scholarly journals Subcellular localization of EEN/endophilin A2, a fusion partner gene in leukaemia

2004 ◽  
Vol 383 (1) ◽  
pp. 27-35 ◽  
Author(s):  
Ngai CHEUNG ◽  
Chi Wai SO ◽  
Judy W. P. YAM ◽  
C. K. C. SO ◽  
Randy Y. C. POON ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Subcellular Localization ◽  
Fusion Partner ◽  
Nucleocytoplasmic Shuttling ◽  
Cellular Functions ◽  
Protein Levels ◽  
Mixed Lineage Leukaemia ◽  
Partner Gene ◽  
M Phase ◽  
Endocytic Vesicles

EEN (extra eleven nineteen), also known as EA2 (endophilin A2), a fusion partner of the MLL (mixed-lineage leukaemia) gene in human acute leukaemia, is a member of the endophilin A family, involved in the formation of endocytic vesicles. We present evidence to show that EEN/EA2 is localized predominantly in nuclei of various cell lines of haemopoietic, fibroblast and epithelial origin, in contrast with its reported cytoplasmic localization in neurons and osteoclasts, and that EEN/EA2 exhibits nucleocytoplasmic shuttling. During the cell cycle, EEN/EA2 shows dynamic localization: it is perichromosomal in prometaphase, co-localizes with the bipolar spindle in metaphase and anaphase and redistributes to the midzone and midbody in telophase. This pattern of distribution coincides with changes in protein levels of EEN/EA2, with the highest levels being observed in G2/M-phase. Our results suggest that distinct subcellular localization of the endophilin A family members probably underpins their diverse cellular functions and indicates a role for EEN/EA2 in the cell cycle.

scholarly journals A Novel Mammalian, Mitotic Spindle–associated Kinase Is Related to Yeast and Fly Chromosome Segregation Regulators

1997 ◽  
Vol 138 (3) ◽  
pp. 643-656 ◽  
Author(s):  
Ganesan Gopalan ◽  
Clarence S.M. Chan ◽  
Peter J. Donovan
Keyword(s):  
Cell Cycle ◽  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Mammalian Cells ◽  
Proximal Part ◽  
Yeast Cells ◽  
Mutant Form ◽  
Wild Type ◽  
Protein Levels ◽  
M Phase

We describe a novel mammalian protein kinase related to two newly identified yeast and fly kinases—Ipl1 and aurora, respectively—mutations in which cause disruption of chromosome segregation. We have designated this kinase as Ipl1- and aurora-related kinase 1 (IAK1). IAK1 expression in mouse fibroblasts is tightly regulated temporally and spatially during the cell cycle. Transcripts first appear at G1/S boundary, are elevated at M-phase, and disappear rapidly after completion of mitosis. The protein levels and kinase activity of IAK1 are also cell cycle regulated with a peak at M-phase. IAK1 protein has a distinct subcellular and temporal pattern of localization. It is first identified on the centrosomes immediately after the duplicated centrosomes have separated. The protein remains on the centrosome and the centrosome-proximal part of the spindle throughout mitosis and is detected weakly on midbody microtubules at telophase and cytokinesis. In cells recovering from nocodazole treatment and in taxol-treated mitotic cells, IAK1 is associated with microtubule organizing centers. A wild-type and a mutant form of IAK1 cause mitotic spindle defects and lethality in ipl1 mutant yeast cells but not in wild-type cells, suggesting that IAK1 interferes with Ipl1p function in yeast. Taken together, these data strongly suggest that IAK1 may have an important role in centrosome and/ or spindle function during chromosome segregation in mammalian cells. We suggest that IAK1 is a new member of an emerging subfamily of the serine/threonine kinase superfamily. The members of this subfamily may be important regulators of chromosome segregation.

scholarly journals The Cak1p Protein Kinase Is Required at G1/S and G2/M in the Budding Yeast Cell Cycle

Genetics ◽  
1997 ◽  
Vol 147 (1) ◽  
pp. 57-71 ◽  
Author(s):  
Ann Sutton ◽  
Richard Freiman
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Cell Cycle Progression ◽  
Budding Yeast ◽  
Protein Kinase Activity ◽  
Synthetic Lethal ◽  
Protein Levels ◽  
M Phase

