metallothionein and Urinary-Bladder-Neoplasms

Metallothioneins (MTs) were reported to be associated with many kinds of tumors' prognosis, although MTs expression varied greatly among tumors. To assess the prognostic value of Metallothioneins (MTs) in different kinds of tumors, comprehensive literature search was conducted to perform a meta-analysis.. Eligible studies were identified by PubMed, MEDLINE, Web of Science (WOS), the Cochrane Library of Systematic Reviews, EMBASE, China National Knowledge Infrastructure (CNKI), WANFANG database and SinoMed database up to December 2017, which was designed to assess the prognostic value of MTs in different kinds of tumors. The main endpoint events were overall survival (OS) and disease-free survival (DFS). Hazard ratios (HRs) and its variance were retrieved from the original studies directly or calculated using Engauge Digitizer version 4.1. Random or fixed effects model meta-analysis was employed depending on the heterogeneity. Publication bias was evaluated by funnel plots, Begg and Egger tests.. A total of 22 studies were enrolled in this meta-analysis, including 2843 tumor tissues (1517 were MTs negative/low, and 1326 were MTs high). Results showed that there was significant association between MTs expression and tumors' OS (HR = 1.60; 95%CI 1.34∼1.92, P < .00001). Subgroup analysis showed that high level of MTs expression was associated with prolonged OS in liver cancer (HR = 0.65, 95%CI 0.48∼0.89, P = .007), but it was on the contrary in the tumor of ovary (HR = 1.47, 95%CI 1.01∼2.14, P = .04), bladder (HR = 1.71, 95%CI 1.21∼2.42, P = .002), intestine (HR = 3.13, 95%CI 1.97∼4.97, P < .00001), kidney (HR = 3.31, 95%CI 1.61∼6.79, P = .001). However, there was no significant association between MTs expression and OS in breast (HR = 1.02, 95%CI 0.69∼1.51, P = .93).. MTs could be taken as a potential prognostic biomarker for tumors, and uniqueness of MTs prognostic value in liver cancer deserved further study.

Topics: Biomarkers, Tumor; Case-Control Studies; Disease-Free Survival; Female; Humans; Intestinal Neoplasms; Kaplan-Meier Estimate; Kidney Neoplasms; Liver Neoplasms; Male; Metallothionein; Neoplasms; Ovarian Neoplasms; Predictive Value of Tests; Prognosis; Proportional Hazards Models; Urinary Bladder Neoplasms

In oncology patients and clinicians are confronted with the search for measures which could help to elicit the individual patient's risk of future outcome, such as recurrence of disease after primary treatment, response to chemo-therapy or a general outline on the aggressiveness of a given lesion. In patient counselling, the emerging role of evidence based treatment choices reveals with cumulative certainty that the available information is inconclusive. This review will focus on current investigations of determinants to predict response to chemotherapy in advanced bladder cancer or to define prognosis of patients prior to any definite treatment. It will discuss the current evidence for the current systemic treatment options and highlight the many promising approaches of implementing markers either as a basis for a clinical decision in combination with other prognosticators (to better detect individuals at risk or to avoid unnecessary invasive procedures) or as a possible part of relevant pathways to be targeted. It will also discuss the role of biological markers with regards to the relevant clinical question and provide the current evidence to each field. It will highlight the need to further harmonize terminology, approaches and circumstances under which markers are evaluated and will provide suggestions for general methodological principles and guidelines for design, conduct, analysis and reporting of marker studies. The exploration of the current aspects of marker research may outline why collaborative, multicentre, and multidisciplinary efforts should be an integral part of future studies.

Topics: Antineoplastic Combined Chemotherapy Protocols; ATP Binding Cassette Transporter, Subfamily B, Member 1; Biomarkers, Tumor; Chemotherapy, Adjuvant; Clinical Trials as Topic; Drug Resistance, Neoplasm; Glutathione; Humans; Meta-Analysis as Topic; Metallothionein; Predictive Value of Tests; Prognosis; Proto-Oncogene Proteins c-bcl-2; Randomized Controlled Trials as Topic; Treatment Outcome; Tumor Suppressor Protein p53; Urinary Bladder Neoplasms

Topics: Carcinoma, Transitional Cell; Humans; Metallothionein; Prognosis; Retrospective Studies; Urinary Bladder Neoplasms; Urologic Neoplasms

Therapeutic efficacy of cisplatin-based treatment of late stage urothelial carcinoma (UC) is limited by chemoresistance. To elucidate underlying mechanisms and to develop new approaches for overcoming resistance, we generated long-term cisplatin treated (LTT) UC cell lines, characterised their cisplatin response, and determined the expression of molecules involved in cisplatin transport and detoxification, DNA repair, and apoptosis. Inhibitors of metallothioneins and Survivin were applied to investigate their ability to sensitise towards cisplatin. Cell growth, proliferation, and clonogenicity were examined after cisplatin treatment by MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, EdU (5-ethynyl-2'-deoxyuridine) incorporation assay, and Giemsa staining, respectively. Cell cycle distribution and apoptosis were quantified by flow cytometry. mRNA and protein expressions were measured by real-time quantitative (qRT)-PCR, western blot, or immunofluorescence staining. LTTs recovered rapidly from cisplatin stress compared to parental cells. In LTTs, to various extents, cisplatin exporters and metallothioneins were induced, cisplatin adduct levels and DNA damage were decreased, whereas expression of DNA repair factors and specific anti-apoptotic factors was elevated. Pharmacological inhibition of Survivin, but not of metallothioneins, sensitised LTTs to cisplatin, in an additive manner. LTTs minimise cisplatin-induced DNA damage and evade apoptosis by increased expression of anti-apoptotic factors. The observed diversity among the four LTTs highlights the complexity of cisplatin resistance mechanisms even within one tumour entity, explaining heterogeneity in patient responses to chemotherapy.

Topics: Antineoplastic Agents; Apoptosis; Carcinoma; Cell Cycle; Cell Line, Tumor; Cisplatin; DNA Damage; Drug Resistance, Neoplasm; Humans; Metallothionein; Urinary Bladder Neoplasms; Urothelium

Cadmium is discussed as being involved in the development of transitional cell carcinoma (TCC) of the bladder and can be observed in urine of these patients. Investigations of urinary samples from bladder cancer patients and normal controls were carried out with special emphasis on metallothionein (MT)-bound cadmium. Compounds that are constituents of urine were separated in urine samples by means of size exclusion chromatography and cadmium was monitored continuously with a hyphenated inductively coupled plasma mass spectrometry (ICP-MS) system. MT-bound cadmium was quantified by peak area integration, taking into account the intensity of the rhodium signal which was added continuously before ICP-MS detection. The obtained results show that urinary cadmium is predominantly bound to the observed MT-fraction. The median of the MT-bound cadmium concentration in the control group was found to be 0.8 microgL(-1) whereas the cancer group has a median of 1.8 microgL(-1). The variance of the data in the cancer group is much higher than in the controls. However, the urinary MT-bound cadmium is significantly elevated in the cancer group; odds-ratio test: 7.11 (95% C.I.: 1.89-26.80), taking into account the total protein content. Due to the fact that only one main cadmium-containing fraction was observed, there is no necessity to separate the MT-fraction before cadmium determination in urine samples in future studies.

