Microarray analysis of B-cell lymphoma cell lines with the t(14;18)

Abstract

The t(14;18) is the most common genetic alteration in follicular lymphoma, and is detectable in a subset of diffuse large B-cell lymphomas (DLBCL), resulting in over-expression of the anti-apoptotic protein BCL-2. Although the t(14;18)-induced over-expression of BCL-2 is an important step in lymphomagenesis, this aberration alone is not sufficient to produce malignant lymphoma. Further analysis of these tumors is needed to identify additional genes that might be involved in the genesis of follicular lymphoma and progression to DLBCL. To address this issue, we analyzed the gene expression profiles of four t(14;18)-positive cell lines and two t(11;14)-positive mantle-cell lymphoma cell lines using cDNA microarrays containing 4364 genes, and compared them to the genetic profile of phenotypically purified B-cells obtained from hyperplastic tonsils. A total of 137 genes were differentially expressed by approximately twofold or more in the t(14;18) cell lines relative to tonsillar B-cells. 68 genes were up-regulated, 69 genes were down-regulated, and approximately 20% of the differentially regulated genes had no known function. The up-regulated genes included a number of genes involved in the promotion of cellular proliferation and survival, as well as cell metabolism. Down-regulated genes included mediators of cell adhesion and negative regulators of cell activation and growth. Hierarchical clustering analysis separated the t(14;18) and mantle-cell lines into distinct groups based on their gene expression profiles. We confirmed the differential expression of approximately 80% of selected up- and down-regulated genes identified by microarray analysis by quantitative real-time fluorescence reverse transcriptase polymerase chain reaction (RT-PCR) analysis and/or immunoblotting. This study demonstrates the utility of cDNA microarray analysis for the assessment of global transcriptional changes that characterize t(14;18)-positive cell lines, and also for the identification of novel genes that could potentially contribute to the genesis and progression of non-Hodgkin's lymphomas with this translocation.

Figure 1.

Unsupervised hierarchical clustering of gene expression data from t(14;18) and mantle-cell lymphoma cell lines using the total complement of 4364 genes. The dendrogram lists the various cell lines and also provides a measure of the relatedness of gene expression in each sample so that samples showing closely related expression profiles are juxtaposed in adjacent branches of the hierarchical dendrogram. Karpas 422, SUDHL-4, SUDHL-6, and OCI-LY1 are t(14;18)-containing B-cell lymphoma cell lines, and NCEB-1 and Granta 519 are t(11;14)-containing mantle-cell lymphoma cell lines. The t(11;14)-containing mantle-cell lymphoma cell lines are closely related and arise from the same branch of the dendrogram. The t(14;18)-containing cell lines arise from a different node and cluster separately from the t(11;14)-cell lines.

Figure 2.

Hierarchical clustering of gene expression data from the four t(14;18)-positive cell lines using the list of 137 previously identified differentially expressed genes contained in Tables 2 and 4 . These genes include all genes over-expressed by approximately twofold or more in at least three of the four t(14;18) cell lines and under-expressed by twofold or more in all four t(14;18) cell lines relative to tonsillar B-cells. The genes corresponding to each horizontal row of the expression matrices are listed on the right side of the figure. Genes that were validated by quantitative fluorescence PCR are denoted by asterisks. The gene expression scale is the same as for Figure 1 .

Figure 3.

Quantitative fluorescence RT-PCR analysis of selected differentially expressed genes identified by microarray analysis in the four t(14;18)-positive cell lines. Expression of the housekeeping gene GAPDH was used as a control for input cDNA in each RT-PCR reaction. Once the level of GAPDH expression was determined for each cDNA sample, it was used to normalize all other genes tested from the same cDNA samples as described in the Materials and Methods. RT-PCR results are depicted as amplification profiles with relative fluorescence on the y axis and cycle number on the x axis. Cell lines with the highest expression of a given gene will have a lower crossing threshold (CT), and therefore, will show an earlier onset of the exponential phase of the amplification curve for that gene. Amplification profiles are included for: (A) stearoyl-CoA desaturase, (B) v-Myb, (C) neurotrophic tyrosine kinase, receptor-related 1, (D) cyclin-dependent kinase 8, (E) human receptor-like tyrosine kinase (H-Ryk), (F) EST 1, (G) EST 2, (H) EST 3, (I) JunB, (J) JunD, (K) FosB, and (L) glyceraldehyde-3-phosphate dehydrogenase control.

Figure 4.

Comparison of cDNA microarray expression data and quantitative fluorescence RT-PCR data for selected differentially expressed genes identified by microarray analysis in the four t(14;18)-positive cell lines. RT-PCR results were calculated as described in the Materials and Methods. We have consistently observed a larger dynamic range of expression for our RT-PCR data relative to the corresponding cDNA microarray expression data. Results are included for: (A) stearoyl-CoA desaturase (SCD), (B) v-Myb, (C) neurotrophic tyrosine kinase, receptor-related 1 (NTRKR1), (D) cyclin-dependent kinase 8 (CDK8), (E) human receptor-like tyrosine kinase (H-RYK), (F) EST 1, (G) EST 2, (H) EST 3, (I) JunB, (J) JunD, and (K) FosB. Note that no microarray data were available for SCD/Karpas 422 (A), SUDHL-6/CDK8 (D), and SUDHL-4/H-RYK (E).