Hematopoietic stem cell expansion and distinct myeloid developmental abnormalities in a murine model of the AML1-ETO translocation - PubMed (original) (raw)

Hematopoietic stem cell expansion and distinct myeloid developmental abnormalities in a murine model of the AML1-ETO translocation

Cristina G de Guzman et al. Mol Cell Biol. 2002 Aug.

Abstract

The t(8;21)(q22;q22) translocation, which fuses the ETO gene on human chromosome 8 with the AML1 gene on chromosome 21 (AML1-ETO), is one of the most frequent cytogenetic abnormalities associated with acute myelogenous leukemia (AML). It is seen in approximately 12 to 15% of AML cases and is present in about 40% of AML cases with a French-American-British classified M2 phenotype. We have generated a murine model of the t(8;21) translocation by retroviral expression of AML1-ETO in purified hematopoietic stem cells (HSC). Animals reconstituted with AML1-ETO-expressing cells recapitulate the hematopoietic developmental abnormalities seen in the bone marrow of human patients with the t(8;21) translocation. Primitive myeloblasts were increased to approximately 10% of bone marrow by 10 months posttransplant. Consistent with this observation was a 50-fold increase in myeloid colony-forming cells in vitro. Accumulation of late-stage metamyelocytes was also observed in bone marrow along with an increase in immature eosinophilic myelocytes that showed abnormal basophilic granulation. HSC numbers in the bone marrow of 10-month-posttransplant animals were 29-fold greater than in transplant-matched control mice, suggesting that AML1-ETO expression overrides the normal genetic control of HSC pool size. In summary, AMLI-ETO-expressing animals recapitulate many (and perhaps all) of the developmental abnormalities seen in human patients with the t(8;21) translocation, although the animals do not develop leukemia or disseminated disease in peripheral tissues like the liver or spleen. This suggests that the principal contribution of AML1-ETO to acute myeloid leukemia is the inhibition of multiple developmental pathways.

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Figures

FIG. 1.

FIG. 1.

Retroviral transduction of murine hematopoietic stem cells. (A) Schematic diagram of MSCV retroviral constructs (control MSCV IRES GFP and MSCV _AML1_-ETO IRES GFP). (B) Gating used for sorting the HSC phenotype c-Kit+Sca-1+Lin− (where Lin represents a cocktail of antibodies to the mature blood cell antigens Mac-1, Gr-1, Ter119, B220, CD3, CD4, CD5, and CD8). (C) Flow cytometric analysis of HSC 24 h after retroviral transduction. Approximately 300 Ly-5.2+ HSC from control or AML1-ETO transductions were transplanted with a radioprotective dose of 2 × 105 Ly-5.1+ whole bone marrow cells into each Ly-5.1+ recipient animal. (D) Western blot analysis of GFP+ (lane 1) or GFP− (lane 2) myeloid scatter-gated cells FACS-sorted from the bone marrow of an 8-week-posttransplant AML1-ETO animal probed with a polyclonal anti-AML1 antibody.

FIG. 2.

FIG. 2.

Abnormal myelopoiesis and decreased B lymphopoiesis in AML1-ETO/GFP+ peripheral blood cells. (A) Flow cytometric analysis of peripheral blood cells from animals at 2.5 months posttransplantation stained with an antibody to the Ly-5.2 donor marker. Peripheral blood cells were gated as GFP− or GFP+ and analyzed for (B) simultaneous Mac-1 and Gr-1 or (C) B220 expression. Note the reduction in Mac-1loGr-1hi cells that represent mature neutrophils and an overrepresentation of Mac-1hiGr-1int cells in the AML1-ETO/GFP+ population compared to the GFP+ control. FACS plots are representative of all cocultured whole bone marrow transplants of control GFP (n = 21) and AML1-ETO-expressing (n = 26) mice and all purified HSC transplants of control GFP (n = 5) and AML1-ETO-expressing (n = 3) mice.

FIG. 3.

FIG. 3.

