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In view of the recent report of a myeloproliferative syndrome in mice that had received an MDR-1-transduced haemopoietic graft, we have investigated the potential effects of MDR-1 expression on primitive haemopoietic cell growth and differentiation. Retroviral gene transfer was used to achieve exogenous expression of either MDR-1 or truncated nerve growth factor receptor (tNGFR) in the multipotent murine haemopoietic progenitor cell line, FDCP-mix. Following gene transfer, clonal lines were derived and FACS analysis confirmed appropriate expression of each transgene. MDR-1 (but not tNGFR) expression was associated with verapamil-sensitive rhodamine efflux and resistance to killing by etoposide. When growth factor responsiveness, proliferative capacity and differentiation capacity were examined, MDR-1 expressing FDCP-mix cells exhibited a normal phenotype and mimicked the response of tNGFR-expressing or untransduced FDCP-mix cells. Thus, in the model system we have used, MDR-1 does not perturb haemopoietic cell growth and development and our data do not support a myeloproliferative role for MDR-1.

Human haemopoietic stem and progenitor cells may be distinguished by the pattern of cell surface markers they display. The cells defined as 'stem' cells are heterogeneous and lack specific markers for their detection. However, they may be identified in in vitro assays such as the long-term culture initiating cell (LTC-IC) and in transplant assays involving immunosuppressed NOD/SCID mice. It is still not clear to what extent, if any, these cell populations overlap. The chemokine macrophage inflammatory protein-1alpha (MIP-1alpha) prolongs survival of LTC-IC in suspension cultures and we now show that in longterm bone marrow cultures (LTBMC) maintenance of haemopoiesis was significantly better from the CD34+ cells which possess MIP-1alpha receptors (P < 0.006). We examined one MIP-1alpha receptor, CCR1, which is present on CD34+ cells from haemopoietic tissues. In LTBMC the production of GM-CFC from CD34+CCR1- cells was significantly higher (P < 0.02) than that from CD34+CCR1+ cultures and the incidence of LTC-IC was 3- to 6-fold higher in the CD34+CCR1- cell fraction. In contrast, the cells responsible for high levels of engraftment in NOD/SCID mice were contained in the CD34+CCR1+ cell fraction. The CD34+CCR1+ cells engrafted to high levels in NOD/SCID and generated large numbers of progenitor cells. Therefore, we conclude that LTC-IC and SRC may be distinguished on the basis of expression of the chemokine receptor CCR1.

We have studied the influence of metabolites of chlorinated pesticides (lindane, pentachlorophenol, hexachlorobenzene) on proliferation and differentiation in two stroma-free murine bone marrow culture models, a multipotent progenitor cell line (FDCP-mix) and primary lineage-depleted bone marrow cells. Tetrachlorohydroquinone (Cl(4)pHQ), tetrachloro-p-benzoquinone (Cl(4p)BQ), but not their positional isomers, tetrachlorocatechol (Cl(4)oHQ) and tetrachloro-o-benzoquinone (Cl(4)oBQ), nor 2,4,6-trichlorophenol (2,4,6-Cl(3)P), were much more toxic to FDCP-mix cells cultured under conditions which lead to self-renewal than under conditions which lead to granulocyte-macrophage differentiation. Under the latter conditions, Cl(4)pHQ and Cl(4p)BQ even stimulated growth at intermediate concentration levels. In the primary cell cultures, pronounced differences were observed in the sensitivity between individual developmental pathways and between the different compounds. The percent of cells differentiating into the granulocytic lineage was increased at high concentration levels of each test compound. However, stimulatory effects on the macrophage lineage were observed at intermediate concentration levels of Cl(4)pHQ, Cl(4p)BQ and 2,4,6-Cl(3)P, and differentiation into erythrocytes was stimulated at low concentrations of 2,4,6-Cl(3)P. It is concluded that chlorinated monocyclic pesticides, after biotransformation to quinoid metabolites, may interact directly with haemopoietic progenitor cells with differential effects on self-renewal and differentiation. These mechanisms could lead to myeloplastic disorders.

