Rigosertib and Cholangiocarcinoma: A Cell Cycle Affair

Rigosertib is multi-kinase inhibitor that could represent an interesting therapeutic option for non-resectable patients with cholangiocarcinoma, a very aggressive hepatic cancer with limited effective treatments. The Western blotting technique was used to evaluate alterations in the expression of proteins involved in the regulation of the cell cycle of cholangiocarcinoma EGI-1 cells. Our results show an increase in EMI1 and Cyclin B protein levels after Rigosertib treatment. Moreover, the phosphorylation of CDK1 is significantly reduced by Rigosertib, while PLK1 expression increased after 24 h of treatment and decreased after 48 h. Finally, we evaluated the role of p53. Its levels increase after Rig treatment, and, as shown in the cell viability experiment with the p53 inhibitor Pifithrin, its activity is necessary for the effects of Rigosertib against the cell viability of EGI-1 cells. In conclusion, we hypothesized the mechanism of the action of Rigosertib against cholangiocarcinoma EGI-1 cells, highlighting the importance of proteins involved in the regulation of cell cycles. The CDK1-Cyclin B complex and p53 play an important role, explaining the Block in the G2/M phase of the cell cycle and the effect on cell viability

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Evolution of CDK1 paralog specializations in a lineage with fast developing planktonic embryos

10.1101/817783 ◽

2019 ◽

Author(s):

Xiaofei Ma ◽

Jan Inge Øvrebø ◽

Eric M Thompson

Keyword(s):

General Rule ◽

Embryonic Cell ◽

Cyclin B ◽

Gene Duplications ◽

Structural Elements ◽

Oikopleura Dioica ◽

Cell Cycles ◽

Protein Levels ◽

Binding Interface ◽

M Phase

AbstractThe active site of the essential, eukaryotic CDK1 kinase is generated by core structural elements, among which the PSTAIRE motif in the critical αC-helix, is universally conserved in metazoans. The CDK2 kinase, sharing the PSTAIRE, arose early in metazoan evolution and permitted subdivision of tasks along the S-M-phase axis. The marine chordate, Oikopleura dioica, is the only metazoan known to possess more than a single CDK1 ortholog, and all of its 5 paralogs show sequence divergences in the PSTAIRE. Through assessing CDK1 gene duplications in the appendicularian lineage, we show that the CDK1 activation loop substrate binding platform, ATP entrance site, hinge region, and main Cyclin binding interface, have all diversified under positive selection. Three of the 5 CDK1 paralogs are required for embryonic divisions and knockdown phenotypes illustrate further subdivision of functions along the S-M-phase axis. In parallel to CDK1 gene duplications, there has also been amplification in the Cyclin B complement. Among these, the CDK1d:Cyclin Ba pairing is required for oogenic meiosis and early embryogenesis and shows evidence of coevolution of an exclusive interaction. In an intriguing twist on the general rule that Cyclin B oscillations on a background of stable CDK1 levels regulate M-phase MPF activity, it is CDK1d protein levels that oscillate, rather than Cyclin Ba levels, to drive rapid, early embryonic cell cycles. Strikingly, the modified PSTAIRE of odCDK1d shows convergence over great evolutionary distance with plant CDKB, and in both O. dioica, and plants, these variants exhibit increased specialization to M-phase.

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A Genetic Screen for Suppressors and Enhancers of the Drosophila PAN GU Cell Cycle Kinase Identifies Cyclin B as a Target

Genetics ◽

10.1093/genetics/158.4.1545 ◽

2001 ◽

Vol 158 (4) ◽

pp. 1545-1556 ◽

Cited By ~ 3

Author(s):

Laura A Lee ◽

Lisa K Elfring ◽

Giovanni Bosco ◽

Terry L Orr-Weaver

Keyword(s):

