Mitotic Shake-off Research Articles

Preparation of Primary Acute Lymphoblastic Leukemia Cells in Different Cell Cycle Phases by Centrifugal Elutriation.

The ability to synchronize cells has been central to advancing our understanding of cell cycle regulation. Common techniques employed include serum deprivation; chemicals which arrest cells at different cell cycle phases; or the use of mitotic shake-off which exploits their reduced adherence. However, all of these have disadvantages. For example, serum starvation works well for normal cells but less well for tumor cells with compromised cell cycle checkpoints due to oncogene activation or tumor suppressor loss. Similarly, chemically-treated cell populations can harbor drug-induced damage and show stress-related alterations. A technique which circumvents these problems is counterflow centrifugal elutriation (CCE), where cells are subjected to two opposing forces, centrifugal force and fluid velocity, which results in the separation of cells on the basis of size and density. Since cells advancing through the cycle typically enlarge, CCE can be used to separate cells into different cell cycle phases. Here we apply this technique to primary acute lymphoblastic leukemia cells. Under optimal conditions, an essentially pure population of cells in G1 phase and a highly enriched population of cells in G2/M phases can be obtained in excellent yield. These cell populations are ideally suited for studying cell cycle-dependent mechanisms of action of anticancer drugs and for other applications. We also show how modifications to the standard procedure can result in suboptimal performance and discuss the limitations of the technique. The detailed methodology presented should facilitate application and exploration of the technique to other types of cells.

Synchronization of Mammalian Cell Cultures by Serum Deprivation.

Mammalian cells are amenable to the study of regulatory mechanisms dictating cell cycle progression in vitro by shifting them into the same phase of the cycle. Procedures to arrest cultured cells in specific phases of the cell cycle may be termed in vitro synchronization. The procedure described here was developed for the study of primary astrocytes and a glioma cell line, but is broadly applicable to other mammalian cells. Its application allows astrocytes to re-enter the cell cycle from a state of quiescence (G0) under carefully defined experimental conditions to move together into subsequent phases such as the G1 and S phases. A number of methods have been established to synchronize mammalian cell cultures, which include counterflow centrifugal elutriation, mitotic shake off, chemically induced cell cycle arrest, and newer live cell methods, such as cell permeable dyes. Yet, there are intrinsic limitations associated with these methods. In the present protocol, we describe a simple, reliable, and reversible procedure to synchronize astrocyte and glioma cultures from newborn rat brain by serum deprivation. The procedure is similar, and generally applicable, to other mammalian cells. This protocol consists essentially of two parts: (1) proliferation of astrocytes under optimal conditions in vitro until reaching desired confluence; and (2) synchronization and G0 phase arrest of cultures by serum down-shift. This procedure has been utilized to examine cell cycle control in astroglioma cells and astrocytes from injured adult brain. It has also been employed in precursor cloning studies in developmental biology, suggesting wide applicability.

Mechanisms controlling the temporal degradation of Nek2A and Kif18A by the APC/C-Cdc20 complex.

The Anaphase Promoting Complex/Cyclosome (APC/C) in complex with its co-activator Cdc20 is responsible for targeting proteins for ubiquitin-mediated degradation during mitosis. The activity of APC/C-Cdc20 is inhibited during prometaphase by the Spindle Assembly Checkpoint (SAC) yet certain substrates escape this inhibition. Nek2A degradation during prometaphase depends on direct binding of Nek2A to the APC/C via a C-terminal MR dipeptide but whether this motif alone is sufficient is not clear. Here, we identify Kif18A as a novel APC/C-Cdc20 substrate and show that Kif18A degradation depends on a C-terminal LR motif. However in contrast to Nek2A, Kif18A is not degraded until anaphase showing that additional mechanisms contribute to Nek2A degradation. We find that dimerization via the leucine zipper, in combination with the MR motif, is required for stable Nek2A binding to and ubiquitination by the APC/C. Nek2A and the mitotic checkpoint complex (MCC) have an overlap in APC/C subunit requirements for binding and we propose that Nek2A binds with high affinity to apo-APC/C and is degraded by the pool of Cdc20 that avoids inhibition by the SAC.

