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At least 19 recordsLinked to original sources
PubMed2006

Demecolcine-induced enucleation of sheep meiotically maturing oocytes.

The objective of this study was to investigate the possible effect of demecolcine, a microtubule-disrupting reagent, on induced enucleation (IE) of sheep meiotically maturing oocytes. Immunofluorescent staining with anti-tubulin antibodies was used to examine the spindle status of the oocytes. When the oocytes with intact germinal vesicles (GV) were cultured in the medium containing various concentrations of demecolcine (0.01 to 0.4 microg.mL-1) for 20 to 22 h, the spindle microtubule organization and first polar body (PB1) extrusion were inhibited by demecolcine in a dose-dependent manner. The highest IE rate (58.1%) was from the treatment with 0.04 microg.mL-1 demecolcine. Demecolcine treatment applied after germinal vesicle breakdown (GVBD) or at metaphase (M) yielded a PB1 extrusion rate and IE efficiency similar to the treatment applied at the onset of maturation. Analysis by immunofluorescence showed that both nonspindle microtubules and spindle microtubules were significantly disorganized by demecolcine. Combination treatment with demecolcine and cycloheximide (CHX) or 6-dimethylaminopurine (6-DMAP) led to single pronuclear formation rather than PB1 extrusion. When demecolcine-treated oocytes were transferred into demecolcine-free medium, the ability to extrude PB1 was quickly restored and a 72.1% IE rate was obtained following such treatment. These results demonstrate that demecolcine can be used as a potential reagent for induced enucleation of sheep meiotically maturing oocytes and may greatly facilitate research in nuclear transfer.

Animals↗
PubMed2003

Effect of demecolcine and nocodazole on the efficiency of chemically assisted removal of chromosomes and the developmental potential of nuclear transferred porcine oocytes.

Brief treatment of metaphase II (MII) stage porcine oocytes with 0.4 microg/mL demecolcine in the presence of 0.05 M sucrose produces a membrane protrusion that contains a condensed chromosome mass. The present study examined the optimal conditions for demecolcine and nocodazole treatment in chemically assisted removal of chromosomes. When matured oocytes were treated with 0.1-0.4 microg/mL demecolcine for 60 min or with 0.4 microg/mL demecolcine for 30 min or 3 microg/mL nocodazole for 30 or 60 min, more than 70% of oocytes had a membrane protrusion containing condensed chromosomes were located. There was no difference in the in vitro developmental potential of enucleated oocytes assisted by 0.1 and 0.4 microg/mL demecolcine or 3 microg/mL nocodazole that received porcine somatic cells. After transfer to 10 recipients, however, two of six recipients that received demecolcine-treated enucleated eggs produced four healthy cloned piglets, but none of the four recipients of nocodazole-treated enucleated eggs produced piglets. Further studies are required to increase the successful development to term because the proportion of live piglets was low (4/2, 672, 0.15%).

Animals↗
PubMed2004

[Demecolcine-induced enucleation of Kunming mouse oocyte].

On the basis of exiting technique pathway of demecolcine-induced enucleation(IE), several factors (Demecolcine concentration, time of demecolcine inition and treatment, oocytes age) affecting the IE rate were tested using Kunming mouse oocytes. The experiments' results demonstrated that: In experiment 1,activated oocytes could be enucleated efficiently by treating with KSOM medium containing 0.4 microg/mL or 0.5 microg/mL demecolcine for 60 min, but 0.5 microg/mL group gained the higher IE rate(33.3%). In experiment 2, maximum IE rate (31.9% -approximately 24.5%) were obtained when oocytes were exposed to 0.5 microg/mL demecolcine between 0 and 5 min postactivation and treated for 60 -approximately 180 min. In experiment 3,oocytes collected from Kunming mouse at 17 -approximately 18h after hCG administration were favoriate to demecolcine-IE(27.1%). By comparison and analysis of the data, we established the optimized IE procedure.

Animals↗
PubMed2005

Activated bovine cytoplasts prepared by demecolcine-induced enucleation support development of nuclear transfer embryos in vitro.

