epidermal-growth-factor and Carcinoma--Transitional-Cell

We provide a contemporary review of bladder tumor markers and summarize their role as prognostic indicators.. A comprehensive review of the literature on prognostic markers for transitional cell carcinoma of the bladder was performed.. Intense research efforts are being made to identify and characterize better various bladder cancers and their true biological potential. The need to predict which superficial tumors will recur or progress and which invasive tumors will metastasize has led to the identification of a variety of potential prognostic markers. Blood group antigens, tumor associated antigens, proliferating antigens, oncogenes, peptide growth factors and their receptors, cell adhesion molecules, tumor angiogenesis and angiogenesis inhibitors, and cell cycle regulatory proteins have recently been identified. The potential clinical applications of these tumor markers are under active investigation. Recent attention has focused on which tumor markers may predict the responsiveness of a particular bladder cancer to systemic chemotherapy.. At present conventional histopathological evaluation of bladder cancer (tumor grade and stage) cannot predict accurately the behavior of most bladder tumors. With a better understanding of the cell cycle, and cell to cell and cell to extracellular matrix interactions as well as improved diagnostic techniques (immunohistochemistry), progress is being made to identify and characterize other potential prognostic markers for transitional cell carcinoma of the bladder. The ultimate goal is to develop reliable prognostic markers that will accurately predict not only the course but also the response of a tumor to therapy. This information may then be used to dictate more aggressive treatment for tumors that are likely to progress and less aggressive treatment for those that are unlikely to progress. In the future these biological markers may also be used in gene therapy for the treatment of bladder cancer.

Topics: Antigens, Neoplasm; Biomarkers, Tumor; Carcinoma, Transitional Cell; Cell Adhesion Molecules; Cell Cycle Proteins; Epidermal Growth Factor; Forecasting; Humans; Neovascularization, Pathologic; Oncogenes; Prognosis; Urinary Bladder Neoplasms

Transitional-cell carcinoma of the bladder is believed to arise through a series of genetic changes affecting cell growth and proliferation. Two basic types of such genes have been described: protooncogenes and tumor suppressor genes. The former have not been studied extensively in bladder cancer, although there is evidence that c-erb B-2/neu is overexpressed. Loss of specific chromosomal regions, which is common in bladder tumors, may inactivate tumor suppressor genes, of which p53 has received the most attention. Work also has been done on epidermal growth factor and its receptor, yielding evidence that malignant and normal urothelium have different sensitivities to its action. Although several advances must be made before genetic changes come to the clinical forefront, the information now being gained with such speed holds considerable promise for diagnosis and treatment.

Topics: Alleles; Carcinoma, Transitional Cell; Epidermal Growth Factor; ErbB Receptors; Genes, Tumor Suppressor; Humans; Oncogenes; Proto-Oncogenes; Urinary Bladder; Urinary Bladder Neoplasms

Cystoscopic visualization of bladder cancer is an essential method for initial bladder cancer detection and diagnosis, transurethral resection, and monitoring for recurrence. We sought to develop a new intravesical imaging agent that is more specific and sensitive using a polypeptide based NIR (near-infrared) probe designed to detect cells bearing epidermal growth factor receptors (EGFR) that are overexpressed in 80% of urothelial carcinoma (UC) cases. The NIR imaging agent consisted of an elastin like polypeptide (ELP) fused with epidermal growth factor (EGF) and conjugated to Cy5.5 to give Cy5.5-N24-EGF as a NIR contrast agent. In addition to evaluation in human cells and tissues, the agent was tested in canine cell lines and tissue samples with naturally occurring invasive UC. Flow cytometry and confocal microscopy were used to test cell-associated fluorescence of the probe in T24 human UC cells, and in K9TCC-SH (high EGFR expression) and K9TCC-Original (low EGF expression) canine cell lines. The probe specifically engages these cells through EGFR within 15 min of incubation and reached saturation within a clinically relevant 1 h timeframe. Furthermore,

Topics: Animals; Carbocyanines; Carcinoma, Transitional Cell; Cell Line, Tumor; Contrast Media; Dogs; Elastin; Epidermal Growth Factor; ErbB Receptors; Humans; Mice, Nude; Peptides; Recombinant Fusion Proteins; Urinary Bladder Neoplasms

The mechanisms whereby cyclooxygenase-2 (COX-2) overexpression may contribute to bladder carcinogenesis remain unknown. We recently developed a transgenic mouse model overexpressing COX-2 under the control of a bovine keratin 5 (BK5) promoter causing a high incidence of transitional cell hyperplasia (TCH) in the bladder with a proportion of lesions progressing to invasive carcinoma. Microarray gene analysis was employed to determine the effects of COX-2 overexpression on gene expression profiles in the urinary bladder. Statistical analysis revealed that 70 genes were upregulated and 60 were downregulated by twofold or more in bladders from transgenic compared to wild-type mice. Expression Analysis Systematic Explorer (EASE) analysis revealed that genes associated with Immune/Stress Response and Cell Cycle/Proliferation biological processes were overexpressed in the transgenic mice. Relevant downregulated genes included three transforming growth factor (TGF)-beta related genes, Tgfb2, Tgfb3, and Tgfbi. The growth factor epiregulin was the most highly induced gene among those validated by qRT-PCR in TCH of BK5.COX-2 mouse bladders in parallel with increased staining for Ki67. Prostaglandin E(2) (PGE(2)) directly induced the expression of epiregulin mRNA in bladders from wild-type FVB mice ex vivo. We further determined that recombinant epiregulin increased both cell proliferation and Erk phosphorylation in UMUC-3 bladder cancer cells. These results indicate that the response of the mouse urinary bladder to elevated COX-2 expression includes enhanced inflammatory response and induction of cell proliferation. The growth factor epiregulin may play a role in bladder carcinogenesis and may serve as a novel target for the prevention and treatment of bladder cancer.

