|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 90 No. 4 (August 15), 1997:
pp. 1448-1457
Anandamide, a Natural Ligand for the Peripheral Cannabinoid Receptor Is a Novel Synergistic Growth Factor for Hematopoietic Cells
By
Peter Valk,
Sandra Verbakel,
Yolanda Vankan,
Samantha Hol,
Shanta Mancham,
Rob Ploemacher,
Angelique Mayen,
Bob Löwenberg, and
Ruud Delwel
From Erasmus University, Institute of Hematology, Rotterdam, The Netherlands.
 |
ABSTRACT |
We recently demonstrated that the gene encoding the peripheral cannabinoid receptor (Cb2) may be a proto-oncogene involved in murine myeloid leukemias. We show here that Cb2 may have a role in hematopoietic development. RNAse protection analysis showed that Cb2 is normally expressed in spleen and thymus. Cb2 mRNA is also expressed in 45 of 51 cell lines of distinct hematopoietic lineages, ie, myeloid, macrophage, mast, B-lymphoid, T-lymphoid, and erythroid cells. The effect of the fatty acid anandamide, an endogenous ligand for cannabinoid receptors, on primary murine marrow cells and hematopoietic growth factor (HGF )-dependent cell lines was then investigated. In vitro colony cultures of normal mouse bone marrow cells showed anandamide to potentiate interleukin-3 (IL-3)-induced colony growth markedly. Whereas HGFs alone stimulate proliferation of the various cell lines in serum-free culture only weakly, anandamide enhances the proliferative response of the cell lines to HGFs profoundly. This was apparent for responses induced by IL-3, granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor, and erythropoietin. Anandamide was already effective at concentrations as low as 0.1 to 0.3 µmol/L and plateau effects were reached at 0.3 to 3 µmol/L. The addition of anandamide as single growth factor had no effect. The costimulatory effect of anandamide was not evident when cells were cultured with fetal calf serum (FCS), suggesting that FCS contains anandamide or another ligand capable of activating the peripheral cannabinoid receptor. Other cannabinoid ligands did not enhance the proliferative responsiveness of hematopoietic cells to HGFs. Transfection experiments of Cb2 in myeloid 32D cells showed that anandamide specifically activates proliferation through activation of the peripheral cannabinoid receptor. Anandamide appears to be a novel and synergistic growth stimulator for hematopoietic cells.
| |
INTRODUCTION |
PROLIFERATION and differentiation of hematopoietic precursor cells is regulated by hematopoietic growth factors (HGFs).1,2 These cytokines bind and activate receptors that belong to the hematopoietin receptor superfamily.3 Receptors of this superfamily are single transmembrane proteins that, upon binding to their specific ligands, form heterodimeric or homodimeric complexes.3 Ligands that bind hematopoietin receptors are small glycoproteins, eg, interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF ), granulocyte colony-stimulating factor (G-CSF ), erythropoietin (Epo), or macrophage colony-stimulating factor (M-CSF ). Another family of surface membrane receptors is the G-protein-coupled receptors (GPCRs).4,5 This superfamily of receptor molecules is characterized by seven hydrophobic stretches of 20 to 25 amino acids that form seven transmembrane-helices connected by alternating extracellular and intracellular loops. Up to now, more than 300 GPCRs have been identified.4,5 In contrast to receptors of the hematopoietin family, GPCRs are in most cases not activated by glycoproteins. Ligands of heptahelical GPCRs include amines, amino acids, peptides or proteins, nucleosides or nucleotides, fatty acid derivates, and phospholipid derivates.4,5 Little is known about the role of GPCRs in hematopoietic growth and development. We recently identified the peripheral cannabinoid receptor (Cb2), which encodes a GPCR, in a common ecotropic virus integration site (Evi11).6,7 In vitro transfection studies in 32D cells supported the hypothesis that Cb2 is a proto-oncogene that is involved in leukemogenesis.6 In the present study, we investigated by RNAse protection analysis the expression pattern of the Cb2 gene in comparison to the gene that encodes the central cannabinoid receptor (Cb1)8,9 in different murine tissues and a panel of murine hematopoietic cell lines. We show that Cb2 encodes a hematopoietic receptor that is expressed in myeloid, macrophage, erythroid, lymphoid, and mast cells. In contrast, Cb1 is mainly expressed in brain and in testis.8,9 The effects of the fatty acid N-arachidonylethanolamide or anandamide, an endogenous ligand for cannabinoid receptors, on the proliferative abilities of HGF-dependent hematopoietic cell lines were investigated. The results of this study demonstrate that anandamide is a ligand that stimulates proliferation of hematopoietic cell lines in synergy with IL-3, Epo, GM-CSF, and G-CSF under serum-free conditions.
| |
MATERIALS AND METHODS |
Mouse hematopoietic cell lines.
A list of murine hematopoietic cell lines used in this study is presented in Table 1. Macrophage cell lines were cultured in Dulbecco's modified Eagle's medium (GIBCO, Ghent, Belgium) plus supplements (100 IU/mL penicillin, 100 ng/mL streptomycin, and 10% fetal calf serum [FCS]). The other cell lines were cultured in RPMI-1640 medium plus supplements. Myeloid cell lines were cultured with 10 ng murine IL-3 and CTLL cells were cultured with 10 IU/mL IL-2 (Cetus, Emmeryville, CA). The pro-B-cell line BAF3 and myeloid 32D cell line both transfected with the human G-CSF receptor gene (BAF-G and 32D [G-CSF-R], respectively) were donated by Dr I.P. Touw (Erasmus University Rotterdam, Rotterdam, The Netherlands).36
RNA isolation.
Mouse tissues were homogenized using an Ultratarax T25 shearing device (IKA Labortechnique, Heiterheim, Germany). Total RNA was extracted from murine hematopoietic cell lines with guanidinium thiocyanate followed by phenol extraction.37 RNA samples of several cell lines were kindly donated by Dr J. Cleveland (St Jude Children's Hospital, Memphis, TN).
RNAse protection.
