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. 2012 Dec 20;10(1):43.
doi: 10.1186/1478-811X-10-43.

Global Gene Expression Changes of in Vitro Stimulated Human Transformed Germinal Centre B Cells as Surrogate for Oncogenic Pathway Activation in Individual Aggressive B Cell Lymphomas

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Global Gene Expression Changes of in Vitro Stimulated Human Transformed Germinal Centre B Cells as Surrogate for Oncogenic Pathway Activation in Individual Aggressive B Cell Lymphomas

Alexandra Schrader et al. Cell Commun Signal. .
Free PMC article

Abstract

Background: Aggressive Non-Hodgkin lymphomas (NHL) are a group of lymphomas derived from germinal centre B cells which display a heterogeneous pattern of oncogenic pathway activation. We postulate that specific immune response associated signalling, affecting gene transcription networks, may be associated with the activation of different oncogenic pathways in aggressive Non-Hodgkin lymphomas (NHL).

Methodology: The B cell receptor (BCR), CD40, B-cell activating factor (BAFF)-receptors and Interleukin (IL) 21 receptor and Toll like receptor 4 (TLR4) were stimulated in human transformed germinal centre B cells by treatment with anti IgM F(ab)2-fragments, CD40L, BAFF, IL21 and LPS respectively. The changes in gene expression following the activation of Jak/STAT, NF-кB, MAPK, Ca2+ and PI3K signalling triggered by these stimuli was assessed using microarray analysis. The expression of top 100 genes which had a change in gene expression following stimulation was investigated in gene expression profiles of patients with Aggressive non-Hodgkin Lymphoma (NHL).

Results: αIgM stimulation led to the largest number of changes in gene expression, affecting overall 6596 genes. While CD40L stimulation changed the expression of 1194 genes and IL21 stimulation affected 902 genes, only 283 and 129 genes were modulated by lipopolysaccharide or BAFF receptor stimulation, respectively. Interestingly, genes associated with a Burkitt-like phenotype, such as MYC, BCL6 or LEF1, were affected by αIgM. Unique and shared gene expression was delineated. NHL-patients were sorted according to their similarity in the expression of TOP100 affected genes to stimulated transformed germinal centre B cells The αIgM gene module discriminated individual DLBCL in a similar manner to CD40L or IL21 gene modules. DLBCLs with low module activation often carry chromosomal MYC aberrations. DLBCLs with high module activation show strong expression of genes involved in cell-cell communication, immune responses or negative feedback loops. Using chemical inhibitors for selected kinases we show that mitogen activated protein kinase- and phosphoinositide 3 kinase-signalling are dominantly involved in regulating genes included in the αIgM gene module.

Conclusion: We provide an in vitro model system to investigate pathway activation in lymphomas. We defined the extent to which different immune response associated pathways are responsible for differences in gene expression which distinguish individual DLBCL cases. Our results support the view that tonic or constitutively active MAPK/ERK pathways are an important part of oncogenic signalling in NHL. The experimental model can now be applied to study the therapeutic potential of deregulated oncogenic pathways and to develop individual treatment strategies for lymphoma patients.

