Aurora-A inhibition offers a novel therapy effective against intracranial glioblastoma.

Glioblastoma remains a devastating disease for which novel therapies are urgently needed. Here, we report that the Aurora-A kinase inhibitor alisertib exhibits potent efficacy against glioblastoma neurosphere tumor stem-like cells in vitro and in vivo. Many glioblastoma neurosphere cells treated with alisertib for short periods undergo apoptosis, although some regain proliferative activity upon drug removal. Extended treatment, however, results in complete and irreversible loss of tumor cell proliferation. Moreover, alisertib caused glioblastoma neurosphere cells to partially differentiate and enter senescence. These effects were also observed in glioma cells treated with the Aurora-A inhibitor TC-A2317 or anti-Aurora-A siRNA. Furthermore, alisertib extended median survival of mice bearing intracranial human glioblastoma neurosphere tumor xenografts. Alisertib exerted similar effects on glioblastoma neurosphere cells in vivo and resulted in markedly reduced activated phosphoThr288Aurora-A and increased abnormal mitoses and cellular ploidy, consistent with on-target activity. Our results offer preclinical proof-of-concept for alisertib as a new therapeutic for glioma treatment.

The selective Aurora-A kinase inhibitor MLN8237 (alisertib) potently inhibits proliferation of glioblastoma neurosphere tumor stem-like cells and potentiates the effects of temozolomide and ionizing radiation.

The selective Aurora-A kinase inhibitor MLN8237 is in clinical trials for hematologic malignancies, ovarian cancer and other solid tumors. We previously showed that MLN8237 is potently antiproliferative toward standard monolayer-cultured glioblastoma cells. We have now investigated the effect of MLN8237 with and without temozolomide or ionizing radiation on the proliferation of glioblastoma tumor stem-like cells (neurospheres) using soft agar colony formation assays and normal human astrocytes by MTT assay. Western blotting was utilized to compare MLN8237 IC50s to cellular Aurora-A and phosphoThr(288)Aurora-A levels. MLN8237 was more potently antiproliferative to neurosphere cells than to standard monolayer glioma cells, and was non-toxic to normal human astrocytes. Western blot analysis revealed that MLN8237 treatment inhibits phosphoThr(288)Aurora-A levels providing proof of drug target-hit in glioblastoma cells. Furthermore, phosphoThr(288)Aurora-A levels partially predicted the antiproliferative efficacy of MLN8237. We also found that Aurora-A inhibition by MLN8237 was synergistic with temozolomide and potentiated the effects of ionizing radiation on colony formation in neurosphere glioblastoma tumor stem-like cells. These results further support the potential of Aurora-A inhibitors as primary chemotherapy agents or biologic response modifiers in glioblastoma patients.

Antagonistic activities of the immunomodulator and PP2A-activating drug FTY720 (Fingolimod, Gilenya) in Jak2-driven hematologic malignancies.

FTY720 (Fingolimod, Gilenya) is a sphingosine analog used as an immunosuppressant in multiple sclerosis patients. FTY720 is also a potent protein phosphatase 2A (PP2A)-activating drug (PAD). PP2A is a tumor suppressor found inactivated in different types of cancer. We show here that PP2A is inactive in polycythemia vera (PV) and other myeloproliferative neoplasms characterized by the expression of the transforming Jak2(V617F) oncogene. PP2A inactivation occurs in a Jak2(V617F) dose/kinase-dependent manner through the PI-3Kγ-PKC-induced phosphorylation of the PP2A inhibitor SET. Genetic or PAD-mediated PP2A reactivation induces Jak2(V617F) inactivation/downregulation and impairs clonogenic potential of Jak2(V617F) cell lines and PV but not normal CD34(+) progenitors. Likewise, FTY720 decreases leukemic allelic burden, reduces splenomegaly, and significantly increases survival of Jak2(V617F) leukemic mice without adverse effects. Mechanistically, we show that in Jak2(V617F) cells, FTY720 antileukemic activity requires neither FTY720 phosphorylation (FTY720-P) nor SET dimerization or ceramide induction but depends on interaction with SET K209. Moreover, we show that Jak2(V617F) also utilizes an alternative sphingosine kinase-1-mediated pathway to inhibit PP2A and that FTY720-P, acting as a sphingosine-1-phosphate-receptor-1 agonist, elicits signals leading to the Jak2-PI-3Kγ-PKC-SET-mediated PP2A inhibition. Thus, PADs (eg, FTY720) represent suitable therapeutic alternatives for Jak2(V617F) MPNs.

