Factor-dependent Myeloid Cell Line Research Articles

Cycloheximide Can Induce Bax/Bak Dependent Myeloid Cell Death Independently of Multiple BH3-Only Proteins.

Apoptosis mediated by Bax or Bak is usually thought to be triggered by BH3-only members of the Bcl-2 protein family. BH3-only proteins can directly bind to and activate Bax or Bak, or indirectly activate them by binding to anti-apoptotic Bcl-2 family members, thereby relieving their inhibition of Bax and Bak. Here we describe a third way of activation of Bax/Bak dependent apoptosis that does not require triggering by multiple BH3-only proteins. In factor dependent myeloid (FDM) cell lines, cycloheximide induced apoptosis by a Bax/Bak dependent mechanism, because Bax-/-Bak-/- lines were profoundly resistant, whereas FDM lines lacking one or more genes for BH3-only proteins remained highly sensitive. Addition of cycloheximide led to the rapid loss of Mcl-1 but did not affect the expression of other Bcl-2 family proteins. In support of these findings, similar results were observed by treating FDM cells with the CDK inhibitor, roscovitine. Roscovitine reduced Mcl-1 abundance and caused Bax/Bak dependent cell death, yet FDM lines lacking one or more genes for BH3-only proteins remained highly sensitive. Therefore Bax/Bak dependent apoptosis can be regulated by the abundance of anti-apoptotic Bcl-2 family members such as Mcl-1, independently of several known BH3-only proteins.

Abstract 2282: Rapid identification of targetable CSF3R mutations that define neutrophilic leukemia by combining functional and genomic screens.

Abstract Large scale sequencing efforts are currently being applied to numerous cancers and although significant numbers of mutations are being identified, functional validation to identify driver mutations is lagging. To accelerate progress in the identification of genetic drivers in hematological malignances, we are taking an integrated approach of deep sequencing coupled with functional screens using molecularly targeted drugs and siRNA screening of primary patient leukemia samples. The functional screens allow prioritization of mutations for validation. Using this approach, we identified a CSF3R mutation in a patient with chronic neutrophilic leukemia (CNL). Patients with CNL and the related disorder, atypical chronic myeloid leukemia (aCML), have neoplastic expansion of granulocytic cells with the diagnosis including exclusion of genetic drivers known to occur in other types of World Health Organization-defined myeloproliferative neoplasms (MPN) or myelodysplastic syndrome/MPN overlap disorders. After identifying a CSF3R mutation in our index case, we examined 21 additional cases of CNL and aCML and found that 59% of patients with CNL/aCML harbor CSF3R mutations. Two distinct regions of mutations within the CSF3R gene were identified. Expression of these classes of mutations in a growth factor-dependent myeloid cell line, BaF3, transformed these cells to growth factor independence. Interestingly, these two mutational classes produce preferential downstream kinase signaling (via TNK2/SRC or JAK kinases) and differential sensitivity to kinase inhibitors. Specifically, mutants signaling through TNK2/SRC are sensitive to SRC inhibitors such as dasatinib while mutants signaling thought JAK kinases were sensitive to JAK inhibitors such as ruxolitinib. One patient with CNL carrying a CSF3R mutation that signals through JAK kinases showed a dramatic and durable clinical improvement after treatment with the JAK1/2 kinase inhibitor, ruxolitinib. Mutations in the CSF3R define a large subset of patients with CNL/aCML and testing for these mutations will complement histopathologic diagnosis of CNL and aCML. Patients with CSF3R mutations may benefit from clinical administration of TNK2/SRC inhibitors (such as dasatinib) or JAK inhibitors (such as ruxolitinib). Our studies demonstrate that combining functional screens with genome sequencing can significantly accelerate target validation to define driving mutations that can be matched with therapies. Citation Format: Julia Maxson, Jason Gotlib, Daniel Pollyea, Angela Fleischman, Christopher Eide, Daniel Bottomly, Beth Wilmot, Shannon McWeeney, Cristina Tognon, J. Blake Pond, Robert Collins, Michael Deininger, Bill Chang, Marc Loriaux, Brian Druker, Jeffrey Tyner. Rapid identification of targetable CSF3R mutations that define neutrophilic leukemia by combining functional and genomic screens. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2282. doi:10.1158/1538-7445.AM2013-2282

Up-regulation of Translation Eukaryotic Initiation Factor 4E in Nucleophosmin 1 Haploinsufficient Cells Results in Changes in CCAAT Enhancer-binding Protein α Activity: IMPLICATIONS IN MYELODYSPLASTIC SYNDROME AND ACUTE MYELOID LEUKEMIA

