Properties of polar and apolar cells from the 16-cell mouse morula

Summary The surface properties of newly formed, isolated 1/16 mouse blastomeres have been analyzed over the 10–12 h period prior to their division to 2/32 cells. Two populations of cells are formed at the 8- to 16-cell transition and their surface phenotypes vary with their relative position within the morula. Outer cells are polar, relatively non-adhesive and relatively large; inner cells are apolar, adhesive and smaller. The surface phenotypes of both inner and outer 1/16 cells are stable during culture for 11 h in isolation. However, the surface phenotypes can be induced to change by culture in combination with a second 1/16 cell, in a manner that is dependent upon the identity of the second cell. Two aggregated polar cells never flatten completely against each other, and both cells retain a clearly defined polar phenotype for 11–12 h. In aggregates of two apolar cells, cell outlines are lost as a result of intercellular flattening and microvilli are displaced away from areas of cell contact. However, if the two apolar cells are subsequently separated an even distribution of microvilli is restored. In most aggregates of an apolar and a polar cell, the polar cell envelops the apolar cell completely. These results are discussed in the context of the normal fate and potential of each cell type within the morula.     

The response of malignant B lymphocytes to ionizing radiation: cell cycle arrest, apoptosis and protection against the cytotoxic effects of the mitotic inhibitor nocodazole

Ionizing radiation and mitotic inhibitors are used for the treatment of lymphoma. We have studied cell cycle arrest and apoptosis of three human B-lymphocyte cell lines after X irradiation and/or nocodazole treatment. Radiation (4 and 6 Gy) caused arrest in the G(2) phase of the cell cycle as well as in G(1) in Reh cells with an intact TP53 response. Reh cells, but not U698 and Daudi cells with defects in the TP53 pathway, died by apoptosis after exposure to 4 or 6 Gy radiation (>15% apoptotic Reh cells and <5% apoptotic U698/Daudi cells 24 h postirradiation). Lower doses of radiation (0.5 and 1 Gy) caused a transient delay in the G(2) phase of the cell cycle for the three cell lines but did not induce apoptosis (<5% apoptotic cells at 24 h postirradiation). Cells of all three cell lines died by apoptosis after exposure to 1 microg/ml nocodazole, a mitotic blocker that acts by inhibiting the polymerization of tubulin (>25% apoptotic cells after 24 h). When X irradiation with 4 or 6 Gy was performed at the time of addition of nocodazole to U698 and Daudi cells, X rays protected against the apoptosis-inducing effects of the microtubule inhibitor (<5% and 15% apoptotic cells, respectively, 24 h incubation). U698 and Daudi cells apparently have some error(s) in the signaling pathway inducing apoptosis after irradiation, and our results suggest that the arrest in G(2) prevents the cells from entering mitosis and from apoptosis in the presence of microtubule inhibitors. This arrest was overcome by caffeine, which caused U698 cells to enter mitosis (after irradiation) and become apoptotic in the presence of nocodazole (26% apoptotic cells, 24 h incubation). These results may have implications for the design of clinical multimodality protocols involving ionizing radiation for the treatment of cancer.     

Transfer of nuclei from 8-cell stage mouse embryos following use of nocodazole to control the cell cycle

Mouse 2-, 4-, 8-, and 16-cell embryos were exposed to nocodazole in M16 culture medium. The effect of different concentrations and exposure times on the efficiency of cell cycle synchronization and the development of the treated embyros after release from the drug was determined. The minimum effective concentration (95% of arrested nuclei) for 4-, 8-, and 16-cell embryos was 5μM nocodazole. The effect upon subsequent development of mouse embryos depended upon both the stage of development of the embryo at treatment (P < 0.001) and the length of exposure to nocodazole (P < 0.001). Exposure to any concentration of nocodazole within the range 2.5–10 μM for 12 hr caused a reduction in the proportion of embryos that formed blastocysts. As the period of exposure to 5μM nocodazole increased from 12 to 24 hr, the proportion of embryos developing to the blastocyst stage decreased. The lower proportion of embyros developing to the blastocyst stage and to term (P < 0.01) suggests that the more advanced stages were more susceptible to damage as a result of exposure to nocodazole. The rate of development of 4-cell embryos to blastocysts was not affected when an exposure time of 9 hr was used. Together these results show that it is possible to use nocodazole to arrest mouse embryonic cells in mitosis but that it is not appropriate to culture the embryos in the presence of this drug for prolonged periods. Individual blastomeres completed mitosis at 60–90 min and started DNA synthesis at 120–150 min after release from nocodazole. Nuclei from blastomeres thus synchronized were used to conduct studies on the effect of the cell cycle on nuclear transfer. A signficant effect was found. When nuclei from 8-cell embryos in G1 or S-phase were used as nuclei donors, development to blastocyst was respectively 27% and none. ©Wiley-Liss, Inc.     