We examined the functional differences between G(0) and G(1) cord blood CD34+ cells for up to 24 weeks in serum-free suspension culture, containing Flt-3 ligand, thrombopoietin and stem cell factor. By week 24, there is more than a 1,000-fold difference in granulocyte, macrophage-colony-forming cells (GM-CFC) cumulative production between the two populations, with cultures initiated from G(0) demonstrating an amplification of 1.1 x 10(5)-1.8 x 10(6) of GM-CFC compared to 45-2.7 x 10(3) for the G(1) cells. Cells from the initial G(0) population are able to produce about 250-fold higher numbers of BFU-E than those from G(1) which translates to 3 x 10(3)-1.1 x 10(5)-fold expansion and 25-390-fold expansion for G(0) and G(1), respectively. This amplification of the progenitor cells is reflected in finding that a greater proportion of the progeny of the G(0) population are CD34+, resulting in a 600-fold expansion of CD34+ cells at week 8. As in other in vitro systems, total cell expansion is less discriminatory of stem cell behavior than progenitor cells, and there is no significant difference in total cell numbers between G(0) and G(1) cultures with a mean fold expansion of 2 x 10(7) at 24 weeks.

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.

This work aimed to provide a means of assaying directly the effects of transient expression of introduced genes on the survival, proliferation, lineage commitment and differentiation of haemopoietic progenitor cells. For this purpose, we have developed a system that allows isolation of productively transfected, mulitipotent haemopoietic cells within a few hours of the introduction of test genes. We have shown that FDCP-mix cells productively transfected with expression plasmids encoding green fluorescent protein (GFP) differentiate normally and retain colony-forming potential. We constructed an expression vector consisting of a bicistronic cassette in which a GFP marker gene and a test gene are driven from the same promoter. The vector design has been optimized for co-expression and the test gene was shown to be biologically active. The expression profile from a transiently transfected template under different growth conditions reveals that active expression continues for at least 2 d after transfection. The transient transfection of FDCP-mix cells with the vectors described provides a powerful tool for analysis of the immediate early effects of test gene overexpression during haemopoietic differentiation.

The clonogenic cells of chronic myeloid leukaemia (CML), unlike normal haemopoietic colony forming cells (CFC), are resistant to the growth inhibitory effects of the chemokine, macrophage inflammatory protein-1alpha (MIP-1alpha). Here, we tested the hypothesis that MIP-1alpha protects normal, but not CML, CFC from the cytotoxic effects of the cell-cycle active drug cytosine arabinoside (Ara-C). Using a 24-h Ara-C protection assay we showed that MIP-1alpha confers protection to normal CFC but also sensitizes CML CFC to Ara-C. The differential MIP-1alpha responsiveness was not due to a down-regulation of MIP-1alpha receptors on CML CD34+ cells as flow cytometric analysis showed similar binding of a biotinylated MIP-1alpha molecule to normal and CML CD34+ cells. Flow cytometric analysis of the MIP-1alpha receptor subtype CCR-5 revealed comparable CCR-5 expression levels on normal and CML CD34+ cells. Furthermore, culture of CD34+ cells for 10 h in the presence of TNF-alpha resulted in an increased MIP-1alpha receptor expression on both normal and CML CD34+ cells. Our data suggest that the unresponsiveness of CML CFC to the growth inhibitory effect of MIP-1alpha is not caused by a lack of MIP-1alpha receptor or total uncoupling of the MIP-1alpha responsiveness but may be due to an intracellular signalling defect downstream of the receptors.