RNAse protection experiments were performed as described.37 cDNA fragments were cloned into Bluescript II SK+ (Stratagene, La Jolla, CA) linearized using the proper enzymes and RNA probes synthesized with T3 or T7 polymerase (Promega, Leiden, The Netherlands). For each incubation, 10 µg of RNA and radiolabeled probe (25,000 cpm) were suspended in 30 µL hybridization buffer (80% deionized formamide, 40 mmol/L PIPES, pH 6.4, 0.4 mol/L NaAc, 1 mmol/L EDTA). The samples were heated to 85°C for 5 minutes and then incubated for 16 hours at 50°C anealing temperature. To these mixtures, 300 µL RNAse digestion buffer (10 mmol/L Tris-HCl, pH 7.5, 5 mmol/L EDTA, and 200 mmol/L NaAc) and 1 U RNAse One (Promega) was added. After 1 hour at 37°C, the reaction was stopped by the addition of 3.3 µL 10% sodium dodecyl sulfate and 20 µg carrier tRNA. The reactions were precipitated with ethanol and fractionated by electrophoresis on a 6% polyacrylamide/7 mol/L urea gel and analyzed by autoradiography. A radiolabeled GAPDH RNA fragment was used as a control.
Serum-free culture medium.
A serum-free culture medium was used to study responses to HGFs and cannabinoid ligands.38-40 Iscove's modified Dulbecco's medium (GIBCO) was supplemented with 15 mg/mL bovine serum albumin (Cohn fraction V; Sigma, Bornem, Belgium), 10-7 mol/L sodium selenite (Merck, Darmstad, Germany), 7.7 × 10-6 mol/L iron-saturated human transferrin (Behring Institute, Marburg, Germany), 7.8 µg/mL cholesterol (Sigma), and 2.8 µg/mL linoleic acid (Merck). In normal bone marrow colony cultures, 10 ng/mL insulin (Sigma), 10-4 mol/L  -mercaptoethanol (Merck), and 50 µg/mL of the nucleosides, adenosine, thymidine, guanosine, cytidine, uridine, 2'deoxycytidine, 2'deoxyadenosine, and 2'deoxyguanosine (Sigma) were added to the culture medium.
HGFs and cannabinoid ligands.
IL-3 (10 ng/mL; donated by Dr J.N. Ihle, St Jude Children's Research Hospital, Memphis, TN), G-CSF (100 ng/mL; Amgen, Thousand Oaks, CA), GM-CSF (50 ng/mL; Genetics Institute, Cambridge, MA), and Epo (2 IU/mL; Boehringer Mannheim, Mannheim, Germany) were added to the cultures at optimal concentrations, which had been determined with normal bone marrow colony cultures.39,40 Cannabinoid ligands included anandamide,  -8-tetrahydrocannabinol (8-THC), WIN55212-2, Cannabinol, and Cannabidiol (Sigma) and CP 55,940 (Pfizer, Groton, CT). They were added at final concentrations between 0.1 and 10 µmol/L.
Tritiated thymidine (3H-TdR) incorporation.
DNA synthesis was measured essentially as described.38 Five thousand cells from various cell lines were cultured in 100 µL of serum-free medium, with or without the addition of HGFs or cannabinoid ligands in 96-well round-bottom microtiter trays (Greiner, Nurtingen, Germany) for 90 hours. Four hours before harvesting, 0.1 µCi tritiated thymidine (2 Ci/mmol 3H-TdR; Amersham International, Amersham, UK) was added. Cells were harvested on nitrocellulose using a filtermate 196 harvester (Packerd Instrument Co, Meriden, CT). Radioactivity was determined with a Topcount, microscintillation counter (Packerd).
Colony formation by normal mouse bone marrow.
Normal bone marrow cells isolated from femora and tibae from BCBA mice were collected in Hank's Balanced Salt Solution. Fifty thousand cells were cultured in 1 mL serum-free medium with 1.2% methylcellulose. Dishes were incubated with or without 10 ng/mL IL-3 in the presence or absence of 10 µmol/L anandamide at 37°C and 100% humidity and 5% CO2. Colonies containing 50 cells or more were scored at day 14.
| |
RESULTS |
Cb2 and Cb1 mRNA expression in murine tissues.
The expression of Cb2 and Cb1 transcripts in murine tissues was determined by RNAse protection (Fig 1B). Using Cb2 cDNA probe I (Fig 1A), an expected mRNA fragment of 185 bp was protected in spleen, thymus, and heart (Fig 1B). No Cb2 transcripts were identified in any of the other tissues examined. RNAse protection experiments using a 460-bp Cb1 probe (Fig 1A) demonstrated Cb1 transcripts in brain and to a lesser extent in testis (Fig 1B). No detectable levels of Cb1 mRNA were identified in any of the other organs investigated.
View larger version (17K):
[in this window]
[in a new window]
| Fig 1.
Cb2 and Cb1 mRNA expression in murine organ tissues. (A) Schematic representation of murine Cb2 and Cb1 mRNA (noncoding exon 1 and protein coding exon 2) and the cDNA probes used for RNAse protection experiments. The shaded boxes represent open reading frames. H, HincII; B, BamHI; S, Stu I; N, Nco I; (E), EcoRI in vector. (B) RNAse protection on 10 µg of total RNA of different mouse organs using Cb2 probe I and Cb1 probe I (see [A]). The protected fragments were 185 bp (Cb2) and 460 bp (Cb1). Sp, spleen; Th, thymus; H, heart; S, stomach; G, gut; K, kidney; B, bladder; L, liver; Br, brain; T, testis.
|
|
Cb2 and Cb1 mRNA expression in murine hematopoietic cell lines.
To examine in which hematopoietic lineages the Cb2 gene may be expressed, RNAse protection experiments were performed using mRNA samples from a large panel of murine hematopoietic cell lines (see the Materials and Methods). Using a 187-bp Cb2 cDNA fragment representing exon-1 (137 bp) and exon-2 (50 bp; Fig 1A), Cb2 transcripts of the correct size were identified in 26 of 29 myeloid, 6 of 7 macrophage, 5 of 5 B-lymphoid, 3 of 5 T-lymphoid, 2 of 2 mast cell, and 3 of 3 erythroid cell lines (Table 1). An example of an RNAse protection experiment is presented in Fig 2. Interestingly, a 460-bp Cb1 transcript was shown in a murine CTLL cell line (Fig 2 and Table 1). In the CTLL cell line, no Cb2 transcripts were identified (Fig 2). No detectable Cb1 mRNA levels were found in any of the other cell lines (Table 1 and Fig 2). No Cb1 or Cb2 transcripts were found in 3 myeloid, 1 macrophage, and 1 T-lymphoid cell lines. These results show that the peripheral cannabinoid receptor is a blood cell receptor that may be expressed in all hematopoietic lineages. The central cannabinoid receptor is only incidentally expressed in hematopoietic cell lines.
