scholarly journals M2 Muscarinic Receptor Activation Impairs Mitotic Progression and Bipolar Mitotic Spindle Formation in Human Glioblastoma Cell Lines

Cells ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1727
Author(s):  
Maria Di Bari ◽  
Vanessa Tombolillo ◽  
Francesco Alessandrini ◽  
Claudia Guerriero ◽  
Mario Fiore ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Mitotic Spindle ◽  
Acetylcholine Receptors ◽  
Time Lapse ◽  
Receptor Activation ◽  
Mitotic Progression ◽  
Spindle Formation ◽  
Acetylated Tubulin ◽  
Abnormal Mitosis

Background: Glioblastoma multiforme (GBM) is characterized by several genetic abnormalities, leading to cell cycle deregulation and abnormal mitosis caused by a defective checkpoint. We previously demonstrated that arecaidine propargyl ester (APE), an orthosteric agonist of M2 muscarinic acetylcholine receptors (mAChRs), arrests the cell cycle of glioblastoma (GB) cells, reducing their survival. The aim of this work was to better characterize the molecular mechanisms responsible for this cell cycle arrest. Methods: The arrest of cell proliferation was evaluated by flow cytometry analysis. Using immunocytochemistry and time-lapse analysis, the percentage of abnormal mitosis and aberrant mitotic spindles were assessed in both cell lines. Western blot analysis was used to evaluate the modulation of Sirtuin2 and acetylated tubulin—factors involved in the control of cell cycle progression. Results: APE treatment caused arrest in the M phase, as indicated by the increase in p-HH3 (ser10)-positive cells. By immunocytochemistry, we found a significant increase in abnormal mitoses and multipolar mitotic spindle formation after APE treatment. Time-lapse analysis confirmed that the APE-treated GB cells were unable to correctly complete the mitosis. The modulated expression of SIRT2 and acetylated tubulin in APE-treated cells provides new insights into the mechanisms of altered mitotic progression in both GB cell lines. Conclusions: Our data show that the M2 agonist increases aberrant mitosis in GB cell lines. These results strengthen the idea of considering M2 acetylcholine receptors a novel promising therapeutic target for the glioblastoma treatment.

Centrosome Aberrations, Disturbed Mitotic Spindle Formation and G1 Arrest in Normal and Leukemic Cells Treated with the SRC/ABL Inhibitor Dasatinib.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2167-2167
Author(s):  
Alice Fabarius ◽  
Michelle Giehl ◽  
Alwin Kraemer ◽  
Oliver Frank ◽  
Martin C. Mueller ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Mitotic Spindle ◽  
Cell Cycle Progression ◽  
Dermal Fibroblasts ◽  
G1 Arrest ◽  
Osteosarcoma Cell ◽  
Cycle Progression ◽  
Spindle Formation

