On the influence of 5-Amino uracil on the cell cycle of root tip meristems

To study the influence of 5-AU on the cell cycle of meristematic root tip tissue the following quantities, among others, have been investigated during and after the 5-AU treatment: mitotic index, labeling index, rate of DNA synthesis, and alteration of the frequency of cells with nuclei of certain DNA content. The rate of DNA synthesis was determined by measuring the rate of ³H-TdR incorporation, after a 5-AU/TdR antagonism could be excluded. Following from these investigations, 5-AU reduces the rate of DNA synthesis; it does not block it completely, as FUdR does. The reduction of the rate of DNA synthesis prolongs the S phase, which results in an accumulation of cells in this state, in fact favourably in the last 1/3 of the S phase. In this connection the possibility of a favoured reduction of DNA synthesis in heterochromatic areas is discussed. Although 5-AU also influences G2, no accumulation of cells in this state has been observed. On the basis of the present results we have to deny the assumption made by several authors that 5-AU acts in the same way as FUdR.     

The action of 5-amino uracil on log growth and division-synchronized Tetrahymena

The influence of 5-amino uracil (5-AU) was investigated on the cell cycle of log growth and division-synchronized Tetrahymena pyriformis GL. The division index of log growth phase Tetrahymena was suppressed by 50% after 40 min in 8 mM 5-AU. Cells division-synthronized by one heat shock per generation were also treated with 5-AU. Cells treated either prior to the first synchronous division (80 min EH) or up to 25 min prior to the second synchronous division (after 160 min EH) were not delayed in their progress through the cell cycle. Cells treated during the S phase of the first free running cell cycle, however, were delayed 5-30 min from reaching the second synchronous division. The effect of 5-AU on DNA and RNA synthesis was also examined. Incorporation of [3H]thymidine into acid-precipitable material was reduced in the presence of 5-AU; the rate of DNA synthesis was also reduced. The depression in the rate of DNA synthesis was greater at the beginning of S than at the end of S. The size of the thymidine pool (nucleosides + nucleotides) did not change during 5-AU treatment; however, an accumulation of thymidine tri-phosphate and a decrease in the amount of thymidine nucleoside was observed. A suppression of [14C]uridine incorporation resulting from 5-AU treatment was observed throughout the cell cycle. The rate of RNA synthesis as monitored by [14C]uridine incorporation into acid precipitable material was also reduced during 5-AU treatment. No change in either the size or the composition of the pool of uridine (nucleoside + nucleotide) was detected in 5-AU treated cells as compared to controls.     

5-AMINOURACIL TREATMENT : A Method for Estimating G2

Treatment of Vicia faba lateral roots with a range of concentrations of 5-aminouracil (5-AU) indicate that cells are stopped at a particular point in interphase. The timing of the fall in mitotic index suggests that cells are held at the S - G₂ transition. When cells are held at this point, treatments with 5-AU can be used to estimate the duration of G₂ + mitosis/2 of proliferating cells. Treatment with 5-AU can also be used to demonstrate the presence of subpopulations of dividing cells that differ in their G₂ duration. Using this method, 5-AU-induced inhibition, we have confirmed that in V. faba lateral roots there are two populations of dividing cells: (a) a fast-dividing population, which makes up ~85% of the proliferating cell population and has a G₂ + mitosis/2 duration of 3.3 hr, and (b) a slow-dividing population, which makes up ~15% of dividing cells and has a G₂ duration in excess of 12 hr. These estimates are similar to those obtained from percentage labeled mitosis (PLM) curves after incorporation of thymidine-³H.     

Cytological demonstration of DNA synthesis inhibition at late S by 5-amino-uracil

Meristematic cells of Allium cepa were treated with 5-amino-uracil (5-AU) while incorporating 5-bromodeoxyuridine (BrdU) into DNA until complete inhibition of mitosis was obtained. The pattern of BrdU substitution in interphase nuclei detected by FPG technique in the cells so treated was that known as corresponding to BrdU incorporation in only early and middle replicating DNA. In addition, when the cells were allowed to reach mitosis in the absence of BrdU after the synchronization induced by 5-AU all the metaphase chromosomes showed, likewise, the typical late-replicating pattern. These results are a cytological demonstration of a preferential inhibition of late-replicating DNA synthesis by 5-AU proposed by several authors.     