Topics: Animals; Biomarkers, Tumor; Carcinoma, Transitional Cell; Cell Cycle Proteins; Cell Proliferation; Cells, Cultured; Cyclooxygenase 2; Epidermal Growth Factor; Epiregulin; Female; Gene Expression Profiling; Gene Expression Regulation, Enzymologic; Immunoblotting; Immunoenzyme Techniques; Male; Mice; Mice, Transgenic; Oligonucleotide Array Sequence Analysis; Organ Culture Techniques; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Urinary Bladder Neoplasms

Cleavage of membrane-anchored heparin-binding epidermal growth factor-like growth factor (proHB-EGF) yields a soluble HB-EGF isoform (sHB-EGF), which is an activating epidermal growth factor receptor (EGFR) ligand and a C-terminal fragment HB-EGF-C acting directly in the nucleus. In bladder cancer, overexpression of both HB-EGF and EGFR have been observed, but to the authors' knowledge the prognostic significance of different modes of HB-EGF signaling have remained unclear.. Expression and intracellular localization of HB-EGF and EGFR were examined by immunohistochemistry in paraffin-embedded specimens from 121 patients who underwent cystectomy for bladder cancer. Tumor stage was pTis/pT1 in 7 patients, pT2 in 41 patients, pT3 in 55 patients, and pT4 in 18 patients. Lymph node metastases were present in 32 patients.. Using an antibody directed against the C-terminal domain, HB-EGF expression was detected in the cytoplasm or in the nucleus of tumor cells. EGFR staining was uniform at the plasma membrane. The actuarial 5-year cancer-specific survival of patients with tumors with predominant nuclear HB-EGF staining was 28% compared with 57% if HB-EGF staining was predominantly cytoplasmic (P = .027). Disease outcome of patients with a 'mixed' HB-EGF staining pattern was found to be between that of the 2 former groups. In agreement with previous studies, strong EGFR expression was associated with poor prognosis. Despite strong EGFR expression, predominant cytoplasmic HB-EGF staining was associated with a more favorable outcome, whereas a predominant nuclear pattern defined a subgroup with extremely poor prognosis (5-year tumor-specific survival of 55% vs 13%, respectively; P = .026).. The current study results confirm that EGFR expression is significantly correlated with disease-specific mortality but that the outcome is also influenced by the mode of HB-EGF signaling. Additional nuclear HB-EGF signaling, indicative of increased cleavage of proHB-EGF, appears to enhance the adverse activities. Cytoplasmic HB-EGF staining likely reflects proHB-EGF, which may also exert antiproliferative effects.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoma, Transitional Cell; Cell Nucleus; Cytoplasm; Epidermal Growth Factor; ErbB Receptors; Female; Heparin-binding EGF-like Growth Factor; Humans; Immunohistochemistry; Intercellular Signaling Peptides and Proteins; Male; Middle Aged; Prognosis; Survival Rate; Urinary Bladder Neoplasms

Laminin-5 (LN-5) and cyclooxygenase 2 (COX-2) play important roles in many kinds of cancers. Recently, it has been reported that epidermal growth factor receptor [corrected] (EGFR) and/or human epidermal growth factor receptor [corrected] 2 (HER2) expressions are associated with LN-5 and/or COX-2 expressions in a few carcinoma cell lines and human tumor tissue. LN-5, COX-2, EGFR, and HER2 expressions were examined immunohistochemically in 67 patients with urothelial carcinomas (UCs), and associations among these 4 biomarkers and clinicopathologic characteristics were investigated. Patients were classified into transurethral resection group and cystectomy group based on clinical end points, and prognostic significances of increased expressions were evaluated. Overexpression of LN-5, COX-2, EGFR, and HER2 was observed in 16 (23.9%), 34 (50.7%), 42 (62.7%), and 15 (22.4%) of 67 patients, respectively. LN-5 overexpression was associated high-grade (P = .002), invasive (pTa+1 versus pT2-4, P = .011), and nonpapillary (P = .027) UCs. Concerning EGFR and HER2, high-grade (EGFR, P = .0009; HER2, P = .003) and nonpapillary (EGFR, P = .016; HER2, P = .0002) UCs had a significantly higher overexpression rate. UCs penetrating basal membrane (pT1-4) showed significantly higher overexpression rates than pTa UCs on all biomarkers. In transurethral resection group, LN-5 overexpression could be proved as an independent prognostic parameter for intravesical recurrence (P = .007), whereas in cystectomy group, nodal involvement was an independent prognostic parameter for cause-specific survival (P = .025). The current study showed that the 4 biomarkers were associated with aggressive behaviors of UCs. Above all, LN-5 overexpression was considered to play an important role in intravesical recurrence of superficial UCs.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoma, Transitional Cell; Cyclooxygenase 2; Epidermal Growth Factor; Female; Humans; Immunohistochemistry; Laminin; Lymphatic Metastasis; Male; Membrane Proteins; Middle Aged; Prognosis; Prostaglandin-Endoperoxide Synthases; Receptor, ErbB-2; Survival Analysis; Urinary Bladder Neoplasms; Urologic Surgical Procedures; Urothelium

Heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) accumulates in the nucleus in aggressive transitional cell carcinoma (TCC) cells and this histologic feature is a marker of poor prognosis in human bladder cancer tissues. Here we report that HB-EGF can be exported from the nucleus during stimulated processing and secretion of the growth factor. Production of reactive oxygen species (ROS) resulted in mobilization of the HB-EGF precursor, proHB-EGF, from the nucleus of TCCSUP bladder cancer cells to a detergent-resistant membrane compartment, where the growth factor was cleaved by a metalloproteinase-mediated mechanism and shed into the extracellular space. Inhibition of nuclear export suppressed HB-EGF shedding. Production of ROS resulted in EGF receptor (EGFR) and Akt1 phosphorylation in HB-EGF-expressing cells. HB-EGF also stimulated cell proliferation and conferred cytoprotection when cells were challenged with cisplatin. These findings show that the nucleus can serve as an intracellular reservoir for a secreted EGFR ligand and, thus, can contribute to an autocrine loop leading to cell proliferation and protection from apoptotic stimuli.

