Necdin, a p53 target gene, regulates the quiescence and response to genotoxic stress of hematopoietic stem/progenitor cells

Abstract

We recently defined a critical role for p53 in regulating the quiescence of adult hematopoietic stem cells (HSCs) and identified necdin as a candidate p53 target gene. Necdin is a growth-suppressing protein and the gene encoding it is one of several that are deleted in patients with Prader-Willi syndrome. To define the intrinsic role of necdin in adult hematopoiesis, in the present study, we transplanted necdin-null fetal liver cells into lethally irradiated recipients. We show that necdin-null adult HSCs are less quiescent and more proliferative than normal HSCs, demonstrating the similar role of necdin and p53 in promoting HSC quiescence during steady-state conditions. However, wild-type recipients repopulated with necdin-null hematopoietic stem/progenitor cells show enhanced sensitivity to irradiation and chemotherapy, with increased p53-dependent apoptosis, myelosuppression, and mortality. Necdin controls the HSC response to genotoxic stress via both cell-cycle-dependent and cell-cycle-independent mechanisms, with the latter occurring in a Gas2L3-dependent manner. We conclude that necdin functions as a molecular switch in adult hematopoiesis, acting in a p53-like manner to promote HSC quiescence in the steady state, but suppressing p53-dependent apoptosis in response to genotoxic stress.

Figure 1

Necdin is dispensable for fetal hematopoiesis. (A) The frequency of immunophenotypic HSCs in necdin-null (Ndn-null) fetal livers (Lin⁻Sca-1⁺Mac1⁺CD48⁻CD150⁺ cells) quantified by flow cytometry was normal. The mean percentage (± SD) of Lin⁻Sca-1⁺Mac1⁺CD48⁻CD150⁺ cells in the fetal liver are shown (P = .64, n = 15). (B) Serial replating studies were performed using fetal liver cells. CFUs were quantified by methylcellulose culture using wild-type (WT) and necdin-null fetal liver cells. The methylcellulose cultures were serially replated weekly for 7 weeks. Mean values (± SD) are shown (n = 4). (C) Wild-type and necdin-null Lin⁻Sca1⁺Mac1⁺CD150⁺ cells (5 × 10²) were cultured on MS5 stromal cells for 4 weeks and tested for colony formation in the long-term culture-initiating cell assay. Data shown are the mean relative number of colonies formed relative to wild-type colonies (± SD) from 2 independent studies (P = .025, n = 5). (D) Cell-cycle analysis of Lin⁻Sca-1⁺Mac1⁺CD48⁻CD150⁺ cells was performed by staining with Hoechst 33342 and Ki67 and analyzed by flow cytometry using an FITC mouse IgG1 Ab as the isotype control. Ki67⁻ cells are defined as HSCs in G₀ (left panels). Data shown are the mean values (± SD; P = .5, n = 15, right panel). (E) Apoptosis of wild-type and necdin-null fetal liver HSCs was assessed using DAPI and annexin V staining. Data shown are the mean percentage (± SD) of annexin V⁺/DAPI⁻ Lin⁻Sca-1⁺Mac1⁺CD48⁻CD150⁺ cells (P = .71, n = 7). (F) Wild-type or necdin-null fetal liver cells (5 × 10⁵) were transplanted into lethally irradiated recipients, and spleen colonies were scored on days 8 and 12 after transplantation. Numbers indicate average colony numbers (± SD). CFU-spleen day 8 (CFU-S8), P = .53, n = 5; CFU-spleen day 12 (CFU-S12), P = .86, n = 5.

Figure 2

The role of necdin in adult hematopoiesis. (A) Necdin-null (Ndn-null) fetal liver cells (CD45.2) normally repopulated lethally irradiated recipient mice (CD45.1). The frequency of donor-derived cells (CD45.2) in peripheral blood was measured monthly by flow cytometry at 4 time points. SDs are shown (left panel). Right panels show the data from flow cytometry 16 weeks after transplantation. (B) Recipient mice receiving necdin-null fetal liver cells (1 × 10⁶) showed normal multilineage reconstituting activity, as assessed by the percentage of donor-derived myeloid cells (bottom left panel), B cells (top right panel), and T cells (top left panel) 16 weeks after transplantation using flow cytometry. (C) The frequency of HSCs (Lin⁻Sca-1⁺c-kit⁺CD48⁻CD150⁺ cells) in the BM of mice reconstituted with wild-type (WT) or necdin-null fetal liver cells was measured by flow cytometric analysis using SLAM cell-surface markers. Data shown are the mean percentage of HSCs (± SD; P = .1, n = 14). (D) Analysis of the common myeloid progenitor (CMP), granulocyte monocyte progenitor (GMP), and megakaryocyte erythrocyte progenitor (MEP) compartments showed comparable frequencies for those transplanted mice that received wild-type versus necdin-null fetal liver cells (P = .44, P = .41, P = .33, respectively, n = 6).

