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Biomedical subjects

C M Heyworth

Publications and source records attributed to C M Heyworth.

At least 37 records · Page 2Linked to original sources
PubMed1998

Abl protein kinase abrogates the response of multipotent haemopoietic cells to the growth inhibitor macrophage inflammatory protein-1 alpha.

The clonogenic cells of chronic myeloid leukaemia (CML), unlike normal haemopoietic progenitor cells, are resistant to the growth inhibitory effects of the chemokine macrophage inflammatory protein-1 alpha (MIP-1alpha). CML is also relatively resistant to chemotherapy and the disease is difficult to cure using conventional therapeutic routes. CML is associated with increased abl oncogene protein tyrosine kinase (PTK) activity. Here, we have tested the hypothesis that these aberrant responses to MIP-1alpha and the relative resistance to chemotherapy are directly related to this increased abl PTK activity in primitive haemopoietic cells. To do this we have expressed a temperature sensitive abl PTK in a growth factor dependent, multipotent stem cell line (FDCP-Mix) in which growth is normally suppressed by MIP-1alpha. In FDCP-Mix cells expressing the ts v-abl PTK and grown at the restrictive temperature for PTK activity the cells were relatively sensitive to cytotoxic agents such as cytosine arabinoside and 5-fluorouracil but MIP-1alpha could induce growth inhibition and confer some degree of protection from these agents. At the permissive temperature for abl PTK, the cells were relatively resistant to cytotoxic drugs and MIP-1alpha treatment neither induced growth inhibition nor protected the cells from cytotoxic drug induced cell death. This lack of response to MIP-1alpha was not due to receptor down modulation as neither the affinity nor the number of 125I-MIP-1alpha binding sites was altered by activating Abl PTK. However, MIP-1alpha mediated increases in cytosolic Ca2+ levels were abrogated by switching cells to the permissive temperature for Abl PTK activity. These data suggest that the relative resistance of CML progenitor cells to therapeutic drugs and the lack of response to MIP-1alpha occurs as a direct consequence of abl PTK activity and involves desensitisation of signal transduction events stimulated by MIP-1alpha receptors. Thus one contributory mechanism to transformation of primitive haemopoietic cells is abrogation of response to a growth inhibitor.

Calcium↗
PubMed1998

Differential response of CD34+ cells isolated from cord blood and bone marrow to MIP-1 alpha and the expression of MIP-1 alpha receptors on these immature cells.

Macrophage inflammatory protein-1 alpha (MIP-1alpha) has been shown to have a role in the control of myeloid stem and progenitor cell proliferation. Recent evidence suggests that MIP-1alpha also has a stimulatory effect on proliferation of mature progenitors as well as an inhibitory effect on immature progenitors in vitro. We have compared the effect of MIP-1alpha on myeloid and erythroid colony formation of CD34+ cells isolated from bone marrow and cord blood. In the presence of MIP-1alpha, bone marrow granulocyte-macrophage-colony forming cells (GM-CFC) were inhibited over a dose range of 15 ng/ml to 500 ng/ml, and GM-CFC from cord blood CD34+ cells were stimulated over the same dose range. MIP-1alpha suppressed BFU-E colonies in both bone marrow and cord blood. Using thymidine suicide assays, the influence of MIP-1alpha on the cycling status of the cells was assessed. A good correlation between the effect of MIP-1alpha on colony formation and cell cycle progression was observed. These results suggest that there is a differential response to MIP-1alpha when bone marrow and cord blood CD34+ cells are compared. Using flow cytometry and a biotinylated human MIP-1alpha/avidin fluorescein conjugate, the expression of MIP-1alpha receptors on CD34+ cells was assessed. The data indicated that there was little quantitative difference in overall expression of receptors (82.9% versus 93%) from bone marrow or cord blood, respectively. However, when Northern blot analysis was used, mRNA for two different MIP-1alpha receptors CCR1 and CCR5 could be detected in bone marrow, but only CCR1 mRNA was seen in cord blood CD34+ samples. Therefore, the expression of different receptor subtypes on CD34+ cells may be responsible for the difference in MIP-1alpha responsiveness observed.

Antigens, CD34↗
PubMed1998

Ectopic interleukin-5 receptor expression promotes proliferation without development in a multipotent hematopoietic cell line.

