Human Bladder Carcinoma Cell Line Research Articles

Morphology and DNA degeneration during autoschizic cell death in bladder carcinoma T24 cells induced by ascorbate and menadione treatment

Feulgen and actin-phalloidin staining as well as gel electrophoresis have been employed in conjunction with cell ultrastructure to describe the effects of 1-, 2-, and 4-hr ascorbate (VC), menadione (VK(3)), and ascorbate:menadione (VC:VK(3)) treatments on the T24 human bladder carcinoma cell line. T24 cells exposed to VC alone display blebs and other superficial membrane defects related to membrane alterations and to superficial cytoskeleton changes. VK(3) treatment damages the cell nucleus and organelles, leads to the redistribution of the organelles in the perikaryon as a consequence of cytoskeletal damage, and results in cytoplasmic self-excisions. After VC:VK(3) treatment, the cells show exaggerated alterations characteristic of each vitamin treatment alone, including damaged mitochondria, self-excision of organelle-free pieces of cytoplasm, and extrusion of the perikaryon containing a nucleus surrounded by the damaged organelles. The nuclear envelope appears intact and contains chromatin that decondenses and dissipates. During the cellular demise that concludes with apparent karyolysis, the cells significantly decrease their size and alter their shape. However, the cisterns of rough endoplasmic reticulum are undamaged, but may become dilated. Since the cellular phenomena leading to cell death differ morphologically from apoptosis and necrosis, but entail self-cutting without nuclear bodies, this new form of cell death was called autoschizis. In addition, gel electrophoresis and Feulgen staining demonstrate that autoschizis is accompanied by random DNA degeneration.

Characterization of the oligosaccharide component of α3β1 integrin from human bladder carcinoma cell line T24 and its role in adhesion and migration

Malignant transformation is highly associated with altered expression of cell surface N-linked oligosaccharides. These changes concern integrins, a family of cell surface glycoproteins involved in the attachment and migration of cells on various extracellular matrix proteins. The integrin α 3 β 1 is particularly interesting because of its role in migration and invasion of several types of metastatic tumours. In this study, α 3 β 1 from human bladder T24 carcinoma cells was purified and treated with peptide N-glycosidase F. Then the N-glycans of the α 3 and β 1 subunits were characterized using matrix-assisted laser desorption ionization mass spectrometry (MALDI MS). In α 3 β 1 integrin the presence of high-mannose, hybrid and predominantly complex type N-oligosaccharides was shown. Unlike to normal epithelium cells, in both subunits of α 3 β 1 integrin from cancer cells, the sialylated tetraantennary complex type glycan Hex 7HexNAc 6FucSia 4 was present. In a direct ligand binding assay, desialylated α 3 β 1 integrin exhibited significantly higher fibronectin-binding capability than untreated integrin, providing evidence that sialic acids play a direct role in ligand-receptor interaction. Moreover, α 3 β 1 integrin was shown to take part in T24 cell migration on fibronectin: anti- α 3 antibodies induced ca 30% inhibition of wound closure. Treatment of T24 cells with swainsonine reduced the rate of bladder carcinoma cell migration by 16%, indicating the role of β1,6 branched complex type glycans in this process. Our data show that α 3 β 1 integrin function may be altered by glycosylation, that both subunits contribute to these changes, and that glycosylation may be considered a newly found mechanism in the regulation of integrin function.

Cytotoxic Screening of Some Tanzania Medicinal Plants

Twenty plants that are used in traditional medicine in Iringa, Tanzania, were tested for in vitro cytotoxic activity on human bladder carcinoma (RT-4), colon adenocarcinoma (HT29), and skin carcinoma (A431) cell lines. At 100 pglml Albizia harveyi, Albizia anthelmintica, Dalbergia nitidula, Euphorbia grantii and Rauvolfia caffra reduced cell proliferation to 50% or more of the three cell lines. Albizia harveyi showed a significant cytotoxic activity on the RT-4 cell line (percentage survival 23%) at 10pgIml. It showed a weak cytotoxic activitv on the HT-29 cell line. Dalbergia nitidula. showed a weak cytotoxic activity with percentage death of the RT-4 and HT-29 cell lines of 39 and 34%, respectively, at the 10 pghnl level. These results show that 19 (95%) of the plant extracts tested are non-toxic. One plant (5%), Albizia harveyi showed cytotoxic activity on one of the cell lines used, which was in agreement with the accepted detection level of biological activity by chance. Bioassay guided fractionation of the plant extracts to identify active compound(s) is suggested.