The control of the balance between ceramide and sphingosine-1-phosphate by sphingosine kinase: oxidative stress and the seesaw of cell survival and death.

Sphingolipids are components of all eukaryotic cells that play important roles in a wide variety of biological processes. Ceramides and sphingosine-1-phosphate (S1P) are signaling molecules that regulate cell fate decisions in a wide array of species including yeast, plants, vertebrates, and invertebrates. Ceramides favor anti-proliferative and cell death pathways such as senescence and apoptosis, whereas S1P stimulates cell proliferation and survival pathways. The control of cell fate by these two interconvertible lipids has been called the sphingolipid rheostat or sphingolipid biostat. Sphingosine kinase, the enzyme that synthesizes S1P, is a crucial enzyme in regulation of the balance of these sphingolipids. Sphingosine kinase has been shown to play dynamic roles in the responses of cells to stress, leading to modulation of cell fate through a variety of signaling pathways impinging on the processes of cell proliferation, apoptosis, autophagy and senescence. This review summarizes the roles of sphingosine kinase signaling in these processes and the mechanisms mediating these responses. In addition, we discuss the evidence tying sphingosine kinase-mediated stress responses to the process of aging.

Induction of brain tumor stem cell apoptosis by FTY720: a potential therapeutic agent for glioblastoma.

FTY720 is a sphingosine analogue that down regulates expression of sphingosine-1-phosphate receptors and causes apoptosis of multiple tumor cell types, including glioma cells. This study examined the effect of FTY720 on brain tumor stem cells (BTSCs) derived from human glioblastoma (GBM) tissue. FTY720 treatment of BTSCs led to rapid inactivation of ERK MAP kinase, leading to upregulation of the BH3-only protein Bim and apoptosis. In combination with temozolomide (TMZ), the current standard chemotherapeutic agent for GBM, FTY720 synergistically induced BTSC apoptosis. FTY720 also slowed growth of intracranial xenograft tumors in nude mice and augmented the therapeutic effect of TMZ, leading to enhanced survival. Furthermore, the combination of FTY720 and TMZ decreased the invasiveness of BTSCs in mouse brains. FTY720 is known to cross the blood-brain barrier and recently received Food and Drug Administration approval for treatment of relapsing multiple sclerosis. Thus, FTY720 is an excellent potential therapeutic agent for treatment of GBM.

The role of sphingosine kinase-1 in EGFRvIII-regulated growth and survival of glioblastoma cells.

We have previously shown that high expression levels of the lipid kinase sphingosine kinase-1 (SphK1) correlate with poor survival of glioblastoma (GBM) patients. In this study we examined the regulation of SphK1 expression by epidermal growth factor receptor (EGFR) signaling in GBM cells. As the EGFR gene is often overexpressed and mutated in GBM, and EGFR has been shown to regulate SphK1 in some cell types, we examined the effect of EGF signaling and the constitutively active EGFRvIII mutant on SphK1 in GBM cells. Treatment of glioma cell lines with EGF led to increased expression and activity of SphK1. Expression of EGFRvIII in glioma cells also activated and induced SphK1. In addition, siRNA to SphK1 partially inhibited EGFRvIII-induced growth and survival of glioma cells as well as ERK MAP kinase activation. To further evaluate the connection between EGFR and SphK1 in GBM we examined primary neurosphere cells isolated from fresh human GBM tissue. The GBM-derived neurosphere cell line GBM9, which forms GBM-like tumors intracranially in nude mice, maintained expression of EGFRvIII in culture and had high levels of SphK1 activity. EGFR inhibitors modestly decreased SphK1 activity and proliferation of GBM9 cells. More extensive blockage of SphK1 activity by a SphK inhibitor, potently blocked cell proliferation and induced apoptotic cell death of GBM9 cells. Thus, SphK1 activity is necessary for survival of GBM-derived neurosphere cells, and EGFRvIII partially utilizes SphK1 to further enhance cell proliferation.

