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The Journal of Immunology, 2006, 176: 7051-7061.
Copyright © 2006 by The American Association of Immunologists

Leukotriene A4 Hydrolase Expression in PEL Cells Is Regulated at the Transcriptional Level and Leads to Increased Leukotriene B4 Production1

Meztli Arguello*,{dagger}, Suzanne Paz*,{dagger}, Eduardo Hernandez*, Catherine Corriveau-Bourque*,{dagger}, Lama M. Fawaz{ddagger},§, John Hiscott2,*,{dagger},{ddagger} and Rongtuan Lin2,*,{ddagger}

* Terry Fox Molecular Oncology Group, Lady Davis Institute for Medical Research, Montreal, Quebec, Canada; {dagger} Department of Microbiology and Immunology, {ddagger} Department of Medicine, and § Meakins-Christie Laboratories, McGill University, Montreal, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Primary effusion lymphoma (PEL) is a herpesvirus-8-associated lymphoproliferative disease characterized by migration of tumor cells to serous body cavities. PEL cells originate from postgerminal center B cells and share a remarkable alteration in B cell transcription factor expression and/or activation with classical Hodgkin’s disease cells. Comparative analysis of gene expression by cDNA microarray of BCBL-1 cells (PEL), L-428 (classical Hodgkin’s disease), and BJAB cells revealed a subset of genes that were differentially expressed in BCBL-1 cells. Among these, four genes involved in cell migration and chemotaxis were strongly up-regulated in PEL cells: leukotriene A4 (LTA4) hydrolase (LTA4H), IL-16, thrombospondin-1 (TSP-1), and selectin-P ligand (PSGL-1). Up-regulation of LTA4H was investigated at the transcriptional level. Full-length LTA4H promoter exhibited 50% higher activity in BCBL-1 cells than in BJAB or L-428 cells. Deletion analysis of the LTA4H promoter revealed a positive cis-regulatory element active only in BCBL-1 cells in the promoter proximal region located between –76 and –40 bp. Formation of a specific DNA-protein complex in this region was confirmed by EMSA. Coculture of ionophore-stimulated primary neutrophils with BCBL-1 cells leads to an increased production of LTB4 compared with coculture with BJAB and L-428 cells as measured by enzyme immunoassay, demonstrating the functional significance of LTA4H up-regulation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Human herpesvirus-8 (HHV-8)3 is the causative agent of Kaposi’s sarcoma (KS) (1, 2) and is strongly associated with the development of two B lymphoproliferative diseases: primary effusion lymphoma (PEL) (3) and multicentric Castelman’s disease (4). PEL is an aggressive non-Hodgkin’s lymphoma originally identified in AIDS patients, with a few reported cases of the disease in HIV-1-negative elderly patients (5, 6), reminiscent of the two classes of Kaposi’s sarcoma: AIDS-related and classic. At the clinical level, PEL is characterized by lymphomatous effusions in the serous body cavities in the absence of a solid tumor mass (7, 8). The cancerous cells are of B cell origin, with Ig gene rearrangement in all cases and somatic hypermutation in most cases. However, PEL cells lack expression of most B cell surface Ags, notably the BCR components surface Ig, CD79 a and b, as well as CD19, CD20, CD21, etc. (3, 7, 9). The plasma cell markers CD30 and CD138 (syndecan-1) are expressed in most cases (10), which along with high expression of IRF-4 (11) and the absence of BCL-6 gene expression (12, 13) has led to their classification as postgerminal center (GC), preterminally differentiated B cells with a partial plasma cell phenotype (10, 11, 14), although a GC origin is possible in some cases (15). This differentiation state closely resembles that of classical Hodgkin’s disease (cHD), another lymphoma derived from late- or post-GC B lymphocytes (reviewed in Refs. 16 and 17). The tumor cells in cHD, known as Reed-Sternberg cells, also lack B cell surface marker expression and share with PEL a peculiar ablation in the expression of B cell-specific transcription factors (18) that may explain the null phenotype in both cases.

Investigation of these two B cell lymphomas has revealed a strikingly similar pattern of B cell-specific transcription factor ablation, including PU.1, Oct-2, Bob-1/OCA-B, BSAP/Pax-5, and IRF-8 (18, 19, 20), as well as constitutive activation of the transcription factors NF-{kappa}B (21, 22) and AP-1 (23, 24). However, the similarities at the molecular level between the two diseases do not translate to the clinical level, where their presentation is remarkably different. cHD is confined to the lymph nodes, without involvement of additional organs until the very late stages of the disease (<www.nlm.nih.gov>). PEL is confined to the serous body cavities without lymph node involvement (reviewed in Ref. 8). In classical Hodgkin’s disease, the cancerous Reed-Sternberg cells constitute <5% of the total tumor load, the remaining cells representing infiltrating inflammatory leukocytes. In PEL, >90% of the lymphomatous effusion is constituted by the malignant B cells, although PEL detection usually happens in the advanced stages of the disease (7). At the level of treatment, cHD responds extremely well to conventional chemotherapy, with >80% survival rate (reviewed in Ref. 25). In contrast, chemotherapy of PEL has been largely unsuccessful, and median survival does not exceed 6 mo (7, 8). This divergence at the clinical level between PEL and cHD likely reflects molecular differences that remain uncharacterized.

The establishment of a chronic inflammatory state is emerging as a key player in cancer development (26, 27, 28, 29). The inflammatory process involves the complex interplay of different cell types that express biological proinflammatory agents such as cytokines, chemokines, and lipid mediators. Leukotrienes are part of the family of lipid mediators derived from arachidonic acid known as eicosanoids. Leukotrienes are found at high levels in most inflammatory lesions and contribute to physiological changes characteristic of the inflammatory process (30). In particular, leukotriene B4 (LTB4), first described (31) as a potent chemoattractant of neutrophils, mediates leukocyte migration to the site of inflammation (32). LTB4 acts not only on neutrophils and eosinophils but also on monocytes (32), macrophages (32), T cells (33, 34), and endothelial cells (35) to promote transmigration, activation, and/or effector functions; LTB4 can also induce B cell activation, proliferation (36), and Ab secretion (37).