Abstract Multitargeted ABL inhibitors have been developed to simultaneously inhibit various pathways associated with proliferation in BCR-ABL+ diseases. Dasatinib (Bristol Myers Squibb) is a potent inhibitor targeting ABL, SRC, and other tyrosine kinases. SRC kinases are required for progression through the initial phase of mitosis. Centrosomes play a fundamental role in mitotic spindle organization, chromosome segregation and genetic stability. We sought to evaluate the activity of dasatinib on proliferation, centrosome status, spindle formation, and cell cycle progression in vitro and in vivo. Normal human dermal fibroblasts (NHDF), Chinese hamster embryonal fibroblasts (CHE), and the human osteosarcoma cell line U2OS were treated with serial concentrations (1nM-10μM) of dasatinib for 3 weeks. Effects of dasatinib were compared with data achieved with the ABL inhibitors imatinib (Novartis, 5–20μM) and nilotinib (Novartis, 0.5–20μM), the specific SRC inhibitor PP2 (Calbiochem-Novabiochem, 0.1–2μM), the ABL/LYN inhibitor INNO-406 (Innovive, 0.1–2μM), and solvent control. Bone marrow and peripheral blood samples from 18 patients (pts, 10 m, 8 f; median age 57 yrs, range 26–75) treated with dasatinib (70mg bid) after imatinib failure for a median of 11 mo (range, 3–16) were investigated. 17 pts had chronic myeloid leukemia (CML) in chronic phase. One patient suffered from a gastrointestinal stromal tumor. For comparison, 3 untreated CML pts and 3 healthy individuals were evaluated. Cell proliferation was determined in liquid culture incubated with serial dilutions of the inhibitor. Centrosome morphology and spindle formation were evaluated after pericentrin and α-tubulin staining, respectively. Cell cycle progression was analyzed by FACS and expression of EG5 by immunofluorescence microscopy. Dasatinib induced a G1 cell cycle arrest in all cell lines tested and in pts associated with a shift to 1n DNA ploidy and absence of EG5 as a marker for G2 phase/mitosis. In vitro, centrosomal aberrations and delay of spindle formation were observed in a dose dependent fashion. In pts, centrosome alterations were found in a median of 17% (range, 10–15) of cells. Disturbed spindle formation was observed in 9/18 pts. In comparison, incubation with imatinib and nilotinib was associated with centrosome aberrations but not with defects of spindle formation and G1 arrest. PP2 induced S-phase arrest; centrosome aberrations were observed at higher dosages (1–2 μM) only, spindle formation was not affected. INNO-406 was associated with both centrosome aberrations and disturbed spindle formation. In pharmacological doses, proliferation of BCR-ABL neg. cell lines was inhibited after dasatinib treatment, but not after incubation with imatinib, nilotinib, PP2, or INNO-406. In conclusion, dasatinib blocks the G1/S transition and thereby inhibits cell growth in normal and neoplastic cells. In addition, it induces both centrosomal and spindle aberrations. Effects of dasatinib are not based on SRC inhibition alone but may be associated with the combination of SRC and ABL inhibition or with non-specific effects on multiple kinases. Therefore, dasatinib should be defined as a cytostatic drug with a strong targeted component resulting in a preferential inhibition of cells harboring a specific target, like BCR-ABL.

Downregulation of Bub1 and a Defective Spindle Checkpoint in Acute Myeloid Leukemia.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2846-2846
Author(s):  
Ralph Waesch ◽  
Dominik Schnerch
Keyword(s):  
Cell Cycle ◽  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Mitotic Spindle ◽  
Myeloid Leukemia ◽  
Cyclin B1 ◽  
Mitotic Progression ◽  
Metaphase Arrest ◽  
Altered Expression ◽  
Acute Myeloid

Abstract Chromosomal aberrations are common in leukemia. The spindle assembly checkpoint (SAC) is a surveillance mechanism responsible to ensure proper chromosome segregation. Unattached chromosomes to the mitotic spindle or lack of spindle tension are sensed by the SAC leading to metaphase arrest. The activated SAC inhibits the ordered proteolysis of regulatory proteins and thereby prevents mitotic progression providing time for the repair of spindle defects. Ubiquitin-dependent proteolysis of cell cycle regulators is a central mechanism of cell cycle control. The anaphase-promoting complex (APC) is an essential ubiquitin-ligase responsible for mitotic progression. The activated SAC inhibits the APC preventing degradation of cyclin B1 and the anaphase inhibitor securin and thereby delaying chromosome separation. We found this mitotic delay mechanism to be defective in acute myeloid leukemia (AML). Leukemia cells that were exposed to the microtubule disrupting agent nocodazole were not capable of preventing anaphase onset. This is indicated by a low mitotic index and degradation of the APC targets securin and cyclin B1 in nocodazole treated Kasumi-1 cells compared to the SAC competent burkitt lymphoma cell line DG-75. Thus, unattached chromosomes were unable to induce prolonged metaphase arrest in these cells. They undergo apoptosis instead due to the complete absence of the mitotic spindle. Similar results with defective mitotic arrest and enhanced apoptosis were obtained for several other leukemia cell lines. In vivo SAC malfunction in AML can favor the generation of aneuploidy, consistent with the numerical aberrations observed in these cells. In order to identify the underlying defect we screened for mutations or altered expression of checkpoint proteins and identified the SAC component Bub1 to be posttranscriptionally downregulated in all tested AML cell lines. Our studies suggest that impaired SAC function is an important mechanism to create genetic instability and thus may contribute to rise and progression of AML.

scholarly journals The Nek6 and Nek7 Protein Kinases Are Required for Robust Mitotic Spindle Formation and Cytokinesis