Figure 3

Loss of necdin has no effects on adult HSC self-renewal in vivo. (A) Serial replating studies. CFUs were quantified by methylcellulose culture using BM mononuclear cells from mice reconstituted with wild-type (WT) or necdin-null (Ndn null) fetal liver cells. The methylcellulose cultures were serially replated weekly for 4 weeks. Mean values (± SD) are shown (n = 4). (B) Donor repopulation after serial BM transplantation. Initially, we transplanted 1 × 10⁶ fetal liver cells from wild-type or necdin-null mice (CD45.2) into lethally irradiated B6.SJL mice (CD45.1). Sixteen weeks after transplantation, we harvested 2 × 10⁶ BM cells from mice reconstituted with wild-type and necdin-null fetal liver cells and transplanted the cells into lethally irradiated B6.SJL mice (CD45.1). The frequency of donor-derived cells (CD45.2) in the peripheral blood was measured 16 weeks after primary, secondary, and tertiary transplantation by flow cytometry. No differences were found between the groups (n = 10). (C) Lethally irradiated recipient mice (CD45.1) were transplanted with 1 × 10⁶ wild-type or necdin-null fetal liver cells (CD45.2) plus 1 × 10⁶ competitor fetal liver cells (CD45.1). The graph shows the mean percentage (± SD) of donor-derived (CD45.2) cells in the peripheral blood 16 weeks after transplantation (n = 7, P = .50). (D) BM cells from mice reconstituted with wild-type or necdin-null fetal liver cells were stained for HSC surface markers and assessed for apoptosis using DAPI and annexin V staining. Data shown are mean percentage of annexin V⁺/DAPI⁻ HSCs (Lin⁻Sca1⁺Mac1⁺CD48⁻CD150⁺ cells) ± SD (P = .86, n = 4, left panel). The right panels show representative flow cytometry data: annexin V staining versus DAPI staining. (E-F) Fetal liver cell homing and lodging ability in irradiated and nonirradiated recipient mice was analyzed. Wild-type or necdin-null fetal liver cells (5 × 10⁶; CD45.2) were injected into lethally irradiated (E) or nonirradiated (F) recipient mice (CD45.1). BM cells were harvested 18 hours after injection and CD45.2⁺ donor-derived cells were enumerated on the Lin⁻Sca1⁺Mac1⁺ population by flow cytometry. Data shown are mean percentages (± SD) of cells that homed (E) or lodged (F) within the Lin⁻Sca1⁺Mac1⁺ cells (E: P = .92, F: P = .48, n = 5).

Figure 4

Necdin maintains adult HSC quiescence. (A) Cell-cycle analysis of Lin⁻Sca-1⁺c-Kit⁺CD48⁻CD150⁺ cells (left panels) isolated from mice reconstituted with wild-type (WT) or necdin-null (Ndn null) fetal liver cells was performed by staining with Hoechst 33 342 and Ki67. Data shown are the mean values of G₀ cells (± SD; P = .0025, n = 7). (B) The proliferation of Lin⁻Sca-1⁺c-Kit⁺CD48⁻CD150⁺ cells isolated from mice reconstituted with wild-type or necdin-null fetal liver cells was measured by in vivo BrdU incorporation over 48 hours. Greater proliferation of necdin-null Lin⁻Sca-1⁺c-Kit⁺CD48⁻CD150⁺ cells was observed (38.9% vs 14.8% for wild-type cells; P = .0007, n = 7). (C) Cell-cycle analysis of Lin⁻Sca-1⁺c-Kit⁺CD48⁻CD150⁺ cells isolated from the BM of mice reconstituted with wild-type, necdin-null, p53-null, or necdin/p53 double-null fetal liver cells was performed by staining with Hoechst 33342 and Ki67. Data shown are the mean values of G₀ cells (± SD; P < .0001 by 1-way ANOVA, n = 4). Significant differences were observed between wild-type and necdin-null, wild-type and p53-null, wild-type and necdin/p53 double-null, necdin-null and p53-null, and p53-null and necdin/p53 double-null recipients.

