Combined Treatment with Demecolcine and 6-Dimethylaminopurine during Postactivation Improves Developmental Competence of Somatic Cell Nuclear Transfer Embryos in Pigs.

This study determined the effects of postactivation treatment with demecolcine and/or 6-dimethylaminopurine (6-DMAP) on in vivo and in vitro developmental competence of somatic cell nuclear transfer (SCNT) embryos in pigs. SCNT embryos were treated for 4 hours with 0.4 µg/mL demecolcine, 2 mM 6-DMAP, or both after electric activation, then transferred to surrogate pigs or cultured for 7 days. The formation rate of SCNT embryos with a single pronucleus was higher in combined treatment with demecolcine and 6-DMAP (95.2%) than treatment with demecolcine alone (87.1%). Blastocyst formation of SCNT embryos was significantly increased in combined treatment with demecolcine and 6-DMAP (48.7%) compared with demecolcine (22.2%) or 6-DMAP alone (37.3%). Fluctuation of maturation promoting factor activity showed different patterns among various postactivation treatments. Pregnancy was established in 1 of 5 surrogates after transfer of SCNT embryos that were treated with demecolcine and 6-DMAP. The pregnant surrogate delivered one healthy live piglet. The results of our study demonstrated that postactivation treatment with demecolcine and 6-DMAP together improved preimplantation development and supported normal in vivo development of SCNT pig embryos, probably influencing MPF activity and nuclear remodeling, including induction of single pronucleus formation after electric activation.

The effects of demecolcine, alone or in combination with sucrose on bovine oocyte protrusion rate, MAPK1 protein level and c-mos gene expression level.

AIMS: Our study aims to clarify the effects of demecolcine, alone or in combination with sucrose on bovine oocyte protrusion rate, MAPK1 protein level and c-mos gene expression level. METHODS: The effects of the demecolcine concentration, treatment duration, and synergistic effects with sucrose solution on the rate of membrane protrusions of bovine oocytes were investigated. Using real-time fluorescent quantitative PCR, western blot analysis, and immunofluorescence assays, the expression of the maternal c-mos gene, the protein level of mitogen-activated protein kinase 1 (MAPK1), and the change in the localization of spindles and nuclei during the demecolcine treatment were analyzed in bovine oocytes. RESULTS: Treatment of bovine oocytes with both demecolcine (0.6 µg/mL) and sucrose (0.05 M) for 1 h led to the highest rate of membrane protrusions, and synergistic effects were also observed. Real-time fluorescent quantitative PCR analysis revealed that the demecolcine treatment up-regulated the expression of the maternal c-mos gene. Western blot analysis indicated that the demecolcine treatment enhanced the protein level of MAPK1 in bovine oocytes. Immunofluorescence analysis indicated that the spindles and nuclei were localized at the place of the membrane protrusions. CONCLUSIONS: The present results suggest that demecolcine might contribute to the activation of the Mos/MAPK pathway and affect spindle structure. These results provide a reference for more efficient generation of enucleated bovine oocytes.

The construction of cloned Sika deer embryos (Cervus nippon hortulorum) by demecolcine auxiliary enucleation.

The objective of our study was to establish the feasibility of experimental protocols for cloning sika deer. We performed auxiliary enucleation to improve the efficiency of nuclear transfer operation by optimizing the demecolcine concentration to induce cytoplasmic protrusions in the sika deer oocytes. In the present study,we had studied the impact of different demecolcine concentrations on cytoplasmic protrusions and enucleation rates. We determined that 95.9% of the sika deer oocytes formed cytoplasmic protrusions when treated for 1 h with 0.8 µg/ml demecolcine. The lowest observed rate of protrusion was 19.3% after overnight treatment with demecolcine. When the oocytes aged or had a poor cumulus expansion, they exhibited a significant decrease in the ability to form cytoplasmic protrusions. The rates of enucleation (94.9% vs 85.8%, p < 0.05), cell fusion (84.6% vs 70.1%, p < 0.05) and blastocyst formation (15.4% vs 10.9%, p < 0.05) using demecolcine auxiliary enucleation were significantly higher than those after blind enucleation. These results demonstrated that sika deer oocytes could be enucleated quickly and effectively using demecolcine auxiliary enucleation, which could enhance the enucleation rate, cell fusion rate and blastocyst rate of cloned embryos in vitro.

A simplified approach for oocyte enucleation in mammalian cloning.