View larger version (18K):
[in this window]
[in a new window]
| Fig 2.
Cb2 and Cb1 mRNA expression in murine hematopoietic cell lines. RNAse protection on 10 µg of total RNA of different murine hematopoietic cell lines, using Cb2 probe II and Cb1 probe I (see Fig 1A). The hematopoietic phenotype of the cell lines is indicated at the top of the figure. M, mast cell; B, B lymphoid; My, myeloid; T, T lymphoid; M , macrophage.
|
|
Anandamide potentiates the proliferative response of myeloid cells to IL-3.
The effect of a natural ligand of cannabinoid receptors, anandamide, on the proliferation of an IL-3-dependent myeloid cell line 32Dcl3 was studied in vitro. When cultured with FCS, anandamide did not alter IL-3-induced proliferation of 32D cells (Fig 3A). However, in serum-free medium, anandamide significantly enhanced the proliferative response of 32D cells to IL-3 (Fig 3B). Anandamide as a single factor did not induce a measurable proliferative effect in 32Dcl3 cells. Tritiated thymidine incorporation (3H-TdR) showed that as little as 0.1 to 0.3 µmol/L anandamide was sufficient to augment DNA synthesis in synergy with IL-3 in 32Dcl3, NFS-60, and NFS-78 cells (Fig 4). The maximal stimulative effect was reached at a concentration of 0.3 to 3 µmol/L anandamide. 3H-TdR experiments with other IL-3-dependent myeloid cell lines, ie, DA-13, DA-28, DA-29, DA-31, NFS-36, NFS-56, and NFS-107, showed similar synergistic dose-response relationships between IL-3 and anandamide (data not shown).
View larger version (13K):
[in this window]
[in a new window]
| Fig 3.
The effect of anandamide on the proliferation of the myeloid cell line 32Dcl3. Growth curves of 32Dcl3 cells cultured in the presence of fetal calf serum (A) or serum-free (B). Cells were cultured with no stimulus ( ), with 10 µmol/L anandamide alone ( ), with 10 ng/mL IL-3 alone ( ), or with IL-3 plus anandamide ( ). Mean cell numbers (±1 × SD) of triplicate experiments are plotted against the number of days in culture. The doubling time in serum-free culture without anandamide was 75 hours and with anandamide was 29 hours. 3H-TdR incorporation data (cpm ±1 × SD) of 32Dcl3 cells cultured in serum containing medium (C) or in serum-free medium (SFM) (D).
|
|
View larger version (16K):
[in this window]
[in a new window]
| Fig 4.
The effects of different concentrations of anandamide on IL-3-induced 3H-TdR incorporation of three murine myeloid cell lines. Cells of 32Dcl3 (A), NFS-60 (B), and NFS-78 (C) were cultured with titrated concentrations of anandamide (0 to 10 µmol/L) in the presence (10 ng/mL;  ) or absence of IL-3 (). The mean values ±1 × SD (cpm × 10-3) of triplicate experiments are shown.
|
|
Anandamide enhances colony growth of normal bone marrow progenitors induced with IL-3.
Normal mouse bone marrow colony cultures were performed to investigate whether anandamide enhances IL-3-stimulated colony growth. In two independent experiments, a twofold elevation of colony formation was observed when the cells were cultured with IL-3 plus anandamide as compared with IL-3 alone (Table 2). Anandamide not only increased colony numbers, but also showed an effect on the size of the colonies (Fig 5). More colonies containing 250 cells or more were found in cultures with IL-3 plus anandamide. Mega-size colonies of 1,000 cells or more were found in IL-3 plus anandamide cultures, but none in colony cultures with IL-3 alone.
View larger version (13K):
[in this window]
[in a new window]
| Fig 5.
Effect of ananadamide on the size of IL-3-induced normal bone marrow colonies. Examples of representative IL-3-induced normal bone marrow colonies after 14 days of culture with (IL-3+AN) or without anandamide (IL-3).
|
|
Effects of different cannabinoid ligands on IL-3-induced proliferation of myeloid cells.
To investigate whether various synthetic molecules all capable of binding cannabinoid receptors would be capable of enhancing the proliferative effects of IL-3, 32Dcl3 cells were cultured with IL-3 plus the cannabinoid agonists CP-55,940, WIN 55212-2, 8-THC, Cannabinol, and Cannabidiol. Whereas DNA synthesis of 32Dcl3 cells was augmented significantly when costimulated with IL-3 and anandamide, no enhancement of thymidine uptake was apparent when 32Dcl3 cells were stimulated with IL-3 plus any of the other cannabinoid ligands (Fig 6).
View larger version (K):
[in this window]
[in a new window]
| Fig 6.
Dose effects of ananadamide and other cannabinoid ligands on IL-3-induced 3H-TdR incorporation of 32Dcl3 cells. Cells were cultured serum-free with IL-3 (10 ng/mL) and titrated concentrations (0 to 10 µmol/L) of anandamide (), WIN 55212-2 (), 8-THC (), Cannabinol ( ), Cannabidiol (), and CP55,940 ( ).
|
|
Anandamide enhances DNA synthesis of hematopoietic cell lines in synergy with GM-CSF, Epo, and G-CSF.
3H-TdR incorporation experiments were performed with three selected GM-CSF-responsive (NFS-36), Epo-responsive (32D-Epo), and G-CSF-responsive (BAF-G) cell lines to investigate whether anandamide would potentiate proliferation in synergy with hematopoietic growth factors other than IL-3. NFS-36 cells showed a weak response to GM-CSF alone or anandamide alone, but a significant increase of thymidine incorporation was evident when both ligands were added (Fig 7A). 32D cells cultured with Epo alone, ie, in the absence of anandamide, did not stimulate DNA synthesis at all. 32D-Epo cells became responsive to Epo in the presence of anandamide (Fig 7B). Finally, whereas G-CSF stimulated some DNA synthesis of BAF-G cells, ananadamide supplemented to the culture medium augmented the G-CSF effect by twofold (Fig 7C). These experiments indicate that anandamide may enhance the stimulative activity of various HGFs.
View larger version (K):
[in this window]
[in a new window]
| Fig 7.