Measuring Cell Cycle Progression Kinetics with Metabolic Labeling and Flow Cytometry

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating cells. Cell cycle division is an integral part of growth and reproduction and deregulation of key cell cycle components have been implicated in the precipitating events of carcinogenesis. Molecular agents in anti-cancer therapies frequently target biological pathways responsible for the regulation and coordination of cell cycle division. Although cell cycle kinetics tend to vary according to cell type, the distribution of cells amongst the four stages of the cell cycle is rather consistent within a particular cell line due to the consistent pattern of mitogen and growth factor expression. Genotoxic events and other cellular stressors can result in a temporary block of cell cycle progression, resulting in arrest or a temporary pause in a particular cell cycle phase to allow for instigation of the appropriate response mechanism. The ability to experimentally observe the behavior of a cell population with reference to their cell cycle progression stage is an important advance in cell biology. Common procedures such as mitotic shake off, differential centrifugation or flow cytometry-based sorting are used to isolate cells at specific stages of the cell cycle. These fractionated, cell cycle phase-enriched populations are then subjected to experimental treatments. Yield, purity and viability of the separated fractions can often be compromised using these physical separation methods. As well, the time lapse between separation of the cell populations and the start of experimental treatment, whereby the fractionated cells can progress from the selected cell cycle stage, can pose significant challenges in the successful implementation and interpretation of these experiments. Other approaches to study cell cycle stages include the use of chemicals to synchronize cells. Treatment of cells with chemical inhibitors of key metabolic processes for each cell cycle stage are useful in blocking the progression of the cell cycle to the next stage. For example, the ribonucleotide reductase inhibitor hydroxyurea halts cells at the G1/S juncture by limiting the supply of deoxynucleotides, the building blocks of DNA. Other notable chemicals include treatment with aphidicolin, a polymerase alpha inhibitor for G1 arrest, treatment with colchicine and nocodazole, both of which interfere with mitotic spindle formation to halt cells in M phase and finally, treatment with the DNA chain terminator 5-fluorodeoxyridine to initiate S phase arrest. Treatment with these chemicals is an effective means of synchronizing an entire population of cells at a particular phase. With removal of the chemical, cells rejoin the cell cycle in unison. Treatment of the test agent following release from the cell cycle blocking chemical ensures that the drug response elicited is from a uniform, cell cycle stage-specific population. However, since many of the chemical synchronizers are known genotoxic compounds, teasing apart the participation of various response pathways (to the synchronizers vs. the test agents) is challenging. Here we describe a metabolic labeling method for following a subpopulation of actively cycling cells through their progression from the DNA replication phase, through to the division and separation of their daughter cells. Coupled with flow cytometry quantification, this protocol enables for measurement of kinetic progression of the cell cycle in the absence of either mechanically- or chemically- induced cellular stresses commonly associated with other cell cycle synchronization methodologies. In the following sections we will discuss the methodology, as well as some of its applications in biomedical research.

Measuring Cell Cycle Progression Kinetics with Metabolic Labeling and Flow Cytometry

