demecolcine and Glioma

About 9% of human cancers are brain tumors, of which 90% are gliomas. gamma-Radiation has been identified as a risk factor for brain tumors. In a previous pilot study, we found that lymphocytes from patients with glioma were more sensitive to gamma-radiation than were lymphocytes from matched control subjects. In this larger case-control study, we compared the gamma-radiation sensitivity of lymphocytes from glioma patients with those from control subjects and investigated the association between mutagen sensitivity and the risk for developing glioma.. We used a mutagen sensitivity assay (an indirect measure of DNA repair activity) to assess chromosomal damage. We gamma-irradiated (1.5 Gy) short-term lymphocyte cultures from 219 case patients with glioma and from 238 healthy control subjects frequency matched by age and sex. After irradiation, cells were cultured for 4 hours, and then Colcemid was added for 1 hour to arrest cells in mitosis. Fifty metaphases were randomly selected for each sample and scored for chromatid breaks. All statistical tests were two-sided.. We observed a statistically significantly higher frequency of chromatid breaks per cell from case patients with glioma (mean = 0.55; 95% confidence interval [CI] = 0.50 to 0.59) than from control subjects (mean = 0.44; 95% CI = 0.41 to 0.48) (P<.001). Using 0.40 (the median number of chromatid breaks per cell in control subjects) as the cut point for defining mutagen sensitivity and adjusting for age, sex, and smoking status, we found that mutagen sensitivity was statistically significantly associated with an increased risk for glioma (odds ratio = 2.09; 95% CI = 1.43 to 3.06). When the data were divided into tertiles, the relative risk for glioma increased from the lowest tertile to the highest tertile (trend test, P<.001).. gamma-Radiation-induced mutagen sensitivity of lymphocytes may be associated with an increased risk for glioma, a result that supports our earlier preliminary findings.

Topics: Adult; Animals; Brain Neoplasms; Case-Control Studies; Chromatids; Chromosome Breakage; Demecolcine; DNA; DNA Damage; DNA Repair; DNA, Single-Stranded; Female; Gamma Rays; Genetic Predisposition to Disease; Glioma; Humans; Lymphocytes; Male; Middle Aged; Neoplasms, Radiation-Induced; Odds Ratio; Radiation Tolerance; Risk; Smoking

The effect of the antitubulin agent colcemid on human glioma cells was investigated by sorting cells with different DNA content and subjecting them to confocal laser microscopy and transmission electron microscopy. The human glioma cell line U251MG was exposed to colcemid at a concentration of 0.05 microg for 16 h. Flow cytometric analysis revealed the accumulation of cells in S/G2M phase. Cells harvested from each of G0/G1 and S/G2M peaks were then analyzed by confocal laser microscopy and transmission electron microscopy. Confocal laser microscopy revealed that colcemid-treated cells harvested from the G0/G1 peak contained mitotic and apoptotic cells in addition to interphase cells. Electron microscopy confirmed that colcemid-treated cells in the G0/G1 peak had fragmented nuclei typical of apoptotic cells and mitotic cells with altered chromatin structure. Some mitotic cells obtained by mitotic shake-off after treatment with colcemid showed DNA strand breaks defined by in situ nick end labeling. The present study indicates that mitotic as well as interphase apoptosis occurs in U251MG cells following colcemid treatment.

Topics: Apoptosis; Cell Cycle; Demecolcine; DNA Fragmentation; Flow Cytometry; Glioma; Humans; Interphase; Microscopy, Confocal; Microscopy, Electron; Mitosis; Tumor Cells, Cultured

The parallel effects of simvastatin on cell cycle and PKC activity in rat C6 glioma cells were investigated. Simvastatin, 2.5 microM, for 24 h resulted in cell growth arrest in early G1 phase of the cell cycle and in a significant increase of total PKC activity (283 +/- 42 vs 470 +/- 61 pmoles/min/mg protein p = 0.002 for control cells and simvastatin-treated cells, respectively). The effect of simvastatin was fully prevented by mevalonate. A time dependent increase of PKC activity was observed in control exponentially free-growing C6 cells approaching confluency: a highly significant negative correlation (r = -0.91 p < 0.0001) between PKC activity and growth rate was calculated. PKC activity was high in cells arrested in G0 by serum starvation (0.4%). Following addition of complete medium (17.5% serum) the PKC activity progressively decreased and reached a minimum when cells traversed the G2/M phase, as determined by DNA analysis distribution. PKC activity dropped 30% in simvastatin-arrested early G1 cells; 44% in hydroxyurea-arrested cells at the G1/S boundary; and 73% in Colcemid mitosis-blocked cells. The results show that C6 glioma cell PKC activity is maximal in a G0 quiescent state and varies at different points of the cell cycle.