Topics: Adult; Aged; Aged, 80 and over; Animals; Cadmium; Carcinoma; Chromatography, Gel; Female; Humans; Male; Mass Spectrometry; Metallothionein; Middle Aged; Probability; Rabbits; Urinary Bladder Neoplasms

Previous studies have provided some evidence of a possible association between cancer and metallothioneins. Whether this relates to an exposure to carcinogenic metals remains unclear.. In order to examine the association between the expression of metallothioneins and bladder tumors, and to compare the levels of arsenic, cadmium, chromium, lead and nickel in animals with bladder tumors and animals without bladder tumors, 37 cases of bovine bladder tumors and 17 controls were collected. The detection and quantification of metallothioneins in bladder tissue of both cases and controls was performed by immunohistochemistry. And the quantification of metals in tissue and hair was assessed by inductively coupled plasma - mass spectrometry.. Increased expression of metallothioneins was associated with bladder tumors when compared with non-tumoral bladder tissue (OR = 9.3, 95% CI: 1.0 - 480). The concentrations of cadmium, chromium, lead and nickel in hair of cases were significantly higher than those of controls. However, as for the concentration of metals in bladder tissue, the differences were not significant.. Though the sample size was small, the present study shows an association between bladder tumors and metallothioneins. Moreover, it shows that concentrations of metals such as cadmium, chromium, lead and nickel in hair may be used as a biomarker of exposure.

Topics: Adenoma; Animals; Carcinoma; Cattle; Cattle Diseases; Gene Expression Regulation, Neoplastic; Hair; Hemangioma; Metallothionein; Metals; Papilloma; Risk Factors; Trace Elements; Urinary Bladder Neoplasms

Despite similarities in tumor stage and grade the individual outcome of bladder cancer patients is not predictable. The ideal tool for treatment stratification has not yet been found. Metallothionein (MT) overexpression is correlated with poor tumor differentiation, resistance to chemotherapy, and impaired survival in different malignancies. The clinical relevance of MT expression for defining patients at high risk for recurrence or progression was assessed. MT was detected immunohistochemically and evaluated semiquantitively in tumor specimens of 103 male and 19 female patients (transsurethral resection: n = 94, cystectomy: n = 28). Mean age of the patients was 68 (38-87) yr. According to histopathological features, three groups were distinguished for further analysis (pTa-1G1-2, pTis/pT1G3, and muscle invasive tumors). A cutoff value of 50% immunoreactive cells was used for further analysis. The 5-yr tumor specific survival rate was significantly lower in patients with high MT expression (32 vs. 72%). Accordingly, impaired 5-yr recurrence (90 vs. 58%), and progression rates (78 vs. 54%) were associated with high MT expression. All patients suffering from pTis and pT1G3 tumors with MT expression above the cutoff value showed recurrence within less than 40 mo, whereas 26% of those patients with MT expression below the cutoff value remained long-term recurrence free. Long term progression free survival was detected in 75% of pT1G3 patients with MT expression below the cutoff value. In contrast, 68% of pT1G3 tumor patients with MT expression above the cutoff value progressed, all within the first 12 mo after initial tumor resection. A correlation between high MT expression and prognosis was demonstrated especially in pT1G3 and pTis tumors, where >50% MT expression was linked to shorter tumor-specific survival and increased recurrence/progression rates. Thus, MT expression seems to be a promising marker for further risk stratification in the clinical treatment of bladder cancer patients.

Topics: Adult; Aged; Aged, 80 and over; Disease-Free Survival; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Male; Metallothionein; Middle Aged; Neoplasm Recurrence, Local; Neoplasm Staging; Prognosis; Retrospective Studies; Risk Factors; Urinary Bladder Neoplasms

Cell migration is essential to cancer invasion and metastasis and is spatially and temporally integrated through transcriptionally dependent and independent mechanisms. As cell migration is studied in vitro, it is important to identify genes that both drive cell migration and are biologically relevant in promoting invasion and metastasis in patients with cancer. Here, gene expression profiling and a high-throughput cell migration system answers this question in human bladder cancer. In vitro migration rates of 40 microarray-profiled human bladder cancer cell lines were measured by radial migration assay. Genes whose expression was either directly or inversely associated with cell migration rate were identified and subsequently evaluated for their association with cancer stage in 61 patients. This analysis identified genes known to be associated with cell invasion such as versican, and novel ones, including metallothionein 1E (MT1E) and nicotinamide N-methyltransferase (NNMT), whose expression correlated positively with cancer cell migration and tumor stage. Using loss of function analysis, we show that MT1E and NNMT are necessary for cancer cell migration. These studies provide a general approach to identify the clinically relevant genes in cancer cell migration and mechanistically implicate two novel genes in this process in human bladder cancer.

Topics: Cell Movement; Cell Proliferation; Extracellular Matrix; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Metallothionein; Neoplasm Invasiveness; Neoplasm Staging; Nicotinamide N-Methyltransferase; RNA, Small Interfering; Substrate Specificity; Urinary Bladder Neoplasms; Wound Healing

We examined metallothionein (MT) expression in bladder cancer and its relationship to clinicopathologic factors, survival data, and outcome of chemotherapy. In 97 patients who underwent radical cystectomy for bladder cancer, 34 of whom received cisplatin-based chemotherapy, MT expression was evaluated immunohistochemically. Results were correlated with histopathologic data, survival rates, and outcome of chemotherapy. MT overexpression was present in 33 patients (34.0%): strong in 7 (7.2%) and focal in 26 (26.8%). Overexpression was an independent prognostic factor and was significantly associated with poor survival. Patients undergoing chemotherapy showed worse survival if their tumours were MT-positive than if they were MT-negative. MT overexpression predicts unfavorable survival in bladder cancer patients. In those treated with cisplatin chemotherapy, survival is significantly poorer if tumours express MT. Our results show that MT overexpression may mediate resistance to alkylating agents. Therefore, further studies are warranted to define those patients who need a more aggressive therapy.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Agents; Carcinoma, Squamous Cell; Carcinoma, Transitional Cell; Cisplatin; Female; Humans; Male; Metallothionein; Middle Aged; Retrospective Studies; Survival Rate; Treatment Outcome; Urinary Bladder Neoplasms

To determine whether metallothionein (MT) protein expression is associated with clinical outcomes in patients with transitional cell carcinoma (TCC) of the bladder.. Archival pathologic radical cystectomy and transurethrally resected specimens and medical charts were reviewed for 123 patients with TCC. Patients were divided into groups based on the TNM stage, tumor grade, and MT protein expression in the primary tumor. Survival and disease progression were correlated with MT expression.. The mean patient age was 66 years (range 41 to 92). Of the 123 tumors, 21, 13, 18, 24, 17, and 30 were pathologically staged as pTa, pT1, pT2, pT3, pT4, and pTis, respectively; 28, 15, 14, and 66 tumors had a histologic grade of X, 1, 2, and 3, respectively. On univariate analysis, TNM stage and tumor grade predicted survival and progression outcomes. MT expression was detected in 69 (56.9%) of 123 bladder cancer specimens. Greater MT protein expression was associated with worse overall survival, disease-specific survival, disease-free survival, and disease-free progression (P = 0.0004, P = 0.05, P = 0.0008, and P = 0.0005, respectively).. MT protein expression in the primary tumor of TCC specimens appeared to be associated with overall survival, disease-specific survival, disease-free survival, and disease-free progression. This finding requires additional validation using other data sets.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Transitional Cell; Female; Humans; Male; Metallothionein; Middle Aged; Prognosis; Survival Rate; Urinary Bladder Neoplasms

The goal of this study was to determine if the expression of the metallothionein (MT)-1/2 proteins might serve as a biomarker for the development of bladder cancer. A retrospective analysis of MT-1/2 staining was performed on 343 tissue sections from patients referred for the diagnosis of bladder cancer. The specimens were subdivided into six categories: benign, dysplastic, low-grade cancer, high-grade cancer with no evidence of invasion, high-grade cancer with evidence of invasion, and carcinoma in situ. There was no expression of MT-1/2 in benign lesions and low-grade cancers, a low incidence of expression in dysplastic lesions and high-grade cancers with no evidence of muscle invasion, and a significantly increased incidence of MT-1/2 in high-grade cancers that had invaded the underlying matrix. The expression of MT-1/2 varied in intensity from sample to sample and was focal in its expression. It was concluded from these findings that MT-1/2 may be a prognostic marker for cancers that are progressing to invade the underlying stroma of the bladder wall. The expression of MT-1/2 was also determined in a cell culture model of human urothelium that had been malignantly transformed by Cd2+ and As3+ and shown to be capable of tumor formation in nude mice. It was demonstrated that the expression of MT-1/2 in the tumor heterotransplants was similar to the pattern found in archival specimens of high-grade bladder cancers. The MT-1/2 staining in the heterotransplants was focal in pattern, varied in intensity, and highest in the less differentiated cells of the tumor. These findings indicate that the cell culture model may serve to help define the role of MT-1/2 expression in bladder cancer invasion.