Synergistic activity of anandamide on GM-CSF-, Epo-, or G-CSF-stimulated thymidine incorporation. Cells were cultured with optimal concentrations of GM-CSF (50 ng/mL; [A] NFS-36), Epo (2 IU/mL; [B] 32D-Epo), or G-CSF (100 ng/mL; [C] BAF-G) in the presence of 10 µmol/L anandamide (AN). The mean values ± 1 × SD (cpm × 10-3) of triplicate experiments are shown.
|
|
Effects of anandamide and other cannabinoid ligands on Cb2-overexpressing myeloid cells.
To verify whether the stimulatory effect of anandamide is mediated through activation of the peripheral cannabinoid receptor, we overexpressed Cb2 cDNA in 32D cells that express the G-CSF receptor (G-CSF-R), but do not respond to G-CSF.6 Cb2-transfected cells show high tritiated thymidine incorporation when cultured with G-CSF plus anandamide (Fig 8A). In comparison, anandamide alone or G-CSF alone did not induce DNA synthesis. Furthermore, anandamide plus G-CSF did not induce proliferation in control vector-transfected 32D (G-CSF-R) cells. These data indicate that anandamide stimulates DNA synthesis after the specific activation of Cb2. In addition, 3H-TdR incorporation experiments using various concentrations of the other cannabinoid ligands showed that DNA synthesis of 32D (G-CSF-R/Cb2) cells was not influenced by any of the other ligands (Fig 8B). Anandamide is apparently selectively capable of stimulating hematopoietic cells through activation of the peripheral cannabinoid receptor among several cannabinoid ligands.
View larger version (K):
[in this window]
[in a new window]
| Fig 8.
Effects of anandamide and other cannabinoid ligands on G-CSF-induced thymidine incorporation of Cb2-transfected 32D/G-CSF-R cells. (A) 32D/G-CSF-R were transfected with Cb-2 cDNA (32D/G-CSF-R/Cb2) or with pBabe control vector (32D/G-CSF-R/pB). Cells were cultured without G-CSF with different concentrations anandamide ([] 32D/G-CSF-R/pB; [] 32D/G-CSF-R/Cb2 ) or with G-CSF plus anandamide ([] 32D/G-CSF-R/pB; [] 32D/G-CSF-R/Cb2). (B) 32D/G-CSF-R-Cb2 cells were cultured with G-CSF plus different concentrations of the distinct cannabinoid ligands, anandamide (), WIN 55212-2 (), 8-THC (), Cannabinol (), Cannabidiol (), and CP55,940 ().
|
|
| |
DISCUSSION |
In this study, we show that the gene encoding the peripheral cannabinoid receptor is expressed in hematopoietic cells and appears to be important for the efficiency of stimulation of growth by a variety of HGFs.
Several studies had previously demonstrated the presence of cannabinoid binding sites on hematopoietic cells,41-43 but these studies had not distinguished between the central and the peripheral cannabinoid receptor. Cb2 expression was shown in spleen and in thymus. The Cb2 mRNA expression in heart tissue might be the result of residual blood that was not eliminated before extraction. Others demonstrated Cb2 mRNA expression in spleen, in the myeloid cell line HL60, and in mast cell lines.7,44 We show here that Cb2 may be expressed in myeloid, erythroid, B-lymphoid, T-lymphoid, macrophage, and mast cells. This finding indicates that Cb2 encodes a hematopoietic receptor that may have a function in a broad scale of hematopoietic lineages. By applying reverse transcriptase-polymerase chain reaction, several groups demonstrated Cb1 transcripts in hematopoietic cells.41,43,45,46 RNAse protection studies presented here show that Cb1 could be detected in brain and testis but not in spleen and thymus. Futhermore, Cb1 could be detected in 1 of 51 cell lines only. These data suggest that Cb1 may occasionally be expressed in hematopoietic cells, whereas Cb2 is commonly expressed. These results enforce the notion that Cb2 rather than Cb1 encodes a hematopoietic cannabinoid receptor.
In vitro studies with murine hematopoietic cell lines and normal bone marrow precursors showed that the peripheral cannabinoid receptor has a function in the regulation of proliferation by HGFs. Stimulatory effects of anandamide on hematopoietic cell proliferation had not been reported. Anandamide enhanced the cellular proliferation induced with IL-3, GM-CSF, G-CSF, and Epo when the cells were cultured in serum-free medium. Whether anandamide synergizes with other cytokines remains open to future investigation. Most of the cell lines analyzed for Cb2 mRNA expression in this study are HGF-independent and therefore did not allow for an extended analysis of the stimulative effects of anandamide with other HGFs. However, interestingly, when a panel of HGF independent cell lines were cultured in a serum-free medium, ie, DA-2 (T-lymphoid), DA-25 (B-lymphoid), RED-5 (erythroid), and J774 cells (macrophage), anandamide was required to induce proliferation in vitro (data not shown). This indicates that the Cb2 may have a role in stimulation of growth of several, if not all, hematopoietic lineages. An important modification of the culture conditions used in this study is the elimination of FCS and the use of a serum-free culture system. The effects of anandamide are not evident when cells are cultured with FCS and therefore other investigators may have missed the effects of anandamide stimulation. The results of this study would suggest that anandamide or another ligand for the cannabinoid receptor is present in FCS. In fact, four other fatty acids have recently been identified to bind and activate cannabinoid receptors.44,47,48 The role and presence of anandamide or any of these fatty acids may explain at least in part the serum dependence of various hematopoietic cell lines and primary hematopoietic cells. The in vitro experiments show that, among a selected number of cannabinoid ligands studied, only anandamide was capable of stimulating the proliferation of hematopoietic cells synergistically with HGFs. This is remarkable, because, according to other investigators, WIN 55212-2, CP55,940, and THC bind and activate the peripheral cannabinoid receptors more efficiently than anandamide.7,49,50 Our findings thus suggest strongly that, in hematopoietic cells, anandamide acts as the most potent agonist through the latter receptors. Up to 1 to 10 µmol/L anandamide was needed for optimal activation of proliferation, which is within the concentration range required for optimal binding.7,42,44,45,49 The transfection studies of Cb2 (Fig 8) confirm that anandamide specifically activates proliferation of 32D (G-CSF-R/Cb2) cells through activation of the peripheral cannabinoid receptor. In contrast, the other cannabinoid ligands fail to stimulate these Cb2-transfected cells. In fact, when cultured with serum, the other Cb ligands inhibit proliferation (data not shown). Because the cannabinoid agonists CP55,940 and THC have been shown to stimulate an additional nonreceptor-mediated signal transduction pathway,51 it is perhaps possible that the alternative nonreceptor-mediated signals suppress proliferation. In any case, the current available data would suggest that only anandamide is capable of stimulating proliferation synergistically with HGFs. Whether the other fatty acids are capable of stimulating proliferation will be investigated.