NPM1 is a ubiquitously expressed nucleolar phosphoprotein, the gene for which maps to chromosome 5q35 in close proximity to a commonly deleted region associated with (del)5q, a type of myelodysplastic syndrome (MDS). This region is also a frequent target of deletions in de novo and therapy-related MDS/acute myeloid leukemia. Previous studies have shown that Npm1(+/-) mice develop an MDS-like disease that transforms to acute myeloid leukemia over time. To better understand the mechanism by which NPM1 haploinsufficiency causes an MDS phenotype, we generated factor-dependent myeloid cell lines from the bone marrow of Npm1(+/+) and Npm1(+/-) mice and demonstrated compromised neutrophil-specific gene expression in the MNPM1(+/-) cells. We attribute these observations to increased levels of the shorter, dominant negative leukemogenic isoform (p30) of CCAAT enhancer-binding protein α (C/EBPα). We show that this increase is caused, in part, by elevated levels of the activated translation initiation factor eIF4E, overexpression of which also increases translation of C/EBPαp30 in HEK293 cells. In a positive feedback loop, eIF4E expression is further elevated both at the mRNA and protein levels by C/EBPαp30 but not by the full-length C/EBPαp42. Re-expression of C/EBPαp42 or NPM1 but not C/EBPαp30 in MNPM1(+/-) cells partially rescues the myeloid phenotype. Our observations suggest that the aberrant feed-forward pathway that keeps eIF4E and C/EBPαp30 elevated in NPM1(+/-) cells contributes to the MDS phenotype associated with NPM1 deficiency.

Upregulation of eIF4E in Nucleophosmin 1 (NPM1) Haploinsufficient Cells Alters CCAAT Enhancer Binding Protein Alpha (C/EBPα) Activity: Implications for MDS and AML

Abstract 2432NPM1, is a highly conserved, ubiquitous nucleolar phosphoprotein that belongs to the nucleoplasmin family of nuclear chaperones. NPM1−/− mice die at mid-gestation (E11.5) from anemia, underscoring the gene’s role in embryonic development. NPM1 is one of the most frequently mutated genes in AML. Mutations in NPM1 are found in 50% of normal karyotype AML patients, and mutant NPM1 (NPMc+) is aberrantly located in the cytoplasm of leukemic blasts in about 35% of all AML patients. Furthermore, NPM1 maps to a region on chromosome 5q that is the target of deletions in both de novo and therapy-associated human MDS. NPM1 thus acts as a haploinsufficient tumor suppressor in the hematological compartment, although the mechanism of its contribution to dysmyelopoiesis remains unknown. NPM-1+/− mice develop a hematological syndrome similar to that observed in human MDS, and develop AML over time. The NPM1 deficient model therefore provides a platform to interrogate the molecular basis of MDS.We identified nucleophosmin (NPM1) in a screen for protein binding partners of C/EBPα. C/EBPα is a single exon gene, but is expressed as two isoforms that arise by alternate translation start sites to yield a full length C/EBPα p42 and a truncated dominant negative C/EBPα p30 isoform. Translational control of isoform expression is orchestrated by a conserved upstream open reading frame (uORF) in the 5’ untranslated region (5’UTR) and modulated by the translation initiation factors eIF4E and eIF2. We generated factor-dependent myeloid cell lines from the bone marrow of Npm1+/+ and Npm1+/− mice. These lines are IL-3-dependent and inducible toward neutrophil maturation with GM-CSF and/ or all- trans retinoic acid (ATRA). Neutrophils derived from MNPM1+/− cells display defective neutrophil-specific gene expression, including a cassette of C/EBPα-dependent genes. These observations led us to postulate that myeloid abnormalities in NPM1 deficiency reflect an aberrant NPM1-C/EBPα axis. We show that NPM1 haploinsufficiency upregulates eIF4E (eukaryotic initiation factor 4E) (but not eIF2), which binds the mRNA-Cap (m7-GTP) as part of the mRNA translation initiation complex, eIF4F. Increased eIF4E is observed in about 30% of all malignancies. Initial increased eIF4E levels in MNPM+/− cells likely reflect transcriptional activation by the oncoprotein c-Myc, protein levels of which are also elevated in MNPM1+/− cells. We propose that increased eIF4E then induces increased C/EBPαp30 translation. C/EBPαp30 is a dominant negative inhibitor of full length C/EBPαp42 activity and disrupts normal neutrophil development. Furthermore, we demonstrate that C/EBPαp30 but not C/EBPαp42, activates the eIF4E promoter. We propose a positive feedback loop, wherein increased C/EBPαp30 induced by eIF4E further increases the expression of eIF4E. Our data suggest that NPM1 deficiency modulates neutrophil-specific gene expression by altering C/EBPα. We propose an aberrant feed-forward mechanism that increases levels of both eIF4E and C/EBPαp30 and likely contributes to MDS associated with NPM1 deficiency. Disclosures:No relevant conflicts of interest to declare.

Nucleophosmin-1 Interacts with CCAAT Enhancer Binding Protein Alpha (C/EBPα) to Facilitate Granulocyte Maturation: Implications in MDS and AML.