Induction of apoptosis by Chan Su, a traditional Chinese medicine, in human bladder carcinoma T24 cells

Chan Su is a traditional Chinese medicine prepared from the dried white secretion of the auricular and skin glands of toads, and has been used as an Oriental drug. However, little is known about the effect of Chan Su on the growth of human cancer cells. This study was undertaken to investigate the underlying mechanism of Chan Su-induced apoptosis in a human bladder carcinoma cell line, T24. The effects of this compound were also tested on cyclooxygenase (COX) activity. Treatment of T24 cells with Chan Su resulted in the inhibition of viability and induction of apoptosis in a concentration-dependent manner, which was proved by trypan blue counts, DAPI staining, agarose gel electrophoresis and flow cytometric analysis. Apoptosis of T24 cells by Chan Su was associated with a down-regulation of anti-apoptotic Bcl-2 and Bcl-X(S/L) expression and an up-regulation of pro-apoptotic Bax expression. Chan Su treatment induced the proteolytic activation of caspase-3 and caspase-9, and a concomitant degradation of poly(ADP-ribose)-polymerase and beta-catenin protein. Furthermore, Chan Su decreased the levels of COX-2 mRNA and protein expression without significant changes in the levels of COX-1, which was correlated with an inhibition in prostaglandin E(2) synthesis. Taken together, these findings partially provide novel insights into the possible molecular mechanisms of the anti-cancer activity of Chan Su.

Synthesis, characterization and antiproliferative behavior of tricarbonyl complexes of rhenium(I) with some 6-amino-5-nitrosouracil derivatives: Crystal structure of fac-[ReCl(CO) 3(DANU-N 5,O 4)] (DANU = 6-amino-1,3-dimethyl-5-nitrosouracil)

New complexes of rhenium(I) with some 5-nitrosopyrimidines with general formula [ReCl(CO) 3L] have been prepared and characterized by elemental analysis, conductivity measurements, IR and 1H, 13C and 15N NMR spectroscopic methods. The complexes appear to be monomeric and the pyrimidine ligands act in a neutral form. The structure of [ReCl(CO) 3(DANU)] · CH 3CN has been solved by X-ray diffraction. The coordination environment around the Re(I) may be described as a distorted octahedron in which the ligand behaves in a bidentate fashion through N5 and O4 atoms, making a five-membered chelate ring. The coordination sphere is completed with three carbonyl groups in fac-arrangement and one chlorine atom. The evaluation of the antiproliferative behavior against five human tumor cell lines (human breast cancer MCF-7 and EVSA-T, human neuroblastoma NB69, human glioma H4 and human bladder carcinoma cell line ECV) suggested a modulator behavior of cell growth at low concentrations due to their estrogenic-like characteristics.

Role of the β1-integrin subunit in the adhesion, extravasation and migration of T24 human bladder carcinoma cells

The abilities of tumor cells to extravasate from the blood vessel system and to migrate through the connective tissue are prerequisites in metastasis formation. Both processes are chiefly mediated by integrins, which mediate both cell-cell and cell-matrix interactions. We investigated the role of integrin subunits in the adhesion, extravasation and migration of the highly invasive human bladder carcinoma cell line T24. Here we show that inhibition of the beta(1)-integrin subunit using the specific beta(1)-integrin blocking antibody 4B4 significantly reduces the adhesion to HUVEC and transmigratory activity of T24 cells. The blockade of the beta(1)-integrin subunit also resulted in a significantly reduced locomotory activity of T24 cells. A detailed cell migration analysis on a single cell level revealed that blockade of the beta(1)-integrin subunit leads to an altered migration pattern of single cells but does not influence migration per se. Migration parameters such as time active, velocity and distance migrated were significantly reduced as compared to untreated control cells. Our observations strongly suggest a central role for the beta(1)-integrin subunit in forming the cell-cell and cell-matrix bonds necessary for adhesion, extravasation and migration.