Mice deficient for the transcription factor, interferon consensus sequence binding protein (ICSBP), are immunodeficient and develop disease symptoms similar to human chronic myeloid leukemia (CML). To elucidate the hematopoietic disorder of ICSBP(-/-) mice, we investigated the growth, differentiation, and leukemogenic potential of ICSBP(-/-) myeloid progenitor cells in vitro, as well as by cell-transfers in vivo. We report that adult bone marrow, as well as fetal liver of ICSBP-deficient mice harbor increased numbers of progenitor cells, which are hyperresponsive to both granulocyte macrophage colony-stimulating factor (GM-CSF) and G-CSF in vitro. In contrast, their response to M-CSF is strongly reduced and, surprisingly, ICSBP(-/-) colonies formed in the presence of M-CSF are mostly of granulocytic morphology. This disproportional differentiation toward cells of the granulocytic lineage in vitro parallels the expansion of granulocytes in ICSBP(-/-) mice and correlates with a 4-fold reduction of M-CSF receptor expressing cells in bone marrow. Cell transfer studies showed an intrinsic leukemogenic potential and long-term reconstitution capability of ICSBP(-/-) progenitors. Further experiments demonstrated strongly reduced adhesion of colony-forming cells from ICSBP(-/-) bone marrow to fibronectin. In summary, ICSBP(-/-) myeloid progenitor cells share several abnormal features with CML progenitors, suggesting that the distal parts of signaling pathways of these two disorders are overlapping.

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.

Cytotoxic T cells and early haemopoietic progenitors share the expression of a number of specific genes. Of these, granzyme B has attracted particular interest because of its role in inducing apoptosis during cytotoxic T cell-mediated target cell killing, and its potential role in the mobilisation and homeostasis of haemopoietic stem cells. Studies of granzyme B regulation should therefore yield valuable information concerning the molecular control of these processes, and also identify elements capable of directing gene expression to two cell types of relevance to gene therapy. Here we show that proximal regulatory elements already known to direct promoter activity in T cells are similarly active in haemopoietic progenitors. However, this activity is not strictly specific, since the promoter regions also direct low levels of reporter gene expression in fibroblasts. More importantly, we also report the presence of two previously unidentified clusters of DNaseI hypersensitive sites upstream from the murine granzyme B gene, and show that these regions impart both increased transcriptional activity and the appropriate cell type specificity on the granzyme B promoter. These upstream regulatory regions are therefore likely to play a key role in the coordination of granzyme B expression in vivo.

Chemokines are important regulators of both hemopoietic progenitor cell (HPC) proliferation and adhesion to extracellular matrix molecules. Here, we compared the biological effects of the CC chemokine macrophage inflammatory protein-1alpha (MIP-1alpha) with those of the CXC chemokine stroma-derived factor-1alpha (SDF-1alpha) on immunomagnetically purified CD34+ cells from leukapheresis products (LP CD34+). In particular, studies on chemokine-induced alterations of LP CD34+ cell attachment to fibronectin-coated plastic surfaces, proliferation of these cells in colony-forming cell (CFC) assays and intracellular calcium mobilization were performed. MIP-1alpha but not SDF-1alpha was found to increase the adhesion of LP CD34+ cells to fibronectin in a dose-dependent manner. Both chemokines elicited growth-suppressive effects on LP CD34+ cells in CFC assays. While MIP-1alpha reduced the number of granulomonocytic (CFC-GM) and erythroid (BFU-E) colonies to the same extent, SDF-1alpha showed a significantly greater inhibitory effect on CFC-GM than BFU-E. Finally, we demonstrated that SDF-1alpha but not MIP-1alpha triggers increases in intracellular calcium in LP CD34+ cells. The SDF-1alpha-induced calcium response was rapid and concentration-dependent, with a maximal stimulation observed at > or = 15 ng/ml. In conclusion, our data suggest distinct biological properties of SDF-1alpha and MIP-1alpha in terms of modulation of LP CD34+ cell adhesion to fibronectin and intracellular calcium levels. However, comparable growth-suppressive effects on HPC proliferation were observed, indicating that this feature may be independent of chemokine-induced calcium responses.

