Human Bladder Tumor Cells Research Articles

Internalization of Bacille Calmette-Guerin by Bladder Tumor Cells

Mechanisms by which intravesical bacille Calmette-Guerin (BCG) treatment mediates antitumor activity are currently poorly understood. We have determined that both human bladder tumor (T-24) and mouse bladder tumor (MBT-2) cells are capable of internalizing the BCG and have characterized this process in vitro. The internalization of BCG by T-24 and MBT-2 cells was verified by histochemistry and electron microscopy. Time dependent internalization of BCG was observed with a maximum occurring at three hrs. Internalization was significantly inhibited by both incubation at 4C and cytochalasin B; conditions known to inhibit phagocytosis. Ultrastructural studies suggested that BCG were transported to membrane bound intracellular compartments and were degraded. The compartments containing the degraded mycobacteria labeled with the fluid phase marker HRP which is known to be transported to lysosomes in a variety of cell types. To determine if the internalization process occurred in vivo, we examined bladder washings of patients treated with intravesical BCG. The majority of the cells in the bladder washings were inflammatory cells which contained ingested BCG. In addition, internalized and degraded BCG were identified in urothelial cells. These data demonstrate that BCG are internalized by transitional epithelial cells both in vitro and in vivo. The internalization process is inhibitable by temperature and microfilament disruption. The potential therapeutic implications of this process and its relationship to antitumor activity of BCG therapy are currently being investigated.

Activation by the Protein-Bound Polysaccharide PSK (Krestin) of Cytotoxic Lymphocytes that Act on Fresh Autologous Tumor Cells and T24 Human Urinary Bladder Transitional Carcinoma Cell Line in Patients with Urinary Bladder Cancer

PSK, a protein-bound polysaccharide Kureha, was tested for its ability to modulate the cytotoxicity of lymphocytes that act on autologous tumor cells and T24 human urinary bladder tumor cells in urinary bladder cancer patients in a 6-h 51Cr release assay. In vitro treatment of peripheral blood lymphocytes (PEL) with PSK for 18hours resulted in an augmentation or induction of cytotoxicity against relatively resistant T24 cells in previously reactive and nonreactive cases, respectively. The PSK-treated PBL were able to kill more effectively tumor cells that were freshly isolated from the same cancer patients than non-treated PBL. The effects of PSK were noted with PBL as well as tumor infiltrating lymphocytes (TIL) and with PSK at concentrations of 10 to 100μg./ml., while PSK at higher doses reduced their lytic activities. The addition of PSK to the assay at the same concentrations also enhanced the cytotoxicities. Autologous tumor killing (ATK) activities of both large granular lymphocytes (LGL) and T lymphocytes were enhanced by PSK. Treatment of PBL with PSK did not effect on the proportion of PBL binding to the tumor cells, while it augmented the cytotoxic activity. Cell-free supernatant of PSK-stimulated lymphocyte culture did not contain any detectable amounts of interferon-α (IFN-α), interferon-γ (IFN-γ) and interleukin-2 (IL-2). In addition, anti-IFN-α monoclonal antibody (MAb), anti-IFN-γ MAb and anti-IL-2 MAb did not inhibit PSK-induced agumentation of cytotoxicity against T24. Oral administration of PSK (three gm./day) to patients with urinary bladder cancer daily for seven days before operation resulted in an augmentation of the cytotoxicity against T24 cells in five out of 10 patients and no change of the cytotoxicity In the other five patients. ATK activity was also enhanced by oral administration of PSK in three out of five patients. These results indicate that the antitumor activity of PSK may be in part mediated through activation of tumor killing system independent of IFN-α, IFN-γ and IL-2.

Cellular cytotoxicity of human natural killer cells and lymphokine-activated killer cells against bladder carcinoma cell lines

