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. 2014 Jan 30;9(1):e87299.
doi: 10.1371/journal.pone.0087299. eCollection 2014.

Mitogen-induced B-cell proliferation activates Chk2-dependent G1/S cell cycle arrest

Pavel A Nikitin  1 , Alexander M Price  1 , Karyn McFadden  1 , Christopher M Yan  1 , Micah A Luftig  1
Affiliations

Mitogen-induced B-cell proliferation activates Chk2-dependent G1/S cell cycle arrest

Pavel A Nikitin et al. PLoS One. .

Abstract

B-cell activation and proliferation can be induced by a variety of extracellular stimuli. The fate of an activated B cell following mitogen stimulation can be dictated by the strength or duration of the signal, the expression of downstream signaling components necessary to promote proliferation, and the cell intrinsic sensors and regulators of the proliferative program. Previously we have identified the DNA damage response (DDR) signaling pathway as a cell intrinsic sensor that is activated upon latent infection of primary human B cells by Epstein-Barr virus (EBV). Here we have assessed the role of the DDR as a limiting factor in the proliferative response to non-viral B-cell mitogens. We report that TLR9 activation through CpG-rich oligonucleotides induced B-cell hyper-proliferation and an ATM/Chk2 downstream signaling pathway. However, B-cell activation through the CD40 pathway coupled with interleukin-4 (IL-4) promoted proliferation less robustly and only a modest DDR. These two mitogens, but not EBV, modestly induced intrinsic apoptosis that was independent from the DDR. However, all three mitogens triggered a DDR-dependent G1/S phase cell cycle arrest preventing B-cell proliferation. The extent of G1/S arrest, as evidenced by release through Chk2 inhibition, correlated with B-cell proliferation rates. These findings have implications for the regulation of extra-follicular B-cell activation as it may pertain to the development of auto-immune diseases or lymphoma.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Multiple B-cell mitogens induce an initial burst of hyper-proliferation.
Kinetics of B cell proliferation in response to (A) Epstein-Barr virus infection, (B) constant stimulation with TLR9 ligand CpG (2.5 µg/mL), or (C) CD40L (5 ng/mL) coupled with interleukin-4 (20 pg/mL) treatment. The mean division number based on precursor cohort analysis and plotted over time post infection or treatment is shown. Vertical dashed lines indicate the period of hyper proliferation. (D) The mean division number and time to first division was calculated as in , for the period of hyper-proliferation.
Figure 2
Figure 2. B-cell mitogens activate the ATM signaling pathway in proliferating cells.
(A) Representative FACS plot of stimulated PBMCs showing the distribution of Cell Trace Violet stained cells versus CD19 staining for B cells. The indicated proliferating and non-proliferating populations were isolated by FACS sorting for subsequent analysis. (B) Immunofluorescence microscopy for γ-H2AX (red) of sorted non-proliferating (Non Prolif) or proliferating (Prolif) B cells. The DAPI stains for DNA are shown. (C) Untreated and 5 Gy γ-irradiated controls assayed as described in (B). (D) The average percentage of cells with γ-H2AX intensity >5X over background are plotted for three normal donors. Samples include B cells, untreated (0), 1 Gy, or 5 Gy γ-irradiation, sorted non proliferating (NP) and proliferating (P) cells for EBV, CpG, or αCD40/IL-4 treatments. Quantification is detailed in Figure S1. (E) Western blot analysis of Chk2, Chk2 phosphorylated on Thr68 as well as the control GAPDH. Untreated (Cntr) and 5 Gy irradiated (IR) Burkitt’s lymphoma cell line 2 (BL2) are included as a positive control. Numbers indicate the normalized densitometry value for phospho-Chk2 Thr 68.
Figure 3
Figure 3. Chk2 inhibition increases proliferation of human B cells.
FACS profile of (A) EBV infected (B) CpG or (C) CD40L/IL-4 treated CD19+ cells at the day of peak proliferation post infection/stimulation in the presence of DMSO (red) or 5 µM Chk2i (blue). For EBV this was day 8 post infection, CpG was day 7 post stimulation, and CD40L/IL-4 was day 6 post stimulation. (D) Relative number of proliferating CD19+ cells were gated and counted over internal beads control and normalized to DMSO-treated sample on the days described in (A–C). Experiment was repeated on PBMC from 3–6 normal donors. Error bars are SEM. Significance of Chk2i treatment compared to the DMSO control was calculated via Student’s t test; ***, P-value <0.01; N.S., not significant. CD40L/IL-4 treatment trended towards significance with a P-value of 0.078.
Figure 4
Figure 4. B-cell mitogens, but not EBV, induce caspase 3/7-dependent apoptosis.
(A) Activated apoptosis in proliferating B cells treated with DMSO (top panels) or Chk2i (bottom panels) is measured with DEVD-FAM, a fluorescently labeled soluble caspase 3/7 inhibitor. FACS profiles represent CD19+ single cells. (B) Percent of active caspase-positive cells is plotted for non-proliferating (NP) or proliferating (P) EBV-infected, CpG treated, or αCD40/IL-4 treated CD19+ cells in the presence of DMSO (red) or Chk2i (blue). By comparing the Chk2i treated or DMSO treated conditions we found no statistically significant difference in the percentage of active caspase-positive cells by Student’s t test (P-value >0.05; N.S., Not Significant). Activation of caspases was compared to LCLs treated with 15 µM camptothecin (CPT) or 10 µM Nutlin-3 (Nut-3). (C) Cleavage of caspase targets (PARP and self-processing of caspase-3) revealed by western blot analysis of sorted non-proliferating (NP) or proliferating (P) EBV-infected or mitogen-stimulated B cells.
Figure 5
Figure 5. Mitogen stimulation or EBV infection of human B cells activates Chk2-dependent cell cycle arrest.
(A) Cell cycle profile of EBV-infected or mitogen-induced proliferating CD19+9 B cells treated with DMSO or Chk2i measured by FACS. Cells were pulsed with 25 µM BrdU for 2 hours and subsequently stained with anti-BrdU antibody and propidium iodide (PI). G1, S, and G2/M stages of the cell cycle are indicated. (B) Percent change of cells (from (A)) in S-phase in Chk2 treated over DMSO-treated samples from four to seven normal donors. Error bars are SEM. Significance was calculated with a Student’s t test. N.S., not significant; *, P-value <0.05; **, P-value <0.01; ***, P-value <0.0001.
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