2009 ◽  
Vol 29 (14) ◽  
pp. 3975-3990 ◽  
Author(s):  
Laura O'Regan ◽  
Andrew M. Fry
Keyword(s):  
Mitotic Spindle ◽  
Spindle Assembly Checkpoint ◽  
Mitotic Spindles ◽  
Mitotic Progression ◽  
Serine Threonine Kinase ◽  
Spindle Formation ◽  
Spindle Poles ◽  
And Function ◽  
Interfering Rna ◽  
Reduced Activity

ABSTRACT Nek6 and Nek7 are members of the NIMA-related serine/threonine kinase family. Previous work showed that they contribute to mitotic progression downstream of another NIMA-related kinase, Nek9, although the roles of these different kinases remain to be defined. Here, we carried out a comprehensive analysis of the regulation and function of Nek6 and Nek7 in human cells. By generating specific antibodies, we show that both Nek6 and Nek7 are activated in mitosis and that interfering with their activity by either depletion or expression of reduced-activity mutants leads to mitotic arrest and apoptosis. Interestingly, while completely inactive mutants and small interfering RNA-mediated depletion delay cells at metaphase with fragile mitotic spindles, hypomorphic mutants or RNA interference treatment combined with a spindle assembly checkpoint inhibitor delays cells at cytokinesis. Importantly, depletion of either Nek6 or Nek7 leads to defective mitotic progression, indicating that although highly similar, they are not redundant. Indeed, while both kinases localize to spindle poles, only Nek6 obviously localizes to spindle microtubules in metaphase and anaphase and to the midbody during cytokinesis. Together, these data lead us to propose that Nek6 and Nek7 play independent roles not only in robust mitotic spindle formation but also potentially in cytokinesis.

scholarly journals Modeling reveals cortical dynein-dependent oscillations in bipolar spindle length

2020 ◽  
Author(s):  
Dayna Mercadante ◽  
Amity Manning ◽  
Sarah Olson
Keyword(s):  
Experimental Data ◽  
Mitotic Spindle ◽  
Force Generation ◽  
Mitotic Progression ◽  
Computational Framework ◽  
Spindle Formation ◽  
Bipolar Spindle ◽  
Spindle Dynamics ◽  
Force Components ◽  
Over Time

AbstractProper formation and maintenance of the mitotic spindle is required for faithful cell division. While much work has been done to understand the roles of the key force components of the mitotic spindle, identifying the consequences of force perturbations in the spindle remains a challenge. We develop a computational framework accounting for the minimal force requirements of mitotic progression. To reflect early spindle formation, we account for microtubule dynamics and interactions with major force-generating motors, excluding chromosome interactions that dominate later in mitosis. We directly integrate our experimental data to define and validate the model, and then use simulations to analyze individual force components over time and their relationship to spindle dynamics, making it distinct from previously published models. Rather than achieving and maintaining a constant bipolar spindle length, oscillations in pole to pole distance occur that coincide with microtubule binding and force generation by cortical dynein. In the context of high kinesin-14 (HSET) activity, we identify the requirement of high cortical dynein activity for bipolar spindle formation.

scholarly journals MYC Dysregulates Mitotic Spindle Function Creating a Dependency on TPX2

2018 ◽  
Author(s):  
Julia Rohrberg ◽  
Alexandra Corella ◽  
Moufida Taileb ◽  
Seda Kilinc ◽  
Marie-Lena Jokisch ◽  
...  
Keyword(s):  
Cell Lines ◽  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Cellular Proliferation ◽  
Spindle Assembly ◽  
Tumor Evolution ◽  
Mouse Tumor ◽  
Breast Cancers ◽  
Mitotic Progression ◽  
Myc Oncogene

AbstractThe MYC oncogene promotes tumorigenesis in part by facilitating cell cycle entry thus driving cellular proliferation. Tumors that overexpress MYC frequently demonstrate aneuploidy, numerical chromosome alterations associated with highly aggressive cancers, rapid tumor evolution, and poor patient outcome. While the role of MYC in overcoming the G1/S checkpoint is well established, it remains poorly understood whether MYC induces chromosomal instability (CIN). Here, we identify a direct influence of MYC on mitotic progression. MYC overexpression induces defects in microtubule nucleation and spindle assembly promoting chromosome segregation defects, micronuclei and CIN. We examined which mitotic regulators are required for the survival of MYC-overexpressing cells and found a reliance on high TPX2 expression. TPX2, a master microtubule regulator, is overexpressed together with MYC in multiple cell lines, in mouse tumor models and in aggressive human breast cancers. High TPX2 expression is permissive for mitotic spindle assembly and chromosome segregation in cells with deregulated MYC, whereas TPX2 depletion blocks mitotic progression, induces cell death and prevents tumor growth. Importantly, attenuation of MYC expression reverses the mitotic defects observed, even in established tumor cell lines, implicating an ongoing role for high MYC in the persistence of a CIN phenotype in tumors. Here, we implicate the MYC oncogene as a regulator of spindle assembly and dynamics and identify a new MYC-TPX2 synthetic-lethal interaction that could represent a future therapeutic strategy in MYC-overexpressing cancers. Our studies suggest that blocking MYC activity can attenuate the emergence of CIN and tumor evolution.

