Cell mechanical properties of human breast carcinoma cells depend on temperature.

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.

Automated Dicentric Aberration Scoring for Triage Dose Assessment: 60Co Gamma Ray Dose-response at Different Dose Rates.

The objective of this study was to establish radiation dose-response calibration curves using automated dicentric scoring to support rapid and accurate cytogenetic triage dose-assessment. Blood was drawn from healthy human volunteers and exposed to Co gamma rays at several dose rates (i.e., 1.0, 0.6, and 0.1 Gy min). After radiation, the blood was placed for 2 h in a 37 °C incubator for repair. Blood was then cultured in complete media to which a mitogen (i.e., phytoghemagglutinin, concentration 4%) was added for 48 h. Colcemid was added to the culture at a final concentration of 0.2 µg mL after 24 h for the purpose of arresting first-division metaphase mitotics. Cells were harvested at the end of 48 h. Samples were processed using an automated metaphase harvester and automated microscope metaphase finder equipped with a suite of software including a specialized automated dicentric scoring application. The data obtained were used to create dose-response tables of dicentric yields. The null hypothesis that the data is Poisson-distributed could not be rejected at the significance level of α = 0.05 using results from a Shiny R Studio application (goodness-of-fit Poisson). Calibration curves based on linear-quadratic fits for Co gamma rays at the three different dose rates were generated using these data. The calibration curves were used to detect blind test cases. In conclusion, using the automated harvester and automated microscope metaphase finder with associated automated dicentric scoring software demonstrates high-throughput with suitable accuracy for triage radiation dose assessment.

Anti-Microtubule Drugs.

Small molecule drugs that target microtubules (MTs), many of them natural products, have long been important tools in the MT field. Indeed, tubulin (Tb) was discovered, in part, as the protein binding partner of colchicine. Several anti-MT drug classes also have important medical uses, notably colchicine, which is used to treat gout, familial Mediterranean fever (FMF), and pericarditis, and the vinca alkaloids and taxanes, which are used to treat cancer. Anti-MT drugs have in common that they bind specifically to Tb in the dimer, MT or some other form. However, their effects on polymerization dynamics and on the human body differ markedly. Here we briefly review the most-studied molecules, and comment on their uses in basic research and medicine. Our focus is on practical applications of different anti-MT drugs in the laboratory, and key points that users should be aware of when designing experiments. We also touch on interesting unsolved problems, particularly in the area of medical applications. In our opinion, the mechanism by which any MT drug cures or treats any disease is still unsolved, despite decades of research. Solving this problem for particular drug-disease combinations might open new uses for old drugs, or provide insights into novel routes for treatment.

Effect of demecolcine-assisted enucleation on the MPF level and cyclin B1 distribution in porcine oocytes.

Demecolcine (DEM) treatment of oocytes induces formation of a membrane protrusion containing a mass of condensed maternal chromosomes, which can be removed with minimal damage prior to somatic cell nuclear transfer (SCNT). However, the effect of this method on the distribution of maturation-promoting factor (MPF) in porcine oocytes has not been reported. Here, the level of MPF and the distribution of cyclin B1 were assessed in porcine oocytes following DEM treatment. In addition, the efficiencies of DEM-assisted and mechanical enucleation were compared, as were the development (in vitro and in vivo) of these oocytes following SCNT. MPF was uniformly distributed in oocytes that had been treated with 0.4 µg/ml DEM for 1 h. Immunofluorescence microscopy showed that in untreated oocytes, cyclin B1, the regulatory subunit of MPF, accumulated around the spindle, and was lowly detected in the cytoplasm. DEM treatment disrupted spindle microtubules, induced chromosome condensation, and reduced the level of cyclin B1 in the nuclear region. Cyclin B1 was uniformly distributed in DEM-treated oocytes and the level of MPF was increased. The potential of embryos generated from DEM-treated oocytes to develop in vivo was significantly greater than that of embryos generated from mechanically enucleated oocytes. This is the first study to report the effects of DEM-assisted enucleation of porcine oocytes on the distribution of cyclin B1. MPF in mature oocytes is important for the development of reconstructed embryos and for efficient SCNT.