Abstract 2768Poster Board II-744NPM-1, a gene that resides on chromosome 5q, is a highly conserved, ubiquitously expressed nucleolar phosphoprotein that belongs to the nucloeplasmin family of nuclear chaperones. NPM-1 is a multifunctional protein and has been implicated in ribosome biogenesis and the transport of pre-ribosomal particles, maintenance of genomic stability by the control of cellular ploidy, in DNA repair and in the regulation of DNA transcription by controlling chromatin condensation/decondensation. NPM1−/− mice die at mid-gestation (E11.5) due to severe anemia, underscoring the role of this gene in embryonic development. NPM-1 has been shown to be deleted or involved with chromosomal translocations in several hematologic malignancies. Mutant NPM-1 (NPMc+) has been found to be aberrantly located in the cytoplasm of leukemic blasts in about 35% of all AML patients. Additionally, mutations in the NPM1 gene have been described in 50% of normal karyotype AML patients. This makes NPM-1 one of the most frequently mutated genes in AML. The NPM-1 gene maps to a region on chromosome 5q that is the target of deletions in both denovo and therapy-associated MDS in humans, and is deleted in 50% of MDS associated with 5q deletions. NPM-1+/− mice develop a hematological syndrome similar to that observed in MDS patients. NPM-1 thus appears to act as a haploinsufficient tumor suppressor in the hematological compartment, the molecular basis of which remains unexplored. The NPM-1 deficient model provides an excellent platform to interrogate not only the molecular basis of MDS but also to study AML progression. To further explore the role of NPM1 in myeloid malignancy, we have generated factor-dependent myeloid cell lines from the bone marrow of Npm1+/+ and Npm1+/− mice. We demonstrate compromised neutrophil function and neutrophil-specific gene expression in the NPM1+/− cells similar to that observed in Npm1+/− mouse bone marrow, and attribute these observations to decreased expression of the master myeloid regulator C/EBPα in the Npm1+/− cells. We have demonstrated that overexpression of wildtype NPM1 but not mutant NPM1 (NPMc+) upregulates C/EBPα expression, and that overexpression of C/EBPα in Npm1 deficient cells rescues the myeloid phenotype. We further demonstrate by co-immunoprecipitation and oligonucleotide pulldown assays that NPM1 and C/EBPα physically interact. Our observations suggest that as a result of protein-protein interactions at specific cis elements in the promoters of C/EBPα target genes, C/EBPα and NPM1 act in concert to facilitate normal neutrophil gene expression and function. Interestingly, mutations in the C/EBPα gene have also been implicated in normal karyotype AML. We demonstrate for the first time a connection between these two components of the myeloid compartment, dysregulation of which can lead to disruption of the myeloid maturation program. Taken together, our observations may explain the defective myeloid phenotype observed when NPM1 expression is compromised in MDS and AML. We are now poised to examine the effects of knocking down genes such as RPS14 (the pathogenic gene in the 5q- syndrome) or overexpressing ones such as FLT3-ITD (associated with MDS/AML harboring NPM1 mutations) in our factor-dependent NPM1+/− cell line, to determine if these genes cooperate with NPM1 deficiency to render the cells factor independent. This cell system should be a useful model that will allow us to further dissect the role of NPM-1 in MDS and AML at the molecular level. Disclosures:No relevant conflicts of interest to declare.

Generation of a Factor Dependent Myeloid Cell Line from Nucleophosmin-1 Heterozygous (NPM-1+/−) Mouse Bone Marrow as a Model for 5q- MDS

Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal disorders characterized by ineffective and disordered hematopoiesis with an increased risk of transformation to acute myeloid leukemia (AML). Interstitial deletion of 5q (del 5q) is the most frequent chromosomal abnormality seen in MDS. Two subgroups of MDS with del 5q have been described. The first, the so called “5q-syndrome” is defined by isolated del 5q and no excess blasts in the bone marrow, female predominance, typical dysmegakaryopoiesis, thrombocytosis and a favorable outcome. The second subgroup involves MDS with del 5q associated with an excess of marrow blasts and/or chromosomal abnormalities in addition to del 5q, that usually do not have the typical features of the 5q-syndrome and carry poorer prognosis. Haploinsufficiency for the ribosomal protein RPS14 gene has recently been identified as a compelling candidate gene for causing the 5q- syndrome, but the genes responsible for poor prognosis del 5q MDS remain to be determined. NPM-1, a highly conserved, ubiquitously expressed nucleolar phosphoprotein that belongs to the nucloeplasmin family of nuclear chaperones, maps to a region on chromosome 5q that is the target of deletions in both denovo and therapy-associated MDS in humans.. NPM-1 has been implicated in ribosome biogenesis and the transport of pre-ribosomal particles, maintenance of genomic stability by the control of cellular ploidy, in DNA repair and in the regulation of DNA transcription by controlling chromatin condensation/decondensation. NPM1−/− mice die at mid-gestion (E11.5) due to severe anemia. NPM-1 has been shown to be deleted or involved with chromosomal translocations in hematologic malignancies, and is one of the most frequently mutated genes in AML. NPM-1+/− mice develop a hematological syndrome very similar to that observed in MDS patients. The haploinsufficient NPM-1 model therefore provides an excellent platform to examine not only MDS but also to study AML progression. Studies on the molecular mechanisms underlying 5q- syndrome have been hampered by the lack of in vitro model cell lines. We have generated a factor dependent myeloid cell line from the bone marrow of NPM-1+/+ and NPM-1+/− mice using retroviral transduction of a truncated retinoic acid receptor alpha gene. The resulting cell lines are IL-3-dependent and can be induced to undergo neutrophil maturation by the addition of GM-CSF and/or all- trans retinoic acid (ATRA). We have shown that, like neutrophils derived from the bone marrow of NPM1+/− mice, the NPM-1+/− cell line-derived neutrophils display both defective neutrophil-specific gene expression and abnormal neutrophil function. We found in particular, that the expression of the myeloid master regulator C/EBPα but not that of the related myeloid-specific C/EBPε gene, was significantly decreased in NPM-1+/− cells as compared to their wild type counterparts (NPM-1+/+). While neutrophils derived from NPM1+/− cells appear morphologically normal, the expression of both neutrophil primary granule protein genes (defensins) and secondary granule protein genes (lactoferrin, MMP8 and MMP9) was significantly decreased. The relationship between NPM-1 and C/EBPα is currently being investigated. Additionally, the function of NPM-1+/− derived neutrophils was compromised as assessed by ROS production as well as by chemotaxis assays. The defective oxidative burst may be the result of decreased expression of a key component of the neutrophil NADPH oxidase complex i.e. gp91phox, which is critical for ROS production. We confirmed that these neutrophil-associated defects were also seen in primary neutrophils from the NPM-1+/− mice. We are currently examining the effect of knocking down RPS14 in our factor-dependent NPM-1+/− cells to determine if the two genes cooperate to render the cells factor independent. This cell line will help to delineate the functions of NPM-1 and provide a platform for examining the potential role of haploinsufficiency of NPM-1 in the propensity for 5q- MDS to progress to AML.

Altered Expression of PP2A Regulatory Subunits in Chronic Myelogenous Leukemia: Identifying Targets for Improved Therapies.

Over 90% of chronic myelogenous leukemia (CML) cases are associated with constitutive activation of the BCR/ABL oncoprotein, which induces transformation of hematopoietic stem cells. Inhibition of BCR/ABL with imatinib has shown remarkable success in treating CML patients. However, the emergence of resistance is becoming a major clinical problem. To direct the development of improved therapies, signaling pathways activated by BCR/ABL must be identified. The aim of this project is to determine whether the activity and expression of the tumor suppressor protein phosphatase 2A (PP2A) is altered in the presence of BCR/ABL. PP2A is a serine/threonine phosphatase that is composed of a catalytic subunit (C), a structural subunit (A), and a variable regulatory subunit (B), of which there are three different families. Each family of PP2A-B subunits has a number of isoforms that determine the substrate specificity, subcellular localization and catalytic activity of the enzyme. In this study we show that transfection of BCR/ABL into FDC-P1, a mouse factor-dependent myeloid cell line, increased tyrosine phosphorylation on the PP2A-C subunit, and this correlated with a reduction in enzymatic activity. Imatinib treatment rescued PP2A activity in wild type BCR/ABL+ cells, but not the imatinib-resistant Y253F BCR/ABL+ cells. Importantly, pharmacological enhancement of PP2A by forskolin and FTY720 resulted in reduced proliferation in both cell lines. Furthermore, we have demonstrated for the first time that BCR/ABL increases the expression of three specific PP2A-B subunit isoforms - B55α, B56α, and B56δ. It is predicted that enhanced levels of these subunits cause displacement of alternate PP2A-B isoforms to alter enzyme composition and hence the specificity of PP2A. To determine the functional role of each individual B subunit, shRNA studies are being performed. These findings confirm that functional inactivation of PP2A is an essential component of BCR/ABL-induced leukemogenesis in CML, and provide novel insight into the mechanisms by which this may occur. Elucidating the importance of each regulatory subunit may identify novel targets to develop improved therapies for CML patients.

