Ahmed Abdelbaset-Ismail

Human haematopoietic stem/progenitor cells express several functional sex hormone receptors.

Evidence has accumulated that murine haematopoietic stem/progenitor cells (HSPCs) share several markers with the germline, a connection supported by recent reports that pituitary and gonadal sex hormones (SexHs) regulate development of murine HSPCs. It has also been reported that human HSPCs, like their murine counterparts, respond to certain SexHs (e.g. androgens). However, to better address the effects of SexHs, particularly pituitary SexHs, on human haematopoiesis, we tested for expression of receptors for pituitary SexHs, including follicle‐stimulating hormone (FSH), luteinizing hormone (LH), and prolactin (PRL), as well as the receptors for gonadal SexHs, including progesterone, oestrogens, and androgen, on HSPCs purified from human umbilical cord blood (UCB) and peripheral blood (PB). We then tested the functionality of these receptors in ex vivo signal transduction studies and in vitro clonogenic assays. In parallel, we tested the effect of SexHs on human mesenchymal stromal cells (MSCs). Finally, based on our observation that at least some of the UCB‐derived, CD45− very small embryonic‐like stem cells (VSELs) become specified into CD45+ HSPCs, we also evaluated the expression of pituitary and gonadal SexH receptors on these cells. We report for the first time that human HSPCs and VSELs, like their murine counterparts, express pituitary and gonadal SexH receptors at the mRNA and protein levels. Most importantly, SexH if added to suboptimal doses of haematopoietic cytokines and growth factors enhance clonogenic growth of human HSPCs as well as directly stimulate proliferation of MSCs.

Activation of the complement cascade enhances motility of leukemic cells by downregulating expression of HO-1

As a crucial arm of innate immunity, the complement cascade (ComC) is involved both in mobilization of normal hematopoietic stem/progenitor cells (HSPCs) from bone marrow (BM) into peripheral blood and in their homing to BM. Despite the fact that ComC cleavage fragments alone do not chemoattract normal HSPCs, we found that leukemia cell lines as well as clonogenic blasts from chronic myeloid leukemia and acute myeloid leukemia patients respond robustly to C3 and C5 cleavage fragments by chemotaxis and increased adhesion. This finding was supported by the detection of C3a and C5a receptors in cells from human malignant hematopoietic cell lines and patient blasts at the mRNA (reverse transcriptase-polymerase chain reaction) and protein level (fluorescence-activated cell sorting), and by the demonstration that these receptors respond to stimulation by C3a and C5a by phosphorylation of p42/44 and p38 mitogen-activated protein kinases (MAPK), and protein kinase B (PKB/AKT). We also found that inducible heme oxygenase 1 (HO-1) is a negative regulator of ComC-mediated trafficking of leukemic cells, and that stimulation of leukemic cells by C3 or C5 cleavage fragments activates p38 MAPK, which downregulates HO-1 expression, rendering cells more mobile. We conclude that activation of the ComC in leukemia/lymphoma patients (for example, as a result of accompanying infections) enhances the motility of malignant cells and contributes to their spread in a p38 MAPK–HO-1-dependent manner. Therefore, inhibition of p38 MAPK or upregulation of HO-1 by small-molecule modulators would have a beneficial effect on ameliorating cell migration-mediated expansion of leukemia/lymphoma cells when the ComC becomes activated.

Evidence that a lipolytic enzyme—hematopoietic-specific phospholipase C-β2—promotes mobilization of hematopoietic stem cells by decreasing their lipid raft-mediated bone marrow retention and increasing the promobilizing effects of granulocytes

Hematopoietic stem/progenitor cells (HSPCs) reside in the bone marrow (BM) microenvironment and are retained there by the interaction of membrane lipid raft-associated receptors, such as the α-chemokine receptor CXCR4 and the α4β1-integrin (VLA-4, very late antigen 4 receptor) receptor, with their respective specific ligands, stromal-derived factor 1 and vascular cell adhesion molecule 1, expressed in BM stem cell niches. The integrity of the lipid rafts containing these receptors is maintained by the glycolipid glycosylphosphatidylinositol anchor (GPI-A). It has been reported that a cleavage fragment of the fifth component of the activated complement cascade, C5a, has an important role in mobilizing HSPCs into the peripheral blood (PB) by (i) inducing degranulation of BM-residing granulocytes and (ii) promoting their egress from the BM into the PB so that they permeabilize the endothelial barrier for subsequent egress of HSPCs. We report here that hematopoietic cell-specific phospholipase C-β2 (PLC-β2) has a crucial role in pharmacological mobilization of HSPCs. On the one hand, when released during degranulation of granulocytes, it digests GPI-A, thereby disrupting membrane lipid rafts and impairing retention of HSPCs in BM niches. On the other hand, it is an intracellular enzyme required for degranulation of granulocytes and their egress from BM. In support of this dual role, we demonstrate that PLC-β2-knockout mice are poor mobilizers and provide, for the first time, evidence for the involvement of this lipolytic enzyme in the mobilization of HSPCs.

