Leah A. Marquez

The SDF‐1‐CXCR4 Axis Stimulates VEGF Secretion and Activates Integrins but does not Affect Proliferation and Survival in Lymphohematopoietic Cells

To better define the role HIV‐related chemokine receptor‐chemokine axes play in human hematopoiesis, we investigated the function of the CXCR4 and CCR5 receptors in human myeloid, T‐ and B‐lymphoid cell lines selected for the expression of these receptors (CXCR4+, CXCR4+ CCR5+, and CCR5+ cell lines). We evaluated the phosphorylation of MAPK p42/44, AKT, and STAT proteins and examined the ability of the ligands for these receptors (stromal‐derived factor‐1 [SDF‐1] and macrophage inflammatory protein‐1β [MIP‐1β]) to influence cell growth, apoptosis, adhesion, and production of vascular endothelial growth factors (VEGF), matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in these cell lines. We found that A) SDF‐1, after binding to CXCR4, activates multiple signaling pathways and that in comparison with the MIP‐1β‐CCR5 axis, plays a privileged role in hematopoiesis; B) SDF‐1 activation of the MAPK p42/44 pathway and the PI‐3K‐AKT axis does not affect proliferation and apoptosis but modulates integrin‐mediated adhesion to fibronectin, and C) SDF‐1 induces secretion of VEGF, but not of MMPs or TIMPs. Thus the role of SDF‐1 relates primarily to the interaction of lymphohematopoietic cells with their microenvironment and does not directly influence their proliferation or survival. We conclude that perturbation of the SDF‐1‐CXCR4 axis during HIV infection may affect interactions of hematopoietic cells with the hematopoietic microenvironment.

Differential MMP and TIMP production by human marrow and peripheral blood CD34+ cells in response to chemokines

As stromal cell-derived factor-1 (SDF-1), macrophage inflammatory protein-1alpha (MIP-1alpha), and interleukin-8 (IL-8) are implicated in the homing and mobilization of human hematopoietic progenitors (HPC), we hypothesized that these chemokines mediate the migration of HPC across subendothelial basement membranes by regulating production of matrix metalloproteinases (MMPs) and their natural tissue inhibitors (TIMPs). Assays for migration across reconstituted basement membrane (Matrigel) and chemotaxis were carried out using CD34(+) cells derived from normal human bone marrow (BM) and mobilized peripheral blood (PB). Secretion of MMPs and TIMPs was evaluated by zymography and reverse zymography and gene expression by RT-PCR. We found that an SDF-1 gradient increases the chemotaxis of BM and PB CD34(+) cells across Matrigel (BM > PB), which is blocked by inhibitors of MMPs (o-phenanthroline, rhTIMP-1, rhTIMP-2, and anti-MMP-9 and anti-MMP-2 antibodies) but enhanced by tumor necrosis factor-alpha (TNF-alpha), a strong stimulator of MMPs. Preincubation of these cells with SDF-1 stimulated the secretion of MMP-2 and MMP-9 in BM and PB CD34(+) cells but of TIMP-1 and TIMP-2 only in PB CD34(+) cells. Preincubation with MIP-1alpha and IL-8 also stimulated the secretion of MMP-9 and MMP-2 (BM > PB), but with respect to TIMPs, the effect was reversed (PB > BM), resulting in trans-Matrigel migration of BM but not of PB CD34(+) cells. We therefore propose that MMPs and TIMPs are involved in 1) SDF-1-induced chemotaxis of human HPC across subendothelial basement membranes, and 2) MIP-1alpha- and IL-8-stimulated migration of HPC.

Expression of matrix metalloproteinases (MMP-2 and -9) and tissue inhibitors of metalloproteinases (TIMP-1 and -2) in acute myelogenous leukaemia blasts: Comparison with normal bone marrow cells

We compared the expression of matrix metalloproteinases (MMP‐2 and MMP‐9) and tissue inhibitors of metalloproteinases (TIMP‐1 and TIMP‐2) in bone marrow acute myelogenous leukaemia (AML) blasts and leukaemic cell lines (HEL, HL‐60, K‐562 and KG‐1) with their expression in normal bone marrow cells. All AML samples and leukaemic cell lines tested expressed MMP‐9 and/or MMP‐2 mRNA and, accordingly, these gelatinases were secreted into media. Moreover, TIMP‐1 and TIMP‐2 mRNA and secreted proteins were demonstrated in all the AML samples. Although all the leukaemic cell lines expressed TIMP‐1, the HL‐60 cells also expressed TIMP‐2. In contrast, normal steady‐state bone marrow immature progenitor cells (CD34+ cells) did not express or secrete either MMP‐2 or MMP‐9, but more mature mononuclear cells from normal bone marrow expressed and secreted MMP‐9. Also, normal bone marrow CD34+ cells and mononuclear cells expressed TIMP‐1 and TIMP‐2 mRNA, but these proteins were not detectable by reverse zymography. Furthermore, whereas bone marrow fibroblasts and endothelial cells secreted only latent MMP‐2, the activated form of this enzyme was found in media conditioned by cells obtained from long‐term cultures of normal and AML bone marrow adherent layers. Our finding of up‐regulated production of gelatinases, TIMP‐1 and TIMP‐2 by leukaemic cells suggests that these proteins may be implicated in the invasive phenotype of AML.

