Failure of centromere separation leads to formation of diplochromosomes in next mitosis in okadaic acid treated HeLa cells.

High concentrations of okadaic acid, sufficient to inhibit phosphatase 1 and 2A activities, induces formation of diplochromosomes in HeLa cells. It has been shown that this is due to a failure of sister chromatid separation in earlier mitosis in the presence of okadaic acid in the medium and not due to bypassing of mitosis (endoreduplication). Moreover, it has been demonstrated that the sister chromatid adherence does not depend on any under-replicated chromatin segment shared by the sister chromatids which might happen in okadaic acid induced premature mitosis, but due to the failure of the centromeres to separate at metaphase - anaphase transition. The role of phophatase 1 in sister chromatid separation has been discussed     

Apoptosis and mitotic arrest are two independent effects of the protein phosphatases inhibitor okadaic acid in K562 leukemia cells.

Treatment of human myeloid leukemia K562 cells with the serine/threonine protein phosphatases inhibitor okadaic acid induced mitotic arrest followed by apoptosis in a synchronized manner. The effect was observed at drug concentrations that inhibited the protein phosphatase type 2A but not type 1. We investigated whether apoptosis was a consequence of the preceding mitosis arrest or was induced independently by okadaic acid. We found that (1) apoptosis, but not mitotic arrest, was inhibited in cells with constitutive expression of Bcl-2; (2) pretreatment of cells with the DNA synthesis inhibitor hydroxyurea blocked the mitotic arrest but not the apoptosis mediated by okadaic acid; (3) down-regulation of c-myc gene was associated with apoptosis, but not with mitotic arrest; and (4) inhibition of protein synthesis abrogated mitotic arrest, but not apoptosis. The results suggest that inhibition of protein phosphatase 2A by okadaic acid provokes mitotic arrest and apoptosis of leukemia cells by independent mechanisms.     

The role of Cdc55 in the spindle checkpoint is through regulation of mitotic exit in Saccharomyces cerevisiae

Cdc55, a B-type regulatory subunit of protein phosphatase 2A, has been implicated in mitotic spindle checkpoint activity and maintenance of sister chromatid cohesion during metaphase. The spindle checkpoint is composed of two independent pathways, one leading to inhibition of the metaphase-to-anaphase transition by checkpoint proteins, including Mad2, and the other to inhibition of mitotic exit by Bub2. We show that Cdc55 is a negative regulator of mitotic exit. A cdc55 mutant, like a bub2 mutant, prematurely releases Cdc14 phosphatase from the nucleolus during spindle checkpoint activation, and premature exit from mitosis indirectly leads to loss of sister chromatid cohesion and inviability in nocodazole. The role of Cdc55 is separable from Bub2 and inhibits release of Cdc14 through a mechanism independent of the known negative regulators of mitotic exit. Epistasis experiments indicate Cdc55 acts either downstream or independent of the mitotic exit network kinase Cdc15. Interestingly, the B-type cyclin Clb2 is partially stable during premature activation of mitotic exit in a cdc55 mutant, indicating mitotic exit is incomplete.     

Essential role of protein phosphatase 2A in metaphase II arrest and activation of mouse eggs shown by okadaic acid, dominant negative protein phosphatase 2A, and FTY720

