Biomedical subjects

H E Broxmeyer

Publications and source records attributed to H E Broxmeyer.

At least 163 records · Page 9Linked to original sources

PubMed1995

Cryopreserved cord blood myeloid progenitor cells can serve as targets for retroviral-mediated gene transduction and gene-transduced progenitors can be cryopreserved and recovered.

To determine future possibilities for gene transfer, we evaluated whether myeloid progenitor cells from human umbilical cord blood (CB) could be frozen, thawed in viable form and transduced with a Neomycin resistance (NeoR) gene using retroviral vectors, and if fresh progenitor cells transduced with a Neo gene could be cryopreserved and recovered. Fresh and thawed cryopreserved nonadherent low-density T-lymphocyte depleted (NALT-) CB cells were assayed before and after gene transduction for colony formation in the presence of multiple growth factors in the absence and presence of G418. The results demonstrate that the NeoR gene could be introduced into thawed cryopreserved myeloid progenitor cells at an efficiency similar to that of fresh cells and that fresh cells transduced with the NeoR gene could be frozen in a cryopreserved state and recovered after thawing. Proviral integration, as assessed by PCR/Southern Analysis, confirmed the G418R colony data. Proviral integration was detected not only in primary G418R-colonies, but also in replated colonies in secondary dishes derived from G418R-multipotential progenitor cells (CFU-GEMM) suggesting stable integration of the transduced gene into early subsets of replatable progenitors. This information may be of use clinically.

3T3 Cells↗

PubMed1995

Transgenic mice expressing either bovine growth hormone (bGH) or human GH releasing hormone (hGRH) have increased splenic progenitor cell colony formation and DNA synthesis in vitro and in vivo.

To investigate the potential effects of growth hormone (GH) on the hematopoietic system, mice transgenic for bovine GH (bGH) or human growth hormone releasing hormone (hGRH) genes, each of which can result in the constitutive overproduction of GH, were analyzed for splenic and bone marrow (BM) localized hematopoietic progenitor cells. These transgenic mice had splenic hyperplasia with increased absolute numbers of splenic erythroid and megakaryocytic progenitor cells as assessed by in vitro assay and megakaryocyte development as seen in spleens. As an in vivo indication of multilineage progenitor cell effects in hGRH mice, the number of day-10 CFU-S colonies derived from the donor spleen was significantly higher than in nontransgenic littermate controls. A high proportion (54-71%) of splenic erythroid, granulocyte-macrophage, and megakaryocyte progenitors were in cycle in transgenic mice in contrast to < or = 30% in nontransgenic control littermates. Compared to controls, splenocytes from hGRH mice had a significantly higher proliferative index when infused into irradiated nontransgenic controls. With the exception of the megakaryocyte colony assay and in vivo proliferative index, none of these findings were evident when identical assays were performed on the BM from the same mice. Consistent with the BM data, peripheral blood leukocyte, erythroid, and platelet numbers were comparable in transgenic and nontransgenic control littermates. We conclude that the constitutive expression of bGH or hGRH leads largely to splenic hematopoietic effects involving progenitor cell populations from at least two lineages.

Animals↗

PubMed1995

The combination of Steel factor and GM-CSF blocks apoptosis induced by retinoic acid and upregulates AP-1 in a human growth factor-dependent cell line.

The effects of hematopoietic growth factors were examined on the cellular action of retinoic acid (RA) using the human factor-dependent cell line, MO7e. Treatment of cells with Steel factor (SLF) plus granulocyte-macrophage colony-stimulating factor (GM-CSF) synergistically stimulated cell proliferation compared to that with each factor alone. This synergism was even greater in the presence of RA than in its absence. Treatment of cells with RA resulted in apoptotic cell death associated with internucleosomal DNA fragmentation in the presence of either SLF or GM-CSF. RA-induced apoptosis and DNA fragmentation were completely blocked by treating cells with SLF plus GM-CSF. Northern analysis showed that the inhibition of RA effects on MO7e cells by SLF plus GM-CSF treatment occurred without modulation of expression of RA receptor-alpha (RAR-alpha) gene. Furthermore, a higher amount of AP-1 complex was detected by electrophoretic mobility shift assays in a nuclear extract prepared from cells treated with SLF plus GM-CSF compared to those treated with each factor alone, while the level of RAR-complex remained similar in cells treated with SLF and/or GM-CSF. These data suggest an interaction in signaling pathways among different types of receptors that might be associated with the AP-1 complex.