Abnormal myelopoiesis in AML1-ETO-expressing bone marrow cells. Flow cytometric analysis of bone marrow from a 10-month-posttransplant AML1-ETO mouse. (A) Bone marrow cells were gated on (panel 1) GFP− and (panel 2) AML1-ETO/GFP+ bone marrow cells and analyzed for expression of Mac-1 and Gr-1. The data are representative of all AML1-ETO-transplanted animals between 2 and 10 months posttransplant. The Mac-1/Gr-1 profile in panel 1 is identical to what is seen in bone marrow from control GFP animals. (B) Wright-Giemsa-stained cytospin preparation of AML1-ETO/GFP+, Mac-1hiGr-1int cells gated as shown in panel A (×100 magnification). Arrows indicate (a) a banded neutrophil and (b) a metamyelocyte. (C) Graded levels of AML1-ETO expression show distinct Mac-1/Gr-1 phenotypes in bone marrow. (D) Northern blot analysis of RNA isolated from GFP− and AML1-ETO/GFP+ bone marrow cells from a 3-month-posttransplant AML1-ETO animal. The blot was probed with a 3′ fragment of the C/EBPα cDNA and a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe. Quantitation of transcript levels was done on a phosphoimager. (E) Wright-Giemsa staining of an eosinophil myelocyte, showing abnormal basophilic granulation from the bone marrow of a 10-month-posttransplant AML1-ETO animal.

FIG. 4.

FIG. 4.

Increase in myeloid colony-forming cells in AML1-ETO animals. (A) Myeloid scatter-gated cells were sorted as GFP− or AML1-ETO/GFP+ from a 10-month-old AML1-ETO mouse, and 1,000 cells from each population were plated in triplicate into M3434 methylcellulose medium (10 ng of murine recombinant interleukin-3, 10 ng of human recombinant interleukin-6, and 50 ng of murine recombinant stem cell factor per ml) supplemented with 0.5 ng of granulocyte-macrophage colony-stimulating factor (R & D Systems) per ml. (B) Three independent AML1-ETO animals at 2 and 10 months posttransplant were used in the analysis. Colonies were enumerated (1 colony of >200 cells) and characterized 10 days after plating. (C) Representative FACS plots of individual methylcellulose colonies stained with Mac-1 and Gr-1. Two representative plots are shown for each sample. (D) Cytospin preparations of GFP− and AML1-ETO/GFP+ colonies stained with Wright-Giemsa. Arrows indicate mature, segmented neutrophils among the GFP− cells that were not seen in any AML1-ETO-expressing colonies.

FIG. 5.

FIG. 5.

Expansion of hematopoietic stem cells in AML1-ETO mice. HSC analysis from a 10-month-posttransplant AML1-ETO mouse. Bone marrow cells were stained with c-Kit, lineage marker antibodies (see text), Sca-1, and the Ly-5.2 donor marker. The percentages of cells in individual gated populations are indicated.

FIG. 6.

FIG. 6.

Delayed differentiation in AML1-ETO-expressing stem cells. The percentage of AML1-ETO-expressing (GFP+) cells in the stem cell population and in whole bone marrow was contrasted at early (2 months, n = 3) and late (10 months, n = 3) times postreconstitution. The ratio of GFP+ cells in the stem cell compartment and in the bone marrow of control GFP animals was similar to the ratio seen in older AML1-ETO animals.

FIG. 7.

FIG. 7.

AML1-ETO expression in stem cells is required for maintenance of abnormal myelopoiesis. Bone marrow from one primary recipient AML1-ETO animal was serially transplanted at a dose of 4 × 106 cells into each of four lethally irradiated secondary mice. Flow cytometric analysis of HSC in one of four secondary animals is shown at 5 weeks posttransplant. All secondary transplant animals received 114,000 AML1-ETO-expressing myeloid cells along with approximately 600 AML1-ETO/GFP+ HSC in the bone marrow inoculum. WBM, whole bone marrow.

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