Vertebrate eggs arrest at second meiotic metaphase. The fertilizing sperm causes meiotic exit through Ca(2+)-mediated activation of the anaphase-promoting complex/cyclosome (APC/C). Although the loss in activity of the M-phase kinase CDK1 is known to be an essential downstream event of this process, the contribution of phosphatases to arrest and meiotic resumption is less apparent, especially in mammals. Therefore, we explored the role of protein phosphatase 2A (PP2A) in mouse eggs using pharmacological inhibition and activation as well as a functionally dominant-negative catalytic PP2A subunit (dn-PP2Ac-L199P) coupled with live cell imaging. We observed that PP2A inhibition using okadaic acid induced events normally observed at fertilization: degradation of the APC/C substrates cyclin B1 and securin resulting from loss of the APC/C inhibitor Emi2. Although sister chromatids separated, chromatin remained condensed, and polar body extrusion was blocked as a result of a rapid spindle disruption, which could be ameliorated by non-degradable cyclin B1, suggesting that spindle integrity was affected by CDK1 loss. Similar cell cycle effects to okadaic acid were also observed using dominant-negative PP2Ac. Preincubation of eggs with the PP2A activator FTY720 could block many of the actions of okadaic acid, including Emi2, cyclin B1, and securin degradation and sister chromatid separation. Therefore, in conclusion, we used okadaic acid, dn-PP2Ac-L199P, and FTY720 on mouse eggs to demonstrate that PP2A is needed to for both continued metaphase arrest and successful exit from meiosis.     

SGO1C is a non-functional isoform of Shugoshin and can disrupt sister chromatid cohesion by interacting with PP2A–B56

Shugoshin (SGO1) plays a pivotal role in sister chromatid cohesion during mitosis by protecting the centromeric cohesin from mitotic kinases and WAPL. Mammalian cells contain at least 6 alternatively spliced isoforms of SGO1. The relationship between the canonical SGO1A with shorter isoforms including SGO1C remains obscure. Here we show that SGO1C was unable to replace the loss of SGO1A. Instead, expression of SGO1C alone induced aberrant mitosis similar to depletion of SGO1A, promoting premature sister chromatid separation, activation of the spindle-assembly checkpoint, and mitotic arrest. In disagreement with previously published data, we found that SGO1C localized to kinetochores. However, the ability to induce aberrant mitosis did not correlate with its kinetochore localization. SGO1C mutants that abolished binding to kinetochores still triggered premature sister chromatid separation. We provide evidence that SGO1C-mediated mitotic arrest involved the sequestering of PP2A–B56 pool. Accordingly, SGO1C mutants that abolished binding to PP2A localized to kinetochores but did not induce aberrant mitosis. These studies imply that the expression of SGO1C should be tightly regulated to prevent dominant-negative effects on SGO1A and genome instability.     

Okadaic acid inhibits a protein phosphatase activity involved in formation of the mitotic spindle of GH4 rat pituitary cells.

Okadaic acid, a selective inhibitor of serine/threonine protein phosphatases, was utilized to investigate the requirement for phosphatases in cell cycle progression of GH4 rat pituitary cells. Okadaic acid inhibited GH4 cell proliferation in a concentration-dependent manner with a half-maximal inhibition (IC50) of approximately 5 nM. Treatment of GH4 cells with 10 nM okadaic acid resulted in a 40-60% decrease in phosphatase activity and an increase in the proportion of phosphorylated retinoblastoma (RB) protein. Cell cycle analysis indicated that okadaic acid increased the percentage of cells in G2-M, decreased proportionally the percentage of cells in G1 phase, and had little effect on the percentage of cells in S-phase. The absence of a change in the proportion of S-phase cells indicates that G1-specific phosphatases responsible for dephosphorylation of RB protein were not inhibited by 10 mM okadaic acid. Mitotic index revealed that 10 nM okadaic acid decreased proliferation of GH4 cells specifically by slowing the progression through mitosis. Immunostaining with anti-tubulin demonstrated that 10 nM okadaic acid-treated mitotic cells contained mitotic spindles; however, the spindle apparatus in these cells frequently contained multiple poles. These results suggest that the organization of spindle microtubules during prometaphase requires a protein phosphatase that is sensitive to nanomolar concentrations of okadaic acid. Chromosomes in 10 nM okadaic acid-treated cells appear to be attached to spindle microtubules and the nuclear envelope is absent. The effects of okadaic acid on the spindle differ from those elicited by the calcium channel blocker, nimodipine, indicating that this okadaic acid sensitive phosphatase is not part of the calcium signalling events which participate in mitotic progression.     