Abstract The CAK1 gene encodes the major CDK-activating kinase (CAK) in budding yeast and is required for activation of Cdc28p for cell cycle progression from G2 to M phase. Here we describe the isolation of a mutant allele of CAK1 in a synthetic lethal screen with the Sit4 protein phosphatase. Analysis of several different cak1 mutants shows that although the G2 to M transition appears most sensitive to loss of Cak1p function, Cak1p is also required for activation of Cdc28p for progression from G1 into S phase. Further characterization of these mutants suggests that, unlike the CAK identified from higher eukaryotes, Cak1p of budding yeast may not play a role in general transcription. Finally, although Cak1 protein levels and in vitro protein kinase activity do not fluctuate during the cell cycle, at least a fraction of Cak1p associates with higher molecular weight proteins, which may be important for its in vivo function.

scholarly journals Regulator of Cell Cycle (RGCC) Expression during the Progression of Alzheimer's Disease

2017 ◽  
Vol 26 (4) ◽  
pp. 693-702 ◽  
Author(s):  
Scott E. Counts ◽  
Elliott J. Mufson
Keyword(s):  
Alzheimer’S Disease ◽  
Cell Cycle ◽  
Alzheimer's Disease ◽  
Cognitive Impairment ◽  
Neuronal Cell ◽  
Cyclin B1 ◽  
Multifunctional Protein ◽  
Protein Levels ◽  
Cell Cycle Reentry ◽  
M Phase

Unscheduled cell cycle reentry of postmitotic neurons has been described in cases of mild cognitive impairment (MCI) and Alzheimer's disease (AD) and may form a basis for selective neuronal vulnerability during disease progression. In this regard, the multifunctional protein regulator of cell cycle (RGCC) has been implicated in driving G1/S and G2/M phase transitions through its interactions with cdc/cyclin-dependent kinase 1 (cdk1) and is induced by p53, which mediates apoptosis in neurons. We tested whether RGCC levels were dysregulated in frontal cortex samples obtained postmortem from subjects who died with a clinical diagnosis of no cognitive impairment (NCI), MCI, or AD. RGCC mRNA and protein levels were upregulated by ~50%-60% in MCI and AD compared to NCI, and RGCC protein levels were associated with poorer antemortem global cognitive performance in the subjects examined. To test whether RGCC might regulate neuronal cell cycle reentry and apoptosis, we differentiated neuronotypic PC12 cultures with nerve growth factor (NGF) followed by NGF withdrawal to induce abortive cell cycle activation and cell death. Experimental reduction of RGCC levels increased cell survival and reduced levels of the cdk1 target cyclin B1. RGCC may be a candidate cell cycle target for neuroprotection during the onset of AD.

Induction of Apoptosis in K562 Human Chronic Myeloid Leukemia Cells by Tetra-Arsenic Tetra-Sulfide.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4375-4375
Author(s):  
Qidong Ye ◽  
Long-Jun Gu ◽  
Yanxia Zhao ◽  
Jincai Zhao ◽  
Wengao Chen ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Morphological Changes ◽  
K562 Cells ◽  
Morphologic Change ◽  
Mrna Levels ◽  
Protein Levels ◽  
Reverse Transcription Pcr ◽  
M Phase

Abstract Realgar has been used as a traditional medicine in China for more than 1500 years. Some studies found that tetra-arsenic tetra-sulfide (As4S4), the main ingredient of realgar, used alone was highly effective and safe for all stages of acute promyelocytic leukemia. To explore the effects of As4S4 in treatment of human chronic myeloid leukemia K562 cells, we used microculture MTS assay to measure the growth inhibition of K562 cells. The morphologic change was determined by Wright’s staining and Hoechst33342 assay. Cell apoptosis was evaluated by DNA agarose gel electrophoresis. The apoptotic rate and cell cycle were measured by flow cytometry. The changes of transcript and protein levels were determined by real-time reverse transcription-PCR and Western blot analysis, respectively. As4S4 had signigicant cytotoxicity on K-562 cells. At the concentration of 2.0μmol/L, the cell viability decreased significantly after 24 hours cultured with the reagent. When the concentration was lower than 0.5μmol/L, As4S4 had little effect on K562 cells. The effect of As4S4 on K562 was time and concentration dependent. After cultured with As4S4 at the concentration of 2.0μmol/L for 24 to 48 hours, K562 cells appeared typical morphological changes of apoptosis. At a concentration greater than or equal to 2.0μmol/L, As4S4 could induce apoptosis significantly. After 12 hours of incubation with 2.0μmol/L As4S4, the apoptosis rate increased from 2.05% to 12.03%. At the same time, the percentage of cells in G1 phase decreased from 69.65% to 50.53%, whereas the percentage of G2/M phase increased from 9.56% to 25.91%. The mRNA levels of BCL-2, BCL-XL, BAD and BAX, and the protein levels of Akt and pAkt down-regulated after the inhibition of As4S4. The transcript and protein levels of BCR-ABL had no change after incubation with As4S4. These results indicated that As4S4 can inhibit the growth of K562 cells efficiently through inducing apoptosis and cell cycle arrest. It seems that As4S4 interferes with Akt pathway and down-regulate BCL-2, BCL-XL, BAD and BAX, which may be involved in the response of K562 to this agent. As4S4 could be beneficial for treatment of CML in combination with conventional drugs.

