MAP Kinase Is Required for the Spindle Assembly Checkpoint but Is Dispensable for the Normal M Phase Entry and Exit in Xenopus Egg Cell Cycle Extracts

In Xenopus laevis egg cell cycle extracts that mimic early embryonic cell cycles, activation of MAP kinase and MAP kinase kinase occurs in M phase, slightly behind that of maturation promoting factor. To examine the possible role of MAP kinase in the in vitro cell cycle, we depleted the extracts of MAP kinase by using anti–Xenopus MAP kinase antibody. Like in the mock-treated extracts, the periodic activation and deactivation of MPF occurred normally in the MAP kinase–depleted extracts, suggesting that MAP kinase is dispensable for the normal M phase entry and exit in vitro. It has recently been reported that microtubule depolymerization by nocodazole treatment can block exit from mitosis in the extracts if enough sperm nuclei are present, and that the addition of MAP kinase– specific phosphatase MKP-1 overcomes this spindle assembly checkpoint, suggesting the involvement of MAP kinase in the checkpoint signal transduction. We show here that the spindle assembly checkpoint mechanism cannot operate in the MAP kinase–depleted extracts. But, adding recombinant Xenopus MAP kinase to the MAP kinase–depleted extracts restored the spindle assembly checkpoint. These results indicate unambiguously that classical MAP kinase is required for the spindle assembly checkpoint in the cell cycle extracts. In addition, we show that strong activation of MAP kinase by the addition of a constitutively active MAP kinase kinase kinase in the absence of sperm nuclei and nocodazole, induced mitotic arrest in the extracts. Therefore, activation of MAP kinase alone is sufficient for inducing the mitotic arrest in vitro.

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Comparison of the effects of BPA and BPAF on oocyte spindle assembly and polar body release in mice

Zygote ◽

10.1017/s0967199415000027 ◽

2015 ◽

Vol 24 (2) ◽

pp. 172-180 ◽

Cited By ~ 14

Author(s):

Kei Nakano ◽

Manami Nishio ◽

Norio Kobayashi ◽

Yuuki Hiradate ◽

Yumi Hoshino ◽

...

Keyword(s):

Cell Cycle ◽

Oocyte Maturation ◽

Spindle Assembly Checkpoint ◽

Polar Body ◽

Sharp Decrease ◽

Spindle Assembly ◽

Environmental Chemical ◽

Cell Cycle Delay ◽

A Cell

SummaryBisphenol AF (BPAF), a homolog of bisphenol A (BPA), is a widely used environmental chemical that has adverse effects on reproduction. The aim of this study was to analyse the effects of BPA and BPAF exposure on oocyte maturation in vitro. Oocytes were cultured in the presence of BPA or BPAF (2, 20, 50 or 100 μg/ml) for 18 h. At concentrations of 50 and 100 μg/ml, BPA and BPAF inhibited oocyte maturation, with BPAF treatment causing a sharp decrease in the number of oocytes reaching maturity. Oocytes were exposed to BPA or BPAF at 2 μg/ml and cultured for different durations (6, 9, 12, 15 or 18 h). Both BPAF and BPA caused a cell cycle delay under these conditions. Oocytes cultured in the presence of BPA or BPAF (50 μg/ml) for 21 h were tested for the localization of α-tubulin and MAD2 using immunofluorescence. High concentrations of BPAF induced cell cycle arrest through the activation of the spindle assembly checkpoint. After 12 h of culture in BPAF (50 μg/ml), oocytes were transferred to control medium for 9 h. Only 63.3% oocytes treated in this manner progressed to metaphase II (MII). Oocytes exposed to high doses of BPA experienced a cell cycle delay, but managed to progress to MII when the culture period was prolonged. In addition, MAD2 was localized in the cytoplasm of these oocytes. In conclusion, both BPAF and BPA exposure affected oocyte maturation, however BPAF and BPA have differential effects on SAC activity.