Sphingosine-1-phosphate regulates glioblastoma cell invasiveness through the urokinase plasminogen activator system and CCN1/Cyr61.

Glioblastoma multiforme (GBM) is an aggressively invasive brain neoplasm with poor patient prognosis. We have previously shown that the bioactive lipid sphingosine-1-phosphate (S1P) stimulates in vitro invasiveness of GBM cells and that high expression levels of the enzyme that forms S1P, sphingosine kinase-1 (SphK1), correlate with shorter survival time of GBM patients. We also recently showed that S1P induces expression of CCN1 (also known as Cyr61), a matricellular protein known to correlate with poor patient prognosis, in GBM cells. In this study, we further explored the role of CCN1 as well as the urokinase plasminogen activator (uPA), a protein known to stimulate GBM cell invasiveness, in S1P-induced invasion using a spheroid invasion assay. We also investigated the roles of various S1P receptors in stimulating invasiveness through these pathways. S1P induced expression of uPA and its receptor, uPAR, in GBM cells. Whereas S1P(1), S1P(2), and S1P(3) receptors all contribute, at least partially, S1P(1) overexpression led to the most dramatic induction of the uPA system and of spheroid invasion, even in the absence of added S1P. Furthermore, neutralizing antibodies directed against uPA or CCN1 significantly decreased both basal and S1P-stimulated GBM cell invasiveness. Inhibition of SphK blocked basal expression of uPA and uPAR, as well as glioma cell invasion; however, overexpression of SphK did not augment S1P receptor-mediated enhancement of uPA activity or invasion. Thus, SphK is necessary for basal activity of the uPA system and glioma cell invasion, whereas S1P receptor signaling enhances invasion, partially through uPA and CCN1.

Immunosuppressive human anti-lymphocyte autoantibodies specific for the type 1 sphingosine 1-phosphate receptor.

Anti-lymphocyte antibodies (Abs) that suppress T-cell chemotactic and other responses to sphingosine 1-phosphate (S1P), but not to chemokines, were found in a lymphopenic patient with recurrent infections. Lymphocyte type 1 S1P receptor (S1P(1)) that transduces S1P chemotactic stimulation was recognized by patient Abs in Western blots of T cells, S1P(1) transfectants, and S1P(1)-hemagglutinin purified by monoclonal anti-hemagglutinin Ab absorption. The amino terminus of S1P(1), but not any extracellular loop, prevented anti-S1P(1) Ab suppression of S1P(1) signaling and T-cell chemotaxis to S1P. Human purified anti-S1P(1) Abs decreased mouse blood lymphocyte levels by a mean of 72%, suppressed mouse T-cell chemotaxis to S1P in vivo, and significantly reduced the severity of dextran sodium sulfate-induced colitis in mice. Human Abs to the amino terminus of S1P(1) suppress T-cell trafficking sufficiently to impair host defense and provide therapeutic immunosuppression.

Human cytomegalovirus regulates bioactive sphingolipids.