Topics: Animals; Astrocytoma; Cell Cycle; Demecolcine; Glioma; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Hydroxyurea; Lovastatin; Mevalonic Acid; Protein Kinase C; Rats; Simvastatin; Tumor Cells, Cultured

Reexpression of growth control and differentiation in response to physiological inducers can be demonstrated in some malignant cell lines, showing that they are not irreversibly transformed. This switch in phenotype is likely to reflect a changing pattern of gene expression, but it has not been known whether such cellular transitions involve major or only minor modulation of chromatin structure. We have studied growth control and accessibility of chromatin to DNase I in C6 glioma cells subjected to different growth regimens using an in situ nick translation assay to label the most exposed regions of nuclear chromatin. In fibroblasts and primary glia, exposed chromatin was localized mainly at the nuclear lamina. This readily labeled DNA structure was largely lacking in the malignant C6 glioma. When C6 cells were treated with dibutyryl cyclic AMP, exposed chromatin was reestablished around the nuclear periphery. This restoration of a normal genome exposure pattern required cytoskeletal integrity. Thus large-scale nuclear reorganization events proceed in parallel with phenotypic normalization. The changes in cell morphology, growth control, cytoskeletal organization, and chromatin exposure and localization are similar to the reverse transformation reaction in CHO-K1 cells, which is also regulated by the cyclic nucleotide system. Hydrocortisone and dexamethasone also restored genome exposure in C6 but less markedly than cAMP derivatives. Diverse transformed cells can thus respond to growth control stimuli with similar nuclear restructuring events, which presumably underlie changes in gene expression. Reverse transformation and redifferentiation appear to be alternative terms describing essentially the same biological phenomenon.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Animals; Animals, Newborn; Brain; Bucladesine; Butyrates; Butyric Acid; Cell Division; Cell Nucleus; Chromatin; Culture Media; Cytoskeleton; Demecolcine; Deoxyribonuclease I; Dexamethasone; DNA, Neoplasm; Fibroblasts; Gene Expression Regulation, Neoplastic; Genetic Techniques; Genome; Glioma; Hydrocortisone; Rats; Rats, Sprague-Dawley; Transformation, Genetic; Tumor Cells, Cultured

As part of our research on the anti-tumour effects of calmodulin antagonists, we examined the localization of Ca(2+)-calmodulin in mitotic C6 glioma cells. Monoclonal anticalmodulin antibodies which require Ca2+ for binding and CREST serum which recognizes kinetochores were used to stain ultrathin frozen sections. By indirect immunofluorescence and immunoelectron microscopy of colcemid-treated cells, Ca(2+)-calmodulin was present in the kinetochore region of the cell. By double label indirect immunofluorescence using anticalmodulin antibodies and CREST serum to stain untreated cells, calmodulin was found to colocalize with kinetochores. On the basis of these results, we hypothesize that Ca(2+)-calmodulin in the kinetochore depolymerizes the microtubules which are transported by dynein in the kinetochore during metaphase oscillating and anaphase poleward chromosomal movements. This hypothesis, which is currently under further investigation, may help explain a mechanism for the antitumour effects of calmodulin antagonists in the treatment of brain tumours.

Topics: Animals; Antineoplastic Agents; Brain Neoplasms; Calcium; Calmodulin; Demecolcine; Fluorescent Antibody Technique; Glioma; Microscopy, Immunoelectron; Rats; Spindle Apparatus

The purpose of the present study was to examine the distribution pattern of electron-dense acridine orange (AO) chromatin interaction products in rat glioma C6 cells at different phases of the cell cycle. For synchronization in the early S-phase the cells in logarithmic growth were treated with 3 micrograms/ml aphidicolin, a specific inhibitor of DNA polymerase alpha and then cultured in normal medium. For synchronization in the M-phase the cells cultured with aphidicolin and then returned to normal medium were treated with 0.05 micrograms/ml colcemid. Histoautoradiographic analysis of the C6 cells using the pulse chase method demonstrated approximately 16 h of cell cycle time and about 6.5 h of S-phase. Ultracytochemically, AO chromatin interaction products were found in all phases of the cell cycle except for the mitotic phase, namely in G1, S, and G2. The highest percentage of AO chromatin interaction products was observed in the early S-phase and the lowest in the G2 phase. The mean number of AO chromatin interaction products per nuclear area increased in the course of S-phase parallel with an increase of 3H-uridine uptake during the S-phase. The results show a characteristic distribution pattern of AO label specific for each of the four stages of the cell cycle, however, the significance of the coincident RNA synthetic activity remains to be elucidated.

