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Zhongguo Shi Yan Xue Ye Xue Za Zhi Oct 2016To verify the role of the newly identified mitotic regulator candidate pre-mRNA processing factor 19 (Prp19) in mitosis and to clarify its underlying mechanism.
OBJECTIVE
To verify the role of the newly identified mitotic regulator candidate pre-mRNA processing factor 19 (Prp19) in mitosis and to clarify its underlying mechanism.
METHODS
FACS analyses with propidium iodide (PI) staining were performed to evaluate the effect of Prp19 knockdown on cell cycle distribution. To further clarify the role of Prp19 in mitosis, the effect of Prp19 depletion was monitored by time-lapse imaging of HeLa/GFP-H2B cells. Cold treatment experiment was used to examine the effect of Prp19 knockdown on the attachment of microtubules and kinetochores. To evaluate the effect of Prp19 knockdown on cell apoptosis, the control and Prp19-knockdown cells were analyzed by FACS with annexin V-FITC/-PI double staining. Furthermore, Western blot analysis of cleaved caspase-3 and PARP was also performed.
RESULTS
Prp19 knockdown causesd mitotic arrest. Time-lapse imaging analysis showed that depletion of Prp19 in HeLa cells results in prometaphase arrest and chromosome misalignment. Cold treatment experiment showed that attachment between kinetochore and microtubule was impaired by Prp19 knockdown. Moreover, the depletion of Prp19 leaded to cell apoptosis in cancer cells.
CONCLUSION
Prp19 is a key regulator of mitotic progression, and its inhibition may provide a new strategy for anti-cancer therapy.
Topics: Annexin A5; Caspase 3; Cell Cycle Proteins; DNA Repair Enzymes; Fluorescein-5-isothiocyanate; HeLa Cells; Humans; Kinetochores; Microtubules; Mitosis; Nuclear Proteins; Prometaphase; RNA Precursors; RNA Splicing Factors
PubMed: 27784391
DOI: 10.7534/j.issn.1009-2137.2016.05.048 -
PloS One Oct 2009The protein kinases Mps1 and Polo, which are required for proper cell cycle regulation in meiosis and mitosis, localize to numerous ooplasmic filaments during...
BACKGROUND
The protein kinases Mps1 and Polo, which are required for proper cell cycle regulation in meiosis and mitosis, localize to numerous ooplasmic filaments during prometaphase in Drosophila oocytes. These filaments first appear throughout the oocyte at the end of prophase and are disassembled after egg activation.
METHODOLOGY/PRINCIPAL FINDINGS
We showed here that Mps1 and Polo proteins undergo dynamic and reversible localization to static ooplasmic filaments as part of an oocyte-specific response to hypoxia. The observation that Mps1- and Polo-associated filaments reappear in the same locations through multiple cycles of oxygen deprivation demonstrates that underlying structural components of the filaments must still be present during normoxic conditions. Using immuno-electron microscopy, we observed triple-helical binding of Mps1 to numerous electron-dense filaments, with the gold label wrapped around the outside of the filaments like a garland. In addition, we showed that in live oocytes the relocalization of Mps1 and Polo to filaments is sensitive to injection of collagenase, suggesting that the structural components of the filaments are composed of collagen-like fibrils. However, the collagen-like genes we have been able to test so far (vkg and CG42453) did not appear to be associated with the filaments, demonstrating that the collagenase-sensitive component of the filaments is one of a number of other Drosophila proteins bearing a collagenase cleavage site. Finally, as hypoxia is known to cause Mps1 protein to accumulate at kinetochores in syncytial embryos, we also show that GFP-Polo accumulates at both kinetochores and centrosomes in hypoxic syncytial embryos.
CONCLUSIONS/SIGNIFICANCE
These findings identify both a novel cellular structure (the ooplasmic filaments) as well as a new localization pattern for Mps1 and Polo and demonstrate that hypoxia affects Polo localization in Drosophila.
Topics: Animals; Cell Cycle; Cell Cycle Proteins; Collagenases; Drosophila; Drosophila Proteins; Female; Green Fluorescent Proteins; Hypoxia; Male; Meiosis; Mitosis; Models, Biological; Oocytes; Prometaphase; Protein Kinases; Protein Serine-Threonine Kinases
PubMed: 19847308
DOI: 10.1371/journal.pone.0007544 -
Science in China. Series C, Life... Jun 1996A new model for mitotic dynamics of eukaryotic cells is proposed. In the kinetochore motor-midzone motor model two kinds of motors, the kinetochore motors and the...
