EVI1 induces myelodysplastic syndrome in mice (original) (raw)
EVI1 causes a fatal disease in mice. We used a retroviral system to constitutively express EVI1 in murine BM cells. We used the murine stem cell virus (MSCV) vector because it provides long-lasting low level of expression in vivo and contains the neomycin resistance selection marker suitable for in vitro studies (28). A diagram of the recombinant retroviral vector is shown in Figure 1A. Lineage-negative murine BM cells were infected in vitro and were injected into lethally irradiated syngeneic recipients as described in Methods. In parallel, control animals received BM cells infected with the MSCV vector. To evaluate the efficiency of infection, 70,000–100,000 infected BM cells were cultured in vitro with or without G418, and the number of colonies was determined. We consistently found that infection efficiency with the EVI1-containing retrovirus ranged between 20% and 30% and was 2.5–4 times lower than with the empty retrovirus (80–85% infection efficiency). Because of the large size of the EVI1 cDNA (about 4 kb), a decrease of retrovirus titer and efficiency of infection was expected. To determine the extent of engraftment, we used donor mice with genotype C57BL/Ly5.2 and recipient irradiated mice with genotype C57BL/Ly5.1, and we evaluated by flow-cytometric analysis the fraction of BM cells stained by LY5.2 antibody that identifies the donor cells. The results indicated that the degree of engraftment ranged between 72.1% and 77.8%. The expression of EVI1 in the BM of the infected mice was confirmed by Western blot analysis. The results (Figure 1B) show the expression of EVI1 in the BM of 2 moribund mice (lanes 4 and 5) and in the EVI1-containing retrovirus packaging cells used as positive control (lane 1). As negative controls, we used the empty-retrovirus packaging cells (lane 2) and the BM of a control mouse (lane 3). To determine the clonality of the disease, we analyzed the BM of 1 control and 2 EVI1-positive mice by Southern blot. The genomic DNA was extracted from the BM and digested with the endonuclease HindIII, which cuts once within the EVI1 cDNA. The position of the probe (0.7 kb NcoI fragment) is shown in Figure 1A. The results (Figure 1C) indicate that the disease is mono- or oligoclonal. A band of about 6.6 kb was detected in all 3 samples. This band identifies the endogenous Evi1 gene. To follow the development of hematopoietic disease, periodic peripheral blood (PB) counts were performed on both groups of animals. Initially, all the mice had normal blood profiles (Table 1). However, 8–12 months after BM transplantation (BMT) the mice that received EVI1-BM developed pancytopenia. At this terminal stage, the animals showed a reduction in platelet, rbc, and white blood cell (wbc) counts, as well as in hemoglobin levels (Figure 1D and Table 1). This condition rapidly worsened, eventually leading to death 10–12 months after BMT (Figure 1E). None of the animals progressed to acute leukemia, and we attribute the cause of death to pancytopenia.
EVI1 causes a fatal disease in reconstituted mice. (A) Retroviral DNA constructs used in Phoenix cell line. The 5′ long terminal repeat (LTR) provides the promoter for a transcript that includes EVI1 and a gene encoding resistance to G418 (_Neo_R, indicated by the arrow). Internal ribosome entry site (IRES) is required for the translation of the _Neo_R transcript. HIII is the unique HindIII restriction site present in the EVI1 cDNA. The location of the probe used for Southern blot analysis is indicated. HA, hemagglutinin epitope. (B) Western blot analysis of EVI1-producing packaging Phoenix cells (lane 1), vector-producing packaging Phoenix cells (lane 2), control mouse BM cells (lane 3), and EVI1-positive BM cells (lanes 4 and 5) confirms the appropriate expression of EVI1 only in EVI1-positive samples (lanes 1, 4, and 5). (C) Southern blot analysis of BM cells from reconstituted EVI1-positive mice (lanes 4 and 5) and control mice (lane 3). (D) Counts of wbc’s (diamonds, × 103/μl), rbc’s (squares, × 106/μl), platelets (horizontal bars, × 105/μl), and levels of hemoglobin (crosses, g/dl) in PB of EVI1-positive mice (left) and in control mice (right). (E) The solid line shows the Kaplan-Meier survival curve of EVI1-positive reconstituted mice. All EVI1-positive mice died or were killed because of disease conditions. The dashed line represents the survival of the control mice.