Novel evidence that the mannan-binding lectin pathway of complement activation plays a pivotal role in triggering mobilization of hematopoietic stem/progenitor cells by activation of both the complement and coagulation cascades

Novel evidence that the mannan-binding lectin pathway of complement activation plays a pivotal role in triggering mobilization of hematopoietic stem/progenitor cells by activation of both the complement and coagulation cascades

Extracellular nucleotides as novel, underappreciated pro-metastatic factors that stimulate purinergic signaling in human lung cancer cells

BackgroundOne of the challenging problems of current radio-chemotherapy is recurrence and metastasis of cancer cells that survive initial treatment. We propose that one of the unwanted effects of radiochemotherapy is the release from damaged (“leaky”) cells of nucleotides such as ATP and UTP that exert pro-metastatic functions and can directly stimulate chemotaxis of cancer cells.MethodsTo address this problem in a model of human lung cancer (LC), we employed several complementary in vitro and in vivo approaches to demonstrate the role of extracellular nucleotides (EXNs) in LC cell line metastasis and tumor progression. We measured concentrations of EXNs in several organs before and after radiochemotherapy. The purinergic receptor agonists and antagonists, inhibiting all or selected subtypes of receptors, were employed in in vitro and in vivo pro-metastatic assays.ResultsWe found that EXNs accumulate in several organs in response to radiochemotherapy, and RT-PCR analysis revealed that most of the P1 and P2 receptor subtypes are expressed in human LC cells. EXNs were found to induce chemotaxis and adhesion of LC cells, and an autocrine loop was identified that promotes the proliferation of LC cells. Most importantly, metastasis of these cells could be inhibited in immunodeficient mice in the presence of specific small molecule inhibitors of purinergic receptors.ConclusionsBased on this result, EXNs are novel pro-metastatic factors released particularly during radiochemotherapy, and inhibition of their pro-metastatic effects via purinergic signaling could become an important part of anti-metastatic treatment.

Novel evidence that pituitary gonadotropins directly stimulate human leukemic cells-studies of myeloid cell lines and primary patient AML and CML cells

We recently reported that normal hematopoietic stem cells express functional pituitary sex hormone (SexH) receptors. Here we report for the first time that pituitary-secreted gonadotrophins stimulate migration, adhesion, and proliferation of several human myeloid and lymphoid leukemia cell lines. Similar effects were observed after stimulation of human leukemic cell lines by gonadal SexHs. This effect seems to be direct, as the SexH receptors expressed by leukemic cells responded to stimulation by phosphorylation of MAPKp42/44 and AKTser473. Furthermore, in parallel studies we confirmed that human primary patient-derived AML and CML blasts also express several functional SexH receptors. These results shed more light on the potential role of SexHs in leukemogenesis and, in addition, provide further evidence suggesting a developmental link between hematopoiesis and the germline.

Inducible Nitric Oxide Synthase (iNOS) Is a Novel Negative Regulator of Hematopoietic Stem/Progenitor Cell Trafficking

Nitric oxide (NO) is a gaseous free radical molecule involved in several biological processes related to inflammation, tissue damage, and infections. Based on reports that NO inhibits migration of granulocytes and monocytes, we became interested in the role of inducible NO synthetase (iNOS) in pharmacological mobilization of hematopoietic stem/progenitor cells (HSPCs) from bone marrow (BM) into peripheral blood (PB). To address the role of NO in HSPC trafficking, we upregulated or downregulated iNOS expression in hematopoietic cell lines. Next, we performed mobilization studies in iNOS−/− mice and evaluated engraftment of iNOS−/− HSPCs in wild type (control) animals. Our results indicate that iNOS is a novel negative regulator of hematopoietic cell migration and prevents egress of HSPCs into PB during mobilization. At the molecular level, downregulation of iNOS resulted in downregulation of heme oxygenase 1 (HO-1), and, conversely, upregulation of iNOS enhanced HO-1 activity. Since HO-1 is a negative regulator of cell migration, the inhibitory effects of iNOS identified by us can be at least partially explained by its enhancing the HO-1 level in BM cells.