Cell Cycle ◽

Dna Replication ◽

Genetic Screen ◽

S Phase ◽

Cyclin B ◽

Cell Cycles ◽

Protein Levels ◽

Drosophila Embryogenesis ◽

Giant Nuclei ◽

Cell Cycle Kinase

Abstract The early cell cycles of Drosophila embryogenesis involve rapid oscillations between S phase and mitosis. These unique S-M cycles are driven by maternal stockpiles of components necessary for DNA replication and mitosis. Three genes, pan gu (png), plutonium (plu), and giant nuclei (gnu) are required to control the cell cycle specifically at the onset of Drosophila development by inhibiting DNA replication and promoting mitosis. PNG is a protein kinase that is in a complex with PLU. We employed a sensitized png mutant phenotype to screen for genes that when reduced in dosage would dominantly suppress or enhance png. We screened deficiencies covering over 50% of the autosomes and identified both enhancers and suppressors. Mutations in eIF-5A and PP1 87B dominantly suppress png. Cyclin B was shown to be a key PNG target. Mutations in cyclin B dominantly enhance png, whereas png is suppressed by cyclin B overexpression. Suppression occurs via restoration of Cyclin B protein levels that are decreased in png mutants. The plu and gnu phenotypes are also suppressed by cyclin B overexpression. These studies demonstrate that a crucial function of PNG in controlling the cell cycle is to permit the accumulation of adequate levels of Cyclin B protein.

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Advantages of Tyrosine Kinase Anti-Angiogenic Cediranib over Bevacizumab: Cell Cycle Abrogation and Synergy with Chemotherapy

Pharmaceuticals ◽

10.3390/ph14070682 ◽

2021 ◽

Vol 14 (7) ◽

pp. 682

Author(s):

Jianling Bi ◽

Garima Dixit ◽

Yuping Zhang ◽

Eric J. Devor ◽

Haley A. Losh ◽

...

Keyword(s):

Cell Cycle ◽

Endometrial Cancer ◽

Tyrosine Kinase ◽

Cell Viability ◽

Cell Lines ◽

Cell Cycle Progression ◽

Kinase Inhibitor ◽

Cell Cycle Checkpoint ◽

Mitotic Catastrophe ◽

M Cell

Angiogenesis plays a crucial role in tumor development and metastasis. Both bevacizumab and cediranib have demonstrated activity as single anti-angiogenic agents in endometrial cancer, though subsequent studies of bevacizumab combined with chemotherapy failed to improve outcomes compared to chemotherapy alone. Our objective was to compare the efficacy of cediranib and bevacizumab in endometrial cancer models. The cellular effects of bevacizumab and cediranib were examined in endometrial cancer cell lines using extracellular signal-related kinase (ERK) phosphorylation, ligand shedding, cell viability, and cell cycle progression as readouts. Cellular viability was also tested in eight patient-derived organoid models of endometrial cancer. Finally, we performed a phosphoproteomic array of 875 phosphoproteins to define the signaling changes related to bevacizumab versus cediranib. Cediranib but not bevacizumab blocked ligand-mediated ERK activation in endometrial cancer cells. In both cell lines and patient-derived organoids, neither bevacizumab nor cediranib alone had a notable effect on cell viability. Cediranib but not bevacizumab promoted marked cell death when combined with chemotherapy. Cell cycle analysis demonstrated an accumulation in mitosis after treatment with cediranib + chemotherapy, consistent with the abrogation of the G2/M checkpoint and subsequent mitotic catastrophe. Molecular analysis of key controllers of the G2/M cell cycle checkpoint confirmed its abrogation. Phosphoproteomic analysis revealed that bevacizumab and cediranib had both similar and unique effects on cell signaling that underlie their shared versus individual actions as anti-angiogenic agents. An anti-angiogenic tyrosine kinase inhibitor such as cediranib has the potential to be superior to bevacizumab in combination with chemotherapy.

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A Genetic Screen for Suppressors and Enhancers of the Drosophila Cdk1-Cyclin B Identifies Maternal Factors That Regulate Microtubule and Microfilament Stability

Genetics ◽

10.1093/genetics/162.3.1179 ◽

2002 ◽

Vol 162 (3) ◽

pp. 1179-1195 ◽

Cited By ~ 3

Author(s):

Jun-Yuan Ji ◽

Marjan Haghnia ◽

Cory Trusty ◽

Lawrence S B Goldstein ◽

Gerold Schubiger

Keyword(s):

