Preparative and analytical chromatography of pegylated myelopoietin using monolithic media.

Monolithic media were compared with Q- and SP-Sepharose high performance chromatography for preparative purification and with Q- and SP-5PW chromatography for analysis of a pegylated form of myelopoietin (MPO), an engineered hematopoietic growth factor. The use of either monolithic or Sepharose based supports for preparative chromatography produced highly purified pegylated MPO with the monolithic media demonstrating peak resolution and repeatability at flow rates of 1 and 5 ml/min resulting in run times as much as five-fold shorter compared to Sepharose separations. The monolithic disks also resulted in 10-fold shorter run times for the analytical chromatography, however, their chromatographic profiles and peak symmetry were not as sharp compared to their Q-5PW and SP-5PW counterparts.

Phage display mutagenesis of the chimeric dual cytokine receptor agonist myelopoietin.

Myelopoietins comprise a class of chimeric cytokine receptor agonists consisting of an hIL-3 (human interleukin-3) receptor agonist and an hG-CSF (human granulocyte colony-stimulating factor) receptor agonist linked head-to-tail at their respective carboxy and amino termini. The combination of an early acting cytokine (hIL-3) with a late acting one (hG-CSF) allows efficient hematopoeitic reconstruction following myeloablative insult, and drives differentiation of non-myelocytic lineages (ie thrombocytic lineages) that are inaccessible using hG-CSF alone, in both preclinical models and clinical settings. A myelopoietin species was displayed and mutagenized on filamentous bacteriophage: both component agonists of myelopoietin were presented in biologically functional conformations as each recognized its corresponding receptor. Five amino acid positions in a short region of the hG-CSF receptor agonist module of myelopoietin that had been identified as important for proliferative activity were mutagenized. Display was used because it allows very 'deep' mutagenesis at selected residues: >10(5) substitution variants were affinity-screened using the hG-CSF receptor and 130 new, active variants of myelopoietin were identified and characterized. None of the selected variants were significantly more active than the parental myelopoietin species in a hG-CSF-dependent cell proliferation assay, though many were as active. Many of these relatively high-activity variants contained parental amino acids at several positions, suggesting the parental sequence may already be optimal at these positions for the assays used, and potentially accounting for the failure to identify enhanced bioactivity variants. Analysis of substitutions of high-activity variants complements and extends previous alanine scanning, and other genetic and biochemical data for hG-CSF variants.

Stem cell growth factor: in situ hybridization analysis on the gene expression, molecular characterization and in vitro proliferative activity of a recombinant preparation on primitive hematopoietic progenitor cells.

INTRODUCTION: In situ hybridization of whole mouse fetuses and their tibias with a stem cell growth factor (SCGF) antisense probe demonstrated specific expression of SCGF mRNA around skeletal tissues, particularly in bone marrow cells, proliferating chondrocytes, the perichondrium and periosteum, but little expression in resting or hypertrophic chondrocytes. METHODS: Recombinant human (rh) SCGF-alpha was purified from a conditioned medium of SCGF-alpha gene-transfected CHO cells. The molecular mass of rhSCGF-alpha, 45 kDa, was shifted down to 40 kDa by digestion with endo-O-glycosidase and sialidase, suggesting O-glycosylation of rhSCGF-alpha with sialic acids. RESULTS: For human bone marrow CD34+Lin- cells, rhSCGF-alpha alone did not stimulate colony-formation, but small cluster-formation (10.3 +/- 2.5/1 x 10(3) CD34+Lin- cells). It promoted growth of erythroid and granulocyte/macrophage (GM) colonies in the primary culture with erythropoietin and GM colony-stimulating factor (CSF) or G-CSF, respectively, and further supported GM progenitor cells in a short-term liquid culture. In contrast, rhSCGF-alpha suppressed stem cell factor (SCF)-stimulated erythroid bursts, indicating some competitive interaction between SCGF and SCF. rhSCGF-alpha was synergistic with interleukin-3 and the flt3 ligand to enhance GM colony-growth, but not synergistic with those inducing ex vivo expansion of GM progenitor cells. CONCLUSION: SCGF is selectively produced by osseous and hematopoietic stromal cells, and can mediate their proliferative activity on primitive hematopoietic progenitor cells.

[Studies of a 35 KDa substance from human fetal liver on the regulation of hematopoiesis].