Demecolcine-induced enucleation (IE) of mouse oocytes has been shown to improve development to term of cloned mice. In this study, we characterized the kinetics and morphological progression of bovine oocytes subjected to IE, and evaluated their ability to support embryo development to the blastocyst stage after nuclear transfer (NT). In vitro matured bovine oocytes were parthenogenetically activated and subsequently exposed to demecolcine at various times post-activation. Onset and duration of demecolcine treatment significantly altered activation and IE frequencies, which varied from 7.1% to 100% and 33.3% to 91.7%, respectively, at 5 hr post-activation. A significant decrease in IE frequencies was observed at 17 hr post-activation (3.4%-46.1%), possibly due to reincorporation of chromosomes into the oocyte after incomplete second polar body (PB) extrusion. Oocytes were reconstructed by NT before (treatment 1) or after (treatment 2) activation and demecolcine treatment, and cultured in vitro. Cleavage (48.1%-54.2%) and blastocyst rates (15.7%-19%) were equivalent for the two treatments, as well as the total cell number in NT blastocysts. Furthermore, most of the blastocysts were completely diploid (treatment 2) or heteroploid but with a majority of diploid nuclei (treatment 1). Our results demonstrate that the IE method can be successfully used to produce enucleated bovine cytoplasts that are competent to support development to the blastocyst stage after NT. This technically simple approach may provide a more efficient method to enhance the success rate of NT procedures. Further studies are needed to improve the in vitro development efficiency and to expand our understanding of the mechanism(s) involved in demecolcine-induced enucleation.

Animals↗
PubMedDate not supplied

Action of demecolcine (colcemid) in the murine sarcoma 180 tumor.

Iv infusions of demecolcine (Colcemid) were established in mice bearing the Sarcoma 180 tumor. During demecolcine infusion at a rate of 6 microgram/hr, the tumor labeling index following 3H-thymidine administration and the mean grain count per labeled cell did not differ significantly from control values. Cells in anaphase and telophase disappeared from the tumor sections, and the net number of cells in the earlier stages of mitosis increased for 20 hrs. The rate of mitotic accumulation during the initial 8 hrs of demecolcine infusion was about one third of what would be expected from the known cell cycle values for this tumor. This reduced rate of accumulation does not appear to result from death of mitotic cells, escape of cells from metaphase blockade, or S-phase arrest secondary to inhibition of DNA synthesis; it is suggested that the reduced accumulation rate is secondary to demecolcine damage to interphase cells. Tumor cells that have been gathered by 4-hr demecolcine infusions may be released in synchronous fashion following the end of the infusion. A small proportion of these cells will recycle synchronously during the following mitotic period.

Anaphase↗
PubMed2005

Nuclear transfer of M-phase ferret fibroblasts synchronized with the microtubule inhibitor demecolcine.

The development of reconstructed embryos following nuclear transfer (NT) appears to be dependent upon a variety of factors, including cell cycle synchronization between the donor nucleus and recipient oocyte. Here we use the microtubule inhibitor, demecolcine, to synchronize ferret fibroblasts in metaphase (M-phase) in order to match their cell cycle position with that of the recipient oocyte at the time of NT. The fibroblasts were obtained from 28-day fetuses and cultured for 1-30 days prior to NT. Fibroblast cultures were treated with 0.05 microg/ml of demecolcine for 3 hr or overnight (14-16 hr) after various times in culture to determine the optimal conditions for M-phase synchronization. The percentage of G2/M-phase cells in demecolcine-treated cultures was significantly greater than that found in untreated cultures (P<0.05). Optimally synchronized M-phase fibroblasts were collected by mitotic shake-off and evaluated for their effectiveness in NT. M-phase somatic cell-derived NT embryos reconstituted by electrofusion or microinjection underwent implantation and formed fetuses at similar rates (5.4% vs. 3.4%, and 1.8% vs. 1.2%, respectively); however, no NT embryos developed to term. In summary, these data demonstrate two important points. First, demecolcine treatment effectively synchronizes ferret fibroblasts in M-phase of the cell cycle; and second, these somatic cells are capable of driving embryo development following NT. Our results should facilitate the development of cloned ferrets as an animal model for human lung disease such as influenza and cystic fibrosis.