Apoptosis↗

PubMed1995

Flt3 ligand stimulates/costimulates the growth of myeloid stem/progenitor cells.

The present studies evaluated effects of recombinant human (rhu) and murine (rmu) flt3 ligand (flt3-L) on colony formation by subsets of myeloid stem and progenitor cells present in low-density (LD) and cell-sorted CD34 hu cord blood (CB) and bone marrow (BM) cells and unseparated mu BM cells. By itself, flt3-L had weak colony-stimulating activity. It stimulated small dispersed CFU-GM-type colonies, but not BFU-E, CFU-GEMM, or HPP-CFC colonies, from LD and CD34 huCB and BM. However, flt3-L had additive to greater-than-additive effects on colony number and size by CFU-GM stimulated with GM-CSF or IL-3, with or without Steel factor (SLF); by CFU-G stimulated by G-CSF with or without SLF; by CFU-M stimulated by CSF-1; and by BFU-E, CFU-GEMM, and HPP-CFC stimulated by Epo with or without IL-3 or SLF. Flt3-L enhanced the effects of SLF, alone and in combination with other CSFs. Similar effects were apparent on LD and sorted CD34 cells and also at the level of single sorted and isolated CD34 cells/well. Flt3-L enhanced expansion of immature subsets of huCD34(+)-column separated CB CFU-GM stimulated by the potent combination of SLF and PIXY321 (a GM-CSF/IL-3 fusion protein). While flt3-L did not enhance the replating capacity of CFU-GEMM plated in the presence of Epo and SLF, it enhanced numbers of these CFU-GEMM colonies with the capacity to be replated. Flt3-L effects were not species-specific; rhu and rmu forms were active on huCB/BM and muBM. These results demonstrate the potent direct-acting stimulating/costimulating activities of flt3-L in vitro on myeloid stem/progenitor cells.

Adult↗

PubMed1995

Tyrosine phosphorylation and activation of focal adhesion kinase (p125FAK) by BCR-ABL oncoprotein.

Focal adhesion kinase (p125FAK; FAK) is a protein tyrosine kinase that is tyrosine-phosphorylated in response to v-src-mediated transformation, cell adhesion, and stimulation with neuropeptides. To elucidate a possible functional relationship between FAK and BCR-ABL oncoprotein detected in Philadelphia chromosome-positive (Ph+) leukemias, we investigated the tyrosine phosphorylation state of FAK in a murine growth factor-dependent cell line and in its stable human bcr-abl cDNA transfectant. In interleukin-3 (IL-3)-dependent NFS/N1.H7 cells, tyrosine phosphorylation of FAK was not detected after stimulation with either IL-3 or Steel factor (SLF), both of which involve Ras-mediated signaling pathways. However, stable gene transfection with p210bcr-abl cDNA into H7 cells made these cells growth factor-independent for proliferation and resulted in constitutive tyrosine phosphorylation and kinase activation of FAK. Constitutive phosphorylation and activation of FAK was also observed in all Ph+ leukemia cell lines examined--that is, K562, TS9;22, and YS9;22, which express p210BCR-ABL, and NALM-21 and OM9;22, which express p185BCR-ABL. Ph-negative (Ph-) cell lines, such as MO7e and JM, did not show any detectable tyrosine phosphorylation of FAK. FAK phosphorylation in BCR-ABL-expressing cells was inhibited in a dose-dependent manner by cytochalasin D, a reagent that disrupts the intracellular network of actin filaments. However, no suppression of kinase activity or protein expression of BCR-ABL was observed after treatment with cytochalasin D. A physical association between BCR-ABL and FAK was not apparent. These data suggest that BCR-ABL may be involved in the activation of FAK. Moreover, FAK may be distinct from components in Ras-mediated signaling cascades that are activated by stimulation of myeloid cells with various cytokines.