Topics: Animals; Arsenic; Biomarkers, Tumor; Cadmium; Carcinoma, Transitional Cell; Cell Line, Transformed; Gene Expression Regulation, Neoplastic; Humans; Metallothionein; Mice; Mice, Nude; Neoplasm Invasiveness; Neoplasm Transplantation; Prognosis; RNA, Messenger; Urinary Bladder Neoplasms; Urothelium

This laboratory has proposed that the third isoform of the metallothionein gene family (MT-3) might be a biomarker for the development of human bladder cancer. Immunohistochemical staining of MT-3 on archival diagnostic specimens showed that only 2 of 63 (3.17%) benign bladder specimens had even weak reactivity for the MT-3 protein. In contrast, 103 of 107 (96.26%) high-grade urothelial cancers and 17 of 17 (100%) specimens of carcinoma in situ stained positive for the MT-3 protein. For low-grade bladder cancer it was shown that 30 of 48 specimens (62.5%) expressed the MT-3 protein. Using a cell culture model (UROtsa), it was demonstrated that expression of the MT-3 protein was not required for malignant transformation of urothelial cells by either Cd(+2) or As(+3). In contrast, it was shown that the cells transformed by Cd(+2) and As(+3) that did not express the MT-3 gene in cell culture, gained expression of MT-3 when grown as heterotransplants in nude mice. The gain in MT-3 expression when cells were grown as heterotransplants was also shown to occur for the MCF-7, T-47D, Hs 578t, MDA-MB-231 breast cancer, and the PC-3 prostate cancer cell lines. An analysis of MT-3 mRNA and protein expression suggested that a posttranscriptional mechanism was responsible for accumulation of the MT-3 protein. The results provide strong evidence that MT-3 could be a biomarker for the development of high-grade bladder cancer and that the expression of the MT-3 gene is not involved in the in vitro malignant transformation of UROtsa cells by Cd(+2) and As(+3).

Topics: Animals; Arsenic; Biomarkers, Tumor; Breast Neoplasms; Cadmium; Cell Transformation, Neoplastic; Cells, Cultured; Female; Humans; Immunohistochemistry; Male; Metallothionein; Metallothionein 3; Mice; Neoplasm Transplantation; Prostatic Neoplasms; Protein Isoforms; RNA, Messenger; Transplantation, Heterologous; Urinary Bladder Neoplasms

The platinum-based chemotherapeutic agent cisplatin is involved in a broad spectrum of activities against human systemic malignancies. However, acquired resistance to cisplatin reduces its clinical efficacy. Elucidation of the molecular basis of cisplatin resistance is required to improve the effectiveness of cisplatin. In the present study, the mechanism of acquired resistance to cisplatin was studied in C3H mice inoculated with MBT-2 murine bladder tumor cells.. C3H mice were subcutaneously inoculated with 1.0 x 10(6) MBT-2 cells/mouse on day 0. The mice were given intraperitoneal injections of 10 micro mol/kg cisplatin and subcutaneous injections of 1000 micro mol/kg propargylglycine, an inhibitor of gamma-cystathionase, once a day for 10 consecutive days from day 11 to day 20.. The metallothionein content of the tumors was increased to twice the control level by repeated administration of cisplatin. Co-administration of propargylglycine reduced metallothionein induction in the tumors and markedly enhanced the antitumor activity of cisplatin. In contrast, the glutathione content in the tumors did not change from the control level after cisplatin administration. The platinum accumulation in tumors treated with cisplatin alone was 1.7-fold greater than when propargylglycine was administered concomitantly. The platinum concentrations changed in accordance with the metallothionein contents.. These observations suggest that metallothionein, but not glutathione or reduced platinum accumulation, might play a role in the acquired resistance to cisplatin of C3H mice inoculated with MBT-2. Moreover, reversal of this resistance might be possible by biochemical modulation of metallothionein.

Topics: Alkynes; Animals; Antineoplastic Agents; Carcinoma, Transitional Cell; Cisplatin; Cystathionine gamma-Lyase; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Enzyme Inhibitors; Female; Glutathione; Glycine; Injections, Intraperitoneal; Metallothionein; Mice; Mice, Inbred C3H; Neoplasm Transplantation; Urinary Bladder Neoplasms

Metallothionein, a low molecular weight intracellular protein, binds mitomycin with high affinity protecting the tumor DNA. We prospectively studied the relationship of metallothionein expression in bladder transitional cell carcinoma and resistance to intravesical mitomycin.. A series of 45 consecutive patients with superficial transitional cell carcinoma treated with intravesical mitomycin were studied. Resected tumor tissues were stained with metallothionein monoclonal antibody E9. Two pathologists scored staining intensity and distribution. All patients were followed with regular flexible cystoscopy.. Median patient age was 73 years (range 44 to 89). Tumor grade was 1 to 3 in 6, 33 and 6 cases, respectively. In 20 patients (44.44%) tumor recurred after mitomycin therapy. Median cytoplasmic staining scores for recurrent and nonrecurrent tumors were 5 (range 0 to 61) and 0 (0 to 14), respectively. Median nuclear staining scores for recurrent and nonrecurrent tumors were 3 (range 0 to 56) and 0 (0 to 11), respectively. Median followup of patients without recurrence was 18 months (range 12 to 36). Nuclear and cytoplasmic staining scores were significantly higher in recurrent than in nonrecurrent tumors. There was no significant relationship of metallothionein expression with tumor grade.. Over expression of metallothionein predicts the resistance of bladder transitional cell carcinoma to intravesical mitomycin therapy.

Topics: Administration, Intravesical; Adult; Aged; Aged, 80 and over; Antibiotics, Antineoplastic; Carcinoma, Transitional Cell; Drug Resistance, Neoplasm; Follow-Up Studies; Gene Expression Regulation, Neoplastic; Humans; Metallothionein; Middle Aged; Mitomycin; Urinary Bladder Neoplasms

The metal binding protein metallothionein (MT) confers drug resistance when MT is induced in tumor tissues. Cisplatin is known to induce MT synthesis in tumor tissues, which may lead to drug resistance. We examined whether a difference in the administration schedule of cisplatin affect the efficiency of MT induction.. Balb/c nude mice were inoculated with NMB-1 human bladder tumor tissues and injected with cisplatin (total dose of 64 micromol/kg) in a single injection or fractioned daily injections. Tumor MT concentration was determined 24 hours after the last injection of cisplatin by mercury binding assay. The effect of pretreatment with ZnCl2 on antitumor activity of cisplatin was examined in NMB-1 bearing mice.. Tumor MT levels increased significantly with the increase in the number of cisplatin injections. Pretreatment of NMB-1 bearing mice with ZnCl2 (200 micromol/kg x 2) caused the same level of MT induction (1.6-fold) as that of fractioned injections of cisplatin (4 x 16 micromol/kg). Pretreatment of NMB-1 bearing mice with ZnCl2 (200 micromol/kg x 2) depressed cisplatin antitumor activity by about 50%.. The induction of MT to a moderate extent (1.6-fold) in NMB-1 tumor inoculated in mice conferred cisplatin resistance. This level of MT induction can be achieved by fractioned daily injections of cisplatin but not by a single injection. Therefore, it is preferable to administer cisplatin as a single injection rather than as fractioned injections to achieve effective antitumor activity with minimum MT induction.

