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A D Whetton

Publications and source records attributed to A D Whetton.

At least 19 recordsLinked to original sources
PubMed2006

FMIP controls the adipocyte lineage commitment of C2C12 cells by downmodulation of C/EBP alpha.

Fms interacting protein (FMIP) is a substrate for Fms tyrosine kinase, and a nuclear/cytoplasm shuttling protein with a leucine zipper. As the phosphorylation of FMIP is observed in insulin-stimulated preadipocytes, we examined the role of FMIP in adipocyte differentiation, using the mesenchymal multipotent stem cells, C2C12 cells, that can differentiate into adipocytes, muscle cells and osteoblasts. Ectopic expression of FMIP in C2C12 impairs the adipocyte differentiation induced by treatment with insulin, dexamethasone and 3-isobutyl-1-methylxanthine. These cells exhibit muscle phenotype with multinuclear morphology. Furthermore, knockdown of endogenous FMIP expression by small interfering RNA improves adipocytic lineage commitment of C2C12 cells, while impairing muscle differentiation. Upon stimulation with insulin, CCAAT/enhancer binding protein (C/EBP)beta, but not C/EBPalpha, is upregulated in cells expressing ectopic FMIP, whereas in FMIP knockdown cells, C/EBPalpha is constitutively expressed. Ectopic expression of C/EBPalpha counteracts the effects of FMIP, whereas C/EBPalpha knockdown partially mimics the effects of FMIP in this system. Northern blot analysis and reverse transcriptase-polymerase chain reaction study reveal that ectopic FMIP-expressing cells do not contain the polyadenylated C/EBPalpha mRNA, but contain the C/EBPalpha pre-mRNA, suggesting that FMIP plays a role in RNA processing and/or export. Indeed, a member of the THO complex that plays a role in mRNA export, THOC1, is co-precipitated with FMIP. The data we have acquired on FMIP suggest that it is a target for tyrosine kinase receptors that potentiate mRNA export.

Adipocytes↗
PubMed2006

Role of the C-terminal actin binding domain in BCR/ABL-mediated survival and drug resistance.

Philadelphia chromosome-positive, chronic myeloid leukaemia (CML) stem and progenitor cells have a survival and growth advantage compared with their normal counterparts. The mechanisms through which the BCR/ABL protein tyrosine kinase (PTK) induces these effects and the important domains within this protein are not fully defined. The F- and G-actin binding region of the BCR/ABL C-terminus may be important in BCR/ABL-mediated events, and we have investigated this by expressing a C-terminus deletion mutant of the temperature-sensitive BCR/ABL PTK, in a haemopoietic progenitor cell line, which models the chronic phase of CML. The truncated BCR/ABL PTK displayed similar levels of PTK activity when compared with wild type and activation of second messenger formation (in the form of sn-1,2-diacylglycerol) remains intact. On fibronectin substrata, localisation of the protein to the periphery of the cell was, however, dependent on the C-terminus of BCR/ABL PTK. Deletion of the C-terminus reversed both BCR/ABL-mediated apoptotic suppression and drug resistance although the progenitor cells did retain a proliferative advantage at low concentrations of growth factor. These results demonstrated that the C-terminal actin-binding domain of BCR/ABL is important for some of BCR/ABL PTK-mediated leukaemogenic effects.

Actins↗
PubMed2001

The specific enhancement of interferon alpha induced growth inhibition by BCR/ABL only occurs in multipotent cells.

