Human Urinary Bladder Carcinoma Research Articles

Retinoid signaling in immortalized and carcinoma-derived human uroepithelial cells

This paper investigates the presence and functionality of retinoid signaling pathways in human urinary bladder carcinoma and SV40-immortalized uroepithelial cell lines. Only two of eight cell lines were proliferation-inhibited by 10 μM of either all- trans or 13- cis-retinoic acid. Transactivation of the CAT gene under control of a retinoid-responsive element demonstrated functionality of the signaling pathway in both sensitive cell lines and four of six resistant cell lines. Relative RT-PCR analysis of a panel of retinoid-responsive and inducible genes demonstrated changes in expression levels of all the genes in response to-retinoic acid treatment together with numerous aberrations dysregulations. We conclude that retinoid signaling may be a target for inactivation during tumorigenesis by uncoupling gene expression, proliferation and differentiation. Therefore retinoids are more likely to be effective for chemoprevention than for treatment of bladder carcinomas.

Clonality of Tumor-Infiltrating Lymphocytes in Human Urinary Bladder Carcinoma

Clonality of Tumor-Infiltrating Lymphocytes in Human Urinary Bladder Carcinoma

Clonality of Tumor-Infiltrating Lymphocytes in Human Urinary Bladder Carcinoma

The immune system has been implicated in the control of bladder tumor growth. To evaluate the clonality of bladder tumor-infiltrating T lymphocytes (TILs) in vivo, we studied the T-cell antigen receptor (TCR) repertoire in tumor biopsy specimens from 10 patients with transitional-cell carcinoma (TCC) of the bladder. Nine patients had a primary tumor, and one had a multifocal disease, consisting of two bladder tumors and three bilateral upper urinary tract sites of involvement. The following specimens from the nine patients with a primary tumor also were analyzed: a recurrent tumor from four patients, a metastatic lymph node from one patient, and peripheral blood from five patients. We used a high-resolution polymerase chain reaction (PCR) method to determine CDR3 (complementarity-determining region 3) size lengths of TCR beta-chain transcripts. Oligoclonal T-cell expansion was identified in all specimens, with a larger number of expanded clones in the tumors than in peripheral blood. Expanded clones were identified in several beta-chain variable region (BV) subfamilies and varied from one patient to the next and also in different specimens from the same patient. However, a number of clones with the same VJ combination and the same CDR3 size were identified in a given patient (in specimens collected either simultaneously or at different times), suggesting homogeneity in the immunogenic environment. Clonal T-cell expansion in patients with bladder cancer may reflect prolonged exposure of T lymphocytes to tumor antigens. Our findings provide a basis for functional studies to elucidate T lymphocyte-bladder tumor cell interactions.

Vascular architecture of human urinary bladder carcinoma: a SEM study of corrosion casts.

The vascular architecture of five advanced invasive papillary tumours of the urinary bladder was investigated using corrosion casting and scanning electron microscopy. The superficial vasculature was composed predominantly of capillary systems of two types: dense flat networks with numerous interconnections and tightly packed tortuous loops, forming multiple irregular folds that reflected the papillary morphology of the tumours. The capillaries were supplied and drained by numerous straight nonanastomosing arterioles and venules, which arose by way of multiple branching of larger vessels originating from the mucosal plexus of the bladder. Differences between the tumours in the spatial arrangement of these vessels probably reflect different growth dynamics. The intramural parts of the tumours contained a chaotic network of straight, uniform capillaries with numerous sprouts, which was very different from the superficial capillary system. It is postulated that different angiogenesis-targeted growth factors may be expressed in the phases of exophytic growth and muscularis invasion of the tumour, leading to the formation of different microvascular patterns.

Membrane potassium channels and human bladder tumor cells. I. Electrical properties.

These experiments were conducted to determine the membrane K+ currents and channels in human urinary bladder (HTB-9) carcinoma cells in vitro. K+ currents and channel activity were assessed by the whole-cell voltage clamp and by either inside-out or outside-out patch clamp recordings. Cell depolarization resulted in activation of a Ca(2+)-dependent outward K+ current, 0.57 +/- 0.13 nS/pF at -70 mV holding potential and 3.10 +/- 0.15 nS/pF at 30 mV holding potential. Corresponding patch clamp measurements demonstrated a Ca(2+)-activated, voltage-dependent K+ channel (KCa) of 214 +/- 3.0 pS. Scorpion venom peptides, charybdotoxin (ChTx) and iberiotoxin (IbTx), inhibited both the activated current and the KCa activity. In addition, on-cell patch recordings demonstrated an inwardly rectifying K+ channel, 21 +/- 1 pS at positive transmembrane potential (Vm) and 145 +/- 13 pS at negative Vm. Glibenclamide (50 microM), Ba2+ (1 mM) and quinine (100 microM) each inhibited the corresponding nonactivated, basal whole-cell current. Moreover, glibenclamide inhibited K+ channels in inside/out patches in a dose-dependent manner, and the IC50 = 46 microM. The identity of this K+ channel with an ATP-sensitive K+ channel (KATP) was confirmed by its inhibition with ATP (2 mM) and by its activation with diazoxide (100 microM). We conclude that plasma membranes of HTB-9 cells contain the KCa and a lower conductance K+ channel with properties consistent with a sulfonylurea receptor-linked KATP.