The oncoprotein NPM-ALK of anaplastic large-cell lymphoma induces JUNB transcription via ERK1/2 and JunB translation via mTOR signaling

Blood ◽  
2007 ◽  
Vol 110 (9) ◽  
pp. 3374-3383 ◽  
Author(s):  
Philipp B. Staber ◽  
Paul Vesely ◽  
Naznin Haq ◽  
Rene G. Ott ◽  
Kotaro Funato ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Translational Control ◽  
Cell Cycle Progression ◽  
Cellular Proliferation ◽  
Mrna Translation ◽  
Large Cell ◽  
Protein Levels ◽  
M Phase ◽  
Alk Positive ◽  
Interfering Rna

Abstract Anaplastic large cell lymphomas (ALCLs) are highly proliferating tumors that commonly express the AP-1 transcription factor JunB. ALK fusions occur in approximately 50% of ALCLs, and among these, 80% have the t(2;5) translocation with NPM-ALK expression. We report greater activity of JunB in NPM-ALK–positive than in NPM-ALK–negative ALCLs. Specific knockdown of JUNB mRNA using small interfering RNA and small hairpin RNA in NPM-ALK–expressing cells decreases cellular proliferation as evidenced by a reduced cell count in the G2/M phase of the cell cycle. Expression of NPM-ALK results in ERK1/2 activation and transcriptional up-regulation of JUNB. Both NPM-ALK–positive and –negative ALCL tumors demonstrate active ERK1/2 signaling. In contrast to NPM-ALK–negative ALCL, the mTOR pathway is active in NPM-ALK–positive lymphomas. Pharmacological inhibition of mTOR in NPM-ALK–positive cells down-regulates JunB protein levels by shifting JUNB mRNA translation from large polysomes to monosomes and ribonucleic particles (RNPs), and decreases cellular proliferation. Thus, JunB is a critical target of mTOR and is translationally regulated in NPM-ALK–positive lymphomas. This is the first study demonstrating translational control of AP-1 transcription factors in human neoplasia. In conjunction with NPM-ALK, JunB enhances cell cycle progression and may therefore represent a therapeutic target.

scholarly journals Rigosertib and Cholangiocarcinoma: A Cell Cycle Affair

2021 ◽  
Vol 23 (1) ◽  
pp. 213
Author(s):  
Alessio Malacrida ◽  
Guido Cavaletti ◽  
Mariarosaria Miloso
Keyword(s):  
Cell Cycle ◽  
Cell Viability ◽  
Kinase Inhibitor ◽  
Therapeutic Option ◽  
Cyclin B ◽  
Cell Cycles ◽  
Protein Levels ◽  
M Phase ◽  
Blotting Technique ◽  
A Cell

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

scholarly journals Differential Subcellular Distribution and Translocation of Seven 14-3-3 Isoforms in Response to EGF and During the Cell Cycle

2020 ◽  
Vol 21 (1) ◽  
pp. 318 ◽  
Author(s):  
Abdalla Abdrabou ◽  
Daniel Brandwein ◽  
Zhixiang Wang
Keyword(s):  
Cell Cycle ◽  
Subcellular Localization ◽  
Mammalian Cells ◽  
Subcellular Fractionation ◽  
Cellular Functions ◽  
Broad Distribution ◽  
Hek 293 ◽  
Multiple Functions ◽  
Mcf 7

Multiple isoforms of 14-3-3 proteins exist in different organisms. In mammalian cells, 14-3-3 protein has seven isoforms (α/β, ε, η, γ, σ, θ/τ, and δ/ζ), with α and δ representing the phosphorylated versions of β and ζ, respectively. While the existence of multiple isoforms may represent one more level of regulation in 14-3-3 signaling, our knowledge regarding the isoform-specific functions of 14-3-3 proteins is very limited. Determination of the subcellular localization of the different 14-3-3 isoforms could give us important clues of their specific functions. In this study, by using indirect immunofluorescence, subcellular fractionation, and immunoblotting, we studied the subcellular localization of the total 14-3-3 protein and each of the seven 14-3-3 isoforms; their redistribution throughout the cell cycle; and their translocation in response to EGF in Cos-7 cells. We showed that 14-3-3 proteins are broadly distributed throughout the cell and associated with many subcellular structures/organelles, including the plasma membrane (PM), mitochondria, ER, nucleus, microtubules, and actin fibers. This broad distribution underlines the multiple functions identified for 14-3-3 proteins. The different isoforms of 14-3-3 proteins have distinctive subcellular localizations, which suggest their distinctive cellular functions. Most notably, 14-3-3ƞ is almost exclusively localized to the mitochondria, 14-3-3γ is only localized to the nucleus, and 14-3-3σ strongly and specifically associated with the centrosome during mitosis. We also examined the subcellular localization of the seven 14-3-3 isoforms in other cells, including HEK-293, MDA-MB-231, and MCF-7 cells, which largely confirmed our findings with Cos-7 cells.