The interleukin-5 (IL-5) receptor is a heterodimer that consists of an IL-5 specific alpha subunit and a common ssc chain that is shared with the receptors for granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin-3 (IL-3). In contrast to IL-5, which acts mainly as an eosinophil lineage specific factor in vivo, IL-3 and GM-CSF stimulate the survival, proliferation and development of various hematopoietic cell lineages and also multipotent progenitor cells. IL-5 has little effect on the survival or proliferation of the multipotent stem cell line FDCP-Mix A4 but does promote some eosinophil development. To investigate whether the lineage specificity of IL-5 is due to the restricted expression of the IL-5 receptor alpha subunit we transfected the FDCP-Mix A4 cells with a retroviral vector containing this alpha subunit. The ectopic expression of the IL-5 receptor alpha subunit in the FDCP-Mix cells did not increase the observed eosinophilic development but did stimulate survival and proliferation of the transfected cells when IL-5 was added. IL-5 thus acts like IL-3 in these cells, promoting proliferation and survival. The results suggest that IL-5, whilst having a capacity to promote proliferation, does not influence eosinophilic lineage commitment in these multipotent cells. The results further argue that the observed lineage specificity of IL-5 is probably due to factors in addition to the restricted expression of the IL-5 receptor alpha subunit.

Animals↗
PubMed1998

Investigation of the molecular mechanisms underlying growth factor synergy: the role of ERK 2 activation in synergy.

Stem Cell Factor (SCF), the ligand for the c-kit proto-oncogene, has been shown to synergistically interact with other cytokines, enhancing the responsiveness of haemopoietic precursors. In this study we have examined the effects of SCF, in combination with interleukin-3 (IL-3), on FDCP-Mix A4 cells, a murine, multipotent, haemopoietic progenitor cell line. Low concentration of IL-3 act to enhance cell survival but do not stimulate proliferation in A4 cells. Similarly, SCF when added alone, acts as a good survival stimulus, but is a poor proliferative stimulus. However, in combination with low concentrations of IL-3, SCF stimulates a synergistic enhancement of proliferation leading to a large increase in cell number after seven days. The synergy observed was not due to SCF stimulated alterations in the mRNA, protein levels or affinity of the IL-3 receptors. Therefore, interactions between cytokines at the level of cytoplasmic signalling pathways were investigated. IL-3 stimulated the rapid and transient tyrosine phosphorylation of several proteins (including those of molecular weights 130, 110, 100, 95, 80, 65, 50 and 45 kDa). Some of these proteins were identified as the Src Homology Collagen (SHC) protein, Janus kinase (JAK-2) and the Mitogen Activated Protein Kinase isoforms ERK 1 and ERK 2. IL-3 also stimulated a transient increase in the activity of both ERK 1 and 2. SCF stimulated a rapid and transient increase in the tyrosine phosphorylation of ERK 1 and ERK 2 although, coaddition of SCF with IL-3, caused no gross differences in the phosphorylation of SHC, JAK-2 or ERKs compared to those observed with IL-3 alone. Coaddition of SCF and low concentration of IL-3 stimulated a reproducible synergistic increase in the activity of ERK 2, whereas only an additive increase in the activity of ERK 1 was observed. These results suggest that ERK 2 activation represents a point at which the two pathways, stimulated by IL-3 and SCF, interact synergistically.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Identification of a serpin specifically expressed in multipotent and bipotent hematopoietic progenitor cells and in activated T cells.

We have identified a gene that has a high level of mRNA expression in undifferentiated, multipotential hematopoietic cells (FDCP-Mix) and that downregulates both transcript and protein, as these cells are induced to differentiate into mature myeloid cells. Sequence analysis of this gene has identified it as a serine protease inhibitor EB22/3 (serpin 2A). Constitutive expression of serpin 2A in FDCP-Mix cells was associated with an increase in the clonogenic potential of the cells and with a delay in the appearance of fully mature cells in cultures undergoing granulocyte macrophage differentiation when compared with control cells. Serpin 2A was also found to be expressed in bone marrow-derived bipotent granulocyte macrophage progenitor cells (GM-colony forming cell [CFC]), but not in erythrocyte progenitor cells from day 15 fetal liver. Expression of serpin 2A also showed a marked up regulation during the activation of cytotoxic suppressor CD8+ T cells, with a clear lag between the appearance of transcript and detection of protein.