C-kit associated with the transmembrane 4 superfamily proteins constitutes a functionally distinct subunit in human hematopoietic progenitors

The transmembrane 4 superfamily (TM4SF) has come into prominence for its association with a wide range of cell surface molecules, especially integrins. We report that TM4SF molecules CD9, CD63, and CD81 are physically associated with c-kit receptor tyrosine kinase in the human factor-dependent myeloid cell line, MO7e. We characterized this complex using coimmunoprecipitation and colocalization methods. The c-kit coimmunoprecipitated with anti-TM4SF antibodies showed several distinct phenotypes compared to the total c-kit immunoprecipitated with anti-c-kit antibody. These included: (1) higher basal level of tyrosine phosphorylation without elevated kinase activity in the absence of Steel factor (SLF), (2) deficient enhancement of tyrosine phosphorylation and kinase activity in response to SLF, (3) elevated binding rate of SLF shown in chemical cross-linking studies, and (4) little internalization and degradation after SLF treatment. Cocapping studies in living cells showed that c-kit colocalized with TM4SF molecules after SLF stimulation, suggesting confirmation of the biochemical data obtained by the coimmunoprecipitation studies. Colocalization of c-kit with CD81 by SLF was also observed in cord blood CD34(+) cells, suggesting the existence of functional units of c-kit in TM4SF complexes in primary hematopoietic cells. This suggests that some TM4SF members may negatively modulate function of c-kit receptor tyrosine kinase and thus regulate receptor sensitivity to SLF in hematopoietic progenitors.

Estrogen-dependent E2a/Pbx1 myeloid cell lines exhibit conditional differentiation that can be arrested by other leukemic oncoproteins

AbstractThe molecular pathways of normal myeloid differentiation, as well as the mechanisms by which oncogenes disrupt this process, remain poorly understood. A major limitation in approaching this problem has been the lack of suitable cell lines that exhibit normal, terminal, and synchronous differentiation in the absence of endogenous oncoproteins and in response to physiologic cytokines, and whose differentiation can be arrested by ectopically expressed human oncoproteins. This report describes clonal, granulocyte-macrophage colony-stimulating factor-dependent myeloid cell lines that exhibit these properties. The cell lines were established by conditional immortalization of primary murine marrow progenitors with an estrogen-regulated E2a/Pbx1-estrogen receptor fusion protein. Clones were identified that proliferated as immortalized blasts in the presence of estrogen, and that exhibited granulocytic, monocytic, or bipotential (granulocytic and monocytic) differentiation on estrogen withdrawal. Differentiation was normal and terminal as evidenced by morphology, cell surface markers, gene expression, and functional assays. The differentiation of the cells could be arrested by heterologous oncoproteins including AML1/ETO, PML/RARα, PLZF/RARα, Nup98/HoxA9, and other Hox proteins. Furthermore, the study examined the effects of cooperating oncoproteins such as Ras or Bcr/Abl, which allowed for both factor-independent proliferation and differentiation, or Bcl-2, which permitted factor-independent survival but not proliferation. These myeloid cell lines provide tools for examining the biochemical and genetic pathways that accompany normal differentiation as well as a system in which to dissect how other leukemic oncoproteins interfere with these pathways.

HIV-1 Nef Promotes Survival of Myeloid Cells by a Stat3-dependent Pathway

Human immunodeficiency virus Nef is a small myristylated protein that plays a critical role in AIDS progression. Nef binds with high affinity to the SH3 domain of the myeloid-restricted tyrosine kinase Hck in vitro, identifying this Src-related kinase as a possible cellular target for Nef in macrophages. Here we show that Nef activates endogenous Hck in the granulocyte-macrophage colony-stimulating factor-dependent myeloid cell line, TF-1. Unexpectedly, Nef induced cytokine-independent TF-1 cell outgrowth and constitutive activation of the Stat3 transcription factor. Induction of survival required the Nef SH3 binding and membrane-targeting motifs and was blocked by dominant-negative Stat3 mutants. Nef also stimulated Stat3 activation in primary human macrophages, providing evidence for Stat3 as a Nef effector in a target cell for human immunodeficiency virus.

Constitutive activation of FLT3 in acute myeloid leukaemia and its consequences for growth of 32D cells.

The receptor tyrosine kinase Flt3 is expressed on leukaemic blasts of most cases with acute myeloid leukaemia (AML). In order to evaluate the presence and significance of constitutive activation of Flt3 for leukaemogenesis, we (1) analysed the expression and activation status of the receptor in AML blasts; and (2) evaluated the functional consequences of constitutively active Flt3 in a myeloid progenitor cell line. Immunoprecipitation studies revealed Flt3 expression in a high proportion of AML cases (27/32) with ligand-dependent Flt3 autophosphorylation in 18, constitutive autophosphorylation in three and no autophosphorylation in six cases. Only one out of three samples with constitutively active Flt3 but 3/18 samples with ligand-dependent autophosphorylated Flt3 contained the recently described internal tandem repeat (ITR) mutations. To test the significance of Flt3 activation in myeloid cell function, we also characterized the biochemical and biological effects of the activating mutation D838V of Flt3 (FLt3D838V) on the factor-dependent myeloid progenitor cell line 32Dcl3: cells transfected with wild-type Flt3 (32D/Flt3) grew FLt3 ligand (FL) dependent, and the receptor was ligand dependently autophosphorylated. In contrast, the receptor was constitutively autophosphorylated in 32D/Flt3D838V cells, which grew independently of FL. We conclude that, in some AML samples, Flt3 is constitutively activated and that this does not correlate with ITR mutations in the juxtamembrane domain. Furthermore, constitutively active Flt3 confers factor independence to the myeloid progenitor cell line 32D. It remains to be determined whether activation of Flt3 is leukaemogenic in vivo and whether strategies aimed at inhibition of Flt3 activation could inhibit leukaemogenesis.