Analysis of Variance↗
PubMed2006

Demecolcine-assisted enucleation for bovine cloning.

The present study demonstrated that demecolcine treatment for at least 30 min produces a membrane protrusion in metaphase II-stage bovine oocytes. The maternal chromosome mass is condensed within the protrusion, which makes it easy to remove the maternal chromosomes for nuclear transfer (NT). Maturation promoting factor activity, but not mitogen-activated protein kinase activity, increased up to 30% in oocytes during demecolcine treatment. One normal healthy calf was obtained after transfer of four NT blastocysts produced following demecolcine treatment. Demecolcine treatment did not increase the potential of NT oocytes to develop into blastocysts. The present study demonstrated that chemically-assisted removal of chromosomes is effective for bovine cloning.

Actins↗
PubMed2002

Demecolcine-induced oocyte enucleation for somatic cell cloning: coordination between cell-cycle egress, kinetics of cortical cytoskeletal interactions, and second polar body extrusion.

Studies were designed to further explore the use of pharmacological agents to produce developmentally competent enucleated mouse oocytes for animal cloning by somatic cell nuclear transfer. Metaphase II oocytes from CF-1 and B6D2F1 strains were activated with ethanol and subsequently exposed to demecolcine at various times postactivation. Chromosome segregation, spindle dynamics, and polar body (PB) extrusion were monitored by fluorescence microscopy using DNA-, microtubule-, and microfilament-selective probes. Exposure to demecolcine did not affect rates of oocyte activation induced by ethanol but did disrupt the coordination of cytokinesis and karyokinesis, suppressing the extent and completion of spindle rotation and second PB extrusion in a strain-dependent manner. Moreover, strain- and treatment-specific variations in the rate of oocyte enucleation were also detected. In particular, CF1 oocytes were more efficiently enucleated relative to B6D2F1 oocytes, and demecolcine treatments initiated early after activation resulted in higher enucleation rates than when treatment was delayed. The observed strain differences are possibly caused by a combination of factors, such as the time course of meiotic cell-cycle progression after ethanol activation, the degree of spindle rotation, and the extent of second PB extrusion. These results suggest that developmentally competent cytoplasts can be produced by timely exposure of activated oocytes to agents that disrupt spindle microtubules. However, the utility of the demecolcine-induced enucleation protocol will require further investigation into factors linking karyokinesis to cytokinesis at the levels of cell-cycle control and oocyte cytoskeletal remodeling following artificial or natural means of egg activation.

Animals↗
PubMed1999

Lack of synchrony among multiple nuclei induces partial DNA fragmentation in V79 cells polyploidized by demecolcine.

The nuclear morphology of polyploidized cells was examined in V79 Chinese hamster cells polyploidized by demecolcine or K-252a, inhibitors of spindle fibre formation and protein kinases, respectively. A variety of nuclear morphologies, including multinuclei, were observed in V79 cells polyploidized by demecolcine but not by K-252a, which produced mononuclear cells. A lack of synchrony in the nuclear cycle was observed among nuclei in multinuclear polyploidized cells. Partial DNA fragmentation, defined as DNA fragmentation of a nucleus in a multinuclear cell, was detected using the TUNEL method in V79 cells polyploidized by demecolcine but not by K-252a. Apoptosis occurred earlier in cell populations treated with demecolcine than in these treated with K-252a once the drugs were removed from the medium, suggesting that polyploidized cells with separate nuclei tend to apoptose earlier than those with mononuclei.

Animals↗
PubMed1994

Apoptosis by demecolcine in V79 cells.

Demecolcine (Colcemid), an inhibitor of spindle fiber formation in M phase, induced apoptosis in V79 cells. At a concentration of 0.01 microgram/ml demecolcine, V79 cells proliferated exponentially as well as controls, although temporal M phase accumulation occurred 6 h after the addition of demecolcine. At 0.1 microgram/ml, the cells became hyperploid after remaining in the M phase for some time. Apoptosis occurred in V79 cells exposed to demecolcine at a concentration of 0.03 microgram/ml. Apoptosis was defined as the appearance of a sub-G1 peak in DNA histograms and a ladder pattern of fragmented DNA in gelelectrophoresis.