Production of Intracellular Superoxide Mediates Dithiothreitol- Dependent Inhibition of Apoptotic Cell Death

Apoptotic cell death proceeds from the activation of cysteine proteinases called caspases. As full enzymatic activity of caspases requires reduction of cysteine residues in and around the catalytic site of the proteases, cysteine- reducing agents such as dithiothreitol (DTT) are expected to facilitate caspase activity upon induction of apoptosis. However, DTT has been shown to efficiently protect cells from apoptosis. The mechanism involved in DTT-mediated inhibition of apoptosis has been attributed to its antioxidant activity. Interestingly, under physiological conditions, thiol-mediated antioxidant reaction has also been shown to result in intracellular generation of superoxide (O(2) (.-)). In line with our earlier findings implicating a slight prooxidant state in resistance to apoptosis, we set out to investigate if the death-inhibitory activity of DTT could be mediated by intracellular O2 (.-). Our results show that incubation of human melanoma cell line M14TF or human bladder carcinoma cell line T24 with DTT induced an increase in intracellular O2 (.-) with concomitant inhibition of apoptosis triggered by CD95 signaling, staurosporine, or hydrogen peroxide. Moreover, preincubation of either cells with Tiron, a specific O2 (.-) scavenger, reverted DTT-induced inhibition of apoptosis. These results show that the apoptosis-inhibitory activity of DTT may not be due to its antioxidant property, but instead linked to its ability to induce an increase in intracellular O2 (.-) level.

Histone deacetylase inhibitors upregulate plakoglobin expression in bladder carcinoma cells and display antineoplastic activity in vitro and in vivo

Histone deacetylase inhibitors (HDACis) are emerging as a promising new class of anticancer agents displaying growth-inhibitory activity and low toxicity in vivo. In this study, we examined the effect of sodium butyrate (NaB) and trichostatin A (TSA) on the growth of human bladder carcinoma cell lines in culture and TSA on the growth of EJ and UM-UC-3 human bladder xenografts in nude mice. NaB and TSA suppressed the growth of bladder cell lines at millimolar (1.5-4.3 mM) and micromolar (0.03-0.33 microM) concentrations, respectively, inducing concentration-dependent cell death. Bladder carcinoma cells within the experimental panel displayed the phenotype of late-stage bladder lesions expressing N-cadherin in the absence of E-cadherin accompanied by low levels of plakoglobin expression. Exposure of these cells to HDACis resulted in upregulation of plakoglobin with no change in E-cadherin expression. A 2-hr exposure to TSA was the minimal time required to upregulate plakoglobin in cells with downregulation to baseline levels occurring within 24 hr following drug removal. In mice bearing EJ and UM-UC-3 bladder xenografts, TSA (500 microg/kg/day) caused suppression of tumor growth compared with mice receiving vehicle alone. A > 70% reduction in mean final tumor volume was recorded in both bladder xenograft models with no detectable toxicity. The results suggest that TSA inhibits bladder carcinoma cell growth and may be a useful, relatively nontoxic agent for consideration in the treatment of late-stage bladder tumors.

Evaluation of the antitumoral potential of different nitric oxide-donating non-steroidal anti-inflammatory drugs (NO-NSAIDs) on human urological tumor cell lines

Our work aimed at identifying the antitumoral potential of new nitric oxide (NO)-releasing non-steroidal anti-inflammatory drug (NSAID) derivatives on human prostate and bladder carcinoma cell lines. Among all molecules tested, two sulindac derivatives, NCX 1102 ((Z)-5-fluoro-2-methyl-1-[[4-(methylsulfinyl)phenyl] methylene]-1H-indene-3-acetic acid 4-(nitrooxy)butyl ester) and NCX 1105 ((Z)-5-fluoro-2-methyl-1-[[4-(methylsulfinyl)phenyl] methylene]-1H-indene-3-acetic acid 6-(nitrooxymethyl)-2-methylpyrydyl ester hydrochloride), were the most cytotoxic compounds. In contrast to its parent molecule sulindac, cell cycle analysis showed that NCX 1102 led to cell accumulation in the G2-M transition stage in all cell lines, and induced apoptosis in five out of the six cell lines. Thus, NO-NSAIDs may be useful for the elaboration of new therapeutic strategies in the management of bladder and prostate cancer.

