Presence Of AMD3100 Research Articles

R4 Rgs Subfamily Proteins Suppress Engraftment and Regulate SDF-1/CXCR4 Signaling in Human CD34+ Hematopoietic Stem/Progenitor Cells

Members of the Regulators of G-protein Signaling (RGS) are GTPase-accelerating proteins and have been implicated in SDF-1-directed trafficking of mature hematopoietic cells. However, their roles in hematopoietic stem and progenitor cells (HSPC) remain largely unknown. In this study, we investigated the expression, functions and mechanism of R4 RGS subfamily members on migration and engraftment of human HSPC. Our results demonstrated that cord blood (CB), bone marrow (BM) and mobilized peripheral blood (MPB) CD34+ cells expressed specific RGS mRNAs, of which RGS1, RGS2, RGS13 and RGS16 were significantly upregulated by SDF-1 (1.6-1.9 fold, n=5, P<0.05). In the presence of AMD3100, a CXCR4 inhibitor, the stimulating effects of SDF-1 on RGS expression were completely abolished (n=6). SDF-1-directed functions (chemotaxis, trans-matrigel migration and calcium flux) and signaling (phosphorylation of Akt, ERK and Stat3) were significantly inhibited in RGS1, RGS13 and RGS16-overexpressing CD34+ cells (n=4-6, P<0.05) but not in RGS2-overexpressing cells, whereas actin polymerization, adhesion and colony formation were unaffected by these RGS members. In the NOD/SCID mouse xenotransplantation model, overexpression of RGS1, RGS13 or RGS16 in CD34+ cells did not impact their short-term homing but substantially compromised their long-term engraftment efficiency in bone marrow and spleens of recipient mice by 91.4%, 83.7% and 71.2%, respectively (n= 8-9; P<0.05). Genome-wide expression microarray and qPCR validation identified 32 common differentially expressed genes (1 upregulated and 31 downregulated) in RGS1, RGS13 or RGS16-overexpressing CD34+ cells. Network analysis revealed the potential mechanisms of RGS1, RGS13 and RGS16 downstream of SDF1/CXCR4 and Gαi protein, leading to compromised Akt, ERK and Stat3 phosphorylation and negative regulation of stem cell functions (CCNA1, SPP1, LPAR5, IL1RL1, HPSE), complement activation (C3AR1, C5AR2, C5AR1), proteolysis (TIMP3, MMP14) and cell migration (THBS1, F2RL2, PROS1, CCL1). Our results highlight the unprecedented functions of R4 RGS proteins in HSPC migration and engraftment, and provide the foundation of future design of RGS-targeting strategies to enhance the efficiency of clinical HSPC transplantation. Disclosures No relevant conflicts of interest to declare.

Using CRISPR/Cas9 and NanoLuc to investigate “endogenous” CXCR4 ligand binding, internalization and β‐arrestin2 recruitment

Ligand binding, receptor internalisation and receptor‐protein interactions are all vital components to receptor signalling that can be investigated using bioluminescence resonance energy transfer (BRET) or fragment complementation assays. However a fundamental limitation of these techniques is the requirement for exogenous expression of fusion proteins, which precludes the application of these methods to study endogenous proteins. Recently, we have demonstrated that BRET can be used to measure protein‐protein interactions when the luciferase NanoLuc was inserted into the genome by CRISPR/Cas9 engineering. Using NanoBRET and the NanoBiT luciferase complementation here we aimed to investigate ligand binding, receptor internalisation and receptor‐β‐arrestin2 interactions when the CXCR4 and/or interacting partners were expressed under endogenous promotion.CRISPR/Cas9‐mediated homology‐directed repair was used to insert Nluc or NanoBiT fragments into the CXCR4, ARRB2 or CXCL12 genomic loci in HEK293 cells. For NanoBRET ligand binding studies, cells expressing HiBiT/ or Nluc/CXCR4 were incubated for 1hr with CXCL12‐AF647 in the absence or presence of AMD3100 (10 μM). Alternatively cells expressing gene‐edited CXCL12/HiBiT were transfected with SNAP/CXCR4, labelled with cell surface SNAP‐AF488 and incubated with or without AMD3100 (10 pM‐10 μM). For internalisation assays cells were incubated with unlabelled ligands only. For β‐arrestin recruitment assays cells expressing both gene‐edited CXCR4/LgBiT and β‐arr2/SmBiT, or cells expressing one gene‐edited protein transfected with the other, were stimulated with CXCL12 (1 pM – 1 μM) and luminescence measured for 1 hr. Luminescence was generated by addition of furimazine and for cells expressing HiBiT 10 nM LgBiT and was measured on a PHERAstar plate reader (BMG).Binding affinities (Kd) of CXCL12‐AF647 were obtained via NanoBRET saturation binding experiments at Nluc/CXCR4 or HiBiT/CXCR4 expressed under endogenous promotion (nM; 22.74 ± 2.05 and 23.47 ± 4.16, n=4 respectively). Binding of gene edited CXCL12/HiBiT to SNAP/CXCR4 was displaced by AMD3100 (pIC50, 7.09 ± 0.16, n=5). In internalization experiments application of CXCL12 to cells expressing gene‐edited HiBiT/CXCR4 resulted in a concentration dependent decrease in luminescence (pEC50, 8.67 ± 0.27, n=5) suggestive of internalization. In cells expressing gene‐edited CXCR4/LgBiT, β‐arr2/SmBiT or both NanoBiT fragments application of CXCL12 resulted in a concentration dependent increase in luminescence respectively that was inhibited by AMD3100, indicative of ligand induced β‐arrestin2 recruitment to CXCR4Using CRISPR/Cas9 mediated genome engineering, NanoBRET and NanoBiT complementation here we have shown that multiple facets of GPCR activation, desensitisation and signalling can be investigated using proteins expressed under endogenous promotion.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Targeting CXC motif chemokine receptor 4 inhibits the proliferation, migration and angiogenesis of lung cancer cells.