A new model for mitotic dynamics of eukaryotic cells is proposed. In the kinetochore motor-midzone motor model two kinds of motors, the kinetochore motors and the midzone motors, play important roles in chromosome movement. Using this model the chromosome congression during prometaphase, the chromosome oscillation during metaphase and the chromatic segregation during anaphase are described in a unified way.
Topics: Chromosomes; Kinetochores; Mitosis; Models, Biological
PubMed: 8760457
DOI: No ID Found -
Cell Cycle (Georgetown, Tex.) Feb 2007Here we show that the human BubR1 and MAD2 genes, which encode inhibitors of the anaphase promoting complex (APC/C), are directly activated by the oncogenic...
Here we show that the human BubR1 and MAD2 genes, which encode inhibitors of the anaphase promoting complex (APC/C), are directly activated by the oncogenic transcription factor c-MYC via E-box sequences in their first introns. In colorectal cancer biopsies elevated expression of c-MYC correlated with increased MAD2 levels. Activation of a conditional c-MYC allele delayed progression through mitosis in pro-metaphase in a MAD2- and BubR1-dependent manner. A fraction of the daughter cells derived from extended mitotic events underwent synchronous apoptosis, which was in part mediated by BubR1. Furthermore, c-MYC activation resulted in CIN (chromosomal instability) in the diploid MIN (microsatellite instability) cell line DLD-1 and further enhanced CIN in the aneuploid CIN-line MCF7. Unexpectedly, c-MYC-induced CIN was independent of c-MYC-induced BubR1/MAD2 expression and mitotic delay. Therefore, c-MYC-induced CIN may be caused be alternative pathways. We observed that activation of c-MYC induced DNA double-strand breaks, as evidenced by formation of gamma-H2AX foci, which colocalized with foci of active DNA replication. Furthermore, c-MYC activation resulted in mitotic chromosomes exhibiting DNA damage. Therefore, oncogenic deregulation of c-MYC prevents repair of replication-stress induced DNA lesions in the G(2)-phase. We suggest that the c-MYC-mediated persistence of DNA lesions throughout mitosis leads to chromosomal missegregation and underlies c-MYC-induced CIN. The effects of deregulated c-MYC on progression through mitosis described here may have important implications for the origin of chromosomal instability in many tumor types and the sensitivity towards cancer therapeutic agents targeting DNA or the mitotic spindle.
Topics: Anaphase; Apoptosis; Calcium-Binding Proteins; Cell Cycle Proteins; Cell Line, Tumor; Chromatin; Chromosomal Instability; Colorectal Neoplasms; DNA Damage; E-Box Elements; Etoposide; Genes, myc; Histones; Humans; Introns; Mad2 Proteins; Mitosis; Prometaphase; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-myc; Repressor Proteins; Transcriptional Activation
PubMed: 17297307
DOI: 10.4161/cc.6.3.3808 -
Oncotarget Jan 2018Cis-trimethoxy resveratrol (cis-3M-RES) induced dose-dependent cytotoxicity and apoptotic DNA fragmentation in Jurkat T cell clones (JT/Neo); however, it induced only...
Cis-trimethoxy resveratrol (cis-3M-RES) induced dose-dependent cytotoxicity and apoptotic DNA fragmentation in Jurkat T cell clones (JT/Neo); however, it induced only cytostasis in BCL-2-overexpressing cells (JT/BCL-2). Treatment with 0.25 μM cis-3M-RES induced G/M arrest, BAK activation, Δψm loss, caspase-9 and caspase-3 activation, and poly (ADP-ribose) polymerase (PARP) cleavage in JT/Neo cells time-dependently but did not induce these events, except G/M arrest, in JT/BCL-2 cells. Moreover, cis-3M-RES induced CDK1 activation, BCL-2 phosphorylation at Ser-70, MCL-1 phosphorylation at Ser-159/Thr-163, and BIM (BIM and BIM) phosphorylation irrespective of BCL-2 overexpression. Enforced G/S arrest by using a G/S blocker aphidicolin completely inhibited cis-3M-RES-induced apoptotic events. Cis-3M-RES-induced phosphorylation of BCL-2 family proteins and mitochondrial apoptotic events were suppressed by a validated CDK1 inhibitor RO3306. Immunofluorescence microscopy showed that cis-3M-RES induced mitotic spindle defects and prometaphase arrest. The rate of intracellular polymeric tubulin to monomeric tubulin decreased markedly by cis-3M-RES (0.1-1.0 μM). Wild-type Jurkat clone A3, FADD-deficient Jurkat clone I2.1, and caspase-8-deficient Jurkat clone I9.2 exhibited similar susceptibilities to the cytotoxicity of cis-3M-RES, excluding contribution of the extrinsic death receptor-dependent pathway to the apoptosis. IC values of cis-3M-RES against Jurkat E6.1, U937, HL-60, and HeLa cells were 0.07-0.17 μM, whereas those against unstimulated human peripheral T cells and phytohaemagglutinin A-stimulated peripheral T cells were >10.0 and 0.23 μM, respectively. These results indicate that the antitumor activity of cis-3M-RES is mediated by microtubule damage, and subsequent prometaphase arrest and prolonged CDK1 activation that cause BAK-mediated mitochondrial apoptosis, and suggest that cis-3M-RES is a promising agent to treat leukemia.