About 30%-40% of hematopoietic stem cells in human fetal liver of 3-5 months are in S phase of cell cycle, much higher than the ratio of 10% of that in adult bone marrow. The existance of highly active hematopoietic stem cell proliferation stimulators is probably its molecular basis. CFU-S "suicide rate" in rats was adopted to detect the effective substance. Through several steps of separation, we obtained a relatively purified substance of 35 kDa, termed it as FLS-4. CD 34 positive cord blood cells were sorted and assayed for their response to FLS-4 in 3H-TdR incorporation assay. The response to FLS-4 alone was approximately 1 times the background response seen with no factor added. In combination with IL-6 and IL-3 produced response that was 2.9 and 6.5 fold respectively greater than that observed with no factor added, but was weakly in comparison with the effects of SCF. In combination with GM-CSF or IL-3, FLS-4 can stimulate the formation of blast-colonies. The results indicate that the FLS-4 is very likely to be a novel hematopoietic stem cell proliferation stimulator. In physical or biological characteristics, it exhibited a unique character different from IL-3, IL-6, GM-CSF, SCF or FLT3 ligand those are known to have hematopoietic stem cell proliferation stimulating activity. During the period of active hematopoiesis in fetal liver, FLS-4 might be the candidate in triggering hematopoietic stem cells from resting G0 to S phase.

Effect of stem cell factor on leukemic progenitor cell growth and sensitivity to cytosine-arabinoside.

Recruitment of quiescent, clonogenic blasts from patients with acute myeloid leukemia (AML) by hematopoietic growth factors (HGFs) may improve the cytotoxic effects of cell-cycle-specific drugs like cytosine-arabinoside (Ara-C). Using the culture methods described by Nara and McCulloch and making a distinction between self-renewing and post-deterministic mitoses, we analyzed the effects of stem cell factor (SCF), a growth factor acting on early hematopoietic progenitor and stem cells. First, we demonstrated that SCF, used in combination with other HGFs included in fetal calf serum (FCS) and/or in 5637 cell line supernatant (5637-CM), stimulated both colony formation and self-renewal of blast progenitors from 10 patients, unlike SCF alone. We tested the effects of SCF on the recruitment of cells in the S-phase by using a bromodeoxyuridine/DNA (BrdUrd/DNA) staining method in flow cytometry (FCM). We showed that SCF stimulated proliferation of AML cells significantly in 9/18 patients with AML. Second, we tested the influence of SCF on the sensitivity to Ara-C of self-renewing leukemic cells from 18 patients with AML. We showed that SCF was efficient in increasing the toxicity of Ara-C on the self-renewing blast progenitors, especially with high concentrations of Ara-C. However, a large patient-to-patient heterogeneity was found and the activity of SCF was not correlated with its effect on the cell cycle. These data indicate that SCF can enhance sensitivity to Ara-C of some leukemic cells with self-renewing capacity.

In vitro characterization of a novel avian haemopoietic growth factor derived from stromal cells.

To better define the role of the chicken haemopoietic microenvironment in supporting haemopoiesis, a continuous cell line was generated by RSV transformation of avian spleen stromal cells (SSL-1). Supernatants from this line were found to contain haemopoietic growth factor activity as measured by the ability to induce proliferation and differentiation of precursor cells present in embryonic and post-hatched haemopoietic tissues. Comparison of cultures grown in the presence of cMGF and SSL-1 conditioned media (CM) revealed that both cytokine sources induced similar types of cell populations. Both sources supported the proliferation of predominantly macrophage-like cells based on colony morphology, differential staining, non-specific esterase staining, and phagocytosis activity. Interestingly, SSL-1 does not express any message for cMGF, nor does it secrete any IL-2 or interferon activities suggesting that the growth factor activities seen in SSL-1 are novel.

The effect of a hematopoietic-promoting factor (HPF) extracted from porcine kidney on the proliferation of human hematopoietic progenitor cells.

Hematopoietic-promoting factor (HPF), which was found in porcine kidney, has been demonstrated to act synergistically with colony-stimulating factor and erythropoietin on murine myeloid colony formation. We investigated the effect of HPF on the proliferation of human hematopoietic progenitor cells prepared from cord blood cells (CB) and peripheral blood cells (PB). HPF enhanced granulocyte colony-stimulating factor plus interleukin-3 and erythropoietin-induced colony formation, where the number of colonies were increased by 7.9-fold in CB and by 1.8-fold in PB, respectively. When we compared the effect of HPF with stem cell factor (SCF) on the colony formation derived from PB in serum-free cultures, HPF enhanced the number of erythroid burst-forming units (BFU-E) to the same extent as SCF. But the effect of HPF on promoting the growth of granulocyte-macrophage colony-forming units (CFU-GM) was less than SCF. When the enriched CD34+ cells from CB and PB were incubated in liquid culture with HPF and IL-3 for 7 days, CFU-GM was increased by 48-fold in CB, and by 25-fold in PB, respectively. The data demonstrate that HPF can potentiate expansion of hematopoietic stem cells to the same extent as SCF, and that the effects of HPF on hematopoitic stem cells differ from that of SCF.