Changes in cell surface and cortical cytoplasmic organization during early embryogenesis in the preimplantation mouse embryo

Membrane topography and organization of cortical cytoskeletal elements and organelles during early embryogenesis of the mouse have been studied by transmission and scanning electron microscopy with improved cellular preservation. At the four- and early eight-cell stages, blastomeres are round, and scanning electron microscopy shows a uniform distribution of microvilli over the cell surface. At the onset of morphogenesis, a reorganization of the blastomere surface is observed in which microvilli becomes restricted to an apical region and the basal zone of intercellular contact. As the blastomeres spread on each other during compaction, many microvilli remain in the basal region of imminent cell-cell contacts, but few are present where the cells have completed spreading on each other. Microvilli on the surface of these embryos contain linear arrays of microfilaments with lateral cross bridges. Microtubules and mitochondria become localized beneath the apposed cell membranes during compaction. Arrays of cortical microtubules are aligned parallel to regions of apposed membranes. During cytokinesis, microtubules become redistributed in the region of the mitotic spindle, and fewer microvilli are present on most of the cell surface. The cell surface and cortical changes initiated during compaction are the first manifestations of cell polarity in embryogenesis. These and previous findings are interpreted as evidence that cell surface changes associated with trophoblast development appear as early as the eight-cell stage. Our observations suggest that morphogenesis involves the activation of a developmental program which coordinately controls cortical cytoplasmic and cell surface organization.     

Cell surface changes during mitosis and cytokinesis of epithelial cells

Summary PtK2 cells were studied with scanning electron microscopy to record changes on the cell surface during mitosis and cytokinesis. During prophase, prometaphase and metaphase, the cells remain very flat with few microvilli on their surfaces. In anaphase cells, there is a marked increase in the number of microvilli, most of which are clumped over the separating chromosomes and polar regions of the mitotic spindle leaving the surface of the interzonal spindle region relatively smooth. Microvilli appear over the interzonal spindle region in telophase and the cells also increase in height. At the beginning of cleavage, the distribution of microvilli is roughly uniform over the surface but it becomes asymmetric at the completion of cleav-age when the daughter cells begin to spread. At this time most microvilli are over the daughter nuclei and the surfaces that border the former cleavage furrow. The regions of the daughter cells distal to the furrow are the first to spread and their surfaces have very few microvilli. When chromosome movement is inhibited by either Nocodazole or Taxol, microvilli formation is inhibited on the arrested cells. Nevertheless cell rounding still takes place in the normal time period. It is concluded from these observations that the signal for the onset of chromosome movement in anaphase is accompanied by a signal for the formation of microvilli. It is suggested that there is also a separate signal for the cell-rounding event in mitosis and that microvilli do not play a role in this contractile process.     

Microvilli and blebs as sources of reserve surface membrane during cell spreading

The increase in surface area that occurs as cells spread from the rounded to the flattened state has been examined in synchronized BHK 21 cells in the scanning electron microscope. Rounded cells, whether in mitosis or dissociated and freshly seeded in culture, are covered with a mixture of folds, blebs, and microvilli. As cells spread, these protuberances disappear, first in the flattening marginal region and progressively submarginally until the entire cell surface is virtually smooth. The estimated surface area of rounded post-mitotic daughter cells, taking microvilli into account, is close to that of fully spread cells 4 h after mitosis. Likewise, rounded early mitotic mother cells, which are also covered with microvilli, have approximately the same surface as fully spread cells just prior to mitosis. These findings suggest that cells possess a membrane reserve in their microvilli and other protuberances which can be utilized for spreading and initiating cell locomotion.     