Regulation of myeloid cell growth by distinct effectors of Ras.

To examine the biochemical pathways by which activated Ha-Ras(G12V) (Ha-RasV12) induces factor-independent growth of myeloid cells, Ha-Ras effector loop mutations, including Y40C, T35S, and E37G, were analysed in a mouse factor-dependent myeloid cell line, WT19. Expression of a single effector loop mutant, Ha-Ras(G12V, Y40C) (Ha-RasV12C40), inhibited factor-withdrawal apoptosis, suggesting that activation of the phosphatidylinositol 3'-kinase (Pl3K) pathway is essential to prevent cell death. Neither Ha-Ras (G12V, T35S) (Ha-RasV12S35), which activates the Rafl signaling pathway, nor Ha-Ras(G12V, E37G) (Ha-RasV12G37), which stimulates the RalGDS pathway, did not have significant effects on factor-withdrawal apoptosis of myeloid cells. Although Ha-RasV12C40 inhibited apoptosis, it did not stimulate entry into the cell cycle. Cell lines containing the combination of Ha-RasV12G37 and Ha-RasV12C40 were capable of factor-independent cell growth, while expression of the other combinations of the Ha-Ras effector mutants were not. The combined expression of Bcl-2 and Ha-RasV12G37 was not sufficient to stimulate factor independent growth, suggesting that Ha-RasV12C40 activates additional signals, besides blocking apoptosis, which are critical for factor-independent growth of myeloid cells. In factor-starved myeloid cells, inducible expression of Ha-RasV12G37 results in decreased level of p27Kip1 protein, a cyclin-dependent kinase inhibitor (CKI). These data suggest that the factor-independent growth of myeloid cells requires the activation of at least two pathways, one inhibiting factor-withdrawal apoptosis, and another causing cell cycle progression.

Human and Xenopus cDNAs encoding budding yeast Cdc7-related kinases: in vitro phosphorylation of MCM subunits by a putative human homologue of Cdc7.

Saccharomyces cerevisiae Cdc7 kinase is essential for initiation of DNA replication, and Hsk1, a related kinase of Schizosaccharomyces pombe, is also required for DNA replication of fission yeast cells. We report here cDNAs encoding Cdc7-related kinases from human and Xenopus (huCdc7 and xeCdc7, respectively). The cloned cDNA for huCdc7 contains an open reading frame consisting of 574 amino acids with a predicted molecular weight of 63,847 that possesses overall amino acid identity of 32% (54% including similar residues) to Cdc7 and Hsk1. huCDC7 is transcribed in the various tissues examined, but most abundantly in testis. Three transcripts of 4.4, 3.5 and 2.4 kb in length are detected. The 3.5 kb transcript is the most predominant and is expressed in all the tissues examined. A cDNA containing a 91 nucleotide insertion at the N-terminal region of huCDC7 is also detected, suggesting the presence of multiple splicing variants. The huCdc7 protein is expressed at a constant level during the mitotic cell cycle and is localized primarily in nuclei in interphase and distributed diffusibly in cytoplasm in the mitotic phase. The wild-type huCdc7 protein expressed in COS7 cells phosphorylates MCM2 and MCM3 proteins in vitro, suggesting that huCdc7 may regulate processes of DNA replication by modulating MCM functions.

The cytoplasmic domain of granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor alpha subunit is essential for both GM-CSF-mediated growth and differentiation.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) regulates differentiation, survival, and proliferation of colony-forming unit-granulocyte-macrophage progenitor cells. The biologic actions of GM-CSF are mediated by binding to a specific receptor consisting of two chains designated as alpha and beta subunits. We have demonstrated that the murine FDC-P1-derived cell line WT-19 transfected with the human GM-CSF receptor alpha and beta subunits (GM-CSFRalpha and beta) can be induced to differentiate by the addition of human GM-CSF (hGM-CSF). By expressing a series of GM-CSFRalpha mutants in WT19 cells, we have determined the amino acid domains of the GM-CSFRalpha cytoplasmic domain that regulate cell differentiation, proliferation, and survival. We found that the membrane proximal proline-rich domain and adjacent 16 residues are essential for both hGM-CSF-dependent cell proliferation and differentiation. In contrast, the C-terminal region of the GM-CSFRalpha cytoplasmic domain was not necessary for cell differentiation mediated by hGM-CSF, but the removal of this region severely impaired the ability of hGM-CSF to support cell survival. While the activation of JAK2, Shc, Erk, and STAT5 proteins correlated with hGM-CSF-mediated cell growth, cellular differentiation occurred in the absence of activation of these signal transduction pathways.

The hemopoietic growth factor, interleukin-3, promotes glucose transport by increasing the specific activity and maintaining the affinity for glucose of plasma membrane glucose transporters.