The new generation of recombinant human hematopoietic cytokines.

In the past year, the most exciting development in the field of hematopoietic growth factors has been the identification of the platelet-inducing factor Mpl ligand. Administration of recombinant Mpl ligand may alleviate the potential for hemorrhagic complications following cancer therapies. Stem cell factor continues to be studied clinically in the mobilization of peripheral blood cells for transplantation.

Effect of a hematopoietic-promoting factor derived from porcine kidney on megakaryocyte colony formation by murine bone marrow cells.

We investigated the effect of a hematopoietic-promoting factor (HPF) purified from porcine kidney on murine megakaryocyte colony formation. Murine bone marrow cells were cultured in agar in Iscove's modified Dulbecco's medium supplemented with 10% fetal calf serum in the presence of interleukin-3 (IL-3) as a source of megakaryocyte colony-stimulating factor. HPF alone exerted only a slight effect on megakaryocyte colony formation, and the combination of HPF with IL-3 enhanced colony formation compared with IL-3 alone. At days 11 and 13, 1.9-fold and 1.7-fold enhancements were observed, respectively. When erythropoietin (Epo) was added to the combinations of IL-3 plus HPF or IL-3 plus HPF plus SCF megakaryocyte colonies were enhanced compared with the cultures without Epo. As the result of determination the ploidy distribution, more than 20% of the megakaryocytes in IL-3 plus HPF showed 32N and above ploidy, but the number of 8N and under megakaryocytes was the same as those stimulated with IL-3 alone. These results suggest that HPF stimulates both the proliferation of megakaryocyte colony-forming units and the maturation of megakaryocytes in the presence of IL-3.

Recombinant growth factors.

Recombinant human growth factors are expected to have a significant impact on the use of allogeneic blood components. For example, subsequent to the approval of recombinant human erythropoietin, blood transfusions in renal dialysis patients declined substantially. Likewise, myeloid growth factors have reduced infections and hospital stay by promoting hematologic recovery after high dose ablative chemotherapy. The high costs of these agents mandate that their use be limited to settings where they are clinically indicated. The use of growth factors may be monitored at medical centers by hospital transfusion committees. This chapter reviews the emerging clinical guidelines for the use of hematopoietic growth factors.

Matrix-assisted laser desorption mass spectrometric analysis of a pegylated recombinant protein.

Matrix-assisted laser desorption ionization mass spectrometry (MALDI) has been investigated as a technique for the characterization of recombinant stem cell factor that had been covalently modified with polyethylene glycol (PEG) chains. The attachment of PEG chains produces a heterogeneous mixture of protein species that differ by 6000 Da. These differences in molecular weight could be readily determined by MALDI. The potential of MALDI as a general strategy for characterizing PEG-modified proteins is discussed.

Mechanism of kit ligand, phorbol ester, and calcium-induced down-regulation of c-kit receptors in mast cells.

The proto-oncogene c-kit is allelic with the white spotting locus (W) on mouse chromosome 5 and it encodes a transmembrane protein tyrosine kinase which belongs to the platelet-derived growth factor and macrophage-colony stimulating factor (CSF-1) receptor subfamily. In an effort to study the function of the c-kit receptor, specifically the physiological mechanism of controlling the signal induced by the ligand, the effect and mechanism of down-regulation of the c-kit receptor by the kit ligand (KL) was investigated in mast cells. Following preincubation with KL, the capacity of mast cells to bind kit antibody was reduced and binding of radiolabeled KL to mast cells decreased with similar kinetics, suggesting that KL stimulates the loss of c-kit receptor from the cell surface. After binding to the c-kit receptor, KL was rapidly internalized, and degradation of the receptor was accelerated. The c-kit receptor was transmodulated by the protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate (TPA) and by the calcium ionophore ionomycin. TPA- and ionomycin-induced down-regulation of the c-kit receptor was accompanied by release of the extracellular domain of the receptor, presumably by proteolytic cleavage near the transmembrane domain. Release of the extracellular domain of the c-kit receptor occurred also in untreated cells but at a slow rate. In addition, ionomycin induced shedding of the intact c-kit receptor. In mast cells depleted of protein kinase C, the c-kit receptor remained sensitive to down-regulation induced by KL and ionomycin, but not by treatment with TPA. Therefore, the down-regulation of the c-kit receptor induced by KL, activated protein kinase C, and an increased level of intracellular calcium is mediated through independent mechanisms.

Association of hematopoietic cell phosphatase with c-Kit after stimulation with c-Kit ligand.