Hematological parameters of EVI1-positive and control mice
EVI1 induces multilineage hematopoietic defects. Postmortem examination of the organs showed no marked difference in the size or appearance of the kidneys, liver, thymus, lungs, or heart. The spleens of all diseased animals showed marked congestion. About half of the diseased mice suffered from splenomegaly; however, a few mice had normal-sized spleens (Table 1). Morphological analysis of tissue paraffin sections of liver and heart tissue showed no abnormality (data not shown). In the control spleen, well-defined white pulp containing lymphocytes and red pulp were preserved as expected (Figure 2A). In contrast, the white pulp of diseased spleens appeared dispersed and the red pulp was expanded (Figure 2B). Furthermore, the red pulp showed increased numbers of erythroid precursors compared with normal controls (Figure 2, C and D). Prussian blue staining revealed large depositions of iron in BM (not shown) and spleen sections (Figure 2F) of the EVI1-positive mice. The iron depositions were not observed in the organs of control mice (Figure 2E). Increased iron depositions suggest increased rbc destruction due to either hemolysis or apoptosis. To rule out the possibility of hemolysis, we measured the total and direct bilirubin levels in the serum of normal and EVI1-positive mice. This assay has been used by other investigators to distinguish between hemolysis and apoptosis in murine studies (29). We found that the level of total and direct bilirubin in the serum of the EVI1-positive mice was not statistically different from the control sera (data not shown), suggesting that hemolysis is not the dominant cause of cell death. To confirm that apoptosis was the major mechanism of cell death, we stained spleen sections of control and EVI1-positive mice with cleaved caspase-3 antibody (Figure 2, G and H at ×10 magnification, and I and J at ×40 magnification). The results clearly show positive immunostaining in sections of the EVI1-positive spleen (Figure 2, H and J) but not in sections of the control spleen (Figure 2, G and I). These results confirm that apoptosis rather than hemolysis is the major cause of cell death.
EVI1 induces BM hypercellularity, dyserythropoiesis, erythroid and megakaryocytic hyperplasia, and apoptosis. Sections of normal spleen tissue (A and C) show normal white and red pulp. In comparison, EVI1-positive spleen tissue shows an expansion of red pulp and erythroid hyperplasia (B and dark cells in D). (E) Prussian blue iron staining of normal spleen tissue does not identify extensive iron deposition. (F) Iron depositions are evident in a section of EVI1-positive spleen tissue stained with Prussian blue. (H and J) Sections of EVI1-positive spleen tissue stained with cleaved caspase-3 antibody demonstrate the presence of apoptosis. (G and I) Normal spleen stained with cleaved caspase-3 antibody. (K and L) Control BM biopsy specimen shows normal cellularity and trilineage hematopoiesis. (M) In contrast, the EVI1-positive BM appears hypercellular, with erythroid and megakaryocytic hyperplasia. The BM dyserythropoiesis in EVI1-positive BM aspirates is shown (N), where the arrows point to nuclear irregularity and nuclear budding of erythroid precursors. (O) A PB smear of a control mouse is shown. In contrast, the PB smear of an EVI1-positive mouse shows anisopoikilocytosis (P), increased number of polychromatophilic rbc’s (Q), and Howell-Jolly bodies (R). Magnification, ×10 (A, B, E, F, G, H, K, M); ×40 (C, D, I, J, L, N, O, P, Q); ×100 (R).
The control BM biopsy specimen showed trilineage hematopoiesis with normal megakaryocytes, myeloid cells, and erythroid precursors (Figure 2, K and L). Trilineage hematopoiesis was also observed in the BM of EVI1-positive mice. However, the BM appeared hypercellular (Figure 2M), with erythroid and megakaryocytic hyperplasia. Dyserythropoiesis as manifested by nuclear irregularity and nuclear budding of erythroid precursors was present in EVI1-positive mice (Figure 2N, yellow arrows). PB smears from EVI1-positive mice were obtained every 1–2 months and compared to smears from age-matched control mice. For approximately 7–8 months after transplantation, the PB morphology appeared normal (data not shown). However, PB from EVI1-positive moribund mice revealed pancytopenia with marked anisopoikilocytosis and increased numbers of polychromatophilic rbc’s (compare normal PB smear, Figure 2O, with EVI1-positive PB smear, Figure 2, P and Q), and presence of Howell-Jolly bodies (Figure 2R).