Enucleation of demecolcine-treated bovine oocytes in cytochalasin-free medium: mechanism investigation and practical improvement.

Demecolcine-assisted/induced enucleation has been used in nuclear transfer cloning procedures for many species, yet its mechanism of action remains unclear. Primarily because oocytoplasm protrusion induced by demecolcine is inhibited by the presence of cytochalasin, its use has had limited application. In this experiment, we investigated the microtubule and microfilament alterations in bovine oocytes after demecolcine and/or cytochalasin B (CB) treatments by immunocytochemical staining. We also examined mechanical enucleation of demecolcine-treated oocytes in cytochalasin-free medium. The results showed that demecolcine-treatment disrupts the balance between microtubule/microfilament interactions primarily by deleting microtubules and with little effect on the microfilaments that we believe accounts for the membrane protrusion. The CB treatment reduced the amount of microfilaments but had little effect on the microtubules. Most demecolcine-induced membrane protrusions disappeared when exposed to CB. Western blotting showed that CB treatment increases G-actin, which indicates a decrease in the microfilaments. High oocyte enucleation, survival, and developmental rates occurred when demecolcine-assisted enucleation was carried out in a CB-free solution. Higher blastocyst development rates and blastocyst cell numbers were achieved compared to control, indicating that CB is not necessary in the enucleation procedure of bovine oocytes. This study provides a clearer understanding of the mechanism for the demecolcine-induced oocyte membrane protrusion, and substantiates the practical use of demecolcine-assisted enucleation in a CB-free environment.

An improved technique for chromosomal analysis of human ES and iPS cells.

Prolonged in vitro culture of human embryonic stem (hES) cells can result in chromosomal abnormalities believed to confer a selective advantage. This potential occurrence has crucial implications for the appropriate use of hES cells for research and therapeutic purposes. In view of this, time-point karyotypic evaluation to assess genetic stability is recommended as a necessary control test to be carried out during extensive 'passaging'. Standard techniques currently used for the cytogenetic assessment of ES cells include G-banding and/or Fluorescence in situ Hybridization (FISH)-based protocols for karyotype analysis, including M-FISH and SKY. Critical for both banding and FISH techniques are the number and quality of metaphase spreads available for analysis at the microscope. Protocols for chromosome preparation from hES and human induced pluripotent stem (hiPS) cells published so far appear to differ considerably from one laboratory to another. Here we present an optimized technique, in which both the number and the quality of chromosome metaphase spreads were substantially improved when compared to current standard techniques for chromosome preparations. We believe our protocol represents a significant advancement in this line of work, and has the required attributes of simplicity and consistency to be widely accepted as a reference method for high quality, fast chromosomal analysis of human ES and iPS cells.

CENP-B interacts with CENP-C domains containing Mif2 regions responsible for centromere localization.

Recently, human artificial chromosomes featuring functional centromeres have been generated efficiently from naked synthetic alphoid DNA containing CENP-B boxes as a de novo mechanism in a human cultured cell line, but not from the synthetic alphoid DNA only containing mutations within CENP-B boxes, indicating that CENP-B has some functions in assembling centromere/kinetochore components on alphoid DNA. To investigate whether any interactions exist between CENP-B and the other centromere proteins, we screened a cDNA library by yeast two-hybrid analysis. An interaction between CENP-B and CENP-C was detected, and the CENP-C domains required were determined to overlap with three Mif2 homologous regions, which were also revealed to be involved in the CENP-C assembly of centromeres by expression of truncated polypeptides in cultured cells. Overproduction of truncated CENP-B containing no CENP-C interaction domains caused abnormal duplication of CENP-C domains at G2 and cell cycle delay at metaphase. These results suggest that the interaction between CENP-B and CENP-C may be involved in the correct assembly of CENP-C on alphoid DNA. In other words, a possible molecular linkage may exist between one of the kinetochore components and human centromere DNA through CENP-B/CENP-B box interaction.