Biological significance of MAPK, AKT and JAK-STAT protein activation by various erythropoietic factors in normal human early erythroid cells

The aim of this study was to identify signal transduction pathways activated by erythropoietin (EpO) and erythropoietin co‐stimulatory factors (kit ligand), insulin‐like growth factor, thrombopoietin, interleukin 3 and granulocyte‐macrophage colony‐stimulating factor) in normal human bone marrow CD34+ cells and d 11 erythroid burst forming unit derived glycophorin+ cells. The activation of these signal transduction pathways was further correlated with various biological effects such as (i) cell proliferation, (ii) inhibition of apoptosis, (iii) activation of adhesion and (iv) secretion of the matrix metalloproteinases (MMPs) MMP‐9 and MMP‐2, and vascular endothelial growth factor (VEGF). We found that in human CD34+ cells and erythroblasts erythropoietic factors may activate similar but different signalling pathways, and that activation of each of the JAK‐STAT, MAPK p42/44 or PI‐3K‐AKT axes alone is not sufficient either to stimulate cell proliferation or inhibit apoptosis, suggesting that these processes are regulated by orchestrated activation of multiple signalling cascades. Accordingly, we found that although cell proliferation was more related to simultaneous activation of JAK‐STAT and MAPK p42/44, the effect on cell survival correlated with activation of PI‐3K‐AKT, MAPK p42/44 and JAK‐STAT proteins. We also demonstrated that differentiating normal human erythroid cells lose their adhesive properties and secrete angiopoietic factors such as MMP‐9, MMP‐2 and VEGF, and we postulate that this secretion by early erythroid cells may play a role in their maturation and egress from the haematopoietic niches of the bone marrow.

Thrombopoietin, but not cytokines binding to gp130 protein-coupled receptors, activates MAPKp42/44, AKT, and STAT proteins in normal human CD34+ cells, megakaryocytes, and platelets

OBJECTIVE The development of megakaryocytes is regulated by thrombopoietin (TPO), which binds to the c-mpl receptor, and by several other cytokines such as interleukin (IL)-6, IL-11, leukemia inhibitory factor (LIF), cilliary neurotropic factor (CNTF), and oncostatin (OSM), which bind to gp130 protein-coupled receptors. We attempted to identify signal transduction pathways activated by these factors in normal human megakaryocytes. MATERIALS AND METHODS To better understand the role of these factors in normal human megakaryopoiesis we studied their effect on 1) purified human bone marrow-derived CD34+ cells, 2) human alpha(IIb)beta3+ cells (shown by immunophenotypical and morphological criteria to be megakaryoblasts), which had been expanded ex vivo from CD34+ cells in chemically defined artificial serum, and 3) gel-filtered human peripheral blood platelets. Further, in an attempt to correlate the influence of these factors on cell proliferation and survival with activation of signal transduction pathways, we evaluated their effect on the phosphorylation of MAPK p42/44 and activation of PI-3K-AKT and JAK-STAT proteins in these various cell types. RESULTS Using serum-free liquid cultures, we found that only TPO and IL-6 protected CD34+ cells and megakaryocytes from undergoing apoptosis (decrease in annexin-V binding, PARP cleavage, and activation of caspase-3). Moreover, only TPO when used alone and IL-6 only when used in combination with TPO, stimulated the growth of human colony-forming unit-megakaryocytes (CFU-Meg) in semisolid serum-free medium. We also observed that while TPO efficiently activated various signaling pathways in CD34+ cells, megakaryocytes, and platelets (MAPK p42/44, PI-3K-AKT, STAT proteins), IL-6 stimulated phosphorylation of STAT-1, -3, and -5 proteins only in CD34+ cells and megakaryoblasts. To our surprise, none of the other gp130 protein-related cytokines tested (IL-11, LIF, CNTF, and OSM) activated these signaling pathways in CD34+ cells, megakaryoblasts, or platelets. CONCLUSIONS Our signal transduction studies explain why TPO, by simultaneously activating several signaling pathways, is the most potent megakaryopoietic regulator and why of all five gp130 protein-related cytokines tested, only IL-6, through activation of STAT proteins, plays a role in normal human megakaryopoiesis.