Topics: Active Transport, Cell Nucleus; Apoptosis; Carcinoma, Transitional Cell; Cell Growth Processes; Cell Nucleus; Epidermal Growth Factor; ErbB Receptors; Flow Cytometry; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Oxidative Stress; Protein Precursors; Reactive Oxygen Species; Signal Transduction; Urinary Bladder Neoplasms

The aim of this study was to compare the profile of EGFr expression in transitional cell carcinoma of the bladder (TCC) and in oral squamous cell carcinoma (OSCC). In addition, to study the influence of EGF stimulation on the expression of major histocompatibility complex class I antigens, placental alkaline phosphatase (PLAP) as well as changes in tumour cell sensitivity to cisplatin using immunocytochemical staining, a colorimetric assay and SDS-gel electrophoresis. The results showed that: a) strong EGFr expression could be seen in 22/88 (27%) cases of TCCs. In oral tumours the values for non-invasive ameloblastoma and invasive OSCC were 4/25 (16%) and 30/41 (73%) respectively. b) EGFr expression in tumour cell lines paralleled that of tumour biopsies. The number of lines expressing high and low EGFr expression amongst TCCs were 4 and 4 and in OSCCs were 3 and 1 respectively. c) Exposure of tumour cell lines to EGF led to: i) an increase in EGFr expression (stimulatory indices SI, ranged from 1.06 to 2.58) for TCCs but a decrease in the case of OSCCs (SI ranged from 0.01 to 0.85). The corresponding SI values for class I antigens were 0.95-1.16 and 0.10-0.84. ii) A significant reduction in expression of PLAP by OSCC cell lines. iii) An increased susceptibility of OSCC cell lines to cisplatin by as much as 14% (p<0.001). These data demonstrated the overexpression of EGFr in a significant proportion of TCCs. As for oral tumours it depended on whether they were of an invasive or non-invasive type. In the invasive cases the majority overexpressed EGFr. The exposure of OSCC but not TCC tumour cells to EGF resulted in down regulation of EGFr and class I antigens. The expression of PLAP was also significantly reduced. Exposure of OSCC cells to EGF resulted in their increased susceptibility to cisplatin. The data supports the notion that the mitogenic activation of some tumour cells by EGF resulted in a reduction of their immune visibility, differentiation status and an increase in chemosensitivity.

Topics: Alkaline Phosphatase; Antineoplastic Agents; Biopsy; Carcinoma, Squamous Cell; Carcinoma, Transitional Cell; Cisplatin; Dose-Response Relationship, Drug; Drug Interactions; Epidermal Growth Factor; ErbB Receptors; Histocompatibility Antigens Class I; Humans; Isoenzymes; Mouth Neoplasms; Neoplasms; Placenta; Tumor Cells, Cultured; Urinary Bladder; Urinary Bladder Neoplasms

The nm23 gene was initially cloned as a metastasis suppressor gene, but the clinical relevance of nm23-H1 as a metastasis suppressor or prognostic indicator for human cancers remains enigmatic. Given that gene expression is regulated at the tissue-specific level, we studied the molecular mechanisms of nm23-H1 expression in human bladder cancer cell lines and the clinical importance of protein product (NM23-H1) in association with patient outcome (n = 257) by immunohistochemistry. We demonstrated that nm23-H1 is expressed in bladder cancer cells without genomic alterations. High NM23-H1 expression was found in 39 cases (15.2%), intermediate expression in 119 cases (46.3%), and low NM23-H1 in 99 cases (38.5%). NM23-H1 was inversely related to staging classification or tumor size (P < 0.05), with the most significant difference being observed between pTa tumors and those of pT1-pT3 bladder cancer (P = 0.01). Reduced NM23-H1, defined as intermediate and low levels of expression, tended to have a higher risk of tumor metastasis (P = 0.06) or poor longtime survival (P = 0.07). In the subset of grade 2 bladder tumors, reduced NM23-H1 significantly correlated with the occurrence of tumor metastasis or poor patient survival (P < 0.05). These findings overall suggest that nm23-H1 may play an important role in suppressing the early step of carcinogenesis and thus act as an invasion suppressor for human bladder cancer. A prospective study is required to clarify the potential of the molecular marker in prediction of disease progression.

Topics: Carcinoma, Papillary; Carcinoma, Transitional Cell; Cell Division; Cohort Studies; Disease Progression; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Humans; Monomeric GTP-Binding Proteins; NM23 Nucleoside Diphosphate Kinases; Nucleoside-Diphosphate Kinase; Prognosis; ras Proteins; RNA, Messenger; Survival Analysis; Transcription Factors; Tumor Cells, Cultured; Up-Regulation; Urinary Bladder Neoplasms

Although pathologic level of invasion and histologic grade are helpful in predicting the clinical outcome of transitional cell carcinoma of the bladder, they also create uncertainty. Immunohistochemical staining for p53, MIB-1, epidermal growth factor receptor (EGFR), c-erb B-2, and bcl-2 have shown promise as prognostic factors when evaluated singly, although multivariate analyses that include histologic grade and the interactive effects of these markers have not been studied extensively. The authors have initiated a prospective study to determine whether these markers add prognostic information to that provided by level of invasion and histologic grade. This initial report details how these five markers relate to invasion of the bladder after controlling for the effects of histologic grade.. The authors evaluated 229 transitional cell carcinomas in 229 patients using the World Health Organization grading schema and immunohistochemical staining with antigen retrieval for p53, MIB-1, EGFR, c-erb B-2, and bcl-2, and they related these markers to invasion after controlling for grade with a multivariate logistic regression model.. Although Grades 2 and 3 were the most important for predicting invasion, Grade 2 tumors that stained for either MIB-1 or p53 indicated a significantly greater probability of invasion than suggested by grade alone. bcl-2 and p53 had an opposing and interactive effect: when p53 was absent, the presence of bcl-2 implied less probability of invasion; but when both bcl-2 and p53 were present, the protective effect of bcl-2 was no longer observed. Although neither EGFR nor c-erb B-2 were as important as the other three markers in determining the risk of invasion, Grade 3 tumors that stained for one, and especially both, of these markers were less likely to be invasive.. These five markers sort into three interactive pairs: MIB-1 and p53, bcl-2 and p53, and EGFR and c-erb B-2. MIB-1 and p53 together imply a greater probability of invasion. bcl-2 appears to have a dual role, which depends on the presence of accumulated p53. Finally, EGFR and c-erb B-2 related closely to each other and in Grade 3 tumors imply a lesser probability of invasion. It is likely that combinations of markers, or correlations between markers and grades, will yield prognostic information that is more powerful than what histologic grade alone can provide.

