Loss of C/EBP alpha cell cycle control increases myeloid progenitor proliferation and transforms the neutrophil granulocyte lineage - PubMed (original) (raw)

Loss of C/EBP alpha cell cycle control increases myeloid progenitor proliferation and transforms the neutrophil granulocyte lineage

Bo T Porse et al. J Exp Med. 2005.

Abstract

CCAAT/enhancer binding protein (C/EBP)alpha is a myeloid-specific transcription factor that couples lineage commitment to terminal differentiation and cell cycle arrest, and is found mutated in 9% of patients who have acute myeloid leukemia (AML). We previously showed that mutations which dissociate the ability of C/EBP alpha to block cell cycle progression through E2F inhibition from its function as a transcriptional activator impair the in vivo development of the neutrophil granulocyte and adipose lineages. We now show that such mutations increase the capacity of bone marrow (BM) myeloid progenitors to proliferate, and predispose mice to a granulocytic myeloproliferative disorder and transformation of the myeloid compartment of the BM. Both of these phenotypes were transplantable into lethally irradiated recipients. BM transformation was characterized by a block in granulocyte differentiation, accumulation of myeloblasts and promyelocytes, and expansion of myeloid progenitor populations--all characteristics of AML. Circulating myeloblasts and hepatic leukocyte infiltration were observed, but thrombocytopenia, anemia, and elevated leukocyte count--normally associated with AML-were absent. These results show that disrupting the cell cycle regulatory function of C/EBP alpha is sufficient to initiate AML-like transformation of the granulocytic lineage, but only partially the peripheral pathology of AML.

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Figures

Figure 1.

Myeloproliferative disease in BRM2 mice. (A) Splenomegaly in BRM2 mouse compared with WT mouse, both 36 wk of age. (B, C) May-Grünwald-Giemsa–stained cytospin preparations of cells from WT (B) and BRM2 (C) spleens shown in (A). Note the prevalence of neutrophil granulocytes (cells with condensed, doughnut-shaped nuclei) in the BRM2 spleen. Original magnification, 100. Flow cytometric analysis of erythroid and myeloid cells in BM from the above WT (D, E) and BRM2 (G, H) mice. Cells were triple-stained with Mac–1-FITC, TER119-PE, and Gr-1-APC or isotype-matched control antibodies, and analyzed on a FACSCalibur flow cytometer. The percentage of Mac-1+/Gr-1+ (granulocytic) and TER119+ (erythroid) cells in each sample is indicated. Cytospins (original magnification, 100) of BM samples confirmed the abundance of neutrophil granulocytes in the BRM2 BM (I) compared with the WT control (F).

Figure 2.

Development of myeloproliferative disease. (A) Flow cytometric analysis of BM from control (+/+, BRM2/+) and BRM2 (BRM2/BRM2) mice at 8, 12, 24, 36, 60, and 75 wk of age, performed as in Fig. 1. For each mouse analyzed, a pair of columns shows the percentage of Mac-1+/Gr-1+ and TER119+ cells. Mice in each genotype/age group are organized according to the number of Mac-1+/Gr-1+ cells present (increasing left to right). The cut-offs used to define the BRM2 phenotypes are indicated (BRM2-A: <12% Mac-1+/Gr-1+; BRM2-B: >55% Mac-1+/Gr-1+ BM cells). These cut-off values differ from the mean value (38.2%; n = 30) observed in control mice by more than two standard deviations (SD = 7.1%), and were observed in the control group only once. The level of Ter119+ cells in BRM2-A mice and of Mac-1+/Gr-1+ cells in BRM2-B mice were significantly greater than the levels observed in control mice (P < 10−17 and P < 10−16, respectively). (B) Levels of c-Kit positive cells in BM from control mice (n = 11), BRM2-A (n = 4), BRM2-B (n = 4), and BRM2-C (n = 6) mice. **P < 0.005; ***P < 0.00005 compared with control mice. +/?, +/+ and +/BRM2; A, BRM2-A; B, BRM2-B; C, BRM2-C. (C) Phenotypic progression of BRM2 mice.

Figure 3.

Three distinct BRM2 phenotypes. Distinct FACS profiles showing TER119 (a panels) and Mac-1/Gr-1 (b panels) staining and May-Grünwald-Giemsa–stained cytospins (c panels; original magnification, 100) of BM cells from representative mice. The percentage of TER119+ and Mac-1+/Gr-1+ cells are indicated.

Figure 4.