Animals↗

PubMed1995

Adeno-associated virus 2-mediated gene transfer and functional expression of the human granulocyte-macrophage colony-stimulating factor.

It is becoming increasingly clear that the adeno-associated virus 2 (AAV)-based vector system may prove to be useful for high-efficiency gene transfer in human cells as well as for potential gene therapy in humans. A recombinant AAV vector containing the gene for a human hematopoietic growth factor, granulocyte-macrophage colony-stimulating factor (GM-CSF), was constructed and used to infect COS-1 cells, a monkey kidney cell line. COS-1 cells infected with the recombinant virus, but not mock-infected cells, expressed high levels of the human GM-CSF gene transcripts. Furthermore, in co-cultivation experiments with the recombinant virus-infected cells, but not in those with mock-infected cells, active proliferation of a GM-CSF-dependent human megakaryocytic leukemia cell line, M07e, could be obtained in the absence of exogenously added GM-CSF. The recombinant GM-CSF protein released into the supernatant was biologically active in progenitor cell assays carried out with primary human hematopoietic cells, and this activity was specifically abrogated by treatment of the supernatant with anti-GM-CSF antibodies. This recombinant virus may be potentially useful in the management and gene therapy of a variety of malignant disorders in the human hematopoietic system.

Animals↗

PubMed1995

Influence in vitro of IL-3/Epo fusion proteins compared with the combination of IL-3 plus Epo in enhancing the proliferation of single isolated erythroid and multipotential progenitor cells from human umbilical cord blood and adult bone marrow.

Human interleukin-3/erythropoietin (IL-3/Epo) fusion protein have been constructed, expressed, and tested for biological activity. These fusion proteins were previously shown to be active on erythroid progenitors (BFU-E) from unseparated human bone marrow. We evaluated if these fusion proteins could stimulate erythroid and multipotential progenitor cells directly at the single-cell level. Two IL-3/Epo fusion proteins containing short (SL-3E, two amino acids) and long (LL-3E, 23 amino acids) linker sequences as well as a short linker Epo/IL-3 sequence (SL-E3, three amino acids) were tested. Highly enriched CD34 or BFU-E enriched CD34 CD33- cells from human umbilical cord blood or CD34 HLA-DR+CD33- cells from normal adult bone marrow were sorted as single cells into single wells. The combination of Epo plus IL-3 synergized to enhance the proliferation of BFU-E and multipotential progenitors (CFU-GEMM) in comparison to the individual effects of these cytokines. The three fusion proteins also enhanced proliferation of BFU-E and CFU-GEMM at the single-cell level and were at least as active as the combination of Epo and IL-3, demonstrating that IL-3/Epo fusion proteins directly stimulate proliferation of BFU-E and CFU-GEMM and that biological activity of IL-3 and Epo in vitro can be maintained when these proteins are fused. The activity of the combination of Epo and IL-3 or the fusion proteins was partially neutralized by preincubation with monoclonal antibodies to either Epo or IL-3 and was neutralized by greater than 90% by the combination of both antibodies, suggesting that the Epo and IL-3 components of the fusion proteins were both involved in the enhancing activity of these proteins. Additionally, use of monoclonal antibody to the human Epo receptor completely blocked the stimulating/enhancing activity of Epo alone, Epo plus IL-3, or the fusion proteins for stimulation of colony formation by BFU-E and CFU-GEMM but not for granulocyte-macrophage progenitors (CFU-GM), suggesting that the enhancing effects of the fusion proteins are most likely mediated, at least in part, by the Epo receptor.