The cytotoxicity of natural killer (NK) and lymphokine activated killer (LAK) cells against two human bladder tumor cell lines (BT-A and BT-B) was investigated using a fluorometric assay by labeling tumor cell DNA with Hoechst dye No. 33342. Our results demonstrate that BT-A and BT-B cells have low sensitivity to the cytotoxic activity of mononuclear cells (MNC) and NK cells. Cytotoxicity of MNC or NK cells against both tumor cell lines is enhanced during co-culture of the effector cells with the target cells, which suggests that BT-A and BT-B cells provide the signals which could activate MNC to exert cytotoxicity. In contrast to NK cells, IL-2-generated LAK cells showed profound cytotoxicity to BT-A and BT-B within 24 h. In addition to cellular cytotoxicity to bladder tumor cells, we also tested the effect of recombinant interleukin 1 beta (rIL-1 beta), recombinant tumor necrosis factor (rTNF), and the supernatants of co-culture of MNC or LAK cells with bladder tumor cells. The results show no cytotoxic or growth-promoting activity of rIL-1, rTNF, or the crude culture supernatants on bladder tumor cells. We found that LAK cells, but not macrophages or NK cells, may play a major role in cellular cytotoxicity against the two bladder tumor cell lines tested. From this finding we conclude that activation of LAK cells may be one important mechanism induced by adjuvant bacillus Calmette-Guérin (BCG) therapy leading to effective prevention of urothelial bladder carcinoma reappearance.

In Vitro Study of the Interaction of Doxorubicin, Thiotepa, and Mitomycin-C, Agents Used for Intravesical Chemotherapy of Superficial Bladder Cancer

Several cytotoxic agents have been identified as effective in the treatment of superficial transitional cell carcinoma of the bladder, including doxorubicin, thiotepa and mitomycin-C. An in vitro study was conducted to assess the interactions of these three drugs against a well differentiated human bladder tumor cell line, RT-4, to identify and evaluate synergistic combinations among these agents. Cytotoxicity was evaluated by a colorimetric assay based on the capacity of viable cells to metabolize a tetrazolium dye, MTT, to produce a colored formazan product. The analyses of drug interactions were done by the isobolographic method (construction of isoeffect plots). The combination of doxorubicin and thiotepa was found to be the most synergistic, followed by the combination of doxorubicin and mitomycin-C. The combination of mitomycin C and thiotepa demonstrated an unpredictable effect. These findings suggest the combination of doxorubicin and thiotepa has potential advantage for chemotherapy of superficial bladder tumors.

Morphometry of nucleoli as an indicator for grade of malignancy of bladder tumors

To establish a new indicator for the classification of human urinary bladder cancers, the nucleoli of normal epithelial and neoplastic cells were analyzed, using morphometric techniques. By electron microscopy, the nucleolar profiles of cells from grade 2 and 3 transitional cell carcinomas were often small and irregular. Morphometry showed that the nucleolar volumes, nucleolar/nuclear volume ratios, volume densities of various nucleolar components, and the numbers of fibrillar centers (FCs) altered significantly with an increase in tumor grade. In particular, an increase in FC numbers in the nuclei of higher grade tumors was associated with a decrease in individual volume. The number of FCs in intact urothelial cells obtained from patients with bladder tumors is significantly larger than in the normal urothelial cells. This may be related to the multicentric origin of bladder cancers. These results suggest that morphometric analysis of nucleoli is useful in evaluating the degree of differentiation and invasive capacity of human bladder tumor cells. In particular, the number and individual volume of FCs may be an indicator of tumor malignancy.

Clonal Growth Requirements of Human Bladder Tumor Cell Lines

We have serially investigated the role of nutrients and growth factors for their ability to support the clonal growth of human bladder tumor cell lines. Five established human bladder tumor cell lines were selected for study of their requirements for clonogenic growth in semisolid cultures. We identified an “optimal medium” to be Alpha MEM medium, 15% horse serum, fiveμg./ml. of transferrin, fiveμg./ml. of insulin and 30mg./ml. of epidermal growth factor. The “optimal assay” enhanced the growth of human bladder tumor cell lines 2.5 times compared to the standard clonogenic assay. The resulting medium is less complicated to maintain, less expensive and supports the growth of human bladder tumor cell lines better than the standard clonogenic assay. (J. Urol, 143:1049-1052, 1990)

