cytochalasin-b and Brain-Neoplasms

We present a novel, multiparametric microphysiometry system for the dynamic online monitoring of human cancer cell metabolism. The optically transparent, modular, hybrid microsystem is based on a glass chip and combines a cell cultivation chamber, microfluidics and metabolic monitoring with fully integrated chemo- and biosensors. pH and oxygen are measured in the cell culture area, and biosensors for lactate and glucose are connected downstream by microfluidics. The wafer-level fabrication features thin-film platinum and iridium oxide microelectrodes on a glass chip, microfluidics in an epoxy resist, a hybrid assembly and an on-chip reference electrode. The reliable analytical performance of the sensors in cell culture medium was demonstrated. The pH sensors exhibit a long-term stable, linear response. The oxygen sensors show a linear behaviour, which is also observed for low oxygen concentrations. Glucose and lactate measurements show a linear, long-term stable, selective and reversible behaviour in the desired range. T98G human brain cancer cells were cultivated and cell culture metabolism was measured on-chip. Stop/flow cycles were applied and extracellular acidification, respiration, glucose consumption and lactate production were quantified. Long-term metabolic rates were determined and all parameters could be measured in the outlet channel. A placement downstream of the cell cultivation area for biosensors was realised. A highly effective medium exchange and undiluted sampling from the cell culture chamber with low flow rates (2 μl min(-1)) and low volumes (15 μl per cycle) were achieved. The drug screening application was demonstrated by detecting alteration and recovery effects of cellular metabolism induced by the addition of substances to the medium.

Topics: Antineoplastic Agents; Biological Transport; Biosensing Techniques; Brain Neoplasms; Cell Culture Techniques; Cell Line, Tumor; Cytochalasin B; Drug Evaluation, Preclinical; Glucose; Humans; Hydrogen-Ion Concentration; Lactic Acid; Microelectrodes; Microfluidic Analytical Techniques; Oxygen

Cytochalasin B (CB) is known to inhibit a number of cancer types, but its effects on gliomas are unknown. We examined the in vitro effects of CB on the proliferation of human glioma U251 cells, as well as determined its mechanism of action. Cell proliferation was determined using CCK-8. The effect of CB on U251 cell morphology was observed under a transmission electron microscope. Cell cycle distribution was assessed using propidium iodine and Giemsa staining, and cell apoptosis was determined by annexin V-fluorescein isothiocyanate/propidium iodide. Cell cycle-related proteins were determined by Western blot. CB effectively inhibited U251 cell proliferation in a dose- and time-dependent manner. The 24, 48, 72, and 96 h IC50 values were 6.41 x 10(-2), 9.76 x 10(-4), 2.57 x 10(-5), and 2.08 x 10(-5) M, respectively. CB increased the proportion of cells in the G2/M phase in a dose-dependent manner, thus increasing the mitotic index and decreasing cdc2 and cyclin B1 protein levels. CB induced morphological changes in the cytoskeleton. Additionally, 10(-5) M CB induced apoptosis in 23.4 ± 0.5% of U251 cells (P < 0.05 vs control group). Caspase-3, -8, and -9 activities were increased after CB treatment. CB inhibited U251 glioma cell proliferation by damaging the microfilament structure. CB also induced glioma cell apoptosis, suggesting that it may be an effective therapeutic agent against gliomas.

Topics: Antineoplastic Agents; Apoptosis; Brain Neoplasms; Cell Cycle Checkpoints; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cytochalasin B; Gene Expression Regulation, Neoplastic; Glioma; Humans

This assay is based on morphological changes of rat glioma cells treated with db-cAMP. The db-cAMP treatment induces a tubulin-dependent change causing the cells to acquire a spherical shape. Pretreatment with tubulin inhibitors brings about the disintegration of tubulin polymer and/or prevents its polymerization. Cells with inhibited tubulin fail to respond to db-cAMP treatment. Cells treated with inhibitors of tubulin polymerization are then separated from the spherical cells by aspiration. A semiautomated scanning procedure evaluates the final culture density and yields graphical data.

