demecolcine and Breast-Neoplasms

The knowledge of cell mechanics is required to understand cellular processes and functions, such as the movement of cells, and the development of tissue engineering in cancer therapy. Cell mechanical properties depend on a variety of factors, such as cellular environments, and may also rely on external factors, such as the ambient temperature. The impact of temperature on cell mechanics is not clearly understood. To explore the effect of temperature on cell mechanics, we employed magnetic tweezers to apply a force of 1 nN to 4.5 µm superparamagnetic beads. The beads were coated with fibronectin and coupled to human epithelial breast cancer cells, in particular MCF-7 and MDA-MB-231 cells. Cells were measured in a temperature range between 25 and 45 °C. The creep response of both cell types followed a weak power law. At all temperatures, the MDA-MB-231 cells were pronouncedly softer compared to the MCF-7 cells, whereas their fluidity was increased. However, with increasing temperature, the cells became significantly softer and more fluid. Since mechanical properties are manifested in the cell's cytoskeletal structure and the paramagnetic beads are coupled through cell surface receptors linked to cytoskeletal structures, such as actin and myosin filaments as well as microtubules, the cells were probed with pharmacological drugs impacting the actin filament polymerization, such as Latrunculin A, the myosin filaments, such as Blebbistatin, and the microtubules, such as Demecolcine, during the magnetic tweezer measurements in the specific temperature range. Irrespective of pharmacological interventions, the creep response of cells followed a weak power law at all temperatures. Inhibition of the actin polymerization resulted in increased softness in both cell types and decreased fluidity exclusively in MDA-MB-231 cells. Blebbistatin had an effect on the compliance of MDA-MB-231 cells at lower temperatures, which was minor on the compliance MCF-7 cells. Microtubule inhibition affected the fluidity of MCF-7 cells but did not have a significant effect on the compliance of MCF-7 and MDA-MB-231 cells. In summary, with increasing temperature, the cells became significant softer with specific differences between the investigated drugs and cell lines.

Topics: Actins; Biomechanical Phenomena; Breast Neoplasms; Bridged Bicyclo Compounds, Heterocyclic; Cell Line, Tumor; Demecolcine; Female; Fibronectins; Heterocyclic Compounds, 4 or More Rings; Humans; Magnetic Iron Oxide Nanoparticles; MCF-7 Cells; Microtubules; Temperature; Thiazolidines

We report on a new method for the study of radiation-induced chromatid breaks in stimulated human peripheral blood T lymphocytes, involving a combination of a 1-h colcemid block and a short (15 min) calyculin A treatment. We find that this procedure eliminates the problem of centromere splitting when calyculin A is used alone for a longer period and produces metaphase spreads with superior quality. By this procedure, the chromosomes and the chromatid breaks are expanded and thereby make for improved break scoring. In a comparison of the new technique with the conventional colcemid block method, we show a close proportionality between the frequencies of chromatid breaks scored with the two methods. The frequency of chromatid breaks with the new method was found to be significantly higher than that with colcemid alone, adding a higher sensitivity to the assay as an additional advantage.

Topics: Antineoplastic Agents; Breast Neoplasms; Carcinogens; Cells, Cultured; Chromatids; Chromatin Assembly and Disassembly; Chromosome Breakage; Chromosomes, Human; Demecolcine; Humans; Marine Toxins; Mutagenicity Tests; Oxazoles; Radiation Injuries

It has been known for many years that caffeine reduces or eliminates the G2-phase cell cycle delay normally seen in human HeLa cells or Chinese hamster ovary (CHO) cells after exposure to X or gamma rays. In light of our recent demonstration of a consistent difference between human normal and tumor cells in a G2-phase checkpoint response in the presence of microtubule-active drugs, we examined the effect of caffeine on the G2-phase delays after exposure to gamma rays for cells of three human normal cell lines (GM2149, GM4626, AG1522) and three human tumor cell lines (HeLa, MCF7, OVGI). The G2-phase delays after a dose of 1 Gy were similar for all six cell lines. In agreement with the above-mentioned reports for HeLa and CHO cells, we also observed that the G2-phase delays were eliminated by caffeine in the tumor cell lines. In sharp contrast, caffeine did not eliminate or even reduce the gamma-ray-induced G2-phase delays in any of the human normal cell lines. Since caffeine has several effects in cells, including the inhibition of cAMP and cGMP phosphodiesterases, as well as causing a release of Ca(++) from intracellular stores, we evaluated the effects of other drugs affecting these processes on radiation-induced G2-phase delays in the tumor cell lines. Drugs that inhibit cAMP or cGMP phosphodiesterases did not eliminate the radiation-induced G2-phase delay either separately or in combination. The ability of caffeine to eliminate radiation-induced G2-phase delay was, however, partially reduced by ryanodine and eliminated by thapsigargin, both of which can modulate intracellular calcium, but by different mechanisms. To determine if caffeine was acting through the release of calcium from intracellular stores, calcium was monitored in living cells using a fluorescent calcium indicator, furaII, before and after the addition of caffeine. No calcium release was seen after the addition of caffeine in either OVGI tumor cells or GM2149 normal cells, even though a large calcium release was measured in parallel experiments with ciliary neurons. Thus it is likely that caffeine is eliminating the radiation-induced G2-phase delay through a Ca(++)-independent mechanism, such as the inhibition of a cell cycle-regulating kinase.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; 3',5'-Cyclic-GMP Phosphodiesterases; 4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone; Animals; Breast Neoplasms; Caffeine; Calcium Channel Blockers; Calcium Signaling; Carcinoma; Chickens; Demecolcine; Drug Resistance; Female; Fibroblasts; G2 Phase; Gamma Rays; HeLa Cells; Humans; Mitosis; Neoplastic Stem Cells; Neurons; Ovarian Neoplasms; Phosphodiesterase Inhibitors; Quinazolines; Radiation Tolerance; Ryanodine; Ryanodine Receptor Calcium Release Channel; Thapsigargin; Tumor Cells, Cultured