Topics: Animals; Antineoplastic Agents; Carcinoma, Transitional Cell; Cell Line, Tumor; Chlorides; Cisplatin; Drug Administration Schedule; Drug Resistance, Neoplasm; Male; Metallothionein; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Transplantation; Urinary Bladder Neoplasms; Zinc Compounds

Metallothionein (MT) is expressed in various types of human tumors, including transitional cell carcinomas of the urinary bladder, but its biological significance remains unclear. In the present study, the role of MT in urinary bladder carcinogenesis induced by N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) treatment was investigated using C57BL/6 mice. One hundred 5-week-old male C57BL/6 mice were divided into two groups, which were given drinking water with or without 0.05% BBN throughout the experimental period. Subgroups of ten animals from each group were sacrificed at weeks 5, 10, 15, 20 and 25, and urinary bladder samples were examined immunohistochemically for MT, proliferating cell nuclear antigen (PCNA) and apoptosis. MT was found to be abundant in normal-looking mucosa, but decreased with progression from precancerous lesions to invasive carcinoma in the urinary bladder obtained from BBN-treated mice. Lesions could be divided into MT-positive and negative. There was a tendency for greater MT expression in PCNA-positive lesions, while apoptosis was rather associated with MT-negativity. These data suggest that the overexpression of MT may play a role in mouse urinary bladder carcinogenesis.

Topics: Animals; Apoptosis; Butylhydroxybutylnitrosamine; Carcinogens; Immunohistochemistry; Male; Metallothionein; Mice; Mice, Inbred C57BL; Precancerous Conditions; Proliferating Cell Nuclear Antigen; Urinary Bladder; Urinary Bladder Neoplasms

We assessed metallothionein expression and its patterns of distribution as possible prognostic variables in bladder cancer with regard to histopathological parameters.. We stained 91 formalin fixed, paraffin embedded tissue specimens of bladder cancer from 91 patients with no history of treatment using an immunohistochemical technique for metallothionein. Relationships between immunoreactivity for metallothionein and histopathological parameters were examined. In addition, these parameters, including metallothionein, were evaluated as potential prognostic markers.. Metallothionein was detected in 33 of 91 bladder cancers (36.3%). There were significant relationships of metallothionein expression with high grade, high stage and nonpapillary growth pattern tumors. There was no specific correlation of metallothionein expression with the interval to intravesical recurrence. In the 31 patients who underwent radical cystectomy a significant relationship between metallothionein immunoreactivity and cancer specific survival was found.. Our findings suggest that a close correlation exists of metallothionein expression with histopathological parameters and metallothionein expression can be a useful prognostic variable for bladder cancer.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoma, Transitional Cell; Female; Humans; Immunoenzyme Techniques; Male; Metallothionein; Middle Aged; Neoplasm Staging; Prognosis; Survival Rate; Urinary Bladder; Urinary Bladder Neoplasms

The goals of this study were to determine the expression of metallothionein isoform 1 and 2 proteins (MT-1 and MT-2) in bladder cancer and then to determine which MT isoform-specific genes promoted the expression of these proteins. Immunohistochemical analysis disclosed no immunoreactivity for MT-1 and MT-2 (designated as MT-1/2 to reflect the nonspecificity of the antibody for the two isoforms) in cells comprising the normal bladder or in nonmalignant bladder disorders, such as cystitis and interstitial cystitis. Immunohistochemical analysis demonstrated that MT-1/2 was overexpressed in all samples of carcinoma in situ and in high-grade bladder cancer, with variable overexpression in low-grade bladder cancer and dysplastic lesions. The intensity and frequency of MT-1/2 staining correlated with the grade of the tumor. The MT-1 and MT-2 proteins are encoded by a family of eight genes (MT-1A, MT-1B, MT-1E, MT-1F, MT-1G, MT-IH, MT-1X, and MT-2A), and reverse transcriptase-polymerase chain reaction was used to determine which genes were expressed in the normal bladder and in bladder cancer. This analysis demonstrated that both normal and cancerous bladder tissue expressed mRNA for the MT-2A and MT-1X genes. The expression of MT-1E mRNA was variable in both normal bladder and bladder cancer specimens. Comparison of expression relative to that of beta-actin demonstrated that the level of MT-1X mRNA was overexpressed greatly in bladder cancer as compared to the level in normal bladder tissue. In contrast, the level of MT-2A mRNA was similar in both the normal and the bladder cancer specimens. The level of MT-1X expression did not vary with tumor grade. These studies suggest that the overexpression of MT-1/2 protein in bladder cancer is a result of the overexpression of the MT-1X gene.

Topics: Adult; Carcinoma, Transitional Cell; Gene Expression; Humans; Immunohistochemistry; Metallothionein; Protein Isoforms; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Up-Regulation; Urinary Bladder Neoplasms

We studied the susceptibilities to N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN)-induced urinary bladder carcinogenesis of male Long-Evans Cinnamon (LEC), F344 and Long-Evans Agouti (LEA) rats. Male rats (n=21) were given 0.1% BBN in their drinking water from week 6, 8 and 10 for one week, and killed in week 56. The incidences of transitional cell tumors (papillomas plus carcinomas) in BBN-treated LEC and F344 rats were 12% and 76%, respectively (P<0.001, experiment 1), and those in LEC and LEA rats were 11% and 95%, respectively (P<0.001, experiment 2). When male LEC and F344 rats were given 0.1% BBN in their drinking water for 7 days, the intake of BBN and the urinary concentration of its active metabolite, N-butyl-N-(3-carboxypropyl)nitrosamine (BCPN), were higher in the LEC rats (P<0.01). The urinary pHs of untreated LEC and F344 rats were similar between week 6 and 30. The urinary copper concentration was lower in LEC rats before jaundice than in F344 rats, but its concentrations in 28- and 50-week-old LEC rats were 1.7 and 2.3 times those in F344 rats. In a two-stage carcinogenesis study using F344 rats, i.p. injections of cupric nitrilotriacetate increased urinary copper excretion, and inhibited BBN-induced bladder carcinogenesis. In a two-stage carcinogenesis study using LEC rats, oral administration of D-penicillamine decreased urinary copper excretion, and increased BBN-induced bladder cancer, although the difference was not significant. These data show that LEC rats are resistant to bladder carcinogenesis and suggest that urinary copper has a significant role in their resistance.

Topics: Age Factors; Animals; Butylhydroxybutylnitrosamine; Carcinogens; Chelating Agents; Copper; Copper Sulfate; Disease Susceptibility; Hydrogen-Ion Concentration; Iron; Male; Metallothionein; Penicillamine; Rats; Rats, Inbred F344; Rats, Inbred LEC; Rats, Long-Evans; Species Specificity; Urinary Bladder Neoplasms; Urine; Zinc

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

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

Acquired drug resistance is one of the most important problems in cancer chemotherapy. One of the mechanisms proposed to contribute to this phenomenon is the sequestration of alkylating agents by metallothionein (MT) in vivo. In this study cadmium-induced human bladder tumor T24 cells were exposed to the therapeutic agents chlorambucil and melphalan. MT-2a, was shown by capillary electrophoresis to comprise 56% of the MT isoforms in induced cells, and drug adducts of MT-2a were isolated and characterized by HPLC and electrospray ionization mass spectrometry. One to four equivalents of drug were found to be covalently adducted. Major binding sites on metallothionein were located in the C-terminal domain by peptide mapping, consistent with previous studies in vitro.

