Varda Deutsch

CXCR4 Regulates Migration and Development of Human Acute Myelogenous Leukemia Stem Cells in Transplanted NOD/SCID Mice

The chemokine stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 participate in the retention of normal hematopoietic stem cells within the bone marrow (BM) and their release into the circulation. Homing and engraftment of human stem cells in immunodeficient mice are dependent on cell surface CXCR4 expression and the production of BM SDF-1, which acts also as a survival factor for both human and murine stem cells. However, the role of SDF-1/CXCR4 interactions in the control of human acute myelogenous leukemia (AML) cell trafficking and disease progression is poorly understood. In this study, we report that although some AML cells do not express surface CXCR4, all AML cells tested express internal CXCR4 and SDF-1. Culture of AML cells with SDF-1 promoted their survival, whereas addition of neutralizing CXCR4 antibodies, SDF-1 antibodies, or AMD3100 significantly decreased it. Pretreatment of primary human AML cells with neutralizing CXCR4 antibodies blocked their homing into the BM and spleen of transplanted NOD/SCID/B2m(null) mice. Furthermore, weekly administrations of antihuman CXCR4 to mice previously engrafted with primary AML cells led to a dramatic decrease in the levels of human AML cells in the BM, blood, and spleen in a dose- and time-dependent manner. Interestingly, the same treatment did not affect significantly the levels of normal human progenitors engrafted into NOD/SCID mice. Taken together, our findings demonstrated the importance of the SDF-1/CXCR4 axis in the regulation of in vivo motility and development of human AML stem cells and identified CXCR4 neutralization as a potential treatment for AML.

Transfer of Endothelial Progenitor and Bone Marrow Cells Influences Atherosclerotic Plaque Size and Composition in Apolipoprotein E Knockout Mice

Objectives—Recent clinical trials use cell therapy with bone marrow (BM) cells or endothelial progenitor cells (EPCs) for ischemic syndromes. We explored the effect of BM cell– or spleen cell–derived EPC transfer on plaque size and stability markers in the apolipoprotein E knockout (apoE KO) mouse model. Methods and Results—ApoE KO mice aged 10 weeks served as recipients. Labeled BM cells and spleen cell–derived EPCs from age-matched apoE KO mice were injected intravenously to 2 groups of recipient mice each. Additional mice served as controls receiving saline. Both protocols were repeated 3 times at 2 weekly intervals. On killing, plaque size and character were studied, lipid profile analyzed, and serum and aortic cytokines assayed. Spleen cell–derived cells contained a significantly larger number of endothelial cell precursors. Labeled EPCs and BM cells were found abundantly in the spleens, yet also in the lesions of the recipient mice. Aortic sinus lesion size was significantly increased in mice receiving BM cells (n=10) in the EPC-treated group (n=10) compared with controls (n=10; a 54% and a 34% increase in aortic sinus plaque area, respectively). Mice receiving EPCs exhibited plaques with larger lipid cores and thinner fibrous caps and a higher number of infiltrating CD3 cells. RT-PCR analysis of aortas revealed reduced expression of mRNA for interleukin-10 (IL-10) in both cell transfer groups. Higher serum concentrations of IL-6 and monocyte chemoattractant protein-1 were found in sera from BM recipients, whereas lower IL-10 levels were found in mice transfused with spleen-derived EPCs. Conclusions—Transfer of BM cells and EPCs may result in an increase in atherosclerotic lesion size, whereas EPC transfer could also potentially influence plaque stability.

Megakaryocyte development and platelet production

Megakaryocytopoiesis involves the commitment of haematopoietic stem cells, and the proliferation, maturation and terminal differentiation of the megakaryocytic progenitors. Circulating levels of thrombopoietin (TPO), the primary growth‐factor for the megakaryocyte (MK) lineage, induce concentration–dependent proliferation and maturation of MK progenitors by binding to the c‐Mpl receptor and signalling induction. Decreased platelet turnover rates results in increased concentration of free TPO, enabling the compensatory response of marrow MKs to increased platelet production. C‐Mpl activity is orchestrated by a complex cascade of signalling molecules that induces the action of specific transcription factors to drive MK proliferation and maturation. Mature MKs form proplatelet projections that are fragmented into circulating particles. Newly developed thrombopoietic agents operating via c‐Mpl receptor may prove useful in supporting platelet production in thrombocytopenic state. Herein, we review the regulation of megakaryocytopoiesis and platelet production in normal and disease state, and the new approaches to thrombopoietic therapy.