Cleavage of cohesin by the CD clan protease separin triggers anaphase in yeast

In eukaryotic cells, replicated DNA strands remain physically connected until their segregation to opposite poles of the cell during anaphase. This "sister chromatid cohesion" is essential for the alignment of chromosomes on the mitotic spindle during metaphase. Cohesion depends on the multisubunit cohesin complex, which possibly forms the physical bridges connecting sisters. Proteolytic cleavage of cohesin's Sccl subunit at the metaphase to anaphase transition is essential for sister chromatid separation and depends on a conserved protein called separin. We show here that separin is a cysteine protease related to caspases that alone can cleave Sccl in vitro. Cleavage of Sccl in metaphase arrested cells is sufficient to trigger the separation of sister chromatids and their segregation to opposite cell poles.     

Histone hyperacetylation in mitosis prevents sister chromatid separation and produces chromosome segregation defects

Posttranslational modifications of core histones contribute to driving changes in chromatin conformation and compaction. Herein, we investigated the role of histone deacetylation on the mitotic process by inhibiting histone deacetylases shortly before mitosis in human primary fibroblasts. Cells entering mitosis with hyperacetylated histones displayed altered chromatin conformation associated with decreased reactivity to the anti-Ser 10 phospho H3 antibody, increased recruitment of protein phosphatase 1-delta on mitotic chromosomes, and depletion of heterochromatin protein 1 from the centromeric heterochromatin. Inhibition of histone deacetylation before mitosis produced defective chromosome condensation and impaired mitotic progression in living cells, suggesting that improper chromosome condensation may induce mitotic checkpoint activation. In situ hybridization analysis on anaphase cells demonstrated the presence of chromatin bridges, which were caused by persisting cohesion along sister chromatid arms after centromere separation. Thus, the presence of hyperacetylated chromatin during mitosis impairs proper chromosome condensation during the pre-anaphase stages, resulting in poor sister chromatid resolution. Lagging chromosomes consisting of single or paired sisters were also induced by the presence of hyperacetylated histones, indicating that the less constrained centromeric organization associated with heterochromatin protein 1 depletion may promote the attachment of kinetochores to microtubules coming from both poles.     

Genotoxicity of barley stripe mosaic virus in infected host plants

A comparative study of the effect of barley stripe mosaic virus (BSMV) and gamma irradiation on mitotic divisions in barley (Hordeum vulgare L.) roots was performed by evaluating the mitotic index (MI), micronucleus (MN) frequency and sister chromatid exchanges (SCE). Results indicate that, similarly to gamma irradiation at doses of 100, 150 and 250 Gy, BSMV reduces the mitotic activity, increases the micronucleus frequency and the rate of SCE and promotes the formation of C-metaphases. In root meristematic cells of the three barley cultivars studied (Galactic, Sonor and Unirea), the mitotic index of infected plants was found to be 52.5, 54.48 and 64.17%, respectively, lower than the uninfected control. An increase in frequency of sister chromatid exchanges was observed in all the experimental variants. In treatments involving viral infection alone or in combination with gamma irradiation chromosomes with three and more chromatid exchanges were observed, while their percentage in the control or in treatments with gamma irradiation alone was reduced. The results of the study indicate that in plants derived from irradiated seeds, BSMV produces an effect that is correlated nonlinearly with the radiation dose applied. Cytological analysis of mitotic divisions in barley roots revealed the genotoxicity of BSMV infection.     