Topics: Biomarkers, Tumor; Carcinoma, Transitional Cell; Epidermal Growth Factor; Humans; Immunoenzyme Techniques; Ki-67 Antigen; Logistic Models; Neoplasm Invasiveness; Neoplasm Proteins; Neoplasm Staging; Prognosis; Prospective Studies; Proto-Oncogene Proteins c-bcl-2; Receptor, ErbB-2; Tumor Suppressor Protein p53; 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

The recently identified epidermal growth factor-related peptide cripto-1 has been previously implicated in the development of the malignant phenotype. The identification of gene products that can act as prognostic markers in bladder cancer would be value in determining the management of this heterogeneous group of patients. This study examines cripto-1 expression in benign and malignant bladder using immunohistochemical techniques. The expression of cripto-1 protein in benign and malignant bladder was examined in 45 bladder tumours (Ta/T1 n = 26, T2 n = 5, T3/T4 n = 14) and six benign controls. All 45 tumours showed positive cytoplasmic staining for cripto-1, including areas of carcinoma in situ. None of the six benign controls showed any evidence of positive cripto-1 staining. Twenty-three (60 per cent) bladder tumours had areas of papillary tumour that showed strong positive staining for cripto-1 as opposed to six (29 per cent) sections of histologically normal urothelium adjacent to tumour (P < 0.05). There was no association between cripto-1 staining and tumour grade, stage, or clinical outcome. Cripto-1 protein appears to be specifically expressed in malignant and benign adjacent urothelium of patients with bladder cancer. Its clinical significance, however, remains to be determined.

Topics: Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoma in Situ; Carcinoma, Transitional Cell; Epidermal Growth Factor; Female; GPI-Linked Proteins; Growth Substances; Humans; Immunoenzyme Techniques; Intercellular Signaling Peptides and Proteins; Male; Membrane Glycoproteins; Middle Aged; Neoplasm Invasiveness; Neoplasm Proteins; Urinary Bladder; Urinary Bladder Neoplasms

A novel continuous cell line, designated BC3c, was established from a surgical biopsy of an invasive solid transitional cell carcinoma of the bladder derived from an 82-yr-old Caucasian female. BC3c cells were near-triploid bearing multiple structural and numerical chromosome anomalies. The epithelial origin of the cancer cells was indicated by the expression of cytokeratins 8 and 19 as well as by the absence of mesenchymal markers. Polymerase chain reaction-restriction-fragment length polymorphisms and single-strand conformation polymorphism mutation detection assays did not reveal any mutations in H-ras codon 12 and K-ras codons 12 and 13. In addition, no mutation in specific hot-spot codons of the p53 gene and no accumulation of the p53 protein were observed. BC3c cells grew rapidly in vitro, even in the absence of exogenous growth factors, because they were found to stimulate their growth in an autocrine manner. BC3c cells were found to express the epidermal growth factor-receptor (EGF-r) abundantly, but in contrast to other established bladder cancer cell lines, human recombinant epidermal growth factor inhibited the cells' proliferation in vitro. These features render the newly established bladder cancer cell line BC3c a useful tool for further experimentation.

Topics: Aged; Aged, 80 and over; Carcinoma, Transitional Cell; Cell Division; Epidermal Growth Factor; Female; Humans; Karyotyping; Tumor Cells, Cultured; Urinary Bladder Neoplasms

Using the heterotopically transplanted rat urinary bladder (HTB) model that was developed in our laboratory, we examined the relationship between the duration of epidermal growth factor (EGF) treatment and acquisition of EGF-independence of urinary bladder tumors that were induced by EGF stimulation. After treatment with N-methyl-N-nitrosourea (MNU) (0.25 mg/0.5 ml of 0.9% NaCl once a week for 3 consecutive weeks), animals at week 3 received EGF [250 ng/0.5 ml phosphate-buffered saline (PBS)] into the HTBs once a week for 20, 28, or 36 weeks. For examination of the effect of EGF withdrawal, one half of the rats received the vehicle (PBS) only beginning at week 23 or week 31 for 8 weeks. When animals were examined at week 23, the incidence and the mean number of tumors per bladder were low, irrespective of EGF treatment. In the bladders that had been exposed to EGF during the first 20 weeks after MNU administration, however, both the incidence and the mean number of tumors per bladder had increased significantly at week 31, regardless of whether or not EGF treatment was continued beyond week 23. Between weeks 31 and 39, EGF treatment demonstrated no effect; both the incidence of tumors and the mean number of tumors were the same as those at week 31. These results suggest that EGF exerts its promoting effect only during the early phase of MNU-initiated bladder carcinogenesis, but that its effect becomes manifest during the subsequent 8 weeks. EGF independence may be due to establishment of an autocrine growth-stimulatory mechanism in bladder tumors.

Topics: Animals; Carcinogens; Carcinoma, Transitional Cell; Epidermal Growth Factor; Male; Methylnitrosourea; Neoplasm Invasiveness; Rats; Rats, Inbred F344; Time Factors; Urinary Bladder Neoplasms

The expression of cripto, a novel transforming gene of the epidermal growth factor superfamily, was immunohistochemically examined in 20 cases of urinary bladder, one ureter, three renal pelvic, 18 kidney, nine prostate, three adrenal and four testicular tumors. Three cases of urinary bladder carcinomas, two of grade 2 and one of grade 3 transitional cell carcinoma which were T1a and T3b, and INF beta and INF gamma, respectively, showed positive binding of specific antibody, along with one cortical carcinoma of the adrenal gland positive staining. None of the ureter, renal pelvic, kidney, prostate or testicular tumors showed positive staining. These results indicate that cripto is not frequently expressed in urological tumors.

Topics: Adrenal Gland Neoplasms; Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoma, Transitional Cell; Epidermal Growth Factor; Female; GPI-Linked Proteins; Humans; Intercellular Signaling Peptides and Proteins; Male; Membrane Glycoproteins; Middle Aged; Neoplasm Proteins; Prostatic Neoplasms; Testicular Neoplasms; Urologic 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

The clinical course of transitional cell carcinoma of the bladder can be difficult to predict due to its potential to invade the muscle layer and/or develop to a high grade lesion. Bladder carcinoma can arise from genetic changes that may activate the oncogenes (-c-erbB2, c-erbB1, c-myc, ras, etc.) and/or inactivate the suppressor genes (p53, Rb). The aim of the present study is to continue a study protocol on the molecular biology of bladder tumors.. From January, 1993 to January, 1995, 85 patients were studied. These patients were divided into two groups: the first group comprised 14 controls of urothelial tissue and the second comprised 65 cases of transitional cell carcinoma of the bladder. p53 expression was determined by an immunohistochemical method (NCL-p53-DO7 monoclonal antibody). Quantification of the p8 oncoprotein in cytosol and EGFR (epidermal growth factor receptor) in membrane was performed by ELISA (Oncogene Science) and RIA (Vienna Lab), respectively. A statistically significant relationship between the expression of p53 and EGFR with tumor stage and grade was found. Quantification of p185 and EGFR showed higher values in the tumor tissue than in the control samples, but a worse survival could not be determined.. The present study shows that p53 expression can be considered to be a prognostic factor. It provides useful information on the aggressive behaviour of the tumor and has a direct relation with the survival rates.

