Transcription factor CCAAT/enhancer-binding protein alpha and critical circadian clock downstream target gene PER2 are highly deregulated in diffuse large B-cell lymphoma

Abstract

Disturbances of circadian rhythms and mammalian clock genes have been implicated in the etiologies of many chronic illnesses, including cancer. We show that transcription factor CCAAT/enhancer-binding protein alpha (C/EBPalpha)-regulated PER2 activation is a potential tumor suppressor pathway in diffuse large B-cell lymphoma (DLBCL), one of the commonest types of mature B-cell lymphoma. Expression analysis of human B-cell lymphoma samples including DLBCL (n = 50), mantle cell (n = 21), follicular (n = 25) and Burkitt (n = 18) lymphoma revealed markedly down-regulated CEBPA and PER2 mRNA levels exclusively in DLBCL samples compared to control lymphatic tissue. We demonstrated direct regulation of the circadian core clock gene PER2 by C/EBPalpha in the pro-B cell line Ba/F3, and forced expression of PER2 resulted in decreased proliferation, G0/G1 cell cycle arrest and increased rates of apoptosis. Interestingly, treatment of human DLBCL cell lines with the histone deacetylase-inhibitor suberoylanilide hydroxamic acid (SAHA) significantly increased the expression of C/EBPalpha and Per2, accompanied by cell growth inhibition; in contrast, siRNA knockdown of CEBPA reduced the anti-proliferative effect of SAHA treatment. Our results show for the first time that C/EBPalpha with its associated direct core clock gene target, PER2, are highly deregulated in DLBCL, suggesting an important tumor suppressive pathway in the pathogenesis of this lymphoma entity.

Figure 1

PER2 and CEBPA mRNA expression in B-cell lymphoma samples. (A) Expression of CEBPA was significantly down-regulated in B-cell lymphoma (DLBCL, n = 50) samples in comparison to normal tonsils (control; mean of n = 8) as analyzed by qRT-PCR. (B) Expression of PER2 was significantly down-regulated in DLBCL (n = 50) samples as analyzed by qRT-PCR. (C, D) Expression of CEBPA (C) and PER2 (D) was significantly down-regulated in DLBCL (n = 50) samples in comparison to follicular lymphoma (FL, n = 25), mantle cell lymphoma (MCL, n = 21) and Burkitt lymphoma (BL; n = 18) samples as analyzed by qRT-PCR. Normal tonsils (n = 8) were used as external controls, GAPDH as internal control. Results represent mean ± SD (indicated by bars); ***p < 0.001.

Figure 2

C/EBPalpha directly regulates the PER2 promoter and induces its expression in a murine pro-B cell line. (A) Chromatin immunoprecipitation was performed from Ba/F3 cells using rabbit C/EBPalpha antibody. IgG was used as negative control, input as positive control. Samples were analyzed by PCR using primers specific for the C/EBP site in the murine PER2 promoter, showing that C/EBPalpha protein bound to the promoter region of PER2. Chromatin was analyzed for GAPDH mRNA as an internal control. (B) Reporter gene assay: Ba/F3 cells were co-transfected with the murine Per2 reporter vector (pGL3_Per2) and pMT_C/alpha vector, and showed a mean of 3.5-fold higher transcriptional activity of pGL3_Per2 in comparison to pGL3_Per2 alone (p = 0.01), as well as in comparison to Ba/F3 cells transfected with empty vector pGL3 either with or without pMT_C/alpha. Results represent mean + SD of triplicate transfection; **p < 0.01. (C) Western blot indicates the expression of Per2 in Ba/F3 cells either with empty vector (pMT_ empty) or with pMT_C/alpha. 293T cells transfected with pMT_C/alpha were used as external positive control, GAPDH as internal control.

Figure 3

Influence of PER2 forced expression on proliferation and apoptosis of Ba/F3 cells. (A, B) PER2 forced expression leads to decreased levels of cell proliferation and viability: after transfection of either pcDNA3.1_empty vector or human pcDNA3.1_PER2 into Ba/F3 cells and G418 selection for 5 days, cell proliferation was determined by (A) MTT-assay (day 1 to day 5; results represent mean ± SD; ***p < 0.001; two-way ANOVA; and (B) trypan blue cell counting on day 1 and day 5; untreated Ba/F3 cells (WT) were used as further control; results represent mean + SD; **p < 0.01. (C) PER2 forced expression leads to increased levels of apoptosis in Ba/F3 cells: Ba/F3 cell line was transfected with either pcDNA3.1_empty vector (left panel) or human pcDNA3.1_PER2 (right panel). After selection with G418 (neomycin) for 5 days, annexin V apoptosis assay with FITC/PI labeling was performed by flow cytometry. Lower left quadrant: FITC/PI negative cells; lower right quadrant: FITC positive and PI negative cells, representing early apoptotic cells; upper right quadrant: FITC/PI positive cells, representing late apoptotic and necrotic cells. (D, E) After transfection of either pcDNA3.1_empty vector or human pcDNA3.1_PER2 into Ba/F3 cells, and selection with G418, (D) Western blot was performed using antibodies against indicated proteins on days 0, 3 and 5 of selection; (E) cell cycle was determined by flow cytometry after 5 days of selection. Analysis represents one of three independent experiments, each with comparable results.

Figure 4

Histone deacetylase inhibitor, SAHA, increases mRNAlevels of CEBPA and PER2 inhuman DLBCLcell lines. (A) Proliferation of DLBCL cell lines and Ba/F3 exposed to suberoylanilide hydroxamic acid (SAHA; 0.5,1.25,2.5 and 5 µM) for 4 days was tested via MTT assay, ED₅₀:1.5–2.0 µM (p < 0.01). Exposing respective cells to DMSO as control for 4 days resulted in no significant change of proliferation as measured by MTT assay (data not shown). (B) The DLBCL cell line, Ly4, was cultured in medium ± SAHA (2.0 µM) and cells were harvested after 0,12 and 24 h. Chromatin immunoprecipitation was performed using Ac-H3 antibody to measure H3 acetylation in the CEBPA promoter region by PCR. IgG was used as negative control, input as positive control; chromatin was analyzed for GAPDH mRNA as internal control. (C, D) mRNA and protein were prepared and expression of CEBPA and PER2 was analyzed by qRT-PCR. Results represent mean ± SD; **p < 0.01; for Western blot analysis, antibodies against either C/EBPalpha or Per2 and GAPDH were applied. (E–G) For siRNA experiments, Ba/F3 and SUDHL-6 cells were transfected with plasmid-derived siRNA against CEBPA, siRNA_C/EBPalpha or control (scrambled DNA), and cultured in medium ± SAHA (2.0 µM). (E, F) Trypan blue cell counting was performed on days 1–5 under phleomycin selection. Results represent mean ± SD; **p < 0.01; **p < 0.001 (two-way ANOVA). (G) SUDHL-6 cells were harvested on day 5 of phleomycin selection and Western blot analysis was performed either with anti-C/EBPalpha, anti-Per2 or anti-GAPDH antibody. Western blot analysis shown is representative for one out of three independently performed experiments. Number ± SD indicates fold change of either C/EBPalpha or Per2 as measured by densitometry out of three independent experiments; vehicle-treated SUDHL6 cells transfected with vector control were set as 1. SAHA-treated cells transfected with control showed significantly increased expression of both C/EBPalpha (p = 0.02) and Per2 (p = 0.018), whereas SAHA-treated cells transfected with siRNA_C/EBPalpha showed no significant change in protein expression (both p > 0.05).