Most mammalian cells rely on an external supply of glucose for survival, proliferation, and function. Glucose enters cells through specific transporter molecules at the plasma membrane by a facilitative process that does not expend energy. Regulation of glucose transport into cells is thought to occur largely through transporter expression at the cell surface, but the extent to which the intrinsic properties of glucose transporters are regulated is at present controversial. Using a bone marrow-derived cell line that responds to the hemopoietic growth factor, interleukin-3 (IL-3), we investigated IL-3 regulation of glucose transport. IL-3 significantly increased 2-deoxyglucose (2-DOG) uptake within 1 h (26 +/- 8.0%, n = 11) with a maximum 73% increase after 6 h. Withdrawal of IL-3 resulted in decreased uptake within 1 h and this continued to decline to 43% of initial uptake by 16 h. To determine whether these changes in 2-DOG uptake were associated with corresponding changes in glucose transporter expression, subtype-specific antisera against Glut-1 and Glut-3 were used. Little change in membrane expression of these transporters was observed prior to 16 h. Fractionation of cell membranes on Nycodenz gradients showed that the majority of each transporter subtype was associated with the plasma membrane (63-93%) and that transporter distribution did not change markedly in response to addition or withdrawal of IL-3. These results demonstrate that IL-3 regulates glucose uptake by modulating the intrinsic transporting ability of glucose transporters. Decreased transporter affinity for 2-DOG and 3-O-methylglucose was observed following IL-3 withdrawal. Similar affinity changes were observed with 2-DOG following exposure of IL-3-stimulated cells to the protein kinase inhibitors, genistein and staurosporine. In contrast, the tyrosine phosphatase inhibitor, vanadate, acted like IL-3 to increase transporter affinity for glucose. Together these results demonstrate that IL-3 acts to maintain the intrinsic transport properties of glucose transporters without markedly affecting their expression or translocation.

Involvement of p21cip-1 and p27kip-1 in the Molecular Mechanisms of Steel Factor-Induced Proliferative Synergy In Vitro and of p21cip-1 in the Maintenance of Stem/Progenitor Cells In Vivo

Steel factor (SLF) is a hematopoietic cytokine that synergizes with other growth factors to induce a greatly enhanced proliferative state of hematopoietic progenitor cells and factor-dependent cell lines. Even though the in vivo importance of SLF in the maintenance and responsiveness of stem and progenitor cells is well documented, the molecular mechanism involved in its synergistic effects are mainly unknown. Some factor-dependent myeloid cell lines respond to the synergistic proliferative effects of SLF plus other cytokines in a manner similar to that of normal myeloid progenitor cells from bone marrow and cord blood. We show here that SLF can synergize with granulocyte-macrophage colony-stimulating factor (GM-CSF) to induce an enhanced phosphorylation of the retinoblastoma gene product and a synergistic increase in the total intracellular protein level of the cyclin-dependent kinase inhibitor, p21cip-1, which is correlated with a simultaneous decrease in p27kip-1 in the human factor-dependent myeloid cell line, M07e. Moreover, these cytokines synergize to increase p21cip-1 binding and decrease p27kip-1 binding to cyclin-dependent kinase-2 (cdk2), an enzyme required for normal cell cycle progression; these inverse events correlated with increased cdk2 kinase activity. It is also shown that exogenous purified p21cip-1 can displace p27kip-1 already bound to cdk2 in vitro. These data implicate increased p21cip-1 and decreased p27kip-1 intracellular concentrations and their stoichiometric interplay in the enhanced proliferative status of cells stimulated by the combination of SLF and GM-CSF. In support of these findings, it is shown that hematopoietic progenitor cells from mice lacking p21cip-1 are defective in SLF synergistic proliferative response in vitro. Moreover, the cycling status of marrow and spleen progenitors and absolute numbers of marrow progenitors were significantly decreased in the p21cip-1 -/-, compared with the +/+ mice. We conclude that the cdk threshold regulators p21cip-1 and p27kip-1 play a critical role in the normal mitogenic response of M07e cells and murine myeloid progenitor cells to these cytokines and particularly in the SLF synergistic proliferative response that is important to the normal maintenance of the stem/progenitor cell compartment.