Despite its success in almost all farm and laboratory animals, somatic cell nuclear transfer (SCNT) is still a low-efficiency technique. In this investigation, we determined the impact of each enucleation step on oocyte viability (assessed by parthenogenetic activation): Hoechst (HO) staining, cytochalasin B, ultraviolet (UV) exposure, and demecolcine. Our data showed that of all the factors analyzed, UV exposure impaired oocyte development (cleavage, 59% for untreated oocytes vs. 8% UV exposed; blastocyst stage, 32% untreated vs. 0% UV exposed). A minor toxicity was detected following demecolcine treatment (cleavage, 62%; blastocyst stage, 13%). Next, we compared HO/UV (canonical) and demecolcine-assisted enucleation (DAE), with a straight removal of metaphase chromosomes without any chemical or physical aid (straight enucleation). DAE improved the preimplantation development of sheep cloned embryos compared to HO/UV enucleation (cleavage, 38% vs. 19%; blastocysts, 17% vs. 4%), yet straight enucleation resulted in the highest cleavage and blastocysts rates (61% and 30%, respectively). We concluded that: (1) UV exposure harms sheep oocyte and embryo development; (2) DAE may represent an alternative approach, especially for unskilled operators; and (3) straight enucleation remains, in our estimation, the most reliable and least harmful protocol for SCNT.

Demecolcine- and nocodazole-induced enucleation in mouse and goat oocytes for the preparation of recipient cytoplasts in somatic cell nuclear transfer procedures.

Treatment of pre-activated oocytes with demecolcine (DEM) has been shown to induce the extrusion of all oocyte chromosomes within the second polar body (PB2). However, induced enucleation (IE) rates are generally low and the competence of these cytoplasts to support embryonic development following somatic cell nuclear transfer (SCNT) is impaired. Here, we explored whether short treatments with DEM or another antimitotic, nocodazole (NOC), improve IE efficiency, and determined the most appropriate timing for nuclear transfer in the cytoplasts produced. We show, for the first time, that IE can be accomplished in mouse and goat oocytes using NOC and that short treatments with DEM or NOC result in similar IE rates, which proved to be strain- and species-specific. Because enucleation induced by both antimitotic drugs is reversible, the IE protocol was combined with the mechanical aspiration of PB2s to increase permanent enucleation rates in mouse oocytes. None of the cloned mouse embryos produced from the resultant cytoplasts developed to the blastocyst stage. However, when they were reconstructed prior to the activation and antimitotic treatment, their in vitro embryonic development was similar to that of cloned embryos produced from mechanically-enucleated oocytes.

Cloned mice derived from embryonic stem cell karyoplasts and activated cytoplasts prepared by induced enucleation.

Our objective was to induce enucleation (IE) of activated mouse oocytes to yield cytoplasts capable of supporting development following nuclear transfer. Fluorescence microscopy for microtubules, microfilaments, and DNA was used to evaluate meiotic resumption after ethanol activation and the effect of subsequent transient treatments with 0.4 micro g/ml of demecolcine. Using oocytes from B6D2F1 (C57BL/6 x DBA/2) donors, the success of IE of chromatin into polar bodies (PBs) was dependent on the duration of demecolcine treatment and the time that such treatment was initiated after activation. Similarly, variations in demecolcine treatment altered the proportions of oocytes exhibiting a reversible compartmentalization of chromatin into PBs. Treatment for 15 min begun immediately after activation yielded an optimized IE rate of 21% (n = 80) when oocytes were evaluated after overnight recovery in culture. With this protocol, 30-50% of oocytes were routinely scored as compartmentalized when assessed 90 min postactivation. No oocytes could be scored as such following overnight recovery, with 66% of treated oocytes cleaving to the 2-cell stage (n = 80). Activated cytoplasts were prepared by mechanical removal of PBs from oocytes whose chromatin had undergone IE or compartmentalization. These cytoplasts were compared with mechanically enucleated, metaphase (M) II cytoplasts whose activation was delayed in nuclear transfer experiments using HM-1 embryonic stem cells. Using oocytes from either B6D2F1 or B6CBAF1 (C57BL/6 x CBA) donors, the in vitro development of cloned embryos using activated cytoplasts was consistently inferior to that observed using MII cytoplasts. Live offspring were derived from both oocyte strains using the latter, whereas a single living mouse was cloned from activated B6CBAF1 cytoplasts.

Synergistic enhancement of apoptosis by DNA- and cytoskeleton-damaging agents: a basis for combination chemotherapy of cancer.