INTRODUCTION: The causative oncogene in CML is the BCR/ABL protein tyrosine kinase. This stem cell disease is often treated with interferon alpha (IFN-alpha) which can initiate haematological and cytological remission which is associated with increased survival. There is however no clear indication of why CML cells are more responsive to IFN-alpha. MATERIALS AND METHODS: To establish if BCR/ABL increases the sensitivity of multipotent cells to IFN-alpha a temperature sensitive mutant of BCR/ABL was expressed in the multipotent haemopoietic stem cell line FDCP-Mix. The effect of IFN-alpha in terms of proliferation, induction of apoptosis, changes in cell cycle inhibitor proteins, and differentiation was assessed by [3H]thymidine incorporation, Annexin V and Western blot analysis. RESULTS: When the BCR/ABL tyrosine kinase was activated, the IFN-alpha-induced inhibition on the growth rate of the FDCP-Mix cell population was more marked than in control populations. The BCR/ABL-mediated effect was due to decreased rates of DNA synthesis. There was no IFN-alpha-mediated induction of apoptosis. This enhanced BCR/ABL mediated growth inhibition occurred over a range of growth factor concentrations and was independent of changes in p21(Cip1) and p27(Kip) levels. When FDCP-Mix cells were induced to differentiate into mature macrophages and neutrophils in the presence of IFN-alpha, there was increased sensitivity to IFN-alpha that was independent of BCR/ABL activity. CONCLUSION: BCR/ABL PTK expression in this primitive multipotent haematopoietic cell line results in an enhanced response to IFN-alpha. In contrast, the more mature myeloid progenitor cells are equally responsive to this growth inhibitor. This data may explain some of the clinical effects of IFN-alpha.

Apoptosis↗
PubMed2001

Glucose transport regulation by p210 Bcr-Abl in a chronic myeloid leukaemia model.

The regulation of nutrient transport by both cytokines and oncogenes has been linked to haemopoietic cell survival. In this study, we found that activation of Bcr--Abl protein tyrosine kinase was associated with the stimulation of glucose transport in the multipotent haemopoietic cell line FDCP-mix, a cell model for chronic-phase chronic myeloid leukaemia (CML). Bcr--Abl upregulation of glucose transport was mediated by phosphatidylinositol-3-kinase. The observation that Bcr--Abl can regulate glucose transport in a CML cell model raises the possibility that glucose transport regulation may have a role to play in the aberrant survival of stem cells in the chronic phase of CML.

Biological Transport↗
PubMed2000

Transforming growth factor-beta 1 induces apoptosis independently of p53 and selectively reduces expression of Bcl-2 in multipotent hematopoietic cells.

Transforming growth factor-beta1 (TGF-beta1) can inhibit cell proliferation or induce apoptosis in multipotent hematopoietic cells. To study the mechanisms of TGF-beta1 action on primitive hematopoietic cells, we used the interleukin-3 (IL-3)-dependent, multipotent FDCP-Mix cell line. TGF-beta1-mediated growth inhibition was observed in high concentrations of IL-3, while at lower IL-3 concentrations TGF-beta1 induced apoptosis. The proapoptotic effects of TGF-beta1 occur via a p53-independent pathway, since p53(null) FDCP-Mix demonstrated the same responses to TGF-beta1. IL-3 has been suggested to enhance survival via an increase in (antiapoptotic) Bcl-x(L) expression. In FDCP-Mix cells, neither IL-3 nor TGF-beta1 induced any change in Bcl-x(L) protein levels or the proapoptotic proteins Bad or Bax. However, TGF-beta1 had a major effect on Bcl-2 levels, reducing them in the presence of high and low concentrations of IL-3. Overexpression of Bcl-2 in FDCP-Mix cells rescued them from TGF-beta1-induced apoptosis but was incapable of inhibiting TGF-beta1-mediated growth arrest. We conclude that TGF-beta1-induced cell death is independent of p53 and inhibited by Bcl-2, with no effect on Bcl-x(L). The significance of these results for stem cell survival in bone marrow are discussed.

Animals↗
PubMed2000

v-Abl protein-tyrosine kinase up-regulates p21WAF-1 in cell cycle arrested and proliferating myeloid cells.