EVI1 represses in vitro growth of BM from EVI1-positive mice. Even though moribund EVI1-positive mice consistently had very low PB counts, their BM appeared hypercellular and contained iron deposition; this result, combined with normal bilirubin levels and caspase-3 activation, strongly suggests that apoptosis is the cause of cell death. This apparently contradictory coexistence of hypercellular BM, cell death, and overall pancytopenia is a puzzling feature of human MDS. To determine whether EVI1 impairs the response of BM precursors to differentiation cytokines, we plated equal numbers of lineage-negative BM cells obtained from moribund EVI1-positive mice and from age-matched control mice in either Epo or GM-CSF as growth factor, and colonies were counted after 3 days (Epo) or 7 days (GM-CSF). The results show that EVI1-positive BM cells form a significantly lower number of colonies than do control cells (Figure 3A). To determine whether, in addition to the presence of EVI1, other defects must occur in the hematopoietic organs of the EVI1-positive mice to repress BM growth in vitro, we freshly infected lineage-negative normal BM cells with empty retrovirus or EVI1-containing retrovirus and plated an equal number of selected cells with either Epo or GM-CSF. The colonies and cells were counted after 3 days (Epo) and 7 days (GM-CSF). The results (Figure 3B) confirm that EVI1 has an immediate repressive effect on the growth of erythroid colonies as previously reported (9). However, if GM-CSF is added to the culture medium instead of Epo, then EVI1 induces a marked increase in the number and size of the colonies, indicating that in response to GM-CSF, EVI1 not only doubles the proliferation rate of the cells but also doubles the clonogenicity of the hematopoietic progenitors. To determine whether EVI1 impairs the response to Epo in the early stages of reconstitution, lineage-negative BM cells were isolated from EVI1-positive mice and controls 3 months after transplantation. At this time, the EVI1-positive mice did not show visible signs of disease and their PB appeared normal (Table 1 and data not shown). As shown in Figure 3C, at this point in time there was already a significant reduction in the number of colonies stimulated by Epo, indicating that impairment of Epo response is an early effect of EVI1 in vivo as well as in vitro and that it occurs in the absence of obvious PB dyserythropoiesis. To determine whether EVI1 affects the differentiation of freshly infected BM, control and EVI1-positive colonies grown in GM-CSF were isolated and sequentially replated. At each step cytospin preparations were made to evaluate cell differentiation. The results confirm that in freshly infected BM cells EVI1 enhances colony formation (data not shown). Furthermore, EVI1 considerably delays the terminal differentiation of the cells from about 28 days (controls) to about 49 days. After 28 days in culture, the control cells appear differentiated, and only macrophages, characterized by small compact nuclei and large vacuolated cytoplasm, were observed (Figure 3D). In contrast, after 28 days in culture, the EVI1 cells appeared small with scarce cytoplasm and large nuclei, indicative of a more immature state (Figure 3D).
EVI1 alters the response to cytokines and significantly increases the number of immature erythroid cells. (A_C) A total of 15,000 lineage-negative cells were isolated from control mice BM (black bars) or BM cells of moribund EVI1-positive mice (white bars). The cells were plated in duplicate in methylcellulose and were cultured with Epo (E) or GM-CSF (GM). After 3 days (Epo) or 7 days (GM-CSF) in culture, the colonies (left panels) and the cells (right panels) in each plate were isolated and counted. (A) The decrease in the number of colonies and cells of EVI1-positive mice shows that the BM cells of these animals have impaired in vitro differentiation. (B) The same assay was carried out with lineage-negative normal BM cells freshly infected with empty retrovirus (black bars) or EVI1-containing retrovirus (white bars). In contrast to the cells obtained from the moribund mice, EVI1 represses only the response to Epo in freshly infected BM cells. (C) The same assay was carried out with lineage-negative BM cells isolated from EVI1-positive mice 3 months after transplantation (white bars) or from age-matched controls (black bars). EVI1 represses only the response to Epo. (D) Cytospin preparations of control murine BM cells (left) or BM cells of moribund EVI1-positive mice (right) stained with Wright-Giemsa stain show that EVI1 delays in vitro differentiation as indicated by the smaller size and larger, less compact nuclei of the cells. (E) The spleens and BM of EVI1-positive mice (E, top panels) have a higher number of Ter119-positive cells than the organs of a control animal (C, bottom panels). Cells were stained with Ter119-PE and CD34-FITC. The percentage of positive cells for each quadrant is noted in the upper left corner of the quadrants. (F) RT-PCR analysis shows the expression of EVI1 in total BM cells of 3 moribund mice (lanes 1, 2, and 3), but not in the BM of a control mouse (lane 4). Analysis of sorted Ter119-positive cells of a moribund mouse (lane 5) confirms the expression of EVI1. EVI1 was not detected in Ter119-positive cells of the control (lane 6). For lane 7, no cDNA was added to the reaction. Gapdh was used as an internal standard.