An increasing volume of data indicates that disrupting the interaction between CXC motif chemokine receptor 4 (CXCR4) and its specific ligand, CXC motif chemokine 12 (CXCL12), may reduce tumor growth and metastasis. However, the translation from bench to bedside must be performed with extreme caution, as the CXCR4/CXCL12 axis is crucial for the normal development and maintenance of tissues and organs. In the present study, Cell Counting Kit-8 and Transwell migration assays were used to detect in vitro proliferation and chemotaxis of CXCR4-expressing A549 cells, a cell strain originating from human non-small-cell lung cancer (NSCLC), with or without the presence of AMD3100, a small-molecule inhibitor specific to CXCR4 signaling. In a xenograft model established by injecting nude mice with A549 cells, tumor growth, CXCR4 expression and microvessel density (MVD) in the tumor mass were determined through tumor size measurements and immunohistochemical staining following intraperitoneal administration of AMD3100 or vehicle. The results demonstrated that CXCR4 blockade inhibited the proliferation of A549 cells and their migration towards CXCL12 in vitro. Tumor growth, CXCR4 expression and MVD were markedly reduced in nude mice treated with AMD3100 compared with mice treated with the vehicle. In conclusion, the present data demonstrated that CXCR4 targeting impaired NSCLC cell growth, angiogenesis and metastatic spread, indicating that it may represent a novel treatment strategy for NSCLC.

Endogenous Membrane Receptor Labeling by Reactive Cytokines and Growth Factors to Chase Their Dynamics in Live Cells

Summary Cytokine and growth factor receptors localized on cell membranes play key roles in many biological events. Because the dynamic behaviors of receptors are deeply involved in cytokine- and growth-factor-dependent signaling pathways, reliable techniques for real-time imaging of receptors in live cells are strongly desired. Here, we describe a method of covalently labeling and imaging membrane receptors in live cells by using chemically reactive cytokines and growth factors coupled with 4-dimethylaminopyridine (DMAP), an organocatalyst that facilitates acyl transfer. Construction of the DMAP cytokines and growth factors relies on non-covalent coordination chemistry between an oligo-histidine tag and dimeric Ni(II)-nitrilotriacetate in an in situ manner. This supramolecular approach is relatively simple and flexible and can be applied to labeling not only transiently but also endogenously expressed receptors. Moreover, given the benefit of this traceless labeling of the receptors, our technique allows real-time imaging of the ligand-induced dynamics of endogenously expressed receptors in live cells.

CXCL12 And Vascular Maturation Of The Coronary System During Heart Development

Elucidating the mechanisms involved in the patterning of coronary blood vessels in the embryo will help to improve therapies for coronary perfusion in the injured heart. In the past few years, there have been significant efforts and advances in the understanding of coronary vessel development. The major endothelial cell sources that provide coronary endothelial progenitors to the heart are the sinus venosus (the vein that delivers the venous return) and the endocardium, although the respective contribution from each source is debated. Angiogenic sprouts from the sinus venosus reach the ventricles starting at E11.5 in the mouse and form a vascular plexus in the subepicardial space. This primitive plexus undergoes a complex and incompletely understood process of vascular maturation that involves pruning, branching and reorganization into a hierarchical network of superficial veins and intramyocardial arteries. The main branches of the coronary arteries ultimately connect to the aorta through the coronary ostia by E13.5 – E14.0.CXCL12 (also known as stromal derived factor 1, SDF1) is a chemokine that acts through the G protein‐coupled receptor CXCR4. Global deficiency of Cxcl12 or Cxcr4 as well as endothelial deletion of Cxcr4 result in vascular defects in the mouse kidney and gut, but these defects do not explain the associated late gestation lethality. We hypothesized that coronary vascular defects might underlie the embryonic lethality of CXCL12 deficiency.Cxcl12 and Tie2Cre/Cxcr4 mutants form an initial coronary vascular plexus, which then fails to remodel into a hierarchical network of mature vessels, resulting in the absence of the major intramyocardial coronary arteries, hemorrhage and reduced ventricular wall perfusion. The coronary veins were mostly unaffected, and the basis of the selectivity of CXCL12 on the arterial side of the vasculature is still unclear.In vitro, CXCL12 does not induce sprouting of endothelial cells from beads embedded in a fibrin gel matrix, but instead promotes maturation of vascular sprouts preformed in response to VEGF, by increasing tube elongation, vessel branching, anastomosis and intussusception, all features of mature blood vessel networks. These features were abolished in the presence of AMD3100, a CXCR4 inhibitor. Thus, CXCL12 does not initiate vasculogenesis but rather promotes further remodeling of a plexus that is initially induced by VEGF.We conclude that failed maturation of the coronary system explains the late‐gestation lethality of Cxcl12 mutants, and highlight the role of CXCL12 as an organ‐specific arterial maturation factor of particular relevance in the coronary vasculature. Our current studies are aimed at defining the role of CXCL12 in reprogramming of venous endothelial cells from the sinus venosus into coronary arterial endothelium.Support or Funding InformationThis work was supported by NIH grant HL070123 to H.M.S. and grant 14BGIA20500059 to S.R.K. from the American Heart Association. S.C. was supported by a Postdoctoral Fellowship from the American Heart Association. H.S. was supported by a Postdoctoral Fellowship from the California Institute for Regenerative Medicine.