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating cells. Cell cycle division is an integral part of growth and reproduction and deregulation of key cell cycle components have been implicated in the precipitating events of carcinogenesis 1,2. Molecular agents in anti-cancer therapies frequently target biological pathways responsible for the regulation and coordination of cell cycle division 3. Although cell cycle kinetics tend to vary according to cell type, the distribution of cells amongst the four stages of the cell cycle is rather consistent within a particular cell line due to the consistent pattern of mitogen and growth factor expression. Genotoxic events and other cellular stressors can result in a temporary block of cell cycle progression, resulting in arrest or a temporary pause in a particular cell cycle phase to allow for instigation of the appropriate response mechanism. The ability to experimentally observe the behavior of a cell population with reference to their cell cycle progression stage is an important advance in cell biology. Common procedures such as mitotic shake off, differential centrifugation or flow cytometry-based sorting are used to isolate cells at specific stages of the cell cycle 4-6. These fractionated, cell cycle phase-enriched populations are then subjected to experimental treatments. Yield, purity and viability of the separated fractions can often be compromised using these physical separation methods. As well, the time lapse between separation of the cell populations and the start of experimental treatment, whereby the fractionated cells can progress from the selected cell cycle stage, can pose significant challenges in the successful implementation and interpretation of these experiments. Other approaches to study cell cycle stages include the use of chemicals to synchronize cells. Treatment of cells with chemical inhibitors of key metabolic processes for each cell cycle stage are useful in blocking the progression of the cell cycle to the next stage. For example, the ribonucleotide reductase inhibitor hydroxyurea halts cells at the G1/S juncture by limiting the supply of deoxynucleotides, the building blocks of DNA. Other notable chemicals include treatment with aphidicolin, a polymerase alpha inhibitor for G1 arrest, treatment with colchicine and nocodazole, both of which interfere with mitotic spindle formation to halt cells in M phase and finally, treatment with the DNA chain terminator 5-fluorodeoxyridine to initiate S phase arrest 7-9. Treatment with these chemicals is an effective means of synchronizing an entire population of cells at a particular phase. With removal of the chemical, cells rejoin the cell cycle in unison. Treatment of the test agent following release from the cell cycle blocking chemical ensures that the drug response elicited is from a uniform, cell cycle stage-specific population. However, since many of the chemical synchronizers are known genotoxic compounds, teasing apart the participation of various response pathways (to the synchronizers vs. the test agents) is challenging. Here we describe a metabolic labeling method for following a subpopulation of actively cycling cells through their progression from the DNA replication phase, through to the division and separation of their daughter cells. Coupled with flow cytometry quantification, this protocol enables for measurement of kinetic progression of the cell cycle in the absence of either mechanically- or chemically- induced cellular stresses commonly associated with other cell cycle synchronization methodologies 10. In the following sections we will discuss the methodology, as well as some of its applications in biomedical research.

Abstract 9107: Pim-1 Kinase Increases Non-Random Chromatid Segregation in Cardiac Progenitor Cells

Rationale: A cell has DNA strands of different replication ages because DNA replication is semi-conservative. Double-stranded DNA molecules possess one DNA strand that is one generation older than the other. Non-sister chromatids containing newly synthesized DNA and ‘old’ DNA strands are non-randomly partitioned between mother and daughter cells. Stem cells are presumed to non-randomly segregate chromatids to determine cell fate of daughter cells, although experimental evidence is lacking. Resident cardiac progenitor cells (CPC) present in the adult heart have been used for cell-based treatment of myocardial damage but the factors determining the stemness and lineage commitment of these cells are poorly understood. Cardio protective kinase Pim-1 increases asymmetric cell division in vivo, proliferation and commitment of CPCs after adaptive transfer in pathological injury model but its role in non-random segregation is unknown. Objective: Establish role of Pim-1 on non-random chromatid segregation and correlate with daughter cell fate determination in cardiac progenitor cells. Methods and Results: Non-random segregation was tracked by incorporation of bromodeoxyuridine (BrdU), a thymidine analogue labeling newly synthesized DNA strands. For label retention assay, cells were labeled for many generations and blocked in second cytokinesis during chase in BrdU free media. Two daughter nuclei within a cell body had different BrdU intensity. Density measurements showed that 4.6% of CPCs outside the 95% confidence interval for equal BrdU intensity between daughter nuclei indicating non-random segregation of BrdU labeled chromatids. Genetic engineering of CPCs to express Pim-1 kinase greatly enhanced non-random segregation (12.6% vs 5.6% GFP expressing cells). Non-random segregation was further confirmed by asymmetric BrdU segregation in anaphase cells from second mitotic shake off during chase. Conclusions: CPCs non-randomly segregate chromatids as demonstrated by selective segregation of BrdU labeled chromatids which is twofold increased in cells overexpressing Pim-1 kinase. Future Directions: The relationship of non-random chromatid segregation to identity of stem cells fate remains to be determined.

