OBJECTIVES: The development of an useful method for obtaining metaphase chromosomes from a biopsied blastomere would allow differentiation between embryos with balanced and normal chromosome complements in the preimplantation genetic diagnosis for chromosomal translocations. This study was performed to evaluate the effects of microtubule depolymerizing agents (MTDAs) on the blastomeres of mouse and human preimplantation embryos, and to establish an effective method for obtaining metaphase chromosomes of biopsied blastomeres in human early embryos. MATERIALS AND MEHTODS: Early embryos (2-4 cell stage) from superovulated mice (ICR strain) were collected and treated with single or mixture MTDAs, such as vinblastine, nocodazole and colcemid. After the treatment of MTDAs for 16 hours, the metaphase aquisition (MA) rates were evaluated by the observation of chromosome status with bis-benzimide or DAPI staining. The optimal condition from the above experiment was applied to human embryos, which were developed from abnormal fertilization (3-pronuclei). Fluorescence in-situ hybridization (FISH) with whole chromosome probes was conducted on the human metaphase chromosomes by the MTDAs. RESULTS: In mouse embryos, the effective concentrations of each MTDAs for obtaining metaphase chromosomes were 1.0 micrometer of vinblastine (20.3%), 5.0 micrometer of nocodazole (28.1%) and 1.0 micrometer colcemid (55.6%), respectively. The highest MA rate (91.2%) in the mouse embryos was obtained by a mixture of vinblastine (1.0 micrometer) and nocodazole (1.0 micrometer). In the human embryos, the metaphase chromosomes of blastomeres were obtained in 44 of 113 blastomeres (38.9%) by treatment of the mixture of vinblastine and nocodazole. FISH signals of the metaphase chromosomes were successfully observed in human individual blastomeres. CONCLUSIONS: The treatment of a mixture MTDAs for obtaining metaphase chromosomes was an efficient method, and the MA rate was above 90% in the mouse embryos. However, only a relatively small proportions of the blastomeres yielded metaphase chromosomes by the MTDAs in the human embryos. The inconsistent effects of MTDAs may be related to the variation of different species and the poor developmental potency of abnormally fertilized human embryos. We should develop more reliable and efficient methods for obtaining the metaphase chromosomes in the biopsied blastomeres of human preimplantation embryos.
Animals ; Blastocyst ; Blastomeres* ; Complement System Proteins ; Demecolcine ; Embryonic Structures* ; Fertilization ; Fluorescence ; Humans* ; Metaphase* ; Mice* ; Microtubules* ; Nocodazole ; Preimplantation Diagnosis ; Translocation, Genetic ; Vinblastine

OBJECTIVES: The development of an useful method for obtaining metaphase chromosomes from a biopsied blastomere would allow differentiation between embryos with balanced and normal chromosome complements in the preimplantation genetic diagnosis for chromosomal translocations. This study was performed to evaluate the effects of microtubule depolymerizing agents (MTDAs) on the blastomeres of mouse and human preimplantation embryos, and to establish an effective method for obtaining metaphase chromosomes of biopsied blastomeres in human early embryos. MATERIALS AND MEHTODS: Early embryos (2-4 cell stage) from superovulated mice (ICR strain) were collected and treated with single or mixture MTDAs, such as vinblastine, nocodazole and colcemid. After the treatment of MTDAs for 16 hours, the metaphase aquisition (MA) rates were evaluated by the observation of chromosome status with bis-benzimide or DAPI staining. The optimal condition from the above experiment was applied to human embryos, which were developed from abnormal fertilization (3-pronuclei). Fluorescence in-situ hybridization (FISH) with whole chromosome probes was conducted on the human metaphase chromosomes by the MTDAs. RESULTS: In mouse embryos, the effective concentrations of each MTDAs for obtaining metaphase chromosomes were 1.0 micrometer of vinblastine (20.3%), 5.0 micrometer of nocodazole (28.1%) and 1.0 micrometer colcemid (55.6%), respectively. The highest MA rate (91.2%) in the mouse embryos was obtained by a mixture of vinblastine (1.0 micrometer) and nocodazole (1.0 micrometer). In the human embryos, the metaphase chromosomes of blastomeres were obtained in 44 of 113 blastomeres (38.9%) by treatment of the mixture of vinblastine and nocodazole. FISH signals of the metaphase chromosomes were successfully observed in human individual blastomeres. CONCLUSIONS: The treatment of a mixture MTDAs for obtaining metaphase chromosomes was an efficient method, and the MA rate was above 90% in the mouse embryos. However, only a relatively small proportions of the blastomeres yielded metaphase chromosomes by the MTDAs in the human embryos. The inconsistent effects of MTDAs may be related to the variation of different species and the poor developmental potency of abnormally fertilized human embryos. We should develop more reliable and efficient methods for obtaining the metaphase chromosomes in the biopsied blastomeres of human preimplantation embryos.
Animals ; Blastocyst ; Blastomeres* ; Complement System Proteins ; Demecolcine ; Embryonic Structures* ; Fertilization ; Fluorescence ; Humans* ; Metaphase* ; Mice* ; Microtubules* ; Nocodazole ; Preimplantation Diagnosis ; Translocation, Genetic ; Vinblastine