Differentiation profile of granulocytes and hematologic parameters in BRM2 mice. (A) Differential counts on cytospins of BM from representative mice. The percentage of nucleated cells at different granulocytic maturation stages is shown. *A leukemic phenotype (M2 AML; >20% myeloblasts [30]). >300 cells were counted per sample. (inset) Myeloid blast in peripheral blood smear from BRM2-C mouse (May-Grünwald-Giemsa stain). (B–D) Leukocyte infiltration in the liver of BRM2-C mice. Hematoxylin-eosin–stained paraffin sections of WT control (B) and BRM2-C mice (C, D). Infiltrating leukocytes form blue clusters; immature myeloid cells (myeloblasts/promyelocytes) are indicated by arrows in (D). Original magnification, 40 (B, C) and 100 (D). (E–H) Control (+/? ,+/+ and BRM2/+: n =11) and BRM2 mice (A, BRM2-A: n = 4; B, BMR2-B: n = 4; C, BRM2-C: n = 6) of 60–75 wk of age were analyzed for peripheral blood levels of (E) platelets, (F) hemoglobin (HGB), (G) erythrocytes (RBC, red blood cells), and (H) leukocytes (WBC, white blood cells). Error bars indicate standard deviations. *P < 0.05 compared with controls. (I) 10 μg total BM RNA from mice with the indicated genotypes were subjected to sequential Northern blotting with probes specific for the G-CSF-R, GM-CSF-Rα, M-CSF-R, and 18S rRNA. White lines indicate that intervening lanes have been spliced out. (J) Receptor expression from (I) was quantified using a Fuji BAS2500 phosphorimager. Average expression levels and the percentage of Mac1+/Gr1+ cells (36.5% in WT), are shown normalized to WT levels for the three BRM2 subtypes.

Figure 5.

Maintenance and progression of BRM2 phenotypes during BM transplantation. Analysis of recipient BM, from mice transplanted with BM from mice displaying the BRM2-A (A), -B (B), and -C phenotypes (C), as well as control BM (D) (panels d-f). The parallel analysis of the donor BM is shown for comparison (panels a–c). Panels show staining for Mac-1 and Gr-1 (panels a, d, and g); for c-Kit and Mac-1 (panels b, e, and h); and cytospins stained with May-Grünwald-Giemsa (panels c, f, and i). In the case of BRM2-A and BRM2-B, examples are shown of mice in which phenotypic progression toward the BRM2-C phenotype was observed (panels g–i). In all surviving recipients >90% of hematopoietic cells were of donor origin.

Figure 6.

Myeloid progenitor levels and proliferation in BRM2 mice. (A) Myeloid CFUs observed in successive replatings of BM from representative WT (n = 4), BRM2-A (n = 2), BRM2-B (n=2), and BRM2-C (n = 2) mice. 5–20,000 cells were plated in methylcellulose-based semisolid medium allowing outgrowth of all myeloid colony types. The number of day 7 myeloid colonies obtained per 10,000 cells plated is shown for each of five successive platings. (B) Myeloid colony types obtained from BM of WT (n = 8), BRM2-A (n = 4), BRM2-B (n = 5), and BRM2-C (n = 3) mice. 5–20,000 BM cells were plated as in (A) and the number of multipotent (CFU-GEMM), committed myeloid (CFU-GM), and committed erythroid (BFU-E) progenitors were scored after 10–12 d. Colony numbers obtained per 10,000 cells are shown. Error bars indicate standard deviations. *P < 0.05 for BRM2-C mice compared with control mice and nontransformed BRM2-A and -B mice.

Figure 7.

HSC and progenitor compartments in BRM2 mice. Flow cytometric determination of BM progenitors. Progenitor analysis of BM cells was performed as described previously (52). The percentage of nucleated BM cells with the immunophenotype of HSCs, CMPs, GMPs, and megakaryocyte-erythroid progenitors (MEP) is indicated. The diagram in the (A) panel shows the phenotyping strategy. Typical examples of analyses of WT (B), BRM2-A (C), BRM2-B (D), and BRM2-C (E) mice are shown.

Figure 8.

HSC and progenitor compartments are expanded in BRM2-C mice. (A) Summary of flow cytometric determination of BM progenitors from WT (n = 8), BRM2-A (n = 3), BRM2-B (n = 4), and BRM2-C (n = 4) mice, determined as in Fig. 6. The average percentage of nucleated BM cells with the immunophenotype of HSC, common lymphoid progenitor cells (CLP), CMP, GMP, and megakaryocyte-erythroid progenitors (MEP) is shown. Error bars indicate standard deviations. *P < 0.05 for BRM2-C mice compared with nonleukemic BRM2-A and -B mice. **P < 0.05 for WT mice compared with BRM2-A/B mice. (B) Competitive repopulation of irradiated recipients using 200,000 WT CD45.1 competitor BM cells and 20,000 CD45.2 BM cells from control mice (WT) or mice with the BRM2-B and -C phenotypes. Standard deviations are indicated by error bars. There was no difference between repopulation using BRM2-B or -C BM; overall, the level of repopulation observed with BRM2 donors was significantly less (*P < 0.05) than that seen with WT controls, which repopulated with the same efficiency as the CD45.1 competitor cells.

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Loss of C/EBP alpha cell cycle control increases myeloid progenitor proliferation and transforms the neutrophil granulocyte lineage - PubMed (original) (raw)