SUBCELLULAR LOCALIZATION OF HEMATOPORPHYRIN DERIVATIVE IN BLADDER TUMOR CELLS IN CULTURE

Mitochondria have been implicated as a primary subcellular site of porphyrin localization and photodestruction. However, other organelles including the cell membrane, lysosomes and nucleus have been shown to be damaged by hematoporphyrin derivative (HpD) photosensitized destruction as well. In this study we attempted to follow the translocation of the fluorescent components of HpD in human bladder tumor cells (MGH-U1) in culture to determine whether specific subcellular localization occurs over time. Following a 30 min exposure to HpD the cellular fluorescence was examined immediately and 1, 2, 4, and 24 h after HpD removal using fluorescence microscopy and an interactive laser cytometer. The in vitro translocation of dye appeared to be fairly rapid with fluorescence present at the cell membrane and later (1-2 h) within a perinuclear area of the cytoplasm. To determine whether HpD had become concentrated into a specific subcellular organelle, these fluorescence distribution patterns were compared with fluorescent marker dyes specific for mitochondria, endoplasmic reticulum and other membranous organelles. The HpD fluorescence did not appear to be as discrete as the dyes specific for mitochondria or endoplasmic reticulum but appeared similar to the diffuse cytomembrane stain. Finally, the interaction between the fluorescent components of HpD and the cellular constituents was evaluated using a "fluorescence redistribution after photobleaching" technique. The results indicated that the mean lateral diffusion for HpD in MGH-U1 cells was 1.05 x 10(-8) cm2/s, a rate closer to that of lipid diffusion (10(-8)) than that of protein diffusion (10(-10)).(ABSTRACT TRUNCATED AT 250 WORDS)

Development and characterization of cisplatin-resistant human testicular and bladder tumour cell lines

Cisplatin-resistant cells were derived in vitro from a human bladder carcinoma cell line (RT112) and a testicular tumour cell line (SuSa) by continuous exposure to increasing concentrations of cisplatin for 14 and 11 months, respectively. Both resistant cell lines had a four-fold level of resistance relative to their parental cell lines, comparing the cisplatin concentration to inhibit colony forming ability by 70%. These levels of resistance were retained in the absence of cisplatin for at least 3 months. In each case, four-fold fewer micronuclei were produced in the resistant lines by the same cisplatin concentrations. Cross-resistance to carboplatin and methotrexate was observed in both resistant cell lines, but neither line was resistant to doxorubicin. Isozyme and DNA analysis with hypervariable probes confirmed the origin of each resistant cell line from its parental line. Population doubling times and intermitotic times were similar in each of the pairs of cell lines. Karyotyping showed that the resistant cell lines had gained and lost marker chromosomes, but there were no changes common to both resistant cell lines.

Detection of tumor-associated membrane proteins in prostate and bladder carcinomas by means of protein blotting.

Analysis of membrane proteins by Western blotting has revealed both overexpression of proteins of molecular weight 10-200 kD (in particular, of proteins of MW less than 43 kD) and increased glycosylation in a xenografted human small cell undifferentiated prostatic carcinoma, and in two xenografted human bladder tumor cell lines compared with preparations from normal human tissue. Of potential functional significance were: a) a 43 kD protein in the bladder line, UCRU-BL-13, which demonstrated increased synthesis and a marked increase in the degree of glycosylation, and b), a 28 kD ConA-binding protein in prostatic tissue which was absent in normal tissue, present in intermediate quantity in a benign hyperplasia and greatly overexpressed in small cell carcinoma. This study demonstrates the utility of the protein blotting/autoradiography technique for the investigation of tumor membrane proteins.

Evaluation of human bladder tumors by ultrastructural morphometric techniques

To estimate the malignancy of the human urinary bladder tumors, various cellular components of normal and neoplastic bladder epithelial cells were examined using morphometric techniques at the electron microscopic level. Nuclear to cytoplasmic ratios were found to be 1:5 in normal epithelial cells, 1:4 in superficial grade 1 (G1) tumor cells, 1:3 in superficial grade 2 (G2) tumor cells and 1:2 in invasive grade 3 (G3) tumor cells. Nuclear volumes were largest in G3 tumor cells, but no differences were seen in cell volumes between normal epithelial and tumor cells. The volumes and surface areas of the rough-surfaced endoplasmic reticulum (rER) and lysosomes per cell decreased progressively from G1 to G3 tumor cells. The volume density of tonofilaments was increased in G1, but decreased in G2 and G3 tumor cells. The cisternal thickness of the rER and Golgi complex was also increased in G1 tumor cells. From these results, it was concluded that morphometric analysis may be useful in evaluating the degree of differentiation and invasive potential of human bladder tumor cells in the low and intermediate risk groups.

Establishment and Characterization of a Doxorubicin-Resistant Human Bladder Cancer Cell Line (MGH-U1R)

A doxorubicin-resistant bladder cancer cell line has been established. This was accomplished by exposing an established human bladder tumor cell line, MGH-U1, to progressively higher concentrations of doxorubicin over a period of 12 months. The resistant cells, MGH-U1R, are nine times more resistant to doxorubicin and 30 times more resistant to daunorubicin than the parent cells. The MGH-U1R and the MGH-U1 cells have identical isozyme phenotypes. Compared to the parent cells, the resistant cells have a slower growth rate, lower confluent density, are more heterogeneous morphologically, and exhibit more chromosomal aberrations and rearrangements. The resistant cells may now be used as an experimental system for the search of means to overcome drug resistance in human bladder cancer.

