Expression of Circadian Core Clock Gene Per2 Is Markedly Down-Regulated in Diffuse Large B-Cell Lymphoma

Abstract

Non-Hodgkin's lymphoma (NHL) is a heterogeneous clinicopathologic entity characterized by distinct cells of origin, cytogenetic and molecular aberrations. In vivo studies recently showed that mice deficient in Period (PER) 2 developed lymphomas. Furthermore, we and others have recently suggested that the core circadian clock genes Period (Per)1 and Per2 have been linked to DNA damage response pathways. We showed that both genes are involved in tumor suppression by regulating cell cycle- and apoptosis-related genes. In mammals and other vertebrates, heterodimers of the basic helix-loop-helix-PAS transcription factors, CLOCK and BMAL1, activate transcription of the Period and Cryptochrome (Cry1 and Cry2) genes via binding to E-box sequences in their promoters. Subsequently, the PER and CRY repressor proteins accumulate in the nucleus and inhibit CLOCK:BMAL1 complexes, thereby inhibiting their own gene expression. This forms the major negative circadian feedback loop. In the present study, we focused on Per1 and Per2 and their role in different types of NHL. Real-time reverse transcriptase-polymerase chain reaction (real time RT-PCR) was performed to determine the mRNA expression levels of Per1 and Per2 in patients with diffuse large B-cell lymphoma (DLBCL; n = 37), mantle cell lymphoma (MCL; n = 12), follicular lymphoma (FL; n = 12), and Burkitt's Lymphoma (n = 6) compared to human normal tonsil samples (n = 10). We further tested their expression in 14 different human lymphoma cell lines. The relative expression of Per2 was markedly down-regulated in all DLBCL samples compared to normal tonsils (mean fold change: − 20.5 ± 28.7; p < 0.001). On the other hand, levels of expression of Per2 in samples of either MCL (2.5 ± 2.6; p = 0.1), FL (1.1 ± 10; p = 0.9), or Burkitt's Lymphoma (− 1.1 ± 2; p = 0.07) showed no significant change compared to the normal tonsils. Moreover, we found no significant regulation of the mRNA levels of Per1 in any of the four NHL subtypes. We confirmed our results by showing concordant results in human lymphoma cell lines. Again, Per2 expression levels were persistently down-regulated in the 7 DLBCL cell lines (OCI-Ly1, -Ly4, -Ly7, -LY10, and SUDHL-4, -6, -16; mean fold change: − 8.6 ± 5.7; p < 0.001) compared to normal tonsils, but not in MCL (SP-49, Jeko-1, NCEB-1), FL (FLK-1), Burkitt's lymphoma (Daudi), and B-precursor acute lymphoblastic leukemia (Nalm-6, BALL-1) cell lines (p > 0.05). Western blot protein analyses showed easily detectable levels of PER2 in tonsil samples, but the protein was either not expressed or significantly reduced in expression in human DLBCL cell lines. In summary, our results strongly suggest for the first time that the disruption of the key circadian clock gene Per2 may play a role in the development of DLBCL.