Cdk1-Dependent Phosphorylation ofNPM Overrides G2/M Checkpoint and Increases Leukemic Blasts in Mice

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1322-1322
Author(s):  
Wei Du ◽  
Yun Zhou ◽  
Suzette Pike ◽  
Qishen Pang
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Cell Cycle Control ◽  
Xenograft Model ◽  
Phosphorylation Sites ◽  
M Cell ◽  
Cycle Progression ◽  
Hematopoietic Stem ◽  
Cycle Arrest ◽  
Regulation Of Cell Cycle

Abstract An elevated level of nucleophosmin (NPM) is often found in actively proliferative cells including human tumors. To identify the regulatory role for NPM phosphorylation in proliferation and cell cycle control, a series of mutants targeting the consensus cyclin-dependent kinase (CKD) phosphorylation sites was created to mimic or abrogate either single-site or multi-site phosphorylation. Cells expressing the phosphomimetic NPM mutants showed enhanced proliferation and G2/M cell-cycle transition; whereas nonphosphorylatable mutants induced G2/M cell-cycle arrest. Simultaneous inactivation of two CKD phosphorylation sites at Ser10 and Ser70 (S10A/S70A, NPM-AA) induced phosphorylation of Cdk1 at Tyr15 (Cdc2Tyr15) and increased cytoplasmic accumulation of Cdc25C. Strikingly, stress-induced Cdk1Tyr15 and Cdc25C sequestration were completely suppressed by expression of a double phosphomimetic NPM mutant (S10E/S70E, NPM-EE). Further analysis revealed that phosphorylation of NPM at both Ser10 and Ser70 sites were required for proper interaction between Cdk1 and Cdc25C in mitotic cells. Moreover, the NPM-EE mutant directly bound to Cdc25C and prevented phosphorylation of Cdc25C at Ser216 during mitosis. Finally, NPM-EE overrided stress-induced G2/M arrest, increased peripheral-blood blasts and splenomegaly in a NOD/SCID xenograft model, and promoted leukemia development in Fanconi mouse hematopoietic stem/progenitor cells. Thus, these findings reveal a novel function of NPM on regulation of cell-cycle progression, in which Cdk1-dependent phosphorylation of NPM controls cell-cycle progression at G2/M transition through modulation of Cdc25C activity.

RUNX1/AML1 Is Phosphorylated at Both Its N- and C-Terminus by cdk6/cyclin D3 or cdk1/cyclin B.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1360-1360
Author(s):  
Florence Bernardin Fried ◽  
Alan D. Friedman
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Cyclin B ◽  
Cyclin D3 ◽  
Phosphorylation Sites ◽  
Runt Domain ◽  
Cycle Progression ◽  
Hematopoietic Stem ◽  
Exogenous Dna ◽  
Cdk Phosphorylation

