AML1/RUNX1 Increases During G1 to S Cell Cycle Progression Independent of Cytokine-dependent Phosphorylation and Induces Cyclin D3 Gene Expression

Abstract

AML1/RUNX1, a member of the core binding factor (CBF) family stimulates myelopoiesis and lymphopoiesis by activating lineage-specific genes. In addition, AML1 induces S phase entry in 32Dcl3 myeloid or Ba/F3 lymphoid cells via transactivation. We now found that AML1 levels are regulated during the cell cycle. 32Dcl3 and Ba/F3 cell cycle fractions were prepared using elutriation. Western blotting and a gel shift/supershift assay demonstrated that endogenous CBF DNA binding and AML1 levels were increased 2-4-fold in S and G2/M phase cells compared with G1 cells. In addition, G1 arrest induced by mimosine reduced AML1 protein levels. In contrast, AML1 RNA did not vary during cell cycle progression relative to actin RNA. Analysis of exogenous Myc-AML1 or AML1-ER demonstrated a significant reduction in G1 phase cells, whereas levels of exogenous DNA binding domain alone were constant, lending support to the conclusion that regulation of AML1 protein stability contributes to cell cycle variation in endogenous AML1. However, cytokine-dependent AML1 phosphorylation was independent of cell cycle phase, and an AML1 mutant lacking two ERK phosphorylation sites was still cell cycle-regulated. Inhibition of AML1 activity with the CBFβ-SMMHC or AML1-ETO oncoproteins reduced cyclin D3 RNA expression, and AML1 bound and activated the cyclin D3 promoter. Signals stimulating G1 to S cell cycle progression or entry into the cell cycle in immature hematopoietic cells might do so in part by inducing AML1 expression, and mutations altering pathways regulating variation in AML1 stability potentially contribute to leukemic transformation. AML1/RUNX1, a member of the core binding factor (CBF) family stimulates myelopoiesis and lymphopoiesis by activating lineage-specific genes. In addition, AML1 induces S phase entry in 32Dcl3 myeloid or Ba/F3 lymphoid cells via transactivation. We now found that AML1 levels are regulated during the cell cycle. 32Dcl3 and Ba/F3 cell cycle fractions were prepared using elutriation. Western blotting and a gel shift/supershift assay demonstrated that endogenous CBF DNA binding and AML1 levels were increased 2-4-fold in S and G2/M phase cells compared with G1 cells. In addition, G1 arrest induced by mimosine reduced AML1 protein levels. In contrast, AML1 RNA did not vary during cell cycle progression relative to actin RNA. Analysis of exogenous Myc-AML1 or AML1-ER demonstrated a significant reduction in G1 phase cells, whereas levels of exogenous DNA binding domain alone were constant, lending support to the conclusion that regulation of AML1 protein stability contributes to cell cycle variation in endogenous AML1. However, cytokine-dependent AML1 phosphorylation was independent of cell cycle phase, and an AML1 mutant lacking two ERK phosphorylation sites was still cell cycle-regulated. Inhibition of AML1 activity with the CBFβ-SMMHC or AML1-ETO oncoproteins reduced cyclin D3 RNA expression, and AML1 bound and activated the cyclin D3 promoter. Signals stimulating G1 to S cell cycle progression or entry into the cell cycle in immature hematopoietic cells might do so in part by inducing AML1 expression, and mutations altering pathways regulating variation in AML1 stability potentially contribute to leukemic transformation. Core binding factor (CBF) 1The abbreviations used are: CBF, core binding factor; G-CSF, granulocyte-colony stimulating factor; ERK, extracellular signal-related kinase; IL-3, interleukin-3; MT, metallothionein; FACS, fluorescence-activated cell sorting; HI-FBS, heat-inactivated fetal bovine serum; MPO, myeloperoxidase; cdk, cyclin-dependent kinase. is a family of transcription factors containing one of three CBFα subunits, RUNX1/AML1, AML2, or AML3, and a CBFβ subunit (1Friedman A.D. Leukemia. 