Sphingolipids are present in membranes of all eukaryotic cells. Bioactive sphingolipids also function as signaling molecules that regulate cellular processes such as proliferation, migration, and apoptosis. Human cytomegalovirus (HCMV) exploits a variety of cellular signaling pathways to promote its own replication. However, whether HCMV modulates lipid signaling pathways is an essentially unexplored area of research in virus-host cell interactions. In this study, we examined the accumulation of the bioactive sphingolipids and the enzymes responsible for the biosynthesis and degradation of these lipids. HCMV infection results in increased accumulation and activity of sphingosine kinase (SphK), the enzyme that generates sphingosine 1-phosphate (S1P) and dihydrosphingosine 1-phosphate (dhS1P). We also utilized a mass spectrometry approach to generate a sphingolipidomic profile of HCMV-infected cells. We show that HCMV infection results in increased levels of dhS1P and ceramide at 24 h, suggesting an enhancement of de novo sphingolipid synthesis. Subsequently dihydrosphingosine and dhS1P decrease at 48 h consistent with attenuation of de novo sphingolipid synthesis. Finally, we present evidence that de novo sphingolipid synthesis and sphingosine kinase activity directly impact virus gene expression and virus growth. Together, these findings demonstrate that host cell sphingolipids are dynamically regulated upon infection with a herpes virus in a manner that impacts virus replication.

Subtype-specific residues involved in ligand activation of the endothelial differentiation gene family lysophosphatidic acid receptors.

Lysophosphatidic acid (LPA) is a ligand for three endothelial differentiation gene family G protein-coupled receptors, LPA(1-3). We performed computational modeling-guided mutagenesis of conserved residues in transmembrane domains 3, 4, 5, and 7 of LPA(1-3) predicted to interact with the glycerophosphate motif of LPA C18:1. The mutants were expressed in RH7777 cells, and the efficacy (E(max)) and potency (EC(50)) of LPA-elicited Ca(2+) transients were measured. Mutation to alanine of R3.28 universally decreased both the efficacy and potency in LPA(1-3) and eliminated strong ionic interactions in the modeled LPA complexes. The alanine mutation at Q3.29 decreased modeled interactions and activation in LPA(1) and LPA(2) more than in LPA(3). The mutation W4.64A had no effect on activation and modeled LPA interaction of LPA(1) and LPA(2) but reduced the activation and modeled interactions of LPA(3). The R5.38A mutant of LPA(2) and R5.38N mutant of LPA(3) showed diminished activation by LPA; however, in LPA(1) the D5.38A mutation did not, and mutation to arginine enhanced receptor activation. In LPA(2), K7.36A decreased the potency of LPA; in LPA(1) this same mutation increased the E(max). In LPA(3), R7.36A had almost no effect on receptor activation; however, the mutation K7.35A increased the EC(50) in response to LPA 10-fold. In LPA(1-3), the mutation Q3.29E caused a modest increase in EC(50) in response to LPA but caused the LPA receptors to become more responsive to sphingosine 1-phosphate (S1P). Surprisingly micromolar concentrations of S1P activated the wild type LPA(2) and LPA(3) receptors, indicating that S1P may function as a weak agonist of endothelial differentiation gene family LPA receptors.

Sphingolipid signaling pathways as potential therapeutic targets in gliomas.

Glioblastoma multiforme (GBM) is a highly malignant brain tumor. The interconvertible bioactive sphingolipids sphingosine-1-phosphate (S1P) and ceramide have profound effects on GBM cells, with ceramide causing cell death and S1P leading to cell survival, proliferation and invasion. This review will examine the effects of ceramide and S1P on glioma cells and the therapeutic potential of these pathways.

Sphingosine 1-phosphate pKa and binding constants: intramolecular and intermolecular influences.