Asynchronous entry into anaphase induced by okadaic acid: spindle microtubule organization and microtubule/kinetochore attachments

Summary We have found that a brief treatment of either PtK₂ cells or stamen hair cells ofTradescantia virginiana during metaphase with okadaic acid, a potent protein phosphatase inhibitor, results in asynchronous entry into anaphase. After this treatment, the interval for the separation of sister chromatids can be expanded from a few seconds to approximately 5 min. We have performed a series of immunolocalizations of cells with anti-tubulin antibodies and CREST serum, asking whether okadaic acid induces asynchronous entry into anaphase through changes in the organization of the spindle microtubules or through a loss in the attachment of spindle microtubules to the kinetochores. Our experiments clearly indicate that asynchronous entry into anaphase after phosphatase inhibitor treatment is not the result of either altered spindle microtubule organization or the long-term loss of microtubule attachment to kinetochores. The kinetochore fiber bundles for all of the separating chromosomes are normally of uniform length throughout anaphase, but after asynchronous entry into anaphase, different groups of kinetochore fiber bundles have distinctly different lengths. The reason for this difference in length is that once split apart, the daughter chromosomes begin their movement toward the spindle poles, with normal shortening of the kinetochore fiber bundle microtubules. Thus, okadaic acid treatment during metaphase does not affect anaphase chromosome movement once it has begun. Our results suggest that one or more protein phosphatases appear to play an important role during metaphase in the regulatory cascade that culminates in synchronous sister chromatid separation.     

Cdc14 Inhibition by the Spindle Assembly Checkpoint Prevents Unscheduled Centrosome Separation in Budding Yeast

The spindle assembly checkpoint (SAC) is an evolutionarily conserved surveillance mechanism that delays anaphase onset and mitotic exit in response to the lack of kinetochore attachment. The target of the SAC is the E3 ubiquitin ligase anaphase-promoting complex (APC) bound to its Cdc20 activator. The Cdc20/APC complex is in turn required for sister chromatid separation and mitotic exit through ubiquitin-mediated proteolysis of securin, thus relieving inhibition of separase that unties sister chromatids. Separase is also involved in the Cdc-fourteen early anaphase release (FEAR) pathway of nucleolar release and activation of the Cdc14 phosphatase, which regulates several microtubule-linked processes at the metaphase/anaphase transition and also drives mitotic exit. Here, we report that the SAC prevents separation of microtubule-organizing centers (spindle pole bodies [SPBs]) when spindle assembly is defective. Under these circumstances, failure of SAC activation causes unscheduled SPB separation, which requires Cdc20/APC, the FEAR pathway, cytoplasmic dynein, and the actin cytoskeleton. We propose that, besides inhibiting sister chromatid separation, the SAC preserves the accurate transmission of chromosomes also by preventing SPBs to migrate far apart until the conditions to assemble a bipolar spindle are satisfied.     

Failure of sister chromatid separation leads to formation of diplochromosomes in colcemid treated PtK1 cells

The origin of diplochromosomes has been traced in multinucleate rat kangaroo cells (PtK1) obtained after colcemid treatment. In these cells the diplochromosomes were shown to originate from restitution nuclei, indicating that they were formed due to the omission or failure of sister chromatid separation and not due to endoreduplication. In this context the mechanism of sister chromatid separation has been discussed. The independence of this mitotic event from other associated processes, such as chromosome condensation, nuclear envelope breakdown or spindle formation has been stressed.     

Acute inhibition of insulin-stimulated glucose transport by the phosphatase inhibitor, okadaic acid.