Endothelial cells were isolated from the aortic intima of Sprague-Dawley species, rat. These cells and each cancer cell line (HeLa, Hep G2, A549, L929 and NIH/3T3 cells) were co-cultivated in alpha-MEM with 3 micrometer or 30 micrometer nocodazole. To investigate the influences induced by nocodazole, the morphological changes were observed under inverted microscope and transmission electron microscope, the amounts of fibronectin produced by vascular endothelial cells and cancer cell lines and the activities of nitric oxide synthetase synthesized mainly by endothelial cells were analyzed in the aspects associated with fine structural changes. The vascular endothelial cells of control group at the 1st, 2nd and 3rd days extended the cell processes, which contacted with cells from all cell lines investigated, but the endothelial cells of nocodazole-treated groups didn't possess the processes. All cell lines in nocodazole-treated groups had a large number of micronucleated cells, but endothelial cells didn't show micronuclei. Compared with control group, the endothelial cells of nocodazole-treated groups at the 1st, 2nd and 3rd days showed the decrease of amounts of fibronectin because of the increase of heterochromatin area. The amounts of fibronectin increased in all cell lines of nocodazole-treated groups at the 2nd and 3rd days whereas the nuclear folding or the dilatation/numerical increase of rough endoplasmic reticulum didn't appear. The activities of nitric oxide synthetase heightened in endothelial cells of nocodazole-treated groups, and therefore the considerable changes in fine structures such as vesicles, lysosomes, liposomes, pyknosis and cell lysis occurred even though the extent of changes differed among the cell lines. Taken together, the materials such as fibronectin or nitric oxide synthetase produced by endothelial cells directly or indirectly acted on cancer cells, and the amounts of fibronectin and the vesicles, lysosomes, liposomes and cell lysis seemed to be much more increased or enforced. Therefore, co-culture system seemed to work better for the investigation of actions of nocodazole and the role of endothelial cells in cancer cells research. Also, the co-culture system was closer to the in vivo state and more favorable in studies for proliferation or metastasis of cancer cells.
Animals ; Cell Line* ; Coculture Techniques ; Endoplasmic Reticulum, Rough ; Endothelial Cells* ; Fibronectins ; Heterochromatin ; Liposomes ; Lysosomes ; Neoplasm Metastasis ; Nitric Oxide Synthase ; Nocodazole* ; Rats ; Rats, Sprague-Dawley