Topics: Aged; Aged, 80 and over; Carcinoma, Transitional Cell; Epidermal Growth Factor; Female; Gene Expression Regulation, Neoplastic; Humans; Male; Middle Aged; Neoplasm Staging; Oncogene Proteins v-erbB; Prognosis; Receptor, ErbB-2; Tumor Suppressor Protein p53; Urinary Bladder; Urinary Bladder Neoplasms; Urothelium

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

The high recurrence rate of bladder cancer is probably due to an efficient repopulation of the bladder by residual transformed cells after resection of the tumour. However, the regenerating capacity of the normal urothelial cells is very high. To study the balance between regenerating normal urothelium and out-growth of transformed urothelial cells, we recently developed an in vitro co-cultivation model. With this model system we studied the effects of growth factors and extracellular-matrix components on the intra-epithelial expansion of human T24 bladder-carcinoma cells in primary mouse-bladder explants. Exposure of the cultures to acidic fibroblast growth factor (aFGF) and laminin led to a dramatic increase in the number of invasive T24 cells into the primary urothelium. Epidermal growth factor (EGF) and collagen types I and IV counteracted the infiltration of individual T24 cells. EGF, aFGF, laminin and collagen types I and IV did not directly affect the migration and proliferation of T24 cells. Apparently, the efficacy of invasion of transformed urothelial cells into primary urothelium is not only dependent on the intrinsic characteristics of the transformed cells, but can be influenced to a considerable extent by exogenous components exerting their influence on the normal urothelium. The clinical relevance of this observation needs to be studied further.

Topics: Animals; Carcinoma, Transitional Cell; Collagen; Epidermal Growth Factor; Epithelium; Female; Fibroblast Growth Factor 1; Fibronectins; Humans; Laminin; Mice; Mice, Inbred C3H; Mucous Membrane; Neoplasm Invasiveness; Tumor Cells, Cultured; Urinary Bladder Neoplasms

To confirm our recent finding that epidermal growth factor (EGF) appeared to contribute to the tumor-enhancing effect demonstrated by normal rat urine, we conducted 2 experiments using our heterotopically transplanted rat urinary bladder model. In experiment 1, after a single dose (0.25 mg) of N-methyl-N-nitrosourea (MNU), we intravesically administered EGF (0.05 ml of 500 ng/ml phosphate-buffered saline) once a week for 30 weeks. Instillation of EGF induced a significantly larger number of tumors than did instillation of the vehicle (P = 0.03). EGF without MNU initiation did not induce tumors. In experiment 2, 2 groups received instillation of killed Escherichia coli (5 x 10(8) cells)/0.5 ml phosphate-buffered saline once a week for 4 weeks to expand the MNU-initiated cell population. Subsequent EGF treatment significantly increased the incidence of tumors (P = 0.01). In the groups which did not receive killed E. coli, EGF treatment induced a significantly higher number of tumors than did vehicle treatment (P < 0.001). All of the tumors were low-grade, superficial transitional cell carcinomas. These observations indicate that EGF acts as a growth-stimulating factor on dormant neoplastic cells and thereby increases the number of tumors.

Topics: Animals; Carcinoma, Transitional Cell; Cell Division; Drug Synergism; Epidermal Growth Factor; Male; Methylnitrosourea; Neoplasm Transplantation; Rats; Rats, Inbred F344; 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

Epidermal growth factor (EGF), a potent mitogen and tumor promoter, is excreted in urine permitting it to incubate with urothelial cells. We have previously shown that the distribution of receptors for EGF (EGF-Rs) on malignant urothelium changes to favor contact between EGF-Rs and intraluminal EGF. OBJECTIVES. To determine if concentrations of EGF in voided urine are different in patients with transitional cell carcinoma (TCC) of the bladder than in those without this condition. METHODS. EGF was measured by radioimmunoassay in the urine of 54 patients with newly diagnosed TCC (16 with grade 1, Stage Ta lesions, and 38 with grade 3, Stage T > or = 2 lesions) and 66 pathologic and normal controls without TCC. Subjects were matched for age and good renal function. RESULTS. EGF concentrations were significantly reduced in patients with TCC compared with controls either when uncorrected (p < 0.0001) or corrected (p < 0.000000002) for urinary creatinine excretion. CONCLUSIONS. The reduced concentration of EGF in voided urine supports previous evidence that the urinary EGF/urothelial EGF-R interaction is important for TCC development and growth, and has the potential to serve as a marker of tumor persistence, recurrence, and therapeutic response.

Topics: Age Factors; Aged; Biomarkers, Tumor; Carcinoma, Renal Cell; Carcinoma, Transitional Cell; Case-Control Studies; Creatinine; Epidermal Growth Factor; Female; Humans; Male; Middle Aged; Neoplasm Invasiveness; Neoplasm Staging; Regression Analysis; Sex Factors; Urinary Bladder Neoplasms

Previous studies have demonstrated quantitation of epidermal growth factor receptors (EGFR) to be of prognostic significance in breast, bladder, esophageal and other neoplasms. However, the relatively large quantity of unfixed tissue required for epidermal growth factor radioligand binding assays (RLBA) has precluded its application to cytologic specimens and small biopsy specimens. For this reason we evaluated reverse transcription intron differential polymerase chain reaction (RTIDPCR) as an assay of EGFR gene expression. Squamous cell carcinoma (A431 and SiHa), transitional cell carcinoma (HT1376, T24, RT4), mammary (MCF7) and endocervical (HeLa) adenocarcinoma, and leukemia (K562) cell lines were used to compare RTIDPCR and RLBA. RTIDPCR involved reverse transcription of RNA and amplification of cDNA using primers for beta-actin and EGFR. Good agreement was observed between the RLBA and RTIDPCR results. RNA extracted from fresh cells, Diff-Quik-stained smears and formalin-fixed, paraffin-embedded cell pellet sections yielded similar results. These data suggest that RTIDPCR may be useful in evaluating gene expression by cells processed as cytologic specimens.