Insulin is thought to exert its effects on cellular function through the phosphorylation or dephosphorylation of specific regulatory substrates. We have analyzed the effects of okadaic acid, a potent inhibitor of type 1 and 2A protein phosphatases, on the ability of insulin to stimulate glucose transport in rat adipocytes. Insulin and okadaic acid caused a 20-25- and a 3-6-fold increase, respectively, in the rate of 2-deoxyglucose accumulation by adipose cells. When added to cells previously treated with okadaic acid, insulin failed to stimulate 2-deoxyglucose accumulation beyond the levels observed with okadaic acid alone. Treatment of cells with okadaic acid did not inhibit the effect of insulin to stimulate tyrosine autophosphorylation of its receptor. These results indicate that okadaic acid potently inhibits the effects of insulin to stimulate glucose uptake and/or utilization at a step after receptor activation. To clarify the mechanism of inhibition by okadaic acid, the intrinsic activity of the plasma membrane glucose transporters was analyzed by measuring the rate of uptake of 3-O-methylglucose by adipose cells, and the concentration of adipocyte/skeletal muscle isoform of the glucose transporter (GLUT-4) in plasma membranes isolated from these cells. Insulin caused a 15-20-fold stimulation of 3-O-methylglucose uptake and a 2-3-fold increase in the levels of GLUT-4 detected by immunoblotting of isolated plasma membranes; okadaic acid caused a 2-fold increase in 3-O-methylglucose uptake, and a 1.5-fold increase in plasma membrane GLUT-4. Pretreatment of cells with okadaic acid blocked the effect of insulin to stimulate 3-O-methylglucose uptake and to increase the plasma membrane concentration of GLUT-4 beyond the levels observed with okadaic acid alone. These results indicate that the effect of okadaic acid to inhibit the effect of insulin on glucose uptake is exerted at a step prior to the recruitment of glucose transporters to the cell surface, and suggest that a phosphatase activity may be critical for this process.     

Mitotic phosphorylation rescues Abl from F-actin-mediated inhibition

We have previously shown that F-actin exerts a negative effect on Abl tyrosine kinase activity. This inhibition results from a direct association of F-actin with the C terminus of Abl and accounts, in part, for the loss of Abl activity in detached fibroblasts. We report here that Abl from mitotic cells or cells treated with the protein phosphatase inhibitor okadaic acid remains active when detached from the extracellular matrix. Aspartic acid substitution of Thr(566), which is phosphorylated in mitotic or okadaic acid-treated cells, is sufficient to abolish F-actin-mediated inhibition and to maintain Abl activity despite cell detachment. A recent crystal structure of the Abl N-terminal region has revealed autoinhibitory interactions among the Src homology 3 (SH3), SH2, and kinase domains. We found that deletion of the SH2 domain also abolished the negative effect of F-actin on kinase activity. Immediately following the kinase domain in Abl is a proline-rich linker (PRL) that binds to several SH3 adaptor proteins. Interestingly, binding of the Crk N-terminal SH3 domain to the PRL also disrupted F-actin-mediated inhibition of Abl kinase. These results suggest that F-actin may reinforce the autoinhibitory interactions to regulate Abl kinase and that inhibition can be relieved through phosphorylation and/or protein interactions with the Abl PRL region.     

Mitotic cytosol inhibits invagination of coated pits in broken mitotic cells

Receptor-mediated endocytosis is inhibited during mitosis in mammalian cells and earlier work on A431 cells suggested that one of the sites inhibited was the invagination of coated pits (Pypaert, M., J. M. Lucocq, and G. Warren. 1987. Eur. J. Cell Biol. 45: 23-29). To explore this inhibition further, we have reproduced it in broken HeLa cells. Mitotic or interphase cells were broken by freeze-thawing in liquid nitrogen and warmed in the presence of mitotic or interphase cytosol. Using a morphological assay, we found invagination to be inhibited only when mitotic cells were incubated in mitotic cytosol. This inhibition was reversed by diluting the cytosol during the incubation. Reversal was sensitive to okadaic acid, a potent phosphatase inhibitor, showing that phosphorylation was involved in the inhibition of invagination. This was confirmed using purified cdc2 kinase which alone could partially substitute for mitotic cytosol.     