Cell Cycle ◽

Cell Cycle Progression ◽

Genetic Screen ◽

Cyclin B ◽

Gene Products ◽

Nuclear Movement ◽

Cell Cycles ◽

Major Mechanism ◽

Cytoskeletal Dynamics ◽

Chromosome Bridges

Abstract Coordination between cell-cycle progression and cytoskeletal dynamics is important for faithful transmission of genetic information. In early Drosophila embryos, increasing maternal cyclin B leads to higher Cdk1-CycB activity, shorter microtubules, and slower nuclear movement during cycles 5-7 and delays in nuclear migration to the cortex at cycle 10. Later during cycle 14 interphase of six cycB embryos, we observed patches of mitotic nuclei, chromosome bridges, abnormal nuclear distribution, and small and large nuclei. These phenotypes indicate disrupted coordination between the cell-cycle machinery and cytoskeletal function. Using these sensitized phenotypes, we performed a dosage-sensitive genetic screen to identify maternal proteins involved in this process. We identified 10 suppressors classified into three groups: (1) gene products regulating Cdk1 activities, cdk1 and cyclin A; (2) gene products interacting with both microtubules and microfilaments, Actin-related protein 87C; and (3) gene products interacting with microfilaments, chickadee, diaphanous, Cdc42, quail, spaghetti-squash, zipper, and scrambled. Interestingly, most of the suppressors that rescue the astral microtubule phenotype also reduce Cdk1-CycB activities and are microfilament-related genes. This suggests that the major mechanism of suppression relies on the interactions among Cdk1-CycB, microtubule, and microfilament networks. Our results indicate that the balance among these different components is vital for normal early cell cycles and for embryonic development. Our observations also indicate that microtubules and cortical microfilaments antagonize each other during the preblastoderm stage.

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Effects of cell-cycle-dependent expression on random fluctuations in protein levels

Royal Society Open Science ◽

10.1098/rsos.160578 ◽

2016 ◽

Vol 3 (12) ◽

pp. 160578 ◽

Cited By ~ 20

Author(s):

Mohammad Soltani ◽

Abhyudai Singh

Keyword(s):

Cell Cycle ◽

Cell Division ◽

Protein Levels ◽

Stochastic Component ◽

Stochastic Expression ◽

A Cell ◽

Intrinsic Stochasticity ◽

Cycle Dependent ◽

Random Fluctuations ◽

Cell To Cell Variability

Expression of many genes varies as a cell transitions through different cell-cycle stages. How coupling between stochastic expression and cell cycle impacts cell-to-cell variability (noise) in the level of protein is not well understood. We analyse a model where a stable protein is synthesized in random bursts, and the frequency with which bursts occur varies within the cell cycle. Formulae quantifying the extent of fluctuations in the protein copy number are derived and decomposed into components arising from the cell cycle and stochastic processes. The latter stochastic component represents contributions from bursty expression and errors incurred during partitioning of molecules between daughter cells. These formulae reveal an interesting trade-off: cell-cycle dependencies that amplify the noise contribution from bursty expression also attenuate the contribution from partitioning errors. We investigate the existence of optimum strategies for coupling expression to the cell cycle that minimize the stochastic component. Intriguingly, results show that a zero production rate throughout the cell cycle, with expression only occurring just before cell division, minimizes noise from bursty expression for a fixed mean protein level. By contrast, the optimal strategy in the case of partitioning errors is to make the protein just after cell division. We provide examples of regulatory proteins that are expressed only towards the end of the cell cycle, and argue that such strategies enhance robustness of cell-cycle decisions to the intrinsic stochasticity of gene expression.

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Ser68 phosphoregulation is essential for CENP-A deposition, centromere function and viability in mice

10.1101/2021.11.23.469796 ◽

2021 ◽

Author(s):

Yuting Liu ◽

Kehui Wang ◽

Li Huang ◽

Jicheng Zhao ◽

Xinpeng Chen ◽

...

Keyword(s):

Cell Cycle ◽

Cell Viability ◽

Histone H3 ◽

Dependent Manner ◽

Centromere Function ◽

Histone H3 Variant ◽

A Cell ◽

Cycle Dependent ◽

Spatiotemporal Control

Centromere identity is defined by nucleosomes containing CENP-A, a histone H3 variant. The deposition of CENP-A at centromeres is tightly regulated in a cell-cycle-dependent manner. We previously reported that the spatiotemporal control of centromeric CENP-A incorporation is mediated by the phosphorylation of CENP-A Ser68. However, a recent report argued that Ser68 phosphoregulation is dispensable for accurate CENP-A loading. Here, we report that the substitution of Ser68 of endogenous CENP-A with either Gln68 or Glu68 severely impairs CENP-A deposition and cell viability. We also find that mice harboring the corresponding mutations are lethal. Together, these results indicate that the dynamic phosphorylation of Ser68 ensures cell-cycle-dependent CENP-A deposition and cell viability.