Nocodazole is an anticancer agent that acts on microtubules or filaments. HeLa, Hep G2, A549, L929 and NIH/3T3 cell lines were cultivated in alpha-MEM with 3micrometer or 30micrometer nocodazole. To elucidate the associations between nocodazole`s antitumor actions and these effects, the influences of nocodazole on the cellular morphology and the organelles involving synthesis, secretion and destruction of proteins were investigated under light and electron microscopes. The changes of intermediate filaments such as pancytokeratins and vimentins that maybe suggest antimetastatic action of nocodazole were observed using immunocytochemical technique, PAP at light microscopic level. Rounded or micronucleate cells were induced by treatment with 3micrometer and 30micrometer nocodazole for 2 hours to 4 days. Multimicronucleate cells appeared in experimental groups of all cell lines. Nuclear foldings occurred in cells of experimental groups treated with nocodazole for 2-3 days. The numerical increases of rough endoplasmic reticulum were observed in HeLa cells treated with nocodazole for 3 days and the dilatation or numerical increases in L929 cells treated with nofodazole for 1-3 days. The fragmentations or dispersion of Golgi complex were observed in cells treated with nocodazole for 1-3 days. The amount of filaments increased in cells treated with nocodazole for 1-3 days. The number of lysosomes increased in cells treated with nocodazole for 1-3 days. The number of liposomes also increased in Hep G2 cells treated with 30micrometer nocodazole for 3 days and in 3micrometer & 30micrometer, 3 days group of 3T3 cells. The amount of pancytokeratins and vimentins increased in cells treated with nocodazole for 1-3 days. Taken together, depolymerization of microlubules was induced by nocodazole, and then the organization of cells was disintegrated. As a result, the rounded cells, the cells having multimicronuclei, and the changes of golgi complexes occurred. But there were relatively no great changes of rough endoplasmic reticulum. The amount of intermediate filaments that maintain the differentiated states of cells increased by nocodazole treatment. It was suggested that morphological changes of cells could be used in evaluation of actions of nocodazole. Especially, the increase of amount of intermediate filaments by nocodazole changed cells of each cell line from undifferentiated state to differentiated, and therefore the author hope that the changes in amount of intermediate filaments provide an important clue in anticancer and antimetastatic actions of nocodazole.
3T3 Cells ; Animals ; Cell Line ; Dilatation ; Endoplasmic Reticulum, Rough ; Golgi Apparatus ; HeLa Cells ; Hep G2 Cells ; Hope ; Humans ; Intermediate Filaments ; Liposomes ; Lysosomes ; Mice ; Microtubules ; Nocodazole* ; Organelles ; Vimentin

To identify the component(s) involved in cell cycle control in the protozoan Giardia lamblia, cells arrested at the G1/S- or G2-phase by treatment with nocodazole and aphidicolin were prepared from the synchronized cell cultures. RNA-sequencing analysis of the 2 stages of Giardia cell cycle identified several cell cycle genes that were up-regulated at the G2-phase. Transcriptome analysis of cells in 2 distinct cell cycle stages of G. lamblia confirmed previously reported components of cell cycle (PcnA, cyclin B, and CDK) and identified additional cell cycle components (NEKs, Mad2, spindle pole protein, and CDC14A). This result indicates that the cell cycle machinery operates in this protozoan, one of the earliest diverging eukaryotic lineages.
Aphidicolin ; Cell Culture Techniques ; Cell Cycle ; Cell Cycle Checkpoints ; Cyclin B ; Gene Expression Profiling ; Genes, cdc ; Giardia lamblia ; Giardia ; Nocodazole ; Spindle Poles

Endochondral bone formation of the developing cranial base is a complex process. This mechanism requires precise orchestration of many cellular events and cartilage matrix metabolism, such as proliferation, becoming round in shape, termination of proliferation, hypertrophic size-increase, and finally programmed cell death. Active formation and degradation of cartilage matrix take place, in which microtubules are involved for intracellular events; bone apposition follows these events. However, the involvement of microtubules during these changes in the developing cranial base has not been identified yet. Thus, we investigated the involvement of microtubules in the regulation of endochondral bone formation during cranial base development. Using tubulin-binding drug nocodazole, we examined the effects of altering the structure and function of microtubules during in vivo organ culture of the mouse cranial base. Cultured specimens were analyzed with HE staining, immunohistochemistry, and cell counting in order to study the morphological and molecular changes that occurred in the tissues. Disruption of the microtubular array by nocodazole reduced cells expressing proliferation marker Ki67, osteogenic marker BSP, and BMP4 within the sphenooccipital synchondrosis region; chondrocyte hypertrophy was ceased in the hypertrophic zone; degeneration of cartilage matrix and bone matrix apposition was inhibited in the ossification center of the basooccipital cranial base. Our data demonstrated that disruption of microtubules by nocodazole have multiple inhibitory effects on the sequential changes that occur during endochondral bone formation, suggesting the importance of normal microtubule-polymerization in cranial base development.
Animals ; Bone Matrix ; Bone Morphogenetic Protein 4 ; Cartilage ; Cell Count ; Cell Death ; Chondrocytes ; Durapatite ; Hypertrophy ; Hypogonadism ; Immunohistochemistry ; Mice ; Microtubules ; Mitochondrial Diseases ; Nocodazole ; Ophthalmoplegia ; Organ Culture Techniques ; Osteogenesis ; Skull Base