We have recently identified the Cb2 gene in a common virus integration site (Evi11)6 and Cb2 was suggested to be a proto-oncogene. Transfection of Cb2 in 32D (G-CSF-R) cells generated G-CSF-dependent cell lines that could be maintained serum free when cultured with anandamide.6 The studies presented here show that activation of the Cb2 receptor has a profound effect on the proliferation of cells. This adds further support to the notion that this receptor, when aberrantly expressed, may alter the proliferative response of hematopoietic cells and contribute to the development of leukemia. Cb2 transgenic animals are currently being studied to investigate whether abnormal Cb2 expression might contribute to the development of leukemia in vivo.
Cb2 receptor-mediated signal transduction by anandamide has been observed by others.49 How activated cannabinoid receptors may stimulate proliferation synergistically with HGFs is unresolved. The costimulatory effects may occur at different levels of HGF receptor signalling. Stimulation of the peripheral cannabinoid receptor may have the following effects: (1) transactivation of the HGF receptors; (2) potentiation of HGF receptor-mediated signal transduction, eg, JAK/STAT or the p21ras/MAP kinase pathways52-54; and (3) activation of signalling pathways that, in parallel with the HGF receptor signalling routes, enhances cell cycling. In fact, examples in support of each of those possibilities exist for seven other transmembrane receptors.55-62 Whether these alternative mechanisms are activated by Cb2 requires verification in future studies.
The results presented in Table 2 and Fig 5 show that the synergism between IL-3 and anandamide is also evident in normal bone marrow colony formation. A greater number of IL-3-dependent colonies were formed in the presence of anandamide. These results indicate that, in the presence of anandamide, IL-3 stimulates the outgrowth of additional populations of precursor cells. IL-3 plus anandamide also stimulated colonies of greater size, which would indicate that the combination of the two factors augments the production of progeny from individual precursor cells as well. Whether anandamide synergizes with other HGFs in the stimulation of normal marrow precursor cells is currently under investigation.
| |
FOOTNOTES |
Submitted October 25, 1996;
accepted April 14, 1997.
Supported by the Dutch Cancer foundation "Koningin Wilhelmina Fonds," the Netherlands Organisation for Scientific Research "NWO," and the Royal Dutch Academy of Sciences "KNAW."
Address reprint requests to Ruud Delwel, PhD, Erasmus University Rotterdam, Institute of Hematology, PO Box 1738, 3000 DR Rotterdam, The Netherlands.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hearly marked
``advertisment'' in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Dr J. Cleveland (St Jude Children's Hospital, Memphis, TN) for providing RNA samples, Dr T.I. Bonner (National Institute of Mental Health, Bethesda, MD) for donating the Cb1 cDNA, Dr I.P. Touw (Erasmus University Rotterdam, Rotterdam, The Netherlands) for donating BAF-G and 32D (G-CSF-R) cells, and K. van Rooyen for preparation of the figures.
| |
REFERENCES |
1.
Clark SC,
Kamen R:
The human hematopoietic colony stimulating factors.
Science
236:1229,
1987[Abstract/Free Full Text]
2.
Metcalf D:
The molecular control of cell division, differentiation commitment and maturation in haemopoietic cells.
Nature
339:27,
1989[Medline]
[Order article via Infotrieve]
3.
Bazan JF:
Structural design and molecular evolution of a cytokine receptor superfamily.
Proc Natl Acad Sci USA
87:6934,
1990[Abstract/Free Full Text]
4.
Gudermann T,
Nurnberg B,
Schultz G:
Receptors and G proteins as primary components of transmembrane signal transduction. Part 1. G-protein-coupled receptors: Structure and function.
J Mol Med
73:51,
1995[Medline]
[Order article via Infotrieve]
5.
Savarese TM,
Fraser CM:
In vitro mutagenesis and the search for structure-function relationships among G protein-coupled receptors.
Biochem J
283:1,
1992
6.
Delwel R,
Hol S,
Vankan Y,
Löwenberg B,
Ihle JN,
Copeland NG,
Valk PJM:
The cannabinoid receptor-2 is a potential oncogene in myeloid mouse leukemias.
Blood
88:1345,
1996
7.
Munro S,
Thomas KL,
Abu-Shaar M:
Molecular characterization of a peripheral receptor for cannabinoids.
Nature
365:61,
1993[Medline]
[Order article via Infotrieve]
8.
Matsuda LA,
Lolait SJ,
Brownstein MJ,
Young AC,
Bonner TI:
Structure of a cannabinoid receptor and functional expression of the cloned cDNA.
Nature
346:561,
1990[Medline]
[Order article via Infotrieve]
9.
Gerard CM,
Mollereau C,
Vassart G,
Parmentier M:
Molecular cloning of a human cannabinoid receptor which is also expressed in testis.
Biochem J
279:129,
1991
10.
Greenberger JS,
Sakaakeeny MA,
Humphries RK,
Eaves CJ,
Eckner RJ:
Demonstration of permanent factor-dependent multipotential (erythroid/neutrophil/basophil) hematopoietic progenitor cell lines.
Proc Natl Acad Sci USA
80:2931,
1983[Abstract/Free Full Text]
11.
Migliaccio G,
Migliaccio AR,
Kreider BL,
Rovera G,
Adamson JW:
Selection of lineage-restricted cell lines immortalized at different stages of hematopoietic differentiation from the murine cell line 32D.
J Cell Biol
109:833,
1989[Abstract/Free Full Text]
12.
Ihle JN,
Rein A,
Mural R:
Immunological and virological mechanisms in retroviral induced murine leukemogenesis.
Adv Viral Oncol
4:95,
1984
13.
Holmes KL,
Palaszinsky E,
Frederickson TN,
Morse HC III,
Ihle JN:
Correlation of cell-surface phenotype with the establishment of interleukin-3-dependent cell lines from wild-mouse murine leukemia virus-induced neoplasms.
Proc Natl Acad Sci USA
82:6687,
1985[Abstract/Free Full Text]
14.