Bone Marrow Cells↗

PubMed1995

Effects of in vivo treatment with PIXY321 (GM-CSF/IL-3 fusion protein) on proliferation kinetics of bone marrow and blood myeloid progenitor cells in patients with sarcoma.

PIXY321, a stimulator of multipotent colony-forming units (CFU-GEMM), burst-forming unit-erythroid (BFU-E), and colony-forming units granulocyte/macrophage (CFU-GM) progenitor cell proliferation in vitro, is currently being assessed in phase-I/-II clinical trials. We evaluated kinetics of CFU-GEMM, BFU-E, and CFU-GM proliferation in bone marrow (BM), blood, and granulocyte (CFU-G) and macrophage (CFU-M) progenitors in BM of chemotherapy-naive patients with sarcoma-administered PIXY321 (25 to 1000 micrograms/m2/day subcutaneously for 14 days). BM and/or blood cells from three to four patients at each dosage were assessed before, during, 1 to 2 days, and 7 days following treatment with PIXY321. Cells were pulse-treated in vitro with or without high-specific activity tritiated thymidine (3H-dThr) (to assess the percentage of progenitors in S-phase of cell cycle) and cultured for immature and more mature subsets of CFU-GEMM, BFU-E, CFU-GM, and for CFU-G and CFU-M. Despite heterogeneity in baseline cycling status of progenitors in BM, administration of 125 to 500 micrograms PIXY321 to patients at least doubled (p < 0.001) cycling rates of all BM progenitors. The cycling rates of blood progenitors increased from a slow or non-cycling state to > 38% cells in cycle. Within 1 to 2 days after cessation of PIXY321 infusion, all progenitors were in a slow or noncycling phase below that of many of the pre-BM samples (immature and mature CFU-GM and mature BFU-E) or were back to background levels (CFU-G, CFU-M, CFU-GEMM, and immature BFU-E). These cycling effects were similar to those previously noted for BM CFU-GM and BFU-E in patients on clinical trial with GM-CSF. In contrast, higher dosages of PIXY321, especially 1000 micrograms, increased cycling of BM and blood progenitor cells early during treatment, but cycling rates decreased while patients were still being administered PIXY321; decreased cycling was maintained after cessation of PIXY321. PIXY321 is thus highly active in vivo as a stimulator of multipotential and more lineage-restricted progenitors. The kinetics of progenitor cell proliferation noted in this study highlight differences seen with GM-CSF and G-CSF. The effects described here could be of relevance in design of future clinical trials using PIXY321 as an adjunct treatment in patients undergoing cytoreductive therapy.

Blood Cells↗

PubMed1994

The ubiquitously expressed Syp phosphatase interacts with c-kit and Grb2 in hematopoietic cells.

The c-kit proto-oncogene encodes a transmembrane tyrosine kinase receptor, which is important for the normal development of hematopoietic cells, melanoblasts, and germ cells. Autophosphorylation of c-kit receptor on tyrosine creates binding sites for cellular src homology 2 (SH2)-containing signaling molecules. The discovery of phosphotyrosine phosphatases that contain SH2 domains suggests roles for these molecules in growth factor signaling pathways. We found that Syp, a phosphotyrosine phosphatase widely expressed in all the tissues in mammals, associates with c-kit receptor after activation with its ligand, steel factor, in the factor-dependent cell line, M07e. Both NH2-terminal and COOH-terminal SH2 domains of Syp, made as glutathione S-transferase fusion proteins, were able to bind to the activated c-kit receptor in vitro. Furthermore, Syp became marginally phosphorylated on tyrosine upon c-kit receptor activation, and tyrosine-phosphorylated Syp was found to be complexed with Grb2 in steel factor-stimulated M07e cells. Direct binding between Syp and Grb2 was also observed in vitro. Last, Ras and Raf interacts in vitro as a result of steel factor-stimulated Ras activation. These results suggest that Syp may be an important signaling component downstream of the c-kit receptor and involved in activation of the Ras signaling pathway in hematopoietic cells.