Animals↗
PubMed1996

Inhibition of demecolcin-induced DNA synthesis by inhibitors of phospholipase C and protein kinase C.

Exposing normal human breast epithelia (HBE) cells, which were growth arrested by a 3-day culture in EGF-deprived medium, to the microtubule disrupting agent, demecolcin (N-deacetyl-N-methyl-colchicine), for 2 hr significantly stimulated the initiation of DNA synthesis 22 hr later. The demecolcin-induced DNA synthesis was not accompanied by activation of the EGF receptor and it was inhibited by calphostin C, an inhibitor of protein kinase C (PKC), and U-73122, an inhibitor of phospholipase C (PLC). Contrary to this, the EGF-induced DNA synthesis was inhibited by tyrphostin A25, a specific inhibitor of the EGF receptor tyrosine kinase, and calphostin C. The results suggested that the involvement of PLC and PKC in the demecolcin-induced signal transduction pathway leads to the initiation of DNA synthesis.

Androstadienes↗
PubMed1990

Determination of the antimitotic agents N-desacetylcolchicine, demecolcine and colchicine in serum and urine.

In an effort to characterize the pharmacokinetic behavior of the antimitotic agent N-desacetylcolchicine a selective, sensitive high-performance liquid chromatographic method was developed for the determination of N-desacetylcolchicine, demecolcine and colchicine in serum or urine. To 0.5 ml of serum or 0.1 ml of urine diluted to 0.5 ml were added 50 microliters demecolcine (2 micrograms/ml) which serves as the internal standard. The sample was extracted using a C2 reversed-phase solid extraction column. N-Desacetyl-colchicine, colchicine and the internal standard were eluted from the column with methanol. The combined eluates were evaporated to dryness and the residue was reconstituted with water. The reconstituted sample was injected into a C18 reversed-phase column and eluted using a mobile phase consisting of 0.1 M potassium dihydrogenphosphate, 5 mM 1-pentanesulfonic acid in methanol and acetonitrile with a final pH of 6.0, at a flow rate of 1.5 ml/min. N-Desacetylcolchicine, colchicine and the internal standard were detected using a variable-wavelength ultraviolet detector at 254 nm. The limit of detection was 0.4 ng/ml for desacetylcolchicine and 4.0 ng/ml for colchicine. The method is linear over a concentration range of 1.0-200 ng/ml. The method has been shown to be a rapid, reliable method to monitor N-desacetylcolchicine levels in clinical trials in cancer patients.

Chromatography, High Pressure Liquid↗
PubMed2001

Establishment of a tetraploid Meth-A cell line through polyploidization by demecolcine but not by staurosporine, K-252A and paclitaxel.

Polyploid cells are made by DNA reduplication without cell division, however, it is not easy to establish polyploid mammalian cell lines. It is worth studying the difference in cell character between hyperploid and parent cell lines. Meth-A cells were polyploidized by demecolcine, K-252a, staurosporine and paclitaxel. The cell-cycle responses of highly polyploid Meth-A cells after the removal of the drugs were examined by flow cytometry (FCM). Meth-A cells were highly polyploidized by these drugs. The polyploid Meth-A cells gradually decreased in ploidy after the drug release. A tetraploid Meth-A cell line was established only from the demecolcine-induced polyploid Meth-A cells. The duration of G1, S and G2/M phases of the tetraploid cell line were mostly the same as those of the parent diploid cells, except that the G2/M phase was 1.5 h longer. The chromosome number of tetraploid Meth-A cell line was about twice of the diploidy. A tetraploid Meth-A cell line was established.

Animals↗
PubMed2000

Different responses of polyploidized V79 cells after removal of two drugs, demecolcine and K-252a.