Significant increase of the erythroid marker Ter119 in EVI1-positive cells. To characterize the hematopoietic cell populations of the EVI1-positive mice, we analyzed lineage-specific antigens in BM and spleen cells of 3 moribund EVI1-positive mice and 2 control mice by flow cytometry. We tested CD3, CD4, and CD8 T cells, as well as CD45R B cells, CD18 macrophages, Ter119 erythroid cells, LY-6G granulocytes, and CD34 early hematopoietic progenitors. The percentages of most cell lineages in control and EVI1-positive mice were comparable. However, the populations of Ter119-positive cells were consistently 2- and 3-fold higher in the spleens and BM of EVI1-positive mice, respectively (Figure 3E shows representative results for 1 EVI1-positive mouse and 1 control mouse). We used RT-PCR to confirm that the Ter119-positive cells express EVI1. The results indicate that the Ter119-positive cells sorted from the EVI1-positive moribund mice, but not those sorted from the control mice, express EVI1 (Figure 3F). To determine whether the increased proliferation of erythroid cells in the spleen and BM appeared early after transplantation, we repeated the flow cytometry quantitation with the BM of EVI1-positive mice 4 months after reconstitution. At this time, the PB smears of these mice appeared normal (data not shown); however, the number of immature erythrocytes was 3-fold higher than in control animals (data not shown). The back-gating analysis of the cells positive for Ter119 localized them within a population of cells with low forward scatter (cell size) and low side scatter (cell complexity).
EpoR and c-Mpl expression is lower in EVI1 positive mice. Impairment of erythropoiesis and platelet formation are two obvious defects that are consistently observed in PB of the moribund EVI1-positive mice and that contribute to their death. To identify the differentiation steps disrupted by EVI1 in these two lineages, we used semi-quantitative RT-PCR to evaluate the expression of potential genes that regulate terminal erythropoiesis and megakaryopoiesis. We found by RT-PCR that two of them, EpoR and c-Mpl, essential to erythroid differentiation and platelet formation (24–27), were repressed (data not shown). We used real-time PCR (RQ-PCR) to quantify more accurately the expression of EpoR and c-Mpl in the BM of 12 moribund EVI1-positive mice and 4 age-matched control mice (Figure 4). The data were normalized to the endogenous expression of Gapdh. The median normalized level of EpoR and c-Mpl in control samples was 231 for EpoR (range 213–244) and 216 for c-Mpl (range 191–224), whereas in the BM of the moribund EVI1-positive mice the level the EpoR was 102 (range 6–170) and that of c-Mpl was 70 (range 24–91). We found that animals with low expression of EpoR also had low expression of c-Mpl and vice versa. The repression of these two genes was also clearly observed 4 months after BMT at a time when the mice appeared normal, as did their PB morphology and profile (data not shown). At 4 months after BMT the median normalized level of EpoR in the BM was 108 (range 98–115), and the level of c-Mpl was 41 (range 39–51) (Figure 4).
EVI1 represses the expression of EpoR and c-Mpl. Real-time quantification of EpoR (squares) and c-Mpl (circles) normalized to Gapdh shows a significant decrease of these genes’ median expression in the BM of EVI1-positive mice 4 months after BMT or at the time of their disease-induced death. The results are plotted as the ratio between EpoR or c-Mpl and Gapdh multiplied by 100.