Abstract RUNX1/AML1 is a key transcriptional mediator of hematopoiesis and leukemogenesis. AML1 regulates myeloid and lymphoid differentiation via activation of lineage-specific genes such as those encoding myeloperoxidase or the T cell receptor δ and participates in apoptotic response pathways via its ability to tranactivate the p14/p19ARF gene. In addition, AML1 accelerates G1 to S cell cycle progression, via activation of the cyclin D3 and potentially the cdk4 genes. CBF oncoproteins such as AML1-ETO or CBFβ-SMMHC interfere with the activities of AML1 and block myeloid differentiation and slow cell cycle progression, and mutations such as loss of p16 which accelerate G1 prevent cell cycle inhibition and cooperate with CBF oncoproteins to induce acute leukemia in mice. In addition to regulation of the cell cycle by AML1, we have been interested in how AML1 activities vary and may be regulated during cell cycle progression. We recently reported that endogenous AML1 levels increase in hematopoietic cell lines as they progress from G1 to S and then diminish again at the end of mitosis (Bernardin-Fried et al J. Biol. Chem.279:15678, 2004). RNA levels did not vary, but exogenous AML1 mimicked the behaviour of the endogenous protein, suggesting regulation at the level of protein stability. Mutation of two Ras-dependent phosphorylation sites, S276 and S293, to alanine did not prevent cell cycle variation. We have therefore set out to evaluate whether AML1 stability might be regulated by cyclin-dependent kinase (cdk) phosphorylation. AML1 contains 480 amino acids and binds DNA via its N-terminal Runt domain. Both the cdk6/cyclin D3 and the cdk1/cyclin B complex, expressed from baculovirus vectors, phosphorylated GST-AML1(1-290) and GST-AML1(290–480). The Runt domain alone, in GST-AML1(86–217), was not phosphorylated. Interestingly, exogenous DNA-binding domain alone did not vary during the cell cycle. This is the first demonstration that a specific kinase phosphorylates AML1 in vitro. There are three (S/T)PX(K/R) cdk consensus sites in AML1, with serines at residues 48, 303, and 424. Mutation of S424 to alanine did not prevent phosphorylation of GST-AML1(290–480). Additional mutations of these and other serines or threonines adjacent to proline are being generated to further map the cdk phosphorylation sites and to enable in vivo experiments designed to evaluate the effects of these mutations on cell cycle-specific AML1 expression. We propose a model in which accumulated phosphorylation of AML1 during the S and G2/M cell cycle phases leads to ubiquitin-mediated AML1 destabilization at the end of mitosis. The increased stability of AML1 in the presence of proteosome inhibitors supports this model. Phosphorylation-mediated destabilization of AML1 may complement the recent finding that direct interaction of cyclin D3 with AML1 inhibits its activity as a transcriptional activator. Each of these mechanisms may help regulate the proliferation of hematopoietic stem/progenitor cells. Finally, perhaps loss of destabilizing C-terminal phosphorylation sites in the AML1-ETO oncoprotein increases its ability to dominantly repress AML1-target genes during myeloid leukemogenesis.

E2f4 regulates fetal erythropoiesis through the promotion of cellular proliferation

Blood ◽  
2006 ◽  
Vol 108 (3) ◽  
pp. 886-895 ◽  
Author(s):  
Kathryn M. Kinross ◽  
Allison J. Clark ◽  
Rosa M. Iazzolino ◽  
Patrick Orson Humbert
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Cellular Proliferation ◽  
Cell Cycle Control ◽  
Macrocytic Anemia ◽  
Cycle Progression ◽  
Promote Cell Cycle Progression ◽  
Regulation Of Cell Cycle ◽  
Fetal Erythropoiesis

Abstract The E2F proteins are major regulators of the transcriptional program required to coordinate cell cycle progression and exit. In particular, E2f4 has been proposed to be the principal family member responsible for the regulation of cell cycle exit chiefly through its transcriptional repressive properties. We have previously shown that E2f4–/– mice display a marked macrocytic anemia implicating E2f4 in the regulation of erythropoiesis. However, these studies could not distinguish whether E2f4 was required for differentiation, survival, or proliferation control. Here, we describe a novel function for E2f4 in the promotion of erythroid proliferation. We show that loss of E2f4 results in an impaired expansion of the fetal erythroid compartment in vivo that is associated with impaired cell cycle progression and decreased erythroid proliferation. Consistent with these observations, cDNA microarray analysis reveals cell cycle control genes as one of the major class of genes down-regulated in E2f4–/– FLs, and we provide evidence that E2f4 may directly regulate the transcriptional expression of a number of these genes. We conclude that the macrocytic anemia of E2f4–/– mice results primarily from impaired cellular proliferation and that the major role of E2f4 in fetal erythropoiesis is to promote cell cycle progression and cellular proliferation.

scholarly journals Evidence for a Structural Relationship between BRCT Domains and the Helicase Domains of the Replication Initiators Encoded by the Polyomaviridae and Papillomaviridae Families of DNA Tumor Viruses