Using 2-Aminopurine Fluorescence and Mutational Analysis to Demonstrate an Active Role of Bacteriophage T4 DNA Polymerase in Strand Separation Required for 3′ → 5′-Exonuclease Activity

The fluorescence of 2-aminopurine deoxynucleotide positioned in a 3′-terminal mismatch was used to evaluate the pre-steady state kinetics of the 3′ → 5′ exonuclease activity of bacteriophage T4 DNA polymerase on defined DNA substrates. DNA substrates with one, two, or three preformed terminal mispairs simulated increasing degrees of strand separation at a primer terminus. The effects of base pair stability and local DNA sequence on excision rates were investigated by using DNA substrates that were either relatively G + C- or A + T-rich. The importance of strand separation as a prerequisite to the hydrolysis of a terminal nucleotide was demonstrated by using a unique mutant DNA polymerase that could degrade single-stranded but not double-stranded DNA, unless two or more 3′-terminal nucleotides were unpaired. Our results led us to conclude that the reduced exonuclease activity of this mutant DNA polymerase on duplex DNA substrates is due to a defect in melting the primer terminus in preparation for the excision reaction. The mutated amino acid (serine substitution for glycine at codon 255) resides in a critical loop structure determined from a crystallographic study of an amino-terminal fragment of T4 DNA polymerase. These results suggest an active role for amino acid residues in the exonuclease domain of the T4 DNA polymerase in the strand separation step.

Matrix Metalloproteinases in the Hematopoietic Microenvironment

Matrix metalloproteinases (MMPs) are structurally and functionally related zinc-dependent endopeptidases capable of degrading the components of extracellular matrix (ECM) and basement membranes. MMPs participate in many physiological processes and have also been implicated in various pathological conditions including tumor invasion and metastasis. The functions of MMPs are known to be controlled by mechanisms leading to activation of their latent forms and through inhibition of both active and latent forms by natural tissue inhibitors of metalloproteinases (TIMPs). The complex relationships between MMPs and TIMPs within the bone marrow microenvironment during normal hematopoiesis as well as during leukemic growth and dissemination have not been extensively investigated. We reported that primary acute myelogenous leukemia (AML) blasts and leukemic KG-1 cells penetrate reconstituted basement membrane (Matrigel) in an in vitro invasion assay, secrete the gelatinases (MMP-2 and MMP-9) and express active MMP-2 on the cell surface. We also analyzed MMP/TIMP expression in normal bone marrow cells of the myeloid and stromal lineages and showed that MMP-2, MMP-9, TIMP-1 and TIMP-2 are produced in the bone marrow microenvironment. Furthermore, we examined the role of gelatinases in the transmigration of stem/progenitor cells from the bone marrow into peripheral blood. We found that steady-state bone marrow CD34(+) cells, unlike circulating peripheral blood CD34(+) cells, did not express MMP-2 and MMP-9 mRNA transcripts and proteins, and that various cytokines were able to upregulate expression of these MMPs in bone marrow CD34(+) cells and trans-Matrigel migration of these cells. Thus, we now have evidence that MMPs and TIMPs are constituents of the hematopoietic microenvironment although their roles in hematopoiesis have yet to be determined.

The proofreading pathway of bacteriophage T4 DNA polymerase.

The base analog, 2-aminopurine (2AP), was used as a fluorescent reporter of the biochemical steps in the proofreading pathway catalyzed by bacteriophage T4 DNA polymerase. “Mutator” DNA polymerases that are defective in different steps in the exonucleolytic proofreading pathway were studied so that transient changes in fluorescence intensity could be equated with specific reaction steps. The G255S- and D131N-DNA polymerases can hydrolyze DNA, the final step in the proofreading pathway, but the mutator phenotype indicates a defect in one or more steps that prepare the primer-terminus for the cleavage reaction. The hydrolysis-defective D112A/E114A-DNA polymerase was also examined. Fluorescent enzyme-DNA complexes werepreformed in the absence of Mg2+, and then rapid mixing, stopped-flow techniques were used to determine the fate of the fluorescent complexes upon the addition of Mg2+. Comparisons of fluorescence intensity changes between the wild type and mutant DNA polymerases were used to model the exonucleolytic proofreading pathway. These studies are consistent with a proofreading pathway in which the protein loop structure that contains residue Gly255 functions in strand separation and transfer of the primer strand from the polymerase active center to form a preexonuclease complex. Residue Asp131 acts at a later step in formation of the preexonuclease complex.

Kinetic and spectral properties of pea cytosolic ascorbate peroxidase

Sufficient highly purified native pea cytosolic ascorbate peroxidase was obtained to characterize some of its kinetic and spectral properties. Its rate constant for compound I formation from reaction with H2O2 is 4.0 × 107 M−1 s−1, somewhat faster than is typical for peroxidases. Compound I has the typical optical spectrum of an iron(IV)‐porphyrin‐π‐cation radical, despite considerable homology with yeast cytochrome c peroxidase. The rate constant for compound I reduction by ascorbate is extremely fast (8.0 × 107 M−1 s−1 at pH 7.8), again in marked contrast to the behavior of the yeast enzyme. The pH‐rate profile for compound I formation indicates a pK a value of 5.0 for a group affecting the active site reaction.

Reaction of autoxidation products of penicillamine with myeloperoxidase

Spectral evidence is presented which shows that penicillamine is able to initiate the formation of the oxidized intermediates of myeloperoxidase in the absence of exogenous hydrogen peroxide. The autoxidation of penicillamine presumably produces superoxide which dismutates spontaneously to form hydrogen peroxide. Thus, the formation of both compounds II and III of myeloperoxidase was observed. We also report that penicillamine can directly reduce cytochrome c and therefore, it could possibly act as a one-electron donor to myeloperoxidase.