Depletion of p31comet protein promotes sensitivity to antimitotic drugs

Antimitotic spindle poisons are among the most important chemotherapeutic agents available. However, precocious mitotic exit by mitotic slippage limits the cytotoxicity of spindle poisons. The MAD2-binding protein p31(comet) is implicated in silencing the spindle assembly checkpoint after all kinetochores are attached to spindles. In this study, we report that the levels of p31(comet) and MAD2 in different cell lines are closely linked with susceptibility to mitotic slippage. Down-regulation of p31(comet) increased the sensitivity of multiple cancer cell lines to spindle poisons, including nocodazole, vincristine, and Taxol. In the absence of p31(comet), lower concentrations of spindle poisons were required to induce mitotic block. The delay in checkpoint silencing was induced by an accumulation of mitotic checkpoint complexes. The increase in the duration of mitotic block after p31(comet) depletion resulted in a dramatic increase in mitotic cell death upon challenge with spindle poisons. Significantly, cells that are normally prone to mitotic slippage and resistant to spindle disruption-mediated mitotic death were also sensitized after p31(comet) depletion. These results highlight the importance of p31(comet) in checkpoint silencing and its potential as a target for antimitotic therapies.     

Increases in nuclear volume and cell size in meristematic cells arrested by 5-aminouracil

Summary Lateral roots ofVicia faba were treated with a solution of 5-aminouracil (3.93 × 10^–3 M). They were either treated for 6 hours and allowed to recover for up to 10 hours, or were treated continuously for up to 24 hours. Mitotic index decreased as the duration of treatment increased,e.g., it was < 0.5 after 6 hours treatment and 4 hours recovery and 0.23 after 12 hours continuous treatment. During this period of low mitotic activity nuclei and cells increased in size: mean nuclear volume, for example, was 1505±651 m³ 8 hours after the end of a 6 hours treatment. In roots treated continuously, nuclear volume increased from 559±204 m³ at 0 hour to 1272±636 m³ at 12 hours. In the first 3 hours it was the larger nuclei that grew,i.e., nuclei that would have proceeded into mitosis if they had not been blocked by 5-AU. But between 3 and 12 hours of continuous exposure to 5-AU all nuclei increased in volume. Cells, on the other hand, showed no response during the first 6 hours of treatment; their areas did not increase till 6–12 hours had elapsed. It appears that in cells blocked by 5-AU growth continues for about 12 hours. Initially, nuclei grow disproportionately large, suggesting that synthesis of nuclear components is favoured at the expense of cytoplasmic constituents, at least during the first 6 hours of treatment; there is an internal imbalance between nuclear and cell growth and a temporary change in the nuclear cytoplasmic ratio. When cells recover from the 5-AU block and enter mitosis their prophase nuclei are also much larger than those of untreated cells. The response to 5-AU is discussed in terms of internal restrictions on cell growth, due to the presence of cell walls, and the heterogeneity in nuclear volumes.     

Effects of mevinolin on cell cycle progression and viability of tobacco BY-2 cells

Mevinolin, an inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase, was used to study the importance of mevalonic acid (MVA) for cell cycle progression of tobacco (Nicotiana tabacum L.) BY-2 cells. After treatment with 5 microM mevinolin, the cell cycle progression was completely blocked and two cell populations accumulated (80% in phase G0/G1 and 20% in G2/M). The arrest could be released by subsequent addition of MVA. Effects were compared to those caused by aphidicolin, an inhibitor of alpha-like DNA polymerases that blocks cell cycle at the entry of the S phase. The 80% proportion of mevinolin-treated TBY-2 cells was clearly arrested before the aphidicolin-inducible block. By the aid of a double-blocking technique, it was shown that the mevinolin-induced cell arrest of highly synchronized cells was due to interaction with a control point located at the mitotic telophase/entry G1 phase. Depending on the developmental stage, mevinolin induced rapid cell death in a considerable percentage of cells. Mevinolin treatment led to a partial synchronization, as shown by the increase in mitotic index. The following decrease was correlated with the above-mentioned induction of cell death.     