The dissociation constant for an ionizable ligand binding to a receptor is dependent on its charge and therefore on its environmentally-influenced pKa value. The pKa values of sphingosine 1-phosphate (S1P) were studied computationally in the context of the wild type S1P1 receptor and the following mutants: E3.29Q, E3.29A, and K5.38A. Calculated pKa values indicate that S1P binds to S1P1 and its site mutants with a total charge of -1, including a +1 charge on the ammonium group and a -2 charge on the phosphate group. The dissociation constant of S1P binding to these receptors was studied as well. The models of wild type and mutant proteins originated from an active receptor model that was developed previously. We used ab initio RHF/6-31+G(d) to optimize our models in aqueous solution, where the solvation energy derivatives are represented by conductor-like polarizable continuum model (C-PCM) and integral equation formalism polarizable continuum model (IEF-PCM). Calculation of the dissociation constant for each mutant was determined by reference to the experimental dissociation constant of the wild type receptor. The computed dissociation constants of the E3.29Q and E3.29A mutants are three to five orders of magnitude higher than those for the wild type receptor and K5.38A mutant, indicating vital contacts between the S1P phosphate group and the carboxylate group of E3.29. Computational dissociation constants for K5.38A, E3.29A, and E3.29Q mutants were compared with experimentally determined binding and activation data. No measurable binding of S1P to the E3.29A and E3.29Q mutants was observed, supporting the critical contacts observed computationally. These results validate the quantitative accuracy of the model.

Roles of sphingosine-1-phosphate (S1P) receptors in malignant behavior of glioma cells. Differential effects of S1P2 on cell migration and invasiveness.

Sphingosine-1-phosphate (S1P) is a bioactive lipid that signals through a family of five G-protein-coupled receptors, termed S1P(1-5). S1P stimulates growth and invasiveness of glioma cells, and high expression levels of the enzyme that forms S1P, sphingosine kinase-1, correlate with short survival of glioma patients. In this study we examined the mechanism of S1P stimulation of glioma cell proliferation and invasion by either overexpressing or knocking down, by RNA interference, S1P receptor expression in glioma cell lines. S1P(1), S1P(2) and S1P(3) all contribute positively to S1P-stimulated glioma cell proliferation, with S1P(1) being the major contributor. Stimulation of glioma cell proliferation by these receptors correlated with activation of ERK MAP kinase. S1P(5) blocks glioma cell proliferation, and inhibits ERK activation. S1P(1) and S1P(3) enhance glioma cell migration and invasion. S1P(2) inhibits migration through Rho activation, Rho kinase signaling and stress fiber formation, but unexpectedly, enhances glioma cell invasiveness by stimulating cell adhesion. S1P(2) also potently enhances expression of the matricellular protein CCN1/Cyr61, which has been implicated in tumor cell adhesion, and invasion as well as tumor angiogenesis. A neutralizing antibody to CCN1 blocked S1P(2)-stimulated glioma invasion. Thus, while S1P(2) decreases glioma cell motility, it may enhance invasion through induction of proteins that modulate glioma cell interaction with the extracellular matrix.

Identification of the hydrophobic ligand binding pocket of the S1P1 receptor.

Sphingosine 1-phosphate (S1P), a naturally occurring sphingolipid mediator and also a second messenger with growth factor-like actions in almost every cell type, is an endogenous ligand of five G protein-coupled receptors (GPCRs) in the endothelial differentiation gene family. The lack of GPCR crystal structures sets serious limitations to rational drug design and in silico searches for subtype-selective ligands. Here we report on the experimental validation of a computational model of the ligand binding pocket of the S1P1 GPCR surrounding the aliphatic portion of S1P. The extensive mutagenesis-based validation confirmed 18 residues lining the hydrophobic ligand binding pocket, which, combined with the previously validated three head group-interacting residues, now complete the mapping of the S1P ligand recognition site. We identified six mutants (L3.43G/L3.44G, L3.43E/L3.44E, L5.52A, F5.48G, V6.40L, and F6.44G) that maintained wild type [32P]S1P binding with abolished ligand-dependent activation by S1P. These data suggest a role for these amino acids in the conformational transition of S1P1 to its activated state. Three aromatic mutations (F5.48Y, F6.44G, and W6.48A) result in differential activation, by S1P or SEW2871, indicating that structural differences between the two agonists can partially compensate for differences in the amino acid side chain. The now validated ligand binding pocket provided us with a pharmacophore model, which was used for in silico screening of the NCI, National Institutes of Health, Developmental Therapeutics chemical library, leading to the identification of two novel nonlipid agonists of S1P1.

