIH3T3 cells, but inhibit the AI growth of the TR cells. Another inhibitor of this growth is also found in the concentrated medium of the NIH3T3 cells.

Topics: 3T3 Cells; Animals; Cell Division; Cell Line, Transformed; Cell Transformation, Neoplastic; Clone Cells; Culture Media, Serum-Free; Epidermal Growth Factor; Genes, ras; Growth Substances; Humans; Mice; Platelet-Derived Growth Factor; Transfection; Transforming Growth Factor alpha; Urinary Bladder Neoplasms

Expression of the epidermal growth factor (EGF) receptor was evaluated by immunohistochemical staining of formalin-fixed, paraffin-embedded tumour tissues employing two antibodies raised to short synthetic peptides from the cytoplasmic domain of the molecule. Both antibodies gave concordant staining of a series of bladder cancers known to express or lack EGF receptors. There was no cross-reaction with the related c-erbB-2 protein, which was also over-expressed in some cases. Cancers with EGF receptor expression also expressed high levels of TGF-alpha, a receptor agonist.

Topics: Antibodies, Monoclonal; ErbB Receptors; Gene Expression; Humans; Immunohistochemistry; Paraffin; Proto-Oncogene Proteins; Proto-Oncogenes; Receptor, ErbB-2; Transforming Growth Factor alpha; Urinary Bladder Neoplasms

Acidic fibroblast growth factor (aFGF) or transforming growth factor-alpha (TGF-alpha), in addition to being mitogenic, induce individual scattering of NBT-II rat bladder carcinoma cell clusters on tissue culture dishes, suggesting that they may contribute to tumor cell dissemination. To assay their scattering potential and their effect on cell invasiveness in a more complex and physiologically relevant model, we analyzed the behavior of NBT-II spheroids confronted with urinary bladder in organotypic cultures. NBT-II spheroids progressively replaced the urothelium at the site of contact with the bladder explant. In the absence of aFGF or TGF-alpha, inserted cells grew in a pattern suggestive of local hyperplasia, with occasional invasive cell protrusions. Exogenous scattering growth factors elicited a more rapid appearance of these protrusions, which were also more numerous. NBT-II cells transfected with cDNA constructs bearing the gene of aFGF, TGF-alpha or the oncogene hst/KFGF were also used. After exogenous or autocrine stimulation of NBT-II cells with the growth factors, a deeper penetration of the bladder wall in the form of nodular outgrowths and clusters of infiltrating cells was always observed. Altogether these observations suggest that the stimulation of NBT-II clusters by scattering/growth factors can promote cell shedding and amplify invasiveness in the complex extracellular environment of bladder tissues.

Topics: Animals; Fibroblast Growth Factor 1; Gene Expression; Growth Substances; Humans; Male; Models, Biological; Neoplasm Invasiveness; Organ Culture Techniques; Rats; Stimulation, Chemical; Time Factors; Transfection; Transforming Growth Factor alpha; Tumor Cells, Cultured; Urinary Bladder; Urinary Bladder Neoplasms