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. 2010 Apr 29;115(17):3481-8.
doi: 10.1182/blood-2009-11-251835. Epub 2010 Jan 11.

Haploinsufficiency of Apc leads to ineffective hematopoiesis

Jianghong Wang  1 , Anthony A Fernald, John Anastasi, Michelle M Le Beau, Zhijian Qian
Affiliations

Haploinsufficiency of Apc leads to ineffective hematopoiesis

Jianghong Wang et al. Blood. .

Abstract

Loss of a whole chromosome 5 or a deletion of the long arm of chromosome 5, -5/del(5q), is a recurring abnormality in myeloid neoplasms. The APC gene is located at chromosome band 5q23, and is deleted in more than 95% of patients with a -5/del(5q), raising the question of whether haploinsufficiency of APC contributes to the development of myeloid neoplasms with loss of 5q. We show that conditional inactivation of a single allele of Apc in mice leads to the development of severe anemia with macrocytosis and monocytosis. Further characterization of the erythroid lineage revealed that erythropoiesis is blocked at the early stages of differentiation. The long-term hematopoietic stem cell (LT-HSC) and short-term HSC (ST-HSC) populations are expanded in Apc-heterozygous mice compared with the control littermates; however, the HSCs have a reduced capacity to regenerate hematopoiesis in vivo in the absence of a single allele of Apc. Apc heterozygous myeloid progenitor cells display an increased frequency of apoptosis, and decreased in vitro colony-forming capacity, recapitulating several characteristic features of myeloid neoplasms with a -5/del(5q). Our results indicate that haploinsufficiency of Apc impairs hematopoiesis, and raise the possibility that loss of function of APC contributes to the development of myelodysplasia.

