ATG5 overexpression is neuroprotective and attenuates cytoskeletal and vesicle-trafficking alterations in axotomized motoneurons.

Injured neurons should engage endogenous mechanisms of self-protection to limit neurodegeneration. Enhancing efficacy of these mechanisms or correcting dysfunctional pathways may be a successful strategy for inducing neuroprotection. Spinal motoneurons retrogradely degenerate after proximal axotomy due to mechanical detachment (avulsion) of the nerve roots, and this limits recovery of nervous system function in patients after this type of trauma. In a previously reported proteomic analysis, we demonstrated that autophagy is a key endogenous mechanism that may allow motoneuron survival and regeneration after distal axotomy and suture of the nerve. Herein, we show that autophagy flux is dysfunctional or blocked in degenerated motoneurons after root avulsion. We also found that there were abnormalities in anterograde/retrograde motor proteins, key secretory pathway factors, and lysosome function. Further, LAMP1 protein was missorted and underglycosylated as well as the proton pump v-ATPase. In vitro modeling revealed how sequential disruptions in these systems likely lead to neurodegeneration. In vivo, we observed that cytoskeletal alterations, induced by a single injection of nocodazole, were sufficient to promote neurodegeneration of avulsed motoneurons. Besides, only pre-treatment with rapamycin, but not post-treatment, neuroprotected after nerve root avulsion. In agreement, overexpressing ATG5 in injured motoneurons led to neuroprotection and attenuation of cytoskeletal and trafficking-related abnormalities. These discoveries serve as proof of concept for autophagy-target therapy to halting the progression of neurodegenerative processes.

Microtubule-associated protein 1b is required for shaping the neural tube.

BACKGROUND: Shaping of the neural tube, the precursor of the brain and spinal cord, involves narrowing and elongation of the neural tissue, concomitantly with other morphogenetic changes that contribue to this process. In zebrafish, medial displacement of neural cells (neural convergence or NC), which drives the infolding and narrowing of the neural ectoderm, is mediated by polarized migration and cell elongation towards the dorsal midline. Failure to undergo proper NC results in severe neural tube defects, yet the molecular underpinnings of this process remain poorly understood. RESULTS: We investigated here the role of the microtubule (MT) cytoskeleton in mediating NC in zebrafish embryos using the MT destabilizing and hyperstabilizing drugs nocodazole and paclitaxel respectively. We found that MTs undergo major changes in organization and stability during neurulation and are required for the timely completion of NC by promoting cell elongation and polarity. We next examined the role of Microtubule-associated protein 1B (Map1b), previously shown to promote MT dynamicity in axons. map1b is expressed earlier than previously reported, in the developing neural tube and underlying mesoderm. Loss of Map1b function using morpholinos (MOs) or δMap1b (encoding a truncated Map1b protein product) resulted in delayed NC and duplication of the neural tube, a defect associated with impaired NC. We observed a loss of stable MTs in these embryos that is likely to contribute to the NC defect. Lastly, we found that Map1b mediates cell elongation in a cell autonomous manner and polarized protrusive activity, two cell behaviors that underlie NC and are MT-dependent. CONCLUSIONS: Together, these data highlight the importance of MTs in the early morphogenetic movements that shape the neural tube and reveal a novel role for the MT regulator Map1b in mediating cell elongation and polarized cell movement in neural progenitor cells.

Dominant lethal effects of nocodazole in germ cells of male mice.

The ability of the anticancer drug, nocodazole, to induce dominant lethal mutations in male germ cells was investigated by the in vivo dominant lethal test. Mice were treated with single doses of 15, 30 and 60 mg/kg nocodazole. These males were mated at weekly intervals to virgin females for 6 weeks. Nocodazole clearly induced dominant lethal mutations in the early spermatid stage with the highest tested dose. Mice treated with 60 mg/kg nocodazole showed an additional peak of dominant lethal induction in mature spermatozoa during the first week matings after treatment. The percentage sperm count and sperm motility were significantly decreased after treatment of males with 30 and 60 mg/kg nocodazole. Moreover, the middle and highest doses of nocodazole significantly increased the percentage of abnormal sperm. Our study provides evidence that nocodazole is a germ cell mutagen. Marked alteration in the spermiogram analysis after nocodazole treatment possibly confirms that nocodazole has a significant effect on sperm maturation and development during storage and transit. The demonstrated mutagenicity profile of nocodazole may support further development of effective chemotherapy with less mutagenicity. Moreover, the cancer patients and medical personnel exposed to this drug chemotherapy may stand a higher risk for abnormal reproductive outcomes.