Clonality of Tumor-Infiltrating Lymphocytes in Human Urinary Bladder Carcinoma

The immune system has been implicated in the control of bladder tumor growth. To evaluate the clonality of bladder tumor-infiltrating T lymphocytes (TILs) in vivo, we studied the T-cell antigen receptor (TCR) repertoire in tumor biopsy specimens from 10 patients with transitional-cell carcinoma (TCC) of the bladder. Nine patients had a primary tumor, and one had a multifocal disease, consisting of two bladder tumors and three bilateral upper urinary tract sites of involvement. The following specimens from the nine patients with a primary tumor also were analyzed: a recurrent tumor from four patients, a metastatic lymph node from one patient, and peripheral blood from five patients. We used a high-resolution polymerase chain reaction (PCR) method to determine CDR3 (complementarity-determining region 3) size lengths of TCR beta-chain transcripts. Oligoclonal T-cell expansion was identified in all specimens, with a larger number of expanded clones in the tumors than in peripheral blood. Expanded clones were identified in several beta-chain variable region (BV) subfamilies and varied from one patient to the next and also in different specimens from the same patient. However, a number of clones with the same VJ combination and the same CDR3 size were identified in a given patient (in specimens collected either simultaneously or at different times), suggesting homogeneity in the immunogenic environment. Clonal T-cell expansion in patients with bladder cancer may reflect prolonged exposure of T lymphocytes to tumor antigens. Our findings provide a basis for functional studies to elucidate T lymphocyte-bladder tumor cell interactions.

Decreased expression of the p16/MTS1 gene without mutation is frequent in human urinary bladder carcinomas.

The p16 (CDKN2,MTS1) gene is located at 9p21 and its product, p16, inhibits the cyclin D/CDK4 complex. Loss of heterozygosity on chromosome 9p is very common in human bladder carcinomas and has been found in all stages of lesions, suggesting that it occurs early in bladder tumor progression. Several studies have revealed frequent homozygous deletion of the p16 gene in cell lines, and that such deletions are also common in some types of cancers. In addition, point mutations in the p16 gene have been identified in several types of neoplasia. In the present examination of urinary bladder tumors, no p16 gene mutations were detected, but nine cases out of 23 (39%) showed decreased mRNA expression, revealed by the reverse transcriptase polymerase chain reaction. There were no histological differences apparent between those cases with normal and those with decreased p16 expression. These results indicate that while p16 gene mutations may be rare, changes in the level of the p16 transcripts could play a role in human bladder carcinoma development.

Human urinary bladder-carcinoma cells are non-osteoinductive.

Small pieces (ca. 2-3 x 3-5 mm) of the urinary-tract mucosa from noninvasive papillary transitional-cell carcinomas of the bladder (ca. urotheliale papillare, n = 33), invasive transitional-cell carcinomas of the bladder (ca. urotheliale papillare infiltrans, n = 6, papillary transitional-cell carcinomas of the bladder with squamous metaplasia (ca. urotheliale papillare cum metaplasia planoepitheliale, n = 4), transitional-cell carcinomas in situ (ca. urotheliale in situ, n = 2), and squamous-cell carcinomas of the bladder (ca. planoepitheliale, n = 2) were grafted intramuscularly into cortisone-immunosuppressed mice to test the ability of transformed transitional epithelium to induce heterotopic osteogenesis. Altogether, 156 implants from 47 cases of urinary bladder carcinoma were performed. Histological examination of implants, excised 10-17 days later, revealed relatively good survival of the grafted epithelium, which had proliferated and, in some cases, formed cysts and islands but failed to induce heterotopic osteogenesis in the surrounding host tissues. In nine implants prepared from four cases (noninvasive papillary transitional-cell carcinoma of the bladder and invasive papillary transitional-cell carcinoma of the bladder, two cases each) a small amount of cartilage and/or bone was found in the stroma of grafted tissue. The rarity of this phenomenon--together with the observation that implants of normal human urinary-tract mucosa have never induced the formation of cartilage/bone, whereas in a similar system, dog or guinea-pig grafts are osteogenic--suggests that the cartilage/bone present in the stroma of implanted cancers is the result of metaplasia of the stroma of the neoplasm and not the product of any osteoinductive potency of human urothelium.