Inflammation has been shown to play an important role in the development of HHV-8 induced KS (reviewed in Ref. 38); however, the role of cellular proinflammatory factors in the development of HHV-8-associated PEL is less clear. Increased levels of the Th2 cytokines IL-6 and IL-10 are detectable in PEL samples and cell lines (39, 40). IL-6 is a potent proinflammatory cytokine that also acts as a B cell survival and proliferation factor. However, although HHV-8-encoded viral IL-6 has been shown to act as an autocrine growth factor for PEL cells (40), the role of cellular IL-6 remains controversial (39, 40). Despite recent advances, few other cellular proinflammatory factors have been associated with PEL, and, in particular, the involvement of lipid mediators such as leukotrienes has not been investigated. In this study, we performed a direct comparison of the gene expression pattern of PEL vs cHD cell lines by cDNA microarray, RT-PCR, and immunoblot to identify differentially expressed genes that may constitute novel PEL signature genes. Four factors involved in inflammation and cell migration were prominent: leukotriene A4 hydrolase (LTA4H), TSP-1, IL-16, and PSGL-1. Promoter analysis of the LTA4H gene identified a transcriptional regulatory element that confers high basal activity to the LTA4H promoter in BCBL-1 cells, providing insight into the transcriptional regulation of this gene in PEL. Increased LTA4H expression led to higher LTB4 production and, combined with expression of both LTB4 receptors by PEL cells, suggests a role for this potent leukotriene in PEL pathogenesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell lines

BCBL-1, a primary effusion lymphoma cell line infected with HHV-8 but negative for EBV or HIV-1, and BJAB, an EBV– Burkitt’s lymphoma (BL) cell line, were a gift from Dr. J. U. Jung (Harvard Medical School, Southborough, MA). Both cell lines were cultured in RPMI 1640 (Wisent) supplemented with 10% FBS, 10–5 M 2-ME, and 8 µg/ml gentamicin. BC-3 (ATCC no. CRL-2277) and BCP-1 (ATCC no. CRL-2294) are PEL cell lines positive for HHV-8 but negative for EBV and HIV-1. They were maintained in RPMI 1640 supplemented with 20% FBS and 8 µg/ml gentamicin. CRO-AP6 is an HHV-8+, HIV+, and EBV– PEL cell line (a gift from Dr. A. Carbone, Centro di Referimento Oncologico, Istituti di Ricovero e Cura a Carattere Scientifico, Rome, Italy). KM-H2 and L-428 are two well-characterized Hodgkin’s lymphoma cell lines of B cell origin (a gift from Dr. S. Smola-Hess, Institute of Virology, University of Cologne, Cologne, Germany). They were cultured in RPMI 1640 supplemented with 10% FBS, 1% sodium pyruvate, and 8 µg/ml gentamicin.

B cell purification

Fresh B lymphocytes were isolated from human tonsils discarded following surgery. The tonsils were thoroughly minced, resuspended in wash medium consisting of RPMI 1640 (Invitrogen Life Technologies) supplemented with 2% FCS (HyClone Laboratories), 50 U/ml penicillin, 50 µg/ml streptomycin, and amphotericin B (1/500 w/v) from Invitrogen Life Technologies; and then layered onto a Ficoll-Paque (Pharmacia Biotech) gradient. Tonsil lymphocytes were separated by rosetting with neuraminidase-treated sheep RBC and Ficoll-Paque density centrifugation. Monocytes were removed from the E-rosette-negative fraction by adherence depletion; the remaining B cells were routinely demonstrated to be >98% pure on flow cytometry by CD19 staining, with <1% CD14+ and <1% CD3+.

RNA isolation and microarray analysis

Total RNA was extracted from BCBL-1, L-428, and BJAB cells using the TRIzol method as per the manufacturer’s instructions (Invitrogen Life Technologies). Concentrations were calculated using the OD260 for each sample and RNA quality was determined using the Agilent 2100 Bioanalyzer (Agilent). RNA from BCBL-1 cells was compared against that of L-428 or BJAB cells. Ten micrograms of RNA from each cell line was reverse transcribed, labeled with the appropriate fluorochrome (indocarbocyanine or indodicarbocyanine; PerkinElmer), and hybridized to a human 1.7K chip (versions 4 and 8; University Health Network Microarray Center, Toronto, Canada), a double-spotted array containing 1718 well-characterized human expressed sequence tags. Two independent experiments comprising three hybridization with corresponding reverse labeling were conducted. Data acquisition was performed using GenePix Pro (4000B; Axon Instruments) at photomultiplier tube gain between 600 and 700. Flagging parameters were set to reject spots with (F532 mean—B532 and F635—B635) intensity values lower than 200. Data analysis was performed using Iobion Gene Traffic software (version 3.0; Iobion) for a two-class experiment. Data were normalized with the Lowess (subgrid) method with background subtraction. Statistical analysis for microarray was applied on spot tables with a p value cutoff of 0.05 and a differential expression cutoff of 1.5-fold and significant, differentially expressed genes in each class were selected. Microarray data have been deposited in NCBI Gene Expression Omnibus (GEO, <http://www.ncbi.nlm.nih.gov/geo> and are accessible through GEO series accession number GSE4464.

RT-PCR analysis

Five micrograms of total RNA isolated using the TRIzol method from the PEL cell lines BCBL-1, BC-3, BCP-1, and CRO-AP6, the cHD cell lines L-428 and KM-H2, as well as BJAB cells was subjected to reverse transcription using SuperScript II/RNaseH– reverse transcriptase (Invitrogen Life Technologies), according to the manufacturer’s instructions. Five microliters of the obtained cDNA was amplified by PCR using Taq polymerase (Amersham Biosciences), as per the manufacturer’s instructions. The following primers were used for amplification: LTA4H (product size: 204 bp), forward: 5'-CCC TAA AGA ACT GGT GGC ACT -3', reverse: 5'-GAC TTT TCC ACC TGC TCT TTC -3'; IL-16 (product size: 333 bp), forward: 5'-AAG GGG CAT CTC CAA CAT CAT CAT-3', reverse: 5'-CTC CTG CCA AGC TGA ACC CAA GAC-3'; TSP-1 (product size: 493 bp), forward: 5'-ACC GCA TTC CAG AGT CTG GC-3', reverse: 5'-ATG GGG ACG TCC AAC TCA GC-3'; BLT-1 (product size: 239 bp), forward: 5'-CAC TGC TCC CTT TTT CCT TCA-3', reverse: 5'-CCA GCA GAA AGG ACA ACA CC -3'; BLT-2 (product size: 173 bp), forward: 5'-ATC ACC CTG CCA GTC TTT TG-3', reverse: 5'-TAA GGG CTT GGG TAC AGG TG-5'; and GAPDH (product size: 376 bp), forward: 5'-CCA TGG AGA AGG CTG GGG-3', reverse: 5'-CAA AGT TGT CAT GGA TGA CC-3'. PCR conditions were as follows for all primers except TSP-1: 94°C for 2 min, cycles: 94°C for 45 s, 56°C for 45 s, 72°C for 45 s, repeat 20–30 times depending on optimal product detection, 72°C for 10 min. For TSP-1 (41), conditions were: 94°C for 2 min, cycles: 94°C for 1 min, 60°C for 1 min, 72°C for 2 min, repeat 28 times, 72°C for 10 min. PRC samples were resolved in a 2% agarose gel and visualized by ethidium bromide staining under UV light.

