The Role of the Iron Transporter ABCB7 in Refractory Anemia with Ring Sideroblasts

Refractory Anemia with Ring Sideroblasts (RARS) is an acquired myelodysplastic syndrome (MDS) characterized by an excess iron accumulation in the mitochondria of erythroblasts. The pathogenesis of RARS and the cause of this unusual pattern of iron deposition remain unknown. We considered that the inherited X-linked sideroblastic anemia with ataxia (XLSA/A) might be informative for the acquired disorder, RARS. XLSA/A is caused by partial inactivating mutations of the ABCB7 ATP-binding cassette transporter gene, which functions to enable transport of iron from the mitochondria to the cytoplasm. Furthermore, ABCB7 gene silencing in HeLa cells causes an accumulation of iron in the mitochondria. We have studied the role of ABCB7 in RARS by DNA sequencing, methylation studies, and gene expression studies in primary CD34⁺ cells and in cultured erythroblasts. The DNA sequence of the ABCB7 gene is normal in patients with RARS. We have investigated ABCB7 gene expression levels in the CD34⁺ cells of 122 MDS cases, comprising 35 patients with refractory anemia (RA), 33 patients with RARS and 54 patients with RA with excess blasts (RAEB), and in the CD34⁺ cells of 16 healthy controls. We found that the expression levels of ABCB7 are significantly lower in the RARS group. RARS is thus characterized by lower levels of ABCB7 gene expression in comparison to other MDS subtypes. Moreover, we find a strong relationship between increasing percentage of bone marrow ring sideroblasts and decreasing ABCB7 gene expression levels. Erythroblast cell cultures confirm the low levels of ABCB7 gene expression levels in RARS. These data provide an important link between inherited and acquired forms of sideroblastic anemia and indicate that ABCB7 is a strong candidate gene for RARS.     

Identification of a Murine Erythroblast Subpopulation Enriched in Enucleating Events by Multi-spectral Imaging Flow Cytometry

Erythropoiesis in mammals concludes with the dramatic process of enucleation that results in reticulocyte formation. The mechanism of enucleation has not yet been fully elucidated. A common problem encountered when studying the localization of key proteins and structures within enucleating erythroblasts by microscopy is the difficulty to observe a sufficient number of cells undergoing enucleation. We have developed a novel analysis protocol using multiparameter high-speed cell imaging in flow (Multi-Spectral Imaging Flow Cytometry), a method that combines immunofluorescent microscopy with flow cytometry, in order to identify efficiently a significant number of enucleating events, that allows to obtain measurements and perform statistical analysis.We first describe here two in vitro erythropoiesis culture methods used in order to synchronize murine erythroblasts and increase the probability of capturing enucleation at the time of evaluation. Then, we describe in detail the staining of erythroblasts after fixation and permeabilization in order to study the localization of intracellular proteins or lipid rafts during enucleation by multi-spectral imaging flow cytometry. Along with size and DNA/Ter119 staining which are used to identify the orthochromatic erythroblasts, we utilize the parameters “aspect ratio” of a cell in the bright-field channel that aids in the recognition of elongated cells and “delta centroid XY Ter119/Draq5” that allows the identification of cellular events in which the center of Ter119 staining (nascent reticulocyte) is far apart from the center of Draq5 staining (nucleus undergoing extrusion), thus indicating a cell about to enucleate. The subset of the orthochromatic erythroblast population with high delta centroid and low aspect ratio is highly enriched in enucleating cells.     

The anti-proliferative effect of neurokinin-A on hematopoietic progenitor cells is partly mediated by p53 activating the 5' flanking region of neurokinin-2 receptor

The bone marrow (BM) is home to at least two stem cells, hematopoietic (HSC) and mesenchymal. Hematopoiesis is partly regulated through neurokinin-1 (NK-1) and NK-2 belonging to the family of G-protein/7-transmembrane receptors. NK-1 and NK-2 show preference for the neurotransmitters, substance P (SP) and neurokinin-A (NK-A), respectively. Hematopoietic suppression mediated by NK-A could be partly explained through the production of TGF-beta1 and MIP-1alpha. This study further characterizes mechanisms by which NK-A inhibits progenitor cell proliferation. The study addresses the hypothesis that p53 is a mediator of NK-A activation and this occurs partly through p53-mediated expression of NK-2. The studies first analyzed two consensus sequences for p53 in supershift assays. Reporter gene assays with NK-2 gene constructs and p53 expressing wild-type and mutant vectors, combined with cell proliferation assays, show NK-A activating p53 to inhibit the proliferation of K562 progenitors. These effects were reversed by hematopoietic stimulators, GM-CSF and SP. Verification studies with human CD34+/CD38- and CD34+/CD38+ BM progenitors show similar mechanisms with the expression of p21. This study reports on p53 as central to NK-A-NK-2 interaction in cell cycle quiescence of hematopoietic progenitors. These effects are reversed by at least two hematopoietic stimulators, SP and GM-CSF, with concomitant downregulation of p53.