scholarly journals Cell Cycle Activation of the Swi6p Transcription Factor Is Linked to Nucleocytoplasmic Shuttling

2003 ◽  
Vol 23 (9) ◽  
pp. 3126-3140 ◽  
Author(s):  
Ethel Queralt ◽  
J. Carlos Igual
Keyword(s):  
Cell Cycle ◽  
Transcription Factor ◽  
Regulatory Subunit ◽  
Transcriptional Level ◽  
Cell Cycle Regulators ◽  
Nucleocytoplasmic Shuttling ◽  
Phenotypic Analysis ◽  
Cell Cycle Activation ◽  
M Phase ◽  
Related Transcription Factor

ABSTRACT The control of the subcellular localization of cell cycle regulators has emerged as a crucial mechanism in the regulation of cell division. In the present work, we have characterized the function of the karyopherin Msn5p in the control of the cell cycle of Saccharomyces cerevisiae. Phenotypic analysis of the msn5 mutant revealed an increase in cell size and a functional interaction between Msn5p and the cell cycle transcription factor SBF (composed of the Swi4p and Swi6p proteins), indicating that Msn5p is involved in Start control. In fact, we have shown that the level of Cln2p protein is drastically reduced in an msn5 mutant. The effect on CLN2 expression is mediated at a transcriptional level, Msn5p being necessary for proper SBF-dependent transcription. On the contrary, loss of MSN5 has no effect on the closely related transcription factor MBF (composed of the Mbp1p and Swi6p proteins). Regulation of SBF by Msn5p is exerted by control of the localization of the regulatory subunit Swi6p. Swi6p shuttles between the nucleus and the cytoplasm during the cell cycle, and we have found that Msn5p is required for Swi6p export from the nucleus during the G2-M phase. What is more important, we have demonstrated that export of Swi6p to the cytoplasm is required for SBF activity, providing evidence for a functional switch of Swi6p linked to its nucleocytoplasmic shuttling during the cell cycle.

scholarly journals Ott1(Rbm15) Regulates p53 Levels during Oncogenic and Proliferative Stress

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1189-1189
Author(s):  
Sakiko Suzuki ◽  
Nathan A Manalo ◽  
Glen D Raffel
Keyword(s):  
Cell Cycle ◽  
P53 Protein ◽  
Transcriptional Level ◽  
Fusion Partner ◽  
Mrna Levels ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Protein Levels ◽  
Dependent Mechanism ◽  
Oncogenic Stress