1999; 13: 1932-1942Crossref PubMed Scopus (85) Google Scholar). The CBFα subunits bind a common DNA consensus site via the N-terminal Runt domain, which also mediates heterodimerization with CBFβ (2Bae S.-C. Yamaguchi-Iwai Y. Ogawa E. Maruyama M. Inuzuka M. Kagoshima H. Shigesada K. Satake M. Ito Y. Oncogene. 1993; 8: 809-814PubMed Google Scholar, 3Meyers S. Downing J.R. Hiebert S.W. Mol. Cell. Biol. 1993; 13: 6336-6345Crossref PubMed Scopus (427) Google Scholar). CBFβ does not contact DNA, but increases the DNA affinity of the α-subunits (4Wang S. Wang Q. Crute B.E. Melnikova I.N. Keller S.R. Speck N.A. Mol. Cell. Biol. 1993; 13: 3324-3339Crossref PubMed Scopus (397) Google Scholar, 5Ogawa E. Inuzuka M. Maruyamna M. Satake M. Naito-Fujimoto M. Ito Y. Shigesada K. Virol. 1993; 194: 314-331Crossref PubMed Scopus (442) Google Scholar). CBF activities are reduced in 30% of acute myeloid leukemia cases, due either to AML1 point mutations, AML1 gene deletion, or, most often, chromosomal abnormalities involving genes encoding AML1 or CBFβ (1Friedman A.D. Leukemia. 1999; 13: 1932-1942Crossref PubMed Scopus (85) Google Scholar). Mice lacking either AML1 or CBFβ do not develop definitive hematopoiesis, indicating a critical role for these factors in pluripotent hematopoietic stem cells (6Okuda T. van Deursen J. Hiebert S.W. Grosveld G. Downing J.R. Cell. 1996; 84: 321-330Abstract Full Text Full Text PDF PubMed Scopus (1606) Google Scholar, 7Wang Q. Stacy T. Binder M. Marin-Padilla M. Sharpe A.H. Speck N.A. Proc. Natl. Acad. Sci., U. S. A. 1996; 93: 3444-3449Crossref PubMed Scopus (1029) Google Scholar, 8Wang Q. Stacy T. Miller J.D. Lewis A.F. Gu T.-L. Huang X. Bushweller J.H. Bories J.-C. Alt F.W. Ryan G. Liu P.P. Wynshaw-Boris A. Binder M. Marin-Padilla M. Sharpe A.H. Speck N.A. Cell. 1996; 87: 697-708Abstract Full Text Full Text PDF PubMed Scopus (544) Google Scholar, 9Sasaki K. Yagi H. Bronson R.T. Tominaga K. Matsunashi T. Deguchi K. Tani Y. Kishimoto T. Komori T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12359-12363Crossref PubMed Scopus (328) Google Scholar, 10Niki M. Okada H. Takano H. Kujo J. Tani K. Hibino H. Asano S. Ito Y. Satake M. Noda T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5697-5702Crossref PubMed Scopus (164) Google Scholar). During early hematopoiesis AML1 regulates myeloid and lymphoid lineage-specific genes, such as those encoding T-cell receptor δ, myeloperoxidase, and the M-CSF receptor (11Redondo J.M. Pfohl J.L. Hernandez-Munain C. Wang S. Speck N.A. Krangel M.S. Mol. Cell. Biol. 1992; 12: 4817-4823Crossref PubMed Google Scholar, 12Suzow J. Friedman A.D. Mol. Cell. Biol. 1993; 13: 2141-2151Crossref PubMed Scopus (95) Google Scholar, 13Nuchprayoon I. Meyers S. Scott L.M. Suzow J. Hiebert S. Friedman A.D. Mol. Cell. Biol. 1994; 14: 5558-5568Crossref PubMed Google Scholar, 14Zhang D.-E. Fujioka K.-I. Hetherington C.J. Shapiro L.H. Chen H.-M. Look A.T. Tenen D.G. Mol. Cell. Biol. 1994; 14: 8085-8095Crossref PubMed Scopus (53) Google Scholar), and AML1 is down-regulated during terminal neutrophilic differentiation (15Bjerregaard M.D. Jurlander J. Klausen P. Borregaard N. Cowland J.B. Blood. 2003; 101: 4322-4332Crossref PubMed Scopus (137) Google Scholar). In addition, CBF/AML1 regulates the G1 to S cell cycle transition. CBFβ-SMMHC is a CBF oncoprotein capable of sequestering CBFα subunits in multimers, which form via its myosin (SMMHC) domain (16Liu P. Tarle S.A. Hajre A. Claxton D.F. Marlton P. Freedman M. Siciliano M.J. Collins F.S. Science. 