LTA4H promoter isolation and plasmid constructions

The LTA4H promoter region was isolated from BJAB genomic DNA using these primers: forward: 5'-CTT TCT CAA TGC TGC ATT CCT C-3' and reverse: 5'-TAC CAG ACT CGT CGA TAG AG-3'. The amplified 2.2-kb fragment was purified and subcloned by blunt-end ligation into the SmaI site of pGL3basic-modified (new NcoI site introduced upstream of the multiple cloning site, missing original fragment spanning from HindIII to NcoI restriction sites) to create LTA4HPRO. The presence of the insert in the correct orientation was verified by analytical digestion with HindIII/XhoI, which excised a 1.3-kb fragment. Deletion fragments S1 (–1702 to +105), S2 (–1196 to +105), and S4 (–123 to +105) were amplified using the following forward primers: 5'-ATT CTG GTGTTC TCT CAG C-3', 5'-CCT ACC TGG AAG CAT ACT GG-3', 5'-TCA GCT CCA GGA GCA CGC TTG G-3', respectively, and blunt-end cloned into the SmaI site of pGL3basic to create S1/pGL3, S2/pGL3, and S4/pGL3. S6/pGL3 (–40 to +105) was constructed by digesting LTA4HPRO with NheI and NcoI followed by Klenow treatment and blunt-end relegation of the plasmid.

Transient transfections and reporter gene assay

Electroporation of BJAB, BCBL-1, and L-428 cells was performed at 950 µF and 250 V. Luciferase assays using full-length LTA4HPRO and deletion constructs was performed following transient transfection of 10 µg of the reporter gene and 1.0 µg of the Renilla internal control (pRL-null) into 10 x 106 cells. The luciferase assay was performed 48 h posttransfection using the Dual Luciferase Reporter Assay System (Promega) as per the manufacturer’s instructions. Transfection efficiency was normalized using the Renilla activity counts. Inhibition of NF-{kappa}B and AP-1 with the chemical inhibitors aspirin (acetylsalicylic acid, 10 mM) or ibuprofen (1 mM) were conducted as follows: 10 x 106 BCBL-1 cells were electroporated with 10 µg of LTA4HPRO and incubated for 42 h, then the appropriate inhibitor or control vehicle (100 mM Tris, pH 7.5) was added and incubated for an additional 6 h before harvesting samples and assaying for luciferase activity.

Whole-cell extract preparation and immunoblot analysis

Whole-cell extracts and immunoblot analysis were performed using standard protocols as described previously (42) ). Briefly, WCE (50–100 µg) prepared using Nonidet P-40 lysis buffer were subjected to electrophoresis on 7.5–12% SDS-PAGE gels. Proteins transferred to Trans-blot nitrocellulose membrane (Bio-Rad) were blocked in TBS containing 5% nonfat milk and 0.05% Tween 20. After washes, primary Ab incubations were conducted for 1–2 h at room temperature. Blots were incubated with an appropriate secondary Ab coupled to HRP (1/5000 dilution, 1 h at room temperature) and visualized using NEN Life Science products. The following primary Abs were used in this study: anti-LTA4H (rabbit polyclonal, a gift from Dr. J. Evans, Merck & Co., Rahway, NJ), was used at a 1/5000 dilution, anti-human Il-16 (goat polyclonal, catalog no. AF-316-PB; R&D Systems) was used at 0.2 µg/ml, anti-thrombospondin-1 (mouse monoclonal, catalog no. MS-419-PO; Medicorp) was used at 1 µg/ml, anti-PSGL-1 (mouse monoclonal, catalog no. sc-13535; Santa Cruz Biotechnology) was used at 1 µg/ml; all dilutions were prepared in blocking solution. Anti-cleaved caspase 3 Ab (rabbit polyclonal; Cell Signaling Technology) was diluted 1/2000 in 5% BSA/TBST. Anti-beta-actin mouse mAb was purchased from Sigma-Aldrich and used at a 1/10,000 dilution.

EMSA

BJAB, BCBL-1, and L-428 nuclear extracts were extracted as described elsewhere (43). S4 and S6 EMSA probes were generated by restriction digestion of S4/pGL3 and S6/pGL3 plasmids with KpnI and BglII. The remaining EMSA probes were generated by annealing of complementary primers and had the following sequences: S8, 5'-ATC ACG CGT CGG CAC CAT GGA ACT TGT AGT TCC TTC ACC CAT CCC CCA ACG CTC GTC TGA AAG CTT ATC C-3'; S9, 5'-ATC ACG CGT TCC CAG GTA GCC AAG CGC CCG CTT GCC GCG CGG CAC CAT GGA ACT TGT AGT TCC TTC ACC CAT CCC CCA ACG CTC GTC TGA AAG CTT ATC C-3'; S91, 5'-TCC CAG GTA GCC AAG CGC CCG CTT GCC-3:prime]'; S92, 5'-GCC GCG CGG CAC CAT GGA ACT TGT AGT-3'; S93, 5'-CCA AGC GCC CGC TTG CCG CGC GGC ACC-3'; and S94, 5'-CAT GGA ACT TGT AGT TCC TTC -3'. EMSA analysis was performed as follows: 2 x 105 cpm of [{gamma}-32P]-ATP of labeled probe was incubated with 5 µg of nuclear extracts in binding buffer (10 mM HEPES (pH 7.5), 50 mM KCl, 5 mM MgCl2, 1 mM EDTA, 1 mM DTT, 0.1 mg/ml poly(dI:dC), 0.1 mg/ml BSA, and 5% glycerol) at room temperature for 30 min. Competition with cold probe was performed using 25- to 50-fold molar excess of unlabeled probe. Binding reactions were subjected to electrophoresis on a 5% polyacrylamide gel in 0.25x TBE at 160 V for 3 h.