Topics: Adenocarcinoma; Base Sequence; Carcinoma, Squamous Cell; Carcinoma, Transitional Cell; Epidermal Growth Factor; ErbB Receptors; Gene Expression; Genes, Neoplasm; Humans; Introns; Leukemia, Myeloid; Molecular Sequence Data; Neoplasms; Polymerase Chain Reaction; RNA, Neoplasm; Staining and Labeling; Tissue Embedding; Tissue Preservation; Tumor Cells, Cultured

Stromal-epithelial interactions may play a key role in tumor growth and metastasis. We have established a model to study the cellular and molecular basis of this paracrine interaction both in vivo and in vitro using a human transitional cell carcinoma cell line (WH). s.c. coinoculation of 1 x 10(6) WH cells with 1 x 10(6) nontumorigenic fetal rat urogenital sinus mesenchymal (rUGM) cells in athymic mice accelerated carcinoma growth 20 times faster than isolated WH cell inoculations and 4 times faster than coinoculations of the same number of NIH-3T3 or human bladder fibroblasts. Characterization of these chimeric tumors with immunohistochemical and DNA dot-blot analyses documented their predominantly human component. To evaluate the underlying mechanisms involved in this paracrine-mediated in vivo tumor growth acceleration, Northern analyses for growth factors (GFs) and extracellular matrix (ECM) expression in the different cell lines, as well as in vitro mitogenic assays, were performed. Northern analysis revealed basic fibroblast growth factor, transforming growth factor alpha, and epidermal growth factor receptor expression by WH cells but not rUGM cells; ECM components (fibronectin and collagens I and IV) were expressed only in the fibroblast cell lines. Cell type-specific paracrine growth factors are produced by cultured stromal and epithelial cells with a 2-3-fold bidirectional increase in WH and rUGM cell growth when cultured with reciprocal cell-type conditioned medium. An autocrine growth loop was observed for WH but not rUGM cells. WH cell growth is stimulated in vitro by low concentrations of transforming growth factor alpha and epidermal growth factor, while rUGM cell growth is stimulated 3-fold by basic fibroblast growth factor. Antiepidermal growth factor receptor antibodies completely inhibited autocrine and paracrine pathways stimulating WH cell growth, while anti-basic fibroblast growth factor antibodies had no inhibitory effect. These observations suggest that autocrine and paracrine growth factor stimulation of WH bladder carcinoma cell growth is most likely mediated by an epidermal growth factor receptor-related pathway. The predominant expression of ECM by fibroblasts in this model suggests that stromal cell ECM components may modulate tumor cell growth and angiogenesis possibly through mechanisms involving cellular adhesion, chemotaxis, or growth factor action.

Topics: 3T3 Cells; Aged; Aged, 80 and over; Animals; Blotting, Northern; Carcinoma, Transitional Cell; Cell Division; Cell Line; Collagen; Culture Media, Conditioned; Epidermal Growth Factor; Epithelium; ErbB Receptors; Fibronectins; Gene Expression; Humans; Male; Mice; Mice, Nude; Neoplasm Transplantation; Rats; RNA, Messenger; Transplantation, Heterologous; Tumor Cells, Cultured; Urinary Bladder Neoplasms

To investigate the proliferative activity (given by the Ki67 index) of the normal, atypical, and neoplastic urothelium and its relation to the cellular reactivity for the epidermal growth factor (EGFr) and transferrin (Tfr) receptors.. The Ki67 index and the level of EGFr and Tfr reactivity were determined on frozen sections from 82 patients with urothelial cancer. Relevant clinical material was reviewed to establish correlations with the degree of atypia and invasion.. Morphologically normal urothelium, whether derived from controls or patients with cancer, exhibited a low Ki67 index (less than 0.1%) and weak receptor reactivity. In transitional cell carcinomas (TCCs) the Ki67 index was increased: it ranged between 0.7% and 10% in non-invasive and exceeded 10% in 88% of the invasive TCCs. Strong positive reactions for EGFr were seen only in invasive TCCs, but in 47% of invasive TCCs the EGFr was not "overexpressed" and did not match the Ki67 index. A better correlation was found between the Ki67 index and the Tfr which was positive in 26% of the non-invasive and in 71% of the invasive tumours. All three variables were increased in severe atypia but varied considerably in lesser degrees of atypia.. Despite the absence of a close correlation, accelerated growth and enhanced receptor expression were characteristic of invasive cancers. These results suggest that the growth rate in TCCs is not causally related to overexpression of growth factor receptors but that the latter is an abnormality which may accompany the malignant phenotype.

Topics: Carcinoma, Transitional Cell; Cell Division; Epidermal Growth Factor; Humans; Male; Middle Aged; Neoplasm Invasiveness; Receptors, Transferrin; Urogenital Neoplasms

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.

Topics: Animals; Carcinoma, Transitional Cell; Cell Division; Collagenases; Epidermal Growth Factor; ErbB Receptors; Glycoproteins; Male; Matrix Metalloproteinase 1; Matrix Metalloproteinase 9; Methylnitrosourea; Mice; Mice, Nude; Neoplasm Invasiveness; Neoplasm Proteins; Rats; RNA, Messenger; RNA, Neoplasm; Tissue Inhibitor of Metalloproteinase-2; Tissue Inhibitor of Metalloproteinases; Transforming Growth Factor beta; Tumor Cells, Cultured; Urinary Bladder Neoplasms; Urine

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

Epidermal growth factor (EGF) concentration in urine was measured with a radioimmunoassay in 40 untreated patients with urothelial tumors and a comparable control group of 25 normals. The concentration of EGF in urine from the patients with bladder, renal pelvic or ureter tumors was lower than normal (p less than 0.05). Patients with T4 tumors had lower concentrations of EGF compared with those of other stages. EGF concentrations after transurethral resection of bladder tumors were significantly elevated compared with the values before operation. These findings suggest that urothelial tumors are related to a low concentration of EGF and that a decrease of EGF is associated with invasion of tumors.