Adaptor Proteins, Signal Transducing↗

PubMed1994

Influence of elevated temperature on natural killer cell activity, lymphokine-activated killer cell activity and lectin-dependent cytotoxicity of human umbilical cord blood and adult blood cells.

PURPOSE: To determine whether hyperthermia is to the benefit or detriment of host immune function, the effect of hyperthermia was evaluated on various functions of T-lymphocytes from human umbilical cord blood and compared to that of adult blood. METHODS AND MATERIALS: Nonadherent mononuclear cells from cord blood or adult blood were used as the effector cells. To generate lymphokine activated killer (LAK) cells, effector cells were kept in culture for 5 days in complete medium containing recombinant human interleukin-2. To activate effector cells to become cytotoxic, cells were kept in culture in complete medium containing Con A. Cytotoxicity was determined in a standard 4-h chromium release assay using K-562 human erythroleukemic cells (in the natural killer cell activity assay) or Daudi cells (in the LAK cell activity or Lectin dependent cytotoxicity assay) as targets. For heat effects, cells in complete medium were heated at the desired temperature in a water bath for 1 h. RESULTS: Lymphokine-activated killer cell activity, lectin-dependent cytotoxicity and T-cell proliferative capacity were not deficient in human cord blood. Cytotoxic activities of T-cells from adult blood as well as from cord blood can be enhanced at febrile range (< or = 40 degrees C), and were significantly decreased by exposure to 1 h at 42 degrees C. CONCLUSION: The febrile responses (< or = 40 degrees C) to infection, in the course of malignant disease and with biological response modifiers treatment, may all be related to host defense mechanisms. Based on these observations, whole body hyperthermia (< or = 40 degrees C), in combination with the appropriate cytokines, may have therapeutic potential in the treatment of neonatal infections and malignancies under certain circumstances. Hyperthermia in febrile range may, therefore, confer an important immunoregulatory advantage to the host. In contrast, tumor killing therapeutic temperature (> 42 degrees C) which inhibits host immunocompetence should probably be used only for local hyperthermia.

Adult↗

PubMed1994

Adeno-associated virus 2-mediated high efficiency gene transfer into immature and mature subsets of hematopoietic progenitor cells in human umbilical cord blood.

Recombinant adeno-associated virus 2 (AAV) virions were constructed containing a gene for resistance to neomycin (neoR), under the control of either the herpesvirus thymidine kinase (TK) gene promoter (vTK-Neo), or the human parvovirus B19 p6 promoter (vB19-Neo), as well as those containing an upstream erythroid cell-specific enhancer (HS-2) from the locus control region of the human beta-globin gene cluster (vHS2-TK-Neo; vHS2-B19-Neo). These recombinant virions were used to infect either low density or highly enriched populations of CD34+ cells isolated from human umbilical cord blood. In clonogenic assays initiated with cells infected with the different recombinant AAV-Neo virions, equivalent high frequency transduction of the neoR gene into slow-cycling multipotential, erythroid, and granulocyte/macrophage (GM) progenitor cells, including those with high proliferative potential, was obtained without prestimulation with growth factors, indicating that these immature and mature hematopoietic progenitor cells were susceptible to infection by the recombinant AAV virions. Successful transduction did not require and was not enhanced by prestimulation of these cell populations with cytokines. The functional activity of the transduced neo gene was evident by the development of resistance to the drug G418, a neomycin analogue. Individual high and low proliferative colony-forming unit (CFU)-GM, burst-forming unit-erythroid, and CFU-granulocyte erythroid macrophage megakaryocyte colonies from mock-infected, or the recombinant virus-infected cultures were subjected to polymerase chain reaction analysis using a neo-specific synthetic oligonucleotide primer pair. A 276-bp DNA fragment that hybridized with a neo-specific DNA probe on Southern blots was only detected in those colonies cloned from the recombinant virus-infected cells, indicating stable integration of the transduced neo gene. These studies suggest that parvovirus-based vectors may prove to be a useful alternative to the more commonly used retroviral vectors for high efficiency gene transfer into slow or noncycling primitive hematopoietic progenitor cells, without the need for growth factor stimulation, which could potentially lead to differentiation of these cells before transplantation.

