metallothionein and Carcinoma--Transitional-Cell

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

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

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

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

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

Human kidneys and their associated tumors (nonneoplastic kidney tissues from patients with a transitional cell carcinoma or an adenocarcinoma and the adenocarcinomas themselves) were evaluated for their Zn, Cd, and Cu contents as well as for their metallothionein (MT) level. The total Cd content was correlated with the MT content, and both values were significantly decreased in the adenocarcinomas in comparison with the other tissues. After extraction and separation by anion-exchange chromatography, MT-0 was identified in the nonneoplastic tissues from both the adenocarcinomas as well as the transitional cell carcinomas. Since until now MT-0 protein was only found in human fetal liver and in Zn-stimulated human monocytes, a possible role for this isoform as an oncofetal marker is hypothesized. Separation of the isoforms of MT by reversed-phase high-performance liquid chromatography and sequence analysis showed besides MT-1e and MT-1l the isoform-MT-1g, which is not expressed in the healthy kidney, and MT-1k, an isoform which is not yet demonstrated in renal tissues. We conclude that the expression profile of the MT isoforms in the kidney changes due to the presence of a tumor.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Amino Acid Sequence; Base Sequence; Cadmium; Carcinoma, Transitional Cell; Copper; DNA; Female; Humans; Kidney; Kidney Neoplasms; Male; Metallothionein; Middle Aged; Molecular Sequence Data; Protein Isoforms; Sequence Homology, Amino Acid; Sequence Homology, Nucleic Acid; Zinc

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

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

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

Metallothioneins (MTs) are small cysteine-rich, metal-binding proteins involved in resistance to heavy-metal toxicity, and are known to bind cisplatin. Several experiments suggest possible involvement of MT in cellular resistance to cisplatin. To investigate the relationship between MT expression and cisplatin resistance in urinary tract transitional cell carcinoma (TCC), immunohistochemical staining for MT was performed in 31 untreated TCCs of the urinary tract. The results were compared with the sensitivity of the tumors to cisplatin as assessed by the microtiter succinate dehydrogenase inhibition (mSDI) test. Variable MT expression was observed in all 31 TCCs, but there was no specific correlation between histopathological parameters and MT expression. Fourteen (87.5%) of the 16 TCCs with less than 10% of their tumor cells positive for MT were sensitive to cisplatin. On the other hand, 6 (75.0%) of the 8 TCCs with MT expression by more than 30% of their tumor cells were resistant to cisplatin, and there was a significant correlation between MT expression and cisplatin resistance (p < 0.01). These results suggest the possible involvement of MT in the intrinsic cisplatin resistance of urinary tract TCC and that immunohistochemical investigation of MT may be useful for predicting the response of these tumors to cisplatin therapy.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Transitional Cell; Cisplatin; Drug Resistance; Drug Screening Assays, Antitumor; Female; Humans; Male; Metallothionein; Middle Aged; Urologic Neoplasms

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

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