v-Abl protein-tyrosine kinase (PTK) promotes cell survival without cell proliferation in interleukin (IL)-3-deprived IC.DP premast cells (1). We now show that in these conditions v-Abl PTK transcriptionally up-regulated the cyclin-dependent kinase inhibitor (CDKI) p21(WAF-1) and inhibited CDK2 and CDK4. When readdition of IL-3 stimulated cell proliferation, p21(WAF-1) was inactivated as a CDKI despite maintenance of elevated protein level. p21(WAF-1) was also up-regulated yet was nonfunctional as a CDKI when v-Abl PTK was activated in cells maintained in IL-3, but this occurred without increased p21(WAF-1) transcription. Using a C-terminal epitope-specific p21(WAF-1) antibody, v-Abl PTK-mediated increase in p21(WAF-1) could be detected in intact cells only in the presence of IL-3. This indicated different binding partners of p21(WAF-1) and/or protein conformation in nondividing or proliferating cells, respectively. The binding of CDK2, CDK4, or proliferating cell nuclear antigen to p21(WAF-1) and its subcellular localization were unchanged in the presence or absence of IL-3. However, two-dimensional analysis revealed different forms of up-regulated p21(WAF-1) in IL-3-deprived, nondividing cells compared with IL-3-stimulated proliferating cells. These data demonstrate that elevation of the CDKI p21(WAF-1) is not always sufficient for cell cycle arrest and indicate an IL-3-sensitive pathway for the inactivation of p21(WAF-1) function as a CDKI.

Animals↗
PubMed2000

Bcr-Abl protein tyrosine kinase activity induces a loss of p53 protein that mediates a delay in myeloid differentiation.

Chronic myeloid leukaemia is a haemopoietic stem cell disorder, the hallmark of which is the expression of the Bcr-Abl Protein Tyrosine Kinase (PTK). We have previously reported that activation of a temperature sensitive Bcr-Abl PTK in the multipotent haemopoietic cell line FDCP-Mix for short periods resulted in subtle changes including, a transient suppression of apoptosis and no inhibition of differentiation. In contrast, activation of the Bcr-Abl PTK for 12 weeks results in cells that display a delay in differentiation at the early granulocyte stage. Flow cytometric analysis also indicates that the expression of cell surface differentiation markers and nuclear morphology are uncoupled. Furthermore, a significant number of the mature neutrophils display abnormal morphological features. Prolonged exposure to Bcr-Abl PTK results in interleukin-3 independent growth and decreased p53 protein levels. FDCP-Mix cells expressing a dominant negative p53 and p53null FDCP-Mix cells demonstrate that the reduction in p53 is causally related to the delay in development. Returning the cells to the restrictive temperature restores the p53 protein levels, the growth factor dependence and largely relieves the effects on development. We conclude that prolonged Bcr-Abl PTK activity within multipotent cells results in a reduction of p53 that drives a delayed and abnormal differentiation.

Animals↗
PubMed2000

Role of phosphatidylinositol 3-kinase and specific protein kinase B isoforms in the suppression of apoptosis mediated by the Abelson protein-tyrosine kinase.

Leukemogenic oncogenes, such as the Abelson protein-tyrosine kinases (PTK), disrupt the normal regulation of survival, proliferation, and differentiation in hemopoietic progenitor cells. In the absence of cytokines, hemopoietic progenitor cells die by apoptosis. Abl PTKs mediate suppression of this apoptotic response leading to aberrant survival. To investigate the mechanism of Abl PTK action, we have used an interleukin-3-dependent murine mast cell line that expresses a temperature-sensitive form of the v-ABL PTK, which is active at the permissive temperature of 32 degrees C and inactive at 39 degrees C. At the permissive temperature, these cells are resistant to apoptosis induced both by the withdrawal of the hemopoietic growth factor (interleukin-3) and the addition of cytotoxic drugs. We demonstrate that v-Abl associates with and stimulates activation of phosphatidylinositol 3-kinase (PI3K) and, crucially, that this activation results in enhanced cellular levels of the mass of the second messenger phosphatidylinositol-3,4,5-trisphosphate. Activation of PI3K leads to enhanced activity of PKB and increased levels of the anti-apoptotic protein Bcl-X(L). Transfection of cells with a dominant negative PKB reduces both the Abl-stimulated PKB activity and the survival effect conferred by activation of this oncogene. Thus, PI3K and PKB are required for the anti-apoptotic effects of Abl PTK.

Animals↗
PubMed2000

Neuropeptide control of bone marrow neutrophil production is mediated by both direct and indirect effects on CFU-GM.