Antiproliferative effect of bcl-2 gene does not concern the control of mitotic events

E1A + c-Ha-ras-transformants overexpressing bcl-2 oncogene are able to be arrested at the G1/S boundary of the cell cycle after DNA damage and upon serum starvation, this cell cycle blockage being accompanied by a decrease in the activity of cyclin E--Cdk2 complexes. Roscovitine-induced inhibition of cyclin-dependent kinases (Cdks) activity does not result in the G1/S arrest of E1A + c-Ha-ras + bcl-2-transformants. Roscovitine treatment causes an accumulation of G2/M cells, mainly at the expense of mitotic cells. However, the expression of Bcl-2 oncoproducts does not re-establish the regulation of mitotic events broken by introduction of E1A and c-Ha-ras oncogenes in normal cells, as revealed by the treatment of E1A + c-Ha-ras + bcl-2-transformants with nocodazole inducing mitotic arrest in normal cells. In spite of the elevated expression of antiapoptotic bcl-2 gene in transformants, nocodazole treatment results in mass apoptotic death preceded by polyploidy. Roscovitine also induces apoptosis with no polyploid cell accumulation being observed. Inhibition of Cdks activity with Roscovitine, as well as violation of microtubule depolymerization with nocodazole result in the apoptotic death in the tested cell lines sensitive (E1A + c-Ha-ras) and resistant (E1A + c-Ha-ras + bcl-2) to damaging agents. Thus, the application of Roscovitine, a specific inhibitor of Cdks, suggests that the decrease in Cdks activity in E1A + c-Ha-ras + bcl-2-transformants is not likely to be responsible for G1/S cell cycle arrest realization after damaging influences. Moreover, an antiproliferative effect of Bcl-2 in E1A + c-Ha-ras-transformants is restricted by restoration of cell cycle events at G1/S and G2/M boundaries, and does not concern the program of mitotic events regulation.     

The differentiation of normal and transformed human fibroblasts in vitro is influenced by electromagnetic fields

We studied the effect of symmetric, biphasic sinusoidal electromagnetic fields (EMF) (20 Hz, 6 mT) on the differentiation of normal human skin fibroblasts (HH-8), normal human lung fibroblasts (WI38), and SV40-transformed human lung fibroblasts (WI38SV40) in in vitro cultures. Cells were exposed up to 21 days for 2 x 6 h per day to EMF. Normal mitotic human skin and lung fibroblasts could be induced to differentiate into postmitotic cells upon exposure to EMF. Concomitantly, the synthesis of total collagen as well as total cellular protein increased significantly by a factor of 5-13 in EMF-induced postmitotic cells. As analyzed by two-dimensional gel electrophoresis of [35S]methionine-labeled polypeptides, EMF-induced postmitotic cells express the same differentiation-dependent and cell type-specific marker proteins as their spontaneously arising counterparts. In SV40-transformed human lung fibroblasts (cell line WI38SV40) the exposure to EMF induced the differentiation of mitotic WI38SV40 cells into postmitotic and degenerating cells in subpopulations of WI38SV40 cell cultures. Other subpopulations of WI38SV40 cells did not show any effect of EMF on cell proliferation and differentiation. These results indicate that long-term EMF exposure of fibroblasts in vitro induces the differentiation of mitotic to postmitotic cells that are characterized by differentiation-specific proteins and differentiation-dependent enhanced metabolic activities.     

Premature onset of mitosis and potentiation of chromosome damage induced by poly-D-lysine in plant cells: evidence for G2 repair

Poly-D-lysine has been reported to induce a triggering of mitosis in plant cells due to a selective stimulatory effect on cells arrested in G2. Root-tip cells of Allium cepa L. were first exposed to maleic hydrazide (MH) early in the cell cycle and posttreated with different concentrations of the polycationic agent while in G2. The result was a dose-dependent potentiation of chromosome damage observed at metaphase without any apparent effect induced by poly-D-lysine itself. The enhancement of the yield of chromosomal aberrations was concomitant with an increase in the frequency of mitosis. In order to test further the stimulatory effect of poly-D-lysine on mitosis, as well as the consequences of a shortening of the time available for repair, cells synchronized by protracted treatment with 5-aminouracil (5-AU), which also induces chromosome damage, were allowed to recover in the presence of the polycationic compound. Our data show that a premature arrival at mitosis resulted in an increase in the frequency of damaged cells observed.     