Macrophage inflammatory protein-1alpha (MIP-1alpha) can stimulate growth inhibitory and potent chemotactic functions in hematopoietic cells. To investigate whether the action of MIP-1alpha may be regulated at the cellular receptor level, we studied the expression and modulation of MIP-1alpha receptors on CD34(+) cells isolated from normal bone marrow (NBM), umbilical cord blood (CB), and leukapheresis products (LP). Expression of MIP-1alpha receptors on CD34(+) cells was analyzed by two-color flow cytometry using a biotinylated MIP-1alpha molecule. The mean percentage of LP CD34(+) cells expressing the MIP-1alpha receptors was 67.7 +/- 7.2% (mean +/- SEM; n = 22) as compared with 89.9 +/- 2.6% (n = 10) and 74.69 +/- 7.04% (n = 10) in CB and NBM, respectively (P = .4). The expression of the MIP-1alpha receptor subtypes on LP CD34(+) cells was studied by indirect immunofluorescence using specific antibodies for the detection of CCR-1, CCR-4, and CCR-5. Microscopical examination revealed a characteristic staining of the cytoplasmic cell membrane for all three receptor subtypes. Detailed analysis of two LP samples showed that 65.8%, 4.4%, and 30.5% of CD34(+) cells express CCR-1, CCR-4, and CCR-5, respectively. Culture of LP CD34(+) cells for 24 to 36 hours in the presence of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) resulted in a significant increase in MIP-1alpha receptor expression. TNF-alpha induced MIP-1alpha receptor upregulation in a time- and concentration-dependent manner. Our results suggest that inhibitory cytokines produced by the bone marrow microenvironment are likely to be involved in the regulation of MIP-1alpha receptor expression on hematopoietic cells., (Copyright 1998 by The American Society of Hematology)

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Multipotential FDCP-Mix A4 (A4) cells can be induced either to self-renew or to differentiate and develop into mature neutrophils in liquid culture, depending on the haemopoietic growth factors with which they are cultured. When cultured in low concentrations of interleukin 3 (IL-3, 1 unit/ml)) plus Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) and Granulocyte-CSF (G-CSF), A4 cells proliferate with accompanying development to form cells which resemble mature, postmitotic neutrophils. The presence of high concentrations of IL-3 (100 units/ml) blocks the development of A4 cells even in the presence of GM-CSF plus G-CSF. A4 cell development to neutrophils is accompanied by major changes in the expression of protein kinase C (PKC) subspecies in these cells. The predominant subspecies present in multipotent A4 cells, as judged by direct chromatographic analysis, was the type III enzyme (alpha) subspecies, whereas in mature A4 cell neutrophils, the type II (beta I + beta II) enzymes were predominant. Phorbol esters added to immature A4 cells resulted in a proliferative response, but when added to postmitotic A4 cells resembling neutrophils they elicited a large increase in reactive oxygen intermediate production. This suggests that the type III (alpha) subspecies may mediate proliferative responses in stem cells, whilst the type II (beta I + beta II) enzymes are more important for the mature cell functions of postmitotic neutrophils. In cultures containing IL-3 (100 units/ml) both the type III, and also the type II subspecies were predominantly membrane-associated for prolonged periods (> 24 hours).(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of the c-kit ligand (stem cell factor [SCF]) on the development of a highly enriched population of granulocyte-macrophage colony-forming cells (GM-CFC) were assessed. In soft agar assays, both in serum-containing and in serum-deprived cultures, SCF promoted the formation of colonies that contained predominantly granulocytic cells with some blast cells also present. The size of these colonies was far smaller than observed in the presence of interleukin-3 (IL-3). In serum-deprived conditions, no colonies were formed in the presence of macrophage colony-stimulating factor (M-CSF), but when M-CSF was combined with SCF, a marked change was noted in that large colonies were produced containing predominantly macrophages. When GM-CFC were cultured in the presence of IL-3 and SCF, colonies were formed that contained blast cells, granulocytes, and macrophages. A synergistic interaction was also seen using a combination of G-CSF plus SCF in either serum-containing or serum-deprived cultures. The addition of SCF to colony-forming assays markedly reduced the concentration of IL-3 or G-CSF required for optimal levels of colony formation. Furthermore, SCF was capable of promoting the survival of GM-CFC for several days, after which large colonies containing mature cells were formed upon the addition of a secondary growth factor such as G-CSF or IL-3. Thus, SCF can directly act on highly enriched committed progenitor cells in serum-deprived conditions to promote survival, proliferation, and development.