Topics: Acridine Orange; Animals; Aphidicolin; Autoradiography; Cell Cycle; Chromatin; Demecolcine; Diterpenes; DNA, Neoplasm; Glioma; Microscopy, Electron; Rats; Staining and Labeling; Tumor Cells, Cultured

Topics: Cell Line; Cytoskeleton; Demecolcine; Fluorescent Antibody Technique; Glial Fibrillary Acidic Protein; Glioma; Humans; Intermediate Filament Proteins; Microscopy, Electron; Vimentin

During the late stages of cell spreading in vitro, the cells extrude a vesicular material into the medium. This phenomenon was observed in human glia and glioma cells as well as in human diploid fibroblasts MRC-5 and WI-38 cells. This extrusion of vesicular material is inhibited by cytochalasin-B and colcemid suggesting the involvement of microfilaments and microtubules and the active nature of this event. It appears that the cells may be excreting damaged surface components by a mechanism similar to patching, capping and endocytosis.

Topics: Cell Line; Cytochalasin B; Cytoskeleton; Demecolcine; Fibroblasts; Glioma; Humans; Microtubules; Neuroglia; Organoids

Cultured C-6 glial cells were utilized to evaluate the effect of antimicrotubular drugs on 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and cholesterol synthesis. Colchicine, Colcemid, and vinblastine (1.0 muM) caused a marked reduction in HMG-CoA reductase activity and, as a consequence, the rate of cholesterol synthesis in these cells. No effect was observed with lumicolchicine, a mixture of colchicine isomers with no effect on microtubules. The effect of colchicine was apparent within 1 h after addition to the culture medium, and, after 6 h, HMG-CoA reductase activity in treated cells was only approximately 15 to 30% of that in untreated cells. Reductase activity was very sensitive to the concentration of drug added, i.e. cells treated with just 0.1 muM colchicine for 6 h exhibited a 50% lower enzymatic activity than did untreated cells. The lack of a generalized, nonspecific toxic effect on the cells was indicated by the finding of no change in the activities of fatty acid synthetase and NADPH-cytochrome c reductase and the rate of total protein synthesis in cells treated with colchicine (1 muM) for 6 h. A close temporal and quantitative correlation was observed between the effects of colchicine on HMG-CoA reductase and on a parameter of microtubular function, i.e. maintenance of glial cell shape. The data suggest that microtubules are involved in the regulation of HMG-CoA reductase and cholesterol synthesis in C-6 glial cells.

Topics: Animals; Cell Line; Cholesterol; Colchicine; Demecolcine; Fatty Acid Synthases; Glioma; Hydroxymethylglutaryl CoA Reductases; Kinetics; Microtubules; NADPH-Ferrihemoprotein Reductase; Rats; Vinblastine

Topics: Animals; Cell Line; Cholesterol; Demecolcine; Glioma; Hydroxymethylglutaryl CoA Reductases; Kinetics; Microtubules; Nerve Tissue Proteins; Rats

Topics: Animals; Brain Neoplasms; Cell Division; Cells, Cultured; Demecolcine; Glioma; Neoplasms, Experimental; Rats

Cellular synchronization using Colcemid as pretreatment for combined chemoradiotherapy was investigated. C6 rat brain tumour was cultured in RPMI medium containing 10(-5)-10(-7) Mol. of Colcemid for 24 hours. The basic cell kinetics were analysed with a Pulse Cytophotometer, which facilitated the analysis of tumour cell cycle phase distribution according to the DNA content. The effect of Colcemid depended on the concentration, and the minimal concentration showing continuous blocking during 48 hours after removal of the drug was 10(-6) Mol. G1 fraction of 2 C DNA content was reduced from 74% to 36%. G2-M phase of 4 C DNA content increased from 9% to 28%. S phase cells increased from 17% to 31%. Polyploid cells in the Tetraploid cell cycle could be recognized. The remaining 36% of cells within the GO + G1 peak of 2 C DNA content were considered to be diploid GO cells.

Topics: Brain Neoplasms; Cell Division; Cells, Cultured; Demecolcine; Glioma; Humans; Neoplasms, Experimental; Premedication; Time Factors