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TheSac3Homologueshd1Is Involved in Mitotic Progression in Mammalian Cells

Journal of Biological Chemistry ◽

10.1074/jbc.m405347200 ◽

2004 ◽

Vol 279 (44) ◽

pp. 46182-46190 ◽

Cited By ~ 10

Author(s):

Sefat-e- Khuda ◽

Mikoto Yoshida ◽

Yan Xing ◽

Tatsuya Shimasaki ◽

Motohiro Takeya ◽

...

Keyword(s):

Cell Cycle ◽

Mammalian Cells ◽

Cell Cycle Progression ◽

Spindle Assembly Checkpoint ◽

Fluorescent Protein ◽

Spindle Assembly ◽

Centrosome Duplication ◽

Cycle Progression ◽

M Phase ◽

Green Fluorescent

SaccharomycesSac3 required for actin assembly was shown to be involved in DNA replication. Here, we studied the function of a mammalian homologue SHD1 in cell cycle progression. SHD1 is localized on centrosomes at interphase and at spindle poles and mitotic spindles, similar to α-tubulin, at M phase. RNA interference suppression of endogenousshd1caused defects in centrosome duplication and spindle formation displaying cells with a single apparent centrosome and down-regulated Mad2 expression, generating increased micronuclei. Conversely, increased expression of SHD1 by DNA transfection withshd1-green fluorescent protein (gfp) vector for a fusion protein of SHD1 and GFP caused abnormalities in centrosome duplication displaying cells with multiple centrosomes and deregulated spindle assembly with up-regulated Mad2 expression until anaphase, generating polyploidy cells. These results demonstrated thatshd1is involved in cell cycle progression, in particular centrosome duplication and a spindle assembly checkpoint function.

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Clb6/Cdc28 and Cdc14 Regulate Phosphorylation Status and Cellular Localization of Swi6

Molecular and Cellular Biology ◽

10.1128/mcb.24.6.2277-2285.2004 ◽

2004 ◽

Vol 24 (6) ◽

pp. 2277-2285 ◽

Cited By ~ 48

Author(s):

Marco Geymonat ◽

Ad Spanos ◽

Glenn P. Wells ◽

Stephen J. Smerdon ◽

Steven G. Sedgwick

Keyword(s):

Cell Cycle ◽

Nuclear Import ◽

Nuclear Export ◽

Cellular Localization ◽

Entry And Exit ◽

Mitotic Exit Network ◽

M Phase ◽

Cdc28 Kinase

ABSTRACT Nuclear export of the transcription factor Swi6 during the budding yeast Saccharomyces cerevisiae cell cycle is known to require phosphorylation of the Swi6 serine 160 residue. We show that Clb6/Cdc28 kinase is required for this nuclear export. Furthermore, Cdc28 combined with the S-phase cyclin Clb6 specifically phosphorylates serine 160 of Swi6 in vitro. Nuclear import of Swi6 occurs concomitantly with dephosphorylation of serine 160 in late M phase. We show that Cdc14 phosphatase, the principal effector of the mitotic exit network, can trigger nuclear import of Swi6 in vivo and that Cdc14 dephosphorylates Swi6 at serine 160 in vitro. Taken together, these observations show how Swi6 dephosphorylation and phosphorylation are integrated into changes of Cdc28 activity governing entry and exit from the G1 phase of the cell cycle.

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Use of the Novel Plk1 Inhibitor ZK-Thiazolidinone to Elucidate Functions of Plk1 in Early and Late Stages of Mitosis

Molecular Biology of the Cell ◽

10.1091/mbc.e07-05-0517 ◽

2007 ◽

Vol 18 (10) ◽

pp. 4024-4036 ◽

Cited By ~ 132

Author(s):

Anna Santamaria ◽

Rüdiger Neef ◽

Uwe Eberspächer ◽

Knut Eis ◽

Manfred Husemann ◽

...