[Study of the therapeutic activity of nocodazole on experimental models of larval alveococcosis].

The therapeutic activity of parenteral nocodazole was studied on four larval alveococcosis models: golden hamsters (Mesocricetus auratus), Djungarian hamsters (Phodopus sungorus campbelli Thomas), albino rats (Rattus rattus albus), and cotton rats (Sigmodon hispidus) at the late stage of E. multilocularis invasion. The agent (aqueous suspension) was intraperitoneally, intramuscularly, and subcutaneously injected in daily doses of 0.125-0.5 g/kg as 1-8 injections. Mebendazole was the drug of comparison. In the golden hamsters, one intraperitoneal nocodazole injection in daily doses of 0.25 and 0.5 g/kg caused death of all alveococcosis larvocysts whereas mebendazole 0.5 g/kg proved to be ineffective. In the Djungarian hamsters, one intraperitoneal nocodazole injection in a dose of 0.25 g/kg was fatal to alveococcosis larvocysts in 3 (75%) of the 4 animals. In the albino rats, nocodazole once administered intraperitoneally in a dose of 0.125 g/kg caused no death of all parasitic larvocysts, but inhibited their growth by 94.4%. The found alveococcus larvocysts were dead in all the cotton rats receiving nocodazole intramuscularly and subcutaneously 8 times in daily doses of 0.125 and 0.2 g/kg, respectively. Larvocystic death induced by nocodazole in animals with intensive invasion was accompanied by the high rate of collapse in larvocysts.

[Ca2+ exit from intracellular stores of growing and fully grown native and devitrified porcine oocytes].

The exit of Ca2+ from intracellular stores in growing and fully grown native and devitrified porcine oocytes stimulated by somatotropin and GTP was investigated using fluorescent dye chlortetracycline. In native as well as in the devitrified porcines oocytes, in their fully grown phase, joint action of somatotropin and GTP stimulated additional freeing of Ca2+ from intracellular stores, but during subsequent processing of the cell with nocodazole (a polymerization inhibitor of microtubules), additional exit of calcium did not occur. In the growing phase of native oocytes during the joint acting of somatotropin and GTP additional exit for Ca2+ from the intracellular stores did not occur. Join action of somatotropin and GTP on growing devitrified oocytes lead to the additional freeing of Ca2+ from intracellular stores. Injection of nocodazole inhibited the exit for calcium in growing devitrified oocytes treated with somatotropin and GTP. The data obtained points to the absence of difference of signal transduction mechanisms in growing and fully grown oocytes after devitrification.

Targeting the microtubular network as a new antimyeloma strategy.

We identified nocodazole as a potent antimyeloma drug from a drug screening library provided by the Multiple Myeloma Research Foundation. Nocodazole is a benzimidazole that was originally categorized as a broad-spectrum anthelmintic drug with antineoplastic properties. We found that nocodazole inhibited growth and induced apoptosis of primary and multiresistant multiple myeloma cells cultured alone and in the presence of bone marrow stromal cells. Nocodazole caused cell-cycle prophase and prometaphase arrest accompanied by microtubular network disarray. Signaling studies indicated that increased expression of Bim protein and reduced X-linked inhibitor of apoptosis protein and Mcl-1(L) levels were involved in nocodazole-induced apoptosis. Further investigation showed Bcl-2 phosphorylation as a critical mediator of cell death, triggered by the activation of c-jun-NH(2) kinase (JNK) instead of p38 kinase or extracellular signal-regulated kinases. Treatment with JNK inhibitor decreased Bcl-2 phosphorylation and subsequently reduced nocodazole-induced cell death. Nocodazole combined with dexamethasone significantly inhibited myeloma tumor growth and prolonged survival in a human xenograft mouse model. Our studies show that nocodazole has potent antimyeloma activity and that targeting the microtubular network might be a promising new treatment approach for multiple myeloma.

Exploring long-term protection of normal human fibroblasts and epithelial cells from chemotherapy in cell culture.