The effects of interferon-gamma (IFN-gamma) on a highly enriched population of granulocyte-macrophage colony-forming cells (GM-CFC) were assessed. When added with myeloid growth factors (interleukin-3 [IL-3], granulocyte-macrophage colony-stimulating factor [GM-CSF], or macrophage-CSF [M-CSF]), IFN-gamma inhibited the formation of colonies in soft agar assays. Furthermore IFN-gamma stimulated an increase in the number of macrophages present in colonies formed in the presence of IL-3. IFN-gamma also inhibited M-CSF-, GM-CSF-, or IL-3-stimulated [3H]-thymidine incorporation in highly enriched GM-CFC. However, when added in the absence of hematopoietic growth factors, IFN-gamma promoted the survival of GM-CFC and had a modest stimulatory effect on DNA synthesis. The direct interaction of the IFN with GM-CFC was confirmed by showing its ability to rapidly activate the sodium/hydrogen antiport in GM-CFC, as do the mitogens GM-CSF, M-CSF, and IL-3. However, the effect of IFN-gamma on intracellular pH and DNA synthesis was transient and pretreatment with IFN markedly inhibited the ability of GM-CSF, M-CSF, and IL-3 to activate the sodium/hydrogen antiport. IFN-gamma has a dual effect on GM-CFC, decreasing the rate of cell death but also limiting the proliferative response to CSFs.

An extract from normal bone marrow (NBME) which inhibits proliferation of spleen colony-forming units CFU-S selectively inhibits interleukin 3 (IL-3)-driven colony formation by primitive hemopoietic progenitors. This activity is distinct from transforming growth factor-beta (TGF beta), which also inhibits development of primitive progenitors. There is evidence that the two activities inhibit proliferation of target cells by different mechanisms and that the bone marrow extract has a direct effect on cell cycling, whereas the effect of TGF beta to suppress proliferation is probably indirect.

A serum-free culture system was used to determine the effects of growth factors on the clonogenic development of a population of cells highly enriched for multipotential day 12 spleen colony forming cells (CFU-S) (FACS-BM). Under these conditions, interleukin-3 (IL-3) was found to be primarily a proliferative stimulus, the progenitor cells developing in the clonal assay systems produced colonies of morphologically undifferentiated cells for up to 20 days. No such induction of proliferation without maturation was observed with other growth factors (eg. granulocyte-macrophage colony stimulating factor (GM-CSF)). However, combinations of IL-3 plus secondary growth factors such as GM-CSF, macrophage colony stimulating factor (M-CSF), granulocyte colony-stimulating factor (G-CSF) or interleukin-1 (IL-1) led to the formation of colonies containing mature haemopoietic cells of the granulocytic, megakaryocytic or monocytic lineages. In contrast, erythroid development did not occur unless the protoporphyrin, haemin, was added to the cultures. Under these conditions mature erythroid cells were produced in cultures containing either IL-3 or GM-CSF (with or without erythropoietin (epo)). In replating experiments it was determined that the FACS-BM cells were able to generate large numbers of clonogenic cells for up to 30-40 free cultures. Such cultures, therefore, may be useful for investigating the biological and basis of the generation of clonogenic cells and of haemopoietic cell differentiation and development in response to growth factors.

Interleukin-3 (IL-3) dependent multipotent haemopoietic stem cells FDCP-Mix A4 (A4) were induced to differentiate and develop into mature neutrophils in response to Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) plus granulocyte CSF (G-CSF). This resulted in an increase in cell number over seven days of culture, following which the cells lost the ability to undergo further proliferation. The effect of GM-CSF on these cells has been assessed at various stages of development. Clonogenic cells, able to respond to GM-CSF, were generated only at days 3, 4 post-induction. From day 5 onwards, mature post-mitotic neutrophils are produced and clonogenic cells are lost. Loss of proliferative potential, in response to GM-CSF, was confirmed using [3H]-thymidine incorporation. Receptors for GM-CSF, were also measured during development using [125I]-GM-CSF binding assays. Although the dissociation constant for GM-CSF binding sites did not vary considerably, the number of such sites increased dramatically from about 20 (day 0, when the cells have a primitive morphology) to about 1000 by day 6 (when the cells are predominantly mature neutrophils). GM-CSF-stimulated Na+/H+ antiport activation was also determined. Although few GM-CSF receptors are expressed at day 0, there is a significant response (63% of maximal) to GM-CSF in terms of intracellular alkalinisation: this response increased markedly until, by day 4 (700 GM-CSF binding sites/cell), there is a maximal activation of the antiport by GM-CSF. By day 7 (greater than 900 GM-CSF binding sites/cell), however, there is significant reduction in activation of the Na+/H+ antiport by GM-CSF. Nonetheless, increased viability of these mature cells is still seen in response to GM-CSF. These results suggest that not only does expression of GM-CSF receptors alter during development of multipotential cells to mature neutrophils, but that these receptors are coupled to different intracellular effector mechanisms as the cells progressively mature.