Expression of a mutant hTERT in human bladder carcinoma cell line T24 and its clinical significance

Objective: To construct a mutant pEGFP- hTERT expression vector, to observe its steady expression in transfected human bladder carcinoma cell line T24 and its role in molecular regulatory mechanisms of telomerase, and to provide a new target gene for bladder cancer. Methods: PCR amplification was performed by using primers based on the known gene sequence of hTERT. PCR production was cloned into plasmid pGEMT-T easy and the sequence of mutant hTERT gene was analyzed. A recombinant mutant hTERT vector (pEGFP-hTERT) was constructed at the EcoR I and Sal I sites of the pEGFP-C1 vector. After transfecting the fusion gene into bladder carcinoma cell line T24 by calcium phosphate-DNA coprecipitation, the steady expression of GFP-hTERT fusion protein was tested by fluorescent light microscopy. The proliferation changes of bladder carcinoma cell line T24 were detected by light microscopy and senescence correlated β-galactosidase staining. Results: Identification of pEGFP-hTERT by enzyme digestion showed that mutant hTERT fragment had been cloned into EcoR I and Sal I sites of the pEGFP-C1 vector. The steady expression of GFP-hTERT fusion protein was localized in the nucleus of transfected cells. Expression of senescence-associated β-galactosidase in transfected cells gradually increased with extended cultured time and cell growth was suppressed. Conclusion: The mutant-type hTERT gene suppresses the proliferation of bladder carcinoma cell line T24 by competitive effect on telomerase activity. This suggests that hTERT gene might be a suitable gene target for bladder cancer therapy.

Antiproliferative effect of nitrosulindac (NCX 1102), a new nitric oxide-donating non-steroidal anti-inflammatory drug, on human bladder carcinoma cell lines

Non-steroidal anti-inflammatory drugs (NSAIDs) are potent antitumoral agents but their side effects limit their clinical use. A novel class of drugs, nitric oxide-donating NSAIDs (NO-NSAIDs), was found to be safer and more active than classical NSAIDs. This study explored the effect of the NO-donating sulindac derivative, NCX 1102, on three human urothelial epithelial carcinoma cell lines (T24, 647V, and 1207) and primary cultures of normal urothelial cells. Cytotoxicity, antiproliferative effect, cell cycle alterations, morphological changes, and apoptosis were investigated after treatment with NCX 1102 in comparison with the native molecule. After treatment, there was a cytotoxic effect (with IC(50) at 48 h of 23.1 micro M on 647V, 19.4 micro M on T24, and 14.5 micro M on 1207) and an antiproliferative effect on all three cell lines with NCX 1102 but not with sulindac. No effect was detected on normal urothelial cells. Flow cytometric analysis showed a differential NCX 1102-induced accumulation of cells in various phases of the cell cycle, depending on cell line and concentration. NCX 1102 induced an occurrence of multinucleated cells in all cell lines and mitotic arrest in 647V and 1207. NCX 1102-treated T24 and 647V cell lines showed a significant difference of apoptotic cell amount when compared to controls. Our results demonstrated a greater antiproliferative potency of NCX 1102 compared to its parent molecule sulindac, and suggested that this new NO-NSAID may have therapeutic impact in the management of bladder cancer.

Enhanced chemosensitivity of bladder cancer cells to cisplatin by suppression of clusterin in vitro

We examined the functional role of clusterin in chemotherapy-induced apoptosis and tested whether anti-sense transfection targeted against clusterin enhances the chemosensitivity in human bladder cancer cells in vitro. Clusterin mRNA and protein expression of 253J cells, a human bladder carcinoma cell line, after treatment with cisplatin were measured by RT-PCR and Western blot analysis. Clusterin expression and cell growth were compared between 253J cells transfected with constructed a clusterin anti-sense plasmid vector (pCR-CLU-AS) and controls. Tumor cell viability was measured with MTT assay after cisplatin treatment. DNA fragmentation and CPP32 assay were performed. Clusterin expression was increased after treatment with cisplatin and highest at 8 h in 253J cells. Clusterin anti-sense transfectants were highly sensitive to apoptotic cell death induced by cisplatin compared with parental 253J cells or control transfectants. Collectively, our results showed that expression of clusterin was increased in the acute phase of cell death caused by cisplatin and that suppressing the expression of clusterin enhanced the susceptibility of apoptosis caused by cisplatin in human bladder cancer cells. These results suggest that lowering the expression of clusterin might increase the sensitivity of bladder cancer cells to chemotherapeutic agents.