SDF‐1α induces differential trafficking of CXCR4‐CXCR7 involving cyclophilin A, CXCR7 ubiquitination and promotes platelet survival

Platelet-derived SDF-1α (CXCL12) mediates inflammatory and regenerative mechanisms. The present study characterizes the effect of SDF-1α ligation in platelets. SDF-1α (0-100 μM) dose and time dependently caused internalization of its receptor CXCR4 (28.9 ± 1.6 vs. 16.1 ± 1.9 in SDF-1α-treated platelets), coupled to the surface externalization of CXCR7 (65.5 ± 8 vs. 162.8 ± 27.6 following SDF-1α treatment), both in vitro and in vivo. This was inhibited in the presence of AMD3100 (100 μM), CXCR4 blocking and vesicular transport inhibitors (brefeldin A, 10 μM; rapamycin, 100 nM). SDF-1α/CXCR-4-mediated CXCR7 translocation was significantly reduced by inhibitors of ERK1/2-(U0126-10 μM) and cyclophilinA (CyPA)-(NIM811-10 μM) by 28 and 46%, respectively. Further, SDF-1α-induced downstream phosphorylation of Erk1/2 led to CyPA-dependent ubiquitination of CXCR7, which is essential for its surface translocation. CyPA-PPIase-activity inhibitor NIM-811, Erk1/2, and E1-ligase inhibitor-(PYR-41-25 μM) significantly abolished SDF-1α-driven CXCR7 ubiquitination and subsequent surface translocation. SDF-1α induced CXCR7 ubiquitination, and its surface exposure was observed in wild-type murine platelets, but not in CyPA-deficient platelets. SDF-1α/CXCR4-CyPA-dependent CXCR7 translocation and its subsequent ligation attenuated activation-induced apoptosis both in vitro and when administered in vivo. This antiapoptotic effect of SDF-1α was abrogated by blocking CXCR7, also significantly affected in Cypa(-/-) platelets. Thus, we decipher a novel mechanism, whereby SDF-1α regulates relative receptor availability in circulating platelets and exerts its prosurvival benefits.-Chatterjee, M., Seizer, P., Borst, O., Schönberger, T., Mack, A., Geisler, T., Langer, H. F., May, A. E., Vogel, S., Lang, F., Gawaz, M. SDF-1α induces differential trafficking of CXCR4-CXCR7 involving cyclophilin A, CXCR7 ubiquitination and promotes platelet survival.

Combination Therapy Accelerates Diabetic Wound Closure

BackgroundNon-healing foot ulcers are the most common cause of non-traumatic amputation and hospitalization amongst diabetics in the developed world. Impaired wound neovascularization perpetuates a cycle of dysfunctional tissue repair and regeneration. Evidence implicates defective mobilization of marrow-derived progenitor cells (PCs) as a fundamental cause of impaired diabetic neovascularization. Currently, there are no FDA-approved therapies to address this defect. Here we report an endogenous PC strategy to improve diabetic wound neovascularization and closure through a combination therapy of AMD3100, which mobilizes marrow-derived PCs by competitively binding to the cell surface CXCR4 receptor, and PDGF-BB, which is a protein known to enhance cell growth, progenitor cell migration and angiogenesis.Methods and ResultsWounded mice were assigned to 1 of 5 experimental arms (n = 8/arm): saline treated wild-type, saline treated diabetic, AMD3100 treated diabetic, PDGF-BB treated diabetic, and AMD3100/PDGF-BB treated diabetic. Circulating PC number and wound vascularity were analyzed for each group (n = 8/group). Cellular function was assessed in the presence of AMD3100. Using a validated preclinical model of type II diabetic wound healing, we show that AMD3100 therapy (10 mg/kg; i.p. daily) alone can rescue diabetes-specific defects in PC mobilization, but cannot restore normal wound neovascularization. Through further investigation, we demonstrate an acquired trafficking-defect within AMD3100-treated diabetic PCs that can be rescued by PDGF-BB (2 μg; topical) supplementation within the wound environment. Finally, we determine that combination therapy restores diabetic wound neovascularization and accelerates time to wound closure by 40%.ConclusionsCombination AMD3100 and PDGF-BB therapy synergistically improves BM PC mobilization and trafficking, resulting in significantly improved diabetic wound closure and neovascularization. The success of this endogenous, cell-based strategy to improve diabetic wound healing using FDA-approved therapies is inherently translatable.