Killing of proliferating normal cells limits chemotherapy of cancer. Several strategies to selectively protect normal cells were previously suggested. Here we further explored the protection of normal cells from cell cycle-specific chemotherapeutic agents such as mitotic inhibitors (MI). We focused on a long-term cell recovery (rather than on a short-term cell survival) after a 3-day exposure to MI (paclitaxel and nocodazole). In three normal human cell types (RPE, NKE, WI-38t cells) but not in cancer cells with mutant p53, pre-treatment with nutlin-3a, a non-genotoxic inducer of wt p53, caused G1 and/or G2 arrest, thus preventing lethal mitotic arrest caused by MI and allowing normal cells to recover after removal of MI. Rapamycin, an inhibitor of the nutrient-sensing mTOR pathway, potentiated the protective effect of nutlin-3a in normal cells. Also, a combination of rapamycin and metformin, an anti-diabetic drug, induced G1 and G2 arrest selectively in normal cells and thereby protected them from MI. A combination of metformin and rapamycin also protected normal cells in low glucose conditions, whereas in contrast it was cytotoxic for cancer cells. Based on these data and the analysis of the literature, we suggest that a rational combination of metformin and rapamycin can potentiate chemotherapy with mitotic inhibitors against cancer, while protecting normal cells, thus further increasing the therapeutic window.

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.

Dynamics of ballistically injected latex particles in living human endothelial cells.

We studied the dynamics of ballistically injected latex particles (BIP) inside endothelial cells, using video particle tracking to measure the mean squared displacement (MSD) as a function of lag time. The MSD shows a plateau at short times and a linear behavior at longer times, indicating that the BIP are trapped into a viscoelastic network. To reveal more about the molecular constituents and the dynamics of this actin network, we added a variety of drugs. Latrunculin and Jasplakinolide aimed at intervening with the actin network caused a strong increase in MSD, whereas Taxol aimed at microtubules gave no measurable change in MSD. Additional corroborating information about these drug effects were obtained from MSD amplitude and exponent distributions and from fluorescent staining images of the actin and microtubule networks. Our evidence strongly suggests that BIP are primarily embedded in the actin network. Additional drug interventions aimed at disabling non-thermal forces could not conclusively resolve the nature of the forces driving BIP dynamics.

TRAIL inactivates the mitotic checkpoint and potentiates death induced by microtubule-targeting agents in human cancer cells.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has attracted interest as an anticancer treatment, when used in conjunction with standard chemotherapy. We investigated the mechanistic basis for combining low-dose TRAIL with microtubule-targeting agents that invoke the mitotic checkpoint. Treatment of T98G and HCT116 cells with nocodazole alone resulted in a robust mitotic block with initially little cell death; low levels of cell death were also seen with TRAIL alone at 10 ng/mL final concentration. In contrast, the addition of low-dose TRAIL to nocodazole was associated with maximally increased caspase-3, caspase-8, and caspase-9 activation, which efficiently abrogated the mitotic delay and markedly increased cell death. In contrast, the abrogation of mitotic checkpoint and increased cell death were blocked by inhibitors of caspase-8 and caspase-9 or pan-caspase inhibitor. The addition of TRAIL to either nocodazole or paclitaxel (Taxol) reduced levels of the mitotic checkpoint proteins BubR1 and Bub1. BubR1 mutated for the caspase cleavage sites, but not wild-type BubR1, was resistant to cleavage induced by TRAIL added to nocodazole, and partially blocked the checkpoint abrogation. These results suggest that adding a relatively low concentration of TRAIL to antimicrotubule agents markedly increases complete caspase activation. This in turn accentuates degradation of spindle checkpoint proteins such as BubR1 and Bub1, contributes to abrogation of the mitotic checkpoint, and induces cancer cell death. These results suggest that TRAIL may increase the anticancer efficacy of microtubule-targeting drugs.

The chemotherapeutic agents nocodazole and amsacrine cause meiotic delay and non-disjunction in spermatocytes of mice.

Aneuploidy of germ cells contributes to reduced fertility, foetal wastage and genetic defects. The possible risk of aneuploidy induction by the cancer chemotherapeutic drugs amsacrine (AMSA) and nocodazole (NOC) was investigated in male mice. Two molecular cytogenetic approaches were used: (1) the BrdU-incorporation assay to test the altered duration of meiotic divisions and (2) the sperm-FISH assay to determine aneuploidy induction during meiosis by observing hyperhaploid and diploid sperm. Sperm were sampled from the Caudae epididymes of treated and solvent control males. Single intraperitoneal injections with NOC (35 mg/kg) and AMSA (15 mg/kg) caused a meiotic delay of 24h. The timing of sperm sampling for the sperm-FISH assay was adjusted accordingly, i.e. 23 days after treatment. Mice were treated with 18, 35 and 50 mg/kg of NOC, or 5, 10, 15 and 20 mg/kg of AMSA. Significant dose-dependent increases above the concurrent controls in the frequencies of hyperhaploid sperm were found with both agents. Significant increases in the frequencies of diploid sperm were found only with AMSA. These results provide a basis for genetic counselling of patients under AMSA or NOC chemotherapy. During a period of 3-4 months after the end of chemotherapy, they may stand a higher risk of siring chromosomally abnormal offspring.