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Figures

Figure 1
Figure 1
Haploinsufficiency of Apc leads to lethality. (A) Analysis of the deletion of Apc in primary mice as determined by semiquantitative polymerase chain reaction of genomic DNA from bone marrow (BM) cells 4 days after induction. (B) Kaplan-Meier survival curve of primary Mx1-Cre+Apcfl/+ (n = 13) and Mx1-Cre−Apcfl/+ (n = 13) mice. (C) Peripheral blood counts of primary mice after induction. The deletion of a single allele of Apc was induced in Mx1-Cre+Apcfl/+ mice treated with 3 doses of pI-pC, referred to as Δ/+ mice. Control Mx1-Cre−Apcfl/+ mice are referred as fl/+ mice. The data were collected when Δ/+ mice became moribund within 3 to 8 months after induction. n = 13; ***P < .001.
Figure 2
Figure 2
Apc-heterozygous mice displayed an alteration in the distribution of CD41+ megakaryocytes and monocytes. Flow cytometric analysis of mature myeloid cells from BM and spleen (SP) from Mx1-Cre+Apcfl/+ mice and the control Mx1-Cre−Apcfl/+ mice was performed 3 to 4 months after induction. The percentage of (A) Gr-1+Mac-1+ and GrlowMac-1+ or (B) CD41+cells is indicated (average ± SD of 4 animals). *P < .05; **P < .01.
Figure 3
Figure 3
Apc-heterozygous mice developed ineffective erythropoiesis with erythroid dysplasia. (A) PB smears (left panels) and representative Giemsa-stained BM smears (right panels). (B) Flow cytometric analysis of BM and SP cells from representative Mx1-Cre+Apcfl/+ and Mx1-Cre−Apcfl/+ mice after pI-pC induction. The single-cell suspensions were simultaneously stained with anti-CD71 and anti-Ter119 antibodies. The numbers indicate the percentages of cells in each population. (C). Hematoxylin and eosin (H&E)–stained sections of adult SP (top panels) and Giemsa-stained spleen touch-preparations (bottom panels) from either control or anemic Apc heterozygous mice. The red arrows identify white pulp. Samples were obtained from both Mx1-Cre+Apcfl/+ and Mx1-Cre−Apcfl/+ mice 4 to 8 months after induction of the Apc deletion by pI-pC. Images were obtained using an Olympus microscope BX45 (Model U-DO3) equipped with an Olympus DP12 digital camera (A panels: 100× oil objective/1.25 NA, C top panels: 10× Plan air objective/0.3 NA, C bottom panels: 50× oil objective/0.9 NA), and processed using Microsoft PowerPoint.
Figure 4
Figure 4
Analysis of the proliferation of erythroblasts (CD71+Ter119+) in BM and SP from Apc-heterozygous mice. (A) Representative histograms of flow cytometric analysis of the cell cycle in splenic erythroblasts. (B-C) Histograms depicting the cell-cycle status of erythroblasts from (B) BM and (C) SP. Samples were obtained from both Mx1-Cre+Apcfl/+ and Mx1-Cre−Apcfl/+ mice with anemia 4 to 6 months after induction of the Apc deletion (average ± SD of 4-6 animals).*P < .01.
Figure 5
Figure 5
LT-HSCs and ST-HSCs are expanded in the absence of a single allele of Apc. (A) Flow cytometric analysis of LSKs (Lin− Sca-1+ Kit+) in primary mice. Comparison of the frequency of LSKs in Mx1-Cre+Apcfl/+ mice and the control Mx1-Cre−Apcfl/+ mice 2 weeks after induction (left panels) and 3 to 4 months after induction (right panels). The percentage of LSK cells is indicated (average ± SD of 3-5 animals).*P < .05; **P < .01. (B) Flow cytometric analysis of the proportion of long-term hematopoietic stem cells (LT-HSC)s, short-term hematopoietic stem cells (ST-HSCs), and multipotential progenitors (MPPs) in the LSK population from representative mice. (C) Total number of LT-HSCs, ST-HSCs and MPPs in BM from Mx1-Cre+Apcfl/+ mice and the control Mx1-Cre−Apcfl/+ mice 3 to 5 months after induction (average ± SD of 3 animals). *P < .05.
Figure 6
Figure 6
Apc haploinsufficiency affects the repopulating capacity of HSCs in vivo. (A) Mice with chimeric BM were generated by transplanting an equal number of wild-type CD45.1+CD45.2+ (CD45 heterozygous) BM cells and CD45.2+ (homozygous) BM cells from Mx1-Cre+Apcfl/+ or Mx1-Cre−Apcfl/+ mice 4 weeks after pI-pC induction of the Apc deletion. (B) Flow cytometric analysis of CD45.1- and CD45.2-stained PB cells from representative chimeric Mx1-Cre+Apcfl/+ or Mx1-Cre−Apcfl/+ mice 1 month after transplantation. The numbers indicate the percentage of cells in each population. (C) Histogram showing the relative ratio of CD45.2+ versus CD45.1+/CD45.2+-stained PB cells in Mx1-Cre−Apcfl/+ versus Mx1-Cre+Apcfl/+ chimeric mice examined at 1 to 4 months after transplantation (mean ± SD of 8-10 animals). *P < .05; **P < .01.
Figure 7
Figure 7
Analysis of in vitro colony-forming capacity, apoptosis, and proliferation of HPCs from Apc-heterozygous mice. (A) In vitro colony-forming assays. The number of BM CFU-GEMM, CFU-GM, CFU-G, CFU-M, and BFU-E was examined in Mx1-Cre+Apcfl/+ or Mx1-Cre−Apcfl/+mice 2 months after induction (mean ± SD of 3 animals). *P < .05. (B-C) Frequency of (B) apoptosis and (C) proliferation in gated HPCs (Lin− Kit+ Sca-1− Il-7R−) stained with Annexin V and DAPI, or DAPI, respectively, from Mx1-Cre+Apcfl/+ and Mx1-Cre−Apcfl/+ mice 3 to 4 months after pI-pC induction (mean ± SD of 3 animals). *P < .05; **P = .057.
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