Plerixafor (AMD3100) induces prolonged mobilization of acute lymphoblastic leukemia cells and increases the proportion of cycling cells in the blood in mice

The CXCR4 antagonist Plerixafor (AMD3100) induces the rapid mobilization of hematopoietic stem and progenitor cells into the blood in mice and humans. AMD3100 similarly induces the mobilization of human acute lymphoblastic leukemia (ALL) cells into the blood in mice. In this study, the temporal response of pre-B ALL cells to AMD3100 was compared with that of normal hematopoietic progenitor cells (HPC) using an NOD/SCID xenograft model of ALL and BALB/c mice, respectively. ALL cells remained in the circulation up to 6 hours after AMD3100 administration, by which time normal HPCs were no longer detectable. AMD3100 also increased the proportion of actively cycling ALL cells in the peripheral blood. Together, these data suggest that ALL cells are more sensitive to the effects of bone marrow disruption than normal progenitors. Using the NOD/SCID xenograft model, we demonstrated that AMD3100 increased the efficacy of the cell cycle specific drug vincristine, resulting in reduced disease levels in the blood and spleens of animals over 3 weeks and extended the survival of NOD/SCID mice with ALL. These data demonstrate that mobilizing agents can increase the therapeutic effect of cell cycle dependent chemotherapeutic agents.

Novel insight into stem cell mobilization-Plasma sphingosine-1-phosphate is a major chemoattractant that directs the egress of hematopoietic stem progenitor cells from the bone marrow and its level in peripheral blood increases during mobilization due to activation of complement cascade/membrane attack complex

Complement cascade (CC) becomes activated and its cleavage fragments play a crucial role in the mobilization of hematopoietic stem/progenitor cells (HSPCs). Here, we sought to determine which major chemottractant present in peripheral blood (PB) is responsible for the egress of HSPCs from the BM. We noticed that normal and mobilized plasma strongly chemoattracts HSPCs in a stromal derived factor-1 (SDF-1)-independent manner because i) plasma SDF-1 level does not correlate with mobilization efficiency, ii) the chemotactic plasma gradient is not affected in the presence of AMD3100, and iii) it is resistant to denaturation by heat. Surprisingly, the observed loss of plasma chemotactic activity after charcoal stripping suggested involvement of bioactive lipids and we focused on sphingosine-1 phosphate (S1P), a known chemoattracant of HSPCs. We found that S1P i) creates in plasma a continuously present gradient for BM-residing HSPCs, ii) is at physiologically relevant concentrations a chemoattractant several magnitudes stronger than SDF-1, and iii) its plasma level increases during mobilization due to CC activation and the interaction of membrane attack complex (MAC) with erythrocytes that are a major reservoir of S1P. We conclude and propose a new paradigm that S1P is a crucial chemoattractant for BM-residing HSPCs and that CC via MAC induces release of S1P from erythrocytes for optimal egress/mobilization of HSPCs.

Mechanism of primitive duct formation in the pancreas and submandibular glands: a role for SDF-1

BackgroundThe exocrine pancreas is composed of a branched network of ducts connected to acini. They are lined by a monolayered epithelium that derives from the endoderm and is surrounded by mesoderm-derived mesenchyme. The morphogenic mechanisms by which the ductal network is established as well as the signaling pathways involved in this process are poorly understood.ResultsBy morphological analyzis of wild-type and mutant mouse embryos and using cultured embryonic explants we investigated how epithelial morphogenesis takes place and is regulated by chemokine signaling. Pancreas ontogenesis displayed a sequence of two opposite epithelial transitions. During the first transition, the monolayered and polarized endodermal cells give rise to tissue buds composed of a mass of non polarized epithelial cells. During the second transition the buds reorganize into branched and polarized epithelial monolayers that further differentiate into tubulo-acinar glands. We found that the second epithelial transition is controlled by the chemokine Stromal cell-Derived Factor (SDF)-1. The latter is expressed by the mesenchyme, whereas its receptor CXCR4 is expressed by the epithelium. Reorganization of cultured pancreatic buds into monolayered epithelia was blocked in the presence of AMD3100, a SDF-1 antagonist. Analyzis of sdf1 and cxcr4 knockout embryos at the stage of the second epithelial transition revealed transient defective morphogenesis of the ventral and dorsal pancreas. Reorganization of a globular mass of epithelial cells in polarized monolayers is also observed during submandibular glands development. We found that SDF-1 and CXCR4 are expressed in this organ and that AMD3100 treatment of submandibular gland explants blocks its branching morphogenesis.ConclusionIn conclusion, our data show that the primitive pancreatic ductal network, which is lined by a monolayered and polarized epithelium, forms by remodeling of a globular mass of non polarized epithelial cells. Our data also suggest that SDF-1 controls the branching morphogenesis of several exocrine tissues.