Antigens, CD↗

PubMed1994

SH2-containing phosphotyrosine phosphatase Syp is a target of p210bcr-abl tyrosine kinase.

The phosphorylation of proteins at tyrosine residues is critical in cellular signal transduction and neoplastic transformation. These mechanisms are regulated by the activities of both protein-tyrosine kinases and protein-tyrosine phosphatases. Recent studies have identified a novel protein-tyrosine phosphatase, termed Syp, that is widely expressed in various tissues. Syp encodes a cytoplasmic phosphatase that contains two Src homology 2 (SH2) domains. Since SH2 domains have been shown to target the association of signal-transducing molecules to activated tyrosine kinases, experiments were performed to determine whether Syp might form specific complexes with p210bcr-abl, a fusion protein believed to be involved in the pathogenesis of chronic myelogenous leukemia and, thus, possibly alter or mediate p210bcr-abl tyrosine kinase activity. We found that Syp was highly and constitutively tyrosine phosphorylated in three different murine cell lines transfected with a p210bcr-abl expression vector. Furthermore, p210bcr-abl, Syp, and Grb2 formed stable complexes in BCR-ABL-expressing cells. Complex formation between p210bcr-abl and Syp was mediated in vitro by the NH2-terminal SH2 domain of Syp. Last, p210bcr-abl tyrosine kinase was effectively dephosphorylated by Syp in vitro. These results suggest an interaction between Syp and BCR-ABL protein, which might play a role in cellular transformation of BCR-ABL.

Animals↗

PubMed1994

Immature human cord blood progenitors engraft and proliferate to high levels in severe combined immunodeficient mice.

Unseparated or Ficoll-Hypaque (Pharmacia, Piscataway, NJ)--fractionated human cord blood cells were transplanted into sublethally irradiated severe combined immunodeficient (SCID) mice. High levels of multilineage engraftment, including myeloid and lymphoid lineages, were obtained with 80% of the donor samples as assessed by DNA analysis, fluorescence-activated cell sorting (FACS), and morphology. In contrast to previous and concurrent studies with adult human bone marrow (BM), treatment with human cytokines was not required to establish high-level human cell engraftment, suggesting that neonatal cells either respond differently to the murine microenvironment or they provide their own cytokines in a paracrine fashion. Committed and multipotential myelo-erythroid progenitors were detected using in vitro colony assays and FACS analysis of the murine BM showed the presence of immature CD34+ cells. In addition, human hematopoiesis was maintained for at least 14 weeks providing further evidence that immature hematopoietic precursors had engrafted the murine BM. This in vivo model for human cord blood-derived hematopoiesis will be useful to gain new insights into the biology of neonatal hematopoietic cells and to evaluate their role in gene therapy. There is growing evidence that there are ontogeny-related changes in immature human hematopoietic cells, and therefore, the animal models we have developed for adult and neonatal human hematopoiesis provide useful tools to evaluate these changes in vivo.

Animals↗

PubMed1994

Coupling between p210bcr-abl and Shc and Grb2 adaptor proteins in hematopoietic cells permits growth factor receptor-independent link to ras activation pathway.