1993; 261: 1041-1044Crossref PubMed Scopus (651) Google Scholar, 17Kummalue T. Lou J. Friedman A.D. Mol. Cell. Biol. 2002; 22: 8278-8291Crossref PubMed Scopus (21) Google Scholar). Expression of CBFβ-SMMHC from the zinc-responsive metallothionein (MT) promoter in the Ba/F3 pro-B lymphoid or 32Dcl3 myeloid cell lines reduces CBF/AML1 DNA binding, leading to the accumulation of hypophosphorylated retinoblastoma (Rb) protein, and slows cell proliferation during G1 (18Cao W. Britos-Bray M. Claxton D.F. Kelley C.A. Speck N.A. Liu P.P. Friedman A.D. Oncogene. 1997; 15: 1315-1327Crossref PubMed Scopus (71) Google Scholar). N-terminal CBFβ residues for with CBFα subunits are for of proliferation by CBFβ-SMMHC W. N. Britos-Bray M. Liu P.P. Friedman A.D. J. Biol. Full Text Full Text PDF Scopus Google Scholar). AML1 DNA binding domain protein in Ba/F3 or 32Dcl3 cells also G1 progression J. W. K. Friedman A.D. Oncogene. PubMed Scopus Google Scholar). a CBF which DNA and slows G1 progression in myeloid cell its to bind DNA T. Lou J. Friedman A.D. Mol. Cell. Biol. 2002; 22: 8278-8291Crossref PubMed Scopus (21) Google Scholar, S.A. N. T. Tenen D.G. Mol. Cell. Biol. PubMed Scopus Google Scholar, J.M. J. H. N. Downing J.R. Meyers S. Hiebert S.W. Mol. Cell. Biol. PubMed Scopus Google Scholar). Inhibition of proliferation by CBFβ-SMMHC or by is by exogenous cyclin or J. W. K. Friedman A.D. Oncogene. PubMed Scopus Google Scholar, Y. Friedman A.D. Biol. 2002; Scopus Google Scholar). AML1 G1 progression in 32Dcl3 cells or as an AML1-ER protein in Ba/F3 cells J. W. K. Friedman A.D. Oncogene. PubMed Scopus Google Scholar, J. Downing J.R. N. Hiebert S.W. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). with AML1-ER proliferation of its domain Friedman A.D. Oncogene. 2002; PubMed Scopus Google Scholar). that of and or of protein, with CBF oncoproteins to acute leukemia in Y. Wang W. M. Friedman A.D. 2002; Google Scholar, Y. M. Friedman A.D. 2002; Google Scholar). the conclusion that AML1 stimulates the G1 to S to AML1 levels are regulated during the cell cycle. 32Dcl3 and Ba/F3 cell cycle fractions were prepared using a that the of or exogenous AML1 levels increased during G1 to S cell cycle as did AML1 DNA binding AML1 lacking two ERK phosphorylation sites cell cycle and induced phosphorylation of endogenous AML1 independent of cell cycle In addition, of AML1 cyclin D3 expression, and AML1 bound and activated the cyclin D3 and of cells M. K. S. G. J. Google were in with heat-inactivated fetal bovine Ba/F3 cells M. Cell. Full Text PDF PubMed Scopus Google were in with HI-FBS, and cells Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google were in with heat-inactivated or mimosine were 32Dcl3 differentiation was induced by with by in with HI-FBS, and 32Dcl3 or Ba/F3 cells were by and 32Dcl3 cells were using cells as and using or (18Cao W. Britos-Bray M. Claxton D.F. Kelley C.A. Speck N.A. Liu P.P. Friedman A.D. Oncogene. 1997; 15: 1315-1327Crossref PubMed Scopus (71) Google Scholar, X. Scott E. Friedman A.D. Blood. 1999; 94: PubMed Google Scholar). cell were by cells were in with and were using as T. Lou J. Friedman A.D. Mol. Cell. Biol. 2002; 22: 8278-8291Crossref PubMed Scopus (21) Google Scholar). the of AML1 S. N. Hiebert S.W. Mol. Cell. Biol. 15: PubMed Scopus Google Scholar), was into the of (18Cao W. Britos-Bray M. Claxton D.F. Kelley C.A. Speck N.A. Liu P.P. Friedman A.D. Oncogene. 1997; 15: 1315-1327Crossref PubMed Scopus (71) Google to was by an encoding for by a and a of the AML1 The with a was The encoding the and is into the of the AML1 also the and the AML1 was by to a with of a of the by H. was used to a in to and Friedman A.D. Oncogene. 