Amino Acid Sequence↗
PubMed1997

How do stem cells decide what to do?

The continuous replenishment of mature blood cells from multipotent stem cells proceeds under the influence of haemopoietic growth factors which clearly regulate both cell survival and proliferation. The extent to which these factors might influence lineage choice is still unclear, however, and it seems likely that resolution of this issue will require direct analysis of multipotent cells undergoing commitment rather than determination of their productivity in colony assays. Chromatin analysis of a multipotent progenitor cell line indicates that many of the genes relevant to alternative lineage fates are maintained in an accessible (primed) state prior to lineage commitment. Furthermore, multipotent cells have been found to co-express a number of lineage-restricted genes, suggesting that commitment proceeds as the consolidation of an existing programme. There are indications that the patterns of gene expression in multipotent progenitors change over time, raising the possibility of temporal priming towards different lineages. In multipotential cell lines, exogenous growth factors are necessary for survival, but not for lineage commitment, implying a largely supportive role in early progenitors. In contrast, recent work on primary bipotent granulocyte/ macrophage progenitors does demonstrate an inductive role for growth factors in these more lineage-restricted cells.

Animals↗
PubMed1996

Hydroquinone stimulates granulocyte-macrophage progenitor cells in vitro and in vivo.

To investigate whether hydroxylated metabolites of benzene may be responsible for the amplification of granulocyte-macrophage progenitor cells (GM-CFC) observed in mice that inhale benzene, groups of six C57BL6 mice were injected with hydroquinone (HQ) (75 mg/kg) or HQ (50 mg/kg) plus phenol (PHE) (50 mg/kg) twice daily for 11 days. Deviations in blood leukocyte and erythrocyte levels by up to one-third were noted in the treated groups; however, the peripheral blood differential counts were unchanged. Although no changes in bone marrow cellularity were observed in mice treated with HQ, cellularity was decreased by a factor of two in the mice that had received HQ plus PHE. The number of GM-CFC per femur was doubled in both treated groups. In vitro experiments using the murine multipotent hematopoietic progenitor cells FDCP mix also showed a duplication of GM-CFC formation in the presence of HQ at concentrations between 10(-6) M and 10(-10) M. When HQ and PHE were present at equimolar concentrations, significantly increased colony formation was still observed with 10(-12) M of metabolites. The effect was independent of the concentration of GM-colony-stimulating factor used. We suggest that HQ is a major mediator of the stimulatory effect of benzene on GM-CFC in mice. In addition, the in vitro data indicate that a direct effect of GM-CFC is involved.

Animals↗
PubMed1995

BB-10010: an active variant of human macrophage inflammatory protein-1 alpha with improved pharmaceutical properties.

The stem cell inhibitor, macrophage inflammatory protein-1 alpha (MIP-1 alpha) or LD78, protects multipotent hematopoietic progenitors in murine models from the cytotoxic effects of chemotherapy. Clinical use of human MIP-1 alpha during chemotherapy could therefore lead to faster hematologic recovery and may allow dose intensification. We have also shown that human MIP-1 alpha causes the rapid mobilization of hematopoietic cells, suggesting an additional clinical use in peripheral blood stem cell transplantation. However, the clinical evaluation of human MIP-1 alpha is complicated by its tendency to associate and form high molecular weight polymers. We have produced a variant of rhMIP-1 alpha, BB-10010, carrying a single amino acid substitution of Asp26 > Ala, with a reduced tendency to form large polymers at physiologic pH and ionic strength. This greatly increases its solubility, facilitating its production and clinical formulation. We confirmed the potency of BB-10010 as a human MIP-1 alpha-like agonist in receptor binding, calcium mobilization, inhibition of colony formation, and thymidine suicide assays. The myeloprotective activity of BB-10010 was shown in a murine model of repeated chemotherapy using hydroxyurea. BB-10010 is therefore an ideal variant with which to evaluate the therapeutic potential of recombinant human MIP-1 alpha.

Amino Acid Sequence↗
PubMed1995

IL-4 promotes macrophage development by rapidly stimulating lineage restriction of bipotent granulocyte-macrophage colony-forming cells.

