Transcriptional profiling by the Lymphochip cDNA microarray (2
) was used to investigate the contribution of both NF-κB and LMP1 to EBV effects on cell gene expression. Three sets of comparisons were made. First, the effects of latency III EBV infection were investigated by comparing RNAs in EBV-negative BL (BL30, BL2, and Ramos) cells with RNAs in EBV-transformed latency III-infected LCLs [IB4 (twice), LCL2, and LCL14]. Another component of this experiment was to compare RNAs in EBV-negative BL (BL41) cells with RNAs in the stable EBV latency III-infected counterpart, BL41/EBV. Second, the effects of conditional LMP1 expression on cell RNAs in BL41 cells were compared to RNAs in BL41. Third, the role of NF-κB in RNA abundance in EBV-transformed latency III-infected LCLs was investigated by comparing RNAs from IB4 LCLs conditionally expressing the dominant IκBα, ΔN-IκBα, with RNAs of the same cells grown under ΔN-IκBα repressed conditions for 0, 8, 16, and 24 h. As a corollary to these latter experiments, RNAs in IB4 and recently established LCL4 cells were compared to RNAs from these LCLs after NF-κB inhibition with BAY11 for 8 h. Changes in gene expression can be compared across the arrays; however, the relative abundance of any array element is only quantitative within the arrays that were mean centered together as indicated by the gray bars above the lanes in Fig. and .
FIG. 4. Genes that change ≥4-fold with EBV infection or LMP1 expression. (A) EBV-induced genes; (B) EBV-repressed genes; (C) LMP1-induced genes. No genes were repressed by LMP1 fourfold. Arrays are arranged as described for Fig. . Groups (more ...)
Labeled cDNA hybridized to 8,843 of 17,856 cDNA array elements by >2-fold over the background level in at least one experiment and to 3,628 cDNA array elements at 2-fold over the background in at least 70% of the arrays (complete data are available [http://kiefflab.bwh.harvard.edu/
]). Using multiclass SAM (86
), 776 (Fig. ) of the 3,628 elements showed changes accepting a Q value of ≤5% (Q values are the lowest false detection rate at which these genes are significantly different from no change). Cluster analysis (24
) based on Pearson correlation coefficients identified EBV latency III-induced genes in LCLs versus BL30, BL2, and Ramos BL cells, EBV latency III-induced genes in BL41, LMP1-induced genes in BL41 cells, and NF-κB induced genes in LCLs (Fig. , blue bar, and Fig. ).
Cluster analysis of all of the array elements revealed that RNAs in IB4 LCLs were most similar to recently derived LCLs despite extensive passage of IB4 LCLs over the past 20 years (Fig. , lanes 4 and 5, are RNAs from early-passage LCLs versus lanes 6 and 7, which are RNAs from extensively passaged IB4). This similarity is consistent with IB4 cells still being dependent on EBNA2 interaction with RBP-Jκ for c-myc
expression and cell proliferation and on NF-κB activation for cell survival (14
). Cell RNAs in the EBV latency III-infected LCLs (Fig. lanes 4 to 7) were also quite similar to cell RNAs in EBV latency III-infected BL41/EBV cells (Fig. , lanes 10 to 11) or LMP1-expressing BL41 cells (Fig. lanes 12 to 14) and differed extensively from RNAs in BL cells (Fig. lanes 1 to 3 and 8 to 9). Thus, latency III EBV infection or merely LMP1 expression substantially alters lymphoblast cell gene expression.
To assess the role of NF-κB activation in EBV-transformed LCL latency III cell RNA abundances, we examined the transcriptional effects of ΔN-IκBα expression on IB4 cells and the effect of BAY11 on both IB4 and recently transformed LCLs. Despite the effect on p38 activation, the transcriptional effects of BAY11 were remarkably similar to those caused by ΔN-IκBα expression. NF-κB inhibition in IB4 LCLs by expression of ΔN-IκBα for 8, 16, or 24 h (Fig. , lanes 18 to 21) was compared to NF-κB inhibition in both IB4 cells and LCL4 by treatment with BAY11 for 8 h (Fig. , lanes 23 and 24 [IB4] and lane 22 [LCL4]). Note that in all lanes labeled D in Fig. , RNAs were directly compared on the same Lymphochip. Overall, two-thirds of the array elements were affected similarly by BAY11 and ΔN-IκB. A total of 424 (55%) array elements decreased and 81 (11%) increased with both treatments. BAY11 treatment at 8 h was most similar to ΔN-IκBα expression at 24 h, reflecting the difference of the kinetics of NF-κB inhibition (Fig. , compare lane 19, which shows ΔN-IκBα induction at 24 h, with lane 24, which shows BAY11 treatment at 8 h). BAY11 treatment (Fig. , lanes 22 to 24) differed from ΔN-IκBα expression (Fig. , lanes 18 to 21) by causing increased ferritin light-chain expression (Fig. , aqua bar). This discrepancy may be due to BAY11-mediated activation of p38.
