Friend virus-induced erythroblastosis in STAT5a/b mutant mice: Properties of isolated erythroblasts in vitro

Abstract

This study examined the roles of the Stat5a and Stat5b gene products in growth and differentiation of mouse erythroid cells. BALB/c mice bearing inactivating mutations in both Stat5a and Stat5b were infected with the anemia-inducing strain of Friend virus. The infected mice developed erythroblastosis in the spleen typical of the early phase of Friend disease. Early erythroblasts purified from the spleens of infected mice (Stat5a/b mutants and BALB/c wild type controls) were cultured in medium containing 2 U/mL of erythropoietin (EPO). During the culture period of 60 h, a significant fraction of the Stat5a/b(−/−) erythroblasts differentiated into reticulocytes. However, the population exhibited several properties that were different from controls. Control cell numbers increased 6-fold during the 60 h culture period and about 90% of the resultant population formed reticulocytes. The Stat5a/b(−/−) cells increased in number only 3.5-fold. About 30% of the cells never initiated hemoglobin synthesis, and about one-half of the remainder did not mature into reticulocytes. After 36 h of culture, the Stat5a/b(−/−) erythroblast population contained a higher fraction than normal of cells which were positive for DNA degradation by a flow cytometric TUNEL assay although morphological apoptosis was not pronounced. This pleotropic phenotype does not resemble the type or timing of apoptosis induced by EPO deprivation. The combined result of the above factors was inefficient erythropoiesis by Stat5a/b(−/−) cells in vitro. Several molecular regulators of apoptosis and of cell cycle were examined. Normal levels of Bcl-x production were observed and no activation of caspase 3 or cleavage of PARP was detected. p27 and p21 were significantly elevated in Stat5a/b(−/−) cells. The fractions of cells in G1 and S phases of the cell cycle were different in Stat5a/b (−/−) and control cells throughout the culture period.