Topics: Alkylation; Animals; Antineoplastic Agents; Cadmium Chloride; Cell Survival; Chromatography, High Pressure Liquid; Humans; Mass Spectrometry; Metallothionein; Methylation; Protein Isoforms; Rabbits; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Trypsin; Tumor Cells, Cultured; Urinary Bladder Neoplasms

The goal of the present study was to determine if the expression of metallothionein isoform 3 (MT-3) might serve as a biomarker for human bladder cancer. To accomplish this goal, we defined the localization and expression of MT-3 protein and mRNA using fresh and archival biopsy specimens obtained from patients undergoing differential diagnosis for a variety of bladder disorders. We used immunohistochemistry, immunoblot, and RT-PCR analysis to define the localization and expression of MT-3 protein and mRNA. Immunohistochemical analysis disclosed no immunoreactivity for MT-3 in normal bladder cells. The absence of MT-3 expression in the normal bladder was further confirmed by demonstrating that MT-3 mRNA could not be detected using reverse transcriptase-polymerase chain reaction (RT-PCR) or MT-3 protein using immunoblot. Immunohistochemistry also disclosed no immunoreactivity for MT-3 in archival biopsy specimens from patients with interstitial cystitis and related disorders. Immunohistochemical analysis demonstrated that MT-3 was expressed in carcinoma in situ (CIS), high-grade bladder cancer, low-grade bladder cancer, and dysplastic lesions. MT-3 immunostaining was intense in both CIS and high-grade bladder cancer, and low to moderate in low-grade bladder cancer and dysplastic lesions. We determined MT-3 mRNA expression in a subset of these bladder cancer specimens; expression was elevated as compared to that of the housekeeping gene, ss-actin. The cDNA from the RT-PCR reaction primed for MT-3 contained a FokI restriction site, a site unique for MT-3 as compared to other MT family members. In conclusion, this study demonstrates that MT-3 is up-regulated in human bladder cancer and that this up-regulation increases with increasing tumor grade. The finding that MT-3 expression is minimal in normal bladder suggests that MT-3 might be developed into an effective biomarker for bladder cancer.

Topics: Adult; Amino Acid Sequence; Base Sequence; Biomarkers, Tumor; Carcinoma, Transitional Cell; DNA Primers; Gene Expression; Humans; Immunohistochemistry; Metallothionein; Protein Isoforms; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Urinary Bladder; Urinary Bladder Neoplasms

Cisplatin is one of the most potent cytotoxic drugs and in chemotherapy has ameliorated numerous tumors. Nevertheless, resistance to cisplatin is a problem that is encountered in the chemotherapy of urologic tumors, especially transitional cell carcinomas. In order to improve definition of the mechanisms of cisplatin-resistance we established a series of cisplatin-resistant sublines from the cell line RT 112 in increasing concentrations of cisplatin. The most resistant subline CP3 is approximately 10 times more resistant than the parental line and shows a 10-fold cross-resistance against methotrexate, whereas vinblastine and doxorubicin are equally effective in the parental and sublines. Combined treatment of CP3 cells with cisplatin and buthionine sulfoximine (BSO) does not result in enhanced cell kill, thereby ruling out glutathione as a resistance mechanism. However, in comparison with parental cells, CP3 cells are about 1.5 times more resistant against cadmium. On the protein level, the cisplatin-resistant cells reveal an enhanced expression of metallothionein II (MTII), but not MTI, suggesting that the cisplatin resistance we observed in these sublines is at least partly mediated by MTII. These sublines will in the future serve as valuable tools for the analysis of cisplatin resistance, especially in view of metallothionein-mediated resistance mechanisms.

Topics: Antineoplastic Agents; Buthionine Sulfoximine; Cadmium; Carcinoma, Transitional Cell; Cell Division; Cisplatin; Doxorubicin; Drug Resistance; Drug Resistance, Multiple; Glutathione; Humans; Metallothionein; Methotrexate; Tumor Cells, Cultured; Urinary Bladder Neoplasms; Vinblastine

We examined the carcinogenicity of N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in transgenic mice deficient in the metallothionein (MT) I and II genes and in control (129/Sv) mice. Both strains of mice were given BBN for 8 weeks with or without Zn treatment. All mice were killed at 12 weeks after the cessation of BBN administration. BBN induced bladder tumors in 75% of MT null mice and in 43% of 129/Sv mice. The average number of bladder tumors per mouse was significantly higher in MT null mice (1. 18 +/- 0.27) than in 129/Sv mice (0.43 +/- 0.20). Zn treatment suppressed the carcinogenicity of BBN in 129/Sv mice but not in MT null mice. Histopathological examination of the tumors revealed that the malignant potential of bladder tumors in 129/Sv mice was greater than that in MT null mice. These results indicate that MT is an important modulator of carcinogenicity of BBN in the bladder of mice.

Topics: Animals; Butylhydroxybutylnitrosamine; Carcinogenicity Tests; Carcinogens; Carcinoma, Transitional Cell; Chlorides; Metallothionein; Mice; Mice, Transgenic; Urinary Bladder Neoplasms; Zinc Compounds

Tissue from primary tumors was analyzed for 118 patients with urothelial cancer who subsequently received cisplatin-based chemotherapy. Immunohistochemical staining was performed for nuclear p53 reactivity; for two proposed mediators of drug resistance, metallothionein (MT) and P-glycoprotein; and for the cell proliferation marker MIB-1. For each marker, immunoreactivity was expressed as a percentage of positively staining cells, and overall intensity of staining was graded on a scale from 0 to 3. The product of these two measurements was calculated to generate a percentage-intensity index. Clinical data were obtained independently via retrospective chart review. Chemotherapy regimens containing cisplatin (cisplatin, methotrexate, and vinblastine or methotrexate, vinblastine, doxorubicin, and cisplatin) were administered for metastatic disease (n = 64), for locally advanced disease (n = 45), or as an adjuvant treatment (n = 9). The overall response rate was 56% among 99 evaluable patients, and median survival was 12.7 months. By univariate analysis, Eastern Cooperative Oncology Group performance status (P = 0.0025), tumor grade (P = 0.03), percentage of MT staining (P = 0.01), and percentage-intensity index of MT staining (P = 0.04) were significant predictors of response to chemotherapy. The first three of these were significant in a multivariate model (P = 0.05, 0.04, and 0.04, respectively). By subgroup analysis, the percentage of MT staining predicted for response in metastatic disease (P = 0.03), but not in locally advanced disease (P = 0.28). Only performance status was significantly related to overall survival (P = 0.0001, log-rank test) in the whole cohort. Overexpression of MT in the 64 patients with metastatic disease was associated with a shorter survival (P = 0.04). Expression of p53, P-glycoprotein, and MIB-1 did not predict for survival. In conclusion, overexpression of MT is associated with a poorer outcome from chemotherapy, possibly due to cisplatin resistance.