Granulocyte macrophage colony-forming cells (GM-CFC) are bipotential progenitor cells that can proliferate and develop into macrophages in response to macrophage CSF or into neutrophils in response to stem cell factor or granulocyte CSF. These cytokines promoted growth and development in highly enriched GM-CFC. In [3H]thymidine suicide assays, IL-4 was shown to stimulate proliferation of GM-CFC to the same degree as IL-3 and other potent mitogens for GM-CFC. IL-4 also maintained the clonogenic potential of enriched GM-CFC over a 2-day period. However, after several days in the presence of IL-4, the GM-CFC began to die and retained blast cell morphology characteristic of the isolated GM-CFC. When a high concentration of IL-4 was added to GM-CFC with neutrophilic stimuli, the response of these cells was altered because macrophages were formed. This effect was achieved by a 4-h preincubation with IL-4, suggesting that an early signal produced by IL-4 promotes lineage restriction, although IL-4 itself cannot promote development. IL-4, like macrophage CSF, translocates PKC-alpha to the nucleus in GM-CFC, this redistribution of protein kinase C alpha (PKC-alpha) being inhibited by calphostin C (a PKC inhibitor). Calphostin C also blocked IL-4-mediated development of macrophages in stem cell factor- and granulocyte-CSF-treated cells. This is further evidence that PKC-alpha translocation is involved in the commitment of GM-CFC to macrophage development. This data also suggests that agonist-stimulated lineage commitment can be uncoupled from development in normal hematopoietic cells.

Animals↗
PubMed1995

Discordant regulation of SCL/TAL-1 mRNA and protein during erythroid differentiation.

The SCL/TAL1 gene was originally identified by virtue of its rearrangement and transcriptional activation in patients with T cell acute lymphoblastic leukaemia. It encodes a helix-loop-helix transcription factor, is not normally expressed in T cells, but is expressed in erythroid, mast, megakaryocytic and progenitor cells. Over-expression of sense and antisense constructs have implicated SCL as a positive regulator of erythroid differentiation. In addition we have previously shown that SCL mRNA levels undergo biphasic modulation during induced erythroid differentiation of murine erythroleukaemia (MEL) cells with a transient early fall followed by a late rise. In this paper we have studied expression of the SCL protein during erythroid differentiation and also the molecular basis for the raised SCL mRNA levels that accompany erythroid differentiation. We have generated an anti-SCL antiserum and used it to demonstrate that an early transient fall in SCL protein does not occur during induced differentiation of MEL cells. Furthermore SCL protein levels underwent a late fall in three different models of erythroid differentiation and in two models of myeloid differentiation. The fall in SCL protein levels during induced erythroid differentiation contrasted with the concomitant marked rise in SCL mRNA levels. These observations have significant implications for the mechanism by which SCL may regulate erythropoiesis. In addition we have demonstrated that the stability of SCL mRNA was only marginally enhanced during erythroid differentiation of MEL cells, whereas the activity of a luciferase reporter construct driven by the SCL promoter was increased 11- to 17-fold. Up-regulation of transcription therefore accounted for most of the increase in SCL mRNA levels during erythroid differentiation.

Animals↗
PubMed1995

Erythroid development of the FDCP-Mix A4 multipotent cell line is governed by the relative concentrations of erythropoietin and interleukin 3.

Conditions are described which promote the erythroid development of the FDCP-Mix A4 (A4) cell line with accompanying proliferation of the cells. The requirements for this development are low concentrations of interleukin 3 (IL-3) plus the presence of erythropoietin (epo) and haemin. When high concentrations of IL-3 are added with erythropoietin and haemin the cells do not differentiate and maintain their blast cell morphology. Addition of haemin, in the absence of erythropoietin, does not promote erythroid development, but the presence of haemin with erythropoietin promotes increased proliferation and maturation. The morphological maturation of A4 cells along the erythroid lineage is accompanied by a gradual loss of clonogenic potential, loss of A4 cell multipotency, increased erythropoietin receptor expression, and an increased expression of the beta-globin gene. An initial increase in mitogenic responsiveness to erythropoietin is followed by a decrease as the cells become refractory to all mitogenic stimuli with the acquisition of a postmitotic, mature erythroid cell phenotype.