Using a population of cells highly enriched for multipotential day 12 spleen colony forming cells (CFU-S) (termed the FACS-BM population), and a serum-free culture system, the requirements for development of multipotential cell have been investigated and compared to previous results using serum containing cultures. In both serum-free and serum supplemented cultures interleukin-3 (IL-3) was a potent colony stimulating factor, although it was more effective in serum free conditions. However, colony stimulation by granulocyte-macrophage colony stimulating factor (GM-CSF) and macrophage-colony stimulating factor (M-CSF) was markedly reduced in the absence of serum. Significantly, the ability of interleukin-1 (IL-1) and granulocyte-colony stimulating factor (G-CSF) to synergise with these two growth factors was retained in serum-free conditions, indicating that these growth factors act directly on the FACS-BM without serum co-factors. Furthermore synergistic interactions between IL-3 plus IL-1, and IL-3 plus M-CSF were only manifest in serum-free conditions. The significance of these results in relation to the ability of these growth factors to act directly on multipotential cells is discussed.

Haemopoietic stem cells in vivo proliferate and develop in association with stromal cells of the bone marrow. Proliferation and differentiation of haemopoietic stem cells also occurs in vitro, either in association with stromal cells or in response to soluble growth factors. Many of the growth factors that promote growth and development of haemopoietic cells in vitro have now been molecularly cloned and purified to homogeneity and various techniques have been described that allow enrichment (to near homogeneity) of multipotential stem cells. This in turn, has facilitated studies at the mechanistic level regarding the role of such growth factors in self-renewal and differentiation of stem cells and their relevance in stromal-cell mediated haemopoiesis. Our studies have shown that at least some multipotential cells express receptors for most, if not all, of the haemopoietic cell growth factors already characterized and that to elicit a response, several growth factors often need to be present at the same time. Furthermore, lineage development reflects the stimuli to which the cells are exposed, that is, some stimuli promote differentiation and development of multipotential cells into multiple cell lineages, whereas others promote development of multipotential cell into only one cell lineage. We suggest that, in the bone marrow environment, the stromal cells produce or sequester different types of growth factors, leading to the formation of microenvironments that direct cells along certain lineages. Furthermore, a model system has been used to show the possibility that the self-renewal probability of multipotential cells can also be modulated by the range and concentrations of growth factors present in the environment. This suggests that discrete microenvironments, preferentially promoting self-renewal rather than differentiation of multipotential cells, may also be provided by marrow stromal cells and sequestered growth factors.

A multipotent hemopoietic cell line has been employed to assess the influence of the hemopoietic growth factors, IL-3, GM-CSF, G-CSF, and CSF-1 on the processes of self-renewal, the generation of lineage restricted progenitor cells and the production of mature neutrophils and macrophages. At a high concentration of IL-3, the cells undergo self-renewal and demonstrate little or no ability to undergo differentiation in the presence of the other growth factors. In the absence of IL-3, the cells show minimal (GM-CSF) or no (G-CSF or CSF-1) ability to respond to these other growth factors. When combined with a low concentration of IL-3, the ability of the cells to respond to GM-CSF, G-CSF, and CSF-1 is enhanced and a selective preference for the neutrophil or macrophage lineage is seen depending on the combination used, i.e., the presence of CSF-1 preferentially promotes macrophage development and G-CSF preferentially promotes neutrophil development. Conditions optimal for neutrophil development were seen using a combination of low IL-3 concentrations plus GM-CSF plus G-CSF. In such conditions, the cells undergo extensive proliferation and progressively lose their clonogenic potential (i.e., differentiation much greater than self-renewal) and acquire the biochemical markers characteristic of fully mature phagocytes.