Topics: Animals; Brain Neoplasms; Bucladesine; Cell Differentiation; Drug Screening Assays, Antitumor; Formazans; Glioma; Rats; Tubulin; Tumor Cells, Cultured

Brain tumor dispersal far from bulk tumor contributes to and, in some instances, dominates disease progression. Three methods were used to characterize brain tumor cell motility in vivo and in vitro: 1) 2 weeks after implantation in rat cerebral cortex, single C6 cells labeled with a fluorescent tag had migrated to brain sites greater than 16 mm distant from bulk tumor; 2) time-lapse videomicroscopy of human brain tumor cells revealed motility of 12.5 microns/hr. Ruffling leading edges and pseudopod formation were most elaborate in more malignant cells; 3) an in vitro assay was devised to quantitatively evaluate motility from a region of high cell density to one of lower cell density. Human brain tumor cells were plated in the center of a petri dish, washed, and refed, establishing a 2-cm circular zone of cells in the dish center. Motility was determined by counting cells daily at predetermined distances from the central zone perimeter. Cells were found 1 cm from the perimeter by 24 hours and 3 cm from the perimeter by 4 days. Increasing serum concentration increased motility; however, neither fibronectin nor arrest of cells in the G0 phase by hydroxyurea altered motility. The addition of cytochalasin B to block cytoskeletal assembly prevented cell motility. Motility increased with increased malignancy. Subpopulations of cells were created by clonal amplification of cells that had migrated most rapidly to the dish periphery. Although morphologically indistinguishable when compared to the original cell line from which they were derived, these subpopulations demonstrated significantly increased motility.

Topics: Amidines; Animals; Astrocytoma; Brain Neoplasms; Cell Movement; Clone Cells; Cytochalasin B; Fibronectins; Fluorescent Dyes; Glioma; Humans; Hydroxyurea; Image Processing, Computer-Assisted; Interphase; Microscopy, Video; Neoplasm Transplantation; Pseudopodia; Rats; Rats, Sprague-Dawley; Tumor Cells, Cultured

We established an in vitro cytokinesis-block micronucleus assay of human tumours for estimation of the proportion of cells undergoing mitosis (the dividing fraction, DF), the time for the number of nuclei to double and the radiosensitivity in terms of the micronucleus frequency, based on a concept described previously. Under certain conditions, the nuclear number doubling time (NNDT) was considered to represent the potential doubling time. Tumour specimens obtained at surgery were disaggregated into single-cell suspensions and were directly cultured in the presence of cytochalasin B with or without irradiation. At various intervals, the percentage of multinucleate cells (the plateau value represented the DF), the average number of nuclei per cell and the number of micronuclei in binucleate cells were determined. DF and NNDT values were obtained in 58 of the 73 tumours investigated, and the micronucleus frequency was obtained in 54 of these 58 tumours. The DF ranged from 4.1% to 71% and the NNDT ranged from 3.1 to 83 days. A DF > or = 20% was associated with a higher recurrence rate in patients undergoing curative operation. A correlation was found between the NNDT and the time to relapse in patients with recurrent disease. The average number of micronuclei per binucleate cell at 2 Gy of irradiation (after subtraction of the value at 0 Gy) ranged from 0.052 to 0.35. Tumours which produced more micronuclei after irradiation showed a better response to radiotherapy. This assay can be readily performed on human tumours and appears to have promise as a predictive assay for radiation therapy.

Topics: Adenocarcinoma; Brain Neoplasms; Carcinoma; Carcinoma, Squamous Cell; Carcinoma, Transitional Cell; Cell Division; Cell Nucleus; Cell Survival; Cytochalasin B; Dose-Response Relationship, Drug; Dose-Response Relationship, Radiation; Humans; Micronucleus Tests; Neoplasms; Predictive Value of Tests; Prognosis; Radiation Tolerance; Remission Induction; Sarcoma; Treatment Outcome; Tumor Cells, Cultured

A permanent cell line has been established from a human intracranial secondary melanoma. During 3 years of continuous growth in vitro the cells have maintained their characteristic phenotypic properties including melanin production. The cultured cells are highly tumorigenic in the athymic mouse and the tumours produced are histologically identical to the human tumour of origin.