2002; PubMed Scopus Google Scholar). The of these the S. N. Hiebert S.W. Mol. Cell. Biol. 15: PubMed Scopus Google Scholar). was prepared by an from P. Y. K. 1996; PubMed Scopus Google into was prepared by the AML1 site with by two of the site and the site The was by DNA and cells were and in of bovine The cells were into a and The of was increased and cell cycle fractions was in by of cells with in by Western and protein were prepared using and to gel and Western blotting as (18Cao W. Britos-Bray M. Claxton D.F. Kelley C.A. Speck N.A. Liu P.P. Friedman A.D. Oncogene. 1997; 15: 1315-1327Crossref PubMed Scopus (71) Google Scholar). to cells were in AML1 was by H. CBFβ was by N. and and D3 and and cyclin were of were and to to protein of were prepared and to gel and assay using a CBF binding site from the promoter and an N-terminal AML1 by S. as I. Meyers S. Scott L.M. Suzow J. Hiebert S. Friedman A.D. Mol. Cell. Biol. 1994; 14: 5558-5568Crossref PubMed Google Scholar). The and mutant were J. Friedman A.D. Mol. Cell. Biol. 1993; 13: 2141-2151Crossref PubMed Scopus (95) Google and In the from the cyclin genes, were to and used as gel AML1 binding sites are and with are were prepared using or the The were to blotting as X. Scott E. Friedman A.D. Blood. 1999; 94: PubMed Google Scholar). cyclin and D3 were by C. and the AML1 by Y. Ito (2Bae S.-C. Yamaguchi-Iwai Y. Ogawa E. Maruyama M. Inuzuka M. Kagoshima H. Shigesada K. Satake M. Ito Y. Oncogene. 1993; 8: 809-814PubMed Google Scholar). was with the CBF DNA and AML1 During G1 to S to 32Dcl3 and Ba/F3 cells into cell cycle fractions In the of the the the cells, an the is cells into to the of to cells in cell which did not by cells by of the as 32Dcl3 cells in cell cycle progression cell cycle and the of cells in are G1 phase cells, S cells, S phase cells, and G2/M phase cells. cycle was in In the cells to as in AML1 is in the cell J. Maruyama M. Satake M. S.-C. Ogawa S. Kagoshima Shigesada K. Ito Y. Mol. Cell. Biol. 15: PubMed Google Scholar). We a gel assay to CBF DNA binding activity during the cell an of protein and CBF DNA binding activity increased as 32Dcl3 or Ba/F3 cells from G1 to S. of protein cell were not the G1 and S phase of CBF gel activity was in that the of the CBF the fractions for cell were to with of CBF or with an point mutations in the CBF binding site was with the CBF DNA from the CBF binding of the 32Dcl3 were to a assay with AML1 or AML1 in the of its was from which increased as cells from G1 to S. assay that Ba/F3 cells CBF DNA binding activity 32Dcl3 cells for to a AML1 with the Ba/F3 the did the of the CBF We prepared from 32Dcl3 and Ba/F3 cell cycle fractions and these for AML1 by Western blotting and was in the 32Dcl3 and protein fractions as a for protein that increased during the cell with of cell AML1 levels increased or from G1 to S or G2/M in the 32Dcl3 relative to and increased or in the Ba/F3 of with 32Dcl3 cells demonstrated a or an of in AML1 during the cell cycle of cell cycle variation is with the for 32Dcl3 cells, but the in CBF DNA binding was that for AML1 in Ba/F3 cells, a of and to CBF cell cycle variation in these phosphorylation does not AML1 during the cell cycle. 32Dcl3 cells were and for in and to with fetal bovine and for the of were to Western blotting for AML1. from cells was also and in the AML1 are by the 32Dcl3 cell cycle fractions were to for or protein prepared to of and for the were to Western blotting for AML1 and to Expression of AML1 in for is with the from the or was not for two of the AML1 was to the or in of the of the in the from the cell cycle fractions were to Western blotting using of these fractions is also in for is the variation in AML1 protein levels during the cell cycle is by variation in AML1 RNA were prepared from 32Dcl3 and Ba/F3 cell cycle fractions and of was to blotting AML1 increased in Ba/F3 cells and 32Dcl3 cells from early G1 to S phase, relative to RNA cell increased during the cell cycle. relative to AML1 RNA increased in to as RNA RNA. AML1 in in of AML1 in early G1 that cells in G1 reduced levels of AML1 compared with cells. 