CEBPalpha and a Critical Circadian Clock Downstream Target Gene, PER2, Are Highly Dysregulated in Diffuse Large B-Cell Lymphoma.

Abstract 3454

Poster Board III-342

Non-Hodgkin's lymphoma (NHL) is a heterogeneous clinicopathologic entity characterized by distinct cells of origin and unique cytogenetic and molecular aberrations. Recently, we found by transcription profiling of CCAAT/enhancer-binding proteins (CEBPs) that they could regulate expression of the core circadian clock gene Period (PER) 2. We now report that C/EBPalpha and PER2 are highly dysregulated specifically in diffuse large B-cell lymphoma (DLBCL), one of the commonest type of high-grade NHL. Real time RT-PCR analysis of human samples of diffuse large B-cell lymphoma (DLBCL; n = 37), mantle cell lymphoma (MCL; n = 12), follicular lymphoma (FL; n = 12), and Burkitt's lymphoma (BL; n = 6) compared to normal tonsil samples (n = 10) revealed a markedly downregulated expression of both CEPBalpha and PER2 specifically in the DLBCL samples. The expression of these two genes was also significantly decreased in the 7 human DLBCL cell lines, OCI-Ly1, -Ly4, -Ly7, -LY10, and SUDHL-4, -6, -16. In contrast, mRNA levels of CEBPalpha and PER2 in samples of MCL, FL or BL showed no significant change compared to the normal tonsils, which were confirmed in cell lines of MCL (SP-49, Jeko-1, NCEB-1), FL (FLK-1) and BL (Daudi). Moreover, PER1, another important circadian rhythm gene of the Period family, showed no differences of expression in any of the NHL samples compared to control lymph nodes. The murine pro-B lymphoid cell line Ba/F3 had low levels C/EBPalpha, and forced expression of the transcription factor resulted in decreased growth and increased apoptosis as measured by trypan blue cell count and Annexin V (FACS), suggesting that this transcription factor may induce apoptosis in lymphoma. Moreover, transfection of Ba/F3 cells with a zinc-inducible CEBPalpha gene increased PER2 expression levels by 7-fold. The analysis of the PER2 promoter region showed several potential CEBPalpha binding sites, and chromatin immunoprecipitation (ChIP) as well as luciferase reporter assay experiments using Ba/F3 cells revealed that CEBPalpha can directly bind to the PER2 promoter and induce its expression. Interestingly, treatment of Ba/F3 and several DLBCL cell lines (Ly-4, SUDHL-4, -6) with the histone deacetylase-inhibitor, SAHA, significantly induced expression of both CEBPalpha and PER2, which was associated with inhibition of cell growth and apoptosis. Our further studies explored the consequences of expressing PER2 on cell proliferation. Forced expression of PER2 in Ba/F3 cells led to substantial growth reduction, G0/G1 cell cycle arrest, and altered protein expression of apoptosis-related genes. The protein level of Bcl-X(L) was downregulated, whereas levels of Bax and PARP cleavage activity were upregulated compared with control cells transfected with empty plasmid. In summary, our results show for the first time that both CEBPalpha and its key downstream target circadian clock gene, PER2, are highly dysregulated in DLBCL. Our data strongly suggest that these genes may play a role in the pathogenesis of this disorder and should be considered for therapeutic manipulation.