Abstract Ott1(Rbm15) is essential for engraftment and maintaining hematopoietic stem cell (HSC) quiescence during proliferative stress; therefore we sought to establish whether Ott1 has a regulatory role within the cell cycle. Ott1 knockout (KO) E14.5 murine embryonic fibroblasts (MEFs) were analyzed using BrdU labelling and demonstrated a higher basal proliferative rate. However, when subjected to oncogenic stress induced by infection with a constitutively active N-Ras expressing retrovirus, Ott1-deleted MEFs undergo immortalization and morphologic transformation in contrast to wild type (WT) MEFs which undergo senescence. Oncogene-induced senescence is a p53-facilitated process. P53 protein levels were shown by western blot to decrease in Ras-infected Ott1 KO MEFs rather than increase as observed in WT Ras-infected MEFs. Consistent with this finding, p16Ink4a, which is a transcriptional target of p53, is not upregulated in Ras-infected Ott1 KO MEFs. Gamma irradiation was still able to induce p53 in Ott1 KO MEFS, demonstrating Ott1 regulation of p53 is specific to the oncogenic stress pathway, but not the DNA damage pathway. Measurement of p53 mRNA levels in Ras-infected Ott1 KO MEFs showed a modest increase compared to WT, indicating the p53 protein decrease must occur at a post-transcriptional level. Classical p53 induction by oncogenic stress occurs through inhibition of ubiquitin-mediated degradation of p53 by ligases such as Mdm2 and Mdm4. To determine why Ras induction of p53 is defective in Ott1 KO MEFS, Ras-infected cells were incubated with the proteasome inhibitor, MG132, which was able to rescue p53 induction, implicating a ubiquitination-dependent mechanism. Furthermore, incubation with Nutlin3, an Mdm2-specific inhibitor, also showed significant rescue of p53 induction, signifying Ott1 is required for Mdm2-mediated degradation of p53 during oncogenic stress. P53 has an essential, non-apoptotic role in HSC function and has also been shown to help maintain HSC quiescence and self-renewal. We previously identified an Ott1-dependent mechanism for down-regulating Thrombopoietin response via its receptor Mpl in Ott1 KO HSCs through expression of a dominant negative alternatively spliced isoform, Mpl-TR. Although Mpl-TR expression is sufficient to reduce Mpl signaling and competitive repopulation in Ott1 KO HSCs, full length Mpl alone is unable to rescue engraftment of Ott1 -deleted HSCs suggesting Ott1 has other critical targets. Based on the Ott1-dependence of p53 function in MEFs, we hypothesized a similar dysfunction of the p53 pathway exists in Ott1 KO HSCs undergoing proliferative stress. Ott1 KO and WT HSCs were analyzed before and after incubation in a cytokine-rich medium to stimulate proliferation. At baseline, Ott1 KO HSCs have similar p53 protein levels as WT HSCs. However, after cytokine stimulation, Ott1 KO HSCs shift into active cell cycle more readily and now demonstrate a significant decrease in p53 protein levels as measured by intracellular flow cytometry. In summary, Ott1 is required for p53 response during oncogenic stress via inhibition of Mdm2. Ott1 is similarly required to maintain p53 levels during proliferative stress in HSCs and may thereby promote quiescence and self-renewal. Moreover, OTT1 is the 5' fusion partner in the chimeric OTT1-MAL (RBM15-MKL1) product in t(1;22)-associated acute megakaryocytic leukemia, raising the possibility that dysregulation of p53 pathways may contribute to the pathogenesis of t(1;22)-derived leukemias. Disclosures No relevant conflicts of interest to declare.

scholarly journals Cell-cycle-dependent phosphorylation and activity of Chinese-hamster ovary topoisomerase II

1993 ◽  
Vol 293 (1) ◽  
pp. 297-304 ◽  
Author(s):  
D A Burden ◽  
L J Goldsmith ◽  
D M Sullivan
Keyword(s):  
Cell Cycle ◽  
Topoisomerase Ii ◽  
Chinese Hamster ◽  
Dna Topoisomerase ◽  
Protein Levels ◽  
Cleavage Activity ◽  
M Phase ◽  
Cycle Dependent

Cell-cycle-dependent protein levels and phosphorylation of DNA topoisomerase II in relation to its catalytic and cleavage activities were studied in Chinese-hamster ovary cells. Immunoreactive topoisomerase II protein levels were maximal in G2-phase cells, intermediate in S- and M-phase cells, and minimal in a predominantly G1-phase population. When the phosphorylation of topoisomerase II in vivo was corrected for differences in specific radioactivity of intracellular ATP, the apparent phosphorylation of S- and M-phase topoisomerase II was altered significantly. Relative phosphorylation in vivo was found to be greatest in M-phase cells and decreased in the other populations in the order: S > G2 > asynchronous. Phosphoserine was detected in every phase of the cell cycle, with a minor contribution of phosphothreonine demonstrated in M-phase cells. Topoisomerase II activity measured in vivo as 9-(4,6-O-ethylidene-beta-D-glucopyranosyl)-4′-demethylepipodophylloto xin (VP-16)-induced DNA double-strand breaks (determined by neutral filter elution) increased in the order: asynchronous < S < G2 < M. Topoisomerase II cleavage activity, assayed in vitro as the formation of covalent enzyme-DNA complexes, was lowest in S phase, intermediate in asynchronous and G2-phase cells, and maximal in M phase. Topoisomerase II decatenation activity was 1.6-1.8-fold greater in S-, G2- and M-phase populations relative to asynchronous cells. Therefore DNA topoisomerase II activity measured both in vivo and in vitro is maximal in M phase, that phase of the cell cycle with an intermediate level of immunoreactive topoisomerase II but the highest level of enzyme phosphorylation. The discordance between immunoreactive topoisomerase II protein levels, adjusted relative phosphorylation, catalytic activity, cleavage activity and amino acid residue(s) modified, suggests that the site of phosphorylation may be cell-cycle-dependent and critical in determining catalytic and cleavage activity.

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