Cell cycle arrest and autoschizis in a human bladder carcinoma cell line following Vitamin C and Vitamin K 3 treatment

Exponentially growing cultures of human bladder tumor cells (T24) were treated with Vitamin C (VC) alone, Vitamin K 3 (VK 3) alone, or with a VC:VK 3 combination for 1, 2, or 4 hr. Flow cytometry of T24 cells exposed to the vitamins for 1 h revealed a growth arrested population and a population undergoing cell death. Cells in G 1 during vitamin treatment arrested in G 1 while those in S phase progressed through S phase and arrested in G 2/M. DNA synthesis decreased to 14 to 21% of control levels which agreed with the percent of cells in S phase during treatment. Annexin V labeling demonstrated the majority of the cells died by autoschizis, but necrosis and apoptosis also were observed. Catalase treatment abrogated both cell cycle arrest and cell death which implicated hydrogen peroxide (H 2O 2) in these processes. Redox cycling of VC and VK 3 increased H 2O 2 production and decreased cellular thiol levels and DNA content, while increasing intracellular Ca 2+ levels and lipid peroxidation. Feulgen staining of treated cells revealed a time-dependent decrease in tumor cell DNA, while electrophoresis revealed a spread pattern. These results suggest that Ca 2+ disregulation activates at least one DNase which degrades tumor cell DNA and induces tumor cell death.

Analyzing changes of chromatin-bound replication proteins occurring in response to and after release from a hypoxic block of replicon initiation in T24 cells.

It was shown previously [Riedinger, H. J., van Betteraey-Nikoleit, M & Probst, H. (2002) Eur. J. Biochem.269, 2383-2393] that initiation of in vivo SV40 DNA replication is reversibly suppressed by hypoxia in a state where viral minichromosomes exhibit a nearly complete set of replication proteins. Reoxygenation triggers fast completion and post-translational modifications. Trying to reveal such fast changes of chromatin-bound replication proteins in the much more complex replication of the cellular genome itself, we developed a protocol to extend these studies using the human bladder carcinoma cell line T24, which was presynchronized in G1 by starvation. Concomitantly with stimulation of the cells by medium renewal, hypoxia was established. This treatment induced T24 cells to contain a large amount of replicons arrested in the 'hypoxic preinitiation state', ready to initiate replication as soon as normal pO2 was restored. Replicons in other stages of replicative activity were not detectable. Consequently the arrested replicons were rapidly released into synchronous initiation and succeeding elongation. Extraction of T24 nuclei with a Triton X-100 buffer yielded a fraction containing the cellular chromatin, including DNA-bound replication proteins, while unbound proteins were removed. The usefulness of this protocol was tested by the proliferation marker PCNA. We demonstrate here that this protein switches from the remainder cellular protein pool into the Triton-extracted nuclear fraction upon reoxygenation. Employing this protocol, analyses of chromatin-bound MCM2, MCM3, Cdc6 and cdk2 suggests that the 'classical' prereplication complex is already formed during hypoxia.

Paclitaxel (taxol) inhibits the arylamine N-acetyltransferase activity and gene expression (mRNA NAT1) and 2-aminofluorene-DNA adduct formation in human bladder carcinoma cells (T24 and TSGH 8301).