Enforced expression of p210bcr-abl transforms interleukin 3 (IL-3)-dependent hematopoietic cell lines to growth factor-independent proliferation. It has been demonstrated that nonreceptor tyrosine kinase oncogenes may couple to the p21ras pathway to exert their transforming effect. In particular, p210bcr-abl was recently found to effect p21ras activation in hematopoietic cells. In this context, experiments were performed to evaluate a protein signaling pathway by which p210bcr-abl might regulate p21ras. It was asked whether Shc p46/p52, a protein containing a src-homology region 2 (SH2) domain, and known to function upstream from p21ras, might form specific complexes with p210bcr-abl and thus, possibly alter p21ras activity by coupling to the guanine nucleotide exchange factor (Sos/CDC25) through the Grb2 protein-Sos complex. This latter complex has been previously demonstrated to occur ubiquitously. We found that p210bcr-abl formed a specific complex with Shc and with Grb2 in three different murine cell lines transfected with a p210bcr-abl expression vector. There appeared to be a higher order complex containing Shc, Grb2, and bcr-abl proteins. In contrast to p210bcr-abl transformed cells, in which there was constitutive tight association between Grb2 and Shc, binding between Grb2 and Shc was Steel factor (SLF)-dependent in a SLF-responsive, nontransformed parental cell line. The SLF-dependent association between Grb2 and Shc in nontransformed cells involved formation of a complex of Grb2 with c-kit receptor after SLF treatment. Thus, p210bcr-abl appears to function in a hematopoietic p21ras activation pathway to allow growth factor-independent coupling between Grb2, which exists in a complex with the guanine nucleotide exchange factor (Sos), and p21ras. Shc may not be required for Grb2-c-kit interaction, because it fails to bind strongly to c-kit.

Adaptor Proteins, Signal Transducing↗

PubMed1994

Ligand-dependent polyubiquitination of c-kit gene product: a possible mechanism of receptor down modulation in M07e cells.

Quantities of proteins in cells are balanced by protein synthesis and degradation. Protein ubiquitination is an important adenosine-triphosphate dependent proteolytic pathway for "short-lived" proteins. We show that soluble steel-factor (SLF) stimulation at 37 degrees C rapidly induced polyubiquitination of c-kit protein in growth-factor-dependent human-myeloid cell line M07e, resulting in smeared, retarded migration of c-kit protein in sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the molecular weight region of 145 kD. Receptor ubiquitination was almost completely absent when cells were treated with SLF at 4 degrees C or at 37 degrees C in the presence of 0.2% sodium azide, or when the cells were pretreated with anti-c-kit monoclonal antibody or genistein, a tyrosine-kinase inhibitor. This suggested that c-kit ubiquitination was ligand dependent and appeared to require intrinsic tyrosine-kinase activation of the c-kit protein. Flow-cytometric analysis of c-kit expression on the cell surface of M07e cells showed down modulation of c-kit within 5 minutes after soluble-SLF treatment at 37 degrees C. However, rapid receptor down modulation was almost completely suppressed when cells were treated with SLF at 4 degrees C or at 37 degrees C in the presence of 0.2% sodium azide, conditions that concomitantly suppressed polyubiquitination of c-kit protein. In addition, these conditions almost completely suppressed radiolabeled SLF (125I-SLF) internalization after ligand-receptor interaction. Pulse-chase studies of 35S-methionine-labeled c-kit protein showed that SLF stimulation at 37 degrees C strikingly enhanced c-kit degradation (T1/2; approximately 20 minutes) compared with that in cells stimulated with SLF at 4 degrees C or at 37 degrees C with 0.2% sodium azide. However, in the presence of chloroquine, which blocks lysosomal degradation, this ligand-induced c-kit degradation at 37 degrees C was only suppressed in part. These data suggest that SLF-induced polyubiquitination of the c-kit receptor protein may play a role in regulation of c-kit-encoded protein-receptor expression in M07e cells.

Amino Acid Sequence↗

PubMed1994

Proliferative and cytotoxic responses of human cord blood T lymphocytes following allogeneic stimulation.