Topics: Animals; Brain Neoplasms; Cell Division; Cell Line; Cell Nucleus; Chromosomes; Cytochalasin B; Humans; Melanins; Melanoma; Mice; Mice, Nude; Neoplasm Transplantation; Transplantation, Heterologous

The dynamics of cell-associated Concanavalin A (Con A) in astrocytes of the newborn rat (RNA), the rat glioma (AC), and the human glioblastoma (GB) were studied in vitro by fluorescence and electron microscopy. Con A receptors on the cell surface were seen usually as a continuous thin layer, and Con A accumulations in fluorescence microscopy were actually Con A receptors on complicatedly infolded cell membrane and collection of Con A pinosomes. No capping occurred in the three types of glial cells. The translational movement of Con A receptors on the cell surface was rapid in the AC, slow in the RNA, and intermediate in the GB, and partly associated with Con A internalization. Con A pinosomes were more numerous in the RNA compared with those in the AC and the GB. Colchicine accelerated the translational mobility of surface Con A receptors more markedly in the AC and the GB than in the RNA. The translational movement Con A receptors, when treated with cytochalasin B, was retarded in the RNA and the GB and rather accelerated in the AC. Con A pinosomes were decreased in the three types of glial cells by treatment with colchicine or cytochalasin B.

Topics: Animals; Astrocytes; Brain Neoplasms; Colchicine; Cytochalasin B; Glioma; Microscopy, Electron; Microscopy, Fluorescence; Neuroglia; Rats; Receptors, Concanavalin A

Tissue culture monolayers of seven human intracranial tumours comprising 2 astrocytomas, 3 meningiomas, 1 secondary squamous cell carcinoma and 1 secondary adenocarcinoma were examined by a double immunofluorescent staining technique to demonstrate Concanavalin A (Con A) surface receptors and cytoplasmic actin in the same cell. Tumour cells, treated with fluoresceinisothiocyanate-labelled Con A (FITC-Con A) showed staining in cell margins or in a random distribution over the cell surface. Incubating the cells with FITC-Con A at 37 degrees for increasing periods of time resulted first in staining of clusters and later of perinuclear globules. Cells, pretreated with 4% paraformaldehyde at 4 degrees for 10 min or with cytochalasin B at 37 degrees for 30 min showed staining restricted to cell margins. In the cytochalasin B-treated cells, the peripheral staining was in the form of coarse clusters. Double fluorochrome studies showed that the anti-actin antibody (AAA) staining occurred in sites closely related to those stained by FITC-Con A both in untreated as well as in cytochalasin B-treated cells. The findings suggest that Con A receptors, as an example of a stable cell membrane determinant in human tumour cells, are associated with actin and that their mobility on the cell surface is dependent on an intact cytoplasmic actin system.

Topics: Actins; Adenocarcinoma; Astrocytoma; Brain Neoplasms; Carcinoma, Squamous Cell; Colchicine; Cytochalasin B; Cytoplasm; Fluorescent Antibody Technique; Humans; In Vitro Techniques; Meningioma; Receptors, Concanavalin A; Receptors, Drug

Primary tissue cultures of human gliomas were treated with cytochalasin B (0.5--60 microgram/ml for 90 min). Cell motility was inhibited irreversibly in glial tumour cells, but the effect was reversible on the mesenchymal cells growing in culture in the lower dose range. Cell adhesion was considerably reduced as the dose was increased, as was the capacity for cells to spread on a surface from suspension. Low concentrations of cytochalasin B caused negligible cell death and little disruption of cell ultrastructure. However, increases in dose were accompanied by a greater predominance of rough endoplasmic reticulum and inclusions and aggregation of microfilament bundles. As seen by scanning electron microscopy, cytochalasin B caused the withdrawal of peripheral cell borders, disappearance of ruffles and the breakdown of cytoplasmic lamellae. Charateristic surface blebs and folds appeared in their place. By comparison, colchicine (1--10 microgram/ml) caused a less marked and non-specific reversible reduction in cell motility on both glial and mesenchymal cells. No significant change in cell adhesion or spreading took place even at high doses, although at all concentrations gross disruption of the cell surface took place with changes in ultrastructure characterised by loss of cytoplasmic microtubules and aggregation of 10 nm filaments.

Topics: Brain Neoplasms; Cell Movement; Cell Survival; Colchicine; Connective Tissue; Cytochalasin B; Cytoskeleton; Endoplasmic Reticulum; Glioma; Humans; Microscopy, Electron; Microscopy, Electron, Scanning; Microtubules; Neuroglia

Topics: Animals; Brain Neoplasms; Cell Line; Cell Nucleus; Cytochalasin B; DNA, Neoplasm; In Vitro Techniques; Neoplasms, Nerve Tissue; Radiation-Sensitizing Agents; Rats; Thymidine; Time Factors