Short-Term Culture of Umbilical Cord Blood-Derived Stem Cells Enhances the Engraftment into NOD/SCID Mice through the Increased Expression of CXCR4.

Human umbilical cord blood (CB) is rich source of hematopoietic stem cells (HSCs) and provides an attractive alternative to bone marrow or mobilized peripheral blood transplantation. However, major disadvantage of CB transplantation is the relatively low number of HSCs in each CB unit, which severely limits its usefulness in clinical transplantation. A part of CD34+ cells expresses cell surface CXCR4, which has been found to be critical for bone marrow engraftment by human hematopoietic stem cells. In the present study, we examined the effect of short-term culture on CXCR4 expression and engraftment in CB-derived CD34+ cells transplanted into non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice. CB-derived CD34+-enriched samples were divided into three aliquots and cultured. The first aliquot was incubated for 2 h in RPMI-1640 medium alone, whereas the second was incubated for 2h in RPMI-1640 medium containing the specific CXCR4 antagonist, AMD3100. As a control, the third sample was neither treated nor incubated. Surface CXCR4 expression on CD34+ cells significantly increased after incubating the cells in medium alone for 2 h (7.7 to 19.8%); this increase was blocked by the addition of AMD3100. No difference in CXCR4 mRNA expression was noted after incubating CD34+ cells for 2 h. The short-term incubation seems to influence the translocation of CXCR4 from the interior of the cell to the cell surface. CD34+ cells cultured for 2 h showed significantly increased transmigrational activity toward SDF-1 and homing activity in NOD/SCID mice. However, those were lower in non-cultured cells and cells treated with AMD3100. Next, we assessed the engraftment of cells cultured into NOD/SCID mice. Engraftment analysis was performed 8 weeks after transplantation. The presence of human cells was detected by the cell surface expression of CD45, CD34, CD33, and CD19. The CD34+ cells cultured demonstrated significantly increased engraftment of human cells in the bone marrow of NOD/SCID mice compared with that of CD34+ cells without culture or with culture in the presence of AMD3100 (p < 0.01). The recipients exhibited CD19+ B-lymphoid cells, CD33+ myeloid cells, and CD34+ immature cells, but neither CD3+ T cells nor CD45+ cells in bone marrow. Finally, we performed a secondary transplant of engrafted cells from previously transplanted mice to confirm long-term engraftment and self-renewal of cultured CB cells. The successful engraftment was observed in mice injected with primary cultured cells, demonstrating that these cultured cells are self-renewing and capable of long-term engraftment. These observations suggest that the increase of the cell surface expression of CXCR4 on CD34 cells resulted in the retention of human umbilical cord blood cells within the bone marrow through the engraftment of homing activity.

Inhibition of CXCR4 activity with AMD3100 decreases invasion of human colorectal cancer cells in vitro

To investigate the effect and mechanism of blockade of the CXC chemokine receptor-4 (CXCR4) signaling pathway by AMD3100, a small non-peptide CXCR4 inhibitor, on invasion and metastasis of colorectal cancer cells in vitro. Human colorectal cancer cell line SW480 was treated with AMD3100 at different final concentrations. 3-(4,5-dimethylthiazole-2-yl)-2.5-dipheny-ltetrazolium bromide (MTT) assay was used to detect the effect of AMD3100 on cell proliferation. The invasion ability of SW480 cells was determined by cell invasion assay kit. In the presence of AMD3100, the CXCL12-mediated migratory response of SW480 cells was tested by classical chemotaxis assays. RT-PCR analysis and Western blotting were used to detect the expression of vascular endothelial growth factor (VEGF), matrix metalloproteinase-2 (MMP-2) and -9 (MMP-9) in SW480 cells. Cell viability was significantly suppressed by AMD3100 in a dose-dependent manner. AMD3100 (100 and 1000 ng/mL) significantly inhibited the invasion ability of SW480 cells. Treatment with AMD3100 markedly reduced the expression of VEGF and MMP-9 but not MMP-2 in SW480 cells. The CXCL12/CXCR4 system is an important mediator of proliferation and invasion of CXCR4-expressing colorectal cancer cells. AMD3100 inhibited invasion and metastasis activity of the colorectal cancer cell line SW480 through down-regulation of VEGF and MMP-9 expression.

In Vivo Mobilization of Multiple Myeloma Cells Out of the Bone Marrow Using the CXCR4 Inhibitor AMD3100 and Bortezomib: Implications for Sensitization of Myeloma Cells to Apoptosis.