The effects of doxorubicin (adriamycin, ADR) and daunorubicin (daunomycin, DAU), two anthracyclinic antibiotics, on a human breast carcinoma cell line (CG5) were studied by cytochemical and morphological methods. Both ADR and DAU were capable of inducing the multinucleation and spreading phenomena, associated with a decrease of the cell growth rate. DAU appeared to be more effective than ADR at the tested concentrations (10(-5), 5 x 10(-5) mM), in affecting the cell growth as well as in inducing multinucleation. As revealed by scanning electron microscopy, spreading and multinucleation were accompanied by a remarkable redistribution of surface structures. Moreover, a dose- and time-dependent rearrangement of the underlying cytoskeletal components was clearly detected. In addition, both ADR and DAU at 5 x 10(-5) mM seemed to favor the rebuilding of microtubules after treatment with colcemid, while a higher dose (10(-4) mM) exerted the opposite effect. Furthermore, both anthracyclines prevented the action of the antimicrotubular agent. When recovered after treatment with cytochalasin B, in presence of ADR (or DAU) (5 x 10(-5), 10(-4) mM), cells showed a microfilament pattern rearranged differently as compared to that of cells recovered in anthracycline-free medium. The results reported here strongly suggest the involvement of actin and tubulin in CG5 cell response to ADR and DAU treatments. Thus, the cytoskeletal apparatus is confirmed as another target involved in the mechanism of action of anthracyclines.

Topics: Actins; Antibiotics, Antineoplastic; Breast Neoplasms; Cell Transformation, Neoplastic; Cytochalasin B; Cytoskeleton; Daunorubicin; Demecolcine; Doxorubicin; Humans; Microscopy, Electron; Microscopy, Electron, Scanning; Microscopy, Fluorescence; Tubulin; Tumor Cells, Cultured

A mitotic cell subset has been identified with nuclear light scatter. Colcemid-treated T-47D human breast cancer cells were permeabilised, stained with ethidium bromide, and analysed by flow cytometry. Cells with G2M DNA content exhibited a unimodal distribution for DNA fluorescence and forward scatter, but two peaks were discernible with 90 degrees light scatter. A discrete low-scattering cell cluster could be distinguished from the G2 cell subset on two-dimensional contour plots of 90 degrees light scatter vs. DNA fluorescence; this cluster was reproduced by mitotic shake-off experiments and varied quantitatively with mitotic indices determined either by microscopy or by stathmokinetic cell-cycle analysis of DNA fluorescence. Cell sorting confirmed that the low-scattering cell cluster comprised predominantly metaphase and anaphase cells. Identification of mitotic cells with this one-step technique enables rapid analysis of drug-induced cell-cycle delay in cell populations with different rates of cell-cycle traverse. Hence, vincristine-induced cytostasis is shown to arise in part because of premitotic G2 arrest, whereas etoposide is shown to affect cycling cells with equal sensitivity in quiescent and activated cell populations. The use of light scatter to discriminate mitotic cells in this way facilitates analysis of drug-induced cell-cycle delay and supplements the information obtainable by conventional cell-cycle analysis.

Topics: Breast Neoplasms; Cell Cycle; Cell Separation; Demecolcine; DNA, Neoplasm; Etoposide; Flow Cytometry; Humans; Light; Microscopy, Fluorescence; Mitosis; Mitotic Index; Scattering, Radiation; Staining and Labeling; Tumor Cells, Cultured; Vincristine

Micronucleation was induced by vincristine, colcemid, and colcemid in combination with cytochalasin B in cells of a human metastatic breast carcinoma cell line (MDA MB 231). Cells treated with the latter combination were enucleated subsequently by centrifugation in the presence of cytochalasin B. The resulting "microcell" fraction was fused with mitotic human primary fibroblasts or mitotic HeLa cells using polyethyleneglycol (PEG). The success of the microcell-mediated chromosome transfer thus could be demonstrated as premature condensation in the mitotic recipient of the transferred micronuclei. This technique of cytogenetic analysis allowed a fast and simple control of the influence of different conditions on micronucleation and fusion of micronuclei with recipient cells. It could be shown that microcell-mediated chromosome transfer from human tumor cells into human normal, as well as human tumor, recipient cells is practicable if the techniques figured out by the present study are employed.

Topics: Breast Neoplasms; Cell Fusion; Cell Line; Cell Nucleus; Chromosomes, Human; Cytochalasin B; Demecolcine; Female; Genetic Engineering; Genetic Vectors; HeLa Cells; Humans; Neoplasms; Polyethylene Glycols; Vincristine

Topics: Breast; Breast Neoplasms; Colchicine; Demecolcine; Humans; Neoplasms; Radiotherapy