Neutrophil isolation and LTB4 enzyme-linked immunoassay (EIA)

Primary human neutrophils were isolated from whole blood as follows: heparinized whole blood was fractionated using Ficoll (Amersham Biosciences), as per the manufacturer’s instructions, and the RBC/PMN fraction was collected. The pellet was resuspended in HBSS to twice the original volume and 6% dextran 500 (Amersham Biosciences) in 0.9% (w/v) NaCl was added to each sample to a final concentration of 1% dextran. The RBC were allowed to settle for 1 h at room temperature, then the PMN-rich supernatant was carefully collected and diluted 1/2 in HBSS and centrifuged down at 600 x g for 10 min. Remaining RBC were lysed in ammonium chloride lysis buffer. The pure PMN fraction was resuspended in PBS and analyzed by FACS for CD16 expression, which showed that >98% of the isolated fraction was neutrophils. Immediately after isolation, the neutrophils were used for transcellular biosynthesis assay. A total of 1 x 107 B cells (BJAB, BCBL-1, or L-428) along with 0.2 x 106 neutrophils were seeded into 6-well plates in 2 ml of PBS. All samples were prepared in triplicate. Positive control was assayed by 1 x 107 neutrophils, as well as B cells alone were assayed as a negative control. Neutrophils were stimulated by addition of 2 mM CaCl2 and 5 nM calcium ionophore A23187 (Sigma-Aldrich). The reaction was allowed to proceed for 5 min at room temperature, then the supernatants were collected and diluted 1/100–1/200 in fresh PBS. Detection of LTB4 was performed by enzyme-linked immunoassay (Leukotriene B4 EIA kit; Cayman Chemical), as per the manufacturer’s instructions.

Transwell migration assay

Migration assay was performed using a 96-well disposable migration chamber (ChemoTx; NeuroProbe) as described elsewhere (44). Briefly, BCBL-1 or BJAB cells were labeled with the fluorescent dye Calcein AM (Invitrogen Life Technologies), as per the manufacturer’s instructions. The labeled cells were resuspended at a density of 3 x 106 cells/ml in RPMI 1640-10% FBS, and 25 µl of the cell suspension (75,000 cells) were placed on top of the polycarbonate filter (PVP-free, 8-µm pore size, and 3.2-mm diameter). The lower chamber was filled with 29 µl of chemoattractant solution (either LTB4, 10–0 M, or a 1/100 dilution of supernatant from transcellular biosynthesis assay) or vehicle alone (PBS and 0.1% human serum albumin (HSA)). The chamber was incubated for 1 h (37°C, 5% CO2), then nonmigrated cells were removed with a cell scraper from the top of the filter. Migrated cells were counted on the bottom side of the membrane using a fluorescence microscope. The experiment was performed in triplicate.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Microarray analysis of the PEL cell line BCBL-1 compared with the cHD cell line L-428

To elucidate the signature genes that distinguish PEL from cHD at the molecular level, microarray analysis was performed using total RNA isolated from the PEL cell line BCBL-1 and compared with the cHD cell line L-428 as well as the BL cell line BJAB (Fig. 1). A subset of ~30 genes of 1700 was differentially regulated exclusively in BCBL-1 cells (Table I); from this list, four genes involved in inflammation at the level of chemotaxis and/or cell motility were significantly up-regulated in BCBL-1. Given that the localization of PEL to serous body cavities involves migration of the tumor B cells from the lymph nodes to the peritoneal, pleural, or pericardial space, it was therefore of interest to further characterize the expression of LTA4H (>5-fold induction), thrombospondin-1 (TSP-1, >6-fold induction), IL-16 (>4-fold induction), and P-selectin glycoprotein ligand (PSGL-1, 4- to 5-fold induction).


Figure 1
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  		FIGURE 1. Gene expression profile of BCBL-1 cells compared with BJAB and L-428 cells. Gene expression is shown as a color representation of ratio values, with red being above and green being below the row/column median level of expression as shown by the scale. Each column represents the average for BCBL-1 cells compared with either BJAB or L-428 cells and each row represents one gene. The dendogram comprises the final gene list after statistical analysis for microarray statistical analysis with a p value cutoff of 0.05, and a differential expression cutoff of 1.5-fold. Genes in bold were confirmed by RT-PCR or immunoblot.

 

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  		Table I. List of genes specifically expressed in BCBL-1 cells compared with L-428 and BJAB cellsa

 
LTA4H, TSP-1, IL-16, and PSGL-1 are overexpressed in PEL cells

To confirm the results obtained by microarray, the expression of LTA4H, IL-16, PSGL-1, and TSP-1 was examined at the mRNA (Fig. 2A, left panel) and/or protein (Fig. 2A, right panel) level by using three additional PEL cell lines (BC-3, BCP-1, and CRO-AP6), and comparing these cells to the cHD cell lines L-428 and KM-H2 as well as the BL cell line BJAB and primary tonsillar B cells. Analysis of the expression of LTA4H mRNA by RT-PCR identified a single 204-bp amplification product, whereas immunoblot analysis revealed the presence of a 69-kDa protein corresponding to LTA4H (Fig. 2A, top row). The mRNA induction level in PEL cells as measured by RT-PCR was >10-fold, whereas the protein level averaged a 5-fold increase compared with cHD and BJAB cells.


Figure 2
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  		FIGURE 2. Four cellular factors involved in chemotaxis and cell migration are up-regulated in PEL cells. A, LTA4H, IL-16, TSP-1, and PSGL-1 mRNA and/or protein levels in PEL cells vs cHD cells. Left-hand panel, Total RNA (5 µg) isolated from four PEL cell lines and two cHD cell lines as well as BJAB cells was analyzed for LTA4H, IL-16, and TSP-1 mRNA levels by RT-PCR. Lane 1, BJAB; lane 2, BCBL-1; lane 3, BC-3; lane 4, BCP-1; lane 5, CRO-AP6; lane 6, L-428; and lane 7, KM-H2. GAPDH amplification was used to ensure equal RNA levels. Right-hand panel, Protein expression of LTA4H, pro-IL-16, TSP-1, and PSGL-1 in PEL cells compared with primary tonsillar B cells, and cHD cells were assessed by immunoblot. Whole-cell extracts (50 µg) were electrophoresed in a 7.5% acrylamide gel and immunoblotted with specific Abs as shown. Lane 1, Primary tonsilar B cells; lane 2, control BJAB cells; lanes 3–6, PEL cell lines (BCBL-1, BC-3, BCP-1, and CRO-AP6); and lanes 7 and 8, cHD cell lines (L-428, KM-H2). An Ab against beta-actin was used to ensure equal loading. B, Immunoblot for cleaved caspase 3 allowed the detection of the active 17-kDa form. Whole-cell extracts (50 µg) were electrophoresed in a 12% acrylamide gel and blotted with an Ab against active caspase 3. Lane 1, Primary tonsillar B cells; lanes 2–5, PEL cell lines (BCBL-1, BC-3, BCP-1, and CRO-AP6); lanes 6 and 7, cHD cell lines (L-428, KM-H2); and lane 8, control BJAB cells. C, Immunoblot against secreted TSP-1 present in growth medium after 24 h of incubation. A total of 1 x 106 cells was seeded into 6-well plates and left for 24 h at 37°C, with 5% CO2. After the incubation period, the supernatants were harvested, run on a 7.5% acrylamide gel, and blotted with a specific Ab against TSP-1. Lanes 1–3, BJAB cells (negative control); lanes 4–6, BCBL-1 cells; and lane 7, medium alone. Experiment was performed in triplicates.

 
RT-PCR amplification of IL-16 mRNA detected the expected 333-bp product in all PEL and BJAB cells but not in cHD cells (Fig. 2A, second row). Induction levels in PEL cells ranged from 2.7- to 21-fold increase compared with the EBV-negative BJAB cells. Other EBV+ BL cell lines analyzed by RT-PCR also exhibited high IL-16 levels (data not shown), indicating that both EBV and HHV-8 can induce expression of this chemokine. IL-16 is synthesized as a large 80-kDa precursor molecule, pro-IL-16, that is cleaved to the bioactive 20-kDa IL-16 molecule by caspase 3 (45). Immunoblot analysis of intracellular precursor protein levels closely mirrored the results obtained at the mRNA level, with high yet variable protein levels in PEL cell lines (Fig. 2A, second row). No band was detected at 20 kDa, as expected for the secreted active IL-16. Immunoblot for activated caspase 3 detected low levels of active,17-kDa caspase 3 protein in all PEL cell lines but not in cHD and BJAB cells (Fig. 2B), thus indicating that bioactive IL-16 is produced and secreted by PEL cells.

TSP-1 levels were also increased in PEL cells compared with cHD and BJAB cells, both at the mRNA and protein levels (Fig. 2A, third row). RT-PCR analysis of TSP-1 mRNA levels identified a single 493-bp product in PEL cells; this transcript was undetectable in BJAB and cHD cells. Immunoblot analysis under reducing conditions revealed the intracellular presence of the 145- to185-kDa protein in PEL cells only; furthermore, TSP-1 protein was detected in supernatants from BCBL-1 cells, demonstrating that this extracellular matrix protein is produced and secreted by PEL cells (Fig. 2C). Similarly, the intracellular PSGL-1 protein level was elevated in PEL cells but not detectable in cHD or BJAB cells (Fig. 2A, fourth row).

Protein expression of LTA4H, TSP-1, PSGL-1, and IL-16 in PEL cells was also compared with expression in primary tonsillar B cells (Fig. 2A, right panel, cf. lane 1 to PEL samples). LTA4H, TSP-1, and PSGL-1 were 5- to 10-fold higher in PEL compared with primary B cells; however, IL-16 levels as well as caspase 3 activation were comparable in PEL and primary tonsillar B lymphocytes, suggesting that during PEL development, malignant B cells have acquired expression of LTA4H, TSP-1, and PSGL-1, while retaining the capacity to produce IL-16.

Isolation of the LTA4H gene promoter

Alteration in the B cell transcriptional program has been shown to play an important role in aberrant gene expression in PEL, particularly in relation to the lack of cell surface markers in these cells (18). For this reason, we sought to investigate whether transcriptional alterations were involved in LTA4H overexpression in PEL. The LTA4H promoter was initially described (46) by Mancini and Evans in 1995, but to date has not been functionally characterized. A 2.1-kb fragment upstream of the transcriptional start site, as well as 105 bp of the 5' untranslated region was cloned adjacent to the luciferase gene (LTA4HPRO; see schematic in Fig. 4A) and the activity of the full-length promoter was investigated. A 130-fold induction in reporter gene activity was observed with the LTA4H promoter relative to the empty vector (pGL3basic) in BCBL-1 cells, compared with 40- and 60-fold in BJAB and L-428 cells, respectively (Fig. 3A). Activity of the LTA4H promoter was not further stimulated in BJAB cells by overexpression of the latent HHV-8 proteins LANA or vFLIP (data not shown).


Figure 4
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  		FIGURE 4. Identification of a positive regulatory region between –123 and –40 bp in the LTA4H promoter active only in BCBL-1 cells but not in BJAB or L-428 cells. A, Schematic representation of LTA4HPRO and deletion constructs. B, Activity of deletion constructs S1, S2, and S4. BCBL-1 or BJAB cells were electroporated with 10 µg of each deletion construct or empty vector (pGL3basic), incubated for 48 h, and assayed for luciferase activity. Fold induction was calculated as the ratio of luciferase activity of the deletion construct to empty vector. Values were normalized using Renilla activity (2 µg of pRLnull) as an internal control. C, Activity of S4 and S6 deletion constructs. BCBL-1, L-428, or BJAB cells were electroporated with S4 or S6 construct and assayed as in B. A representative experiment conducted in duplicate is shown. At least three independent experiments were performed.

 

Figure 3
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  		FIGURE 3. Activity of the LTA4H promoter in BCBL-1 cells vs L-428 and BJAB cells. The putative promoter region and partial 5' untranslated region of the LTA4H gene (–2021 to +105 bp) was subcloned in front of the luciferase gene to create LTA4HPRO. A, Ten micrograms of LTA4HPRO or the empty vector (pGL3basic) was electroporated into BCBL-1, L-428, or control BJAB cells along with 2 µg of Renilla (pRLnull) as internal control. Fold activity of the promoter was calculated as the ratio of LTA4HPRO activity to empty vector. Values were normalized according to Renilla activity. The experiment shown is representative of at least three independent experiments carried out in duplicate. B, Inhibition of NF-{kappa}B and AP-1 with the chemical inhibitors aspirin (acetylsalicylic acid, 10 mM) or ibuprofen (1 mM, NF-{kappa}B inhibitor only) did not affect the activity of LTA4HPRO. BCBL-1 cells were electroporated with 10 µg of LTA4HPRO and incubated for 42 h, then the appropriate inhibitor was added and incubated for an additional 6 h before harvesting samples and assaying for luciferase activity. Fold induction was calculated as the ration between LTA4HPRO and empty vector (pGL3basic) after normalization using Renilla as an internal control (2 µg of pRLnull). A representative experiment performed in duplicate is shown. At least three independent experiments were performed.

 
Both PEL and cHD cells share a constitutive activation of NF-{kappa}B (21, 22) and AP-1 (23, 24) transcription factors and have been linked to disease progression, in particular, to expression of proinflammatory genes such as IL-6. To investigate whether NF-{kappa}B or AP-1 played a role in the overexpression of LTA4H, pharmacological inhibitors of these transcription factors were used during the reporter gene assay with the full-length LTA4H promoter. However, neither ibuprofen, acetylsalicylic acid, nor the specific AP-1 inhibitor SP600125 had a significant influence in promoter activity in BCBL-1 (Fig. 3B and data not shown). Therefore, neither NF-{kappa}B or AP-1 appear to be involved in LTA4H expression in PEL.

Identification of a LTA4H promoter element active only in PEL cells

Four deletion fragments of the LTA4H promoter were constructed to identify potential regulatory elements active in PEL cells (Fig. 4A). In both BJAB and BCBL-1, fragment S1, in which the distal 319 bp were deleted, exhibited roughly the same activity as the full-length promoter. Fragment S2, from –1196 to +105, exhibited a 1.5-fold increase in activity compared with S1, demonstrating the existence of a negative cis-regulatory element between –1702 and –1196. Between S2 and S4 there was a 1.5- to 2-fold loss in activity, indicating the presence of one or more positive element(s) between –1196 and –123 (Fig. 4B). Interestingly, between S4 and S6 a 50% loss of activity was observed exclusively in BCBL-1 cells, whereas no significant change in promoter activity was seen in BJAB or in L-428 cells (Fig. 4C). Thus, at least one positive cis-regulatory element is present in the LTA4H promoter that is active only in BCBL-1 cells; moreover, this element is located in the promoter proximal region between –123 to –40 bp from the transcriptional start site.

Two protein-DNA complexes form in the –76 to –40 region of the LTA4H promoter in BCBL-1 cells

Further investigation of the transcriptional control of the LTA4H promoter was performed by EMSA. Nuclear extracts from BCBL-1, BJAB, and L-428 cells were incubated with probes corresponding to the S4 and S6 regions of the LTA4H promoter (Fig. 5A, left panels). With probe S4, the presence of a protein-DNA complex was observed in BCBL-1 cells but not in BJAB or L-428 cells. This complex disappeared when S6 was used as probe, thus demonstrating that binding occurred in the –123 to –40-bp region. This observation is in agreement with results from the reporter gene assay, in which S4 contained a transactivating element that produced 2-fold higher activity than S6 in BCBL-1 cells.


Figure 5
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  		FIGURE 5. Formation of a specific protein-DNA complex in the –76 to –40-bp region of the LTA4H promoter in BCBL-1 cells. A, Nuclear extracts (5 µg) from BCBL-1, BJAB, or L-428 cells were analyzed by EMSA with different probes spanning the LTA4H promoter proximal region, as indicated, to identify specific complexes formed exclusively in BCBL-1. Probe S4 spans from –123 to +105 bp; probe S6 from –40 to +105 bp; probe S9 from –76 to +5 bp; and probe S8 from –46 to +5 bp. Specific complexes are indicated by an arrow. A nonspecific complex is indicated by an asterisk. B, Shorter probes spanning the region from –76 to +5 were assayed for specific complex formation as in A. Probe S91 contains the –76 to –49-bp region; probe S92, –51 to –25 bp; probe S93, –66 to –40 bp; and probe S94, –40 to –19 bp. •, Lanes 1, 4, 7, and 10; BJAB nuclear extract {circ}, lanes 2, 5, 8, 11, 13–15; BCBL-1 nuclear extracts, gray circles,# lanes 3, 6, 9, and 12, L-428 nuclear extracts. Lanes 13-15, competition with S91, S92, and S93 cold probe, respectively. Specific complexes are indicated by arrows. A nonspecific complex is indicated by an asterisk. C, Schematic representation of binding region for complexes A and B formed exclusively in BCBL-1 cells.

 
To further delineate the factor(s) binding region, smaller probes were designed to fine-tune the position of the binding site. Probe S9 spanned nucleotides –76 to +5 and probe S8 from –46 to +5. Using these two probes in EMSA analysis, formation of a complex could be clearly seen in BCBL-1 cells but not in BJAB nor L-428 with both probes, although S9 probe yielded a sharper band than S8 (Fig. 5A, right panel). Four additional probes, S91 (–76 to –49) and S92 (–51 to –25), S93 (–66 to –40), and S94 (–40 to –19), were used in EMSA analysis (Fig. 5B) to refine the mapping of the binding site(s). S91 showed formation of a faint, diffuse complex in BCBL-1 cells and a stronger higher complex present in all cell lines. S92 lead to the formation of two specific complexes in BCBL-1 cells: complex A was stronger and had lower motility, whereas complex B was very faint and migrated faster than complex A. S93 showed the presence of one strong band that roughly comigrated with complex B. The S94 probe showed no protein binding in any cell line. The specificity of the binding was demonstrated by cold probe competition, which disrupted the formation of the higher specific complexes but not of a lower nonspecific band. Therefore, there are at least two distinct binding sites in this region: binding site A is centered at position –41/42 and truncations beyond –40 bp abrogate binding to this site. Binding site B is upstream of A, centered at position –46/48 (see Fig. 5C for schematic). Identification of the binding protein(s) is still under investigation.

Transcellular biosynthesis of LTB4 is increased in BCBL-1 cells

Leukotriene B4 is a potent lipid mediator that acts as a potent chemoattractant of neutrophils (31), monocytes, macrophages (32), and T cells (33, 34) and can induce B cell proliferation and Ab secretion (37). Because B cells normally lack 5-lipoxygenase (5-LO) activity, we postulated that up-regulation of LTA4H in PEL cells may contribute to increased production of LTB4 by transcellular biosynthesis. To examine this possibility, BCBL-1 cells were cocultured at a 1:50 ratio with primary neutrophils stimulated with calcium ionophore to induce 5-LO activity. In parallel, control BJAB and L-428 cells were assayed. Supernatants were collected and analyzed by EIA for LTB4. Coculture with BCBL-1 cells resulted in LTB4 levels of 620 pg/ml (±65 pg/ml), whereas coculture with BJAB or L-428 produced 295 pg/ml (±44 pg/ml) or 201 pg/ml (±7 pg/ml), respectively (Fig. 6A). Therefore, BCBL-1 cells have the capacity to produce 2- to 3-fold more LTB4 than control B cells in the presence of stimulated neutrophils. B lymphocytes have been shown to express both LTB4 receptors, BLT-1 and BLT-2, and therefore also have the capacity to respond to exogenous LTB4. All PEL cell lines had strong expression of BLT-1 and BLT-2 mRNA as demonstrated by RT-PCR, and these levels were comparable to those of BJAB and L-428 cells (Fig. 6B). Therefore, PEL cells have retained the potential to respond to LTB4 stimulation. Migration experiments demonstrated that BCBL-1 cells migrate 50–100 times more readily across a porous membrane than control BJAB cells (Fig. 7, cf. A and B); however, LTB4 did not further increase the migration of BCBL-1 cells (Fig. 7, C and D). LTB4 also failed to induce BCBL-1 cell proliferation (data not shown) when used alone or in combination with IL-4 or IL-6. The biological effect of LTB4 on PEL cells remains under investigation, although preliminary evidence suggests that LTB4 may lead to activation of the NF-{kappa}B pathway (M. Arguello and S. Paz, unpublished observation).


Figure 6
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  		FIGURE 6. Formation of LTB4 by transcellular biosynthesis is increased in BCBL-1 cells. A, Transcellular biosynthesis of LTB4 measured by EIA. BCBL-1, BJAB, or L-428 cells were incubated with calcium ionophore-stimulated primary neutrophils at a ratio of 50:1 for 10 min. Supernatants were diluted 100-fold and assayed for secreted LTB4 by EIA. Experiment was performed in triplicate. Data significance was assessed using paired Student’s t test; *, p = 0.010; **, p = 0.004. B, RNA levels of the LTB4 receptors BLT-1 and BLT-2 in BJAB, BCBL-1, and L-428 cells. Total RNA (5 µg) from each cell line was subjected to RT-PCR using specific primers for BLT-1 and BLT-2. GAPDH primers were used as a control for equal RNA content. Representative results are shown. At least three independent experiments were performed.

 

Figure 7
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  		FIGURE 7. BCBL-1 cells migrate readily across a porous membrane. A–C, Representative fields of Calcein AM-labeled cells migrated to the bottom side of polycarbonate membranes in the Transwell migration experiment. A, Control BJAB cells plus PBS/0.1% HSA; BCBL-1 cells plus PBS/0.1% HSA (B); and BCBL-1 cells plus LTB4 (10–10 M) (C). D, Quantitation of Transwell migration experiment. The total number of migrated BCBL-1 or BJAB cells (Calcein AM labeled) were counted on the bottom side of polycarbonate filter under a fluorescence microscope. Error bars correspond to SD on triplicate readings.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Chronic inflammation is emerging as a key player in cancer development and this link has been strongly established for certain cancer types such as colon cancer (47), esophageal adenocarcinoma (48), and KS (reviewed in Ref. 49). The present work identified four genes involved in inflammation, LTA4H,IL-16, TSP-1, and PSGL-1, as highly up-regulated in PEL cells. LTA4H synthesizes LTB4, a potent chemotactic stimulus for neutrophils, whereas IL-16 is chemotactic for CD4+ T cells and dendritic cells (DCs). TSP-1 and PSGL-1 are involved in chemotaxis and cell adhesion and extravasation, respectively. Together, these four factors would likely contribute to the establishment of an inflammatory milieu and contribute to PEL development.

The involvement of lipid mediators of inflammation in cancer development has been investigated both at the clinical and basic level. Long-term treatment with nonsteroidal anti-inflammatory drugs, compounds that inhibit synthesis of eicosanoids, reduces the incidence of colorectal cancer in large population-based studies as well as in rodent models of familiar adenomatous polyposis (reviewed in Ref. 50). The leukotriene LTB4, which induces a vigorous inflammatory response, has been equally implicated in cancer development (51, 52), and use of LTA4H or LTB4 receptor inhibitors reduced cancer incidence in animal and in vitro models (51, 52). Initial microarray analysis of the PEL cell line BCBL-1 demonstrated that the enzyme LTA4H was highly expressed in BCBL-1 but not in the cHD cell line L-428. LTA4H up-regulation was evident at the mRNA and protein levels in all PEL cell lines investigated, at least 5-fold higher than in primary tonsillar B cells, BJAB, or cHD cell lines. These results are in agreement with previous microarray studies (53, 54) of PEL biopsy samples and cell lines. Furthermore, the present work focused on the transcriptional elements that regulate LTA4H expression as well as establishing a role for its overexpression in LTB4 synthesis by PEL cells.

Isolation of the LTA4H promoter demonstrated that up-regulation of gene expression in PEL cells is controlled at the transcriptional level. Indeed, the activity of the full-length promoter was >2- to 3-fold higher in BCBL-1 cells than in control BJAB or L-428 cells. The use of truncated promoter constructs identified potential regulatory sites in the LTA4H promoter; the distal region between –1702 and –1196 bp contained a negative regulatory element that was active in all cell lines, whereas the region between –1196 and –123 contained one or more activating elements. Interestingly, the promoter-proximal region between –123 and –40 bp contained a positive cis-acting element that doubled promoter activity in BCBL-1 cells only, with no effect in control cells. EMSA analysis demonstrated the presence of two protein-DNA complexes in the –51 to –40 bp region that are formed in BCBL-1 cells but not in control BJAB or L-428 cells. Thus, we have identified a regulatory site in the proximal promoter that is responsible for the observed up-regulation of LTA4H in PEL. Candidate transcription factors that could potentially bind to this element were identified by computer modeling (55). Supershift analysis and cold probe competition in EMSA assays was then undertaken to identify binding proteins. Abs and/or probes against C/EBP-beta, E2F-1, OCT, myc, myb, and others were assayed without success. Additional attempts to identify the binding factor(s) are currently under way.

Synthesis of LTB4 from arachidonic acid is a multistep catalytic process involving the enzymes 5-LO and LTA4H. Initially, arachidonic acid is transformed into LTA4 by 5-LO, in a process that requires both calcium stimulation of enzymatic activity as well as the presence of the coactivator 5-LO-activating protein. The unstable epoxide LTA4 is then converted into LTB4 by LTA4H, and in this process the enzyme undergoes suicide inactivation (56, 57); thus, the generation of LTB4 is limited by the amount of LTA4H available. Interestingly, whereas 5-LO expression is largely restricted to myeloid-derived cells, LTA4H expression is more ubiquitous and can be found in a variety of tissues (58). There is increasing evidence that LTB4 synthesis in vitro and in vivo can be achieved by a process known as transcellular biosynthesis, in which two cell types, one with 5-LO and another with LTA4H activity, cooperate in the production of LTB4 (30, 59, 60, 61). Transcellular biosynthesis of leukotrienes thus contributes to the amplification of the inflammatory response by increasing the overall amount of LTA4H available to catalyze the last step of the reaction.

The functional significance of LTA4H up-regulation in PEL was demonstrated by an increased ability of BCBL-1 cells to produce LTB4 by transcellular biosynthesis, as demonstrated by EIA experiments. Indeed, incubation of BCBL-1 cells with calcium ionophore-stimulated neutrophils lead to at least 50% more LTB4 production than BJAB or L-428 cells. Thus, even under limited neutrophil infiltration, PEL cells have the ability to magnify LTB4 production. Additionally, the capability of PEL cells to respond to secreted LTB4 was underlined by the expression of both LTB4 receptors (BLT-1 and BLT-2). Preliminary evidence using BCBL-1 suggests that LTB4 stimulation leads to NF-{kappa}B activation (M. Arguello and S. Paz, unpublished observation); at this stage, increased phosphorylation of the I{kappa}B{alpha} inhibitor and NF-{kappa}B p65-specific DNA-binding activity have been detected early (1–2 h) after LTB4 treatment. The potential role of NF-{kappa}B activation in PEL pathogenesis requires further investigation. LTB4 does not trigger PEL cell chemotaxis or proliferation when used alone or in combination with IL-4 or IL-6; however, a role for LTB4 as a growth modulator during the early stages of PEL is possible as LTB4 enhances the proliferation of activated B cells (62, 63). LTA4H also possesses aminopeptidase activity (64) and, although few physiological substrates have been identified, it is thought that LTA4H may participate in extracellular matrix remodeling. A potential role for the aminopeptidase activity of LTA4H in PEL cannot be discarded.

Microarray analysis as well as subsequent PCR and immunoblot analysis identified additional proinflammatory genes that were up-regulated in PEL cells, including TSP-1, IL-16, and PSGL-1. TSP-1 intracellular levels were at least 5-fold higher in PEL cells than in BJAB or cHD cells, and TSP-1 was readily detected in the medium of BCBL-1 cells, as expected for a secreted extracellular matrix protein. The multidomain nature of TSP-1 endows this protein with diverse and sometimes opposing biological roles (reviewed in Ref. 65); however, the chemotactic properties of TSP-1 (66, 67) and the induction of locomotion of leukocytes has been documented (68). Increased levels of the TSP-1 receptor CD36 were identified in KS lesions (69). Overexpression of TSP-1 in PEL could serve as a chemoattractant for tumor B cells or contribute to the invasion of serous body cavities, although these possibilities remain to be investigated.

IL-16 was identified as another up-regulated gene by microarray, PCR, and immunoblot analysis in PEL cells. IL-16 induces a strong chemotactic response in CD4-expressing cells such as CD4+ T cells monocytes, eosinophils, and monocyte-derived DCs (reviewed in Ref. 70). Kaser et al. (71) demonstrated production of IL-16 by mantle zone and GC B cells associated with chemoattraction of Th cells and DCs. HHV-8 infection may induce IL-16 expression during early PEL development and stimulate B maturation and proliferation as well as chemoattraction of Th cells to body cavities to promote establishment of an inflammatory state favorable to lymphomagenesis.

Because PEL is detected usually at advanced stages of disease, the early steps of PEL development remain obscure. The identification of LTA4H, IL-16, TSP-1, and PSGL-1 as proinflammatory molecules up-regulated in PEL suggest the involvement of chemotactic and inflammatory factors in the migration of the PEL B cell of the lymph nodes to body cavities. Indeed, in migration experiments the PEL cell line BCBL-1 exhibited 50- to 100-fold greater motility across a porous membrane than control BJAB cells, even in the absence of exogenous chemotactic stimulus. Although LTB4 may not be a chemoattractant to PEL cells themselves, production of LTB4 by PEL cells could lead to the recruitment of PMNs and monocytes, whereas IL-16 production would permit PEL cells to retain the capacity to attract CD4+ cells such as Th cells, monocytes, and DCs. The creation of an inflammatory milieu would support continued cell growth, a phenomenon reminiscent of early KS development, and the parallel expression of cell motility factors by PEL cells may explain their localization to body cavities. Understanding the molecular alterations that influence PEL development will aid in the identification of novel therapeutic targets, as well as potential risk factors for this disease.


   Acknowledgments
 
We thank Dr. J. Evans (Merck & Co.) for the gift of reagents, Dr. Raphaelle Romieu-Mourez (Lady Davis Institute), Dr. Tuula Nyman (Finnish Institute of Occupational Health, Helsinki, Finland), and Peter Wilkinson (Lady Davis Institute) for their technical help as well as members of the Molecular Oncology Group for helpful discussions.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by a Canadian Institutes of Health Research Operating Grant (to R.L. and J.H.), a grant from the National Cancer Institute of Canada, with the support of the Canadian Cancer Society (to J.H.), a National Sciences and Engineering Research Council postgraduate scholarship, and a Fonds de Recherche en Santé du Québec, and l’Aide á la Recherche Bourse de Formation de 2e Cycle (to M.A.). Back

2 Address correspondence and reprint requests to Drs. Rongtuan Lin and John Hiscott, Lady Davis Institute for Medical Research, Jewish General Hospital, 3755 Chemin de la Côte-Sainte-Catherine, Montreal, Ontario, H3T-1E2, Canada. E-mail addresses, respectively: rongtuan.lin{at}mcgill.ca and john.hiscott{at}mcgill.ca Back

3 Abbreviations used in this paper: HHV-8, human herpesvirus-8; KS, Kaposi’s sarcoma; PEL, primary effusion lymphoma; GC, germinal center; cHD, classical Hodgkin’s disease; BL, Burkitt’s lymphoma; LT, leukotriene; LTA4H, LTA4 hydrolase; EIA, enzyme-linked immunoassay; HSA, human serum albumin; TSP-1, thrombospondin-1; PSGL-1, P-selectin glycoprotein ligand-1; DC, dendritic cell. Back

Received for publication May 12, 2005. Accepted for publication March 16, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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