Abstract
Hodgkin Lymphoma (HL) is unique in its cellular composition, consisting of a minority of tumor cells in an inflammatory background. The tumor cells (Reed-Sternberg cells, RS cells) are thought to be derived from germinal center B cells (centroblasts, CB) that have escaped apoptosis during antigen selection. Global gene expression studies in HL have been hampered by the fact that RS cells only represent less than 1% of the cells in the tumor tissue. Only a few large-scale gene expression studies are available, mostly using cell lines derived from patients with classical HL. To get more insight into the gene expression profile of RS cells we applied the Serial Analysis of Gene Expression Technique (SAGE) on several HL cell lines. SAGE was performed on the cell lines L428 and L1236 (classical cell lines, cHL), DEV (Nodular Lymphocyte Predominant cell line, NLP) and on CB as normal counterparts.
In total, about 100.000 tags were sequenced (Table 1). After normalization and exclusion of tags present only once, 3.635 genes were retained and used for further analyses. A five-fold difference in tag frequency between the HL cell lines and CB was used to select for up- or downregulated genes. Although many genes were upregulated in every individual HL cell line compared to CB, only few genes were consistently upregulated. In contrast, comparison of the downregulated genes revealed that 125 were downregulated in all three HL cell lines compared to CB (Table 2). Tags corresponding to Fascin, TARC/CCL17 and BIC, known to be highly expressed in HL indeed were observed in the SAGE libraries. Among the genes downregulated in L428 a loss of B lineage-specific genes was observed confirming the previous findings in L1236. Interestingly our SAGE results suggest that the NLP cell line DEV has an intermediate loss of B-lineage specific genes compared to CB. To gain more insight in the biology, differentially expressed genes were grouped according to gene ontology’s (based on biological process or molecular function) and pathways. We have now selected more than 70 genes that will be subjected to quantitative RT-PCR analysis to confirm our SAGE results and to study their expression in tumor cell enriched HL samples.
In conclusion: Downregulation of gene expression seems to be important in the pathogenesis of HL since we observed a consistent downregulation of more than 100 genes in HL compared to CB.
Table 1. Overview of Hodgkin lymphoma SAGE libraries
Table 2. Number of up- and downregulated genes in Hodgkin lymphoma compared to centroblasts
Library DEV L428 L1236 CB HL type NLP cHL cHL Centroblasts Tags 16316 20990 30623 29787 Genes 4703 5602 10561 11095 L428 L1236 cHL DEV cHL & NLP HL L428 and L1236 are derived from classical Hodgkin lyphoma (cHL) cases, DEV is derived from a case of Nodular Predominant Hodgkin lymhoma (NLP HL) Upregulated 396 116 14 330 7 Downregulated 329 169 141 309 125
Gene expression studies in radiation-sensitive cell lines