Steel factor induces serine phosphorylation of Stat3 in human growth factor-dependent myeloid cell lines

Steel factor (SLF) acts synergistically with various hematopoietic growth factors that use the Jak-Stat pathways in vivo and in vitro, although the contribution by SLF to this pathway is unknown. We show here that SLF induces time- and dose-dependent phosphorylation of Stat3 in the human growth factor-dependent cell lines MO7e and TF-1. This phosphorylation occurs exclusively on serine residues. Simultaneous stimulation with SLF plus other cytokines that induce tyrosine phosphorylation of Stat3, such as interleukin-9 (IL-9) in MO7e cells or IL-6 in TF-1 cells, resulted in tyrosine phosphorylation and enhanced serine phosphorylation of Stat3. Serine phosphorylation alone did not promote nuclear translocation or DNA binding activity to the sis-inducible element of Stat3. However, costimulation with SLF plus IL-9 in MO7e cells resulted in the nuclear translocation of serine-hyperphosphorylated Stat3. Serine phosphorylation of Stat3 was also observed by the stimulation of cells with granulocyte-macrophage colony-stimulating factor and IL-3, which do not induce tyrosine phosphorylation of Stat3. These results suggest that SLF might modulate the Jak-Stat3 pathway by serine phosphorylation and that the Jak-Stat pathway may be differentially regulated by the combinational stimulation of two or more cytokines.

Steel factor induces serine phosphorylation of Stat3 in human growth factor-dependent myeloid cell lines

Abstract Steel factor (SLF) acts synergistically with various hematopoietic growth factors that use the Jak-Stat pathways in vivo and in vitro, although the contribution by SLF to this pathway is unknown. We show here that SLF induces time- and dose-dependent phosphorylation of Stat3 in the human growth factor-dependent cell lines MO7e and TF-1. This phosphorylation occurs exclusively on serine residues. Simultaneous stimulation with SLF plus other cytokines that induce tyrosine phosphorylation of Stat3, such as interleukin-9 (IL-9) in MO7e cells or IL-6 in TF-1 cells, resulted in tyrosine phosphorylation and enhanced serine phosphorylation of Stat3. Serine phosphorylation alone did not promote nuclear translocation or DNA binding activity to the sis- inducible element of Stat3. However, costimulation with SLF plus IL-9 in MO7e cells resulted in the nuclear translocation of serine- hyperphosphorylated Stat3. Serine phosphorylation of Stat3 was also observed by the stimulation of cells with granulocyte-macrophage colony- stimulating factor and IL-3, which do not induce tyrosine phosphorylation of Stat3. These results suggest that SLF might modulate the Jak-Stat3 pathway by serine phosphorylation and that the Jak-Stat pathway may be differentially regulated by the combinational stimulation of two or more cytokines.

Granulocyte-Macrophage Colony-stimulating Factor Induces the Transcriptional Activation of egr-1 through a Protein Kinase A-independent Signaling Pathway

Granulocyte-macrophage colony-stimulating factor (GM-CSF) rapidly and transiently induces the transcriptional activation of the early growth response gene-1 (egr-1) in the human factor-dependent myeloid leukemic cell line, TF-1. We previously demonstrated that the cAMP response element (CRE) is required for GM-CSF-induced egr-1 expression and that phosphorylation of CREB on serine 133 plays a critical role during GM-CSF signal transduction. To determine whether GM-CSF activates signaling pathways through a protein kinase A-dependent or -independent pathway, we measured cAMP levels following GM-CSF or forskolin treatment of TF-1 cells. Forskolin but not GM-CSF stimulation resulted in an increase in cAMP levels. Transient transfection assays with TF-1 cells were also performed with a -116-nucleotide egr-1 promoter construct and the protein kinase inhibitor, PKI. Although PKI inhibited forskolin induction of the -116-nucleotide construct, it did not affect GM-CSF stimulation of this construct. In the present study, we demonstrated that GM-CSF induces egr-1 expression through a protein kinase A-independent pathway.

Membrane-Bound Steel Factor Induces More Persistent Tyrosine Kinase Activation and Longer Life Span of c-kit Gene-Encoded Protein Than Its Soluble Form

Alternative splicing of exon 6 results in the production of two isoforms of Steel factor (SLF): the membrane-bound and soluble forms. To investigate differences in the kinetics of c-kit tyrosine kinase activated by these two isoforms, we used a stromal cell line (SI/SI4) established from SI/SI homozygous murine embryo fetal liver and its stable transfectants containing either hSCF248 cDNA (including exon 6; secreted form) or hSCF220 cDNA (lacking exon 6; membrane-bound form) as the source of each isoform. Interaction of factor dependent myeloid cell line MO7e with stromal cells producing either isoform resulted in activated c-kit tyrosine kinase and induction of the same series of tyrosine phosphorylated cellular proteins in MO7e cells. However, SI4-h220 (membrane-bound form) induced more persistent activation of c-kit kinase than SI4-h248 (soluble form) did. Flow cytometric analysis and pulse-chase studies using [35S]methionine showed that SI4-h248 induced rapid downmodulation of cell-surface c-kit expression and its protein degradation in MO7e cells, whereas SI4-h220 induced more prolonged life span of c-kit protein. Addition of soluble recombinant human SLF to SI4-h220 cultures enhanced reduction of cell-surface c-kit expression and its protein degradation. Because the kinetics of c-kit inactivation strikingly fits with the protein degradation rates of c-kit under the conditions described above, rapid proteolysis of c-kit protein induced by soluble SLF stimulation may function as a "turn-off switch" for activated c-kit kinase.