Text mining of full-text journal articles combined with gene expression analysis reveals a relationship between sphingosine-1-phosphate and invasiveness of a glioblastoma cell line.

BACKGROUND: Sphingosine 1-phosphate (S1P), a lysophospholipid, is involved in various cellular processes such as migration, proliferation, and survival. To date, the impact of S1P on human glioblastoma is not fully understood. Particularly, the concerted role played by matrix metalloproteinases (MMP) and S1P in aggressive tumor behavior and angiogenesis remains to be elucidated. RESULTS: To gain new insights in the effect of S1P on angiogenesis and invasion of this type of malignant tumor, we used microarrays to investigate the gene expression in glioblastoma as a response to S1P administration in vitro. We compared the expression profiles for the same cell lines under the influence of epidermal growth factor (EGF), an important growth factor. We found a set of 72 genes that are significantly differentially expressed as a unique response to S1P. Based on the result of mining full-text articles from 20 scientific journals in the field of cancer research published over a period of five years, we inferred gene-gene interaction networks for these 72 differentially expressed genes. Among the generated networks, we identified a particularly interesting one. It describes a cascading event, triggered by S1P, leading to the transactivation of MMP-9 via neuregulin-1 (NRG-1), vascular endothelial growth factor (VEGF), and the urokinase-type plasminogen activator (uPA). This interaction network has the potential to shed new light on our understanding of the role played by MMP-9 in invasive glioblastomas. CONCLUSION: Automated extraction of information from biological literature promises to play an increasingly important role in biological knowledge discovery. This is particularly true for high-throughput approaches, such as microarrays, and for combining and integrating data from different sources. Text mining may hold the key to unraveling previously unknown relationships between biological entities and could develop into an indispensable instrument in the process of formulating novel and potentially promising hypotheses.

Signal transduction of sphingosine-1-phosphate G protein-coupled receptors.

Sphingosine-1-phosphate (S1P) is a bioactive lipid capable of eliciting dramatic effects in a variety of cell types. Signaling by this molecule is by a family of five G protein-coupled receptors named S1P1-5 that signal through a variety of pathways to regulate cell proliferation, migration, cytoskeletal organization, and differentiation. These receptors are expressed in a wide variety of tissues and cell types, and their cellular effects contribute to important biological and pathological functions of S1P in many processes, including angiogenesis, vascular development, lymphocyte trafficking, and cancer. This review will focus on the current progress in the field of S1P receptor signaling and biology.

Type 1 sphingosine 1-phosphate G protein-coupled receptor signaling of lymphocyte functions requires sulfation of its extracellular amino-terminal tyrosines.

The type 1 sphingosine 1-phosphate (S1P) G protein-coupled receptor (S1P1) transduces signals from S1P that mediate thymocyte emigration, T cell transmigration of lymph nodes, and T cell chemotaxis in tissues. Alterations in expression of functional S1P1 receptors by lymphocytes are the major mechanisms controlling their responses to S1P and were thought to be solely a consequence of the balance between surface down-regulation and insertion. However, results now show that lack of sulfation of tyrosines 19 and 22 of the extracellular N terminus of S1P1 diminishes high-affinity S1P binding and decreases S1P signaling of T cell migration and other functions. Non-sulfatable mutant (Y19,22F)S1P1 endows T cells with lower-affinity binding of [32P]S1P than wild-type S1P1 and transduces lesser effects of S1P on chemotaxis, chemokine-elicited chemotaxis, and T cell receptor-mediated proliferation and cytokine generation. Inhibition of S1P1 tyrosine sulfation or sulfatase removal of S1P1 sulfate in mouse CD4 T cells suppresses immune functional effects of S1P. Tyrosine sulfation of S1P1 may be a major controller of S1P effects on T cell traffic.