Buchberg AM,
Bedigian HG,
Jenkins NA,
Copeland NC:
Evil2, a common integration site involved in murine myeloid leukemogenesis.
Mol Cell Biol
10:4658,
1990[Abstract/Free Full Text]
15.
Trakhtenbrot L,
Krauthgamer R,
Resnitzky P,
Haran-Ghera N:
Deletion of chromosome 2 is an early event in the development of radiation-induced myeloid leukemia in SJL/J mice.
Leukemia
2:545,
1988[Medline]
[Order article via Infotrieve]
16.
Azumi JI,
Sachs L:
Chromosome mapping of the genes that control differentiation and malignancy in myeloid leukemic cells.
Proc Natl Acad Sci USA
74:253,
1977[Abstract/Free Full Text]
17.
De Both NJ,
Hagenmeijer A,
Rhijnsburger EH,
Vermey M,
Van't Hul E,
Smit EME:
DMSO induced terminal differentiation and trisomi 15 in myeloid cell line transformed by the Rauscher murine leukemia virus.
Cell Differ
10:13,
1981[Medline]
[Order article via Infotrieve]
18.
Leenen PJM,
Jansen AMAC,
van Ewijk W:
Murine macrophage cell lines can be ordered in a linear differentiation sequence.
Differentiation
32:157,
1986[Medline]
[Order article via Infotrieve]
19.
Shen-Ong GLC,
Potter M,
Mushinsky JF,
Lavu S,
Reddy EP:
Activation of the c-myb locus by viral insertional mutagenesis in plasmacytoid lymphosarcomas.
Science
226:1077,
1984[Abstract/Free Full Text]
20.
Dudek H,
Reddy EP:
Murine myeloid leukemias with aberrant myb loci show heteroneous expression of novel myb proteins.
Oncogene
4:1489,
1989[Medline]
[Order article via Infotrieve]
21.
Pierce JH,
Di Fiore PP,
Aaronson SA,
Potter M,
Pumphrey J,
Scott A,
Ihle JN:
Neoplastic transformation of mast cells by Abelson-MuLV: Abrogation of IL-3 dependence by nonautocrine mechanism.
Cell
41:685,
1985[Medline]
[Order article via Infotrieve]
22.
Raschke WC,
Baird S,
Ralph P,
Nakoinz I:
Functional macrophage cell lines transformed by Abelson leukemia virus.
Cell
15:261,
1978[Medline]
[Order article via Infotrieve]
23.
Ralph P,
Prichard J,
Cohn M:
Reticulum cell sarcoma: An effector cell in antibody-dependent cell mediated immunity.
J Immunol
114:898,
1975[Abstract/Free Full Text]
24.
Ralph P,
Ho M-K,
Litcofsky PB,
Springer TA:
Expression and induction in vitro of macrophage differentiation antigens on murine cell lines.
J Immunol
130:108,
1983[Medline]
[Order article via Infotrieve]
25.
Warner NL,
Moore MAS,
Metcalf D:
A transplantable myelomonocytic leukemia in BALB/c mice: Cytology, karyotype and muramidase content.
J Natl Cancer Inst
43:963,
1969
26.
Hagemeijer A,
Smit EME,
Govers F,
de Both NJ:
Trisomy 15 and other nonrandom chromosome changes in Rauscher murine leukemia virus-induced leukemia cell lines.
J Natl Cancer Inst
69:945,
1982
27.
Lanier LL,
Warner NL:
Cell cycle related heterogeneity of Ia antigen expression on a murine B lymphoma cell line: Analysis by flow cytometry.
J Immunol
126:626,
1981[Abstract]
28.
Warner NL,
Daley MJ,
Richey J,
Spellman C:
Flow cytometry analysis of murine B cell lymphoma differentiation.
Immunol Rev
48:197,
1979[Medline]
[Order article via Infotrieve]
29.
Palacios R,
Steinmetz M:
IL-3 dependent mouse clones that express B-220 surface antigen, contain Ig genes in germline configuration, and generate B-lymphocytes in vivo.
Cell
41:727,
1985[Medline]
[Order article via Infotrieve]
30.
Decleve A,
Lieberman M,
Ihle JN,
Rosenthal NPN,
Lung ML,
Kaplan HS:
Physiochemical, biological and serological properties of a leukemogeneic virus isolated from cultured RadLV-induced lymphomas of C57BL/Ka mice.
Virology
90:23,
1978[Medline]
[Order article via Infotrieve]
31.
Harris AW,
Bankhurst AD,
Mason S,
Warner NL:
Differentiated functions expressed by cultured mouse lymphoma cells. II. Theta antigen, surface immunoglobulin and a receptor for antibody on cells of a thymoma cell line.
J Immunol
110:431,
1973[Abstract/Free Full Text]
32.
Ichikawa Y:
Differentiation of a cell line of myeloid leukemia.
J Cell Physiol
74:223,
1969[Medline]
[Order article via Infotrieve]
33.
Koren HS,
Handwerger BS,
Wunderlich JR:
Identification of macrophage-like characteristics in a cultured murine tumor line.
J Immunol
114:894,
1975[Abstract/Free Full Text]
34.
Shevach EM,
Stobo JD,
Green I:
Immunoglobin and theta-bearing murine leukemias and lymphomas.
J Immunol
108:1146,
1972[Abstract/Free Full Text]
35.
Gillis S,
Smith KA:
Long term culture of tumour-specific cytotoxic T cells.
Nature
268:154,
1977[Medline]
[Order article via Infotrieve]
36.
Dong F,
Van Buitenen C,
Pouwels K,
Hoefsloot LH,
Löwenberg B,
Touw IP:
Distinct cytoplasmic regions of the human granulocyte colony-stimulating factor receptor involved in induction of proliferation and maturation.
Mol Cell Biol
13:7774,
1993[Abstract/Free Full Text]
37. Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual (ed 2). Cold Spring Harbor, NY, Cold Spring Harbor Laboratory, 1989
38.
Salem M,
Delwel R,
Touw I,
Mahmoud L,
Löwenberg B:
Human AML colony growth in serum-free culture.
Leuk Res
12:157,
1988[Medline]
[Order article via Infotrieve]
39.
Ploemacher RE,
van Soest PL,
Boudewijn A:
Autocrine transforming growth factor 1 blocks colony formation and progenitor cell generation by hematopoietic cells stimulated with steel factor.