A change in sister chromatid behavior precedes nuclear division in Saccharomyces cerevisiae

We used a genetic assay to monitor the behavior of sister chromatids during the cell cycle. We show that the ability to induce sister chromatid exchanges (SCE) with ionizing radiation is maximal in budded cells with undivided nuclei and then decreases prior to nuclear division. SCE can be induced in cells arrested in G2 using either nocodazole or cdc mutants. These data show that sister chromatids have two different states prior to nuclear division. We suggest that the sister chromatids of cir. III, a circular derivative of chromosome III, separate (anaphase A) prior to spindle elongation (anaphase B). Other interpretations are also discussed. SCE can be induced in cdc mutants that arrest in G2 and in nocodazole-treated cells, suggesting that mitotic checkpoints arrest cells prior to sister chromatid separation.     

Evidence for the regulation of exocytic transport by protein phosphorylation

We investigated the effects of the protein phosphatase inhibitors okadaic acid and microcystin-LR upon transport of newly synthesized proteins through the exocytic pathway. Treatment of CHO cells with 1 microM okadaic acid rapidly inhibited movement of a marker protein (vesicular stomatitis virus G protein) from the endoplasmic reticulum to the Golgi compartment. Both okadaic acid and microcystin-LR also inhibited transport in an in vitro assay reconstituting movement to the Golgi compartment, at concentrations equivalent to those required to inhibit phosphorylase phosphatase activity. Inhibition both in vivo and in vitro could be antagonized by protein kinase inhibitors, suggesting that protein phosphorylation was directly responsible for this effect. An early stage in the transport reaction associated with vesicle formation or targeting was inhibited by protein phosphorylation, which could be reversed by fractions enriched in protein phosphatase 2A. Protein kinase antagonists did not inhibit transport between sequential compartments of the exocytic pathway in vitro, suggesting that protein phosphorylation is not itself required for vesicular transport. During mitosis, vesicular transport is inhibited simultaneous to the activation of maturation-promoting factor. It is proposed that the inhibition caused by okadaic acid and microcystin-LR involves a similar mechanism to that responsible for the mitotic arrest of vesicular transport.     

Separase is Required at Multiple Pre-Anaphase Cell Cycle Stages in Human Cells

The yeast separase proteins Esp1 and Cut1 are required for loss of sister chromatid cohesion that occurs at the moment of anaphase onset. Circumstantial evidence has linked human separase to centromere separation at anaphase, but a direct test that the role of this enzyme is functionally conserved with the yeast proteins is lacking. Here we describe the effects of separase depletion from human cells using RNA interference. Surprisingly, HeLa cells lacking separase are delayed or arrest at the G2/M phase transition. This arrest is not likely due to the activation of a known checkpoint control, but may be a result of a failure to construct a mitotic chromosome. Without separase, cells also have a prolonged prometaphase, perhaps resulting from defects in spindle assembly or dynamics. In cells that reach mitosis, sister arm resolution and separation are perturbed, whereas in anaphase cells sister centromeres do appear to separate. These data indicate that separase function is not restricted to anaphase initiation and that its role in promoting loss of sister chromatid cohesion might be preferentially at arms but not centromeres.     

Pharicin A,a novel natural ent-kaurene diterpenoid,induces mitotic arrest and mitotic catastrophe of cancer cells by interfering with BubR1 function

In this study, we report the functional characterization of a new ent-kaurene diterpenoid termed pharicin A, which was originally isolated from Isodon xerophilus, a perennial shrub frequently used in Chinese folk medicine for tumor treatment. Pharicin A induces mitotic arrest in leukemia and solid tumor-derived cells identified by their morphology, DNA content, and mitotic marker analyses. Pharicin A-induced mitotic arrest is associated with unaligned chromosomes, aberrant BubR1 localization, and deregulated spindle checkpoint activation. Pharicin A directly binds to BubR1 in vitro, which is correlated with premature sister chromatid separation in vivo. Pharicin A also induces mitotic arrest in paclitaxel-resistant Jurkat and U2OS cells. Combined, our study strongly suggests that pharicin A represents a novel class of small molecule compounds capable of perturbing mitotic progression and initiating mitotic catastrophe, which merits further preclinical and clinical investigations for cancer drug development.