Figure 5

Necdin-null hematopoietic cells are highly sensitive to chemotherapy. (A) Survival after weekly 5-FU administration. 5-FU was administered intraperitoneally weekly (the initial dose was 125 mg/kg, with subsequent doses of 90 mg/kg) and the survival rates of mice repopulated with wild-type (WT) or necdin-null (Ndn null) fetal liver cells were measured. Results were analyzed with a log-rank nonparametric test and expressed as Kaplan-Meier survival curves (P = .0387, n = 10). (B) Hematopoietic reconstitution was monitored by serial peripheral blood count of mice injected with a single dose of 5-FU (200 mg/kg intraperitoneally). WBC counts are shown at each point after 5-FU administration as a percentage of the initial values for each group of mice (mean ± SD; n = 3 for each time point). (C) Gross morphology of femurs from untreated mice and mice 14 days after 5-FU administration is shown. Slides were stained with H&E. The slides were analyzed under a Zeiss Axioplan 2 Upright Wide-field Microscope (Carl Zeiss) equipped with a Zeiss AxiCam HRc Camera (Carl Zeiss; original magnification ×100 with a 10× objective). The images were acquired by a Volocity software (PerkinElmer). (D) HSCs isolated from the mice repopulated with wild-type or necdin-null fetal liver cells were assessed for apoptosis 60 hours after a dose of 5-FU (200 mg/kg intraperitoneally) using DAPI and annexin V staining. Data shown are mean percentage (± SD) of annexin V⁺/DAPI⁻ Lin⁻Sca-1⁺c-Kit⁺CD48⁻CD150⁺ cells (P = .015, n = 5). (E) The proliferation of Lin⁻Sca-1⁺c-Kit⁺CD48⁻CD150⁺ cells isolated from mice reconstituted with wild-type or necdin-null fetal liver cells 4 days after 5-FU administration was measured by BrdU incorporation in vivo over 48 hours (P = .76, n = 5).

Figure 6

Necdin-null hematopoietic cells are highly sensitive to irradiation. (A) Survival curves for mice reconstituted with wild-type (WT) or necdin-null (Ndn null) fetal liver cells, given total body irradiation (6.5 Gy), and monitored regularly for survival (P = .0017, n = 10). (B) Survival curves for mice reconstituted with wild-type or necdin-null fetal liver cells given TBI (8 Gy) and monitored regularly for survival (P = .0091, n = 8). (C) HSCs (Lin⁻Sca-1⁺c-Kit⁺CD48⁻CD150⁺ cells) from the BM of the mice repopulated with wild-type or necdin-null fetal liver cells were assessed for apoptosis 12 hours after a dose of total-body irradiation (6.5 Gy) using DAPI and annexin V staining (left panels). Data shown are the mean percentage (± SD) of the annexin V⁺/DAPI⁻ population in Lin⁻Sca-1⁺c-Kit⁺CD48⁻CD150⁺ cells (P = .0098, n = 5, right panel). (D) Cell-cycle analysis of HSCs after G-CSF treatment (200 μg/kg daily). Lin⁻Sca-1⁺c-Kit⁺CD48⁻CD150⁺ cells isolated from mice reconstituted with wild-type or necdin-null fetal liver cells after 5 days of G-CSF treatment were analyzed by staining with Hoechst 33342 and Ki67. Data shown are the mean values of G₀ cells (± SD; P = .75, n = 5). (E) Apoptosis of HSCs after G-CSF treatment (200 μg/kg daily). HSCs from the BM of the 5-day G-CSF–treated mice repopulated with wild-type or necdin-null fetal liver cells were assessed for apoptosis 12 hours after a dose of total body irradiation (6.5 Gy) using DAPI and annexin V. Data shown are the mean percentage (± SD) of the annexin V⁺/DAPI⁻ population in Lin⁻Sca-1⁺c-Kit⁺CD48⁻CD150⁺ cells (P < .005, n = 4). (F) HSCs (Lin⁻Sca-1⁺c-Kit⁺CD48⁻CD150⁺) from the BM of mice repopulated with wild-type, necdin-null, p53-null, or necdin/p53 double-null fetal liver cells were assessed for apoptosis 12 hours after a dose of total body irradiation (6.5 Gy) using DAPI and annexin V staining. Data shown are the mean percentage ± SD of the annexin V⁺/DAPI⁻ population within HSCs (P < .0001 by 1-way ANOVA, n = 4). Significant differences were observed between wild-type and necdin-null, wild-type and p53-null, necdin-null and p53-null, necdin-null and necdin/p53 double-null, and p53-null and necdin/p53 double-null recipients.