Keyword(s):

Spindle Assembly Checkpoint ◽

Spindle Assembly ◽

Cleavage Furrow ◽

The Novel ◽

Mitotic Progression ◽

Binding Partner ◽

Molecule Inhibitor ◽

Interaction Partners ◽

Late Stages

Polo-like kinase 1 (Plk1) is a key regulator of mitotic progression and cell division in eukaryotes. It is highly expressed in tumor cells and considered a potential target for cancer therapy. Here, we report the discovery and application of a novel potent small-molecule inhibitor of mammalian Plk1, ZK-Thiazolidinone (TAL). We have extensively characterized TAL in vitro and addressed TAL specificity within cells by studying Plk1 functions in sister chromatid separation, centrosome maturation, and spindle assembly. Moreover, we have used TAL for a detailed analysis of Plk1 in relation to PICH and PRC1, two prominent interaction partners implicated in spindle assembly checkpoint function and cytokinesis, respectively. Specifically, we show that Plk1, when inactivated by TAL, spreads over the arms of chromosomes, resembling the localization of its binding partner PICH, and that both proteins are mutually dependent on each other for correct localization. Finally, we show that Plk1 activity is essential for cleavage furrow formation and ingression, leading to successful cytokinesis.

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The Polo-like kinase Plx1 is a component of the MPF amplification loop at the G2/M-phase transition of the cell cycle in Xenopus eggs

Journal of Cell Science ◽

10.1242/jcs.111.12.1751 ◽

1998 ◽

Vol 111 (12) ◽

pp. 1751-1757 ◽

Cited By ~ 16

Author(s):

A. Abrieu ◽

T. Brassac ◽

S. Galas ◽

D. Fisher ◽

J.C. Labbe ◽

...

Keyword(s):

Phase Transition ◽

Cell Cycle ◽

Cyclin A ◽

Cyclin B ◽

Cdc2 Kinase ◽

C Terminus ◽

M Phase ◽

Egg Extracts ◽

Amplification Loop

We have investigated whether Plx1, a kinase recently shown to phosphorylate cdc25c in vitro, is required for activation of cdc25c at the G2/M-phase transition of the cell cycle in Xenopus. Using immunodepletion or the mere addition of an antibody against the C terminus of Plx1, which suppressed its activation (not its activity) at G2/M, we show that Plx1 activity is required for activation of cyclin B-cdc2 kinase in both interphase egg extracts receiving recombinant cyclin B, and cycling extracts that spontaneously oscillate between interphase and mitosis. Furthermore, a positive feedback loop allows cyclin B-cdc2 kinase to activate Plx1 at the G2/M-phase transition. In contrast, activation of cyclin A-cdc2 kinase does not require Plx1 activity, and cyclin A-cdc2 kinase fails to activate Plx1 and its consequence, cdc25c activation in cycling extracts.

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Novel Thiadiazoline Spiro Quinoline Analogues Induced Cell death in MCF-7 cells via G2/M Phase Cell Cycle Arrest

10.21203/rs.3.rs-289802/v1 ◽

2021 ◽

Author(s):

Selvaraj Shyamsivappan ◽

Raju Vivek ◽

Thangaraj Suresh ◽

Adhigaman Kaviyarasu ◽

Sundarasamy Amsaveni ◽

...

Keyword(s):

Cell Cycle ◽

Cell Death ◽

Cell Cycle Arrest ◽

Spectroscopic Studies ◽

Phase Cell ◽

Quinoline Derivatives ◽

Cycle Arrest ◽

M Phase ◽

Mcf 7

Abstract A progression of novel thiadiazoline spiro quinoline derivatives were synthesized from potent thiadiazoline spiro quinoline derivatives . The synthesized compounds portrayed by different spectroscopic studies and single X-ray crystallographic studies. The compounds were assessed for in vitro anticancer properties towards MCF-7 and HeLa cells. The compounds showed superior inhibition action MCF-7 malignant growth cells. Amongst, the compound 4a showed significant inhibition activity, the cell death mechanism was evaluated by fluorescent staining, and flow cytometry, RT-PCR, and western blot analyses. The in vitro anticancer results revealed that the compound 4a induced apoptosis by inhibition of estrogen receptor alpha (ERα) and G2/M phase cell cycle arrest. The binding affinity of the compounds with ERα and pharmacokinetic properties were confirmed by molecular docking studies.