A chemical genetic screen for cell cycle inhibitors in zebrafish embryos.

Chemical genetic screening is an effective strategy to identify compounds that alter a specific biological phenotype. As a complement to cell line screens, multicellular organism screens may reveal additional compounds. The zebrafish embryo is ideal for small molecule studies because of its small size and the ease of waterborne treatment. We first examined a broad range of known cell cycle compounds in embryos using the mitotic marker phospho-histone H3. The majority of the known compounds exhibited the predicted cell cycle effect in embryos. To determine whether we could identify novel compounds, we screened a 16 320-compound library for alterations of pH3. This screen revealed 14 compounds that had not been previously identified as having cell cycle activity despite numerous mitotic screens of the same library with mammalian cell lines. With six of the novel compounds, sensitivity was greater in embryos than cell lines, but activity was still detected in cell lines at higher doses. One compound had activity in zebrafish embryos and cell lines but not in mammalian cell lines. The remaining compounds exhibited activity only in embryos. These findings demonstrate that small molecule screens in zebrafish can identify compounds with novel activity and thus may be useful tools for chemical genetics and drug discovery.

Proteasome inhibitors abolish cell death downstream of caspase activation during anti-microtubule drug-induced apoptosis in leukemia cells.

PURPOSE: Anti-microtubule drugs and proteasome inhibitors are currently among the most intensively studied anti-tumor agents, however little is known about their pharmacological interactions at the cellular level. MATERIALS AND METHODS: The human promyelocytic leukemia cell line, HL-60, was exposed to nocodazole or etoposide in combination with proteasome or caspase inhibitors. Apoptotic cell death was detected by flow cytometry as sub-G1 population. Caspase and proteasome activities were monitored by the fluorogenic substrates Ac-DEVD-AMC and Suc-LLVY-AMC, respectively, in cell lysate. Heat shock protein 70 (HSP70) expression was determined by Western blotting. RESULTS: Nocodazole, a microtubule inhibitor, induced caspase-dependent apoptosis in the HL-60 cell line. At sub-cytotoxic concentrations, proteasome inhibitors, including MG-132 or clasto-beta-lactone, decreased nocodazole-induced apoptotic DNA fragmentation without affecting the induction of caspase-3 activity. In contrast, MG-132 decreased both DNA fragmentation and caspase activation induced by etoposide, a topoisomerase-II inhibitor. HSP70 had previously been found to inhibit apoptosis independently from caspase activation. In this study, MG-132 up-regulated HSP70 protein expression, both in the presence or absence of nocodazole. CONCLUSION: Proteasome inhibitors decreased anti-microtubule agent-induced apoptotic DNA fragmentation downstream of caspase-3 activation, possibly due to increased HSP70 expression. The results indicate that combination treatment with these novel anti-tumor agents in leukemia requires careful evaluation of their molecular interaction at the level of apoptosis induction.

Spindle checkpoint and apoptotic response in alpha-particle transformed human bronchial epithelial cells.

The mitotic spindle checkpoint and apoptosis in response to nocodazole, a microtubule-disrupting agent, were investigated in the alpha-particle transformed human bronchial epithelial cell lines BERP35T1, BERP35T4 and the parental BEP2D cell line. When treated with 0.2 microg/ml of nocodazole, BEP2D and BERP35T1 cells were efficiently arrested in the mitotic phase, whilst BERP35T4, a transformed cell line showing chromosomal instability, failed to be arrested as evidenced by a low G2/M fraction. BERP35T4 cells also showed a higher proportion of aneuploids when treated with nocodazole or not. Thus, the BERP35T4 cell line has a defect in spindle checkpoint function. The extent of apoptosis induced by nocodazole (0.3 microg/ml) was significantly higher (2-fold to 2.5-fold) in BEP2D cells than in the two transformed cell lines. Furthermore, the induced apoptosis was found to occur predominantly before mitotic division in BEP2D cells. In BERP35T4 cells, however, 50% of induced apoptosis occurred before mitotic division and 50% occurred after division in binucleated cells when co-treated with cytochalasin B. The 5'-CpG island of the Chfr gene, a mitotic checkpoint gene that functions in entry into metaphase, was found to be methylated in BERP35T4 cells but not in BEP2D cells. Consistent with methylation, the expression of the Chfr gene was markedly suppressed in BERP35T4 cells. Our results suggest that the impaired spindle checkpoint and abnormal apoptotic response may be related to the oncogenic progression of human bronchial epithelial cells initiated by exposure to alpha-particles.

