Profiling of Hodgkin's lymphoma cell line L1236 and germinal center B cells: identification of Hodgkin's lymphoma-specific genes

Abstract

The malignant cells of classical Hodgkin's lymphoma (cHL), Hodgkin and Reed-Sternberg (HRS) cells, appear to be derived from germinal center (GC) B cells in most cases of the disease. Apart from recent findings of constitutive activation of some transcription factors and autocrine stimulation by cytokine receptors, the mechanisms of malignant transformation in cHL still remain poorly understood. We performed a large scale gene expression study using serial analysis of gene expression (SAGE), comparing the cHL cell line L1236 and human GC B cells. Semiquantitative RT-PCR was used to confirm results from the SAGE and to analyze gene expression in 3 additional cHL cell lines. To investigate expression of some genes in cHL cases, we applied RT-PCR on microdissected HRS cells. In total, 464 genes showed a change in expression level of 5-fold or higher. For 12 genes (out of 177) identified as upregulated in L1236 cells, RT-PCR confirmed the SAGE results and also showed elevated expression in 3 other cHL cell lines. For 3 of the upregulated genes, expression by HRS cells in the tissue also was confirmed. Several of the differentially expressed genes may play a role in the pathogenesis of cHL because they represent potential oncogenes, such as rhoC, L-myc, and PTP4A, or transcription factors, such as ATF-5, ATBF1, and p21SNFT. The genes that showed significantly deregulated expression in HRS cells should be helpful not only for the identification of genes involved in the pathogenesis of cHL but also for discovering potential prognostic markers or therapeutic targets.

Figure 1

FACS analysis of human tonsillar mononucleated cells after Ficoll purification (A) and after MACS enrichment of CD77⁺ cells (B). The purity is higher than 95%. The figure shows the result for 1 of 8 tonsils used for the SAGE profile.

Figure 2

Semiquantitative RT-PCR analysis for 12 genes in L1236 and GC B cells (CB, centroblasts). cDNA of L1236 and GC B cells was normalized to β-actin by real-time PCR. Equal amounts of cDNA were introduced in RT-PCR analysis using 4 different cDNA dilutions for L1236 (left panel) and GC B cells (right panel). RT-PCR was performed until a faint band was detectable in the lowest dilution of the L1236 cDNA. By comparing the intensity of the bands of L1236 and GC B cells on an agarose gel, the difference of expression for a given gene was estimated. Normal RT-PCR analysis of β-actin is given in the lowest panel and shows the same amount of PCR product for both cell populations. In total, 12 genes found to be upregulated in the SAGE analysis were analyzed. For some cDNAs primerdimers are visible and represent the lower bands. Tag numbers are normalized to 30000 tags.

Figure 3

Semiquantitative RT-PCR analysis for 12 genes in 4 HL cell lines (L428, KMH2, HDLM2, and L1236). The cDNA of the 4 HL cell lines was normalized to β-actin by real-time PCR. RT-PCR analysis of β-actin is given in the lowest panel and shows the same amount of PCR product for all cell populations. A limited cycle number was performed to retain the differences in band intensities in the gel analysis. For each gene, the PCR results are shown for the lowest cycle number at which a PCR product was obtained for all 4 cell lines. These cycle numbers were between 28 and 35 cycles. For some cDNAs primerdimers are visible and represent the lower bands. For 2 transcripts, PTP4A and L-myc, L1236 is the cell line which shows the highest expression of the 4 HL cell lines. Therefore, for these genes it cannot clearly be stated that the expression level in the 3 other HL cell lines is much higher than that in GC B cell because GC B cells were not included in this analysis.