Morphology of Ras-Transformed Cells Becomes Apparently Normal Again with Tyrosine Kinase Inhibitors without a Decrease in the Ras-GTP Complex1

Radicicol, an inhibitor of protein-tyrosine kinase, was found to cause morphological reversion of v-Ha-ras-transformed NIH3T3 fibroblasts and T24 human urinary bladder carcinoma cells that contain an activated ras mutation. The network of actin stress fibers was restored during the treatment with radicicol. A similar morphological change was observed with another protein-tyrosine kinase inhibitor, herbimycin A. Radicicol did not cause any changes in the proportion of the active GTP binding form of p21ras or its subcellular localization. These results rule out the possibility that the morphological reversion by radicicol is due to direct or indirect inhibition of the p21ras function. Cycloheximide and actinomycin D inhibited the morphological change by radicicol, suggesting that the induced transcription of a gene(s) followed by de novo protein synthesis is required for suppression of the transformed phenotype in ras-transformed cells by tyrosine kinase inhibitors.

Stimulation of TIMP-1 and metalloproteinase production in co-cultures of human tumor cells and human fibroblasts

The co-cultures of five different human tumor cell lines with human normal fibroblasts significantly stimulated the production of tissue inhibitors of metalloproteinases-1 (TIMP-1) when compared to cultures of individual cells. In the co-culture of T24 human urinary bladder carcinoma cells and CCD18 human fibroblasts, production of both TIMP-1 and metalloproteinases was stimulated, and the stimulatory effects were dependent on the cellular ratio between the fibroblasts and carcinoma cells. On day 6 of culture, collagenase and stromelysin were stimulated at a ratio of CCD18 fibroblasts to T24 cells of 1:0.1, while the maximum TIMP-1 production occurred at a ratio of 1:1. Thus, the cellular ratio in the interaction of carcinoma cells with host fibroblasts affects the production of TIMP-1 and metalloproteinases and hence modulates the balance between them.

P53 mutations in early neoplastic lesions of the urinary bladder in rats treated with N-butyl-N-(4-hydroxybutyl)nitrosamine.

Rat experimental models using N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) as an initiating agent have been widely used to study carcinogenic processes in the urinary bladder. In this study, early neoplastic lesions from 10 male F344 rats treated with 0.05% BBN for 16 weeks were analyzed for changes in the H-ras or p53 genes by polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) analysis and subsequent DNA sequencing. Lesions were pooled for each of the 10 rats and six showed point mutations in the p53 gene and one in the H-ras gene. These results would indicate that BBN-induced rat urinary bladder carcinomas are similar to human urinary bladder carcinomas with respect to alterations in the p53 and H-ras genes and that p53 gene alterations are relatively early events in rat urinary bladder carcinogenesis induced by BBN treatment.

Inhibition of fibroblast growth by polyanions; effects of dextran sulfate and lignin derivatives

When macrophages prepared from rat bone marrow were cultured for more than one week, contaminating fibroblasts grew and formed large colonies which were observable with the naked eye after Giemsa staining. The addition of dextran sulfate or watersoluble lignin derivatives (acetyl or sulfonyl) to the culture medium almost completely inhibited the growth of fibroblasts at 10 g/ml. These polyanions also inhibited the growth of chick embryo fibroblasts in a dose-dependent manner: at 5 g/ml the inhibitory effect was 20–30% of the control and at 100 g/ml it was 50–70%. In addition, these polyanions inhibited the mitosis of hepatocytes, although their inhibitory effects on the growth of hepatocytes were smaller than those observed on the growth of fibroblasts. On the other hand, these polyanions did not affect the growth of human urinary bladder carcinoma cells (HUB-4 and HUB-15). In conclusion, dextran sulfate and lignin derivatives may be useful in eliminating fibroblasts from certain cells.