The greatest decrease in expression levels after NF-κB inhibition occurred for 47 cDNA array elements (Fig. ). These array elements were also EBV latency III and LMP1 induced. On average, these cDNAs were 1.6-fold less abundant in LCLs in which NF-κB was inhibited, 4-fold more abundant with LMP1 expression, and 1.9-fold more abundant in EBV latency III-infected cells than in BL cells. These 47 array elements were significantly changed in all of the arrays, with an average Q value (false detection rate) of 1%. The 47 array elements are encoded by 21 unique genes. The abundance of these RNAs began to decrease after only 8 h of ΔN-IκBα induction. Newly identified EBV-induced, LMP1-induced, and NF-κB-induced genes in LCLs include the pleckstrin, Jun-B, c-FLIP, NF-κB1 and -κB2, IκB
, and CIP4 genes. Other genes in this cluster have been identified as EBV or LMP1 induced (RANTES, CD54, TRAF1, BFL-1, CD40, EBI3, CD83, CD95, and A20 genes) (22
), as NF-κB induced in LCLs (A20, TRAF1, EBI3, CD54, CD95, BFL-1, and c-IAPs genes) (14
), or as NF-κB induced in other cells (c-FLIP, Jun-B, c-IAP, CD83, BCL-XL, NF-κB1 and -κB2, and IκBα genes) (4
). Many of these genes regulate NF-κB (e.g., IκBα, IκB
, and NF-κB1 and -κB2 genes), protect cells from apoptosis (e.g., c-FLIP, A20, c-IAPs, BFL-1, BCL-XL, and TRAF1 genes), or regulate other cellular responses (e.g., RANTES, CD54, CD83, pleckstrin, and CIP4 genes).
NF-κB has a more significant role in EBV regulation of cell gene expression than is evident from consideration of the 47 most highly NF-κB upregulated RNAs. Of all NF-κB-upregulated array elements, 277 were EBV upregulated, and 84 were EBV downregulated. As anticipated, most NF-κB- and EBV-induced array elements were also LMP1 induced (Fig. ). NF-κB-induced and EBV-repressed array elements were usually LMP1 repressed (Fig. ). Only MIP1α and MIP1β, both EBV- and LMP1-induced genes, showed increased expression with NF-κB inhibition (Fig. , small pink bar, and Fig. ).
Of the 776 significantly changed array elements, 480 were EBV latency III induced since they were expressed at higher levels in EBV latency III converted BL cells than in EBV-negative BL cells (Fig. , red bar) and 296 were EBV latency III repressed since they were expressed at lower levels in EBV latency III-converted BL cells than in EBV-negative cells (Fig. , green bar). Overall, the effects were modest. Only 383 array elements were EBV induced more than twofold; of these, 134 were induced more than fourfold. Similarly, only 219 array elements were EBV repressed more than twofold; of these, only 28 were repressed more than fourfold.