Topics: Adult; Aged; Antigens, Nuclear; Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily B, Member 1; Autoantigens; Carcinoma, Transitional Cell; Cisplatin; Drug Resistance, Neoplasm; Female; Humans; Immunohistochemistry; Ki-67 Antigen; Male; Metallothionein; Middle Aged; Muscle, Skeletal; Neoplasm Invasiveness; Nuclear Proteins; Predictive Value of Tests; Probability; Prognosis; Retrospective Studies; Survival Rate; Tumor Suppressor Protein p53; Urinary Bladder Neoplasms; Urothelium

Metallothionein (MT), a major zinc-binding intracellular protein thiol, has been associated with cytoprotection from heavy metals, antineoplastic drugs, mutagens, and cellular oxidants. Despite its small mass (7 kDa), nuclear partitioning of MT has been observed in both normal and malignant tissues. The factors controlling MT sequestration are unknown. Thus, we examined the regulation of MT subcellular distribution in human cancer cell lines that exhibit prominent nuclear MT. The nuclear disposition of MT was unaltered during cell cycle passage in synchronized cells. MT redistributed to the cytoplasm when cells were exposed to reduced temperature. Cytoplasmic redistribution was also seen in DU-145 and HPC36M prostatic cancer cells after ATP depletion, but not in PC3-MA2 and SCC25/CP cells. Pretreatment with 10 microM CdCl2 did not significantly alter MT distribution but did render all cells sensitive to cytoplasmic redistribution after either reduced temperature or ATP depletion. Thus, nuclear retention of MT is energy requiring and this ability of MT to accumulate in subcellular compartments against its concentration gradient may be important in the capacity of MT to supply Zn or other metals to target sites within the cell.

Topics: Adenosine Triphosphate; Biological Transport; Carcinoma, Transitional Cell; Cell Cycle; Cell Nucleus; Energy Metabolism; Humans; Male; Metallothionein; Molecular Weight; Nuclear Envelope; Prostatic Neoplasms; Temperature; Tumor Cells, Cultured; Urinary Bladder Neoplasms

This study investigated the presence of zinc and expression of metallothionein (MT) in different pathological changes of the rat bladder induced by administration of N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN). Using the Timm staining method, the presence of zinc was observed in normal and benign hyperplastic epithelial cells of the rat bladder, particularly in the malignant bladder tumor, induced by the administration of BBN. Immunohistochemically, MT expression was detected only in noninvasive (30%) and invasive transitional cell carcinoma (80%) of the rat bladder where the tumor cells were rich in zinc. Our data suggest that: (1) growth and development of the rat bladder tumor, especially malignant tumors, may have a high requirement for zinc and (2) MT synthesis may be induced by a high zinc concentration in rat bladder tumor cells.

Topics: Animals; Butylhydroxybutylnitrosamine; Carcinogens; Carcinoma, Transitional Cell; Epithelium; Histocytochemistry; Hyperplasia; Immunohistochemistry; Male; Metallothionein; Neoplasm Invasiveness; Rats; Rats, Wistar; Urinary Bladder; Urinary Bladder Neoplasms; Zinc

In this paper we demonstrate the relationship between the antitumor activity of cis-diamminedichloroplatinum (II) (CDDP) and inducibility of metallothionein (MT) and glutathione (GSH) of genitourinary tumors.. The chemosensitivity test was performed in athymic mice bearing tumors derived from the human tumor cell lines: ACHN (renal cell carcinoma), NMB-1 (urinary bladder transitional cell carcinoma), and NMT-1 (testicular embryonal cell carcinoma). A single dose of CDDP (25 mumol/kg body weight), was administered i.p. to athymic tumor bearing mice. Concentrations of platinum, MT, and GSH were measured in organ and tumor homogenates 24 h after CDDP administration.. We observed that tumors derived from NMB-1 and NMT-1 were very sensitive to CDDP, but ACHN derived tumors were resistant to CDDP. Measurement of platinum concentrations in tumor tissues revealed no correlation to the observed chemosensitivities of the tumors. Furthermore, 24 h after CDDP administration, the levels of MT and GSH in NMB-1 and NMT-1 derived tumors were lower them or equal to those of control mice. In contrast, mice bearing tumors derived from ACHN exhibited a 1.7-fold and a 2.1-fold increase in MT and GSH, respectively, as compared to control mice.. These findings suggest that the inducibility of MT and GSH in tumor tissues following CDDP administration may be a contributing factor in the development of CDDP resistance in renal cell carcinoma.

Topics: Animals; Cisplatin; Drug Resistance, Neoplasm; Glutathione; Humans; Kidney Neoplasms; Male; Metallothionein; Mice; Mice, Nude; Neoplasm Transplantation; Testicular Neoplasms; Tumor Cells, Cultured; Urinary Bladder Neoplasms; Urogenital Neoplasms

Metallothionein (MT) is an intracellular metal-binding protein which has been implicated in various biological roles, including heavy-metal detoxification and zinc and copper homeostasis, and has putative antioxidant properties. High levels of MT have been detected in certain human tumours, but its functions are unclear. The presence of tumour may cause stress conditions along with alterations in host metabolism, such as the redistribution of metals and, subsequently, in changes in hepatic MT isoforms. The distribution of basal levels of MT-1 and MT-11 isoforms in livers of different strains of mice and their induction in mice inoculated with tumour cells are investigated. While Balb-c, C57/BL and CD1 mice strains had an equal distribution of both hepatic MT isoforms, MT-I and MT-II. In addition, MT-I was the predominant isoform synthesised (> 88%) in the livers of all strains of mice at 24 h after injection with either cadmium or zinc salts. After inoculation with human testicular T7800 or T7799 tumour cells, the major form of MT induced in the livers of nude (nu/nu) mice was Zn-MT-I, and its concentration was positively correlated with the size of the inoculated tumours (r2 = 0.85). A similar positive relation was found in the livers of Balb-c mice inoculated with MM45T mouse bladder tumour cells (r2 = 0.96). Following surgical removal of T7800 tumour, hepatic MT concentrations returned to basal values. There was an increase in plasma MT levels in tumour-bearing mice and it was positively correlated with the increase in hepatic MT levels. These results demonstrate a specific increase in hepatic MT-I isoform in tumour-bearing mice, and this may be due to a generalised stress during tumour growth.

Topics: Analysis of Variance; Animals; Cell Line; Chromatography, Gel; Copper; Cytosol; Humans; Liver; Male; Metallothionein; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Nude; Spectrophotometry, Atomic; Teratocarcinoma; Testicular Neoplasms; Transplantation, Heterologous; Tumor Cells, Cultured; Urinary Bladder Neoplasms; Zinc

This study was undertaken to elucidate the mechanism(s) of cross-resistance to cisplatin (CDDP) in a mitomycin C (MMC)-resistant human bladder cancer cell line, J82/MMC. The J82/MMC cell line displayed 2- to 3-fold cross-resistance to CDDP and carboplatin when compared to the parental J82/WT cells. Drug uptake studies revealed that cross-resistance to CDDP in the J82/MMC cell line was independent of reduced platinum accumulation. The J82/MMC cell line exhibited approximately a 1.5-fold resistance to cadmium chloride, an indicator for increased metallothionein (MT) content, when compared to the J82/WT cells. Northern blot analysis showed a 2.7-fold higher level of MT-IIA mRNA in the J82/MMC cell line compared with J82/WT. We have reported previously that, whereas glutathione (GSH) level is comparable in these cells, GSH transferase (GST) activity is significantly higher in the J82/MMC cell line compared with J82/WT. Results of the present study showed that the elevated GST activity in the J82/MMC cell line was due to an over-expression of pi-type GST protein. Although buthionine-S,R-sulfoximine (BSO)-induced GSH depletion significantly enhanced CDDP cytotoxicity in both cell lines, the magnitude of potentiation was markedly higher in J82/MMC cells (about 2.1-fold) relative to J82/WT (about 1.6-fold). Our results suggest that cross-resistance to CDDP in the J82/MMC cell line may be due to alterations in cellular thiols.

