Identification of Cyclin D1-Dependent Genes by Gene Expression Profiling in MCL Cell Lines and Primary Cases.

The hallmark of MCL is overexpression of cyclin D1 due to the t(11;14). As cyclin D1 plays a central role in cell cycle regulation and influences the activity of several transcription factors, its overexpression is believed to initiate MCL lymphomagenesis. To dissect the transcriptional network regulated by cyclin D1, and to assess the relationship of cyclin D1 to genes comprising the recently described MCL proliferation signature, the consequences of cyclin D1 knockdown were analyzed by comparative gene expression profiling.

Four MCL cell lines, Granta 519, Jeko-1, Rec-1 and Z-138 were transduced with lentivirus containing the cyclin D1 shRNA or with empty lentivirus in triplicates. At day seven after infection, RNA was extracted and used for Gene Chip expression analysis (U133 Plus 2.0 arrays/ Affymetrix). After robust multi-array average (RMA) preprocessing, genes which were differentially expressed in all four cell lines were identified using BioConductor software. Validation was performed by correlating cyclin D1 levels with candidate gene expression by qRT-PCR in 10 primary MCL cases.

To identify genes regulated by cyclin D1 in all four cell lines, statistical analysis applying a false discovery rate of 10% was performed, thereby accepting only genes with a >1,1 and <0,9 fold ratio and an average expression >25. We identified 344 genes regulated after cyclin D1 knockdown (93 upregulated, 251 downregulated). Classification of the differentially expressed genes to biological processes revealed overrepresentation of genes involved in replication (23,0%), chromatin packaging and remodelling (12,5%), signal transduction (10,5%), metabolism (9,9%), transcription (9,6%) and cell cycle (6,4%). Of the 20 genes comprising the MCL proliferation signature (Rosenwald et al., Blood 2003), 10 were identified as cyclin D1 dependent. These genes were validated in MCL with high vs. low levels of cyclin D1 mRNA. Although there was a direct correlation between these 10 genes and cyclin D1 levels, it is notable that there was no correlation between the proliferation rate as assessed with MiB1 and the level of cyclin D1 expression

Comparative gene expression profiling before and after knockdown of cyclin D1 in MCL cell lines reveals a complex transcriptional network influenced by cyclin D1 expression levels. We also show that 10 of the 20 genes comprising the MCL proliferation signature are cyclin D1-dependent. These genes showed good correlation with cyclin D1 mRNA levels in primary cases, however, no correlation with proliferation rate was identified, suggesting cyclin D1-independent mechanisms governing proliferation in MCL. The identification of cyclin D1-dependent genes assists in our understanding of the contribution of cyclin D1 overexpression to the biology and clinical course of MCL.

No relevant conflicts of interest to declare.