Mutant N-RAS Induces Erythroid Lineage Dysplasia in Human CD34+ Cells
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RAS mutations arise at high frequency (20–40%) in both acute myeloid leukemia and myelodysplastic syndrome (which is considered to be a manifestation of preleukemic disease). In each case, mutations arise predominantly at the N-RAS locus. These observations suggest a fundamental role for this oncogene in leukemogenesis. However, despite its obvious significance, little is known of how this key oncogene may subvert the process of hematopoiesis in human cells. Using CD34+ progenitor cells, we have modeled the preleukemic state by infecting these cells with amphotropic retrovirus expressing mutant N-RAS together with the selectable marker gene lacZ. Expression of the lacZ gene product, β-galactosidase, allows direct identification and study of N-RAS–expressing cells by incubating infected cultures with a fluorogenic substrate for β-galactosidase, which gives rise to a fluorescent signal within the infected cells. By using multiparameter flow cytometry, we have studied the ability of CD34+ cells expressing mutant N-RAS to undergo erythroid differentiation induced by erythropoietin. By this means, we have found that erythroid progenitor cells expressing mutant N-RAS exhibit a proliferative defect resulting in an increased cell doubling time and a decrease in the proportion of cells in S + G2M phase of the cell cycle. This is linked to a slowing in the rate of differentiation as determined by comparative cell-surface marker analysis and ultimate failure of the differentiation program at the late-erythroblast stage of development. The dyserythropoiesis was also linked to an increased tendency of the RAS-expressing cells to undergo programmed cell death during their differentiation program. This erythroid lineage dysplasia recapitulates one of the most common features of myelodysplastic syndrome, and for the first time provides a causative link between mutational activation of N-RAS and the pathogenesis of preleukemia.Blood cell
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This study was undertaken to determine the effect of short-course high-dose methylprednisolone (HDMP) treatment on peripheral blood (PB) CD34+ progenitor cells during remission induction treatment in 11 children with newly diagnosed acute leukemia (7 with ALL, 4 with AML) whose bone marrow (BM) cells expressed fewer than 5% CD34 at the time of diagnosis. All children who had no infection were given HDMP as a single daily oral dose of 30 mg/kg for the first four days of induction therapy. The number of CD34+ progenitor cells were determined by flow cytometry before and after four days of HDMP treatment. While the number of PB blast cells significantly decreased after only a four-day course of HDMP treatment, the number of PB CD34+ progenitor cells increased in all patients. In addition, after four days of HDMP treatment polymorphonuclear leukocytes (PMN) and mononuclear cells (MNC) increased significantly (p < 0.05). We suggest that the potential beneficial effects of HDMP in the induction treatment of acute leukemia may occur partly by the stimulation of PB CD34+ hematopoietic progenitor cells in a short period of time.
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If 'sensitivity' is defined as the ratio of haem synthesis in erythropoietin (Ep)-stimulated cultures to that in cultures without added Ep, then haematopoietic tissue of ex-hypoxic mice is less sensitive to Ep in vitro than similar tissue from normal donors. This appears to be due primarily to the loss of erythropoietin responsive cells from the marrow and spleen. In addition, saline extracts of mature erythrocytes inhibited erythropoiesis in normal bone marrow cultures in a manner directly related to the 'haematocrits' used during their preparation. These data support recent suggestions that the preference of erythrocytotic mice over normal animals for Ep assay probably can be attributed to their low baseline level of erythropoiesis rather than an increased sensitivity to Ep.
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Abstract Background: Despite treatment advances, systemic lupus erythematosus (SLE) patients frequently experience disease flares, which can lead to organ damage and premature death. Therefore, assessing disease activity in SLE patients is crucial for adjusting treatment and preventing further organ damage. The aim of this study was to investigate progenitor cells and circulating endothelial cells levels in relation to SLE activity and accumulate organ damage. Methodos: A case-control study was conducted. CD34 + CD45 low/- progenitor cells, CD34 + CD45 low/- CD133 + progenitor, Endothelial Progenitor cells (EPC) and Circulating Endothelial cells (CEC) levels in peripheral blood were assessed by flow cytometry. Results: Thirty-two SLE patients and 28 matched controls were included. SLE patients had lower levels of CD34 + CD45 low/- progenitor cells (p=0.001), CD34 + CD45 low/- CD133 + progenitor cells (p=0.016), EPC (p=0.018) and CEC (p<0.001) compared to controls. In addition, cell subpopulations studied correlate with SLE activity biomarkers. CD34 + CD45 low/- progenitor cells showed a moderate negative correlation with levels of both C3 and C4. We also found significantly higher levels of CD34 + CD45 low/- progenitor cells, CD34 + CD45 low/- CD133 + progenitor cells, EPC and CEC in patients with SLE with SDI scores ≥1 versus those without organ damage (p=0.0073, p=0.018, p=0.018 and p=0.020, respectively). Conclusion: We found that CD34 + CD45 low/- progenitor cells, CD34 + CD45 low/- CD133 + progenitor cells, CPE and CEC were significantly reduced in patients with SLE as well as associated with disease activity and organ damage. Our observations suggest that CD34 + CD45 low/- progenitor cells could serve as a potential biomarker for disease activity and organ damage in SLE patients. It should be confirmed in a prospective study.
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Polypotential hemopoietic stem cell numbers were monitored by means of the splenic colony-forming unit (CFU-S) method in bone marrow and spleen of CBA mice in condition of suppressed erythropoiesis (after syngeneic red blood cell hypertransfusion and chronic hypoxia). Erythropoietic activity determined by radioactive isotope 59Fe uptake in erythroid cells of spleen, bone marrow and erythrocytes of peripheral blood sharply decreased after these treatments. CFU-S contents in bone marrow and spleen increased during a week after syngeneic red blood cell hypertransfusion and after chronic hypoxia. It is assumed that the increase in hemopoietic stem cell contents in hemopoietic organs of mice under inhibited erythropoiesis may be interpreted due to the appearance of vacant differentiation potencies of stem cells, which in the normal state is realized in production of erythroid cells.
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