Noradrenaline- and peptide-containing nerve fibres project into the bone marrow and terminate in association with stromal cells and within the parenchyma. Peptidergic nerve terminals are also associated with antigen-processing and -presenting cells throughout the body and have been shown to be important in leucocyte trafficking and wound healing, as well as haemopoiesis. Here, we tested the in vivo effects of deleting the peripheral neuropeptide network on haemopoiesis and also investigated whether the target cell population for these substances was myeloid progenitor cells (colony-forming unit-granulocyte/macrophage, CFU-GM). Deletion of the neuropeptides, substance P (SP) and calcitonin gene-related peptide (CGRP) by capsaicin abrogates normal blood cell production. These neuropeptides produced significant stimulation of colony formation from unfractionated bone marrow and elicited production of soluble factors capable of stimulating highly enriched CFU-GM. CGRP also had a direct stimulatory effect on highly enriched CFU-GM. Noradrenaline elicited factors that inhibited colony formation and had no direct effect on CFU-GM. We conclude that the neuropeptides form the positive arm of a neural control system and that noradrenaline acts as a negative regulator.

Animals↗
PubMed2000

The effect of Bcr-Abl protein tyrosine kinase on maturation and proliferation of primitive haematopoietic cells.

BACKGROUND: Chronic Myeloid Leukaemia (CML) is characterised by the chromosomal translocation resulting in expression of the Bcr-Abl protein tyrosine kinase (PTK) in early stem cells and their progeny. However the precise nature of Bcr-Abl effects in primitive CML stem cells remains a matter of active debate. MATERIALS AND METHODS: Extremely primitive Bcr-Abl fusion positive cells were purified from patients with CML using multiparameter flow cytometric analysis of CD34, Thy, and lineage marker (Lin) expression, plus rhodamine-123 (Rh-123) brightness. Progenitor cells of increasing maturity were examined for cycling status by flow cytometry and their proliferative status directly correlated with cell phenotype. The activation status of a key transcription factor, signal transducers and activators of transcription (STAT-5), was also analyzed by immunocytochemistry. RESULTS: The most primitive stem cells currently defined (CD34+Lin-Thy+ Rh-1231o) were present as a lower proportion of the stem cell compartment (CD34+Lin-) of CML patients at presentation than of normal individuals (2.3% +/- 0.4 compared with 5.1% +/- 0.6 respectively). Conversely there was a significantly higher proportion of the more mature cells (CD34+Lin-Thy-Rh-123 hi) in CML patients than in normal individuals (79.3 +/- 1.8 compared with 70.9 +/- 3.3). No primitive subpopulation of CML CD34+Lin- cells was cycling to a significantly greater degree than cells from normal donors, in fact, late progenitor cells (CD34+Lin+) were cycling significantly less in CML samples than normal samples. STAT5, however, was observed to be activated in CML cells. CONCLUSIONS: We conclude that no subpopulation of CML stem cells displays significantly increased cell cycling. Thus, increased cycling cannot be a direct consequence of Bcr-Abl PTK acquisition in highly enriched stem cells from patients with CML. In vivo CML need not be considered a disease of unbridled stem cell proliferation, but a subtle defect in the balance between self renewal and maturation.

Cell Division↗
PubMed1999

Activation of granulocyte-macrophage colony-stimulating factor and interleukin-3 receptor subunits in a multipotential hematopoietic progenitor cell line leads to differential effects on development.