PURPOSE: Nocodazole, a microtubule disrupting reagent, is known to arrest cells in the M phase, To gain insight on the regulatory mechanism of H2B histone gene expression by nocodazole in HL-60 cell, the binding pattern of nuclear proteins to cis element in the human H2B histone gene promoter has been investigated with DNase I footprinting and DNA mobility shift assay. MATERIALS AND METHODS: Northern blot hybridization was performed by the method of Virca et al. A Hinc II-Sac I fragment of pSPH28 was used as probe for Northern blot analysis of H2B histone mRNA. DNase I footprinting and DNA mobility shift assay were performed by the method of Lim et al. End labeled DNA oligomer (upper strand, 5'-CTTCACCTTATTTGCATAA GCGATTC-3') for octamer binding activity was mixed with nuclear extracts in a 20 ul reaction volume containing 60 mM KC1, 12 mM HEPES, pH 7.9, 5 mM MgCl2, 0.2 mM EDTA, 0.2 mM DTT, 12% glycerol, and 2 ug of poly [dI-dC]. RESULTS: The level of H2B histone mRNA rapidly was reduced at 24 hours in nocodazole-treated HL-60 cells and the mRNA was repressed in proportion to the concentration of nocodazole. Nocodazole-dependent repression of H2B histone gene was restored by replacement with nocodazole-free media. In DNase I footprinting analysis, one nuclear factor bound at 42 bp site (octamer motif) in the absence of nocodazole. In the presence of nocodazole, the binding of nuclear factor on octamer motif partially vanished. In DNA mobility shift assay, one DNA-protein complex (Octl) was formed when octamer motif was incubated with nuclear extract of HL-60 cell. After nocodazole treatment, Octl binding activity was reduced by time dependent manner. CONCLUSION: These results suggest that nocodazole-dependent repression of H2B histone gene is correlated with reduction of Octl binding activity in HL-60 cell.
Blotting, Northern ; Cell Division ; Deoxyribonuclease I ; DNA ; Edetic Acid ; Electrophoretic Mobility Shift Assay ; Gene Expression* ; Glycerol ; HEPES ; Histones* ; HL-60 Cells* ; Humans ; Hydrogen-Ion Concentration ; Magnesium Chloride ; Microtubules ; Nocodazole* ; Nuclear Proteins ; Repression, Psychology ; RNA, Messenger

Nocodazole is an anticancer agent, well-known for its antimetastatic activity that acts on microtubules, microfilaments and extracellular matrix proteins. Hela, Hep G2, A549, L929, and NIH/3T3 cell lines were cultivated in alpha-MEM with 3microM or 30microM nocodazole. To investigate the mechanism of nocodazole preventing tumor cell metastasis, the influences of nocodazole on the amounts of glycoprotein, fibronectin, laminin and actin were investigated using PAS staining and PAP technique at light microscopic level. Two designed models ; coverglass and 3-day-old rat heart fragments models, were used in observing the invasiveness of cancer cells. Partitularly the three-dimensional model coculturing cell lines and heart fragment was used in evaluating the migration and/or proliferation or the invasiveness of cell around the fragment, and observed under inverted or bright field light microscope. The amount of glycoprotein of all cell-lines increased in cells of groups treated with nocedazole for 1, 2 and 3 day. The amounts of fibronectin usually increased in cells of groups treated with nocodazole for 1, 2 and 3 day. The amounts of laminin increased in cells of groups treated with nocodazole. The amounts of actin usually increased in cells of groups treated with nocodazole for 1, 2 and 3 days. With the prolonging of nocodazole-treatment time in two dimensional model using coverglass, the cells of control group except Hep G2 cells formed monolayer in cell-free zone according to migration or proliferation of many cells. But only a few cells of experimental groups migrated or proliferated into cell-free zone. In rat heart fragment model the cells of control group showed the invasiveness into the fragment but few or none of the cells from experimental groups attached around the fragment. Taken together, nocodazole increased the synthesis of fibronectin and laminin in cells in place of depolymerizing microtubules. Therefore, the amounts of extracellular matrix proteins in the extracellular space increased. And the increase amounts of actin connected to the extracellular matrix proteins through the integrin of plasma membrane seemed to strengthen cell attachment because of accordance between the orientation of actin and extracellular matrix proteins. Since it is important for cancer cells` metastasis to secrete various enzymes to pass through extracellular matrix proteins, it is expected more difficult for the cells to metastasize into other regions due to the increase of extracellular matrix proteins. As a result of confirmation of antiinvasive actions using two kinds of model, nocodazole seems to be a valuable anti-metastatic agent by supressing the cell motility and consequently, the invasiveness into the fragment. Nocodazole at concentration of 3microM will be probably anticipated antimetastatic activity reflecting that the effects of nocodazole between 3microM and 30microM groups had no differences.
Actin Cytoskeleton ; Actins ; Animals ; Cell Line* ; Cell Membrane ; Cell Movement ; Extracellular Matrix Proteins* ; Extracellular Matrix* ; Extracellular Space ; Fibronectins ; Glycoproteins ; Heart ; Hep G2 Cells ; Laminin ; Microtubules ; Neoplasm Metastasis ; Nocodazole* ; Rats