The 134 array elements induced by EBV more than fourfold are encoded by 68 genes (Fig. ). All 68 genes changes were significant, with Q values of <2%, except for CYB5, which had a Q value of <5% (Fig. ). Of these 68 gene changes, 34 were induced by LMP1 and 18 were NF-κB induced. Most other EBV-induced genes are probably upregulated by EBNA2. Many of these 68 genes had been identified as EBV induced by comparison of EBV-positive and -negative BL cells by FACS, previous microarray studies, or by subtractive hybridization; these genes include MIP1α, DNASE1L3, CCR7, EBI2, MARCKS, CD23, CD39, MHC class I and II molecules, A20, TRAF1, CD58, vimentin, CD83, CD95, SHP-1, SLAM, STAT1, CD21, NK4, MXA, and the interferon (IFN)-induced genes 1-8D, 1-8U, and 9-27 (7
). Some of the new EBV-induced genes encode components of the IFN pathway (IRF-4 and IFN-γRα), B-lymphocyte receptor-mediated NF-κB activation (PKCβ) (82
), tyrosine kinase signaling (SKAP55) (62
), mitogen-activated protein kinase signaling (DUSP5 and p62Dok) (44
), vesicular transport (RAB27A and RAB22B) (45
), or chemotaxis (RGS1) (69
EBV repressed 28 array elements more than fourfold. These 28 elements were encoded by 17 genes (Fig. ). Most genes changes were highly significant, with Q values of <2%; only Ig-λ-like 1, GLUT5, and CXCR5 had Q values of between 2 and 5%. PKA-RIIβ, CD10, and Ig-λ-like 1 had been identified as EBV repressed (16
). New EBV-repressed genes included transcription factors B-Myb and MEF2B, cyclin D3, chemokine receptor CXCR5, the GαiGTPase-activating protein RGS13, the B-lymphocyte receptor signaling component CD79B, the Campath1 antigen CD52, and the TAP-independent MHC class I peptide delivery protein JAW1 (Fig. ). Genes such as CDC25C, HMG-I, PCNA, RFC, E2F5, ZAP70, SOD1, and 14-3-3
, which have been reported to be EBV repressed more than twofold, were confirmed (data not shown) (7
LMP1 significantly contributes to EBV effects and induced at least 210 of the 480 EBV-induced array elements (Fig. , yellow striped bars); 161 were induced ≥2-fold. LMP1 also repressed 172 of 296 EBV-repressed array elements (Fig. , purple areas). Only 24 elements were repressed more than twofold, and none were repressed more than fourfold. LMP1 effects were concordant with EBV effects except for FcRγII and Glutaredoxin (Fig. and data not shown), which were EBV induced and LMP1 repressed and CXCR5 (Fig. ), which was EBV repressed and LMP1 induced.
LMP1 induced 74 array elements ≥4-fold; these 74 array elements are encoded by 35 genes (Fig. ). Newly identified LMP1-induced genes include IRF-4, HVEM, NK4, FGFR2, RGS1, pleckstrin, MIP1β, SLAM, and SH3-SAM. Many of the 35 are known targets of the NF-κB pathway and are NF-κB induced in LCLs (Fig. ).
LCLs resemble antigen-activated B lymphoblasts in the expression of adhesion and activation markers. The extent to which LCL gene expression resembles that of antigen-activated B cells was assessed by comparing LCL RNA abundances with those of anti-IgM-stimulated peripheral blood B cells, BL cells, purified germinal-center cells, and four diffuse large cell lymphoma cell lines, two of which have a GC-like phenotype (SUDHLs) and two of which have an activated phenotype (OCI-LYs). Multiclass SAM analysis indicated that 3,399 cDNA array elements were significantly different between cell types, with Q values of <5%. These 3,399 were further characterized by Pearson correlation coefficients by using cluster analysis of both the genes and the arrays. LCLs clustered with IgM-stimulated B cells and were separate from germinal-center cells or B-cell lymphomas with a germinal-center origin (Fig. , array tree).
FIG.5. LCLs are more similar to IgM-stimulated B cells than germinal-center B cells or diffuse large-cell lymphomas (DLCLs). mRNAs abundance for 3,399 cDNAs was compared among LCLs (lanes 1 to 4), IgM-stimulated B cells (lanes 5 to 9), germinal-center B cells (more ...)
Two nodes, comprising 633 array elements encoded by 440 genes, clearly show the distinction between activated lymphocytes and LCLs versus GC cells (Fig. ). The first node encompasses genes that are expressed at relatively high level in LCLs and IgM-simulated B cells. Within this group are genes that were previously characterized as differentially expressed in activated versus germinal-center cells (Fig. , orange text), including TCPTP, APR, BCL-2, CYP2a1, PBEF, and Id2 (3
). Many EBV-induced genes (Fig. , pink text), LMP1-induced genes (Fig. , green text), and EBV- and LMP1-induced genes (Fig. , purple text) are in this node. Some, such as EBI2, SP100, and PP2Aβ, are activation signature genes and EBV induced (Fig. , black text), and others, such as IRF-4 and interleukin-6, are activation signature genes and both EBV and LMP1 induced (Fig. , asterisks). The second node encompasses genes that are expressed at lower levels in LCLs and IgM-stimulated B cells than in germinal-center B cells or germinal-center-derived lymphomas. TTG-2, PKCδ, A-myb, BCL-7, OGG1, and BCL-7A are germinal-center signature genes that are expressed at low levels in LCLs (Fig. , blue text) (3
). Some genes in this cluster, such as MEF2b, CD79B, and GLUT5, were identified as EBV repressed in these studies and are not part of the germinal-center signature (Fig. , purple text), whereas JAW1, CD10, and RGS13 are EBV-repressed and germinal-center signature genes (Fig. , black text). Overall, EBV latency III gene expression is similar to that of IgM-stimulated B cells.