Topics: Carcinoma, Transitional Cell; Epidermal Growth Factor; Female; Humans; Kidney Neoplasms; Male; Radioimmunoassay; Ureteral Neoplasms; Urinary Bladder Neoplasms

Recent studies have shown that orthotopic (transurethral) transplantation of human bladder cancer cell lines into nude mice permits tumor growth that more accurately reflects their clinical malignant status in the original host. We have previously demonstrated that transfection and overexpression of normal or mutated c-Ha-ras genes into a noninvasive human papillary transitional cell carcinoma cell line confer upon these cells an invasive phenotype in vivo with behavior remarkably similar to the clinical behavior of high grade bladder carcinomas. Since elevated expression levels of the epidermal growth factor receptor (EGF-R), in addition to that of c-Ha-ras, have been correlated with transitional cell carcinoma progression, we sought to determine whether up-regulation of the EGF-R had occurred in the invasive high ras expressors and if so, what functional significance this might have. Our results show that invasive cell lines which overexpress the c-Ha-ras gene also have increased epidermal EGF-R expression. This was found to occur at both the protein and mRNA levels, and analysis of the EGF-R promoter/enhancer sequences has revealed a putative AP-1 site which may possibly enhancer sequences has revealed a putative AP-1 site which may possibly serve as a ras response element. In addition, we found that the cells overexpressing the EGF-R had acquired a positive sensitivity to the stimulatory mitogenic effects of EGF. Hence, the results obtained suggest a role for either a normal or a mutated overexpressing Ha-ras in up-regulating the surface EGF-R, possibly through an AP-1 site during human bladder carcinoma progression; they also highlight the potential that EGF may have in cooperating with this EGF-R up-regulation to help mediate enhanced tumor growth.

Topics: Blotting, Northern; Carcinoma, Transitional Cell; Cell Division; Cell Line; Epidermal Growth Factor; ErbB Receptors; Genes, ras; Humans; Neoplasm Invasiveness; Phenotype; RNA, Neoplasm; Up-Regulation; Urinary Bladder Neoplasms

We previously demonstrated that the specific component of rat urine designated as Fraction I (Fr.I), which has been known to enhance carcinogenesis in the rat urinary bladder, contains epidermal growth factor (EGF) and transferrin (TF). The present study was designed to determine whether EGF or TF is responsible for the tumor-enhancing effect of Fr.I. The heterotopically transplanted rat urinary bladder (HTB), which has been developed in our laboratory, was used for the study. Fr.I was prepared from normal rat urine by a method published previously. Fr.I deficient in EGF or TF was prepared by passing this fraction through an Affi-Gel Hz column coupled with anti-rat EGF or TF antibodies, respectively. EGF and TF eluted from the column (designated as eluted EGF and eluted TF) were also tested for tumor-enhancing activity. Fr.I passed through the column coupled with nonimmune rabbit IgG served as control (Fr.I column control). After initiation of carcinogenesis in HTBs by instillation of a single dose of 0.25 mg of N-methyl-N-nitrosourea, test materials were administered into these HTBs once a week for 30 weeks. The results showed that removal of EGF significantly reduced the tumor-enhancing effect of Fr.I (P less than 0.001 as compared to that of the Fr.I column control) and that eluted EGF by itself significantly enhanced the carcinogenesis as compared to that of the vehicle control (P less than 0.006). Removal of TF from Fr.I also reduced the tumor-enhancing effect of Fr.I (P less than 0.01). However, removal of both EGF and TF from Fr.I did not enhance the inhibitory effect demonstrated by the Fr.I which was deficient in EGF. Likewise, combined use of TF and EGF did not exceed the tumor-promoting effect of EGF. The results indicate that EGF in Fr.I may play a significant role in the promotion of bladder carcinogenesis by urine.

Topics: Animals; Carcinoma, Transitional Cell; Drug Synergism; Epidermal Growth Factor; Hyperplasia; Male; Rats; Rats, Inbred F344; Transferrin; Urinary Bladder; Urinary Bladder Neoplasms

To evaluate the distribution and density of epidermal growth factor (EGF) receptors (EGF-Rs) on urothelium, immunohistological studies using a monoclonal antibody to the binding portion of the human EGF-R were performed on frozen specimens of normal urothelium (N = 20), urothelium from patients with nonurothelial urological malignancies (N = 15) and inflammatory diseases (N = 8), low grade superficial transitional cell carcinomas (TCC) (N = 13), high grade superficial or invasive TCC (N = 28), and endoscopically normal appearing urothelium from patients with low grade superficial (N = 5) or high grade (N = 21) TCC elsewhere in the bladder (or ipsilateral renal pelvis/ureter). EGF-Rs are found only on the basal layer of epithelial cells (with scattered representation on intermediate cells) in 95% of normal urothelial specimens and 100% of pathological specimens without urothelial malignancy. Alternatively, 92.3% of specimens of low grade superficial TCC and 100% of high grade TCCs had EGF-Rs richly expressed on the superficial as well as the deeper layers of urothelium. This "malignant" distribution of EGF-Rs was also found on all specimens of endoscopically normal appearing urothelium in patients with TCC elsewhere. The density of EGF-Rs correlated closely with tumor grade on both "premalignant" and frankly neoplastic urothelium. We conclude that the expression of EGF-Rs on urothelium favors the interaction of premalignant and malignant tissue with urinary EGF. To determine if altering the physiochemical environment of urine could interfere with this interaction, the effects of pH on the binding of and growth responses to EGF were assessed on four human TCC cell lines. Scatchard plots demonstrated that varying pH from 5.0 to 7.5 did not significantly change the total number of receptors, but EGF-R affinity was reduced approximately 20-fold as pH decreased from 7.5 to 5 in each TCC target. Similarly, significant growth stimulation by EGF at pH 7.5 was abrogated at pH less than or equal to 7.0 while growth rates in the absence of EGF remained unchanged at lower pHs. It thus appears that urinary acidification may hold promise in the management and prevention of recurrent bladder cancer.