Radiosensitivity of Early and Late M-Phase HeLa Cells Isolated by a Combination of Fluorescent Ubiquitination-Based Cell Cycle Indicator (Fucci) and Mitotic Shake-Off

The mitotic shake-off method revealed the remarkable variation of radiosensitivity of HeLa cells during the cell cycle: M phase shows the greatest radiosensitivity and late S phase the greatest radioresistance. This method harvests all M-phase cells with a round shape, making it impossible to further subdivide M-phase cells. Recently, the fluorescent ubiquitination-based cell cycle indicator (Fucci) was developed; this system basically causes cells in G(1) to emit red fluorescence and other cells to emit green fluorescence. Because the green fluorescence rapidly disappears at late M phase, two-dimensional flow cytometry analysis can usually detect a green(high)/red(low) fraction including S-, G(2)- and early M-phase cells but not a transitional fraction between green(high)/red(low) and green(low)/red(low) including late M-phase cells. However, combining the shake-off method concentrated the transitional fraction, which enabled us to separate early and late M-phase cells without using any drugs. Here we demonstrate for the first time that cells in early M phase are more radiosensitive than those in late M phase, implying that early M phase is the most radiosensitive sub-phase during the cell cycle.

The Effect of Cell Cycle Synchronization on Tumor Sensitivity to Reovirus Oncolysis

The potential for increased sensitivity of tumor cells to oncolytic reovirus by altering the normal cell cycle using clinically available pharmacological agents was investigated. B16.F10 mouse melanoma cells were partially synchronized with hydroxyurea, thymidine, or by mitotic shake-off. Cell survival was determined using MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)- 2-(4-sulfophenyl)-2H-tetrazolium)] survival assay and virus yield in tumors by plaque assay. An enhanced sensitivity to reovirus was observed following the removal of either hydroxyurea or thymidine from the culture medium (P < 0.0001). The greatest survival difference compared to normal cycling cells was noted when the majority of cells were in S and G2/M phases, and was associated with increased viral replication. Cells collected by mitotic shake-off were nearly devoid of cells in S phase and were less susceptible to reovirus-induced cell kill than their nonsynchronized counterparts (P < 0.0001). In vivo combination of hydroxyurea followed by intratumoral reovirus resulted in reduced tumor growth and increased survival compared to monotherapy (P = 0.0041) at 15 days. Increased amounts of virus were retrieved from tumors from mice treated with sequential hydroxyurea/reovirus compared to concomitant treatment or reovirus monotherapy. These data justify clinical evaluation of this approach supported by the extensive experience, low cost, simple administration, and availability of hydroxyurea.

Interaction of Hepatitis B Viral Oncoprotein with Cellular Target HBXIP Dysregulates Centrosome Dynamics and Mitotic Spindle Formation

Hepatitis B virus infection is associated with hepatocellular carcinoma, claiming 1 million lives annually worldwide. To understand the carcinogenic mechanism of hepatitis B virus-encoded oncoprotein HBx, we explored the function of HBx interaction with its cellular target HBXIP. Previously, we demonstrated that viral HBx and cellular HBXIP control mitotic spindle formation, regulating centrosome splitting. By using various fragments of HBx, we determined that residues (137)CRHK(140) within HBx are necessary for binding HBXIP. Mutation of the (137)CRHK(140) motif in HBx abolished its ability to bind HBXIP and to dysregulate centrosome dynamics in HeLa and immortal diploid RPE-1 cells. Unlike wild-type HBx, which targets to centrosomes as determined by subcellular fractionation and immunofluorescence microscopy, HBx mutants failed to localize to centrosomes. Overexpression of viral HBx wild-type protein and knockdown of endogenous HBXIP altered centrosome assembly and induced modifications of pericentrin and centrin-2, two essential proteins required for centrosome formation and function, whereas HBXIP nonbinding mutants of HBx did not. Overexpression of HBXIP or fragments of HBXIP that bind HBx neutralized the effects of viral HBx on centrosome dynamics and spindle formation. These results suggest that HBXIP is a critical target of viral HBx for promoting genetic instability through formation of defective spindles and subsequent aberrant chromosome segregation.

Inhibitors of phosphatidylinositol 3-kinase activity prevent cell cycle progression and induce apoptosis at the M/G1 transition in CHO cells.