Vitamin D3 stimulates embryonic stem cells but inhibits migration and growth of ovarian cancer and teratocarcinoma cell lines

BackgroundDeficiency in Vitamin D3 (cholecalciferol) may predispose to some malignancies, including gonadal tumors and in experimental models vitamin D3 has been proven to inhibit the growth of cancer cells. To learn more about the potential role of vitamin D3 in cancerogenesis, we evaluated the expression and functionality of the vitamin D receptor (VDR) and its role in metastasis of ovarian cancer cells and of murine and human teratocarcinoma cell lines.MethodsIn our studies we employed murine embrynic stem cells (ESD3), murine (P19) and human (NTERA-2) teratocarcimona cells lines, human ovarian cancer cells (A2780) as well as purified murine and human purified very small embryonic like stem cells (VSELs). We evaluated expression of Vitamin D3 receptor (VDR) in these cells as well as effect of vitamin D3 exposure on cell proliferation and migration.ResultsWe here provide also more evidence for the role of vitamin D3 in germline-derived malignancies, and this evidence supports the proposal that vitamin D3 treatment inhibits growth and metastatic potential of several germline-derived malignancies. We also found that the ESD3 murine immortalized embryonic stem cell line and normal, pluripotent, germline-marker-positive very small embryonic-like stem cells (VSELs) isolated from adult tissues are stimulated by vitamin D3, which suggests that vitamin D3 affects the earliest stages of embryogenesis.ConclusionsWe found that however all normal and malignant germ-line derived cells express functional VDR, Vitamin D3 differently affects their proliferation and migration. We postulate that while Vitamin D3 as anticancer drug inhibits proliferation of malignant cells, it may protect normal stem cells that play an important role in development and tissue/organ regeneration.

Human rhabdomyosarcoma cells express functional pituitary and gonadal sex hormone receptors: therapeutic implications

Evidence has accumulated that sex hormones play an important role in several types of cancer. Because they are also involved in skeletal muscle development and regeneration, we were therefore interested in their potential involvement in the pathogenesis of human rhabdomyosarcoma (RMS), a skeletal muscle tumor. In the present study, we employed eight RMS cell lines (three fusion positive and five fusion negative RMS cell lines) and mRNA samples obtained from RMS patients. The expression of sex hormone receptors was evaluated by RT-PCR and their functionality by chemotaxis, adhesion and direct cell proliferation assays. We report here for the first time that follicle-stimulating hormone (FSH) and luteinizing hormone (LH) receptors are expressed in established human RMS cell lines as well as in primary tumor samples isolated from RMS patients. We also report that human RMS cell lines responded both to pituitary and gonadal sex hormone stimulation by enhanced proliferation, chemotaxis, cell adhesion and phosphorylation of MAPKp42/44 and AKT. In summary, our results indicate that sex hormones are involved in the pathogenesis and progression of RMS, and therefore, their therapeutic application should be avoided in patients that have been diagnosed with RMS.

The Involvment of Hematopoietic-Specific PLC -β2 in Homing and Engraftment of Hematopoietic Stem/Progenitor Cells

Migration and bone marrow (BM) homing of hematopoietic stem progenitor cells (HSPCs) is regulated by several signaling pathways, and here we provide evidence for the involvement in this process of hematopoietic-specific phospholipase C-β2 (PLC-β2). This enzyme is involved in release of intracellular calcium and activation of protein kinase C (PKC). Recently we reported that PLC-β2 promotes mobilization of HSPCs from BM into peripheral blood (PB), and this effect is mediated by the involvement of PLC-β2 in the release of proteolytic enzymes from granulocytes and its role in disintegration of membrane lipid rafts. Here we report that, besides the role of PLC-β2 in the release of HSPCs from BM niches, PLC-β2 regulates the migration of HSPCs in response to chemotactic gradients of BM homing factors, including SDF-1, S1P, C1P, and ATP. Specifically, HSPCs from PLC-β2-KO mice show impaired homing and engraftment in vivo after transplantation into lethally irradiated mice. This decrease in migration of HSPCs can be explained by impaired calcium release in PLC-β2-KO mice and a high baseline level of heme oxygenase 1 (HO-1), an enzyme that negatively regulates cell migration.