Figure 7

Molecular mechanisms of necdin in regulating the response of HSCs to irradiation. (A) Transcript profiling of LSK cells from mice reconstituted with wild-type (WT) and necdin-null (Ndn null) fetal liver cells after a sublethal dose of irradiation (6.5 Gy) were analyzed by Affymetrix oligonucleotide array. Genes that are differentially expressed between wild-type and necdin-null HSPCs are shown. (B) The relative mRNA expression level of Gas2L3 in LSK cells isolated from mice reconstituted with wild-type or necdin-null fetal liver cells after a sublethal dose of irradiation (6.5 Gy) was evaluated by quantitative PCR and normalized to hypoxanthine-guanine phosphoribosyltransferase expression. Data shown are the mean ratio (± SD) of transcript levels relative to that of wild-type cells (n = 2). (C) Effect of down-regulating Gas2L3 expression on the HSPC response to irradiation. Wild-type or necdin-null LSK cells were nucleofected with control or Gas2L3-directed siRNAs. Twenty-four hours after nucleofection, the cells, which showed efficient Gas2L3 knockdown in LSK cells by quantitative PCR (left panel), were irradiated at 2 Gy and their apoptosis was measured by annexin V and DAPI staining. Data shown are mean values (± SD; P < .0001 by 1-way ANOVA, n = 6, right panel). Significant differences were observed between control/wild-type and Gas2L3 knockdown/wild-type, control/wild-type and control/necdin-null, control/wild-type and Gas2L3 knockdown/necdin-null, Gas2L3 knockdown/wild-type and control/necdin-null, and control/necdin-null and Gas2L3 knockdown/necdin-null. (D) Effect of Gas2L3 overexpression on the HSPC response to irradiation. Gas2L3 overexpression in LSK cells was confirmed by quantitative PCR (n = 2, left panel); the cells were then irradiated at 2 Gy and their apoptosis was measured by annexin V and DAPI staining. Data shown are mean values (± SD; P = .01, n = 5, right panel). (E) Green fluorescent protein–positive HSCs (Lin⁻Sca-1⁺c-Kit⁺CD48⁻CD150⁺) from the BM of the mice repopulated with wild-type control, wild-type Gas2L3 knocked-down 1 (KD1), wild-type Gas2L3 KD2, necdin-null control, necdin-null Gas2L3 KD1, or necdin-null Gas2L3 KD2 fetal liver cells were assessed for apoptosis 12 hours after a dose of total-body irradiation (6.5 Gy) using DAPI and annexin V staining. Data shown are the mean percentage ± SD of the annexin V⁺/DAPI⁻ population within HSCs (P < .0001 by 1-way ANOVA). Significant differences were observed between wild-type control and wild-type Gas2L3 KD1, wild-type control and wild-type Gas2L3 KD2, wild-type control and necdin-null control, wild-type control and necdin-null Gas2L3 KD1, wild-type control and necdin-null Gas2L3 KD2, wild-type Gas2L3 KD1 and necdin-null control, wild-type Gas2L3 KD2 and necdin-null control, necdin-null control and necdin-null Gas2L3 KD1, and necdin-null control and necdin-null Gas2L3 KD2. (F) The relative mRNA expression levels of Gas2L3 in CD48⁻CD150⁺LSK cells, CD34⁻LSK cells, and CD34⁺LSK cells isolated from mice before and after a sublethal dose of irradiation (6.5 Gy) was evaluated by quantitative PCR and normalized to hypoxanthine-guanine phosphoribosyltransferase expression. Data shown are the mean ratio of transcript levels relative to the wild-type cells (± SD; n = 3).