Frequency and mechanism of Lewis antigen expression in human urinary bladder and colon carcinoma patients

Changes in the expression of Lewis antigens have been associated with cancer diseases, and recent results have pointed at a possible increased risk of cancer development among Lewis negative patients. The frequency of the erythrocyte Lewis phenotypes Le(a- b+), Le(a+ b-) and Le(a- b-) was analysed in patients suffering from urinary bladder cancer (82), colon cancer (21), and benign urological diseases (45). An increased frequency of Lewis negative individuals was found among colon cancer patients (P less than 0.004) and bladder cancer patients (P = 0.05). The Lewis negative phenotype was shown to be associated with unfavourable disease parameters: invasion (P less than 0.02) and high grade of atypia (P less than 0.01) in bladder cancer patients, and high Dukes stage (P less than 0.05) in colon cancer patients. alpha 1-4fucosyltransferase activity (Lewis transferase) was shown to be present in saliva from four out of eight erythrocyte Lewis negative cancer patients, indicating that some patients with advanced cancer disease may have converted from a Lewis positive to a Lewis negative phenotype.

Immunohistochemical Analysis of c‐erbB‐2 Oncogene Product and Epidermal Growth Factor Receptor Expression in Human Urinary Bladder Carcinomas

Expression of the product of the c-erbB-2 gene, a proto-oncogene related to, but distinct from c-erbB-1 encoding the epidermal growth factor receptor (EGF-R), was investigated in human urinary bladder carcinomas. In addition, levels of EGF-R and transferrin receptor were also analyzed using an immunohistochemical approach, and the results compared with histological pattern and grading, and tumor staging. Increased expression of c-erb B-2 product was found in 32% of cases (7/22), a positive reaction being observed in 60% of transitional cell carcinoma (TCC) Grade 3 lesions (3/5), 20% of Grade 2 TCCs (2/10) and 100% of adenocarcinomas (AC) (2/2), but in none of the cases of squamous cell carcinoma (SCC). Although no statistical correlation with staging was evident, TCCs or SCCs of high grade and stage often showed EGF-R-positive staining, whereas other well differentiated lesions and normal bladder epithelium were generally negative. Most cases of urinary bladder carcinoma were positive for the transferrin receptor, which was not detected in normal bladder. The results thus suggested that a positive reaction for c-erbB-2 product is correlated with TCC histological grading or AC morphology. A high intensity of EGF-R staining in human bladder carcinomas may be associated with poor differentiation and invasion, whereas transferrin receptor expression might reflect tumor growth.

Establishment and Characterization of a Human Urinary Bladder Carcinoma Cell Line (TSGH-8301)

A cell line derived from a well-differentiated human transitional cell carcinoma of the urinary bladder, designated TSGH-8301, was established in vitro. The cultured epithelioid cells exhibited monolayer growth and loss of contact inhibition. The tumorigenicity of TSGH-8301 had been shown by growth in soft agar and tumor induction in athymic nude mice. A reverse ratio of lactate dehydrogenase (LHD) isoenzyme in the cell line and nude mouse-grown tumors was seen predominantly with LDH-V. Chromosomal analysis revealed a heterodiploid stem line with a modal number of 50. Sera of urinary bladder cancer patients reacted with membrane antigens of the TSGH-8301 cells, suggesting the existence of tumor-associated antigens in the cells. In vitro chemosensitivity tests of these cells may provide data valuable in the selection of proper anticancer drugs for the TSGH-8301 donor patient.

Establishment and characterization of a human urinary bladder carcinoma cell line (TSGH-8301).

A cell line derived from a well-differentiated human transitional cell carcinoma of the urinary bladder, designated TSGH-8301, was established in vitro. The cultured epithelioid cells exhibited monolayer growth and loss of contact inhibition. The tumorigenicity of TSGH-8301 had been shown by growth in soft agar and tumor induction in athymic nude mice. A reverse ratio of lactate dehydrogenase (LHD) isoenzyme in the cell line and nude mouse-grown tumors was seen predominantly with LDH-V. Chromosomal analysis revealed a heterodiploid stem line with a modal number of 50. Sera of urinary bladder cancer patients reacted with membrane antigens of the TSGH-8301 cells, suggesting the existence of tumor-associated antigens in the cells. In vitro chemosensitivity tests of these cells may provide data valuable in the selection of proper anticancer drugs for the TSGH-8301 donor patient.

Induction of alkaline phosphatase activity by synergistic action of dibutyryl cyclic adenosine monophosphate, prednisolone, butyrate and sodium chloride in cultured cells.

