APOPTOSIS OF LATE-STAGE ERYTHROBLASTS IN MEGALOBLASTIC-ANEMIA - ASSOCIATION WITH DNA-DAMAGE AND MACROCYTE PRODUCTION

An in vitro model of folate-deficient erythropoiesis has been develope d using proerythroblasts isolated from the spleens of Friend virus-inf ected mice fed an amino acid-based, folate-free diet. Control proeryth roblasts were obtained from Friend virus-infected mice fed the same di et plus 2 mg folic acid/kg diet. Our previous studies showed that, aft er 20 to 32 hours of culture in folate-deficient medium with 4 U/mL of erythropoietin, the folate-deficient proerythroblasts underwent apopt osis, whereas control erythroblasts survived and differentiated into r eticulocytes over a period of 48 hours, The addition of folic acid or thymidine to the folate-deficient medium prevented the apoptosis of th e folate-deficient erythroblasts, thereby implicating decreased thymid ylate synthesis as the main cause of apoptosis in the folate-deficient erythroblasts, In the study reported here, we examined intracellular folate levels, uracil misincorporation into DNA, p53 and p21 proteins, and reticulocyte formation in erythroblasts cultured in folate-defici ent or control medium. In all experiments, the folate-deficient erythr oblasts cultured in folate-deficient medium gave results that varied s ignificantly from folate-deficient erythroblasts cultured in control m edium or control erythroblasts cultured in either folate-deficient or control media. Folate-deficient erythroblasts cultured in folate-defic ient medium had marked decreases in all coenzyme forms of folate that persisted throughout culture, increased uracil misincorporation into D NA, persistent accumulations of p53 and p21, and decreased reticulocyt e production but increased size of individual reticulocytes, A model o f folate-deficient erythropoiesis based on apoptosis of late stage ery throblasts is presented. This model provides explanations for the clin ical findings in megaloblastic anemia. (C) 1997 by The American Societ y of Hematology.