Curcumin-treated cancer cells show mitotic disturbances leading to growth arrest and induction of senescence phenotype

Cellular senescence is recognized as a potent anticancer mechanism that inhibits carcinogenesis. Cancer cells can also undergo senescence upon chemo- or radiotherapy. Curcumin, a natural polyphenol derived from the rhizome of Curcuma longa, shows anticancer properties both in vitro and in vivo. Previously, we have shown that treatment with curcumin leads to senescence of human cancer cells. Now we identified the molecular mechanism underlying this phenomenon. We observed a time-dependent accumulation of mitotic cells upon curcumin treatment. The time-lapse analysis proved that those cells progressed through mitosis for a significantly longer period of time. A fraction of cells managed to divide or undergo mitotic slippage and then enter the next phase of the cell cycle. Cells arrested in mitosis had an improperly formed mitotic spindle and were positive for γH2AX, which shows that they acquired DNA damage during prolonged mitosis. Moreover, the DNA damage response pathway was activated upon curcumin treatment and the components of this pathway remained upregulated while cells were undergoing senescence. Inhibition of the DNA damage response decreased the number of senescent cells. Thus, our studies revealed that the induction of cell senescence upon curcumin treatment resulted from aberrant progression through the cell cycle. Moreover, the DNA damage acquired by cancer cells, due to mitotic disturbances, activates an important molecular mechanism that determines the potential anticancer activity of curcumin.     

High sensitivity of DNA replication to 5-aminouracil in the middle of the S period

Summary Cell distribution in different compartments of the cell cycle (G₁, early, middle and late S, G₂ and mitosis) has been studied during treatment with 0.5 mM 5-aminouracil and recovery inAllium cepa L. root meristems by cytophotometric and autoradiographic methods. At optimum conditions for obtaining mitotic synchronization, 5-aminouracil gives rise to cell accumulation in the S period, preferentially in its middle zone where the relative DNA content is 2.8 ± 0.1 C. After a 14-hour treatment 33% of the proliferative population is accumulated in this particular region.During recovery, a drastic reduction of the S phase and a clear increase of the mitotic frequency are the most important events observed. Apparently, the removal of the drug frees the blockage and the accumulated cells complete their interphase making up the mitotic wave.     

Small ubiquitin-related modifier ligase activity of Mms21 is required for maintenance of chromosome integrity during the unperturbed mitotic cell division cycle in Saccharomyces cerevisiae

The SUMO ligase activity of Mms21/Nse2, a conserved member of the Smc5/6 complex, is required for resisting extrinsically induced genotoxic stress. We report that the Mms21 SUMO ligase activity is also required during the unchallenged mitotic cell cycle in Saccharomyces cerevisiae. SUMO ligase-defective cells were slow growing and spontaneously incurred DNA damage. These cells required caffeine-sensitive Mec1 kinase-dependent checkpoint signaling for survival even in the absence of extrinsically induced genotoxic stress. SUMO ligase-defective cells were sensitive to replication stress and displayed synthetic growth defects with DNA damage checkpoint-defective mutants such as mec1, rad9, and rad24. MMS21 SUMO ligase and mediator of replication checkpoint 1 gene (MRC1) were epistatic with respect to hydroxyurea-induced replication stress or methyl methanesulfonate-induced DNA damage sensitivity. Subjecting Mms21 SUMO ligase-deficient cells to transient replication stress resulted in enhancement of cell cycle progression defects such as mitotic delay and accumulation of hyperploid cells. Consistent with the spontaneous activation of the DNA damage checkpoint pathway observed in the Mms21-mediated sumoylation-deficient cells, enhanced frequency of chromosome breakage and loss was detected in these mutant cells. A mutation in the conserved cysteine 221 that is engaged in coordination of the zinc ion in Loop 2 of the Mms21 SPL-RING E3 ligase catalytic domain resulted in strong replication stress sensitivity and also conferred slow growth and Mec1 dependence to unchallenged mitotically dividing cells. Our findings establish Mms21-mediated sumoylation as a determinant of cell cycle progression and maintenance of chromosome integrity during the unperturbed mitotic cell division cycle in budding yeast.     

Stabilization of P/CAF,as a ubiquitin ligase toward MDM2, suppresses mitotic cell death through p53-p21 activation in HCT116 cells with SIRT2 suppression

We previously reported that the suppression of SIRT2, an NAD + -dependent protein deacetylases, induces p53 accumulation via degradation of p300 and the subsequent MDM2 degradation, eventually leading to apoptosis in HeLa cells. The present study identified a novel pathway of p53 accumulation by SIRT2 suppression in HCT116(p53+/+) cells in which SIRT2 suppression led to escape from mitotic cell death caused by spindle assembly checkpoint activation induced by microtubule inhibitors such as nocodazole but not apoptosis or G1 or G2 arrest. We found that SIRT2 interacts with P/CAF, a histone acetyltransferase, which also acts as a ubiquitin ligase against MDM2. SIRT2 suppression led to an increase of P/CAF acetylation and its stabilization followed by a decrease in MDM2 and activation of the p53-p21 pathway. Depression of mitotic cell death in HCT116(p53+/+) cells with SIRT2 suppression was released by suppression of P/CAF or p21. Thus, the P/CAF-MDM2-p53-p21 axis enables the escape from mitotic cell death and confers resistance to nocodazole in HCT116(p53+/+) cells with SIRT2 suppression. As SIRT2 has attracted attention as a potential target for cancer therapeutics for p53 regulation, the present study provides a molecular basis for the efficacy of SIRT2 for future cancer therapy based on p53 regulation. These findings also suggest an undesirable function of the SIRT2 suppression associated with activation of the p53-p21 pathway in the suppression of mitotic cell death caused by spindle assembly checkpoint activation.     