Raf-1/MEK/MAPK pathway is necessary for the G2/M transition induced by nocodazole

The dynamic balance between polymerization and depolymerization of microtubules is critical for cells to enter and exit mitosis, and drugs that disrupt this balance, such as taxol, colchicine, and nocodazole, arrest the cell cycle in mitosis. Although the Raf/MEK/MAPK pathway can be activated by these drugs, its role in mitosis has not been addressed. Here, we characterize activation of Raf/MEK/MAPK by nocodazole when mitosis is induced. We find that at early time points (up to 3 h) in nocodazole induction, Raf/MEK/MAPK is activated, and inhibition of MAPK activation by a MEK inhibitor, PD98059 or U0126, reduces the number of cells entering mitosis by creating a block at G(2). At later time points and in mitosis, activation of MEK/MAPK is severely inhibited, even though Raf-1 activity remains high and can be further increased by growth factor. This inhibition is reversed when cells are released from metaphase and enter G(0)/G(1) phase. In addition, we find that binding of Raf-1 to 14-3-3 is progressively induced by nocodazole, reaching a maximum in mitosis, and that this binding is necessary to maintain mitotic Raf-1 activity. Our present study indicates that activation of the Raf/MEK/MAPK pathway is necessary for the G(2)/M progression.     

Characterization of the p53-Dependent Postmitotic Checkpoint following Spindle Disruption

The p53 tumor suppressor gene product is known to act as part of a cell cycle checkpoint in G₁ following DNA damage. In order to investigate a proposed novel role for p53 as a checkpoint at mitosis following disruption of the mitotic spindle, we have used time-lapse videomicroscopy to show that both p53^+/+ and p53^−/− murine fibroblasts treated with the spindle drug nocodazole undergo transient arrest at mitosis for the same length of time. Thus, p53 does not participate in checkpoint function at mitosis. However, p53 does play a critical role in nocodazole-treated cells which have exited mitotic arrest without undergoing cytokinesis and have thereby adapted. We have determined that in nocodazole-treated, adapted cells, p53 is required during a specific time window to prevent cells from reentering the cell cycle and initiating another round of DNA synthesis. Despite having 4N DNA content, adapted cells are similar to G₁ cells in that they have upregulated cyclin E expression and hypophosphorylated Rb protein. The mechanism of the p53-dependent arrest in nocodazole-treated adapted cells requires the cyclin-dependent kinase inhibitor p21, as p21^−/− fibroblasts fail to arrest in response to nocodazole treatment and become polyploid. Moreover, p21 is required to a similar extent to maintain cell cycle arrest after either nocodazole treatment or irradiation. Thus, the p53-dependent checkpoint following spindle disruption functionally overlaps with the p53-dependent checkpoint following DNA damage.     

p53-Independent Apoptosis and p53-Dependent Block of DNA Rereplication Following Mitotic Spindle Inhibition in Human Cells

We have studied the response of human transformed cells to mitotic spindle inhibition. Two paired cell lines, K562 and its parvovirus-resistant KS derivative clone, respectively nonexpressing and expressing p53, were continuously exposed to nocodazole. Apoptotic cells were observed in both lines, indicating that mitotic spindle impairment induced p53-independent apoptosis. After a transient mitotic delay, both cell lines exited mitosis, as revealed by flow-cytometric determination of MPM2 antigen and cyclin B1 expression, coupled to cytogenetic analysis of sister centromere separation. Both cell lines exited mitosis without chromatid segregation. K562 p53-deficient cells further resumed DNA synthesis, giving rise to cells with a DNA content above 4C, and reentered a polyploid cycle. In contrast, KS cells underwent a subsequent G1 arrest in the tetraploid state. Thus, G1 arrest in tetraploid cells requires p53 function in the rereplication checkpoint which prevents the G1/S transition following aberrant mitosis; in contrast, p53 expression is dispensable for triggering the apoptotic response in the absence of mitotic spindle.     