Acetylator polymorphism in man results from differential expression of human liver N-acetyltransferase. N-Acetyltransferase enzyme activity has been demonstrated to be involved in some types of chemical carcinogenesis. Paclitaxel (taxol) had been shown to affect N-acetyltransferase activity of human lung cancer cells. In this study, paclitaxel was chosen to investigate the effects of arylamine N-acetyltransferase activity (N-acetylation of substrate), gene expression and 2-aminofluorene-DNA adduct formation in human bladder carcinoma cell lines (T24 and TSGH 8301). The N-acetyltransferase activity (N-acetylation of substrates) was determined by high performance liquid chromatography assaying for the amounts of acetylated 2-aminofluorene and p-aminobenzoic acid and nonacetylated 2-aminofluorene and p-aminobenzoic acid. Intact human bladder carcinoma T24 and TSGH 8301 cells were used for examining N-acetyltransferase activity, gene expression and 2-aminofluorene-DNA adduct formation. The results demonstrated that the N-acetyltransferase activity, gene expression (NAT1 mRNA) and 2-aminofluorene-DNA adduct formation in intact human bladder carcinoma cells were inhibited and decreased by paclitaxel in a dose-dependent manner. The effects of paclitaxel on the apparent values of Km and Vmax of N-acetyltransferase enzyme from intact human bladder carcinoma cells were also determined in these cell lines. A marked influence of paclitaxel was observed on the decreasing apparent values of Km and Vmax from intact human bladder carcinoma cells (T24 and TSGH 8301). Thus, paclitaxel is an uncompetitive inhibitor to the NAT enzyme.

Effects of retinoids in human bladder carcinoma cell lines: Inhibition of growth and induction of apoptosis

Effects of retinoids in human bladder carcinoma cell lines: Inhibition of growth and induction of apoptosis

Use of the comet-FISH assay to demonstrate repair of the TP53 gene region in two human bladder carcinoma cell lines.

The alkaline single-cell gel electrophoresis (comet) assay can be combined with fluorescence in situ hybridization (FISH) methodology to investigate the localization of specific gene domains within an individual cell. The position of the fluorescent hybridization spots in the comet head or tail indicates whether the sequence of interest lies within or in the vicinity of a damaged region of DNA. In this study, we used the comet-FISH assay to examine initial DNA damage and subsequent repair in the TP53 gene region of RT4 and RT112 bladder carcinoma cells after 5 Gy gamma irradiation. In addition to standard comet parameter measurements, the number and location of TP53 hybridization spots within each comet was recorded at each repair time. The results indicate that the rate of repair of the TP53 gene region was fastest during the first 15 min after damage in both cell lines. When compared to overall genomic repair, the repair of the TP53 gene region was observed to be significantly faster during the first 15 min and thereafter followed a rate similar to that for the overall genome. The data indicate that the TP53 domain in RT4 and RT112 cells is repaired rapidly after gamma irradiation. Furthermore, this repair may be preferential compared to the repair of overall genomic DNA, which gives a measure of the average DNA repair response of the whole genome. We suggest that the comet-FISH assay has considerable potential in the study of gene-specific repair after DNA damage.

Realtime visualization of tumor cell/endothelial cell interactions during transmigration across the endothelial barrier.

In cancer the blood-borne spread of tumor cells leads to the formation of secondary tumors at distant loci whereby the extravasation of tumor cells is a prerequisite step during hematogenous metastasis. Here, we describe a novel in vitro realtime model which shows the complete sequence of the extravasation process. We developed an in vitro system allowing us to monitor the sequence of extravasation events of tumor cell clusters across a monolayer of human umbilical cord endothelial cells (HUVEC). Fluorescence markers and laser scanning confocal microscopy were used to visualize the interactions between tumor cells and endothelium. Our model indicates that the extravasation of tumor cell clusters derived from the invasive human bladder carcinoma cell line T24 occurs in a relatively short time-frame up to 4 h after adhesion to the endothelium. We demonstrate that the vascular endothelium is irreversibly damaged at the site of tumor cell extravasation. Realtime laser scanning confocal microscopy leads to a better understanding of the complex and dynamic cell-to-cell and cell-to-matrix interactions during the extravasation process.

Influence of the Microenvironment on Invasiveness of Human Bladder Carcinoma Cell Lines

Influence of the Microenvironment on Invasiveness of Human Bladder Carcinoma Cell Lines

Influence of the Microenvironment on Invasiveness of Human Bladder Carcinoma Cell Lines

Influence of the Microenvironment on Invasiveness of Human Bladder Carcinoma Cell Lines