Figures

Figure 1
Figure 1
Identification of IL21, CD40L, αIgM, BAFF and LPS regulated genes in transformed human germinal centre B cells using microarrays. BL2 cell were stimulated with αIgM F(ab)2 fragments (3 hrs) (A), IL21 (2 hrs) (B), CD40L (6 hrs) (C), LPS (6 hrs) (D) and BAFF (9 hrs) (E). RNAs from these cells were used for gene expression profiling on Affymetrix HGU-133 plus 2.0 microarray chips. The heatmaps show the most highly changed gene expression (TOP100) (adj. p-value ≤ 0.05) in response to each stimulus. Each row in the heatmaps represents a gene and each column represents a microarray sample. Yellow and blue indicate high and low expression, respectively. Control probes (left column) are shown in comparison to stimulated probes (right column). Additional details are summarized within Table 3 and suppl. Tables 1–5.
Figure 2
Figure 2
Expression of a selection of genes after stimulation in transformed human germinal centre B cells as detected by qRT-PCR. BL2 cells were stimulated as described in Figure 2. One representative experiment out of two is shown. All samples were analysed in triplicates. Expression of ICAM1, CD58, CCR7, CXCL10, ID1, BCL6, MYC, RGS1, DUSP2, -10, -5, -22, SLAMF −3, -7, -6, LEF1, BCL9, PYGO1 is shown as 2-ΔΔCT or ΔCT values, relative to abl housekeeper expression and compared to unstimulated control. CXCL10 (D), RGS1 (H), DUSP5 (K) and SLAMF7 (N) show no detectable expression in unstimulated cells (basal ΔCt > 10). Therefore, expression is depicted as ΔCt values. N.E. = Not Expressed.
Figure 3
Figure 3
Overlap in global gene expression of IL-21, CD40L, αIgM, BAFF and LPS affected genes. BL2 cells were stimulated and data were processed as described in the text. The overlaps of differentially expressed gene lists at both ends were compared using the “Ordered List package” [55]. Genes affected by the stimuli assigned on top of each plot are compared. The gene lists are compared in the same (blue line) or reversed order (green line). The y-axis gives the size of the overlap in the number of genes of the top n-genes (x-axis) in both gene lists. The orange line gives the expected size of a random overlap, and the vertical orange lines indicate 95% probability intervals of random overlaps. All overlaps show a high statistical significance (α < 0.005). Additional details are summarized within Additional file 10: Table S6, Additional file 11: Table S7, Additional file 12: Table S8, Additional file 13: Table S9, Additional file 14: Table S10, Additional file 15: Table S11, Additional file 16: Table S12, Additional file 17: Table S13, Additional file 18: Table S14, Additional file 19: Table S15.
Figure 4
Figure 4
Changes in global gene expression separates Burkitt lymphoma and diffuse large B cell lymphoma. The expression of the TOP100 most prominent responding genes upon stimulation of BL2 with αIgM F(ab)2 fragments was investigated in primary lymphoma. Left panel: 220 lymphoma cases [20]; right panel: 99 lymphoma cases [21]. NHL cases were ordered from left to right according to their expression of αIgM affected genes in BL2 cells. The Heatmaps display the expression of target genes (columns) across lymphoma samples (rows). The lower colour bar above the heatmaps marks mBL in red, non-mBL in green and intermediate lymphoma in yellow (molecular diagnosis). The affiliation of samples to ABC/GCB DLBCL subgroups and the presence of an IG-MYC translocation is encoded in the middle and upper bar on top of the map (see legend for colour coding). Relative gene expression is encoded with yellow (high expression) and blue (low expression). As different microarrays were used for the whole genome expression analyses of cell perturbation and patient samples (Affymetrix HGU-133A and HGU133 plus2.0), the list of TOP100 genes had to be adapted to be able to transfer the resulting genes to patient data (see also Table 3, Material and Methods section and Supplementary material for additional details).
Figure 5
Figure 5
Individual diffuse large B cell lymphomas are characterized by a specific activation of gene modules. The TOP100 most prominent responding genes upon stimulation of BL2 with αIgM, CD40L, IL21, LPS and BAFF (see also Table 1) were investigated for their expression in the gene expression profiles of two distinct datasets of primary lymphoma. (A) left panel: 175 DLBCLs [20]; right panel: 99 DLBCL cases [21]. DLBCL cases were ordered from left to right according to the similarity of gene expression to the stimulated status of BL2 cells. The Heatmaps display the expression of target genes (columns) across lymphoma samples (rows). The colour bar above the heatmaps marks mBL in red, non-mBL in green and intermediate lymphoma in yellow. The affiliation of samples to ABC/GCB DLBCL subgroups and the presence of an IG-MYC translocation in encoded in a bar on top of the map (see legend for colour coding). Relative gene expression is encoded with yellow (high expression) and blue (low expression). Additional details on statistical significant differences are summarized within Additional file 7: Figure S1.
Figure 6
Figure 6
Pathways involved in the regulation of a selected set of genes in response to αIgM treatment. (A) Prior knowledge scheme of monitored pathways. Dashed lines implicate the presence of additional signalling molecules interlinking the depicted factors, solid lines reflect a direct link. (B) hierarchical presentation of kinases affected by utilized chemical inhibitors [2]. (C-N) BL2 cell were preincubated for 3 hrs with 2 μM SB203580 (p38), 10 μM SP600125 (JNK), 10 μM U0126 (MAP2K), 100nM 5Z-7-oxozeaenol (TAK), 7 μM ACHP (IKK2) or 10 μM Ly294002 (PI3K) inhibitors and then stimulated by 1.3 μg/ml αIgM F(ab)2 fragments for additional 3 hrs in the presence of respective inhibitors. Cells were harvested to isolate RNA for corresponding qRT-PCR. Expression of the following genes is shown: SGK1, PYGO1, SLAMF3, EGR2, ID3, CCR7, DUSP2, SLAMF6, MYC, LEF1, IRF4 and RGS1. Results are presented as 2-ΔΔCT values, relative to abl housekeeper expression and compared to the corresponding unstimulated inhibitor treated control. As RGS1 (N) expression is below detectable levels in unstimulated probes, only ΔCt values relative to stimulated control without inhibitors were compared. One representative experiment out of three or more biological replicates is shown. Cells were treated with DMSO (1/8) 5Z-7-oxozeaenol (2/9), ACHP (3/10), SB203580 (4/11), SP600125 (5/12), Ly294002 (6/13), U0126 (7/14) in the absence (1–7) or presence of αIgM F(ab)2 fragments (8–14). n.e. – not expressed. Details on pathway activations as measured by immunoblot, kinase activity and Ca2+ influx analysis are summarized in Additional file 24: Figure S3.
Figure 7
Figure 7
Schemes summarizing investigated kinase involvement in αIgM mediated gene expression. αIgM mediated activation / induction of gene expression (A) and suppressive effects of pathway elements onto selected genes affecting basal and αIgM mediated gene expression (B). Within the scheme (A) genes are listed and sorted according to the groups described within the main text in relation to corresponding kinases. Data for gene expression changes of BCL6, BCL9, DUSP5, DUSP10 and SLAMF7 are described within Additional file 25: Figure S4.

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