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. 2014 Jan;5(1):80-9.
doi: 10.1007/s13238-013-0017-9. Epub 2014 Jan 29.

Gadd45a Deletion Aggravates Hematopoietic Stem Cell Dysfunction in ATM-deficient Mice

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Free PMC article

Gadd45a Deletion Aggravates Hematopoietic Stem Cell Dysfunction in ATM-deficient Mice

Yulin Chen et al. Protein Cell. .
Free PMC article

Abstract

Ataxia telangiectasia mutated (ATM) kinase plays an essential role in the maintenance of genomic stability. ATM-deficient (ATM(-/-)) mice exhibit hematopoietic stem cell (HSC) dysfunction and a high incidence of lymphoma. Gadd45a controls cell cycle arrest, apoptosis and DNA repair, and is involved in the ATM-p53 mediated DNA damage response. However, the role of Gadd45a in regulating the functionality of ATM(-/-) HSCs is unknown. Here we report that Gadd45a deletion did not rescue the defects of T-cells and B-cells development in ATM(-/-) mice. Instead, ATM and Gadd45a double knockout (ATM(-/-) Gadd45a(-/-)) HSCs exhibited an aggravated defect in long-term self-renewal capacity compared to ATM(-/-) HSCs in HSC transplantation experiments. Further experiments revealed that the aggravated defect of ATM(-/-) Gadd45a(-/-) HSCs was due to a reduction of cell proliferation, associated with an accumulation of DNA damage and subsequent activation of DNA damage response including an up-regulation of p53-p21 signaling pathway. Additionally, ATM(-/-) Gadd45a(-/-) mice showed an increased incidence of hematopoietic malignancies, as well as an increased rate of metastasis than ATM(-/-) mice. In conclusion, Gadd45a deletion aggravated the DNA damage accumulation, which subsequently resulted in a further impaired self-renewal capacity and an increased malignant transformation in ATM(-/-) HSCs.

Figures

Figure 1
Figure 1
Gadd45a deletion does not rescue the defect of immune system in ATM−/−mice. (A–D) The WT, Gadd45a−/−, ATM−/−, and ATM−/− Gadd45a−/− mice (2–3 months old) were analyzed by FACS. The percentage of T-cells and/or B-cells (relative to WT) is shown in the indicated hematopoietic tissues. (n = 3–4). (E–F) The LSK cells (CD45.2) from WT, Gadd45a−/−, ATM−/−, and ATM−/− Gadd45a−/− mice were transplanted into lethally irradiated recipient mice (CD45.1). The percentage of donor-derived T-cells and B-cells in PB (relative to WT) was analyzed 2 and 4 months post-transplantation by FACS. (n = 3–4)
Figure 2
Figure 2
Gadd45a deletion aggravates the defect of self-renewal capacity of ATM/HSCs. (A–B) HSCs and HPCs from 2–3 months old mice were analyzed by FACS. The number of LSK cells, HPCs, and LT-HSCs (SLAMhigh) were analyzed and are shown (relative to WT). (n = 4). (C) HSCs were clonally sorted into 96-well plates (one cell per well) from WT, Gadd45a−/−, ATM−/−, and ATM−/− Gadd45a−/− mice and cultured in liquid medium for 14 days. The percentage of colonies was calculated by dividing the number of colonies with the original number of single-cell seeded. (n = 3). (D) Four thousand LSK cells (CD45.2) isolated from WT, Gadd45a−/−, ATM−/−, and ATM−/− Gadd45a−/− mice were transplanted into lethally irradiated recipients (CD45.1), along with competitors (CD45.1/2). The chimerism of donor-derived granulocytes in PB was tested monthly. The result is shown at indicated time point post-transplantation. (n = 5–8). (E) The chimerism of donor-derived indicated population in BM was tested 26 weeks post-transplantation. (n = 5–8). (F) The absolute number of the indicated donor-derived cells in BM was analyzed by FACS 26 weeks after transplantation. (n = 5–8)
Figure 3
Figure 3
Reduced proliferation in ATM/Gadd45a/HSCs after transplantation. (A–C) ROS level, apoptosis, and cell cycle of donor-derived cells were detected 26 weeks after transplantation. (A) ROS level was detected by the fluorescence intensity of DCFDA in LSK cells. The representative FACS plots are shown. (n = 3). (B) Apoptosis of the indicated population was tested by Annexin V/DAPI staining. The percentage of Annexin V positive population was compared in each group. (n = 5–7). (C) Cell cycle of HSCs (LSK gated) and HPCs (LKS gated) was examined by BrdU incorporation. The percentage of BrdU positive population was compared in each group. (n = 5–7)
Figure 4
Figure 4
DNA-damage-induced p53-p21 activation in ATM/Gadd45a/HSCs after transplantation. (A–C) ATM−/− and ATM−/− Gadd45a−/− donor-derived LSK cells were isolated by FACS sorting 26 weeks after transplantation and subjected to fluorescence experiments. (A) The DNA damage accumulation was assessed by comet assay. The representative photos and statistical results are shown. The Olive Tail Moment stands for DNA damage level per cell. (n = 117). (B–C) The expression of p53 and p21 in ATM−/− and ATM−/− Gadd45a−/− donor-derived LSK cells in single-cell level was compared by immunofluorescence. The fluorescence intensity stands for expression level in single cell, which was determined by ImageJ (designed by NIH). (n = 40–60)
Figure 5
Figure 5
Gadd45a prevents the incidence of hematopoietic malignancies in ATM/mice. (A) The ratio of malignancy, pathological type, and metastasis in ATM−/− and ATM−/− Gadd45a−/− mice are shown. (B–C) AML with spleen infiltration was observed in one ATM−/− Gadd45a−/− mice (without lymphoma). The FACS plots in the indicated tissues and the pictures of spleen (infiltrated) and thymus (without lymphoma) of the AML mice are shown. (D) Representative FACS plots in the indicated tissues in ATM−/− (lymphoma without BM infiltration) and ATM−/− Gadd45a−/− (lymphoma with BM infiltration) mice with are shown

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