2008 ◽  
Vol 82 (17) ◽  
pp. 8849-8862 ◽  
Author(s):  
Anuradha Kumar ◽  
Woo S. Joo ◽  
Gretchen Meinke ◽  
Stephanie Moine ◽  
Elena N. Naumova ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Repair ◽  
Cell Cycle Progression ◽  
Cell Cycle Control ◽  
Cellular Proteins ◽  
Cycle Progression ◽  
Cellular Processes ◽  
Tumor Viruses ◽  
Cellular Pathways ◽  
Regulation Of Cell Cycle

ABSTRACT Studies of DNA tumor viruses have provided important insights into fundamental cellular processes and oncogenic transformation. They have revealed, for example, that upon expression of virally encoded proteins, cellular pathways involved in DNA repair and cell cycle control are disrupted. Herein, evidence is presented that BRCT-related regions are present in the helicase domains of the viral initiators encoded by the Polyomaviridae and Papillomaviridae viral families. Of interest, BRCT domains in cellular proteins recruit factors involved in diverse pathways, including DNA repair and the regulation of cell cycle progression. Therefore, the viral BRCT-related regions may compete with host BRCT domains for particular cellular ligands, a process that would help to explain the pleiotropic effects associated with infections with many DNA tumor viruses.

Phosphorylation of Runx1 by Cyclin-Dependent Kinases Regulates Its Interaction with HDAC1 and HDAC3.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1381-1381 ◽  
Author(s):  
Hong Guo ◽  
Alan D. Friedman
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Hematopoietic Cells ◽  
Cyclin D3 ◽  
Mrna Levels ◽  
Cycle Progression ◽  
Hematopoietic Stem ◽  
Protein Levels ◽  
Cdk Phosphorylation ◽  
Regulation Of Cell Cycle

Abstract Runx1/AML1 is a key regulator of hematopoiesis and leukemic transformation, as RUNX1(−/−) mice do not develop definitive hematopoietic stem cells, and sever alleukemic oncogenes, e.g. AML1-ETO, CBFβ-SMMHC, or TEL-AML1, inhibit Runx1activities. We have investigated regulation of cell cycle progression by Runx1. Runx1stimulates G1 to S cell cycle progression in hematopoietic cell lines and in transduced myeloid progenitors, and inhibition of Runx1 by CBFβ-SMMHC or AML1-ETO slows G1 progression. Runx1 induces cdk4 and cyclin D3 transcription, and exogenous cdk4, cyclin D2, or c-Myc overcomes inhibition of G1 progression by CBF oncoproteins. In addition to regulating cell cycle progression, Runx1 protein levels are themselves increased as hematopoietic cells progress from G1 to S to G2/M, though mRNA levels remain constant. Runx1 contains three consensus cdk sites, (S/T)PX(R/K), S48, S303, and S424, and using phospho-specific antisera we find that each of these is modified in hematopoietic cells. Mutation of these serines to aspartic acid, mimicking phosphorylation, increases trans-activation of a reporter containing four CBF sites or the TCRβ promoter, whereas mutation to alanine reduces trans-activation. p300 interacts similarly with Runx1(tripleA) and Runx1(tripleD). We have now evaluated interaction of HDACs1–8 with these variants and Runx1 and find that both HDAC1 and HDAC3 have reduced affinity for RUNX1(tripleD), as assessed by co-immunoprecipitation from transiently transfected 293T cells. Evaluation of single serine residue mutants (S48D, S303D, and S424D) demonstrates reduced affinity of HDAC1 or HDAC3 specifically for the Runx1(S424D) mutant, consistent with previous mapping of the Runx1:HDAC1 and Runx1:HDAC3 interactions to this region of Runx1. Thus, cdk phosphorylation of Runx1 S424 reduces affinity for HDAC1 and HDAC3, increasing Runx1 trans-activation potency. Regulation of Runx1 activity by cdks may control key developmental processes, including expansion of definitive HSC during development and regulation of the balance between adult HSC quiescence and proliferation.