Background: MM is characterized by widespread involvement of the bone marrow (BM) as the result of successful homing, engraftment and growth of myeloma cells. The BM provides protection and resistance of MM to therapeutic agents. Therefore, disruption of the interaction of MM cells with their microenvironment should lead to enhanced sensitivity to therapeutic agents. We hypothesized that disrupting CXCR4/SDF-1 axis will induce mobilization of MM cells from the BM into the circulation.Methods: MM.1S cells were co-cultured with bone marrow stromal cells (BMSCs) in the presence of AMD3100 (50uM, Sigma), bortezomib (0–2.5nM, Millennium) or combination of both; and cell proliferation was measured using [3H]-thymidine uptake. We then tested the in vivo AMD3100-induced mobilization of MM cells after they homed to the BM. MM1.S cells that had been fluorescently labeled with DiD (Invitrogen) were injected into mice through their tail veins. Beginning 24 hours later and for three subsequent days, the mice were treated with 5mg/kg AMD3100 sq daily, injected with fluorescently labeled c-kit antibody for HSC detection and immediately monitored for presence of circulating MM1-S cells or HSCs, using in vivo flow cytometry. To test whether AMD3100 induces mobilization of MM cells in established tumors, a GFP+ and luciferase+ osteotropic MM.1S cell line (Luc+GFP+MM.1S) was developed. This mouse model was used for continuous, real-time quantitation of MM cells mobilization. Bioluminescence imaging was used to determine tumor growth in vivo. Mice were treated with AMD3100 (5mg/kg, daily), bortezomib (1mg/kg biweekly) or the combination.Results: There was a significant increase in proliferation of MM cells in co-culture with BMSCs compared to MM cells alone. Moreover, AMD3100 alone did not inhibit proliferation; however, it significantly enhanced the cytotoxic effect of bortezomib in the presence of stromal cells. In vivo studies revealed that, unlike HSC, no appreciable mobilization of MM1S cells occurred after the first AMD3100 injection; however, the second AMD3100 treatment induced a fourfold increase in circulating MM cell numbers above background, while no further remarkable increase in circulating HSC was observed. The effect of AMD on an established tumor model showed that by the end of the first week of AMD3100 or bortezomib treatment, a 2-fold increase of circulating MM cells was observed compared to control mice, a trend which continued for three weeks. Moreover, compared to AMD3100 or bortezomib treatment alone, a further increase of circulating MM cells was observed in the peripheral blood of mice treated with combination of AMD3100 and bortezomib following the second bortezomib treatment. In the AMD3100 only treated mice, the counts of circulating MM cell continued to increase and tumors continued to progress, while in the AMD3100-bortezomib-treated mice, the circulating MM cell count decreased and the tumors regressed.Conclusion: These data support the hypothesis that disruption of the CXCR4/SDF-1 axis mobilizes MM cells, and that AMD3100 can be used to enhance the effects of therapeutic agents such as bortezomib.

Hypoxia-inducible factor 1 and VEGF upregulate CXCR4 in glioblastoma: implications for angiogenesis and glioma cell invasion

Hypoxia and hypoxia-inducible factor-1 (HIF-1) play a critical role in glioblastoma multiforme (GBMs). CXCR4 is involved in angiogenesis and is upregulated by HIF-1α. CXCR4 is a chemokine receptor for stromal cell-derived factor-1 (SDF-1)α, also known as CXCL12. We hypothesized that CXCR4 would be upregulated by hypoxia in GBMs. First, we investigated the expression of HIF-1α and CXCR4 in GBMs. CXCR4 was consistently found colocalized with HIF-1α expression in pseudopalisading glioma cells around areas of necrosis. In addition, angiogenic tumor vessels were strongly positive for CXCR4. Next, we tested the in vitro effect of hypoxia and vascular endothelial growth factor (VEGF) on the expression of CXCR4 in glioma cell lines and in human brain microvascular endothelial cells (HBMECs). Exposure to hypoxia induced significant expression of CXCR4 and HIF-1α in glioma cells, whereas treatment with exogenous VEGF increased CXCR4 expression in HBMECs. We also transfected U87MG glioma cells with an HIF-1α construct and observed that CXCR4 was upregulated in these cells even in normoxic conditions. We then used a lentivirus-mediated shRNA expression vector directed against HIF-1α. When exposed to hypoxia, infected cells failed to show HIF-1α and CXCR4 upregulation. We performed migration assays under normoxic and hypoxic conditions in the presence or absence of AMD3100, a CXCR4 inhibitor. There was a significant increase in the migration of U87MG and LN308 glioma cells in hypoxic conditions, which was inhibited in the presence of AMD3100. These studies demonstrate the critical role played by hypoxia and CXCR4 in glioma cell migration. Based on these studies, we suggest that hypoxia regulates CXCR4 in GBMs at two levels. First, through HIF-1α in the pseudopalisading tumor cells themselves and, secondly, by the VEGF-stimulated angiogenic response in HBMECs. We believe this knowledge may lead to a potentially important two-pronged therapy against GBM progression using chemotherapy targeting CXCR4.