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Akt/Protein Kinase B-Dependent Phosphorylation and Inactivation of WEE1Hu Promote Cell Cycle Progression at G2/M Transition

Molecular and Cellular Biology ◽

10.1128/mcb.25.13.5725-5737.2005 ◽

2005 ◽

Vol 25 (13) ◽

pp. 5725-5737 ◽

Cited By ~ 97

Author(s):

Kazuhiro Katayama ◽

Naoya Fujita ◽

Takashi Tsuruo

Keyword(s):

Cell Cycle ◽

Cell Cycle Progression ◽

Kinase Inhibitor ◽

Phosphorylation Site ◽

Cytoplasmic Localization ◽

M Cell ◽

Serine Threonine Kinase ◽

Cycle Progression ◽

M Phase ◽

Promote Cell Cycle Progression

ABSTRACT The serine/threonine kinase Akt is known to promote cell growth by regulating the cell cycle in G1 phase through activation of cyclin/Cdk kinases and inactivation of Cdk inhibitors. However, how the G2/M phase is regulated by Akt remains unclear. Here, we show that Akt counteracts the function of WEE1Hu. Inactivation of Akt by chemotherapeutic drugs or the phosphatidylinositide-3-OH kinase inhibitor LY294002 induced G2/M arrest together with the inhibitory phosphorylation of Cdc2. Because the increased Cdc2 phosphorylation was completely suppressed by wee1hu gene silencing, WEE1Hu was associated with G2/M arrest induced by Akt inactivation. Further analyses revealed that Akt directly bound to and phosphorylated WEE1Hu during the S to G2 phase. Serine-642 was identified as an Akt-dependent phosphorylation site. WEE1Hu kinase activity was not affected by serine-642 phosphorylation. We revealed that serine-642 phosphorylation promoted cytoplasmic localization of WEE1Hu. The nuclear-to-cytoplasmic translocation was mediated by phosphorylation-dependent WEE1Hu binding to 14-3-3θ but not 14-3-3β or -σ. These results indicate that Akt promotes G2/M cell cycle progression by inducing phosphorylation-dependent 14-3-3θ binding and cytoplasmic localization of WEE1Hu.

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Aurora kinases A and B are up-regulated by Myc and are essential for maintenance of the malignant state

Blood ◽

10.1182/blood-2009-11-251074 ◽

2010 ◽

Vol 116 (9) ◽

pp. 1498-1505 ◽

Cited By ~ 140

Author(s):

Jürgen den Hollander ◽

Sara Rimpi ◽

Joanne R. Doherty ◽

Martina Rudelius ◽

Andreas Buck ◽

...

Keyword(s):

Cell Cycle ◽

Kinase Activity ◽

Kinase Inhibitor ◽

Aurora Kinase ◽

Cell Cycle Regulators ◽

Aurora Kinase Inhibitor ◽

Aurora Kinases ◽

Protein Levels ◽

Malignant State ◽

E Boxes

Myc oncoproteins promote continuous cell growth, in part by controlling the transcription of key cell cycle regulators. Here, we report that c-Myc regulates the expression of Aurora A and B kinases (Aurka and Aurkb), and that Aurka and Aurkb transcripts and protein levels are highly elevated in Myc-driven B-cell lymphomas in both mice and humans. The induction of Aurka by Myc is transcriptional and is directly mediated via E-boxes, whereas Aurkb is regulated indirectly. Blocking Aurka/b kinase activity with a selective Aurora kinase inhibitor triggers transient mitotic arrest, polyploidization, and apoptosis of Myc-induced lymphomas. These phenotypes are selectively bypassed by a kinase inhibitor-resistant Aurkb mutant, demonstrating that Aurkb is the primary therapeutic target in the context of Myc. Importantly, apoptosis provoked by Aurk inhibition was p53 independent, suggesting that Aurka/Aurkb inhibitors will show efficacy in treating primary or relapsed malignancies having Myc involvement and/or loss of p53 function.

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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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