SQSTM1/p62 Is a Necessary Cofactor In MDS/AML With Deletion Of Mir-146a

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 747-747
Author(s):  
Jing Fang ◽  
Brenden Barker ◽  
Lyndsey Bolanos ◽  
Xiaona Liu ◽  
Andres Jerez ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Survival ◽  
Cell Cycle Arrest ◽  
Research Funding ◽  
Cell Cycle Progression ◽  
Leukemic Cells ◽  
Molecular Networks ◽  
M Cell ◽  
Cycle Progression ◽  
Cycle Arrest

Abstract Deletions involving chromosome 5 (del(5q)) are the most common genetic abnormalities in Myelodysplastic Syndrome (MDS) and secondary Acute Myeloid Leukemia (AML). Chromosome 5q deletions extending beyond q34 portend a worse overall survival, are associated with high-risk (HR) disease, and exhibit significant downregulation of miR-146a, a gene residing on the extended deleted region on 5q34. Additional evidence linking miR-146a loss to HR del(5q) MDS/AML comes from mouse genetic studies; miR-146a-/- mice develop a myeloid proliferative disease and myeloid tumors, in part by derepression of TNFR associated factor 6 (TRAF6) and persistent NF-kB activation. To determine the contribution of miR-146a deficiency to HR MDS/AML, we first examined hematopoietic stem/progenitor cells (HSPC) from miR-146a-/- mice. miR-146a-/- HSPC are highly proliferative, and exhibit increased cell survival and altered HSC fitness. In addition, genetic and/or pharmacologic inhibition of TRAF6/NF-kB signaling impairs cell cycle progression and preferentially leads to apoptosis of malignant miR-146a-/- HSPC. Although inhibiting the TRAF6/NF-kB axis may represent a therapeutic opportunity in miR-146alow MDS/AML patients, unfortunately, NF-kB inhibitors in clinical trials have been disappointing and ones for TRAF6 do not exist. Chromosome deletions that target tumor suppressor genes also involve multiple neighboring genes, such as with del(5q), and loss of certain neighboring genes may expose cancer-specific vulnerabilities. To overcome the limitations of NF-kB inhibitors and identify novel therapeutic targets, we examined the expression of all genes residing within the long arm on chr 5 (5q11-q35) from del(5q) MDS and control CD34+ cells and built molecular networks using GeneConnector functionality in NetWalker. A single major intrachromosomal NF-kB signaling node formed corresponding to the overexpressed chr 5q genes. Among the compensated/overexpressed genes residing on chr 5q and within the NF-kB node, SQSTM1/p62 (5q35) emerged as an obvious candidate as it is an essential cofactor for NF-kB activation by binding TRAF6. First, we evaluated the contribution of p62 to the malignant miR-146alow HSPC phenotype. Overexpression of p62 enhanced proliferation of miR-146a-/- HSPC by promoting G2/M cell cycle progression. Conversely, knockdown of p62 in miR-146a-/- HSPC led to cell cycle arrest and rescued defective myeloid engraftment in competitive HSC transplantation assays, suggesting p62 is required in miR-146alow leukemic cells. Furthermore, the importance of p62 was confirmed in MDS/AML cell lines and patient samples. RNAi-mediated knockdown of p62 resulted in a G2/M cell cycle arrest, reduced cell survival, and impaired leukemic progenitor function, underscoring the importance of p62 in MDS/AML. In addition, interfering with p62-TRAF6 binding by overexpressing a small peptide corresponding to the p62-TRAF6 binding interface suppressed TRAF6-mediated NF-kB activation, and similarly inhibited cell cycle progression and induced apoptosis of human miR-146alow leukemic cells. Collectively, these findings reveal an intrachromosomal gene network that not only drives HR del(5q) myeloid malignancies, but also exposes them to cancer-specific therapeutic vulnerability by disrupting the binding between p62 and TRAF6. Disclosures: Makishima: AA & MDS international foundation: Research Funding; Scott Hamilton CARES grant: Research Funding. Maciejewski:NIH: Research Funding; Aplastic anemia&MDS International Foundation: Research Funding.