Use of alternate coreceptors on primary cells by two HIV-1 isolates

Two HIV-1 isolates (CM4 and CM9) able to use alternate HIV-1 coreceptors on transfected cell lines were tested for their sensitivity to inhibitors of HIV-1 entry on primary cells. CM4 was able to use CCR5 and Bob/GPR15 efficiently in transfected cells. The R5 isolate grew in Δ32/Δ32 CCR5 PBMC in the absence or presence of AMD3100, a CXCR4-specific inhibitor, indicating that it uses a receptor other than CCR5 or CXCR4 on primary cells. It was insensitive to the CCR5 entry inhibitors RANTES and PRO140, but was partially inhibited by vMIP-1, a chemokine that binds CCR3, CCR8, GPR15 and CXCR6. The coreceptor used by this isolate on primary cells is currently unknown. CM9 used CCR5, CXCR4, Bob/GPR15, CXCR6, CCR3, and CCR8 on transfected cells and was able to replicate in the absence or presence of AMD3100 in Δ32/Δ32 CCR5 PBMC. It was insensitive to eotaxin, vMIP-1 and I309 when tested individually, but was inhibited completely when vMIP-1 or I309 was combined with AMD3100. Both I309 and vMIP-1 bind CCR8, strongly suggesting that this isolate can use CCR8 on primary cells. Collectively, these data suggest that some HIV-1 isolates can use alternate coreceptors on primary cells, which may have implications for strategies that aim to block viral entry.

SDF-1/CXCL12 Enhances Survival of Murine Embryonic Stem Cells and Their Production of Myeloid Progenitors, but Not Hemangioblasts.

Stromal cell derived factor (SDF)-1/CXCL12 has been shown to promote the survival of embryonic retinal ganglion cells, peritoneal B1a (PerBla) lymphocytes and chronic lymphocytic leukemia B cells. Our previous studies implicated SDF-1 as an important factor in enhancing survival of murine bone marrow (BM) hematopoietic stem cells and, human cord blood and adult human BM myeloid progenitors. Since pluripotent embryonic stem (ES) cells can give rise to differentiated cell types derived from all three primary germ layers (endoderm, mesoderm, and ectoderm) and adult stem cells are generated during embryoid body (EB) formation, we investigated whether SDF-1 has effects on survival of ES cells and EB generation of hematopoietic progenitor cells. In order to establish SDF-1 expression patterns during EB formation, we screened supernatants during day 1–5 EB formation for SDF-1 production by three murine ES cell lines (E14, R1 and CCE). We observed low but detectable SDF-1 secreted in cultures of ES cells and day 1 stage EBs. SDF-1 was increased in the media from day 2 stage EBs and continued to increase through days 4–5. CXCR4, SDF-1 receptor, expression was also analyzed. CXCR4 mRNA expression was low in ES cells and day 1–3 EBs, and increased significantly from Day 4 EBs, reaching maximum levels at day 5, and decreasing after day 6. Surface CXCR4 expression was consistent with mRNA data. To determine if SDF-1 had an effect on ES cell survival, we cultured ES cells without serum, and added serum at either 0, 24, 48 or 96 hrs to each of the following groups: A) Control, B) SDF-1 (100ng/ml) or C) AMD3100 (1 μM), an SDF-1 receptor (CXCR4) antagonist. Colonies were scored 7 days after the addition of serum. SDF-1 enhanced survival of ES cells, while AMD3100 decreased survival. We also checked the apoptosis of ES cells after withdrawing serum for 24, 48, 72 and 96 hours in four groups: A) control, B) SDF-1 (100ng/ml), C) AMD3100 (1 μM) or D) AMD3100 (1 μM) and SDF-1 (100ng/ml). SDF-1 decreased apoptosis and AMD3100 blocked the SDF-1 effect. AMD3100 alone increased apoptosis compared to control. This suggests that AMD3100 blocked endogenous SDF-1 effects. To determine if SDF-1 had an effect on differentiation of hematopoietic progenitor cells, we added SDF-1 (100 ng/ml), AMD3100, or AMD3100 plus SDF-1 (100 ng/ml) at the beginning of EB formation, immediately after removal of LIF, and quantitated primitive erythroid (p-BFU-E), definitive erythroid (d-BFU-E), granulocyte-macrophage (CFU-GM) and multipotential Granulocyte/Erythroid/Macrophage/Megakaryocyte (CFU-GEMM) colony formation. In comparison to control cells (cultured without SDF-1 and AMD3100), SDF-1 increased numbers of p-BFU-E, d-BFU-E, CFU-GM, and CFU-GEMM colonies. Addition of AMD3100 with SDF-1 blocked the enhancing effect of SDF-1. In addition, significantly decreased numbers of colonies were also observed in the presence of AMD3100 alone. This suggests that AMD3100 blocks endogenous SDF-1 actions, consistent with our data on SDF-1 production during EB formation. In order to determine when SDF-1 starts affecting hematopoiesis, hemangioblast colony assays were used. Neither SDF-1 nor AMD3100 influenced hemangioblast colony formation or expression of Flk-1 mRNA, a marker of hemangioblasts. The results suggest a role for SDF-1 in ES cell growth and differentiation.