Activation of specific cytokine receptors promotes survival and proliferation of hematopoietic progenitor cells but their role in the control of differentiation is unclear. To address this issue, the effects of human interleukin-3 (hIL-3) and human granulocyte-macrophage colony-stimulating factor (hGM-CSF) on hematopoietic development were investigated in hematopoietic progenitor cells. Murine multipotent factor-dependent cell-Paterson (FDCP)-mix cells, which can self-renew or differentiate, were transfected with the genes encoding the unique alpha and/or shared beta(c) human hIL-3 receptor (hIL-3 R) or hGM-CSF receptor (hGM R) subunits by retroviral gene transfer. Selective activation of hIL-3 Ralpha,beta(c) or hGM Ralpha,beta(c) transfects by hIL-3 and hGM-CSF promoted self-renewal and myeloid differentiation, respectively, over a range of cytokine (0.1 to 100 ng/mL) concentrations. These qualitatively distinct developmental outcomes were associated with different patterns of protein tyrosine phosphorylation and, thus, differential signaling pathway activation. The cell lines generated provide a model to investigate molecular events underlying self-renewal and differentiation and indicate that the alpha subunits act in combination with the hbeta(c) to govern developmental decisions. The role of the alpha subunit in conferring specificity was studied by using a chimeric receptor composed of the extracellular hIL-3 Ralpha and intracellular hGM Ralpha subunit domains. This receptor promoted differentiation in response to hIL-3. Thus, the alpha subunit cytosolic domain is an essential component in determining cell fate via specific signaling events.

Animals↗
PubMed1999

Macrophage-inflammatory protein-1alpha receptor expression on normal and chronic myeloid leukemia CD34+ cells.

We have assessed expression of MIP-1alpha binding sites on the surface of CD34+ cells from normal bone marrow (NBM) and chronic myeloid leukemia (CML) peripheral blood. This study has highlighted a small subpopulation of CD34+ (15.7 +/- 6.2% in NBM and 9 +/- 4% in CML), which has specific macrophage-inflammatory protein-1alpha (MIP-1alpha) cell surface binding sites. Further phenotypic characterization of the receptor-bearing cells has shown that they do not express the Thy-1 Ag, suggesting that they are committed progenitor cells rather than CD34+ Thy+ stem cells. However, more than 80% of methanol-fixed CD34+ cells do bind MIP-1alpha, suggesting that these cells may possess a pool of internal receptors, although we were unable to induce cell surface expression by cytokine stimulation. The percentage of these CD34+, MIP-1alpha-R+ cells present in the CD34 compartment of NBM is significantly higher than in CML, implicating lack of binding sites as part of the mechanism for the loss of response to this chemokine seen in CML. Specific Ab to the MIP-1alpha receptor implicated in HIV infection, CCR5, revealed that very few CD34+ cells expressed these receptors and that expression was confined to the CD34+ Thy- progenitor population. Data presented in this work suggest that active binding sites for the stem cell growth inhibitor MIP-1alpha are not constitutively expressed on the surface of most resting primitive multipotent cells, and that these cells are not potential targets for HIV-1 infection through CCR5.

Antigens, CD34↗
PubMed1999

Flt3 ligand can promote survival and macrophage development without proliferation in myeloid progenitor cells.

Flt3 ligand elicits a variety of effects on early hemopoietic progenitors by occupying its cognate receptor, Flt3, a member of the type III tyrosine kinase receptor family. The cytokines macrophage colony-stimulating factor (M-CSF) and stem cell factor (SCF) bind to related members of this tyrosine kinase receptors family, c-fms and c-kit, respectively. The relative effects of the cytokines M-CSF, SCF, and Flt3L on the proliferation and development of the late myeloid progenitors granulocyte-macrophage colony-forming cells (GM-CFC) were investigated. Distinct biologic responses were stimulated by ligand binding to these different tyrosine kinase receptors in enriched GM-CFC. M-CSF stimulated GM-CFC to proliferate and develop into macrophages. SCF, on the other hand, stimulated GM-CFC to develop into neutrophils. Flt3 ligand had a relatively small proliferative effect on enriched GM-CFC compared to SCF and M-CSF and had no ability to either stimulate colony formation or synergize with these two cytokines in promoting DNA synthesis, colony formation, or expansion in liquid culture. Flt3 ligand, however, was capable of maintaining the clonogenic potential of GM-CFC and acted as an anti-apoptotic agent as assessed using the Annexin-V apoptosis assay. GM-CFC cultured in Flt3 ligand eventually formed macrophages and neutrophils in liquid culture. Labeling with the membrane-associated cell tracker dye PKH26 indicated that the majority of the enriched GM-CFC responded to Flt3 ligand by undergoing limited proliferation and macrophage development, whereas other cells survived but did not proliferate and differentiate into macrophages. Thus, Flt3 ligand promoted survival and stimulated development without proliferation in primary-enriched myeloid progenitor cells.