PubMed: 29435156
DOI: 10.18632/oncotarget.23576 -
Biochemical and Biophysical Research... Sep 2014Exposure of human Jurkat T cells to JNK inhibitor IX (JNKi), targeting JNK2 and JNK3, caused apoptotic DNA fragmentation along with G2/M arrest, phosphorylation of...
Exposure of human Jurkat T cells to JNK inhibitor IX (JNKi), targeting JNK2 and JNK3, caused apoptotic DNA fragmentation along with G2/M arrest, phosphorylation of Bcl-2, Mcl-1, and Bim, Δψm loss, and activation of Bak and caspase cascade. These JNKi-induced apoptotic events were abrogated by Bcl-2 overexpression, whereas G2/M arrest, cyclin B1 up-regulation, Cdk1 activation, and phosphorylation of Bcl-2 family proteins were sustained. In the concomitant presence of the G1/S blocking agent aphidicolin and JNKi, the cells underwent G1/S arrest and failed to induce all apoptotic events. The JNKi-induced phosphorylation of Bcl-2 family proteins and mitochondrial apoptotic events were suppressed by the Cdk1 inhibitor. Immunofluorescence microscopic analysis revealed that mitotic spindle defect and prometaphase arrest were the underlying factors for the G2/M arrest. These results demonstrate that JNKi-induced mitochondrial apoptosis was caused by microtubule damage-mediated prometaphase arrest, prolonged Cdk1 activation, and phosphorylation of Bcl-2 family proteins in Jurkat T cells.
Topics: Aphidicolin; Apoptosis; DNA Fragmentation; G1 Phase Cell Cycle Checkpoints; G2 Phase Cell Cycle Checkpoints; Gene Expression Regulation; Humans; Jurkat Cells; Membrane Potential, Mitochondrial; Microtubules; Mitochondria; Mitogen-Activated Protein Kinase 10; Mitogen-Activated Protein Kinase 9; Myeloid Cell Leukemia Sequence 1 Protein; Phosphorylation; Prometaphase; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-bcl-2; Signal Transduction
PubMed: 25218503
DOI: 10.1016/j.bbrc.2014.09.015 -
Journal of Cell Science Feb 1991The site of tubulin subunit dissociation was determined during poleward chromosome movement in prometaphase newt lung cell mitotic spindles using fluorescence...
The site of tubulin subunit dissociation was determined during poleward chromosome movement in prometaphase newt lung cell mitotic spindles using fluorescence photobleaching techniques and nocodazole-induced spindle shortening. Synchronous shortening of all kinetochore microtubules was produced by incubating cells in 17 microM nocodazole to block microtubule assembly. Under these conditions the spindle poles moved towards the metaphase plate at a rate of 3.6 +/- 0.4 microns min-1 (n = 3). On the basis of anti-tubulin immunofluorescent staining of cells fixed after incubation in nocodazole, we found that nonkinetochore microtubules rapidly disappeared and only kinetochore fibers were present after 60-90 s in nocodazole. To localize the site of tubulin subunit dissociation, a narrow bar pattern was photobleached across one half-spindle in prometaphase-metaphase cells previously microinjected with 5-(4,6-dichlorotriazin-2-yl) amino fluorescein (DTAF)-labeled tubulin. Immediately after photobleaching, cells were perfused with 17 microM nocodazole to produce shortening of kinetochore microtubules. Shortening was accompanied by a decrease in the distance between the bleach bar and the kinetochores. In contrast, there was little or no decrease in the distance between the bleach bar and the pole. Compared to their initial lengths, the average kinetochore to pole distance shortened by 18%, the bleach bar to kinetochore distance shortened by 28% and the average bleached bar to pole distance shortened by 1.6%. The data provide evidence that tubulin subunits dissociate from kinetochore microtubules at a site near the kinetochore during poleward chromosome movement. These results are consistent with models of poleward force generation for chromosome movement in which prometaphase-metaphase poleward force is generated in association with the kinetochore.