Topics: Antimetabolites, Antineoplastic; Biological Transport; Buthionine Sulfoximine; Cadmium; Cadmium Chloride; Carboplatin; Cell Line; Cell Survival; Chlorides; Cisplatin; Drug Resistance; Gene Expression; Glutathione; Glutathione Transferase; Humans; Isoenzymes; Kinetics; Metallothionein; Methionine Sulfoximine; Mitomycin; RNA, Messenger; Tumor Cells, Cultured; Urinary Bladder Neoplasms

Human metallothioneins (hMTs) are a family of highly homologous proteins thought to be critical in cellular protection against toxins. The comparative function of individual isoforms is, however, obscure. Antibodies to individual MT isoforms might help clarify this issue but their generation has been challenging. We now describe a strategy that has successfully produced an epitope-specific antiserum to a major human isoform, hMT IIA. The immunogen, a conjugated tridecyl amino acid synthetic peptide unique to residues 8-20 in hMT IIA (AAGDSCTCAGSCK), yielded an antiserum (B2) that reacted specifically with hMT IIA in a concentration- and titer-dependent manner and showed no reactivity with human or rabbit liver MT I or horse MTs. This antiserum recognized rabbit liver MT II and, surprisingly, also reacted weakly with chicken MT. MT amino acid sequence comparisons and peptide blocking studies suggested aspartate-11 and threonine-14 are important antigenic determinants for B2. Using confocal immunofluorescence microscopy and B2 antiserum, we observed nuclear localization of hMT IIA with human bladder T24 tumor cells in exponential growth but more cytoplasmic localization at confluence. These results suggest the subcellular location of hMT IIA is a function of cell density in T24 bladder carcinoma cells. The general approach of epitope-specific antibodies may be useful for the generation of antibodies to other MT isoforms and for studying the role of individual MT isoforms in biology and toxicology.

Topics: Amino Acid Sequence; Animals; Carcinoma, Transitional Cell; Cell Count; Cell Nucleus; Cytoplasm; Enzyme-Linked Immunosorbent Assay; Epitopes; Horses; Humans; Immune Sera; Metallothionein; Microscopy, Fluorescence; Molecular Sequence Data; Rabbits; Subcellular Fractions; Tumor Cells, Cultured; Urinary Bladder Neoplasms

Metallothionein (MT) in tumor cells has been implicated as one of the factors involved in mechanisms of resistance to anti-cancer drugs, including cis-diaminedichroloplatinum (CDDP) and adriamycin (ADM). The relationship between the expression of MT and chemotherapy with anti-cancer drugs was studied in CDDP- and ADM-resistant human bladder cancer cell lines and tissue samples from clinical cases. In drug-resistant cell lines (T-24/ADM, CI-7/CDDP) established in our laboratory, MT expression was studied by immunohistochemistry using the avidin-biotin peroxidase complex (ABC) method and radioimmunoassay (RIA), using anti-MT antibody. In addition, other potential mechanisms of drug resistance, such as P-glycoprotein expression were examined and the levels of reduced glutathione (GSH), oxidized glutathione (GSSG) and glutathione-S-transferase (GST) determined in these cell lines. The results of these investigations demonstrate that the expression of MT in resistant cell lines increased 2.1- and 2.5-fold when compared with parent cell lines (CI-7, T-24). GSH, GSSG and GST levels were unchanged and P-glycoprotein was not over-expressed. A total of 120 tissue samples from 35 clinical cases of bladder cancer, before and after chemotherapy, were stained for MT which was detected in 10 of the 35 cases before chemotherapy. The incidence of MT expression was significantly higher (p < 0.05) in cases with lower pathological tumor grades.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily B, Member 1; Drug Resistance; Female; Glutathione; Glutathione Transferase; Humans; Male; Metallothionein; Middle Aged; Retrospective Studies; Tumor Cells, Cultured; Urinary Bladder Neoplasms

The expression of metallothionein (MT) in certain tumor cells has been associated with resistance to anticancer drugs. In the present study, we examined the effects of inhibition of MT synthesis on resistance to anticancer drugs of human bladder tumor which were inoculated in nude mice. The results show that pretreatment of tumor-bearing mice with zinc salts increased MT content, both in normal and tumor tissues, with a marked reduction in the antitumor activity of cisplatin, Adriamycin, and melphalan. Injection of propargylglycine, an inhibitor of cystathionase, decreased MT induction by zinc in the tumor and diminished the resistance to these drugs. These results suggest a role for MT in drug resistance in tumors, and injection of propargylglycine may provide a potential means to overcome drug resistance caused by elevation of MT levels in certain tumors.

Topics: Alkynes; Animals; Cisplatin; Colonic Neoplasms; Cysteine; Doxorubicin; Drug Resistance; Female; Fibrosarcoma; Glycine; Humans; Male; Melphalan; Metallothionein; Mice; Mice, Inbred BALB C; Mice, Inbred ICR; Mice, Nude; Pargyline; Sulfates; Tumor Cells, Cultured; Urinary Bladder Neoplasms; Zinc Compounds; Zinc Sulfate

Pre-chemotherapy bladder tumor tissue was retrospectively analyzed in 21 patients treated with neoadjuvant cisplatin, methrotrexate and vinblastine. Immunohistochemical staining for 2 proposed mediators of drug resistance, p-glycoprotein and metallothionein, was performed and compared to chemotherapeutic response and survival. There was no significant relationship between staining for p-glycoprotein and the response to chemotherapy or survival. Patients with no detectable staining for metallothionein were statistically more likely to sustain a complete pathological response (p = 0.029) after chemotherapy than those with any detectable staining. No relationship between metallothionein immunostaining and survival was apparent. This study offers further evidence linking metallothionein in tumor resistance to cisplatin containing chemotherapy regimens.

Topics: Antineoplastic Combined Chemotherapy Protocols; ATP Binding Cassette Transporter, Subfamily B, Member 1; Carcinoma, Transitional Cell; Chemotherapy, Adjuvant; Cisplatin; Cystectomy; Humans; Lymph Node Excision; Metallothionein; Methotrexate; Retrospective Studies; Survival Analysis; Urinary Bladder; Urinary Bladder Neoplasms; Vinblastine

Some 68% of bladder tumors will respond to cisplatin-based chemotherapy but only 30% will have a durable response. Recent studies have suggested that the metallothionein (MT) gene may produce cisplatin drug resistance in cell lines. To determine the role of MT gene overexpression in human tumors resistant to cisplatin, we evaluated 19 bladder tumors, seven of which had been exposed to cisplatin, for MT mRNA expression. By Northern analysis, four of the seven tumors exposed to cisplatin overexpressed the MT gene compared to untreated tumors. Of the three treated tumors without MT overexpression, one was a relapse 4 yr after the last dose of cisplatin and the other two received only one dose of chemotherapy. MT gene overexpression was found in some tumors that had failed cisplatin chemotherapy and may be a mechanism for drug resistance in bladder cancer.

Topics: Base Sequence; Carcinoma, Transitional Cell; Cisplatin; Drug Resistance; Gene Expression Regulation, Neoplastic; Humans; Metallothionein; Molecular Sequence Data; RNA, Neoplasm; Urinary Bladder Neoplasms

The role of metallothionein (MT) in cisplatin (cis-DDP) resistance and renal toxicity was investigated in C3H mice inoculated with mouse bladder tumor (MBT-2). C3H mice were inoculated s.c. with 1 x 10(6) MBT-2 cells/mouse on day 0. Mice were given injections of proparglyglycine (PPG) (500 mumol/kg s.c.) once a day for 3 days from day 7 to day 9 and with ZnSO4 (200 mumol/kg s.c.) once a day for 2 days from day 8 to day 9. cis-DDP (50 mumol/kg i.p.) was administered 10 days after MBT-2 cell inoculation. Since MT contents in the tumor and kidneys were significantly increased by administration of ZnSO4, both the antitumor activity of cis-DDP and its renal toxicity were reduced. However, coadministration of PPG reduced MT induction in tumor without affecting the level of renal MT. As a result, PPG could clearly overcome the MT-mediated cis-DDP resistance of tumors without compromising the protective effect exerted by renal MT on nephrotoxicity of the drug. It was suggested, therefore, that PPG may be a promising adjunct in cancer chemotherapy to overcome the drug resistance of tumors caused by the elevated level of MT.