The HGF/c-Met Signaling Pathway in Hodgkin Lymphoma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1551-1551
Author(s):  
Chuanhui Xu ◽  
Anke Van Den Berg ◽  
Arjan Diepstra ◽  
Miao Wang ◽  
Debora Jong ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Growth ◽  
Cell Cycle Arrest ◽  
Hodgkin Lymphoma ◽  
Signaling Pathway ◽  
Cell Lines ◽  
Cell Cycle Progression ◽  
M Cell ◽  
Cycle Progression ◽  
Cycle Arrest

Abstract Abstract 1551 Poster Board I-574 Introduction Hodgkin lymphoma (HL) is a B-cell neoplasm characterized by a minority of neoplastic cells, the so-called Hodgkin and Reed-Sternberg (HRS) cells, which are located within an extensive infiltrate of reactive cells. Aberrant signaling of various receptor tyrosine kinases (RTKs) via autocrine or paracrine mechanisms contributes to the survival and proliferation of HRS cells. Activation of the hepatocyte growth factor (HGF)/c-Met signaling pathway has been implicated in the pathophysiology of many cancers, but its role in HL is poorly investigated. In this study, we investigated the expression of c-Met and HGF in HL patient tissues and studied the cell physiological effects of the HGF/c-Met signaling pathway using a c-Met tyrosine kinase inhibitor SU11274 in HL cell lines. Methods The expression of c-Met and HGF in HL patient tissues was studied by immunohistochemistry on a HL tissue microarray. The c-Met expression level was determined by Western blotting, while HGF mRNA and protein levels were measured by quantitative (q)RT-PCR and ELISA in four HL cell lines, i.e. L428, KMH2, L1236 and U-HO1. The effects of SU11274 treatment on the activity of the HGF/c-Met signaling pathway was determined by detection of phosphorylated downstream targets by Western blotting. Effects on cell growth and cell cycle were determined by 3-(4,5- Dimethylthiazol -2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay and by flow cytometry with Propidium iodide (PI), respectively. Results C-Met was detected in HRS cells in 55% (26/47) of HL patient tissues. Expression of HGF was detected in HRS cells in 5 c-Met positive and 2 c-Met negative HL patient tissues. C-Met was highly expressed in L428 compared to three other HL cell lines, whereas HGF was highly expressed in KMH2 and not or only weakly in the other three HL cell lines. Detectable levels of phosphorylated c-Met (p-Met) were observed only in L428 consistent with the high basal expression level of c-Met. Phosphorylation of c-Met, Akt and Erk1/2 were upregulated upon HGF stimulation of L428 cells. This activation could be blocked by inhibiting c-Met activation with SU11274. In functional studies, SU11274 suppressed cell growth in L428, promoted G2/M cell cycle arrest after 24h incubation, and induced tetraploidy after 48h. Washing of the cells after induction of G2/M arrest resulted in normal cell cycle progression indicating that the G2/M cell cycle arrest was reversible. Inhibition of PI3K, MEK1/2 and Erk1/2, three downstream targets of the HGF/c-Met signaling pathway, also induced G2/M cell cycle arrest in L428, indicating that these factors are involved in the G2/M cell cycle arrest induced by SU11274. Conclusion Co-expression of c-Met and HGF in HRS cells was observed in 11% of the HL patient tissues and HGF/c-Met signaling pathway regulates cell growth and cell cycle progression in L428 cells. Disclosures No relevant conflicts of interest to declare.