Transient production of recombinant proteins by Chinese hamster ovary cells using polyethyleneimine/DNA complexes in combination with microtubule disrupting anti-mitotic agents.

We have developed a simple and robust transient expression system utilizing the 25 kDa branched cationic polymer polyethylenimine (PEI) as a vehicle to deliver plasmid DNA into suspension-adapted Chinese hamster ovary cells synchronized in G2/M phase of the cell cycle by anti-mitotic microtubule disrupting agents. The PEI-mediated transfection process was optimized with respect to PEI nitrogen to DNA phosphate molar ratio and the plasmid DNA mass to cell ratio using a reporter construct encoding firefly luciferase. Optimal production of luciferase was observed at a PEI N to DNA P ratio of 10:1 and 5 mug DNA 10(6) cells(-1). To manipulate transgene expression at mitosis, we arrested cells in G2/M phase of the cell cycle using the microtubule depolymerizing agent nocodazole. Using secreted human alkaline phosphatase (SEAP) and enhanced green fluorescent protein (eGFP) as reporters we showed that continued inclusion of nocodazole in cell culture medium significantly increased both transfection efficiency and reporter protein production. In the presence of nocodazole, greater than 90% of cells were eGFP positive 24 h post-transfection and qSEAP was increased almost fivefold, doubling total SEAP production. Under optimal conditions for PEI-mediated transfection, transient production of a recombinant chimeric IgG4 encoded on a single vector was enhanced twofold by nocodazole, a final yield of approximately 5 microg mL(-1) achieved at an initial viable cell density of 1 x 10(6) cells mL(-1). The glycosylation of the recombinant antibody at Asn297 was not significantly affected by nocodazole during transient production by this method.

Insulin promotes formation of polymerized microtubules by a phosphatidylinositol 3-kinase-independent, actin-dependent pathway in 3T3-L1 adipocytes.

Direct demonstrations implicating the microtubule cytoskeleton in insulin-mediated adipose/muscle-specific glucose transporter (GLUT4) translocation are beginning to emerge, and one role of the microtubule network appears to be the provision of a solid support for GLUT4 vesicle movement. In the current study we show that insulin treatment increases total polymerized alpha-tubulin in microtubules in a time- and dose-dependent manner that coincides with established insulin-mediated changes in GLUT4 translocation. Insulin stimulates the growth of microtubules through a pathway that requires tyrosine kinase activity, as indicated by inhibition of the effect after treatment with genistein. Insulin-mediated growth was not inhibited by treatment with the MAPK kinase (MEK) inhibitor, PD98059 or by wortmannin, indicating that the effect does not require activation of extracellular signal-regulated kinase 1/2 or phosphatidylinositide 3-kinase. Depolymerization of the actin cytoskeleton with latrunculin B abrogated the effect of insulin on microtubule polymerization, indicating that an intact actin network is a requirement for insulin-dependent modulation of microtubules. Using methods that measure insulin-dependent GLUT4 translocation in populations of adipocytes as opposed to individual cells, we show a statistically significant reduction in translocation (30% inhibition) in the presence of low concentrations of nocodazole (2 mum). This concentration incompletely depolymerizes the microtubule network, revealing that partial depolymerization of microtubules is sufficient to inhibit GLUT4 translocation. It is likely that stabilization of the microtubule network contributes to insulin stimulation of GLUT4 translocation.

The influence of chromatin structure on initial DNA damage and radiosensitivity in CHO-K1 and xrs1 cells at low doses of irradiation 1-10 Gy.