A Novel Aurora-a Kinase Inhibitor MLN8237 Induces Cytotoxicity and Cell Cycle Arrest in Experimental Multiple Myeloma Models.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1719-1719 ◽  
Author(s):  
Gullu Gorgun ◽  
Elisabetta Calabrese ◽  
Mala Mani ◽  
Teru Hideshima ◽  
Hiroshi Ikeda ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Cell Lines ◽  
Mitotic Spindle ◽  
Kinase Inhibitor ◽  
Thymidine Incorporation ◽  
Significant Inhibition ◽  
Aurora A Kinase ◽  
Aurora A ◽  
Resistant Line

Abstract Multiple myeloma (MM) is an incurable bone marrow derived plasma cell malignancy. Despite significant improvements in treating patients suffering from this disease, MM remains uniformly fatal owing to intrinsic or acquired drug resistance. Thus, additional modalities for treating MM are required. In this study, we examined the anti-tumor activity of MLN8237, a small molecule Aurora-A kinase inhibitor, in experimental models of MM. Aurora-A is a mitotic kinase that localizes to centrosomes and the proximal mitotic spindle and functions in mitotic spindle formation and in regulating chromatid congression and segregation. Aurora-A gene amplification and protein overexpression is a common event in many cancers, and has been experimentally linked to genetic instability and tumorigenesis. In MM, increased Aurora-A gene expression has previously been correlated with centrosome amplification and a worsened disease prognosis. Thus, inhibition of Aurora A in MM may prove to be therapeutically beneficial. Here we show that Aurora-A protein is highly expressed in eight distinct MM cell lines. The affect of Aurora-A inhibition in these cell lines was examined in cytotoxicity (MTT viability) and proliferation (3[H]thymidine incorporation) assays by treating with MLN8237 (0.25 mM −32 mM) for 24, 48 and 72h. Although there was no significant inhibition of cell viability and proliferation at 24h, a marked effect occurred 48 and 72h after compound addition at concentrations as low as 0.25 mM. Interestingly, the melphalan resistant line (LR5) and Doxorubucin resistant line (Dox40) were among the least sensitive to MLN8237 induced cell cytotoxicity. The affect of MLN8237 on peripheral blood mononuclear cells (PBMCs) from healthy donors was also examined at the same concentrations and exposure time used for the MM cell lines. In healthy PBMCs, MLN8237 did not induce cytotoxicity as measured by the MTT assay, but there was a significant inhibition of proliferation at 48 and 72h as measured by the 3[H]thymidine incorporation assay at concentrations above 4uM. To delineate the mechanisms of cytotoxicity and growth inhibitory activity of MLN8237, apoptotic markers and cell cycle profiles were examined in the MM cell lines. Fluorescence conjugated-Annexin V and propidium iodide (PI) co-staining of MM cell lines after culturing in the presence or absence of MLN8237 at 1 mM (IC50) for 24, 48 and 72h demonstrated that MLN8237 induces apoptosis in these lines. This finding was corroborated by demonstrating increased capase-9 expression by Western blot analysis. Cell cycle analysis by flow cytometry demonstrated that MLN8237 results in an accumulation of tetraploid cells, presumably by abrogating G2/M progression. These results suggest that MLN8237 represents a possible novel agent for treating MM patients. Additional studies are ongoing to assess the anti-tumor effects of MLN8237 alone and in combination with other therapeutic agents in xenograft models of MM.