Expression profiles of miRNAs and involvement of miR-100 and miR-34 in regulation of cell cycle arrest in Artemia

2015 ◽  
Vol 470 (2) ◽  
pp. 223-231 ◽  
Author(s):  
Ling-Ling Zhao ◽  
Feng Jin ◽  
Xiang Ye ◽  
Lin Zhu ◽  
Jin-Shu Yang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Cell Cycle Regulation ◽  
Cell Cycle Progression ◽  
Expression Profiles ◽  
Regulatory Mechanisms ◽  
Cycle Progression ◽  
Cycle Arrest ◽  
Regulation Of Cell Cycle ◽  
Cycle Regulation

We established an expression profile of miRNA for cell cycle arrest in Artemia and found that miR-100 and miR-34 promote and prevent cell cycle progression respectively. The regulatory mechanisms of these two miRNAs provide insights into cell cycle regulation.

PTTG has a Dual Role of Promotion-Inhibition in the Development of Pituitary Adenomas

2019 ◽  
Vol 26 (11) ◽  
pp. 800-818
Author(s):  
Zujian Xiong ◽  
Xuejun Li ◽  
Qi Yang
Keyword(s):  
Cell Cycle ◽  
Pituitary Adenoma ◽  
Pituitary Adenomas ◽  
Cell Cycle Progression ◽  
Key Factors ◽  
Cycle Progression ◽  
Sister Chromatids ◽  
Regulation Of Cell Cycle ◽  
Late Stages

Pituitary Tumor Transforming Gene (PTTG) of human is known as a checkpoint gene in the middle and late stages of mitosis, and is also a proto-oncogene that promotes cell cycle progression. In the nucleus, PTTG works as securin in controlling the mid-term segregation of sister chromatids. Overexpression of PTTG, entering the nucleus with the help of PBF in pituitary adenomas, participates in the regulation of cell cycle, interferes with DNA repair, induces genetic instability, transactivates FGF-2 and VEGF and promotes angiogenesis and tumor invasion. Simultaneously, overexpression of PTTG induces tumor cell senescence through the DNA damage pathway, making pituitary adenoma possessing the potential self-limiting ability. To elucidate the mechanism of PTTG in the regulation of pituitary adenomas, we focus on both the positive and negative function of PTTG and find out key factors interacted with PTTG in pituitary adenomas. Furthermore, we discuss other possible mechanisms correlate with PTTG in pituitary adenoma initiation and development and the potential value of PTTG in clinical treatment.

scholarly journals A DNA-Damage-Induced Cell Cycle Checkpoint in Arabidopsis

Genetics ◽  
2003 ◽  
Vol 164 (1) ◽  
pp. 323-334
Author(s):  
S B Preuss ◽  
A B Britt
Keyword(s):  
Cell Cycle ◽  
Gamma Radiation ◽  
Cell Cycle Progression ◽  
Cell Cycle Checkpoint ◽  
Phase Cell ◽  
Cycle Arrest ◽  
Cycle Checkpoint ◽  
Radiation Induced ◽  
High Doses ◽  
Regulation Of Cell Cycle

Abstract Although it is well established that plant seeds treated with high doses of gamma radiation arrest development as seedlings, the cause of this arrest is unknown. The uvh1 mutant of Arabidopsis is defective in a homolog of the human repair endonuclease XPF, and uvh1 mutants are sensitive to both the toxic effects of UV and the cytostatic effects of gamma radiation. Here we find that gamma irradiation of uvh1 plants specifically triggers a G2-phase cell cycle arrest. Mutants, termed suppressor of gamma (sog), that suppress this radiation-induced arrest and proceed through the cell cycle unimpeded were recovered in the uvh1 background; the resulting irradiated plants are genetically unstable. The sog mutations fall into two complementation groups. They are second-site suppressors of the uvh1 mutant's sensitivity to gamma radiation but do not affect the susceptibility of the plant to UV radiation. In addition to rendering the plants resistant to the growth inhibitory effects of gamma radiation, the sog1 mutation affects the proper development of the pollen tetrad, suggesting that SOG1 might also play a role in the regulation of cell cycle progression during meiosis.

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