Showing papers by "Anna Rita Migliaccio published in 1987"

Cloning of human erythroid progenitors (BFU‐E) in the absence of fetal bovine serum

Abstract: Summary We describe culture conditions which enabled us to clone early human erythroid progenitors (BFU-E) in the absence of fetal bovine serum (FBS). Our medium, which is chemically fully defined, supports proliferation and differentiation of erythroid progenitors comparable to that in FBS-supplemented cultures. Furthermore, it allows cloning of BFU-E from all human haemopoietic tissues (adult marrow, embryonic liver and yolk sac) and adult or perinatal blood. This system should facilitate the investigation of human erythroid differentiation in vitro (e.g. cell-cell interaction. mechanism of action of haemopoietins and inhibitors, regulation of Hb synthesis) as well as the mechanisms underlying myeloproliferative disorders. As an example, we have found that recombinant Ep shows, in this culture system, the same dose response as Ep from conventional sources.

Oestradiol stimulates tyrosine phosphorylation and hormone binding activity of its own receptor in a cell-free system.

Abstract: Recent experiments have shown that calf uterus oestrogen receptor exists in a tyrosine-phosphorylated hormone binding form and in non-phosphorylated, non-hormone binding form. We report here that physiological concentrations of oestradiol in complex with the receptor stimulate the calf uterus receptor kinase that converts the non-hormone binding receptor into hormone binding receptor through phosphorylation of the receptor on tyrosine. The activity of this enzyme has been followed by reactivation of hormone binding sites and phosphorylation on tyrosine of calf uterus phosphatase-inactivated receptor. Phosphorylation of the receptor has been demonstrated by interaction of kinase 32P-phosphorylated proteins with anti-receptor antibody followed either by sucrose gradient centrifugation or SDS-PAGE of the immunoprecipitated proteins. Hormone stimulation of the kinase is inhibited by receptor occupancy of the anti-oestrogen tamoxifen. Oestradiol-receptor complex increases the affinity of the kinase for the dephosphorylated receptor. Findings of this report are consistent with the observation that several protein tyrosine kinases that are associated with peptide hormone receptors are stimulated by the binding of the hormone to the receptor. This is the first report on the activation of a tyrosine kinase by a steroid hormone. The finding that hormones can regulate their own receptor binding activity through a tyrosine kinase is also new.

Early hemopoietic differentiation: the action of multi-CSF is complemented by lineage specific growth factors.

Abstract: Although mechanisms controlling differentiation of hemopoietic stem and early progenitor cells are still poorly understood, it is generally conceded that a pivotal role is played by hemopoietic growth factors (HGFs). However, in-vitro analysis of their action on early progenitors may be obscured by cell-cell interaction, as well as by the presence of fetal bovine serum (FBS). To overcome these limitations, we investigated the action of pure multipotent or lineage-specific HGFs on purified progenitors grown in FBS-free cultures. In the murine system, highly purified progenitors were cultured in the presence of multipotent colony-stimulating factor (multi-CSF, also termed interleukin-3), erythropoietin (Ep) and macrophagic-CSF (M-CSF). Each HGF was unable by itself to induce significant colony growth. However, combined addition of multi-CSF and either Ep or M-CSF gave rise only to pure erythroid or macrophagic colonies, respectively. Partly purified human progenitors were challenged by human granulomonocytic-CSF (GM-CSF), pluripotent CSF (PPO, also termed granulocytic-CSF, G-CSF) and Ep. Here again, each HGF was unable per se to promote colony growth, but combined addition of GM-CSF or PPO and Ep gave rise only to pure erythroid colonies. These results support a model of early hemopoietic differentiation according to which multi-lineage HGFs represent "competence" GFs, the action of which is complemented by lineage-specific "progression" HGFs.