Topics: Animals; Chromosomes; Epithelial Cells; Lung; Metaphase; Microinjections; Microscopy, Fluorescence; Microtubules; Nocodazole; Prophase; Salamandridae; Spindle Apparatus; Tubulin; Video Recording
PubMed: 2055954
DOI: 10.1242/jcs.98.2.151 -
Chromosoma 1961
Topics: Animals; Chromosomes; Grasshoppers; Humans; Male; Prometaphase; Sex Chromosomes; Spermatocytes; Spermatozoa
PubMed: 13728791
DOI: 10.1007/BF00328917 -
Three-dimensional maps of all chromosomes in human male fibroblast nuclei and prometaphase rosettes.PLoS Biology May 2005Studies of higher-order chromatin arrangements are an essential part of ongoing attempts to explore changes in epigenome structure and their functional implications...
Studies of higher-order chromatin arrangements are an essential part of ongoing attempts to explore changes in epigenome structure and their functional implications during development and cell differentiation. However, the extent and cell-type-specificity of three-dimensional (3D) chromosome arrangements has remained controversial. In order to overcome technical limitations of previous studies, we have developed tools that allow the quantitative 3D positional mapping of all chromosomes simultaneously. We present unequivocal evidence for a probabilistic 3D order of prometaphase chromosomes, as well as of chromosome territories (CTs) in nuclei of quiescent (G0) and cycling (early S-phase) human diploid fibroblasts (46, XY). Radial distance measurements showed a probabilistic, highly nonrandom correlation with chromosome size: small chromosomes-independently of their gene density-were distributed significantly closer to the center of the nucleus or prometaphase rosette, while large chromosomes were located closer to the nuclear or rosette rim. This arrangement was independently confirmed in both human fibroblast and amniotic fluid cell nuclei. Notably, these cell types exhibit flat-ellipsoidal cell nuclei, in contrast to the spherical nuclei of lymphocytes and several other human cell types, for which we and others previously demonstrated gene-density-correlated radial 3D CT arrangements. Modeling of 3D CT arrangements suggests that cell-type-specific differences in radial CT arrangements are not solely due to geometrical constraints that result from nuclear shape differences. We also found gene-density-correlated arrangements of higher-order chromatin shared by all human cell types studied so far. Chromatin domains, which are gene-poor, form a layer beneath the nuclear envelope, while gene-dense chromatin is enriched in the nuclear interior. We discuss the possible functional implications of this finding.
Topics: Cell Culture Techniques; Cell Nucleus; Child, Preschool; Chromosome Mapping; Chromosomes, Human; Diploidy; Fibroblasts; Humans; In Situ Hybridization, Fluorescence; Male; Probability; Prometaphase; Skin
PubMed: 15839726
DOI: 10.1371/journal.pbio.0030157 -
Biochemical and Biophysical Research... Oct 2014Mitotic catastrophe, a form of cell death that occurs during mitosis and after mitotic slippage to a tetraploid state, plays an important role in the efficacy of cancer...
Mitotic catastrophe, a form of cell death that occurs during mitosis and after mitotic slippage to a tetraploid state, plays an important role in the efficacy of cancer cell killing by microtubule inhibitors. Prolonged mitotic arrest at the spindle assembly checkpoint (SAC) is a well-known requirement for mitotic catastrophe and, thus, for conferring sensitivity to microtubule inhibitors. We previously reported that downregulation of SIRT2, a member of the sirtuin family of NAD+-dependent deacetylases, confers resistance to microtubule inhibitors by abnormally prolonging mitotic arrest and thus compromising the cell death pathway after mitotic slippage. Thus, turning off SAC activation after a defined period is an additional requirement for efficient post-slippage death. Here, we investigated whether SIRT2 deacetylates BubR1, which is a core component of the SAC; acetylation of BubR1 at lysine 250 (K250) during prometaphase inhibits its APC/C-dependent proteolysis and thus regulates timing in anaphase entry. We showed that SIRT2 deacetylates BubR1 K250 both in vitro and in vivo. We also found that SIRT2 knockdown leads to increased levels of BubR1 acetylation at prometaphase; however, this increase is not substantial to elevate the levels of total BubR1 or delay the transition from prometaphase to anaphase. The present study shows that SIRT2 is a deacetylase for BubR1 K250, although the abnormally prolonged SAC activation observed in SIRT2 knockdown cells is not accompanied by a change in BubR1 levels or by delayed progression from prometaphase to anaphase.
Topics: Acetylation; Anaphase; Cell Line; Gene Knockdown Techniques; Humans; Prometaphase; Protein Serine-Threonine Kinases; Proteolysis; Sirtuin 2
PubMed: 25285631
DOI: 10.1016/j.bbrc.2014.09.128