Stem Cells
11:336,
1993[Medline]
[Order article via Infotrieve]
40.
Ploemacher RE,
van Soest PL,
Voorwinden H,
Boudewijn A:
Interleukin-12 synergizes with interleukin-3 and steel factor to enhance recovery of murine hemopoietic stem cells in liquid culture.
Leukemia
7:1381,
1993[Medline]
[Order article via Infotrieve]
41.
Kaminsky NE,
Abood AE,
Kessler FK,
Martin BR,
Schatz AR:
Identification of a functional relevant cannabinoid receptor on mouse spleen cells that is involved in cannabinoid-mediated immune modulation.
Mol Pharmacol
42:736,
1992[Abstract]
42.
Lynn AB,
Herkenham M:
Localization of cannabinoid receptors and nonsaturable high density cannabinoid binding sites in peripheral tissues of the rat: Implications for receptor mediated immune modulation by cannabinoids.
J Pharmacol Exp Ther
268:1612,
1994[Abstract/Free Full Text]
43.
Bouaboula M,
Rinaldi M,
Carayon P,
Carillon C,
Delpech B,
Shire D,
Le Fur G,
Casellas P:
Cannabinoid-receptor expression in human leukocytes.
Eur J Biochem
214:173,
1993[Medline]
[Order article via Infotrieve]
44.
Facci. L,
Dal Toso R,
Buriani A,
Skaper SD,
Leon A:
Mast cells express a peripheral cannabinoid receptor with differential sensitivity to anandamide and palmitoylethanolamide.
Proc Natl Acad Sci USA
92:3376,
1995[Abstract/Free Full Text]
45.
Schwarz H,
Blanco FJ,
Lotz M:
Anandamide, an endogenous cannabinoid receptor agonist inhibits lymphocyte proliferation and induces apoptosis.
J Neuroimmunol
55:107,
1994[Medline]
[Order article via Infotrieve]
46. Daaka Y, Klein TW, Friedman H: Expression of cannabinoid receptor mRNA in murine and human leukocytes, in Sharp B (ed): The Brain Immune Axis and Substance Abuse, Vol 13. New York, NY, Plenum, 1995, p 91
47.
Barg J,
Fride E,
Hanus L,
Levy R,
Matus-Leibovitch N,
Heldman E,
Bayewitch M,
Mechoulam R,
Vogel Z:
Cannabinomimetic behavioral effects of and adenylate cyclase inhibition by two new edogenous anandamides.
Eur J Pharmacol
287:145,
1995[Medline]
[Order article via Infotrieve]
48.
Lee M,
Yang KH,
Kaminski N:
Effects of putative cannabinoid receptor ligands, anandamide and 2-arachidonyl-glycerol, on immune function in B6C3F1 mouse splenocytes.
J Pharmacol Exp Ther
275:529,
1995[Abstract/Free Full Text]
49.
Shire D,
Calandra B,
Rinaldi-Carmona M,
Oustric D,
Pessegue B,
Bonnin-Cabanne O,
Le Fur G,
Caput D,
Ferrara P:
Molecular cloning, expression and function of the murine Cb2 peripheral cannabinoid receptor.
Biochem Biophys Acta
1307:132,
1996[Medline]
[Order article via Infotrieve]
50.
Slipetz DM,
O'Neal GP,
Favreau L,
Dufresne C,
Gallant M,
Gareau Y,
Guay D,
Labelle M,
Metters KM:
Activation of the human peripheral cannabinoid receptor results in inhibition of adenylylcyclase.
Mol Pharmacol
48:352,
1995[Abstract]
51.
Felder CC,
Veluz JS,
Williams HL,
Briley EM,
Matsuda LA:
Cannabinoid agonists stimulate both receptor- and nonreceptor-mediated signal transduction pathways in cells transfected with and expressing cannabinoid receptor clones.
Mol Pharmacol
42:838,
1992[Abstract]
52.
Nicholson SE,
Oates AC,
Harpur AG,
Ziemiecki A,
Wilks AF,
Layton JE:
Tyrosin kinase Jak1 is associated with the granulocyte-colony-stimulating factor receptor and both become tyrosine phosphorylated after receptor activation.
Proc Natl Acad Sci USA
91:2985,
1994[Abstract/Free Full Text]
53.
Tian SS,
Lamb P,
Seidel AM,
Stein RB,
Rosen J:
Rapid activation of the STAT3 transcription factor by granulocyte colony stimulating factor.
Blood
84:1760,
1994[Abstract/Free Full Text]
54.
Bashey A,
Healy L,
Marshall CJ:
Proliferative but not nonproliferative responses to granulocyte colony-stimulating factor are associated with activation of the p21ras/MAP kinase signalling pathway.
Blood
83:949,
1994[Abstract/Free Full Text]
55.
Daub H,
Weiss FU,
Wallasch C,
Ullrich A:
Role of transactivation of the receptor in signalling by G-protein-coupled receptors.
Nature
379:557,
1996[Medline]
[Order article via Infotrieve]
56.
Marrero MB,
Schieffer B,
Paxton WG,
Heerdt L,
Berk BC,
Delafontaine P,
Bernstein KE:
Direct stimulation of Jak/STAT pathway by the angiotensin II AT1 receptor.
Nature
375:247,
1995[Medline]
[Order article via Infotrieve]
57.
Wartmann M,
Campbell D,
Subramanian A,
Burstein SH,
Davis RJ:
The MAP kinase signal transduction pathway is activated by the endogenous cannabinoid anandamide.
FEBS Lett
359:133,
1995[Medline]
[Order article via Infotrieve]
58.
Bouaboula M,
Poinot-Chazel C,
Bourrie B,
Canat X,
Calandra B,
Rinaldi-Carmona M,
Le Fur G,
Casellas P:
Activation of mitogen activated protein kinase by stimulation of the central cannabinoid receptor Cb1.
Biochem J
312:637,
1995
59.
Bouaboula M,
Bourrie B,
Rinaldi-Carmona M,
Shire D,
Le Fur G,
Casellas P:
Stimulation of cannabinoid receptor Cb1 induces Krox-24 expression in human Astroma cells.
J Biol Chem
270:13973,
1995[Abstract/Free Full Text]
60.