32Dcl3 or Ba/F3 cells were to an which induces the cyclin-dependent in hematopoietic cells Q. T. C. Friedman A.D. Oncogene. 2003; 22: PubMed Scopus Google Scholar). was or for as for to the increased from to in Ba/F3 cells and from to in 32Dcl3 cells, with and from these were for endogenous AML1 by Western blotting to mimosine for reduced endogenous AML1 levels in Ba/F3 cells and in 32Dcl3 cells, relative to protein 32Dcl3 cells were from to G-CSF, which induces differentiation with a G1 arrest X. Scott E. Friedman A.D. Blood. 1999; 94: PubMed Google Scholar). The increased from to and to and AML1 levels were reduced and in reduced AML1 levels with G1 arrest are due to reduced AML1 RNA were prepared from 32Dcl3 cells in or in for or and from 32Dcl3 or Ba/F3 cells in mimosine for were to blotting for AML1 and to AML1 RNA increased in 32Dcl3 cells in to to mimosine for or to for and in Ba/F3 cells in to AML1 During G1 to S that endogenous AML1 levels are regulated during the cell that exogenous AML1 protein levels might exogenous a is the of the AML1 of a containing for The lacking endogenous AML1 or was of the zinc-responsive metallothionein promoter in to also the gene to the promoter. was and into 32Dcl3 cells. in the of were by Western a from cells was by Western blotting using an AML1 two were with the with AML1 from 32Dcl3 cells of a AML1 phosphorylation T. M. K. K. Y. K. K. N. Y. Y. T. H. Mol. Cell. Biol. 1996; PubMed Scopus Google Scholar). The increased of exogenous is due to the and to the that exogenous AML1 residues lacking in the endogenous the of and AML1 are S. N. Hiebert S.W. Mol. Cell. Biol. 15: PubMed Scopus Google Scholar). We that was endogenous AML1. of the zinc-responsive promoter not of cell lines during by activity of AML1 but also of via of in the of of the of the is to that in cells to to endogenous AML1 levels. cells were to and to elutriation. cycle fractions were by Western blotting for cyclin and cyclin with early G1 cells Myc-AML1 levels increased by phase and by phase D3 and levels increased Analysis of a 32Dcl3 demonstrated a in exogenous Myc-AML1 during cell cycle progression that AML1 RNA increased relative to with and regulation of AML1 during G1 to S progression the of cell cycle phase the of exogenous AML1 by an independent 32Dcl3 cells AML1-ER from the in J. W. K. Friedman A.D. Oncogene. PubMed Scopus Google Scholar). Western of cell cycle fractions demonstrated that AML1-ER increased by S phase and and by G2/M phase relative to CBFβ which the cell cycle D3 levels increased during G1 to S exogenous AML1 levels to endogenous AML1 during the cell cycle of the AML1 to or the AML1 and RNA to and RNA AML1 DNA during the to the domain of AML1 for its cell cycle 32Dcl3 lines either from the the DNA binding domain and N-terminal or from the the DNA binding domain, were to and Western and In to Myc-AML1 or which increased or increased and increased during the cell cycle. Analysis of a of AML1 by ERK and are of phosphorylation in hematopoietic cells T. M. K. K. Y. K. K. N. Y. Y. T. H. Mol. Cell. Biol. 1996; PubMed Scopus Google Scholar). In these found that exogenous was as in Ba/F3 cells, that the two were of and and that of and to the of the two cytokine-dependent phosphorylation of endogenous AML1 during the cell 32Dcl3 cells from and for by to the used for elutriation. was the cells were to the and protein were prepared were to Western blotting using AML1 the of AML1 was in the of a whereas the of AML1 was in the or the two were of and the a and the cells potentially a not to found that are to cytokine-dependent AML1 to cell cycle 32Dcl3 cell cycle fractions were