Mitotic compaction of chromatin was generated by treatment of cells with nocodazole. Alternatively, chromatin structure was altered by incubating cells in 500 mM NaCl. The irradiation response in the dose range of 1-10 Gy was measured by colony assay and by a modified fluorometric analysis of DNA unwinding (FADU) assay which measures the amount of undamaged DNA by EtBr fluorescence. Cell survival curves of irradiated CHO-K1 cells showed that treatment with nocodazole increases radiosensitivity as indicated by a decrease of the mean inactivation dose (D) from 4.446 to 4.376. Nocodazole treatment increased the initial radiation-induced DNA damage detected by the FADU assay from 7% to 13%. In repair-defective xrs1 cells, the same conditions increased the radiosensitivity from 1.209 to 0.7836 and the initial DNA damage from 43% to 57%. Alterations to chromatin structure by hypertonic medium increased radiosensitivity in CHO-K1 cells from of 4.446 to 3.092 and the initial DNA damage from 7% to 15%. In xrs1 cells these conditions caused radiosensitivity to decrease from 1.209 to 1.609 and the initial DNA damage to decrease from 43% to 36%. Disruption of chromatin structure by hypertonic treatment was found to be time-dependent. A threefold increase of exposure time to hypertonic medium from 40 to 120 min increased the initial DNA damage in CHO-K1 cells from 7% to 18% but decreased initial DNA damage in xrs1 cells from 43% to 21%. Perturbation of chromatin structure with hypertonic treatment has been shown to increase the radiosensitivity and the initial DNA damage in repair-competent CHO-K1 cells and decrease the radiosensitivity and DNA damage in repair-defective xrs1 cells. Hypertonic treatment thus abolishes differences in chromatin structure between cell lines and differences in initial DNA damage. Radiosensitivity and initial DNA damage are correlated ( r(2)=0.92; p=0.0026) and this correlation also holds when chromatin compaction is altered. The experiments demonstrate that initial DNA damage and chromatin structure are major determinants of radiosensitivity.

Determination and synchronisation of G1-phase of the cell cycle in 2- and 4-cell mouse embryos.

Incorporation of [3H]thymidine at different concentrations into mouse embryos at early developmental stages was determined by autoradiography. Methods to synchronise the G1-phase of mouse 2- and 4-cell embryos were also investigated. The results showed that the ability of embryos to incorporate [3H]thymidine increased with development. Embryos at the 4-cell stage were not labelled when the concentration of [3H]thymidine was lower than 5 microCi/ml, whereas the nuclei of embryos at morula and blastocyst stages began to show silver grains at a concentration of 0.1 microCi/ml of [3H]thymidine. After 2- and 4-cell mouse embryos were synchronised at the onset of G1-phase by treatment with low temperature or nocodazole, and DNA synthesis was detected by autoradiography, the duration of G1-phase was estimated. The result showed that 43% of the 2-cell embryos had a G1-phase of < or = 1 h, 22% had a G1-phase of < or = 2 h, 22% had a G1-phase of < or = 3 h and 13% had a G1-phase of < or = 4 h. The G1-phase in 85% of the 4-cell embryos was < or = 3 h, that in 8% of embryos was < or = 4 h and that in 7% of embryos was < or = 5 h. The toxicity of nocodazole on mouse embryo development was assessed based on both blastocyst formation and the number of blastomeres, and the results indicated that the effect of nocodazole on embryo development and cell cycle block was dose-dependent. The minimum concentration of nocodazole for metaphase block of mouse late 2-cell embryos was 0.05 microM, and the appropriate concentrations which did not impair development were 0.05-0.5 microM.

G(1) and G(2) cell-cycle arrest following microtubule depolymerization in human breast cancer cells.

Microtubule-depolymerizing agents are widely used to synchronize cells, screen for mitotic checkpoint defects, and treat cancer. The present study evaluated the effects of these agents on normal and malignant human breast cell lines. After treatment with 1 microM nocodazole, seven of ten breast cancer lines (type A cells) arrested in mitosis, whereas the other three (type B cells) did not. Similar effects were observed with 100 nM vincristine or colchicine. Among five normal mammary epithelial isolates, four exhibited type A behavior and one exhibited type B behavior. Further experiments revealed that the type B cells exhibited a biphasic dose-response curve, with mitotic arrest at low drug concentrations (100 nM nocodazole or 6 nM vincristine) that failed to depolymerize microtubules and a p53-independent p21(waf1/cip1)-associated G(1) and G(2) arrest at higher concentrations (1 microM nocodazole or 100 nM vincristine) that depolymerized microtubules. Collectively, these observations provide evidence for coupling of premitotic cell-cycle progression to microtubule integrity in some breast cancer cell lines (representing a possible "microtubule integrity checkpoint") and suggest a potential explanation for the recently reported failure of some cancer cell lines to undergo nocodazole-induced mitotic arrest despite intact mitotic checkpoint proteins.