Bouaboula M,
Poinot-Chazel C,
Marchand J,
Canat X,
Bourrie B,
Rinaldi-Carmona M,
Calandra B,
Le Fur G,
Casellas P:
Signalling pathway associated with stimulation of Cb2 peripheral cannabinoid receptor. Involvement of both mitogen-activated kinase and induction of Krox-24 expression.
Eur J Biochem
237:704,
1996[Medline]
[Order article via Infotrieve]
61.
Kato J,
Matsuoka M,
Polyak K,
Massague J,
Sherr CJ:
Cyclic AMP induced G1 phase arrest mediated by an inhibitor (p27kip1) of cyclin dependent kinase 4 activation.
Cell
79:487,
1994[Medline]
[Order article via Infotrieve]
62.
Van Biesen T,
Hawes BE,
Luttrell DK,
Krueger KM,
Touhara K,
Porfiri E,
Sakaue M,
Luttrell LM,
Lefkowitz RJ:
Receptor-tyrosine-kinase- and G-mediated MAP kinase activation by a common signalling pathway.
Nature
376:781,
1995[Medline]
[Order article via Infotrieve]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
A. M. Boehmler, A. Drost, L. Jaggy, G. Seitz, T. Wiesner, C. Denzlinger, L. Kanz, and R. Mohle
The CysLT1 Ligand Leukotriene D4 Supports {alpha}4{beta}1- and {alpha}5{beta}1-Mediated Adhesion and Proliferation of CD34+ Hematopoietic Progenitor Cells
J. Immunol.,
June 1, 2009;
182(11):
6789 - 6798.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
W. Gonsiorek, D. Hesk, S.-C. Chen, D. Kinsley, J. S. Fine, J. V. Jackson, L. A. Bober, G. Deno, H. Bian, J. Fossetta, et al.
Characterization of Peripheral Human Cannabinoid Receptor (hCB2) Expression and Pharmacology Using a Novel Radioligand, [35S]Sch225336
J. Biol. Chem.,
September 22, 2006;
281(38):
28143 - 28151.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. V. Siegmund, E. Seki, Y. Osawa, H. Uchinami, B. F. Cravatt, and R. F. Schwabe
Fatty Acid Amide Hydrolase Determines Anandamide-induced Cell Death in the Liver
J. Biol. Chem.,
April 14, 2006;
281(15):
10431 - 10438.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
O. Ofek, M. Karsak, N. Leclerc, M. Fogel, B. Frenkel, K. Wright, J. Tam, M. Attar-Namdar, V. Kram, E. Shohami, et al.
Peripheral cannabinoid receptor, CB2, regulates bone mass
PNAS,
January 17, 2006;
103(3):
696 - 701.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. G. Nackley, A. M. Zvonok, A. Makriyannis, and A. G. Hohmann
Activation of Cannabinoid CB2 Receptors Suppresses C-Fiber Responses and Windup in Spinal Wide Dynamic Range Neurons in the Absence and Presence of Inflammation
J Neurophysiol,
December 1, 2004;
92(6):
3562 - 3574.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. J. Carrier, C. S. Kearn, A. J. Barkmeier, N. M. Breese, W. Yang, K. Nithipatikom, S. L. Pfister, W. B. Campbell, and C. J. Hillard
Cultured Rat Microglial Cells Synthesize the Endocannabinoid 2-Arachidonylglycerol, Which Increases Proliferation via a CB2 Receptor-Dependent Mechanism
Mol. Pharmacol.,
April 1, 2004;
65(4):
999 - 1007.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
S. Hart, O. M. Fischer, and A. Ullrich
Cannabinoids Induce Cancer Cell Proliferation via Tumor Necrosis Factor {alpha}-Converting Enzyme (TACE/ADAM17)-Mediated Transactivation of the Epidermal Growth Factor Receptor
Cancer Res.,
March 15, 2004;
64(6):
1943 - 1950.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M.-T. Samson, A. Small-Howard, L. M. N. Shimoda, M. Koblan-Huberson, A. J. Stokes, and H. Turner
Differential Roles of CB1 and CB2 Cannabinoid Receptors in Mast Cells
J. Immunol.,
May 15, 2003;
170(10):
4953 - 4962.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Orelio and E. Dzierzak
Identification of 2 novel genes developmentally regulated in the mouse aorta-gonad-mesonephros region
Blood,
March 15, 2003;
101(6):
2246 - 2249.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. C. Howlett, F. Barth, T. I. Bonner, G. Cabral, P. Casellas, W. A. Devane, C. C. Felder, M. Herkenham, K. Mackie, B. R. Martin, et al.
International Union of Pharmacology. XXVII. Classification of Cannabinoid Receptors
Pharmacol. Rev.,
June 1, 2002;
54(2):
161 - 202.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Alberich Jorda, S. E. Verbakel, P. J. M. Valk, Y. V. Vankan-Berkhoudt, M. Maccarrone, A. Finazzi-Agro, B. Lowenberg, and R. Delwel
Hematopoietic cells expressing the peripheral cannabinoid receptor migrate in response to the endocannabinoid 2-arachidonoylglycerol
Blood,
April 15, 2002;
99(8):
2786 - 2793.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. W. Klein, B. Lane, C. A. Newton, and H. Friedman
The Cannabinoid System and Cytokine Network
Experimental Biology and Medicine,
October 1, 2000;
225(1):
1 - 8.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
W. Gonsiorek, C. Lunn, X. Fan, S. Narula, D. Lundell, and R. W. Hipkin
Endocannabinoid 2-Arachidonyl Glycerol Is a Full Agonist through Human Type 2 Cannabinoid Receptor: Antagonism by Anandamide
Mol. Pharmacol.,
May 1, 2000;
57(5):
1045 - 1050.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
P. J. M. Valk, Y. Vankan, M. Joosten, N. A. Jenkins, N. G. Copeland, B. Löwenberg, and R. Delwel
Retroviral Insertions in Evi12, a Novel Common Virus Integration Site Upstream of Tra1/Grp94, Frequently Coincide with Insertions in the Gene Encoding the Peripheral Cannabinoid Receptor Cnr2
J. Virol.,
May 1, 1999;
73(5):
3595 - 3602.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
M. Maccarrone, M. van der Stelt, A. Rossi, G. A. Veldink, J. F. G. Vliegenthart, and A. F. Agro
Anandamide Hydrolysis by Human Cells in Culture and Brain
J. Biol. Chem.,
November 27, 1998;
273(48):
32332 - 32339.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
Abstract
Introduction
Abstract