to for or and for AML1 by Western blotting a of but the levels of AML1 to and to vary during the cell cycle. In AML1 levels or in the the with the cell cycle and or in the to an of and during the cell cycle in the two cytokine-dependent phosphorylation during cell cycle and does not to a of AML1. the role of AML1 32Dcl3 lines exogenous from the promoter were AML1 mutant increased during cell cycle progression were was phosphorylation of AML1 does not for the in endogenous or exogenous AML1 during G1 to S The D3 by D3 levels did not relative to in 32Dcl3 or Ba/F3 cell cycle fractions However, a factor with activity in S phase, activated the cyclin D3 and A. as from the of an consensus site P. Y. K. 1996; PubMed Scopus Google Scholar). the that the in CBF DNA binding activity in S might to cyclin D3 in hematopoietic cells. We Ba/F3 cells AML1 oncoproteins T. Lou J. Friedman A.D. Mol. Cell. Biol. 2002; 22: 8278-8291Crossref PubMed Scopus (21) Google Scholar, W. Britos-Bray M. Claxton D.F. Kelley C.A. Speck N.A. Liu P.P. Friedman A.D. Oncogene. 1997; 15: 1315-1327Crossref PubMed Scopus (71) Google Scholar), CBFβ-SMMHC or AML1-ETO from the promoter to the of CBF cyclin and D3 Ba/F3 lines were 32Dcl3 lines for as the promoter is in 32Dcl3 cells, to of these cell was not as for hematopoietic cell lines K. A. J.D. Proc. Natl. Acad. Sci. U. S. A. 1993; PubMed Scopus Google Scholar). did not cyclin or D3 in Ba/F3 cells, but was by the or by with D3 the We AML1 binding sites from the and D3 the AML1 and affinity for AML1 In a AML1 site from the was and with and of a of mutant in the AML1 and of the from the cyclin promoter The site from the promoter did not from the promoter and from the D3 to an as as the in the of the and sites of AML1 the cyclin D3 a promoter the transcription site was to and with and in cells AML1 activated the cyclin D3 promoter in in and of the AML1 binding site reduced to a role for binding the in activity did not of is that endogenous CBF DNA binding and AML1 protein levels to protein during the G1 to S cell cycle transition. In levels of exogenous to cell cycle increased the conclusion that AML1 protein stability is regulated during the cell cycle. of AML1 in cells is with of AML1 transcription also during cell cycle were in two hematopoietic cell We hematopoietic cells as these are most in of the that hematopoiesis (6Okuda T. van Deursen J. Hiebert S.W. Grosveld G. Downing J.R. Cell. 1996; 84: 321-330Abstract Full Text Full Text PDF PubMed Scopus (1606) Google Scholar, 7Wang Q. Stacy T. Binder M. Marin-Padilla M. Sharpe A.H. Speck N.A. Proc. Natl. Acad. Sci., U. S. A. 1996; 93: 3444-3449Crossref PubMed Scopus (1029) Google Scholar). the of AML1 variation in levels and cyclin during cell cycle progression in cells H. H. S.A. 2003; PubMed Scopus Google Scholar). AML1 levels in G2/M phase compared with S phase cells, whereas CBF and AML1 DNA binding was that AML1 DNA binding activity is regulated during S to G2/M lines of using the 32Dcl3 and Ba/F3 cell lines demonstrated that gene by AML1 stimulates G1 to S In of AML1 genes G1 an by of to S phase and exogenous AML1 the G1 to S the AML1 domain (18Cao W. Britos-Bray M. Claxton D.F. Kelley C.A. Speck N.A. Liu P.P. Friedman A.D. Oncogene. 1997; 15: 1315-1327Crossref PubMed Scopus (71) Google Scholar, W. N. Britos-Bray M. Liu P.P. Friedman A.D. J. Biol. Full Text Full Text PDF Scopus Google Scholar, J. W. K. Friedman A.D. Oncogene. PubMed Scopus Google Scholar, Y. Friedman A.D. Biol. 2002; Scopus Google Scholar, Friedman A.D. Oncogene. 