Co2+ is able to substitute for Mn2+ in some exogenous and endogenous galactosyltransferase reactions.

The ability of Co2+ to substitute for Mn2+ in exogenous and endogenous galactosyltransferase reactions was tested. Exogenous transfer was measured towards different high and low molecular weight galactose acceptors using galactosyltransferase from the following sources: crude serum, the serum enzyme partially purified by affinity chromatography and a pure enzyme preparation from milk. Endogenous transfer was estimated in preparations from human urinary bladder tumor cells and from rat liver microsomal fractions. The results show that Co2+ is able to substitute for Mn2+ in some exogenous and endogenous galactosyltransferase reactions. This ability seems to depend on the molecular structure of the galactose acceptor as well as on the nature of the enzyme.

Differential Sensitivities of Human Testicular and Bladder Tumor Cell Lines to Chemotherapeutic Drugs

The in vitro drug sensitivities of 5 human testicular tumor cell lines (Tera II, SuSa, NEC-8, 833K, T3B1) and 5 human bladder carcinoma cell lines (RT4, RT112, T24, HT1197, HT1376) were compared. Cytotoxicities of cisplatin and doxorubicin were assessed by inhibition of colony-forming ability during continuous exposure to a range of drug concentrations. The ranges of the drug concentrations required to kill 70% of clonogenic cells obtained against the testicular cell lines were 1-7 ng/ml and 21-161 ng/ml for doxorubicin and cisplatin, respectively, compared with 4-19 ng/ml and 112-431 ng/ml for the bladder cell lines. This study shows that continuous cell lines retain the relative clinical chemosensitivities of their tumors of origin. The results also indicate that testicular tumor cells are inherently more sensitive to the cytotoxic effects of chemotherapeutic drugs than are bladder cancer cells.

Prevention of Human Bladder Tumor Cell Implantation in an in Vitro Assay

The high recurrence rate of bladder tumors can be reduced by prevention of tumor cell reimplantation on denuded urothelium following transurethral resection. This can be achieved by intravesical chemotherapy immediately after the resection of the bladder tumors. We have demonstrated, in an in-vitro system, the process of human bladder tumor cell implantation on a naturally produced extracellular matrix (ECM) which simulates the exposed bladder basement membrane and submucosa. Using this model we examined the efficacy of various cytotoxic agents in preventing tumor cell adhesion to the ECM. Human bladder tumor cell implantation was prevented following exposure of the cells to distilled water, epodyl or mitocycin C, and significantly reduced following one hour incubation in cisplatinum and doxorubicin. The maximal effect for each of these cytotoxic agents was reached within 30 to 60 minutes of treatment. Mitomycin C reached maximal effect within 10 minutes. In contrast, thiotepa did not cause a significant reduction in cell adherence to ECM as compared to untreated control cells.

In vitro growth inhibition of Dunning rat prostate tumor by bone marrow factor.

Normal Copenhagen rat bone marrow was assayed for growth inhibition of cultured MAT LyLu rat prostate tumor cells. A marrow-derived factor was identified that had significant growth inhibitory activity in vitro against MAT LyLu as well as against DU-145 human prostate tumor and MBT-2 mouse bladder tumor cells but that was noninhibitory to normal rat fibroblasts. The factor was stable to degradation by acid, heat, freezing, trypsin, and carboxypeptidase B. The factor was nonreactive with Coomassie blue, and the molecular weight was estimated as less than 620 daltons. A similar factor was identified in normal human and normal rat sera. The presence of this factor in bone marrow may explain the absence of osseous metastases in the Dunning rat prostate tumor model.