Human urinary bladder carcinoma cells (JTC-32) retain a low alkaline phosphatase activity. Prednisolone or a hypertonic concentration of NaCl caused a moderate increase in the activity (10- to 15-fold of control), but dibutyryl cAMP or butyrate did not. Examination of the combined effect of these four agents revealed that they acted synergistically in any combination. When the cells were incubated with the four agents together, the enzyme activity increased 60- to 250-fold. Serum also contributed to this synergistic increase. These agents slightly inhibited cell growth and protein synthesis. The enzyme induction was completely inhibited by cycloheximide or actinomycin D. The synergistic effect of the four agents on the enzyme activity was also observed in other strains of carcinoma cells, human urinary bladder carcinoma cells (JTC-30) and monkey hepatocarcinoma cells (NCLP-6E). Thus, it is concluded that the coexistence of the four agents provides general and superior conditions for the induction of alkaline phosphatase in cultured carcinoma cells.

The autorelease of alkaline phosphatase from the plasma membrane during the incubation of cultured liver cell homogenates.

When a rat hepatoma cell (R-Y121B) homogenate was incubated at 37 degrees C, 30-70% of the total alkaline phosphatase was released into the supernatant fluid from the precipitate fractions. The release reached a plateau level after 10 h of incubation at 37 degrees C. The optimum pH value for the release was 7.4. Alkaline phosphatase activity increased during the incubation of the cell homogenates, but this increase was independent of the enzyme release. Serum increased not only alkaline phosphatase activity in the cultured cells but also enzyme release in their homogenates. In addition, we examined a rat liver homogenate and the following 11 cell lines: 3 hepatoma cell lines, including the R-Y121B cell line, 4 liver cell lines, 2 human urinary bladder carcinoma cell lines, a kidney cell line, and a mouse adrenal tumor cell line. Only in the cultured liver cell line and hepatoma cell lines, 30-60% of the total enzyme was released into the soluble fraction from the precipitate fractions; the release was not observed in the other cell lines, nor in the rat liver homogenate. The release of alkaline phosphatase took place in both heat-stable and heat-labile alkaline phosphatases. Alkaline phosphatase, extracted from cell homogenates, showed two bands during polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The mobilities of the two bands changed inversely with or without sodium dodecyl sulfate. In general, the alkaline phosphatase which showed slow mobility with sodium dodecyl sulfate was more readily released from the plasma membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Tumorigenicity of T24 urinary bladder carcinoma cell sublines.

Two sublines of the T24 human urinary bladder carcinoma cell line which differ in tumorigenicity in nude mice have been studied (T24A and T24P). T24A obtained directly from the American Type Culture Collection is non-tumorigenic while T24P obtained after multiple passages in several NCI laboratories produces tumors in 100% of inoculated mice. T24P cells differ morphologically from T24A, have a higher saturation density, are less serum-dependent for growth, and are more sensitive to ouabain toxicity. Cytogenetic studies show that the 2 sublines differ significantly in chromosome number, with a modal chromosome range of 76-89 in T24A and a modal chromosome number of 48-51 in T24P. Southern blot analysis of MspI cleaved T24A and T24P DNAs with the H-ras SmaI probe indicates that both contain only the activated mutant allele originally described in T24. Northern blot analysis shows equal amounts of the 1.2kB ras polyadenylated message, and immunoblotting with rasHa antibody demonstrates no significant difference in the amounts of ras proteins. These results indicate that 2 sublines of a ras oncogene-containing tumor cell line can differ greatly in tumorigenicity and other in vitro characteristics of transformation, and yet have similar expression of the ras oncogene. The fact that the tumorigenic cell line contains fewer chromosomes suggests that tumorigenicity may be related to the loss of some regulatory gene.

In vivo localisation of human urinary bladder carcinoma xenograft in nude mice using radiolabelled monoclonal antibody.

Monoclonal antibody 1G3.10 (mouse IgG3) against the human urinary bladder carcinoma TSGH-8301 was isolated from hybridoma ascites and labelled with radioiodine. The antibody reacts with a cell surface antigen preferentially expressed in human bladder carcinoma. Binding studies in vitro demonstrated that its specificity for antigen was retained after iodination. The clearance of the radiolabelled monoclonal antibody was not modified by the presence of the tumour. In vivo localisation of the radiolabelled antibody and control normal IgG to tumour xenograft was determined by counting the tissue radioactivity and by external gamma ray scintigraphy with computer analysis of the region of interest at various times after the intravenous injection of radiolabelled antibodies. Maximum tumour-to-blood radioactivity (4.5) was obtained 4 days after antibody injection. There was no tumour localisation of radiolabelled normal IgG. Specificity of the localisation was also confirmed by using a non-reactive colon carcinoma xenograft. Distinct tumour images were obtained without the use of subtraction techniques. These studies of human bladder tumour localisation using this monoclonal antibody show its obvious potential for clinical use.