Phosphatidylinositol 3-kinase (PI3-kinase) activity has been implicated in regulating cell cycle progression at distinct points in the cell cycle by preventing cell cycle arrest or apoptosis. In this study, the role of PI3-kinase activity during the entire G1 phase of the ongoing cell cycle was studied in Chinese hamster ovary (CHO) cells synchronized by mitotic shake-off. We show that inhibition of PI3-kinase activity during and 2 h after mitosis inhibited cell cycle progression into S phase. In the presence of the PI3-kinase inhibitor wortmannin or LY294002, cells were arrested during early G1 phase, leading to the expression of the cleaved caspase-3, a central mediator of apoptosis. These results demonstrate that PI3-kinase activity is required for progression through the M/G1 phase. In the absence of PI3-kinase activity, cells are induced for apoptosis in this particular phase of the cell cycle.

Membrane Type-1 Matrix Metalloproteinase (MT1-MMP) Exhibits an Important Intracellular Cleavage Function and Causes Chromosome Instability

Elevated expression of membrane type-1 matrix metalloproteinase (MT1-MMP) is closely associated with malignancies. There is a consensus among scientists that cell surface-associated MT1-MMP is a key player in pericellular proteolytic events. Now we have identified an intracellular, hitherto unknown, function of MT1-MMP. We demonstrated that MT1-MMP is trafficked along the tubulin cytoskeleton. A fraction of cellular MT1-MMP accumulates in the centrosomal compartment. MT1-MMP targets an integral centrosomal protein, pericentrin. Pericentrin is known to be essential to the normal functioning of centrosomes and to mitotic spindle formation. Expression of MT1-MMP stimulates mitotic spindle aberrations and aneuploidy in non-malignant cells. Volumes of data indicate that chromosome instability is an early event of carcinogenesis. In agreement, the presence of MT1-MMP activity correlates with degraded pericentrin in tumor biopsies, whereas normal tissues exhibit intact pericentrin. We believe that our data show a novel proteolytic pathway to chromatin instability and elucidate the close association of MT1-MMP with malignant transformation.

In vitro synchronization of mammalian astrocytic cultures by serum deprivation

The study of the regulation of cell division cycle in vitro requires cell cultures growing in the same phase of the cycle. The procedure by which cells are arrested in specific phases of the cell cycle is termed synchronization. Synchronization is particularly important in the study of astrocyte biology, as its application allows astrocytes to re-enter the cell cycle from a state of quiescence (G 0), and, under carefully defined experimental conditions, move together into subsequent phases such as the G 1 and S phases. A number of methods have been established to synchronize mammalian cell cultures, including centrifugal elutriation, mitotic shake-off, and chemically induced cell cycle arrest. Yet there are intrinsic limitations associated with these methods. In the present protocol, we describe a simple, reliable, and reversible procedure to synchronize astrocytic cultures from newborn rat brains by serum deprivation. This protocol consists essentially of two parts: (1) proliferation of astrocytes under optimal conditions in vitro until reaching desired confluence; and (2) synchronization of cultures by serum down-shift and arrested in the G 0 phase of the cell cycle. This procedure has recently been extended toward the study of cell cycle control in astroglioma cells and astrocytes from injured adult brains. Since it was also employed in recent precursor cloning studies in developmental biology, this procedure will certainly find increasing use in future research.

Cytosolic phospholipase A(2) activity during the ongoing cell cycle.

Cytosolic phospholipase A(2) (cPLA(2)) is of special interest because it selectively releases arachidonic acid from membrane phospholipids. Arachidonic acid has been implicated to play an important role in various cellular responses. Recently arachidonic acid release and prostaglandin synthesis have been shown to be cell cycle dependent and therefore the activity of cPLA(2) during the ongoing cell cycle was investigated, using the mitotic shake off method for cell synchronisation. cPLA(2) activity was high in mitotic cells and decreased rapidly in the early G1 phase. A strong increase in activity was measured following the G1/S transition in both neuroblastoma and Chinese hamster ovary cells. The changes in activity were not due to a difference in cPLA(2) expression but due to phosphorylation of cPLA(2). Phosphorylation of cPLA(2) occurs through MAPK since the use of a specific MAPK kinase inhibitor and serum depletion of synchronised cells inhibited cPLA(2) activity.