2002; PubMed Scopus Google Scholar). is not that hematopoietic cells increased AML1 during G1 and to AML1 during the G1 arrest with differentiation is a the reduced AML1 protein levels those to AML1 during the cell cycle. is that hematopoietic cells cyclin and and that the and D3 which bind AML1. its AML1 activated the D3 promoter the to with to contribute most of the AML1 binding site indicating from AML1 binding or also contribute to regulation of the cyclin D3 as with AML1 did not cell cycle variation of CBF DNA binding activity in part for to of AML1 with the cyclin D3 promoter by chromosomal the of or into the AML1 in hematopoiesis Y. T. T. Blood. 2003; Scholar). regulation of the cyclin D3 gene by CBF family AML1 is in pluripotent and hematopoietic stem cells Blood. 1997; PubMed Google Scholar, T. Gu T.-L. Stacy T. Wang Q. Binder M. Marin-Padilla M. Speck N.A. 1999; PubMed Google Scholar), that of AML1 protein stability or RNA also a role in the entry of these cells into cell cycle from as to hematopoietic or during In and RNA levels cells from to and during proliferation protein levels are during S phase due to phosphorylation C. Y. Oncogene. 1996; 12: Google Scholar, S.R. PubMed Scopus Google Scholar, J. 1993; 12: PubMed Scopus Google Scholar). cyclin levels cell cycle entry C.J. Science. 1996; PubMed Scopus Google Scholar). Ba/F3 and 32Dcl3 cells the reduced levels of cyclin D3 in early cell fractions that these lines a and are to We found that CBFβ-SMMHC G1 to S progression in myeloid as does 32Dcl3 and Ba/F3 cells J. Friedman A.D. Blood. 2003; Scholar), and AML1-ETO the proliferation of and Hetherington C.J. N. Liu Tenen D.G. Blood. PubMed Google Scholar, J. Blood. 2002; PubMed Scopus Google Scholar). In addition, reduced in of cell cycle progression W. Downing J. Blood. 2003; Scholar). the role of AML1 in regulation of the cell cycle and cell cycle entry in hematopoietic stem cells. might to AML1 protein is that phosphorylation of AML1 by such as or increases its stability during AML1 three phosphorylation sites that are in the and and of the phosphorylation sites phosphorylation by regulates cell cycle activity of the transcription factor in cells Y. P. S. J. H. P. H. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). variation during the cell cycle by a which in and X. K. C. Science. PubMed Scopus Google Scholar). AML1 levels in 32Dcl3 cells were found to increased by the G. Shigesada K. Ito K. T. Ito Y. J. PubMed Scopus Google Scholar). We using cells significant cell cycle might a or protein, which AML1 in of and AML1-ER that residues to the DNA binding domain are in the regulation of AML1 cyclin D3 with AML1 via A. S. Blood. 2002; PubMed Scopus Google Scholar), potentially We found that cytokine-dependent phosphorylation of and by ERK did not vary during the cell cycle AML1 AML1 or protein its stability during cell cycle progression or in to cell cycle entry or In is to the or phosphorylation of a AML1 during the cell cycle as the or form AML1 to might which levels the cell cycle. In a of acute myeloid AML1 activities are reduced due to or of potentially due to of differentiation reduced I. Meyers S. Scott L.M. Suzow J. Hiebert S. Friedman A.D. Mol. Cell. Biol. 1994; 14: 5558-5568Crossref PubMed Google Scholar, M. M. W. Wang X. Liu P.P. Friedman A.D. Blood. PubMed Google Scholar, C. van M. J. van J.D. J. J.H. P.P. Hiebert S.W. 2002; 8: PubMed Scopus Google Scholar). mutations, which AML1 via protein using the to AML1 in G1 phase cells. In these mutations stimulating G1 progression Y. Wang W. M. Friedman A.D. 2002; Google Scholar, Y. M. Friedman A.D. 2002; Google Scholar). acute increased of AML1 genes due to gene or to T. J. K. S. Scholar, P. C. S. S.A. A. PubMed Scopus Google Scholar, M. PubMed Scopus Google Scholar, N. G. Leukemia. 2002; PubMed Scopus Google Scholar). In these AML1 by stimulating cell cycle which AML1 levels the AML1 cell cycle variation might pathways to We for H. for the C. for the cyclin and H. N. and S. Hiebert for