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. 2016 Jul;101(7):e272-6.
doi: 10.3324/haematol.2015.139675. Epub 2016 Apr 1.

Tumor suppressors BTG1 and BTG2 regulate early mouse B-cell development

Esther Tijchon  1 , Liesbeth van Emst  1 , Laurensia Yuniati  1 , Dorette van Ingen Schenau  1 , Jørn Havinga  1 , Jean-Pierre Rouault  2 , Peter M Hoogerbrugge  3 , Frank N van Leeuwen  1 , Blanca Scheijen  4
Affiliations

Tumor suppressors BTG1 and BTG2 regulate early mouse B-cell development

Esther Tijchon et al. Haematologica. 2016 Jul.
No abstract available

Keywords: aggressive non-Hodgkin lymphoma; hematopoiesis; lymphocytes; pediatric acute lymphoblastic leukemia.

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Figures

Figure 1.
Figure 1.
Decreased B-cell numbers in mice deficient for Btg1 and Btg2. (A–B) Flow cytometric analysis was performed on mononuclear cells isolated from bone marrow (BM) and spleen of wild-type (WT) control (n=16), Btg1−/− (n=7), Btg2−/− (n=8) and Btg1−/−;Btg2−/− mice (n=11). The fractions of B220+ BM cells (A) and B220+IgM+ splenocytes (B) are indicated. Each dot represents an individual sample and the horizontal line indicates the mean. (C) Competitive repopulation assay of wild-type and Btg1−/−;Btg2−/− donor cells (CD45.2) derived of BM mixed 1:1 with CD45.1 wild-type cells which were transplanted into 9Gy (Gray) irradiated C57BL6/J mice (CD45.1). Flow cytometric analysis was performed on blood and spleen. The fraction of B220, CD3 and Mac-1 of WT:WT (CD45.1:CD45.1) (n=4) and WT: Btg1−/−; Btg2−/− (CD45.1:CD45.2) (n=6) cells are indicated. (D) Schematic representation of the different B-cell developmental stages according to Hardy nomenclature. Cell surface markers that distinguish specific Hardy fractions are indicated. (E) The percentage for the different Hardy B-cell fractions within BM is indicated as determined in WT control (n=16), Btg1−/− (n=7), Btg2−/− (n=8), and Btg1−/−; Btg2−/− (n=11) mice. Each bar represents the mean of each group of mice ± the standard error of the mean (SEM). *P<0.05, **P<0.01, ***P<0.001.
Figure 2.
Figure 2.
Btg1 stimulates B-cell progenitor outgrowth in response to IL-7. (A) Bone marrow (BM) mononuclear cells (1 × 105 cells) were isolated from wild-type (WT) control (n=18), Btg1−/− (n=7), Btg2−/− (n=8) and Btg1−/−; Btg2−/− mice (n=11) and added in methylcellulose containing 10 ng/mL IL-7. (B) BM mononuclear cells were isolated from WT control (n=10), Btg1−/− (n=7), Btg2−/− (n=4) and Btg1−/−; Btg2−/− mice (n=4) and added after CD19 isolation (2 × 104 CD19+ cells) in methylcellulose containing 10ng/ml IL-7. (A, B) Mean colony counts (and SEM) were determined (>30 cells/colony) after seven days of culture. Data are representative of at least two independent experiments. **P<0.01, ***P<0.001. (C) BM cells from WT, Btg1−/−, Btg2−/−, and Btg1−/−; Btg2−/− mice (all n=2) were stained with B220 and IL-7Rα/CD127 antibodies and the percentage of IL-7Rα-positive cells within the B220+ fraction was determined. The black line indicates IL-7Rα expression in the WT cells, while the blue peak shows the IL-7Rα expression in the Btg1−/−, Btg2−/− and Btg1−/−; Btg2−/− double knockout cells. (D) Apoptosis assay of CD19+ BM cells isolated from WT (n= 4) and Btg1−/− (n=7) cultured for 4 days in B-cell specific medium, by measuring Annexin V using flow cytometry. The percentage of Annexin V-positive cells in CD19+ BM cells is indicated. (E) Proliferation assay of CD19+ BM cells isolated from WT (n= 3) and Btg1−/− (n=3) cultured for 4 days in B-cell specific medium, measuring Ki-67 by flow cytometry. The cell cycle distribution according to Ki-67 staining in CD19+ BM cells is indicated. Each bar represents the mean of each group of mice ± the standard error of the mean (SEM). **P<0.01, ***P<0.001.
Figure 3.
Figure 3.
Aberrant T-lineage gene expression in progenitor B-cells deficient for Btg1 and Btg2. (A) Relative expression levels of B-cell transcription factors E2A (Tcf3), Foxo1, Ebf1 and Pax5 was determined using cDNA generated from B220+ bone marrow (BM) cells of wild type control (WT), Btg1−/−, Btg2−/− and Btg1−/−; Btg2−/−mice by qRT-PCR and normalized to the expression of the housekeeping gene TBP. (B) RNA was isolated from B220+CD43+ and B220+CD43− cells derived from wild type control and Btg1−/−; Btg2−/− mice (n=4) and analyzed by microarray using the Illumina BeadArray platform. The data obtained were RMA normalized and relative gene expression differences of >1.6-fold was determined. KEGG pathway analysis was performed using the online Gene Set Enrichment Analysis (GSEA) tool and relevant pathways with highest P-value that are differentially expressed between WT and Btg1−/−; Btg2−/− cells are indicated. (C) Relative expression levels of Cd4, Ikzf2, Tcf7, Gata3 and Notch1 were determined on cDNA generated from B220+ BM cells of the different genotypes by real-time PCR and normalized to TBP expression. (A and C) Data represent the mean and SEM of three independent experiments containing cDNA derived from 2 different biological samples. *P<0.05, **P<0.01, ***P<0.001.
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