ARP, a peptide derived from the stress-associated acetylcholinesterase variant has hematopoietic growth promoting activities

BackgroundPsychological stress induces rapid and long-lasting changes in blood cell composition, implying the existence of stress-induced factors that modulate hematopoiesis. Here we report the involvement of the stress-associated “readthrough” acetylcholinesterase (AChE-R) variant, and its 26 amino acid C-terminal domain (ARP) in hematopoietic stress responses.Materials and MethodsWe studied the effects of stress, cortisol, antisense oligonucleotides to AChE, and synthetic ARP on peripheral blood cell composition and clonogenic progenitor status in mice under normal and stress conditions, and on purified CD341 cells of human origin. We employed in situ hybridization and immunocytochemical staining to monitor gene expression, and 5-bromo-2-deoxyuridine (BrdU), primary liquid cultures, and clonogenic progenitor assays to correlate AChE-R and ARP with proliferation and differentiation of hematopoietic progenitors.ResultsWe identified two putative glucocorticoid response elements in the human ACHE gene encoding AChE. In human CD341 hematopoietic progenitor cells, cortisol elevated AChE-R mRNA levels and promoted hematopoietic expansion. In mice, a small peptide crossreacting with anti-ARP antiserum appeared in serum following forced swim stress. Ex vivo, ARP was more effective than cortisol and equally as effective as stem cell factor in promoting expansion and differentiation of early hematopoietic progenitor cells into myeloid and megakaryocyte lineages.ConclusionsOur findings attribute a role to AChE-R and ARP in hematopoietic homeostasis following stress, and suggest the use of ARP in clinical settings where ex vivo expansion of progenitor cells is required.

The CXCR4 antagonist AMD3100 impairs survival of human AML cells and induces their differentiation

The chemokine stromal cell-derived factor-1 (SDF-1) and its receptor, CXCR4, participate in the retention of acute myeloblastic leukemia (AML) cells within the bone marrow microenvironment and their release into the circulation. AML cells also constitutively express SDF-1-dependent elastase, which regulates their migration and proliferation. To study the molecular events and genes regulated by the SDF-1/CXCR4 axis and elastase in AML cells, we examined gene expression profiles of the AML cell line, U937, under treatment with a neutralizing anti-CXCR4 antibody or elastase inhibitor, as compared with non-treated cells, using DNA microarray technology. Unsupervised hierarchical clustering analysis demonstrated similar gene expression profiles of anti-CXCR4 antibody or elastase inhibitor-treated cells, as compared with control. Pathway and functional analysis showed a greater tendency toward differentiation in cells under either one of both treatment modalities. Thus given, we further analyzed the effects of CXCR4 inhibition on AML cell growth and differentiation using the antagonist AMD3100. AMD3100 arrested proliferation in AML cell lines and triggered changes that mimicked differentiation, including morphological changes and the expression of myeloid differentiation antigens. Inhibition of elastase also triggered the differentiation of AML cells. Our study defines a new role for the SDF-1/CXCR4 axis in the regulation of leukemic cell survival and differentiation.

Heparanase expression in human leukemias is restricted to acute myeloid leukemias

OBJECTIVE Matrix metalloproteinases and an endo-beta-D-glucuronidase (heparanase) are enzymes that degrade the protein and carbohydrate constituents of basement membranes, thereby facilitating transendothelial migration of blood-borne cells. Heparanase activity was found to correlate with the metastatic potential of solid tumors. We evaluated heparanase expression, at the levels of gene and protein expression and activity in a variety of leukemias, and compared it with normal hematopoietic cells. MATERIALS AND METHODS Heparanase expression was evaluated in leukocytes isolated from peripheral blood of 71 patients with myeloid and lymphoid leukemias, or non-Hodgkins lymphoma. Analysis was performed at two levels: heparanase RNA was determined by reverse transcriptase polymerase chain reaction, and heparanase protein was evaluated by immunocytochemistry and flow cytometry. RESULTS In eight peripheral blood samples from normal donors, heparanase RNA was detected, and protein was found within the cytoplasm of granulocytes. In mononuclear cells derived from various leukemias, heparanase RNA was expressed in 14 of 15 acute myeloid leukemia (AML) samples. In contrast, cells derived from all 33 chronic lymphoblastic leukemia, all 7 non-Hodgkins lymphoma, 7 of 8 chronic myeloid leukemia, and 6 of 8 acute lymphoblastic leukemia patients showed no detectable expression of the heparanase RNA. Heparanase protein was detected primarily within the cytoplasm of AML cells, indicating that the enzyme is produced and stored within the cytoplasm of myeloid cells, with limited expression on the cell surface. CONCLUSION We propose that heparanase expression is associated with the myeloid lineage and may serve as an independent marker to support the identification of AMLs.

Hydrolytic and Nonenzymatic Functions of Acetylcholinesterase Comodulate Hemopoietic Stress Responses

Glucocorticoid-initiated granulocytosis, excessive proliferation of granulocytes, persists after cortisol levels are lowered, suggesting the involvement of additional stress mediator(s). In this study, we report that the stress-induced acetylcholinesterase variant, AChE-R, and its cleavable, cell-penetrating C-terminal peptide, ARP, facilitate granulocytosis. In postdelivery patients, AChE-R-expressing granulocyte counts increased concomitantly with serum cortisol and AChE activity levels, yet persisted after cortisol had declined. Ex vivo, mononuclear cells of adult peripheral blood responded to synthetic ARP26 by overproduction of hemopoietically active proinflammatory cytokines (e.g., IL-6, IL-10, and TNF-α). Physiologically relevant ARP26 levels promoted AChE gene expression and induced the expansion of cultured CD34+ progenitors and granulocyte maturation more effectively than cortisol, suggesting autoregulatory prolongation of ARP effects. In vivo, transgenic mice overexpressing human AChE-R, unlike matched controls, showed enhanced expression of the myelopoietic transcription factor PU.1 and maintained a stable granulocytic state following bacterial LPS exposure. AChE-R accumulation and the consequent inflammatory consequences can thus modulate immune responses to stress stimuli.