Stem-Loop Binding Protein, the Protein That Binds the 3′ End of Histone mRNA, Is Cell Cycle Regulated by Both Translational and Posttranslational Mechanisms

The expression of the replication-dependent histone mRNAs is tightly regulated during the cell cycle. As cells progress from G(1) to S phase, histone mRNA levels increase 35-fold, and they decrease again during G(2) phase. Replication-dependent histone mRNAs are the only metazoan mRNAs that lack polyadenylated tails, ending instead in a conserved stem-loop. Much of the cell cycle regulation is posttranscriptional and is mediated by the 3' stem-loop. A 31-kDa stem-loop binding protein (SLBP) binds the 3' end of histone mRNA. The SLBP is necessary for pre-mRNA processing and accompanies the histone mRNA to the cytoplasm, where it is a component of the histone messenger RNP. We used synchronous CHO cells selected by mitotic shakeoff and HeLa cells synchronized at the G(1)/S or the M/G(1) boundary to study the regulation of SLBP during the cell cycle. In each system the amount of SLBP is regulated during the cell cycle, increasing 10- to 20-fold in the late G(1) and then decreasing in the S/G(2) border. SLBP mRNA levels are constant during the cell cycle. SLBP is regulated at the level of translation as cells progress from G(1) to S phase, and the protein is rapidly degraded as they progress into G(2). Regulation of SLBP may account for the posttranscriptional component of the cell cycle regulation of histone mRNA.

Integrin Signaling at the M/G1 Transition Induces Expression of Cyclin E

The activities of the mammalian G1 cyclins, cyclin D and cyclin E, during cell cycle progression (G1/S) are believed to be regulated by cell attachment and the presence of growth factors. In order to study the importance of cell attachment and concomitant integrin signaling on the expression of G1 cyclins during the natural adhesion process from mitosis to interphase, protein expression was monitored in cells that were synchronized by mitotic shake off. Here we show that in Chinese hamster ovary (CHO) and neuroblastoma (N2A) cells, expression of cyclin E at the M/G1 transition is regulated by both growth factors and cell attachment, while expression of cyclin D seems to be entirely dependent on the presence of serum. Expression of cyclin E appears to be correlated with the phosphorylation of the retinoblastoma protein, suggesting a link with the activity of the cyclin D/cdk4 complex. Expression of the cdk inhibitors p21cip1/Waf1 and p27Kip1 is not changed upon serum depletion or detachment of cells during early G1, suggesting no direct role for these CKIs in the regulation of cyclin activity. Although inhibition of cyclin E/cdk2 kinase activity has been reported previously, this is the first time that cyclin E expression is shown to be dependent on cell attachment.

Half-Lives of Ornithine Decarboxylase and S-Adenosylmethionine Decarboxylase Activities during the Cell Cycle of Chinese Hamster Ovary Cells

Cells in mitosis were seeded immediately after being harvested by the mitotic shake off technique from a culture of exponentially growing Chinese hamster ovary cells. At 2, 5, 7, 10, and 12 h after seeding, cycloheximide was added. Cells were sampled at various times after cycloheximide addition and the ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC) activities were determined. Flow cytometric analysis showed that cells sampled at 2, 5, 7, 10, and 12 h after seeding were found in mid G1, at the G1/S transition, in mid S phase, at the S/G2 transition, and in late G2, respectively. The half-lives of ODC and AdoMetDC activities varied during the cell cycle. The half-life of ODC activity showed a biphasic pattern with increases in connection to the G1/S and S/G2 transitions while the half-life of AdoMetDC activity increased only at the G1/S transition.

Nuclear translocation of mitogen‐activated protein kinase p42MAPK during the ongoing cell cycle

Mitogen-activated protein (MAP) kinases are serine/threonine protein kinases that are activated rapidly in cells stimulated by various extracellular signals. With stimulation of quiescent cells by growth factors, activated p42/p44 MAP kinases rapidly translocate to the nucleus, where they induce immediate early gene transcription. The MAP kinase signal transduction pathway represents an important mechanism by which growth factors regulate cellular events such as cell cycle progression or cell growth. In the present study, p42MAPK (ERK2) was studied during the ongoing cell cycle of Chinese hamster ovary cells synchronized by mitotic shake-off. We show that protein expression of p42MAPK increased in mid-G1 and that MAP kinase is phosphorylated during G1, as visualized by a gel-mobility shift and by the use of phosphospecific antibodies. This phosphorylation appeared to occur in the cytoplasm rather than at the plasmamembrane. In addition, phosphorylated p42MAPK was found to translocate to the nucleus during late/mid-G1. Treatment of cells with MEK inhibitor PD098059 prevented the phosphorylation and nuclear translocation of MAP kinase and DNA synthesis. Thus, nuclear translocation of p42MAPK is not restricted to the G0/G1 transition but occurs in every cell cycle and seems to be required for cell cycle progression. J. Cell. Physiol. 180:325–333, 1999. © 1999 Wiley-Liss, Inc.