Animals↗
PubMed1999

Homing and mobilization in the stem cell niche.

All mature blood cells are derived from the haemopoietic stem cell (HSC). In common with all other haemopoietic cells, stem cells are mobile, and it is this property of mobility that has allowed bone marrow transplantation to become a routine clinical option. Successful transplantation requires haemopoietic stem cells to home to the bone marrow, leave the peripheral circulation and become stabilized in regulatory niches in the extravascular space of the bone marrow cavity. This homing and tethering process is reversible - haemopoietic stem cells can be released from their bone marrow tethering through changes in molecular interactions, which are also important in homing following transplantation. The molecular mechanisms regulating this two-way flow of stem cells are beginning to be elucidated, and much recent data has emerged that sheds light on the processes and molecules involved in these complex physiological events. This article reviews current knowledge of the adhesive, homing and proliferative influences acting on HSCs and progenitor cells.

Animals↗
PubMed1998

p210 Bcr-Abl expression in a primitive multipotent haematopoietic cell line models the development of chronic myeloid leukaemia.

Chronic myeloid leukaemia (CML) is a clonal disorder of the pluripotent haemopoietic stem cell, the hallmark of which is the constitutively activated Bcr-Abl protein tyrosine kinase. During the initial chronic phase of CML the primitive multipotent leukaemic progenitor cells remain growth factor dependent and are capable of producing terminally differentiated cells. Although the available evidence suggests that Bcr-Abl directly affects signalling pathways involved in controlling the development of primitive haemopoietic progenitors the identification of the specific biological consequences of Bcr-Abl activity in these progenitors has been hampered by the lack of suitable systems modelling CML. By transfecting the multipotent haemopoietic cell line FDCP-Mix with a temperature sensitive mutant of Bcr-Abl we have developed the first working model that mirrors the chronic phase of CML. FDCP-Mix cells expressing Bcr-Abl tyrosine kinase activity remain growth factor dependent and retain their ability to differentiate. Normal neutrophilic cells are formed in response to G-CSF and GM-CSF. In addition, the transfected FDCP-Mix cells grown at the permissive temperature for Bcr-Abl tyrosine kinase activity display enhanced survival and proliferation in low concentrations of growth factor. These findings are consistent with the initial subtle changes seen in CML progenitor cells during the chronic phase and confirm that Bcr-Abl effects are context specific, i.e. they depend on the origin and developmental potential of the transfected cells. This questions the significance of studies in non-haemopoietic and differentiation blocked haemopoietic cells.

Cell Division↗
PubMed1998

An activated protein kinase C alpha gives a differentiation signal for hematopoietic progenitor cells and mimicks macrophage colony-stimulating factor-stimulated signaling events.

Highly enriched, bipotent, hematopoietic granulocyte macrophage colony-forming cells (GM-CFC) require cytokines for their survival, proliferation, and development. GM-CFC will form neutrophils in the presence of the cytokines stem cell factor and granulocyte colony-stimulating factor, whereas macrophage colony-stimulating factor leads to macrophage formation. Previously, we have shown that the commitment to the macrophage lineage is associated with lipid hydrolysis and translocation of protein kinase C alpha (PKCalpha) to the nucleus. Here we have transfected freshly prepared GM-CFC with a constitutively activated form of PKCalpha, namely PKAC, in which the regulatory domain has been truncated. Greater than 95% of the transfected cells showed over a twofold increase in PKCalpha expression with the protein being located primarily within the nucleus. The expression of PKAC caused macrophage development even in the presence of stimuli that normally promote only neutrophilic development. Thus, M-CSF-stimulated translocation of PKCalpha to the nucleus is a signal associated with macrophage development in primary mammalian hematopoietic progenitor cells, and this signal can be mimicked by ectopic PKAC, which is also expressed in the nucleus.

Animals↗