The process of injury and repair in airway epithelium involves cell spreading and migration followed by cell proliferation. IQ domain GTPase-activating protein 1 (IQGAP1) acts in a series of cell processes, but has not been clarified in lung epithelial cells. In this study, a widely used model of injury and repair in vitro by scratching bronchial epithelial cells (BECs) was utilized to investigate the function of IQGAP1. The results showed that IQGAP1 was abundant in BECs of mouse, rat, pig and human. IQGAP1 was colocalized with tubulin cytoskeleton, but was destroyed by nocodazole, a microtubule disassembly reagent. IQGAP1 mRNA and protein expressions increased at 6-9 h after scratching. In addition, overexpression of IQGAP1 translocated β-catenin from the cytoplasm into the nucleus and activated the Tcf/Lef signal. Scratching altered the associations of IQGAP1 with β-catenin, adenomatous polyposis coli (APC) and cytoplasmic linker protein-170 (CLIP-170). Silencing IQGAP1 expression by small interference RNA (siRNA) blocked the wound closure. It is concluded that IQGAP1 signal is involved in the wound closure of BECs induced by scratching.
Adenomatous Polyposis Coli Protein ; metabolism ; Animals ; Bronchi ; cytology ; Cell Proliferation ; Cells, Cultured ; Cytoskeleton ; metabolism ; Epithelial Cells ; cytology ; pathology ; Humans ; Mice ; Microtubule-Associated Proteins ; metabolism ; Neoplasm Proteins ; metabolism ; Nocodazole ; pharmacology ; Rats ; Swine ; Tubulin ; metabolism ; beta Catenin ; metabolism ; ras GTPase-Activating Proteins ; metabolism

Sec13p has been known as an endoplasmic reticulum-Golgi transport protein. Recently, it has also been shown to be required for the formation of septation in the fission yeast Schizosaccharomyces pombe. In the present study, we focused on the role of a human homolog of Saccharomyces cerevisiae SEC13, Sec13 protein during mitosis in U2OS cells. We found that the expression of Sec13 was constant throughout the cell cycle, and localized to the kinetochores at metaphase during mitosis. By using green fluorescent protein technology, we observed that Sec13 is required for evasion of mitotic arrest in response to spindle damage, leading to G1-like phase and apoptotic cell death. In addition, cells expressing exogenous Sec13 showed giant nuclei compared to endogenous ones in the absence of nocodazole. These results demonstrate that Sec13 is involved in the regulation of the metaphase/anaphase transition and may be functionally associated with mitotic machinery to maintain genomic stability during mitosis.
Anaphase ; Antineoplastic Agents/pharmacology ; Cell Line, Tumor/drug effects/metabolism/pathology ; *G1 Phase ; *Genomic Instability ; Green Fluorescent Proteins/metabolism ; Humans ; Kinetochores/metabolism ; Membrane Proteins/*genetics/metabolism ; Metaphase ; Mitosis/*physiology ; *Mitotic Spindle Apparatus ; Nocodazole/pharmacology ; Osteosarcoma/genetics/metabolism/pathology ; Research Support, Non-U.S. Gov't