In the adult, blood cell production or haemopoiesis takes place mainly in the bone marrow. The blood cell types produced are a reflection of the needs of the organism at any moment, for example bacterial infection leads to a large increase in neutrophil production. The rate and scale of blood cell production in vivo are regulated, at least in part, by the synthesis and release of specific cytokines both within the bone marrow and also from other tissues. Here we detail the range of cytokines which act directly on haemopoietic stem cells and myeloid progenitor cells. Also cellular systems which will permit the elucidation of the specific interactions between these various cytokines which regulate stem cell self-renewal, differentiation and proliferation are described.

Stromal cells of the bone marrow can provide the growth-promoting and differentiation-inducing molecules which are necessary for haemopoiesis. While the nature of these stimuli is largely unknown, the development of haemopoietic cells in association with stromal cells requires intimate cell contact. Molecules of the extracellular matrix, such as heparan sulphate, are able to bind growth factors and in this way the stromal cells may form microenvironmental niches which preferentially promote development of multipotent and committed cells along discrete lineages. Cells from some patients with acute and chronic myeloid and lymphoid leukaemias are defective in their ability to interact with stromal cells and consequently cannot survive in stromal cell-mediated long-term marrow cultures. We have exploited this phenomenon to obtain normal haemopoietic cells from patients with leukaemia, and to use these cells for successful autografting in patients with acute and chronic myeloid leukaemias.

Two potent inhibitors of mono-adenosine diphosphate (ADP) ribosylation have recently been described and characterized, named p-methoxylbenzylaminodecamethylene guanidine sulfate (MBAMG) and benzylaminododecylguanine hydrochloride (BADGH). We have used these agents to investigate the role of ADP ribosylation in hematopoiesis using long-term marrow cultures. The addition of MBAMG (10(-6) mol/L) or BADGH (5 X 10(-4) mol/L) led to both an inhibition of mature cell production and the development of colony-stimulating factor (CSF-1)-responsive GM-CFC, but had no effect upon spleen colony-forming units (CFU-S) or on progenitor cells which respond to the multilineage stimulating factor present in WEHI-3B cell-conditioned medium. These data indicate that these inhibitors of mono-ADP ribosylation can block the commitment and/or differentiation of stem cells and infers that ADP ribosylation may be of some importance in the hematopoietic process.

Interleukin 3 (IL-3) is an important regulator of haemopoietic stem cell proliferation both in vivo and in vitro. Little is known about the possible mechanisms whereby this growth factor acts on stem cells to stimulate cell survival and proliferation. Here we have investigated the role of intracellular pH and the Na+/H+ antiport in stem cell proliferation using the multipotential IL-3-dependent stem cell line, FDCP-Mix 1. Evidence is presented that IL-3 can stimulate the activation of an amiloride-sensitive Na+/H+ exchange via protein kinase C activation. IL-3-mediated activation of the Na+/H+ exchange is not observed in FDCP-Mix 1 cells where protein kinase C levels have been down-modulated by treatment with phorbol esters. Also the protein kinase C inhibitor H7 can inhibit IL-3-mediated increases in intracellular pH. This activation of Na+/H+ exchange via protein kinase C has been shown to occur with no measurable effects of IL-3 on inositol lipid hydrolysis or on cytosolic Ca2+ levels. Evidence is also presented that this IL-3-stimulated alkalinization acts as a signal for cellular proliferation in stem cells.

Haemopoietic cell growth factors are normally assayed using unfractionated marrow cells (NBM). However, using this population it is difficult to distinguish between direct versus indirect effects, because of the low incidence of colony forming cells (CFC) and the presence of possible accessory cells (which may themselves be acted upon by the growth factors and stimulated to produce other growth stimulatory or inhibitory molecules that influence the development of the CFC). Furthermore, NBM contain the whole spectrum of multipotent and lineage-restricted CFC and it is often difficult to determine precisely which populations are being stimulated to develop. This latter problem can be solved, in part, by using marrow from mice previously treated with 5-fluorouracil (5-FU): an agent that preferentially kills the more mature, actively cycling CFC but spares the proliferatively quiescent multipotent stem cells. Since the 5-FU-treated marrow also contains many possible accessory cells, however, it is again not clear whether or not the responses elicited by growth factors are due to direct or indirect effects upon the CFC. To circumvent this problem we have obtained a highly enriched population of multipotent stem cells (FACS-BM) that is free of accessory cells, and have compared the responses of these cells to NBM and to 5-FU-BM in the presence of a variety of growth factors.(ABSTRACT TRUNCATED AT 250 WORDS)

