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 ma y improve haemoglobin levels in patients with ringsideroblastic anaemia (RA RS) and reduce bone marrow apoptosis. We studied bone marrow from 10 RARS p atients, two of whom were also investigated after successful treatment. Mon onuclear, erythroid and CD34(+) cells were analysed with regard to prolifer ation, apoptosis, clonogenic capacity and oncoprotein expression, in the pr esence or absence of Fas-agonist, Fas-blocking antibody 2 and caspase-3 inh ibitor. During culture, RARS bone marrow cells showed higher spontaneous ap optosis (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 eryth roid colony-forming units (CFU-E) (< 10% of control) and granulocyte-macrop hage CFU (CFU-GM (< 50% of control) from CD34(+) cells. Fas ligation increa sed apoptosis and decreased colony growth equally in RARS and controls, but caused significantly more caspase activation in RARS (P < 0.01). Fas-block ing antibody showed no significant inhibitory effect on spontaneous apoptos is or ineffective haematopoiesis, as measured using phosphatidylserine expo sure, the terminal deoxynucleotide transferase-mediated dUTP-biotin nick-en d labelling technique, caspase activity or clonogenic growth. Caspase inhib ition reduced apoptosis, increased proliferation and enhanced erythroid col ony growth from CD34(+) cells in RARS, but showed no effect on normal cells . CFU-E improved > 1000% after successful treatment. Thus, erythroid apopto sis in RARS is initiated at the CD34(+) level and growth factor treatment m ay improve stem cell function. Enhanced caspase activation at the stem cell level, albeit not mediated through endogenous activation of the Fas recept or, contributes to the erythroid apoptosis in RARS.