The antiviral activity of the CXCR4 antagonist AMD3100 is independent of the cytokine-induced CXCR4/HIV coreceptor expression level.

The chemokine receptor CXCR4 is the main coreceptor used by T-tropic X4 HIV-1 strains to infect its target T cells. It has been proven that the CXCR4 expression level in T cells is strongly up-regulated by interleukin (IL)-4, a Th2-type cytokine that is secreted preferentially in HIV-infected patients in a later stage of disease. This results in an enhancement of HIV-1 replication in CD4+ T-lymphocytes. We have now evaluated the potency of the CXCR4 antagonist AMD3100 in phytohemagglutinin (PHA)/IL-2- versus PHA/IL-4-activated T cells in order to determine whether the compound has comparable CXCR4-antagonistic and anti-HIV-1 effects under these different cytokine treatments. We analyzed the CXCR4 expression level and the dose-dependent inhibition of CXCR4 expression by AMD3100, by monitoring the binding of an anti-CXCR4 monoclonal antibody (clone 12G5). We also determined stromal cell-derived factor (SDF)-1-induced intracellular calcium signaling and HIV-1 replication in these cells in the absence and presence of AMD3100. The CXCR4 expression level in PHA/IL-4-stimulated cells was much higher than in PHA/IL-2-stimulated cells. However, the potency of the bicyclam AMD3100 to block anti-CXCR4 mAb binding, SDF-1-induced intracellular calcium signaling, and HIV-1 replication of the X4 NL4.3 strain and three primary isolates remained unchanged. Our data indicate that CXCR4 antagonists such as AMD3100 act independently of the HIV-1 coreceptor expression level. These compounds should therefore be useful in suppressing HIV-1 infection in all stages of the disease.

HIV-1 gp41 six-helix bundle formation occurs rapidly after the engagement of gp120 by CXCR4 in the HIV-1 Env-mediated fusion process.

The onset of cell fusion mediated by HIV-1 IIIB Env is preceded by a lag phase of 15-20 min. Fusion mediated by the CD4-independent HIV-1 Env 8x, which is capable of interacting directly with CXCR4, proceeds with a greatly reduced lag phase. We probed the intermediate steps during the lag phase in HIV-1 IIIB Env-mediated fusion with Leu3-a, an inhibitor of attachment of gp120 to CD4, AMD3100, an inhibitor of attachment of gp120 to CXCR4, and C34, a synthetic peptide that interferes with the transition of gp41 to the fusion active state. Inhibitions of fusion as a function of time of addition of C34 and of AMD3100 were equivalent, indicating that engagement of gp120 by CXCR4 and formation of the gp41 six-helix bundle follow similar kinetics. The initial steps in fusion mediated by the CD4-independent Env 8x are too rapid for these inhibitors to interfere with. However, when 8x Env-expressing cells were incubated with target cells at 25 degrees C in the presence of AMD3100 or C34, prior to incubation at 37 degrees C, these inhibitors were capable of inhibiting 8x Env-mediated fusion. To further examine engagement of gp120 by CXCR4 and exposure of binding sites for C34, we have reversibly arrested the fusion reaction at 37 degrees C by adding cytochalasin B to the medium. We show that CXCR4 engagement and six-helix bundle formation only occur after the release of the cytochalasin arrest, indicating that a high degree of cooperativity is required to trigger the initial steps in HIV-1 Env-mediated fusion.

Shift of clinical human immunodeficiency virus type 1 isolates from X4 to R5 and prevention of emergence of the syncytium-inducing phenotype by blockade of CXCR4.

The emergence of X4 human immunodeficiency virus type 1 (HIV-1) strains in HIV-1-infected individuals has been associated with CD4(+) T-cell depletion, HIV-mediated CD8(+) cell apoptosis, and an impaired humoral response. The bicyclam AMD3100, a selective antagonist of CXCR4, selected for the outgrowth of R5 virus after cultivation of mixtures of the laboratory-adapted R5 (BaL) and X4 (NL4-3) HIV strains in the presence of the compound. The addition of AMD3100 to peripheral blood mononuclear cells infected with X4 or R5X4 clinical HIV isolates displaying the syncytium-inducing phenotype resulted in a complete suppression of X4 variants and a concomitant genotypic change in the V2 and V3 loops of the envelope gp120 glycoprotein. The recovered viruses corresponded genotypically and phenotypically to R5 variants in that they could no longer use CXCR4 as coreceptor or induce syncytium formation in MT-2 cells. Furthermore, the phenotype and genotype of a cloned R5 HIV-1 virus converted to those of the R5X4 virus after prolonged culture in lymphoid cells. However, these changes did not occur when the infected cells were cultured in the presence of AMD3100, despite low levels of virus replication. Our findings indicate that selective blockade of the CXCR4 receptor prevents the switch from the less pathogenic R5 HIV to the more pathogenic X4 HIV strains, a process that heralds the onset of AIDS. In this article, we show that it could be possible to redirect the evolution of HIV so as to impede the emergence of X4 strains or to change the phenotype of already-existing X4 isolates to R5.