Blotting, Northern↗
PubMed1995

Macrophage inflammatory protein-1 alpha mediated growth inhibition in a haemopoietic stem cell line is associated with inositol 1,4,5 triphosphate generation.

Macrophage Inflammatory Protein-1 alpha (MIP-1 alpha) can inhibit the proliferation of multipotent haemopoietic cells. Using the FDCP-Mix A4 multipotent stem cell line, MIP-1 alpha was shown to inhibit 1L-3 stimulated cell cycling (assessed using the [3H]-thymidine "suicide" assay). Furthermore, MIP-1 alpha can inhibit 1L-3-stimulated [3H]-thymidine incorporation in FDCP-Mix cells, with half maximal inhibition observed at 3 ng/ml MIP-1 alpha. Prostaglandin E2, but not MIP-1 alpha was able to elevate cyclic AMP levels in FDCP-Mix A4 cells although both agents can cause growth inhibition. However, MIP-1 alpha addition resulted in a pertussis-toxin-insensitive increase in the level of the second messenger inositol 1,4,5 triphosphate (Ins 1,4,5P3). This response was both rapid (maximal at 5 seconds) and transient. A half maximal effect was observed at 5 ng/ml MIP-1 alpha and the dose dependency correlated with that for MIP-1 alpha mediated growth inhibition. A rapid increase in cytosolic Ca2+ levels was also observed in response to MIP-1 alpha. Inositol lipid hydrolysis and an increase in cytosolic Ca2+ (signals normally associated with proliferation) may therefore be implicated in growth inhibitory mechanisms in multipotent cells.

Animals↗
PubMed1994

Growth factors and the molecular control of haematopoiesis.

In the absence of appropriate growth factors, for example interleukin-3 or GM-CSF, cultured bone marrow stem cells die by a process known as apoptosis or programmed cell death. Apoptosis may occur in vivo when concentrations of specific growth factors are limiting and may be a means of regulating cell numbers. Growth factors are also essential for proliferation of bone marrow stem cells but differentiation can occur, provided there is a survival stimulus in the absence of growth factors. Combinations of growth factors may be synergistic in stimulating the survival and proliferation of multipotent stem cells. Although neither stem cell factor, nor GM-CSF alone can significantly induce the proliferation of stem cells, the combination induces the proliferation of these cells. Committed progenitor cells such as granulocyte-macrophage colony-forming cells, however, are stimulated to proliferate by GM-CSF alone, while stem cell factor in combination with GM-CSF results in only a slight additive effect. To date, most research has concentrated on the growth stimulatory factors. GM-CSF has an important role in the reversal of chemotherapy-induced myelosuppression in cancer patients and in other bone marrow disorders. A number of growth inhibitory molecules have now been identified, such as macrophage inhibitory protein-1 alpha. In the future, it is possible that improvements in cure rates may be achieved in cancer patients by combining the growth inhibitory factors with the stimulatory factors. Inhibitory factors may be given before chemotherapy to prevent toxicity and stimulatory factors may be given afterwards to treat neutropenic patients.

Cell Differentiation↗
PubMed1994

Cytokine-mediated protein kinase C activation is a signal for lineage determination in bipotential granulocyte macrophage colony-forming cells.

Granulocyte macrophage colony-forming cells (GM-CFC) have the potential to develop into either macrophages and/or neutrophils. With a highly enriched population of these cells we have found that although GM-CFC are equally responsive to macrophage colony stimulating factor (M-CSF) and stem cell factor (SCF) in terms of DNA synthesis, M-CSF stimulated the development of colonies containing macrophages in soft gel assays, while SCF promoted neutrophilic colony formation. When SCF and M-CSF were combined, mainly macrophage development was stimulated both in soft agar colony-forming assays and liquid cultures. An analysis of some potential signaling mechanisms associated with cytokine-mediated developmental decisions in GM-CFC revealed that M-CSF, but not SCF, was able to chronically stimulate phosphatidylcholine breakdown and diacylglycerol production, indicating that protein kinase C (PKC) may be involved in the action of M-CSF. Furthermore, M-CSF, but not SCF, can increase the levels of PKC alpha (PKC alpha) expression and stimulate the translocation of PKC alpha to the nucleus. When the PKC inhibitor, calphostin C, was added to GM-CFC cultured in M-CSF then predominantly neutrophils were produced, conversely PKC activators added with SCF stimulated macrophage development. The data indicate a role for PKC in M-CSF-stimulated macrophage development from GM-CFC.