Mechanisms of protective action of wheat germ agglutinin on cell growth in wheat seedling roots under salinity

Effects of 20 nM wheat germ agglutinin (WGA) on relative growth rate, mitotic index (MI) and the cell area in the root extension zone were investigated in seedling of Triticum aestivum L. under the influence of 2% NaCl. It was elucidated that pretreatment of wheat seedling with WGA prevented a salinity induced inhibition of root cell growth, and accelerated the restoration of cell growth after stress removal. The protective WGA effect on root cell growth may be due, presumably, to reorganization of phytohormone balance caused by WGA treatment, which could lead to accumulation of LAA and decrease in the ABA level.     

Selection of mitotic Chinese hamster ovary cells from microcarriers. Cell cycle-dependent induction of mutation by 5-bromo-2'-deoxyuridine and ethyl methanesulfonate

Large quantities of mitotic cells may be collected by mitotic detachment from a population of Chinese hamster ovary cells growing on positively charged dextran microcarriers in suspension culture. Exponentially growing cells are treated for 2.5 h with colcemid and mitotic cells are detached from the microcarriers by increasing the stirring speed. A yield of 4-6% of the total population is obtained and, of the cells collected, 85-95% are arrested in metaphase. Using this means to synchronize cells we have determined the cell cycle dependence of the toxic and mutagenic effects of 5-bromo-2'-deoxyuridine (BUdR) and ethyl methanesulfonate (EMS). Mutation was measured at two independent loci: resistance to 6-thioguanine and resistance to ouabain. Both mutagens were more toxic during S phase as compared to G1 or G2 or mitosis. BUdR induced significant mutation only during S phase. The maximum induction of 6-thioguanine resistance was observed in cultures treated 10 h after plating of mitotic cells (2 h into S phase), while the maximum induction of ouabain resistance was observed in cultures treated 10-12 h after plating of mitotic cells (2-4 h into S phase). EMS induced significant mutation at all points in the cell cycle. Mutation induction reached a minimum during S phase but the magnitude of difference between any two points in the cell cycle was found to be less than two-fold.     

UV-C irradiation delays mitotic progression by recruiting Mps1 to kinetochores

The effect of UV irradiation on replicating cells during interphase has been studied extensively. However, how the mitotic cell responds to UV irradiation is less well defined. Herein, we found that UV-C irradiation (254 nm) increases recruitment of the spindle checkpoint proteins Mps1 and Mad2 to the kinetochore during metaphase, suggesting that the spindle assembly checkpoint (SAC) is reactivated. In accordance with this, cells exposed to UV-C showed delayed mitotic progression, characterized by a prolonged chromosomal alignment during metaphase. UV-C irradiation also induced the DNA damage response and caused a significant accumulation of γ-H2AX on mitotic chromosomes. Unexpectedly, the mitotic delay upon UV-C irradiation is not due to the DNA damage response but to the relocation of Mps1 to the kinetochore. Further, we found that UV-C irradiation activates Aurora B kinase. Importantly, the kinase activity of Aurora B is indispensable for full recruitment of Mps1 to the kinetochore during both prometaphase and metaphase. Taking these findings together, we propose that UV irradiation delays mitotic progression by evoking the Aurora B-Mps1 signaling cascade, which exerts its role through promoting the association of Mps1 with the kinetochore in metaphase.     