A NOVEL Aurora A Kinase INHIBITOR MLN8237 Induces Cytotoxicity and CELL Cycle Arrest IN MULTIPLE MYELOMA.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3830-3830
Author(s):  
Gullu Gorgun ◽  
Elisabetta Calabrese ◽  
Teru Hideshima ◽  
Jeffrey Ecsedy ◽  
Giada Bianchi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Board Of Directors ◽  
Cell Lines ◽  
Mitotic Spindle ◽  
Research Funding ◽  
Thymidine Incorporation ◽  
Aurora A Kinase ◽  
Advisory Committees

Abstract Abstract 3830 Poster Board III-766 Multiple myeloma (MM) is an incurable bone marrow derived plasma cell malignancy. Despite significant improvements in treating patients suffering from this disease, MM remains uniformly fatal due to intrinsic or acquired drug resistance. Thus, additional modalities for treating MM are required. Targeting cell cycle progression proteins provides such a novel treatment strategy. Here we assess the in vivo and in vitro anti-MM activity of MLN8237, a small molecule Aurora A kinase (AURKA) inhibitor. AURKA is a mitotic kinase that localizes to centrosomes and the proximal mitotic spindle, where it functions in mitotic spindle formation and in regulating chromatid congression and segregation. In MM, increased AURKA gene expression has been correlated with centrosome amplification and a worse prognosis; thus, inhibition of AURKA in MM may prove to be therapeutically beneficial. Here we show that AURKA protein is highly expressed in eight MM cell lines and primary patient MM cells. The affect of AURKA inhibition was examined using cytotoxicity (MTT viability) and proliferation (3[H]thymidine incorporation) assays after treatment of these cell lines and primary cells with MLN8237 (0.0001 μM – 4 μM) for 24, 48 and 72h Although there was no significant inhibition of cell viability and proliferation at 24h, a marked effect on both viability and proliferation occurred after 48 and 72h treatment at concentrations as low as 0.01 μM. Moreover, MLN8237 inhibits cell growth and proliferation of primary MM cells and cell lines even in the presence of bone marrow stromal cells (BMSCs) or cytokines IL-6 and IGF1. Similar experiments revealed that MLN8237 did not induce cytotoxicity in normal peripheral blood mononuclear cells (PBMCs) as measured by MTT assay, but did inhibit proliferation at 48 and 72h, as measured by the 3[H]thymidine incorporation assay. To delineate the mechanisms of cytotoxicity and growth inhibitory activity of MLN8237, apoptotic markers and cell cycle profiles were examined in both MM cell lines and primary MM cells. Annexin V and propidium iodide staining of MM cell lines cultured in the presence or absence of MLN8237 (1 μM) for 24, 48 and 72h demonstrated apoptosis, which was further confirmed by increased cleavage of PARP, capase-9, and caspase-3 by immunoblotting. In addition, MLN8237 upregulated p53-phospho (Ser 15) and tumor suppressor genes p21 and p27. Cell cycle analysis demonstrated that MLN8237 treatment induces an accumulation of tetraploid cells by abrogating G2/M progression. We next determined whether combining MLN8237 with conventional (melphalan, doxorubucin, dexamethasone) and other novel (VELCADE®) therapeutic agents elicited synergistic/additive anti-MM activity by isobologram analysis using CalcuSyn software. Combining MLN8237 with melphalan, dexamethasone, or VELCADE® induces synergistic/additive anti-MM activity against MM cell lines in vitro (p≤0.05, CI<1). To confirm in vivo anti-MM effects of MLN8237, MM.1S cells were injected s.c. into g-irradiated CB-17 SCID mice (n=40, 10 mice EA group). When tumors were measurable (>100 mm3), mice were treated with daily oral doses of vehicle alone or 7.5mg/kg, 15mg/kg, 30mg/kg MLN8237 for 21 days. Overall survival (defined as time between initiation of treatment and sacrifice or death) was compared in vehicle versus- MLN8237- treated mice by Kaplan-Meier method. Tumor burden was significantly reduced (p=0.02) and overall survival was significantly increased (p=0.02, log-rank test) in animals treated with 30mg/kg MLN8237. In vivo anti-MM effects of MLN8237 were further validated by performing TUNEL apoptosis-cell death assay in tumor tissues excised from control or treated animals. Importantly, a significant dose-related increase in apoptotic cells was observed in tumors from animals that received MLN8237 versus controls. These results suggest that MLN8237 represents a promising novel targeted therapy in MM. Disclosures: Ecsedy: Millennium Pharmaceutical: Employment. Munshi:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Richardson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees. Anderson:Millennium: Research Funding; Novartis: Research Funding; Celgene: Research Funding.

scholarly journals Mutations in alpha- and beta-tubulin affect spindle formation in Chinese hamster ovary cells.