Protein tyrosine phosphorylation and dephosphorylation have been implicated in the growth and functional responses of hematopoietic cells. Recent studies have identified a novel protein tyrosine phosphatase, termed hematopoietic cell phosphatase (HCP) or PTP1C, that is predominantly expressed in hematopoietic cells. HCP 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 with activated growth factor receptors containing intrinsic protein kinase activity, we assessed the association of HCP with two hematopoietic growth factor receptors, c-Kit and c-Fms. The results demonstrate that HCP transiently associates with ligand-activated c-Kit but not c-Fms and that this association occurs through the SH2 domains. In both colony-stimulating factor 1- and stem cell factor-stimulated cells, there is a marginal increase in tyrosine phosphorylation of HCP. Lastly, HCP can dephosphorylate autophosphorylated c-Kit and c-Fms in in vitro reactions. The potential role of HCP in stem cell factor signal transduction is discussed.

Analysis of the heat-induced denaturation of proteins using temperature gradient gel electrophoresis.

A new technique, temperature gradient gel electrophoresis (TGGE), was applied to the study of heat-induced protein denaturation. The gels used contained 30 mM Borax + 75 mM boric acid, pH 8.4, and various concentrations of urea. When this technique was applied to bovine serum albumin, the protein showed discontinuous bands upon melting, indicating that the thermal transition is irreversible. The apparent melting temperature, calculated based on the relative intensity of two bands in the transition region, was 58 degrees C in 2 M urea. When the thermal denaturation of bovine serum albumin was analyzed spectroscopically, the transition was again irreversible, with an apparent transition temperature of 57 degrees C, consistent with the TGGE results. Recombinant stem cell factor, recombinant granulocyte macrophage-colony stimulating factor, and catalase were also analyzed by TGGE, indicating that the technique can be used to analyze denaturation of monomeric and multimeric proteins.

Soluble stem cell factor in human serum.

Stem cell factor (SCF) is a recently described factor active in the early stages of hematopoiesis. It can exist in membrane-bound form and in proteolytically released soluble form. The levels and nature of SCF in human serum are described. As determined by an enzyme-linked immunosorbent assay performed for 257 samples, SCF level in serum averaged 3.3 +/- 1.1 ng/mL. The serum SCF was partially purified by immunoaffinity chromatography and analyzed by glycosidase treatments in conjunction with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. The results show that the SCF has N-linked and O-linked carbohydrate and corresponds to the soluble form, at or about 165 amino acids in length. The findings suggest functional importance for soluble SCF in humans.

[Partial purification of bone marrow cell-stimulating activity from the parenchymal liver cell supernatant].

Mouse bone marrow cell-stimulating activity has been found in the supernatants of mouse parenchymal liver cells. In order to clarify the character, we attempted to purify the activity by a four-step purification procedure involving concentration and chromatographies on DEAE-cellulose, Sephacryl S-300, and Superose 6. By DEAE-cellulose chromatography, the activity was found to be eluted stepwisely with 0.1 M (Peak 1) and 0.2 M (Peak 2) NaCl. Gel filtration revealed that the activities in Peak 1 and Peak 2 had molecular weights of 170,000 and 600,000, respectively. Both preparations of the activity finally obtained derived the cells which spread over the plastic dish from mouse bone marrow cells, but did not stimulate the proliferation of IL-3/GM-CSF dependent cell line, IC2. These results suggest the presence of the bone marrow cell-stimulating activities, which are different from GM-CSF and IL-3, in the parenchymal liver cell supernatants.

Support of human hematopoiesis in long-term bone marrow cultures by murine stromal cells selectively expressing the membrane-bound and secreted forms of the human homolog of the steel gene product, stem cell factor.

The maintenance and differentiation of hematopoietic stem cells is influenced by cells making up the hematopoietic microenvironment (HM), including bone marrow-derived stromal cells. We and several other investigators have recently demonstrated the molecular basis of abnormal HM observed in the steel mutant mouse and cloned the normal cDNA products of this gene (termed SCF, KL, or MCF). In this report, we focus on the human counterpart of the mouse Steel (Sl) gene. Alternative splicing of the human SCF pre-mRNA transcript results in secreted and membrane-bound forms of the protein. To investigate the role of these two forms of human SCF, we targeted an immortalized stromal cell line derived from fetal murine homozygous (Sl/Sl) SCF-deficient embryos for gene transfer of various human cDNAs encoding SCF. We report that stable stromal cell transfectants can differentially process the two forms of human SCF protein product. We also demonstrate that both soluble SCF and membrane-bound SCF are active in increasing the number of human progenitor cells in the context of stromal cell cultures, although in a qualitatively different manner. Hence, the membrane-bound form of SCF may play an important role in the cell-cell interactions observed between stromal and hematopoietic cells both in vitro and in vivo.