Apoptosis in refractory anaemia with ringed sideroblasts is initiated at the stem cell level and associated with increased activation of caspases.
Treatment with granulocyte colony-stimulating factor plus erythropoietin may improve haemoglobin levels in patients with ringsideroblastic anaemia (RARS) and reduce bone marrow apoptosis. We studied bone marrow from 10 RARS patients, two of whom were also investigated after successful treatment. Mononuclear, erythroid and CD34+ cells were analysed with regard to proliferation, apoptosis, clonogenic capacity and oncoprotein expression, in the presence or absence of Fas-agonist, Fas-blocking antibody 2 and caspase-3 inhibitor. During culture, RARS bone marrow cells showed higher spontaneous apoptosis (P < 0.05) and caspase activity (P < 0.05)) than bone marrow cells from healthy donors. Eight out of nine patients had reduced growth of erythroid colony-forming units (CFU-E) (< 10% of control) and granulocyte-macrophage CFU (CFU-GM) (< 50% of control) from CD34+ cells. Fas ligation increased apoptosis and decreased colony growth equally in RARS and controls, but caused significantly more caspase activation in RARS (P < 0.01). Fas-blocking antibody showed no significant inhibitory effect on spontaneous apoptosis or ineffective haematopoiesis, as measured using phosphatidylserine exposure, the terminal deoxynucleotide transferase-mediated dUTP-biotin nick-end labelling technique, caspase activity or clonogenic growth. Caspase inhibition reduced apoptosis, increased proliferation and enhanced erythroid colony growth from CD34+ cells in RARS, but showed no effect on normal cells. CFU-E improved > 1000% after successful treatment. Thus, erythroid apoptosis in RARS is initiated at the CD34+ level and growth factor treatment may improve stem cell function. Enhanced caspase activation at the stem cell level, albeit not mediated through endogenous activation of the Fas receptor, contributes to the erythroid apoptosis in RARS.
Department of Medicine, Division of Haematology, Karolinska Institutet at Huddinge University Hospital, 141 86 Huddinge, Sweden. Eva.Hellstrom-Lindberg@medhs.ki.se
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Hellström-Lindberg
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Schmidt-Mende
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Forsblom
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Christensson
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Fadeel
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Zhivotovsky
D000368:Aged; D000756:Anemia, Sideroblastic / Q000097:blood* / Q000188:drug therapy / Q000276:immunology; D018952:Antigens, CD34 / Q000378:metabolism; D017209:Apoptosis; D016022:Case-Control Studies; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D004789:Enzyme Activation; D004921:Erythropoietin / Q000627:therapeutic use*; D005434:Flow Cytometry; D016179:Granulocyte Colony-Stimulating Factor / Q000627:therapeutic use*; D006412:Hematopoietic Stem Cells / Q000473:pathology*; D006801:Humans; D020287:In Situ Nick-End Labeling; D016014:Linear Models; D008875:Middle Aged; D019014:fas Receptor / Q000378:metabolism
D018952:Antigens, CD34; D019014:fas Receptor; D004921:Erythropoietin; D016179:Granulocyte Colony-Stimulating Factor; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases