1. Induction of autotaxin by the Epstein-Barr virus promotes the growth and survival of Hodgkin lymphoma cells
by Karl R. N. Baumforth, Joanne R. Flavell, Gary M. Reynolds, Gillian Davies, Trevor R. Pettit, Wenbin Wei, Susan Morgan, Tanya Stankovic, Yasuhiro Kishi, Hiroyuki Arai, Marketa Nowakova, Guy Pratt, Junken Aoki, Michael J. O. Wakelam, Lawrence S. Young, and Paul G. Murray
Blood
Volume 106(6):
September 15, 2005
©2005 by American Society of Hematology

2. Increased growth and survival of EBV-positive HL cells expressing a limited repertoire of EBV genes is associated with the transcriptional up-regulation of autotaxin.
Increased growth and survival of EBV-positive HL cells expressing a limited repertoire of EBV genes is associated with the transcriptional up-regulation of autotaxin. (A) RT-PCR analysis demonstrates a pattern of virus gene expression in EBV-infected KM-H2 (KM-H2-Akata) cells predominated by expression of the EBERs, Qp-driven EBNA1, and the BamH1A transcripts. There was also very low-level transcription from Cp/Wp and associated low levels of EBNA2 and LMP2 transcripts in these cells. Neither LMP1 RNA nor protein (data not shown) could be detected in these cells. (B) Loss of EBER expression in 2 clones (SD3 and SD5) of EBV-negative L591 cells derived from the EBV-positive parental line by serial dilution. Quantitative PCR was also used to confirm the loss of the EBV genome from these cells (data not shown). Clone SD3 was used in later experiments. (C) (upper panels) WST1 assays showing relative metabolic activity (cell growth) of control cells (KMH2-neo) adjusted to a value of 1 compared with EBV-infected KM-H2 cells (KM-H2 + Akata) in 10% and 1% sera. A clear and significant difference in cell growth between control and EBV-infected cells is observed. (lower panels) Cell viability assays at 10% and 0.1% serum, demonstrating consistent and significant increases in the viability of EBV-infected KM-H2 cells compared with controls. ▪ indicates KM-H2-neo; and □, KM-H2 + Akata. (D) L591 SD3 cells showed dramatically reduced viability compared with EBV-positive parental L591 cells in 10% and 1% sera. Cell proliferation was similarly affected by EBV loss (data not shown). ▪ indicates L591; and □, L591-SD3. (E) Heat map showing gene expression differences across 4 replicates of EBV-negative KM-H2 cells and EBV-infected KM-H2 cells (KM-H2 Akata); 26 probe sets met the criteria of a positive or negative change of 2.5-fold or greater and a false discovery rate of 5% or less. Eleven probe sets were up-regulated and 15 were down-regulated after EBV infection. The most highly up-regulated probe set (mean fold increase, 4.16) was autotaxin (ENPP2). (F) Confirmation of increased autotaxin mRNA in EBV-infected KM-H2 (KM-H2 Akata) and L591 cells by RT-PCR analysis compared with EBV-negative parental KM-H2, KM-H2 neo control, and L591-SD3 cells. Other EBV-negative HL cell lines (L428, L540, L1236) and the EBV-transformed lymphoblastoid cell line X50-7 expressed lower levels of autotaxin mRNA.
Karl R. N. Baumforth et al. Blood 2005;106:
©2005 by American Society of Hematology

3. Generation of autotaxin-specific monoclonal antibodies and the specific up-regulation of autotaxin protein by EBV-infection of HL cells.
Generation of autotaxin-specific monoclonal antibodies and the specific up-regulation of autotaxin protein by EBV-infection of HL cells. (A) Immunoblotting of sf9 cells infected with autotaxin (ATX)-baculovirus using 2A12 antibody demonstrates strong reactivity, whereas sf9 cells infected with wild-type (wt) baculovirus show no reactivity. Also shown here is reactivity against autotaxin in human serum, human plasma, culture supernatant and cells of breast cancer cell line MBA-MD-231, and supernatant and cells of glioma cell line SF539. (B) Western blotting using 2A12 antibody demonstrates increased autotaxin protein in EBV-infected KM-H2 cells (KM-H2 Akata) compared with uninfected KM-H2 cells (left panel). Most other EBV-negative HL cell lines (L428, L540, L1236, HD-MyZ, HDLM2) expressed lower levels of autotaxin protein. (right panel) Down-regulation of autotaxin protein in L591 cells after the loss of the EBV genome. (C) Autotaxin could not be detected in EBV-negative BL cells (Ramos, DG75, BJAB) or in a number of EBV-positive BL cells, including those displaying a type 1 form of EBV latency (Akata, Rael, Elijah), a type 3 form of EBV latency (Raji, Namalwa), or a Wp-restricted pattern of EBV latency (Daudi) that expresses EBNA1 and the EBNA3 family. BL lines are negative for autotaxin, though there is an unidentified lower molecular weight band in the DG75, BJAB, Akata, Elijah, and Rael BL lines and in the IB4 LCL line. Furthermore, of several lymphoblastoid cells lines (X50-7, B95.8 LCL, IB4), only B95.8 cells expressed detectable levels of autotaxin. (D) NPC cells, including EBV-positive C666-1 and EBV-negative HONE-1 cells, lacked autotaxin protein. HONE-1 cells infected with the same Akata-derived recombinant EBV used to generate EBV-positive KM-H2 cells failed to up-regulate autotaxin expression after infection.
Karl R. N. Baumforth et al. Blood 2005;106:
©2005 by American Society of Hematology

4. Expression of autotaxin in lymphoid tissues.
Expression of autotaxin in lymphoid tissues. (A-B) Strong reactivity for autotaxin in HRS cells from EBV-positive primary HL tumors. (C) Lower levels of autotaxin expression in EBV-negative HRS cells despite strong labeling of nontumor cells (arrow). The difference in expression between EBV-negative and EBV-positive tumors was highly significant (see “Expression of autotaxin in primary lymphoma tissues”). In B-NHL, autotaxin expression was largely restricted to CD30+ ALCL (D), whereas most high-grade B-NHLs, such as BL (E) and CD30- DLBCL (F), and low-grade B-NHLs, such as chronic lymphocytic lymphoma (CLL) (G), were negative. (F-G, arrows) Strong labeling of endothelial cells. This restricted pattern may be partly attributed to the finding that among normal B cells, autotaxin expression was absent from germinal center (GC) and mantle zone (MZ) (H) but was expressed by CD138+ postgerminal center B cells (I). Images were acquired using Nikon TE2000 with 60 ×/1.4 NA oil immersion lens (Nikon, Kingston-upon-Thames, United Kingdom). Cells were stained with immunoperoxidase. Images were captured with a Nikon Coolpix 2100, and Paint Shop Pro 8.0 (Jase Software, Maidenhead, United Kingdom).
Karl R. N. Baumforth et al. Blood 2005;106:
©2005 by American Society of Hematology

5. Increased soluble autotaxin production by EBV-infected HL cells is associated with increased use of LPC and generation of LPA. (A) (upper panel) Immunoblotting demonstrates increase in autotaxin protein levels in the supernatant of EBV-infected KM-H2 cells ...
Increased soluble autotaxin production by EBV-infected HL cells is associated with increased use of LPC and generation of LPA. (A) (upper panel) Immunoblotting demonstrates increase in autotaxin protein levels in the supernatant of EBV-infected KM-H2 cells compared with controls. Other EBV-negative HL cells produced lower but detectable levels of soluble autotaxin compared with EBV-positive variants. (lower panel) EBV-negative KM-H2 cells cultured in supernatant from EBV-infected KM-H2 cells (neo cells, EBV media) showed a transient increase in growth compared with control (EBV-negative KM-H2 cells grown in their own supernatant; neo cells, neo media). EBV-positive KM-H2 cells grown in medium from EBV-negative KM-H2 cells (EBV cells, neo media) showed reduced growth compared with control (EBV-positive KM-H2 cells grown in their own supernatant EBV cells, EBV media). ▪ indicates Neo cells, Neo media; ▦, Neo cells, EBV media; ▨, EBV cells, Neo media; and □, EBV cells, EBV media. (B) KM-H2 cells treated with exogenously supplied LPAfollowed by cell growth assay (WST-1). LPA significantly increased the growth of EBV-negative KM-H2 cells (KM-H2-neo) but not of EBV-positive cells (KM-H2-EBV). ▪ indicates KM-H2-neo; ▦, KM-H2-neo + LPA; ▨, KM-H2-EBV; and □, KM-H2-EBV + LPA. (C) (top panel) Microarray analysis of LPA/SIP receptor (EDG receptor) expression in EBV-negative KMH-2 cells and EBV-positive KM-H2 cells (KM-H2 Akata) represented as Affymetrix “calls.” P indicates mRNA present; A, mRNA absent; M, mRNA called marginal. Although EBV-negative and EBV-positive KM-H2 cells expressed the S1PR1 receptor (EDG1), they lacked expression of the major LPA receptor, LPAR1 (EDG2). The other known LPA or S1P receptors (EDG3-8) were lacking in these cells. (bottom panel) These findings were confirmed by RT-PCR analysis of a range of EBV-positive and EBV-negative HL cells (data for EDG1 and EDG2 shown). (D) Hydrolysis of 20 μM LPC by serum-free-conditioned medium, collected after 16 hours of culture over a 5-hour incubation. (E) Generation of LPA by incubation of 20 μM LPC for 3 hours with KM-H2 or KM-H2-EBV cell-conditioned medium. (F) Acyl species analysis of LPA generated by incubation of egg LPC with conditioned media. The 18:1 and 18:0 LPAs were generated by the cells without added LPC. □ indicates KM-H2 neo; and ▪, KMH2-EBV. See the “Statistical analysis” section under “Materials and methods” for explanation of error bars and asterisks.
Karl R. N. Baumforth et al. Blood 2005;106:
©2005 by American Society of Hematology

6. Autotaxin promotes the growth and survival of EBV-infected HL cells.
Autotaxin promotes the growth and survival of EBV-infected HL cells. (A) (top left) Semiquantitative RT-PCR analysis of equivalent starting amounts of cDNA (shown here are results of 30 cycles of amplification) from untreated EBV-positive KM-H2 cells or these cells treated for 48 hours with transfection reagent alone (Ribojuice only), Ribojuice plus scrambled siRNA, or autotaxin (ATX)-specific siRNAs. A clear difference in autotaxin mRNA levels between siRNA-transfected and control cells was detectable. (bottom panel) GAPDH mRNA levels were unaffected by these treatments. (B) Immunoblotting demonstrates knockdown of autotaxin protein in EBV-positive KM-H2 cells treated with autotaxin-specific siRNAs compared with controls. (C) Treatment of EBV-infected KM-H2 cells with autotaxin-specific siRNAs resulted in significant reduction in proliferation after 48 hours (top panel) and cell viability after 72 hours (bottom panel) compared with cells treated with transfection reagent alone or with scrambled siRNA (data not shown). RJ indicates ribojuice transfection reagent. (D) Down-regulation of autotaxin expression in EBV-infected KM-H2 cells resulted in reduced generation of LPA from LPC.
Karl R. N. Baumforth et al. Blood 2005;106:
©2005 by American Society of Hematology