Interleukin-3 (IL-3)-dependent cell lines (FDCP-mix) were cloned and isolated from long-term bone-marrow cultures infected with src-MoMuLV. These cell lines have many of the characteristics of hematopoietic stem cells. Early isolates of the FDCP-mix cells form spleen colonies in irradiated mice and establish long-term hematopoiesis on irradiated marrow stroma in vitro in the absence of IL-3. These two properties of the cells are lost within 15 weeks of establishing the cell lines, but the cell lines retain their ability to differentiate in a multilineage response to hematopoietic growth factors and to hematopoietic stromal cells, as well as to self-renew in the presence of IL-3. The choice between differentiation and self-renewal in FDCP-mix cells can clearly be modified by culture conditions: in particular, cultures containing horse serum preferentially promote self-renewal, whereas cultures containing fetal calf serum preferentially promote differentiation. The FDCP-mix cell lines are not leukemic, nor do they contain the src oncogene. Their ability to respond to hematopoietic growth factors and stroma in a similar manner to normal hematopoietic cells makes them a valuable model for studying the regulation of hemopoietic cell self-renewal and differentiation.

The in vitro differentiation of multipotent stem cells in long-term marrow cultures can be blocked by treatment with agents that modify cholera toxin induced ADP-ribosylation of proteins. The latter agents also inhibit the growth and development of progenitor cells in soft gels in response to interleukin-3 but have little effect upon the development of progenitor cells that respond to the macrophage colony stimulating factor (CSF-1). Cholera toxin, in the same system, inhibits the development of CSF-1 responsive progenitor cells but has little effect on the development of cells that respond to IL-3. Similarly, progenitor cells that respond to IL-3 are relatively more resistant to pertussis toxin than cells that respond to CSF-1. These data indicate that ADP-ribosylation may be an important post-translational modification of regulatory proteins concerned with hemopoietic cell differentiation and growth in response to stromal cells or growth factors.

Interleukin 3 (IL-3) promotes the survival, proliferation and development of progenitor cells from several distinct haemopoietic lineages and can also stimulate the self-renewal of stem cells. We have explored the mode of action of this growth factor in promoting survival and proliferation, using a multipotent haemopoietic stem cell line FDC-Mix 1. In the absence of IL-3 these cells died within 16-48 h. However, this requirement for IL-3 could be replaced by 12-O-tetradecanoylphorbol-13-acetate (TPA) plus Ca2+ ionophore, which promoted not only survival but also DNA synthesis with no concomitant loss of the multipotential nature of these cells. TPA and Ca2+ ionophore, respectively, could also interact synergistically with IL-3 to promote DNA synthesis. Both IL-3 and TPA stimulated the translocation of protein kinase C (PK-C) from the cytosol to a membrane-bound form in FDC-Mix 1 cells. Previously we suggested that IL-3 can activate the primary metabolism of IL-3-dependent cells so that increased glucose transport and glycolysis lead to maintenance of ATP levels and cellular survival. To investigate whether TPA and, or, Ca2+ ionophore could also influence cellular survival via an activation of glucose uptake we assessed the effects of these agents on hexose transport. TPA +/- Ca2+ ionophore activated hexose transport to the same degree as does IL-3 but these agents cannot superstimulate FDC-Mix 1 hexose transport in cells that already exhibit an activated transport system from preincubation with IL-3. We conclude that IL-3 maintains FDC-Mix 1 cells via its ability to activate PK-C and increase cytosolic levels of Ca2+, and that an IL-3-mediated activation of PK-C may promote cellular survival via its ability to enhance hexose uptake by phosphorylating the glucose transport protein.