Actinomycin D (AD)-induced apoptosis in CMK-7 cells is greatly accelerated by cytoskeletal poisons such as colcemid (CL) and cytochalasin D (CD). This phenomenon is important in the combination chemotherapy of cancer so that its generality was investigated. Four human leukemia and two human solid tumor cell lines were treated with combinations of one DNA-damaging agent [AD, mitomycin C (MMC), or etoposide (VP- 16)] and one cytoskeletal poison [CL, CD, or vinblastine (VBL)]. The apoptosis was monitored by assaying caspase-3 activity and the DNA cleavage ratio. The caspase-3 activation in all leukemia and HeLa S3 cell lines was, except for a few cases, 1.3-to 6.0-fold enhanced by combinations of the DNA-damaging agent with a cytoskeletal poison. The DNA cleavage ratio as well as the dead cell ratio was also 1.4-to 23.7-fold enhanced in CMK-7, U-937, HeLa S3, and Colo320 DM cell lines by the combinations of AD with CL, CD, or VBL. The combination index for caspase-3 activation by AD and CL in U-937 cells was smaller than 1 at Fa of more than 0.03. Thus, apoptosis in many tumor cell lines is synergistically enhanced by various combinations of DNA- and cytoskeleton-damaging agents.

Effects of colcemid concentration on chromosome aberration analysis in human lymphocytes.

As a part of technical improvements of chromosome aberration analysis on human peripheral lymphocytes for biological radiation dosimetry, we examined the optimal conditions for the use of colcemid in chromosome preparation in order to obtain enough number of cells at metaphase in the first cell division. When treated with colcemid at concentrations below 0.01 microgram/ml from the beginning of culture, cultures harvested at 48 hours had low mitotic indices. Colcemid treatment at 0.025 to 0.05 microgram/ml during 48 hours resulted in high mitotic indices (8 to 15%) and almost of the mitotic cells remaining in the 1st cell division, suggesting that this range of colcemid concentration was appropriate for continuous treatment with colcemid. We further examined the effect of colcemid concentration on the quantitative consistency of the yields of radiation-induced chromosome aberration. Repeated experiments showed that the yield of dicentrics and centric rings in the culture having colcemid at 0.025 microgram/ml concentration were larger than that at 0.05 microgram/ml. These data indicate the importance of assuring the accuracy of colcemid concentration in the lymphocyte culture for cytogenetic radiation dosimetry.

The labelling index is not always reliable. Discrepancies between the labelling index and the mitotic rate in the rat corneal epithelium after intraperitoneal and topical administration of tritiated thymidine and colcemid.

Many cell kinetic studies are based on the assumption that tritiated thymidine injected into an animal is available for incorporation into DNA for only a short time, and that it labels all cells in the S phase. The present study indicates that this is not the fact for the rat corneal epithelium. The labelling index (LI) declines considerably from the limbal area to the central cornea, while the mitotic rate is almost constant all over the corneal epithelium. The LI should therefore not be used as the only criterion in the assessment of proliferation rate.

[Immediate results of polychemotherapy in esophageal cancer patients]. / Neposredstvennye rezul'taty polikhimioterapii u bol'nykh rakom pishchevoda.

Short-term results of intensive 5-day polychemotherapy given to patients with esophageal cancer are presented. The drug therapy was mainly indicated for dramatic disorders of esophageal patency for liquid food (18 patients) or for its complete lack (5 patients). As a result of the treatment esophageal patency improved in 17 of the 23 patients (73.3%). This made it possible to give up palliative surgery and to carry out x-ray treatment.

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.

[In vivo cell cycle synchronization of the murine sarcoma 180 by continuous colcemid infusion (author's transl)].