Recombinant human granulocyte-colony stimulating factor administration for treating amyotrophic lateral sclerosis: A pilot study

Granulocyte-colony stimulating factor (G-CSF) is used to mobilize CD34+ haematopoietic stem cells from the bone marrow to the peripheral blood. We proposed to use cell subsets induced by G-CSF to slow down disease progression in patients with amyotrophic lateral sclerosis (ALS). Patients with definite or probable ALS were assigned in a double-blind manner to receive G-CSF or placebo every three months for a year. The primary outcome measure was the functional decline, measured by the revised ALS Functional Rating Scale, Revised (ALSFRS-R) score. Secondary outcome measures included vital capacity, manual muscle strength, compound muscle action potential amplitudes, neurophysiological index, and McGill single item quality of life score (QoL). Thirty-nine patients were enrolled. Seventeen patients who received G-CSF and 18 who received placebo were evaluated. G-CSF was effective in mobilizing CD34+ to blood. The outcome measures used showed no statistically significant benefit, although there was a trend of slowing disease progression following two G-CSF treatments, as shown by lower slopes of ALSFRS-R and QoL in the first six treatment months. The treatment had no major side-effects. G-CSF administration in ALS patients caused successful mobilization of autologous bone marrow cells, but was not effective in slowing down disease deterioration.

Advances in megakaryocytopoiesis and thrombopoiesis: from bench to bedside

Megakaryocytopoiesis involves the commitment of haematopoietic stem cells, proliferation and terminal differentiation of megakaryocytic progenitors (MK‐p) and maturation of megakaryocytes (MKs) to produce functional platelets. This complex process occurs in specialized niches in the bone marrow where MKs align adjacent to vascular endothelial cells, form proplatelet projections and release platelets into the circulation. Thrombopoietin (THPO, TPO) is the primary growth factor for the MK lineage and necessary at all stages of development. THPO is constitutively produced in the liver, and binds to MPL (c‐Mpl) receptor on platelets and MKs. This activates a cascade of signalling molecules, which induce transcription factors to drive MK development and thrombopoiesis. Decreased turnover rate and platelet number result in increased levels of free THPO, which induces a concentration‐dependant compensatory response of marrow‐MKs to enhance platelet production. Newly developed thrombopoietic agents operating via MPL receptor facilitate platelet production in thrombocytopenic states, primarily immune thrombocytopenia. Other drugs are available for attenuating malignant thrombocytosis. Herein, we review the regulation of megakaryocytopoiesis and platelet production in normal and disease states, and the innovative drugs and therapeutic modalities to stimulate or decrease thrombopoiesis.

Celecoxib But Not Rofecoxib Inhibits the Growth of Transformed Cells in Vitro

Purpose: Nonsteroidal anti-inflammatory drugs reduce the risk of colorectal cancer. The cyclooxygenase (COX) pathway of arachidonic acid metabolism is an important target for nonsteroidal anti-inflammatory drugs. Increased expression of COX-2 was recently shown to be an important step in the multistep process of colorectal cancer carcinogenesis. The new COX-2-specific inhibitors offer the benefit of cancer protection without the gastrointestinal toxicity reported for the old drugs. The purpose of this study was to compare the growth effects of two specific COX-2 inhibitors, celecoxib (Pfizer, Inc., New York, NY), and rofecoxib (Merck, White House Station, NJ) in normal and transformed enterocytes. Experimental Design: Cultures of normal rat intestinal epithelial cell line, IEC-18, vector control cells, c-K-ras, c-K-ras-bak, and antisense-bak derivatives were treated with different dosages of celecoxib (0–60 μm) and rofecoxib (0–20 μm). Cell cycle analysis and apoptosis were assessed by fluorescence-activated cell sorting analysis. Protein expression was assessed by Western blot analysis and caspases 3 and 8 activities by ELISA. Results: Celecoxib inhibited cell growth and induced apoptosis in a time- and dose-dependent manner. IEC18 parental cells were two to four times more resistant to celecoxib than ras, ras-bak, and antisense bak transformed cells that overexpress the COX-2 protein. The induction of apoptosis by celecoxib involved the caspase pathways. Rofecoxib, up to its maximal concentration of 20 μm, did not inhibit cell growth or induce apoptosis. Conclusions: Celecoxib may prove to be a very efficient component in the prevention and treatment of gastrointestinal tumors because it inhibits the growth of cancerous cells without affecting the growth of normal cells.