On the basis of the success of recent cord blood transplants, we undertook a comparison of the proliferative and cytotoxic potential, following allogeneic stimulation, of E-rosetted T cells from cord blood or adult peripheral blood (PB3) or bone marrow (BM). The magnitude of the proliferative response of cord blood (CB) T cells to alloantigen was greater than or equal to that of adult PB or BM T cells. Proliferation following culture with IL-2, IL-4, or IL-7 produced a similar result. In contrast, the cytotoxic activity in 1(0), 2(0), or 3(0) mixed leukocyte culture of CB T cells was minimal, typically less than 20% at an effector:target ratio of 100:1. Cytometric analysis of CB T cell populations before and after culture with allostimulation demonstrated similar ratios of CD4+ and CD8+ cells in cultures of CB and adult T cells. Down-regulation of CD45RA antigen expression, a measure of CD4+ cell activation, was comparable in adult and CB cultures. Activation of CD8+ CTL effectors was assessed by the acquisition of CD28 antigen expression. After culture, a smaller proportion of CD8+CD28+ effector cells was present in CB cultures as compared to adult T cell cultures. Thus, following in vitro priming with alloantigen CB T cells generate a vigorous proliferative response yet produce little antigen-specific cytotoxicity. This diminished cytotoxicity may, in part, be related to the low incidence of graft vs host disease thus far noted in human CB transplants.

Adult↗

PubMed1994

Physical characteristics and polymerization during iron saturation of lactoferrin, a myelopoietic regulatory molecule with suppressor activity.

Lactoferrin (LF) has been implicated in normal regulation of myeloid blood cell production in vitro and in vivo and abnormalities in LF-cell interactions have been associated with progression of leukemia and other hematopoietic disorders. LF may be clinically useful and for this reason we studied selected biochemical characteristics of LF. Purified human milk LF was saturated with iron from solution and analyzed by gel electrophoresis, ion-exchange and gel filtration chromatography. The metalloprotein was found to contain several molecular weight species on polyacrylamide gels. High resolution ion-exchange chromatography demonstrated the binding of LF to both anionic and cationic media under identical conditions indicating a bipolar charge distribution. Gel filtration studies revealed a tetramerized form of LF, the formation and stability of which was dependent on the ionic strength of the solution.

Buffers↗

PubMed1994

Myeloid cell proliferation stimulated by Steel factor is pertussis toxin sensitive and enhanced by cholera toxin.

Effects of G-protein toxins on Steel factor (SLF) and granulocyte-macrophage colony stimulating factor (GM-CSF) stimulated proliferation of human factor-dependent cell line, M07e, were evaluated. Pertussis toxin pretreatment suppressed GM-CSF- or Steel factor-induced proliferation by 54 +/- 8%; however, proliferation induced by the combination of GM-CSF plus Steel factor was suppressed to a much lesser extent (14 +/- 8%). Pretreatment of M07e cells with cholera toxin, suppressed GM-CSF- and GM-CSF plus Steel factor-stimulated proliferation by 57 +/- 6% and 79%, respectively, but increased the proliferative response to Steel factor alone by twofold. Similar effects of pertussis toxin and cholera toxin were observed on proliferation of normal myeloid progenitor cells from human umbilical cord blood. Pertussis toxin treatment of M07e cells for 4 h resulted in the ADP-ribosylation of 40-42 kDa protein band but did not significantly increase cyclic AMP levels. Cholera toxin pretreatment was associated with a 10-fold increase in intracellular cyclic AMP levels. These results implicate pertussis toxin sensitive pathways for both GM-CSF and Steel factor, but suggest that these pathways may not be required for synergistic proliferation stimulated by the combination. In addition, proliferation stimulated by GM-CSF, +/- Steel factor, is sensitive to cholera toxin pretreatment; whereas cholera toxin pretreatment enhanced proliferation stimulated by Steel factor, possibly via increased cyclic AMP. This suggests divergent signal transduction pathways for the two cytokines.

Adenylyl Cyclases↗