To examine whether or not cells polyploidized by different mechanisms behave in a different manner after drug removal, V79 Chinese hamster cells were assessed by flow cytometry (FCM) after their polyploidization by demecolcine and K-252a, inhibitors of spindle-fiber formation and protein kinase, respectively. Cell cycle analysis of DNA histograms of V79 cells before and after the drug release was performed. With both drugs, the ploidy of V79 cells increased just after the drug removal and was maintained for a week. A difference was evident 10 days after the release. Tetraploid cells were the main population from 10 to 18 days after the release of K-252a, but not demecolcine. Cell cycle parameters were almost the same in pseudo diploid and tetraploid V79 cells, except for the tetraploid S phase which was 2h longer.

Animals↗
PubMed1991

Expulsion of demecolcine-induced micronuclei from mouse bone marrow polychromatic erythrocytes.

The mouse micronucleus test is a valuable tool for evaluating in vivo chromosome damage produced by test articles in polychromatic erythrocytes of bone marrow. Compounds that are clastogens, such as cyclophosphamide, induce micronuclei that are smaller than those induced by compounds that are spindle poisons, such as demecolcine. In vitro studies have previously shown that the frequency of mitomycin C- and vincristine-induced micronuclei in mouse L-929 cells was reduced due to micronuclear extrusion following treatment with cytochalasin B. The current study shows that micronuclei are also expelled in vivo, that expulsion is dependent upon micronuclear size, and that observation of these extruded micronuclei is dependent upon the method of sample preparation.

Animals↗
PubMed1986

Effect of cytochalasin B and demecolcine on freeze-thaw survival of murine embryos in vitro.

The effects of cytochalasin B (CB), a microfilament inhibitor, and demecolcine (DC), a microtubule inhibitor, on freeze-thaw survival and culture survival of early cleavage stage mouse embryos, was evaluated. In the first experiment, eight-cell mouse embryos were frozen in dimethylsulfoxide (DMSO) or DMSO + 0.1 microgram/ml DC + 7.5 micrograms/ml CB. In the second experiment, eight-cell embryos were dehydrated and cultured in the presence of either DMSO, DMSO + DC, DMSO + CB, or DMSO + DC + CB for 45 min prior to rehydration and culture to stimulate the osmotic and chemical changes encountered during the dehydration and rehydration procedures, but without the consequences of freezing and thawing. In the third experiment, additional eight-cell embryos were frozen in either DMSO, DMSO + DC, DMSO + CB, or DMSO + DC + CB. The survival of embryos frozen in DMSO (75%) was significantly higher (P less than 0.01) than that of embryos frozen in DMSO + DC + CB (55%). No differences (P = 0.55) were observed after a 48-hr culture period in the development of embryos dehydrated, cultured, and rehydrated but not frozen. Embryos frozen in the presence of both DC and CB had a lower (P = 0.06) survival rate (55%) than that of embryos frozen in the presence of DMSO, DC, or CB (approximately 70%). These results suggest that both microfilaments and microtubules have a role in maintaining the structural integrity of the plasma membrane during the freeze-thaw process and that the loss of either loss does not seem to be detrimental to survival, but the loss of both results in lower survival.

Animals↗
PubMed1977

The formation of free axonal sprouts from a dorsal root ganglion-nerve preparation maintained in organotypic culture, and the effects of demecolcine.

Nerve-ganglion preparations from rat dorsal spinal nerve roots were maintained in organotypic culture for 20 h. Free axonal sprouts formed at the cut tips. Clear and dense-core vesicles, mitochondria and smooth endoplasmic reticulum accumulated in the axons for a distance of 500 micronm behind the cut, as has previously been described in dorsal roots sectioned in vivo. Sprouting did not occur in dorsal roots maintained in culture without their ganglia attached. Sprouting was also prevented by demecolcine (3 x 10(7) M) which reduced the number of microtubules in non-myelinated, small myelinated and large myelinated axons to respectively 45, 30 and 20% of control values. The sprouts contained several types of vesicle including small clear vesicles, large and small dense-core vesicles and flattened vesicles. The possible relevance of the vesicles to transmitter mechanisms in these neurones is discussed.

Animals↗