In recent years, partial synchronization in vivo of normal tissues or solid tumors has been achieved by some investigators. A study of synchronization in vivo is valuable for not only analysis of the cell cycle kinetics, but also chemotherapy. Most of the chemotherapeutic agents and radiation show a specific reaction to a specific phase of cell cycle. This concept supports the value of chemotherapy using synchronization of tumor cells. In other words, the chemotherapeutic effect is enhanced by killing greater numbers of tumor cells which are gathered in a specific phase after synchronization. At the same time toxic effects are reduced by allowing normal tissue escaping from the effective phase. Colcemid is one of the most useful metaphase arrest agents. Colcemid blocks cells at metaphase by disrupting the mitotic spindle, and has been found to be less toxic than colchicine. Colcemid, when used in optimum dosage and duration of exposure, will accumulate tumor cells in metaphase with less toxic effects on normal tissues. Synchronization of the accumulated cells will be achieved after colcemid release. The purpose of this study is to examine the effects of colcemid on the cell cycle and to maximize synchrony after release of colcemid in sarcoma 180 tumor bearing mice. A mitotic linear accumulation was obtained by continuous colcemid infusion at 5.82 microgram/hr. Low dose colcemid infusion (0.582 and 1.455 microgram/hr) for 14 hours did not accumulated mitotic cells, but doses more than 5.82 microgram/hr of colcemid blocked it completely, accumulating 25.5% of cells after a 20 hours infusion. Therefore, mitotic accumulate by using colcemid is thought to be dose and time dependent within the limited range. However, the rates of mitotic accumulation were underestimated probably because of the increment of pyknoses and inhibition of DNA synthesis by high dose and long exposure of colcemid. A four hours colcemid infusion at 5.82 microgram/hr accumulated 11% of tumor cells without severe damage on cell cycle timing. The cells accumulated in mitotic phase recovered 3-4 hours after release of colcemid, and partial tumor synchronization was achieved in the following next one cycle. The rate of synchronization was thought to be about 30%, which was compatible with that of synchronization in vivo. Therefore, colcemid infusion at optimal dosage and duration of exposure can synchronize tumor cells reasonably. It is possible that therapeutic potential will be elevated if G1 or S phase specific drugs are used as killing agents after synchronization with colcemid. Moreover, higher synchrony will be obtained after several intermittent infusions of colcemid.

[Intraneoplastic iontophoresis of cytostatics in the overall treatment of esophageal cancer]. / Vnutriopukholevyi ionoforez tsitostatikov v kompleksnom lechenii raka pishchevoda.

The authors report the results of clinical elaboration and testing of the technic of intraesophageal intratumor sarcolysin iontophoresis associated with radiotherapy in 40 inoperable patients with cancer of the thoracic esophagus. A total focal dose of irradiation was on the average 1/3 less compared with the routine one. Iontophoresis was performed by a specially designed electrode with 5-10 mA during 60 minutes. The iontophoresis procedure was followed by irradiation. The immediate good results of the treatment were twice as frequent as in control groups. The treatment proceeded with less pronounced radiation response. An average survival of patients while using chemical drugs intophoresis proved to be equal to control data that is likely to be due to the far-advanced stage of the disease in most patients. An average survival in patients with less tumor proliferation was found to be longer than in analogous control series.

The effects of colcemid on hematopoiesis in the mouse.

Colcemid was found to induce a dose and schedule dependent marrow magakaryocytosis and peripheral thrombocytosis. The response could be divided into early and late components. The early component appears to have been due to a direct stimulatory effect, probably by enhancement of endoreduplication in metaphase arrested megakaryocyte precursors. The ealy stimulatory response was blunted on toxic drug schedules. In contrast, the late component of the thrombopoietic response was demonstrated best on the most toxic drug schedules. It coincided temporally with the reactive restoration of the mononuclear marrow and blood cell elements, respectively. Thus, the late component appears to be a nonspecific rebound phenomenon. On comparing the thrombopoietic properties of Colcemid with those of the vinca alkaloids in experimental systems, the former appears to have a more favorable therapeutic index. The data suggest that colchicine and its derivatives may be useful agents in the treatment of clinical thrombocytopenic states.

Demecolcin and vinblastine sulphate as stathmokinetic agents for different tissues of the hairless mouse.

The dose/response relationship of the kinetostatic effect of demecolcin (ColcemidR) and vinblastine sulphate (VelbeR) was estimated in the epidermis, the forestomach, the jejunum and the colon of the hairless mouse (hr/hr) at 3 hours after intraperitoneal (i.p.) injection. A dose of 0.15 mg Colcemid per animal was found to be the lowest effective dose in all the organs tested. The value did not differ much from the corresponding effective dose of Velbe. This dose of Colcemid was then used to study the accumulation of arrested mitoses during the first 4 hours after i.p. injection. An almost linear increase in the mitotic count was found up to 4 hours in the epidermis, 3.5 hours in the forestomach and 3 hours in the jejunum and the colon. A dose of 0.15 mg Colcemid per animal i.p. with an observation time of 3 hours is thus a reliable method for estimation of the mitotic rate in many organs. It is also concluded that both Colcemid and Velbe can be used as reliable stathmokinetic drugs for both epidermis, forestomach and jejunum and colon mucosa provided that correct doses and short enough accumulation time (3 hours) is used.