The differentiation of normal and transformed human fibroblasts in vitro is influenced by electromagnetic fields

We studied the effect of symmetric, biphasic sinusoidal electromagnetic fields (EMF) (20 Hz, 6 mT) on the differentiation of normal human skin fibroblasts (HH-8), normal human lung fibroblasts (WI38), and SV40-transformed human lung fibroblasts (WI38SV40) in in vitro cultures. Cells were exposed up to 21 days for 2 × 6 h per day to EMF. Normal mitotic human skin and lung fibroblasts could be induced to differentiate into postmitotic cells upon exposure to EMF. Concomitantly, the synthesis of total collagen as well as total cellular protein increased significantly by a factor of 5–13 in EMF-induced postmitotic cells. As analyzed by two-dimensional gel electrophoresis of [³⁵S]methionine-labeled polypeptides, EMF-induced postmitotic cells express the same differentiation-dependent and cell type-specific marker proteins as their spontaneously arising counterparts. In SV40-transformed human lung fibroblasts (cell line WI38SV40) the exposure to EMF induced the differentiation of mitotic WI38SV40 cells into postmitotic and degenerating cells in subpopulations of WI38SV40 cell cultures. Other subpopulations of WI38SV40 cells did not show any effect of EMF on cell proliferation and differentiation. These results indicate that long-term EMF exposure of fibroblasts in vitro induces the differentiation of mitotic to postmitotic cells that are characterized by differentiation-specific proteins and differentiation-dependent enhanced metabolic activities.     

Cellular parameters of the shoot apical meristem in Arabidopsis.

The shoot apical meristem (SAM) is a small group of dividing cells that generate all of the aerial parts of the plant. With the goal of providing a framework for the analysis of Arabidopsis meristems at the cellular level, we performed a detailed morphometric study of actively growing inflorescence apices of the Landsberg erecta and Wassilewskija ecotypes. For this purpose, cell size, spatial distribution of mitotic cells, and the mitotic index were determined in a series of optical sections made with a confocal laser scanning microscope. The results allowed us to identify zones within the inflorescence SAM with different cell proliferation rates. In particular, we were able to define a central area that was four to six cells wide and had a low mitotic index. We used this technique to compare the meristem of the wild type with the enlarged meristems of two mutants, clavata3-1 (clv3-1) and mgoun2 (mgo2). One of the proposed functions of the CLV genes is to limit cell division rates in the center of the meristem. Our data allowed us to reject this hypothesis, because the mitotic index was reduced in the inflorescence meristem of the clv3-1 mutant. We also observed a large zone of slowly dividing cells in meristems of clv3-1 seedlings. This zone was not detectable in the wild type. These results suggest that the central area is increased in size in the mutant meristem, which is in line with the hypothesis that the CLV3 gene is necessary for the transition of cells from the central to the peripheral zone. Genetic and microscopic analyses suggest that mgo2 is impaired in the production of primordia, and we previously proposed that the increased size of the mgo2 meristem could be due to an accumulation of cells at the periphery. Our morphometric analysis showed that mgo2 meristems, in contrast to those of clv3-1, have an enlarged periphery with high cell proliferation rates. This confirms that clv3-1 and mgo2 lead to meristem overgrowth by affecting different aspects of meristem function.     

Two LXXLL motifs in the N terminus of Mps1 are required for Mps1 nuclear import during G2/M transition and sustained spindle checkpoint responses

Spindle assembly checkpoint kinase Mps1 is spatially and temporally regulated during cell cycle progression. Mps1 is predominately localized to the cytosol in interphase cells, whereas it is concentrated on kinetochores in prophase and prometaphase cells. The timing and mechanism of Mps1 redistribution during cell cycle transition is currently poorly understood. Here, we show that Mps1 relocates from the cytosol to the nucleus at the G₂/M boundary prior to nuclear envelope breakdown (NEB). This timely translocation depends on two tandem LXXLL motifs in the N terminus of Mps1, and mutations in either motif abolish Mps1 nuclear accumulation. Furthermore, we found that phosphorylation of Mps1 Ser80 (which is located between the two LXXLL motifs) also plays a role in regulating timely nuclear entry of Mps1. Mps1 that is defective in LXXLL motifs has near wild-type kinase activity. Moreover, the kinase activity of Mps1 appears to be dispensable for nuclear translocation, as inhibition of Mps1 by a highly specific small-molecule inhibitor did not perturb its nuclear entry. Remarkably, translocation-deficient Mps1 can mediate activation of spindle assembly checkpoint response; however, it fails to support a sustained mitotic arrest upon prolonged treatment with nocodazole. The mitotic slippage can be attributed to precocious degradation of Mps1 in the arrested cells. Our studies reveal a novel cell cycle-dependent nuclear translocation signal in the N terminus of Mps1 and suggest that timely nuclear entry could be important for sustaining spindle assembly checkpoint responses.