Differential effects of vitamin E and three hydrophilic antioxidants on the actinomycin D-induced and colcemid-accelerated apoptosis in human leukemia CMK-7 cell line.

The actinomycin D (AD)-induced apoptosis in human leukemia CMK-7 cell line is greatly accelerated by microtubule disruption with colcemid (CL). We studied the effect of antioxidants on this apoptosis in order to learn how the universal signal mediators, reactive oxygen species (ROS), are involved. Caspase-3 activation and DNA fragmentation were both suppressed by vitamin E (VE), t-butylhydroxyanisole, and luteolin. The ROS formation in the AD treatment was evidenced by flow cytometry, and further supported by suppression of caspase-3 activation by superoxide radical-forming enzyme inhibitors (TTFA, rotenone, and DPI). The inhibition of apoptosis by VE was completed during the initial 1-h treatment with AD, but it did not appear when VE was added with CL to washed cells after AD treatment. Luteolin, an iron chelator PDTC, and a water-soluble VE analogue, trolox, inhibited the apoptosis when added with CL after the AD treatment. Western blot analysis showed that the proteolytic cleavage of procaspase-9 and procaspase-3 were both inhibited when VE was added with AD or when luteolin was added with CL, and that the cytochrome c liberation was suppressed by both antioxidants. This result implies that the ROS are initially formed in lipophilic environments (e.g. mitochondrial membrane) and then they diffuse into an aqueous environment (i.e. cytoplasm) where they promote the apoptotic process in combination with the cytoskeletal disruption. Thus, the different antioxidants are effective to scavenge ROS for preventing the apoptosis in its different phases.

NBD-isocolcemid-tubulin interaction: a novel one-step reaction involving no conformational adjustment of reactants.

Isocolcemid, a colcemid analogue in which the positions of the C-ring methoxy and carbonyl are exchanged, is virtually inactive in binding to tubulin and inhibiting the formation of microtubule assembly. We have found that the substitution of a NBD group in the side chain of the B-ring of isocolcemid can reverse the effect of these structural alterations (at the C-ring) and the newly synthesized NBD-isocolcemid restores the lost biological activity. It inhibits microtubule assembly with an IC(50) of 12 microM and competes efficiently with [(3)H]colchicine, for binding to tubulin. NBD-isocolcemid has two binding sites on tubulin; one is characterized by fast binding, whereas the binding to the other site is slow. These two sites are independent and unrelated to each other. Colchicine and its analogues compete with NBD-isocolcemid for the slow site. Association and dissociation rate constants for the fast site, obtained from the stopped-flow measurements, are (7.37 +/- 0. 70) x 10(5) M(-1) s(-1) and 7.82 +/- 2.74 s(-1), respectively. While the interaction of colchicine and its analogues with tubulin involves two steps, NBD-isocolcemid binding to tubulin at the slow site has been found to be a one-step reaction. This is evident from the linear dependence of the observed rate constant (k(obs)) with both NBD-isocolcemid and tubulin concentrations. The interaction of NBD-isocolcemid with tubulin does not involve the conformational change of NBD-isocolcemid, as is evident from the unchanged CD spectra of the drug. The absence of enhanced GTPase activity of tubulin and the native-like protease cleavage pattern of the NBD-isocolcemid-tubulin complex suggest an unaltered conformation of tubulin upon NBD-isocolcemid binding to it as well. Implications of this on the mechanism of polymerization inhibition have been discussed.

Molecular dynamics simulation of colchicinoids.