Animals↗
PubMed1994

Combinations of colony-stimulating factors promote enhanced proliferative potential in enriched granulocyte-macrophage colony-forming cells.

The effects of combinations of colony-stimulating factors (CSFs) have been assessed using a highly enriched population of murine granulocyte-macrophage colony-forming cells (GM-CFC). Unlike the situation observed with more primitive myeloid progenitor cells, little or no effect on the numbers of colonies formed from GM-CFC in response to specific combinations of growth factors was observed; however, the size of the majority of colonies formed was greatly increased. The largest increase in the number of cells per colony was observed when interleukin-3 (IL-3) was present with either granulocyte-macrophage colony-stimulating factor (GM-CSF), GM-CSF plus granulocyte-colony-stimulating factor (G-CSF), or macrophage colony-stimulating factor (M-CSF); there was a > five-fold increase when compared to colony size in the presence of IL-3 alone. The combination of G-CSF with IL-3 was not able to promote an increase in mean colony size; however, G-CSF plus GM-CSF did give a significant increase. Where combinations of hematopoietic growth factors led to increased numbers of cells per colony, the delayed addition of one of the cytokines to soft gel assays for a period > 2 days led to a loss of the observed enhancement in the number of cells per colony. In cultures of progenitor cells enriched by centrifugal elutriation and that contained combinations of CSFs, there was an increase in the number of GM-CFC over a 2-day incubation period. The distinct effects observed with GM-CSF, IL-3, and G-CSF on GM-CFC suggest that they influence different molecular signaling mechanisms within common target progenitor cells.

Bone Marrow Cells↗
PubMed1994

Expression of lineage restricted transcription factors precedes lineage specific differentiation in a multipotent haemopoietic progenitor cell line.

Lineage commitment and differentiation are likely to be coordinated by the combined effects of multiple transcription factors acting on numerous different target genes. The mechanisms by which lineage-restricted patterns of transcription factor expression are established are therefore of particular relevance to our understanding of the role of transcription factors both in normal development and in oncogenesis. Here, we report that the genes for the lineage-restricted transcription factors SCL, GATA-1 and GATA-2 are expressed in all multipotent, IL-3-dependent, haemopoietic progenitor cell lines tested. Moreover, a liquid differentiation assay has been used to demonstrate down regulation of SCL, GATA-1, GATA-2 and PU-1 during differentiation into non-expressing lineages. These data support the concept that multiple lineage-restricted transcription factors are expressed prior to lineage commitment.

Cell Line↗
PubMed1993

Protein kinase C activators can interact synergistically with granulocyte colony-stimulating factor or interleukin-6 to stimulate colony formation from enriched granulocyte-macrophage colony-forming cells.

The effects of direct activators of protein kinase C (PKC) (the phorbol ester tetradecanoyl phorbol myristic acid [TPA] or bryostatin) on the ability of a highly enriched population of granulocyte-macrophage colony-forming cells (GM-CFC) to proliferate and develop in soft agar was assessed. In the absence of colony stimulating factors, the PKC activators did not stimulate colony formation. However, in the presence of optimal concentrations of granulocyte colony-stimulating factor (G-CSF) or interleukin-6 (IL-6), TPA or bryostatin markedly elevated the number of colonies formed from the GM-CFC. In the absence of TPA, IL-6, and G-CSF, respectively, both stimulated the formation of about 3% of the colonies observed when IL-3 was present. When TPA plus G-CSF or IL-6 were added together, this figure increased to 48% and 54%, respectively. In both instances, the types of mature cells formed was altered from colonies of mature neutrophilic cells to a mixture consisting predominantly of macrophages with some neutrophils. Similar results were observed when bryostatin replaced TPA in these assays. When single cell colony-forming assays were performed, the same results were obtained. The presence of G-CSF, or IL-6, and the activator of PKC used (TPA or bryostatin) was required throughout the colony-forming assay for an optimal synergistic effect to be observed. These data indicate that agents that activate PKC can promote the proliferation and development of GM-CFC via a synergistic interaction with G-CSF or IL-6. Furthermore, there is an apparent role for PKC in development and possibly lineage commitment of GM-CFC.

Animals↗