1983 ◽  
Vol 97 (4) ◽  
pp. 1055-1061 ◽  
Author(s):  
I Abraham ◽  
M Marcus ◽  
F Cabral ◽  
M M Gottesman
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Nonpermissive Temperature ◽  
Spindle Formation ◽  
Interphase Cell ◽  
Alpha Tubulin ◽  
Mutant Cells

Two Chinese hamster ovary cell lines with mutated beta-tubulins (Grs-2 and Cmd-4) and one that has a mutation in alpha-tubulin (Tax-1) are temperature sensitive for growth at 40.5 degrees C. To determine the functional defect in these mutant cells at the nonpermissive temperature, they were characterized with respect to cell cycle parameters and microtubule organization and function after relatively short periods at 40.5 degrees C. At the nonpermissive temperature all the mutants had normal appearing cytoplasmic microtubules. Premature chromosome condensation analysis failed to show any discrete step in the interphase cell cycle in which these mutants are arrested. These cells, however, show several defects at the nonpermissive temperature that appear related to the function of microtubules during mitosis. Time-lapse studies showed that mitosis was lengthened in the three mutant lines at 40.5 degrees C as compared with the wild-type cells at this temperature, resulting in a higher proportion of cells in mitosis after temperature shift. There was also a large increase in multinucleated cells in mutant populations after incubation at the nonpermissive temperature. Immunofluorescent studies using a monoclonal anti--alpha-tubulin antibody showed that the mutant cells had a high proportion of abnormal spindles at the nonpermissive temperature. The two altered beta-tubulins and the altered alpha-tubulin all were found to cause a similar phenotype at the high temperature that results in mitotic delay, defective cytokinesis, multinucleation, and ultimately, cell death. We conclude that spindle formation is the limiting microtubule function in these mutant cell lines at the nonpermissive temperature and that these cell lines will be of value for the study of the precise role of tubulin in mammalian spindle formation.

scholarly journals Subcellular localization and mitotic interactome analyses identify SIRT4 as a centrosomally localized and microtubule associated protein

2020 ◽  
Author(s):  
Laura Bergmann ◽  
Alexander Lang ◽  
Christoph Bross ◽  
Simone Altinoluk-Hambüchen ◽  
Iris Fey ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Tumor Suppressor ◽  
Mitotic Spindle ◽  
Cell Cycle Progression ◽  
Mitotic Progression ◽  
Cycle Progression ◽  
Functional Roles ◽  
The Family ◽  
Spindle Apparatus ◽  
Regulation Of Cell Cycle

AbstractThe stress-inducible and senescence-associated tumor suppressor SIRT4, a member of the family of mitochondrial sirtuins (SIRT3, SIRT4, and SIRT5), regulates bioenergetics and metabolism via NAD+-dependent enzymatic activities. Next to the known mitochondrial location, we found that a fraction of endogenous or ectopically expressed SIRT4, but not SIRT3, is located at the mitotic spindle apparatus in the cytosol. Confocal spinning disk microscopy revealed that SIRT4 localizes during the cell cycle dynamically at centrosomes with an intensity peak in G2 and early mitosis. Moreover, SIRT4 binds to microtubules and interacts with structural (α,β-tubulin, γ-tubulin, TUBGCP2, TUBGCP3) and regulatory (HDAC6) microtubule components as detected by co-immunoprecipitation and mass spectrometric analyses of the mitotic SIRT4 interactome. Overexpression of SIRT4 resulted in a pronounced decrease of acetylated α-tubulin (K40) associated with altered microtubule dynamics in mitotic cells. SIRT4 or the N-terminally truncated variant SIRT4(ΔN28), which is unable to translocate into mitochondria, delayed mitotic progression and reduced cell proliferation. This study extends the functional roles of SIRT4 beyond mitochondrial metabolism, and suggests that SIRT4 acts as a novel centrosomal / microtubule-associated protein in the regulation of cell cycle progression. Thus, stress-induced SIRT4 may exert its role as tumor suppressor through mitochondrial as well as extramitochondrial functions, the latter associated with its localization at the mitotic spindle apparatus.

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