Nuclear translocation of mitogen-activated protein kinase p42MAPK during the ongoing cell cycle.

Mitogen-activated protein (MAP) kinases are serine/threonine protein kinases that are activated rapidly in cells stimulated by various extracellular signals. With stimulation of quiescent cells by growth factors, activated p42/p44 MAP kinases rapidly translocate to the nucleus, where they induce immediate early gene transcription. The MAP kinase signal transduction pathway represents an important mechanism by which growth factors regulate cellular events such as cell cycle progression or cell growth. In the present study, p42MAPK (ERK2) was studied during the ongoing cell cycle of Chinese hamster ovary cells synchronized by mitotic shake-off. We show that protein expression of p42MAPK increased in mid-G1 and that MAP kinase is phosphorylated during G1, as visualized by a gel-mobility shift and by the use of phosphospecific antibodies. This phosphorylation appeared to occur in the cytoplasm rather than at the plasma-membrane. In addition, phosphorylated p42MAPK was found to translocate to the nucleus during late/mid-G1. Treatment of cells with MEK inhibitor PD098059 prevented the phosphorylation and nuclear translocation of MAP kinase and DNA synthesis. Thus, nuclear translocation of p42MAPK is not restricted to the G0/G1 transition but occurs in every cell cycle and seems to be required for cell cycle progression.

Down-Regulation of Nucleophosmin/B23 mRNA Delays the Entry of Cells into Mitosis

In investigating the regulation of nucleophosmin/B23 mRNA expression at the entry of mitosis, the results of Northern gel blot analysis showed that the nucleophosmin/B23 mRNA levels significantly increased in prometaphase (nocodazole-arrested) or metaphase (colchicine-arrested) cells collected by mitotic shake-off. A higher level of nucleophosmin/B23 mRNA was detected in all the collected mitotic cells arrested by treatment with nocodazole for 10-18h as compared to that in G2cells. An attempt was then made to determine whether the regulation of nucleophosmin/B23 mRNA plays a role in the control of entry into mitosis. Down-regulation of nucleophosmin/B23 mRNA by transfection of its antisense construct resulted in the delay of cells entering mitosis. The demonstration of the characteristic changes in the mRNA level of nucleophosmin/B23 during the entry of cells into mitosis implicates the importance of nucleophosmin/B23 in the control of the mitotic fate of nucleoli and cell growth.

Chemically induced micronucleus formation in V79 cells—comparison of three different test approaches

The in vitro micronucleus test (MNT) is a useful assay for the detection of mutagenic events on both the chromosomal and the genomic level. The main disadvantage for introducing the in vitro MNT into official test guidelines seems to be the disparity of existing protocols. To contribute to the aim of standardisation, three different methodological approaches of the in vitro MNT with V79 cells were compared: the standard assay using an asynchronically growing mixed cell population, the cytokinesis block (CB) assay and a modified MNT, the so-called mitotic shake-off (MSO) method. V79 cells were thus treated with two known aneugens (colcemide and griseofulvin) and two clastogens (mitomycin C and cyclophosphamide) over various time periods. The cultures of the CB assay were additionally exposed to cytochalasin B (Cyt-B), an inhibitor of cell, but not of nucleus division. After treatment, the cells were harvested and analysed for the appearance of micronuclei (MN). All three assays yielded positive results for all test substances. These results support the suitability of the MNT with V79 cells with regard to the ability to detect the genotoxic potential of both clastogens and aneugens independent of the test protocol applied. Thus, all three methods are appropriate for MN detection, but due to the fact that the application of Cyt-B has no advantages for a cell line like V79 in which nearly all cells undergo a normal cell cycle, its use is not recommended.