Sphingosine kinase-1 expression correlates with poor survival of patients with glioblastoma multiforme: roles of sphingosine kinase isoforms in growth of glioblastoma cell lines.

Sphingosine-1-phosphate is a bioactive lipid that is mitogenic for human glioma cell lines by signaling through its G protein-coupled receptors. We investigated the role of sphingosine-1-phosphate receptors and the enzymes that form sphingosine-1-phosphate, sphingosine kinase (SphK)-1, and -2 in human astrocytomas. Astrocytomas of various histologic grades expressed three types of sphingosine-1-phosphate receptors, S1P1, S1P2, and S1P3; however, no significant correlation with histologic grade or patient survival was detected. Expression of SphK1, but not SphK2, in human astrocytoma grade 4 (glioblastoma multiforme) tissue correlated with short patient survival. Patients whose tumors had low SphK1 expression survived a median 357 days, whereas those with high levels of SphK1 survived a median 102 days. Decreasing SphK1 expression using RNA interference or pharmacologic inhibition of SphK significantly decreased the rate of proliferation of U-1242 MG and U-87 MG glioblastoma cell lines. Surprisingly, RNA interference to knockdown SphK2 expression inhibited glioblastoma cell proliferation more potently than did SphK1 knockdown. SphK knockdown also prevented cells from exiting G1 phase of the cell cycle and marginally increased apoptosis. Thus, SphK isoforms may be major contributors to growth of glioblastoma cells in vitro and to aggressive behavior of glioblastoma multiforme.

An IgM-kappa rat monoclonal antibody specific for the type 1 sphingosine 1-phosphate G protein-coupled receptor with antagonist and agonist activities.

Sphingosine 1-phosphate (S1P) type 1G protein-coupled receptors (S1P1 GPCRs) are specific high-affinity transducers for this lipid growth factor and cellular mediator. S1P1 GPCRs are widely-expressed and physiologically critical in the cardiovascular and immune systems. Functional rat monoclonal antibodies (MoAbs) have been generated against human S1P1 GPCRs expressed in rat null-cell transductants to provide bioavailable agents capable of stimulating or suppressing the S1P-S1P1 GPCR axis. The rat IgM-kappa anti-S1P1 GPCR MoAb designated 4B5.2 binds specifically to native human or mouse S1P1 GPCRs in cell membranes, but not to solubilized and denatured S1P1 GPCRs. Specific binding of 32P-S1P to cellular S1P1 GPCRs is not blocked by 4B5.2. T cell chemotactic responses to S1P and S1P suppression of T cell chemotaxis to chemokines both are inhibited selectively by 4B5.2. In contrast, generation of gamma-interferon by stimulated T cells is diminished by 4B5.2 as by S1P. T cell S1P1 GPCR-selective antagonist and agonist effects of 4B5.2 in vivo may alter immune responses as distinctively as the available poly-S1P GPCR-directed pharmacological agents, without the undesirable side-effects attributable to actions of these agents on other S1P GPCRs.

Sphingosine-1-phosphate stimulates motility and invasiveness of human glioblastoma multiforme cells.

Sphingosine-1-phosphate (S1P) is a bioactive lipid which is a potent mitogen for glioblastoma multiforme cells. Here we show that S1P also potently enhances the in vitro motility of glioblastoma cells by signaling through receptors coupled to G(i/o) proteins. Moreover, S1P also enhanced in vitro invasion of glioblastoma cells through Matrigel. S1P had no effect on matrix metalloproteinase secretion but did enhance glioblastoma cell adhesion. S1P is present at high levels in brain tissue. Thus it is possible that autocrine or paracrine signaling by S1P through its G protein-coupled receptors enhances both glioma cell proliferation and invasiveness.