EVI1 induces myelodysplastic syndrome in mice
J. Clin. Invest. Silvia Buonamici, et al. 114:713 doi:10.1172/JCI21716 [
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Figure 3EVI1 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.