Gene silencing of CENP-E by small interfering RNA in HeLa cells leads to missegregation of chromosomes after a mitotic delay

Centromeric protein-E (CENP-E) is a kinesin-like motor protein required for chromosome congression at prometaphase. Functional perturbation of CENP-E by various methods results in a consistent phenotype, i.e., unaligned chromosomes during mitosis. One unresolved question from previous studies is whether cells complete mitosis or sustain mitotic arrest in the presence of unaligned chromosomes. Using RNA interference and video-microscopy, we analyzed the dynamic process of mitotic progression of HeLa(H2B)-GFP cells lacking CENP-E. Our results demonstrate that these cells initiated anaphase after a delayed mitotic progression due to the presence of unaligned chromosomes. In some dividing cells, unaligned chromosomes are present during anaphase, causing nondisjunction of some sister chromatids producing aneuploid daughter cells. Unlike in Xenopus extract, the loss of CENP-E in HeLa cells does not impair gross checkpoint activation because cells were arrested in mitosis in response to microtubule-interfering agents. However, the lack of CENP-E at kinetochores reduced the hyperphosphorylation of BubR1 checkpoint protein during mitosis, which may explain the loss of sensitivity of a cell to a few unaligned chromosomes in the absence of CENP-E. We also found that presynchronization with nocodazole sensitizes cells to the depletion of CENP-E, leading to more unaligned chromosomes, longer arrest, and cell death.     

Nocodazole induces mitotic cell death with apoptotic-like features in Saccharomyces cerevisiae

We investigated the fate of budding yeast treated with nocodazole, a microtubule-depolymerizing drug. Cells died after mitotic arrest while staying in mitosis, suggesting that mitotic cell death, but not mitotic slippage, mainly occurs in nocodazole-treated cells. Nocodazole-treated cells showed features of apoptotic-like cell death, but not those of cell lysis or autophagy. Consistently, mitochondria-dependent production of reactive oxygen species was involved in the cell death. Similar cell death was also seen in cells after mitotic arrest by perturbation of the anaphase-promoting complex/cyclosome. In addition, caspase activity was found in nocodazole-treated cells, which was independent of the metacaspase, Mca1. Our results suggest that budding yeast can be a model to study mitotic cell death in cancer treatment with antimitotic drugs.     

Microtubule dependency of p34cdc2 inactivation and mitotic exit in mammalian cells

The protein kinase inhibitor 2-aminopurine induces checkpoint override and mitotic exit in BHK cells which have been arrested in mitosis by inhibitors of microtubule function (Andreassen, P. R., and R. L. Margolis. 1991. J. Cell Sci. 100:299-310). Mitotic exit is monitored by loss of MPM-2 antigen, by the reformation of nuclei, and by the extinction of p34cdc2-dependent H1 kinase activity. 2-AP-induced inactivation of p34cdc2 and mitotic exit depend on the assembly state of microtubules. During mitotic arrest generated by the microtubule assembly inhibitor nocodazole, the rate of mitotic exit induced by 2-AP decreases proportionally with increasing nocodazole concentrations. At nocodazole concentrations of 0.12 microgram/ml or greater, 2-AP induces no apparent exit through 75 min of treatment. In contrast, 2-AP brings about a rapid exit (t1/2 = 20 min) from mitotic arrest by taxol, a drug which causes inappropriate overassembly of microtubules. In control mitotic cells, p34cdc2 localizes to kinetochores, centrosomes, and spindle microtubules. We find that efficient exit from mitosis occurs under conditions where p34cdc2 remains associated with centrosomal microtubules, suggesting it must be present on these microtubules in order to be inactivated. Mitotic slippage, the natural reentry of cells into G1 during prolonged mitotic block, is also microtubule dependent. At high nocodazole concentrations slippage is prevented and mitotic arrest approaches 100%. We conclude that essential components of the machinery for exit from mitosis are present on the mitotic spindle, and that normal mitotic exit thereby may be regulated by the microtubule assembly state.     