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Synthesis and In Vitro Antitumor Activity of Naringenin Oxime and Oxime Ether Derivatives

International Journal of Molecular Sciences ◽

10.3390/ijms20092184 ◽

2019 ◽

Vol 20 (9) ◽

pp. 2184 ◽

Cited By ~ 4

Author(s):

Ahmed Dhahir Latif ◽

Tímea Gonda ◽

Máté Vágvölgyi ◽

Norbert Kúsz ◽

Ágnes Kulmány ◽

...

Keyword(s):

Cell Cycle ◽

Antioxidant Activities ◽

Biological Activities ◽

Gynecological Cancer ◽

Human Leukemia ◽

Oxime Ether ◽

M Phase ◽

Induced Apoptosis ◽

Mcf 7

Naringenin is one of the most abundant dietary flavonoids exerting several beneficial biological activities. Synthetic modification of naringenin is of continuous interest. During this study our aim was to synthesize a compound library of oxime and oxime ether derivatives of naringenin, and to investigate their biological activities. Two oximes and five oxime ether derivatives were prepared; their structure has been elucidated by NMR and high-resolution mass spectroscopy. The antiproliferative activity of the prepared compounds was evaluated by MTT assay against human leukemia (HL-60) and gynecological cancer cell lines isolated from cervical (HeLa, Siha) and breast (MCF-7, MDA-MB-231) cancers. Tert-butyl oxime ether derivative exerted the most potent cell growth inhibitory activity. Moreover, cell cycle analysis suggested that this derivative caused a significant increase in the hypodiploid (subG1) phase and induced apoptosis in Hela and Siha cells, and induced cell cycle arrest at G2/M phase in MCF-7 cells. The proapoptotic potential of the selected compound was confirmed by the activation of caspase-3. Antioxidant activities of the prepared molecules were also evaluated with xanthine oxidase, DPPH and ORAC assays, and the methyl substituted oxime ether exerted the most promising activity.

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Microtubule Cytoskeleton Remodeling by Acentriolar Microtubule-organizing Centers at the Entry and Exit from Mitosis in Drosophila Somatic Cells

Molecular Biology of the Cell ◽

10.1091/mbc.e09-01-0011 ◽

2009 ◽

Vol 20 (11) ◽

pp. 2796-2808 ◽

Cited By ~ 32

Author(s):

Sara Moutinho-Pereira ◽

Alain Debec ◽

Helder Maiato

Keyword(s):

Cell Cycle ◽

Mitotic Spindle ◽

De Novo ◽

Animal Cell ◽

Somatic Cells ◽

Spindle Assembly ◽

Entry And Exit ◽

Down Regulation ◽

Microtubule Organizing Centers ◽

Cytoskeleton Remodeling

Cytoskeleton microtubules undergo a reversible metamorphosis as cells enter and exit mitosis to build a transient mitotic spindle required for chromosome segregation. Centrosomes play a dominant but dispensable role in microtubule (MT) organization throughout the animal cell cycle, supporting the existence of concurrent mechanisms that remain unclear. Here we investigated MT organization at the entry and exit from mitosis, after perturbation of centriole function in Drosophila S2 cells. We found that several MTs originate from acentriolar microtubule-organizing centers (aMTOCs) that contain γ-tubulin and require Centrosomin (Cnn) for normal architecture and function. During spindle assembly, aMTOCs associated with peripheral MTs are recruited to acentriolar spindle poles by an Ncd/dynein-dependent clustering mechanism to form rudimentary aster-like structures. At anaphase onset, down-regulation of CDK1 triggers massive formation of cytoplasmic MTs de novo, many of which nucleated directly from aMTOCs. CDK1 down-regulation at anaphase coordinates the activity of Msps/XMAP215 and the kinesin-13 KLP10A to favor net MT growth and stability from aMTOCs. Finally, we show that microtubule nucleation from aMTOCs also occurs in cells containing centrosomes. Our data reveal a new form of cell cycle–regulated MTOCs that contribute for MT cytoskeleton remodeling during mitotic spindle assembly/disassembly in animal somatic cells, independently of centrioles.

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