Topics: Adenocarcinoma; Carcinoma, Transitional Cell; Cell Division; Epidermal Growth Factor; Epithelium; ErbB Receptors; Female; Humans; Kinetics; Male; Neoplasm Invasiveness; Prostatic Hyperplasia; Prostatic Neoplasms; Tumor Cells, Cultured; Ureter; Urinary Bladder; Urinary Bladder Neoplasms

We surveyed the tumor-related proteins present in the urine specimens of 118 bladder cancer patients to seek a possible marker enabling future diagnosis and prognosis of this disease. We identified a protein of 180 kDa. by sodium dodecyl sulfate polyacrylamide gel electrophoresis in urine samples subjected to prior adsorption by protein-A conjugated to a sepharose bead. This protein appears to be a glycoprotein because it binds to concanavalin A-conjugated sepharose and can be eluted by alpha-methyl D-mannoside. It does not react immunochemically with antibodies prepared against either carcinoembryonic antigen or epidermal growth factor receptor, both of which have an apparent molecular weight close to 180 kDa. We found this protein in the urine of 74.3% of the patients with transitional cell carcinoma. It was not present in age-matched controls, patients with benign prostatic hyperplasia or patients with 10 other cancers. There was 1 false positive result in a patient with prostate cancer. It does not appear to be associated with urinary tract infection, blood contamination, premedication or anesthesia.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoembryonic Antigen; Carcinoma, Transitional Cell; Electrophoresis, Polyacrylamide Gel; Epidermal Growth Factor; Female; Humans; Male; Middle Aged; Molecular Weight; Proteinuria; Urinary Bladder Diseases; Urinary Bladder Neoplasms; Urinary Tract Infections

Epidermal growth factor (EGF) concentration and 24-hour excretion in urine were measured with a radioimmunoassay in 18 patients previously treated for various types of urinary bladder tumors and a comparable control group of 18 normals. The median concentration of EGF in urine from the patients was 1.50 nmol/l and from normals 3.03 nmol/l. The median 24-hour excretion of EGF in urine from the patients was 1.96 nmol and from normals 3.33 nmol. These differences between patients and normals were statistically significant (p less than 0.05 and p less than 0.005) although there was an overlap in individual values of EGF concentration and excretion in urine from patients and normals. This study suggests that urothelial neoplasia is related to a low concentration and excretion of EGF.

Topics: Aged; Carcinoma, Transitional Cell; Epidermal Growth Factor; Female; Humans; Male; Radioimmunoassay; Urinary Bladder Neoplasms

Epidermal growth factor (EGF) is a cell-regulating polypeptide that appears important to the maintenance and function of some benign tissues and to the transformation and proliferation of certain malignancies. In humans the highest concentrations of EGF are found in the urine. We investigated possible interactions between EGF and normal and neoplastic tissues of the urinary system with indirect immunohistochemical staining of paraffin-embedded tissue sections. A polyclonal antibody directed against mouse EGF but shown to react with human EGF was used in the assays. Positive staining was granular in nature and confined to the cytoplasm. Staining of the renal parenchyma (N = 5) was observed in the epithelium of the proximal and distal tubules and the collecting ducts. There was staining of clear cell (N = 6) and papillary (N = 3) carcinomas of the kidney. Staining of the normal urothelium (N = 5) was limited to superficial cells. All transitional cell (N = 21) and squamous (N = 2) carcinomas of the bladder stained. Subjectively, the staining intensity of the transitional cell carcinomas correlated inversely with tumor differentiation. In light of evidence that internalized, receptor-bound EGF is rapidly degraded, the striking immunohistochemical demonstration of cytoplasmic EGF suggests active synthesis. EGF synthesized by urothelial and renal carcinomas may be involved in an autocrine mechanism of malignant proliferation.

Topics: Adenocarcinoma; Carcinoma; Carcinoma, Papillary; Carcinoma, Squamous Cell; Carcinoma, Transitional Cell; Epidermal Growth Factor; Humans; Immunoenzyme Techniques; Kidney; Kidney Neoplasms; Urinary Bladder; Urinary Bladder Neoplasms

Cultures of human normal urothelial (HU) cells and transitional cell carcinoma (TCC) cell lines were tested for their ability to specifically bind and respond to epidermal growth factor (EGF). The criteria of response investigated were stimulation of growth and induction of ornithine decarboxylase (ODC) activity. All four human TCC cell lines tested were stimulated to grow by EGF (1-100 ng/ml) in a dose related fashion while only one of four HU cell cultures was similarly stimulated, and growth stimulation of this cell line occurred only at early passage. TCC and HU cells bound EGF to similar degrees with Kds of 0.86 nM for TCC and 2.54 nM for HU (P greater than 0.05) and 91637 +/- 9816 (SD) (high affinity) receptors/cell for TCC and 124275 +/- 16841 for HU (P greater than 0.10). Baseline ODC activity was statistically similar in the two TCC and the three HU cell cultures tested. However, EGF induced dose related ODC activity only in the TCC cell lines and not in any of the HU cell cultures. Thus, while both malignant and normal urothelium bind EGF equally well in vitro, only TCC responds to EGF in two parameters relevant to neoplasia: growth and induction of ODC activity.

Topics: Carcinoma, Transitional Cell; Cell Line; Cells, Cultured; Endothelium; Enzyme Induction; Epidermal Growth Factor; ErbB Receptors; Humans; In Vitro Techniques; Kinetics; Ornithine Decarboxylase; Urinary Bladder Neoplasms

Growth promoting properties have been identified in the conditioned serum-free medium from cultures of the human transitional cell carcinoma cell line 647V. This activity appears to reside in a molecule of more than 5000 MW. The factor responsible for 647V growth is distinguished from epidermal growth factor, since it fails to inhibit the specific binding of epidermal growth factor by 647V cells, and 647V cells are not stimulated to grow by epidermal growth factor. It also does not appear to be an insulin-like growth factor-like molecule since it fails to competitively bind with human insulin-like growth factor-carrier protein.

Topics: Binding, Competitive; Carcinoma, Transitional Cell; Carrier Proteins; Cell Line; Culture Media; Epidermal Growth Factor; ErbB Receptors; Growth Substances; Humans; Insulin; Insulin-Like Growth Factor Binding Proteins; Molecular Weight; Peptides; Receptors, Cell Surface; Somatomedins; Urinary Bladder Neoplasms