Effects of chemical manipulation of mitotic arrest and slippage on cancer cell survival and proliferation

Microtubule-targeting cancer therapies interfere with mitotic spindle dynamics and block cells in mitosis by activating the mitotic checkpoint. Cells arrested in mitosis may remain arrested for extended periods of time or undergo mitotic slippage and enter interphase without having separated their chromosomes. How extended mitotic arrest and mitotic slippage contribute to subsequent cell death or survival is incompletely understood. To address this question, automated fluorescence microscopy assays were designed and used to screen chemical libraries for modulators of mitotic slippage. Chlorpromazine and triflupromazine were identified as drugs that inhibit mitotic slippage and SU6656 and geraldol as chemicals that stimulate mitotic slippage. Using the drugs to extend mitotic arrest imposed by low concentrations of paclitaxel led to increased cell survival and proliferation after drug removal. Cells arrested at mitosis with paclitaxel or vinblastine and chemically induced to undergo mitotic slippage underwent several rounds of DNA replication without cell division and exhibited signs of senescence but eventually all died. By contrast, cells arrested at mitosis with the KSP/Eg5 inhibitor S-trityl-L-cysteine and induced to undergo mitotic slippage were able to successfully divide and continued to proliferate after drug removal. These results show that reinforcing mitotic arrest with drugs that inhibit mitotic slippage can lead to increased cell survival and proliferation, while inducing mitotic slippage in cells treated with microtubule-targeting drugs seems to invariably lead to protracted cell death.     

The Saccharomyces cerevisiae spindle pole body duplication gene MPS1 is part of a mitotic checkpoint

M-phase checkpoints inhibit cell division when mitotic spindle function is perturbed. Here we show that the Saccharomyces cerevisiae MPS1 gene product, an essential protein kinase required for spindle pole body (SPB) duplication (Winey et al., 1991; Lauze et al., 1995), is also required for M-phase check-point function. In cdc31-2 and mps2-1 mutants, conditional failure of SPB duplication results in cell cycle arrest with high p34CDC28 kinase activity that depends on the presence of the wild-type MAD1 checkpoint gene, consistent with checkpoint arrest of mitosis. In contrast, mps1 mutant cells fail to duplicate their SPBs and do not arrest division at 37 degrees C, exhibiting a normal cycle of p34CDC28 kinase activity despite the presence of a monopolar spindle. Double mutant cdc31-2, mps1-1 cells also fail to arrest mitosis at 37 degrees C, despite having SPB structures similar to cdc31-2 single mutants as determined by EM analysis. Arrest of mitosis upon microtubule depolymerization by nocodazole is also conditionally absent in mps1 strains. This is observed in mps1 cells synchronized in S phase with hydroxyurea before exposure to nocodazole, indicating that failure of checkpoint function in mps1 cells is independent of SPB duplication failure. In contrast, hydroxyurea arrest and a number of other cdc mutant arrest phenotypes are unaffected by mps1 alleles. We propose that the essential MPS1 protein kinase functions both in SPB duplication and in a mitotic checkpoint monitoring spindle integrity.     

Activation of JNK triggers release of Brd4 from mitotic chromosomes and mediates protection from drug-induced mitotic stress

Some anti-cancer drugs, including those that alter microtubule dynamics target mitotic cells and induce apoptosis in some cell types. However, such drugs elicit protective responses in other cell types allowing cells to escape from drug-induced mitotic inhibition. Cells with a faulty protective mechanism undergo defective mitosis, leading to genome instability. Brd4 is a double bromodomain protein that remains on chromosomes during mitosis. However, Brd4 is released from mitotic chromosomes when cells are exposed to anti-mitotic drugs including nocodazole. Neither the mechanisms, nor the biological significance of drug-induced Brd4 release has been fully understood. We found that deletion of the internal C-terminal region abolished nocodazole induced Brd4 release from mouse P19 cells. Furthermore, cells expressing truncated Brd4, unable to dissociate from chromosomes were blocked from mitotic progression and failed to complete cell division. We also found that pharmacological and peptide inhibitors of the c-jun-N-terminal kinases (JNK) pathway, but not inhibitors of other MAP kinases, prevented release of Brd4 from chromosomes. The JNK inhibitor that blocked Brd4 release also blocked mitotic progression. Further supporting the role of JNK in Brd4 release, JNK2-/- embryonic fibroblasts were defective in Brd4 release and sustained greater inhibition of cell growth after nocodazole treatment. In sum, activation of JNK pathway triggers release of Brd4 from chromosomes upon nocodazole treatment, which mediates a protective response designed to minimize drug-induced mitotic stress.     