Colchicine, a tricyclic alkaloid, has a remarkable range of biological activities. It binds with tubulin and prevents the formation of microtubules. This compound consists of a six membered aromatic ring (A ring), a seven membered troponoid ring (C ring) and another seven membered aliphatic ring (B ring). Using molecular mechanics and molecular dynamics simulations as tools, conformational analysis of colchicine and its several important analogs were done. Molecular mechanics studies show that conformational space of these molecules have one low energy region. Taking the low energy minima as the starting conformation, molecular dynamics simulation for 100 pico seconds is done for each of the analogs and molecular dynamics simulation in solution is done for three representative compounds colchicine,isocolchicine and A-C compound. Internal coordinate trajectories show that the value of the dihedral angle C9-C7-C1-C14 (phi), (C7-C1 bond connects the A and C ring), is within 40 degrees to 50 degrees for all the compounds with fluctuations less than 15 degrees. These calculations indicate that there is an overall similarity in the dynamically averaged structure of all the drugs. The A ring and B ring of the compounds are more or less rigid. The C ring is somewhat flexible, the average conformation and motional properties show overall similarity. The potential energy curve and dynamics behaviour of colchicine and isocolchicine suggests that the difference in binding property of colchine and isocolchicine may originate from the positional difference of carbonyl oxygen and methoxy group of C ring, which is the only difference in the structures of the two compounds and this has no effect on the motional property and average conformations of these two compounds. From our study it is proposed that the movements occuring at various positions of the drug molecules are significantly correlated. It is suggested that such correlated motion may play an important role in the biological property of these compounds.

In vitro induction of micronuclei in lymphocytes: the use of bromodeoxyuridine as a proliferation marker.

A simple method to determine the induction of micronuclei in cultured lymphocytes is described as an alternative to the cytochalasin-B method. It is proposed for use in the evaluation of the genotoxic potential of agents in vitro. It allows the recording of events only in the proliferating population of cells and at the same time it eliminates the possibility of recording combined effects with a cytokinesis-blocking agent. 16 microM bromodeoxyuridine (BrdU) was used to label proliferating cells that were treated with colcemid or mitomycin C at different concentrations. A monoclonal antibody against BrdU incorporated in the DNA and a peroxidase-diaminobenzidine brown stain were used to identify those cycling cells in a slide. To obtain the maximum yield of micronuclei, the best time for the addition of bromodeoxyuridine was found to be at 40 h from the initiation of cultures, 8 h before treating cells with the chemicals. Identification of micronuclei was easy, fast and unequivocal. In addition, the formation of structures similar to micronuclei, but that still are part of the nucleus could be observed. It is not clear if these structures are an intermediate stage in the formation of MN, but this methodology provides the possibility of observing and studying them.

Thermodynamics of colchicinoid-tubulin interactions. Rrol of B-ring and C-7 substituent.

The quenching of tryptophan fluorescence has been used to determine the kinetic and thermodynamic parameters of binding of B-ring analogs of colchicine to tubulin. The on rate, activation energy, off-rate, and thermodynamics of binding reaction have been found to be controlled at different points of analog structure. The on-rate and off-rate of deacetamidocolchicine (DAAC) binding with tubulin is 17 times slower than that of 2-methoxy-5-(2',3',4'-trimethoxyphenyl)tropone-tubulin (AC-tubulin) interaction, although both reactions have very similar activation energies. The presence of B-ring alone does not significantly affect the thermodynamics of the binding reactions either, since both AC-tubulin and DAAC-tubulin interactions are enthalpy driven. Introduction of a NH2 group at C-7 position of the B-ring, as in deacetylcolchicine (NH2-DAAC) lowers the on-rate further with a significant rise in the value of the activation energy. However, bulkier substitutions at the same position, as in demecolcine (NHMe-DAAC) and N-methyldemecolcine (NMe2-DAAC) have no significant additional effect either on the on-rate or on the value of activation energy. Introduction of NH2 group in the C-7 position of B-ring also increases the positive entropy of the binding reaction to a significant extent, and it is maximum when NMe2 is substituted instead of NH2 group. Thus, interaction of NH2-DAAC, NHMe-DAAC, and NMe2-DAAC with tubulin are entropy driven. Our results suggest that the B-ring side chain of aminocolchicinoids makes contact(s) with dimeric tubulin molecules.