Topics: Alkynes; Animals; Cisplatin; Cystathionine gamma-Lyase; Drug Resistance; Female; Glutathione; Glycine; Kidney; Metallothionein; Mice; Mice, Inbred C3H; Neoplasm Transplantation; Pargyline; Sulfates; Urinary Bladder Neoplasms; Zinc; Zinc Sulfate

Metallothionein (MT) is a low molecular-metal binding protein with multiple biological functions. Recently, MT has been implicated as a factor involved in resistance to anticancer drugs, which presumably inactivates anticancer drugs, including cisplatin, and doxorubicin. In this report, we investigated the relationship of MT expression with the clinical features in bladder cancer and renal cell carcinoma. In 35 cases of bladder cancer, 10 cases of renal cell carcinoma and 3 cases of normal mucosa of bladder, the expression of MT was immunohistologically examined by avidinebiotin-peroxidase (ABC) staining of paraffin-embedded tissue specimens with anti-MT antibody. Intense MT expression was noted in all cases of normal mucosa of bladder. MT was detected in 10 of 35 cases of bladder cancer, with the incidence of MT expression being significantly higher increases with lower pathological tumor grade. MT was detected in 8 of 10 cases of renal cell carcinoma, and all of the their normal renal tubules showed more intense staining. A number of hypotheses can be proposed from these observations. First, our observation of decreased MT expression in poorly differentiated carcinomas, which are the more proliferating tumors, this suggests correlation of MT expression with proliferative status of cancer. Second, the higher incidence of MT expression in renal cell carcinoma than in bladder cancer may suggest that it is a factor responsible for the lower efficacy of chemo-therapy in renal cell carcinoma than in bladder cancer.

Topics: Aged; Antineoplastic Agents; Carcinoma, Renal Cell; Drug Resistance; Female; Humans; Immunoenzyme Techniques; Inactivation, Metabolic; Kidney Neoplasms; Male; Metallothionein; Middle Aged; Neoplasm Invasiveness; Neoplasm Staging; Urinary Bladder Neoplasms

Metallothionein (MT) in tumor cell has been indicated as one of the factors involved in the mechanism of resistance to anticancer drugs. The relationship between expression of MT and chemotherapy with anticancer drugs was studied with bladder cancer culture cell lines and tissue samples from clinical cases. In drug-resistance cell lines, MT expression was studied by immunohistological staining of the avidin -biotin peroxidase complex (ABC) method, and by the radioimmunoassay (RIA) method, using an anti-MT antibody. As for tissue samples from clinical cases, 114 paraffin embedded samples of 29 cases before and after chemotherapy were subjected to immunohistological staining of MT. Human-bladder cancer cell lines with resistance to anticancer drugs (C1-7/CDDP, T-24/ADM) showed increased of expression of MT compared to each parental cell lines (C1-7, T-24), suggesting relationship of resistance to anticancer drugs and MT expression. In the clinical cases, those with continuous administration such as intra-vesicle chemotherapy or oral administration chemotherapy showed greater incidence of positive staining of MT expression in comparison with cases with fewer administrations, such as intra-arterial infusion therapy or intravenous administration chemotherapy. These results demonstrated that repetitive and continuous administration of anticancer drugs cause increase of MT in bladder cancer cell, which may be a possible mechanism of acquired resistance to anticancer drugs.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Agents; Drug Administration Routes; Drug Resistance; Female; Humans; Immunoenzyme Techniques; Male; Metallothionein; Middle Aged; Radioimmunoassay; Tumor Cells, Cultured; Urinary Bladder Neoplasms

The effects of bismuth nitrate pretreatment on the toxicity and antitumor activity of cis-diamminedichloroplatinum (cisplatin, CDDP) were examined in nude mice that had been inoculated with human bladder-tumor tissue. Pretreatment with bismuth nitrate depressed the renal toxicity of CDDP without compromising its activity against a transplantable human bladder tumor. Renal metallothionein (MT) and bismuth (Bi) levels in nude mice were markedly increased by Bi preadministration, but no significant MT induction was observed in inoculated human bladder-tumor tissue in which only a trace amount of Bi was incorporated. Furthermore, it was confirmed that tumor platinum (Pt) concentrations in CDDP-treated mice were not affected by Bi pretreatment. Thus, the administration of Bi compounds prior to chemotherapy with CDDP may provide an effective mode of treatment for advanced bladder tumors.

Topics: Animals; Bismuth; Cisplatin; Drug Evaluation, Preclinical; Drug Interactions; Drug Therapy, Combination; Humans; Kidney; Male; Metallothionein; Mice; Mice, Nude; Neoplasm Transplantation; Nitrates; Premedication; Urinary Bladder Neoplasms

Metallothionein is a metal binding protein thiol found in high concentrations in the liver and kidney. Recent evidence has linked overexpression of cellular metallothionein with tumor cell resistance to chemotherapeutic drugs, such as alkylating agents and cis-diamminedichloroplatinum (II) (cisplatin). We studied the metallothionein content of 9 human transitional cell carcinomas of the bladder with immunohistochemical methods. All tumors stained positive for metallothionein and the staining was localized almost exclusively to the cytoplasm. Uroepithelium displaying dysplastic changes or carcinoma in situ demonstrated the greatest intensity of staining, while staining in the invasive portions of the tumor was weak and variable. These findings were of interest, since combination chemotherapy of invasive transitional cell carcinoma of the bladder often is ineffective against carcinoma in situ. Normal uroepithelium stained strongly in all 3 patients who experienced disease progression and death, and in only 1 of the 5 who are currently without evidence of disease.

Topics: Aged; Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Transitional Cell; Cisplatin; Cytoplasm; Doxorubicin; Epithelium; Humans; Immunohistochemistry; Metallothionein; Methotrexate; Middle Aged; Urinary Bladder Neoplasms; Vinblastine

When Chinese hamster lung (CHL) cells were cultured in medium containing 25 microM cadmium chloride, resistant cells appeared at a frequency of 0.04%. When one of three tumor promoters, 12-O-tetradecanoylphorbol-13-acetate (TPA), aplysiatoxin and dihydroteleocidin B, was added during selection with cadmium chloride, the frequency of appearance of resistant cells increased more than 50-fold. Two of the resistant clones obtained were characterized. Both clones produced much higher levels of metallothionein I mRNA than the parental CHL cells. Southern blot analysis showed that in these resistant cells, metallothionein I genes were amplified approximately 5-fold. Therefore, it seems that tumor promoters can enhance the frequency of gene amplification. One possible mechanism of the action of tumor promoters in oncogenesis is amplification of activated c-onc genes. Consistent with this idea, it has been reported that c-onc genes are amplified in various cancer cells. We also found that the c-Ha-ras and c-myc genes were amplified in a bladder cancer removed surgically and in a transplanted rat hepatocellular carcinoma, Morris hepatoma 7794A, respectively.

Topics: Animals; Cadmium; Carcinogens; Cells, Cultured; Cricetinae; Cricetulus; Drug Resistance; Gene Amplification; Humans; Liver Neoplasms, Experimental; Metallothionein; Oncogenes; Tetradecanoylphorbol Acetate; Urinary Bladder Neoplasms