CDK1 inhibitors antagonize the immediate apoptosis triggered by spindle disruption but promote apoptosis following the subsequent rereplication and abnormal mitosis

Spindle-disrupting agents and CDK inhibitors are important cancer therapeutic agents. Spindle toxins activate the spindle-assembly checkpoint and lead to sustained activation of CDK1. Different published results indicate that CDK1 activity is either important or dispensable for the cytotoxicity associated with spindle disruption. Using live cell imaging and various approaches that uncoupled mitotic events, we show that apoptosis was induced by both prolonged nocodazole treatment as well as by inhibition of CDK1 activity after a transient nocodazole block. However, distinct mechanisms are involved in the two types of cell death. The massive apoptosis triggered by nocodazole treatment requires the continue activation of cyclin B1-CDK1 and is antagonized by premature mitotic slippage. By contrast, apoptosis induced by nocodazole followed by CDK inhibitors occurred after rereplication and multipolar mitosis of the subsequent cell cycle. The presence of dual mechanisms of cytotoxicity mediated by spindle disruption and CDK inhibition may reconcile the various apparent inconsistent published results. These data underscore the essential role of cyclin B1-CDK1 as the basis of apoptosis during mitotic arrest, and the role of mitotic slippage and abnormal mitosis for apoptosis at later stages.     

Brd4 is required for recovery from antimicrotubule drug-induced mitotic arrest: preservation of acetylated chromatin

The mammalian bromodomain protein Brd4 interacts with mitotic chromosomes by binding to acetylated histone H3 and H4 and is thought to play a role in epigenetic memory. Mitotic cells are susceptible to antimicrotubule drugs. These drugs activate multiple response pathways and arrest cells at mitosis. We found that Brd4 was rapidly released from chromosomes upon treatment with antimicrotubule drugs, including the reversible agent nocodazole. Yet, when nocodazole was withdrawn, Brd4 was reloaded onto chromosomes, and cells proceeded to complete cell division. However, cells in which a Brd4 allele was disrupted (Brd4+/-), and expressing only half of the normal Brd4 levels, were defective in reloading Brd4 onto chromosomes. Consequently, Brd4+/- cells were impaired in their ability to recover from nocodazole-induced mitotic arrest: a large fraction of +/- cells failed to reach anaphase after drug withdrawal, and those that entered anaphase showed an increased frequency of abnormal chromosomal segregation. The reloading defect observed in Brd4+/- cells coincided with selective hypoacetylation of lysine residues on H3 and H4. The histone deacetylase inhibitor trichostatin A increased global histone acetylation and perturbed nocodazole-induced Brd4 unloading. Brd4 plays an integral part in a cellular response to drug-induced mitotic stress by preserving a properly acetylated chromatin status.     

Checkpoint genes required to delay cell division in response to nocodazole respond to impaired kinetochore function in the yeast Saccharomyces cerevisiae.

Inhibition of mitosis by antimitotic drugs is thought to occur by destruction of microtubules, causing cells to arrest through the action of one or more mitotic checkpoints. We have patterned experiments in the yeast Saccharomyces cerevisiae after recent studies in mammalian cells that demonstrate the effectiveness of antimitotic drugs at concentrations that maintain spindle structure. We show that low concentrations of nocodazole delay cell division under the control of the previously identified mitotic checkpoint genes BUB1, BUB3, MAD1, and MAD2 and independently of BUB2. The same genes mediate the cell cycle delay induced in ctf13 mutants, limited for an essential kinetochore component. Our data suggest that a low concentration of nocodazole induces a cell cycle delay through checkpoint control that is sensitive to impaired kinetochore function. The BUB2 gene may be part of a separate checkpoint that responds to abnormal spindle structure.