The Mechanism of Human Bladder Tumor Implantation in an in Vitro Model

Implantation of tumor cells in the bladder following transurethral resection of superficial bladder tumors is believed to be one factor in the etiology of bladder tumor recurrences. Using an in vitro model system we have studied the initial interaction between bladder carcinoma cells and a naturally produced basement membrane-like substrate.Minced explants of superficial low grade human bladder tumors from 10 patients were plated into culture dishes coated with a naturally produced extracellular matrix (ECM). This ECM has been shown to resemble the human urothelial basement membrane and submucosa in its macromolecular composition and ultrastructural appearance. It was found that a firm attachment of the human bladder tumor cells occurred within one hour, reached a maximal value within 24 hours and was followed by flattening and proliferation of the plated cells. These results indicate that prevention of tumor implantation should be initiated in the first hour after transurethral resection of the bladder tumors. This assay can be used for the investigation of various treatments to prevent tumor implantation.

Characterization of cisplatin-induced DNA damage in human bladder tumour cell lines

sera and aliquots assayed for EH activity. None of the clones expressed more activity than the mouse control cells. When extracts were treated with the anti-human antiserum, however, no greater reduction in EH activity occurred than occurred with the mouse cell pellets. It can be concluded, therefore, that none of the hybrids was expressing human EH activity. There are several possible explanations for this. Firstly, the gene coding for human EH could be located on chromosome 6. It has proved impossible to test for this to date since all clones examined segregated this chromosome. It could also be possible that either chromosome 3 or 22 was segregated from these clones. Finally, it may be that the human EH gene was present in some of these clones but was not expressed. This has been reported for tyrosine aminotransferase expression in hybrid cells but is not the case for aryl hydrocarbon hydroxylase or glutathione S-epoxide transferases (Benedict er a/., 1972; Silberstein & Shows, 1982). Further work is in progress to test these possibilities.

Photodynamic destruction of cultured human bladder tumor cells by hematoporphyrin derivative: effects of porphyrin molecular aggregation

Photodynamic destruction of cultured human bladder tumor cells by hematoporphyrin derivative: effects of porphyrin molecular aggregation

Differential Growth Regulation of Human Bladder Tumor Cells (J82) in Culture by Normal Bladder Cells from Different Aged Human Donors

This study used an agarose gel culture system to study growth regulatory interactions between normal and neoplastic human bladder cells. The effects of normal human fibroblasts on tumor cell cloning efficiency in agarose was studied using 2 experimental protocols. In 1 system, the fibroblasts proliferated as anchored cells beneath the agarose, and in the other the fibroblasts were nongrowing cells embedded within the agarose as nonanchored cells. In contrast to the largely inhibitory effects of anchored fetal fibroblasts on tumor cell growth, their nonanchored counterparts were exclusively stimulatory. Similar experiments with normal fibroblasts derived from the bladders of increasingly older donors showed a progressive decline in their capacity to inhibit tumor cell growth in agarose. Further experiments modulating the growth rate of neonatal fibroblasts with fetal bovine serum have indicated that the diverse tumor responses elicited by anchored versus nonanchored cells, as well as those observed for the aged bladder fibroblasts, were a function of fibroblast growth rate. We concluded that there was an inverse regulatory relationship between the growth of normal and tumor cells; rapidly growing fibroblasts were inhibitory whereas nongrowing fibroblasts stimulated tumor cell growth in agarose culture.

Lack of learning and memory impairment in mice treated with lithium and haloperidol in combination

The influence of 13 anticancer alkylating agents on cell proliferation, cell cycle parameters, and morphonuclear characteristics was monitored in vitro on three neoplastic cell lines. This monitoring was carried out by means of the digital cell image analysis of Feulgen-stained nuclei. This computer-assisted microscope analysis of chromatin texture made it possible to assess 15 morphonuclear parameters. These 15 parameters were submitted to multivariate analyses, that is, principal-components analyses followed by the canonical transformation of the data. The 13 alkylating agents included four nitrogen mustards (chlormethine, chlorambucil, melphalan, and cyclophosphamide), two nitrosoureas (carmustine and lomustine), two platinum analogues (cisplatine and carboplatine), two ethyleneimine derivatives (thiotepa and investigational PE1001), one antibiotic (mitomycin C), one alkylsulfonate (busulfan), and one triazene (dacarbazine). The mouse MXT mammary and the human J82 and T24 bladder tumor cell lines were used in this study. The results show that these alkylating agents induced specific modifications to the chromatin pattern according to the subclass to which they belong. In other words, the multivariate statistical analyses of the 15 parameters made it possible to identify, at least partly, distinct subclasses of alkylating agents according to their mechanisms of action. As a validation of the methodology, the results also show that most of the alkylating agents induced an increase in the percentage of cells in the G2 phase, while some sometimes induced an increase in the percentage of cells in the S phase of the cell cycle.