T Papayannopoulou

Fetal Hb Production during Acute Erythroid Expansion: I. OBSERVATIONS IN PATIENTS WITH TRANSIENT ERYTHROBLASTOPENIA AND POST‐PHLEBOTOMY

Summary. In order to study fetal haemoglobin production during acute erythroid expansion we did sequential measurements of Hb F‐containing erythrocytes (F‐cells) and of relevant haematological parameters in 10 subjects recovering from erythroid aplasia, iron deficiency anaemia or following phlebotomy. An increased production of F‐cells was consistently observed during the acute marrow expansion, but there were significant differences in the maximum F‐cell response among individuals. These differences could not be explained by differences in the degree of anaemia alone, nor could they be correlated with the level of peak reticulocytosis. Two patients who reached the highest F‐cell numbers were probably carriers of heterocellular hereditary persistence of Hb F, suggesting that this gene may play a role in determining the magnitude of F‐cell production in anaemic patients.

Integrin alpha4 blockade sensitizes drug resistant pre-B acute lymphoblastic leukemia to chemotherapy

Bone marrow (BM) provides chemoprotection for acute lymphoblastic leukemia (ALL) cells, contributing to lack of efficacy of current therapies. Integrin alpha4 (alpha4) mediates stromal adhesion of normal and malignant B-cell precursors, and according to gene expression analyses from 207 children with minimal residual disease, is highly associated with poorest outcome. We tested whether interference with alpha4-mediated stromal adhesion might be a new ALL treatment. Two models of leukemia were used, one genetic (conditional alpha4 ablation of BCR-ABL1 [p210(+)] leukemia) and one pharmacological (anti-functional alpha4 antibody treatment of primary ALL). Conditional deletion of alpha4 sensitized leukemia cell to nilotinib. Adhesion of primary pre-B ALL cells was alpha4-dependent; alpha4 blockade sensitized primary ALL cells toward chemotherapy. Chemotherapy combined with Natalizumab prolonged survival of NOD/SCID recipients of primary ALL, suggesting adjuvant alpha4 inhibition as a novel strategy for pre-B ALL.

The role of CS1 moiety of fibronectin in VLA4‐mediated haemopoietic progenitor trafficking

In vitro the integrin VLA4 mediates the adhesion of haemopoietic progenitors to bone marrow stroma through an interaction with its ligands VCAM‐1 and the CS1 moiety of fibronectin. The VLA4/VCAM‐1 pathway has been implicated in haemopoietic trafficking in vivo since antibodies to both VLA4 and VCAM‐1 decrease the homing (lodgement) of transplanted progenitors and mobilize progenitors. However, the role of the CS1 domain of fibronectin in progenitor trafficking in vivo has not been explored. We studied the effect of competitive inhibition of the VLA4/CS1 pathway on progenitor homing and mobilization in mice. Pre‐incubation of bone marrow cells with a CS1 inhibitor did not alter the number of CFU‐C or CFU‐S12 lodged to the bone marrow of lethally irradiated mice 3 h after transplantation. In addition, continuous administration of a CS1 inhibitor did not increase the number of CFU‐C in the peripheral blood. In order to study the role of the VLA4/CS1 pathway in trafficking of more primitive progenitors we studied whether administration of a CS1 inhibitor mobilized radioprotective cells. In contrast to the effect of anti‐VCAM‐1 which mobilized cells capable of rescuing 100% of lethally irradiated mice, administration of a CS1 inhibitor did not increase the number of radioprotective cells in the peripheral blood. Haemopoietic progenitors also bind to the RGD motif of fibronectin through an interaction with VLA5 and we therefore also studied the effect of antibodies to VLA5 on progenitor homing and mobilization. Antibody to VLA5 did not alter bone marrow lodgement at 3 h or increase the number of circulating haemopoietic progenitors. These studies therefore imply that, in contrast to VCAM‐1, the CS1 moiety of fibronectin is not a significant ligand in VLA4 mediated progenitor trafficking in vivo.

Coexpression of embryonic, fetal, and adult globins in erythroid cells of human embryos: Relevance to the cell-lineage models of globin switching☆

The cellular control of the switch from embryonic to fetal globin formation in man was investigated with studies of globin expression in erythroid cells of 35- to 56-day-old embryos. Analyses of globins synthesized in vivo and in cultures of erythroid progenitors (burst-forming units, BFUe) showed that cells of the yolk sac (primitive) erythropoiesis, in addition to embryonic chains, produced fetal and adult globins and that cells of the definitive (liver) erythropoiesis, in addition to fetal and adult globins, produce embryonic globins. That embryonic, fetal, and adult globins were coexpressed by cells of the same lineage was documented by analysis of globin chains in single BFUe colonies: all 67 yolk sac-origin BFUe colonies and 42 of 43 liver-origin BFUe colonies synthesized epsilon-, gamma-, and beta-chains. These data showed that during the switch from embryonic to adult globin formation, embryonic and definitive globin chains are coexpressed in the primitive, as well as in the definitive, erythroid cells. Such results are compatible with the postulate that the switch from embryonic to fetal globin synthesis represents a time-dependent change in programs of progenitor cells rather than a change in hemopoietic cell lineages.

Effects of butyrate and glucocorticoids on gamma- to beta-globin gene switching in somatic cell hybrids.

Butyrate and its analogs have been shown to induce fetal hemoglobin in humans and primates and in erythroid cell cultures. To obtain insights concerning the cellular mechanisms of butyrate action, we analyzed the effects of butyrate on human globin gene expression in hybrids produced by fusing mouse erythroleukemia cells (MEL) with human fetal erythroid cells (HFE). These hybrids initially express human fetal hemoglobin but subsequently switch to adult globin expression after several weeks in culture. We found that alpha-aminobutyric acid, a butyrate analog which does not induce terminal maturation, strikingly delays the rate of the gamma- to beta-globin gene (gamma-to-beta) switch in the HFE x MEL hybrids. The effect of butyrate on globin expression is transient, with the result that the delay of globin gene switching requires the continuous presence of this compound in culture. Furthermore, butyrate fails to induce fetal hemoglobin expression in hybrids which have switched, suggesting that the effect of this compound on gamma-globin expression is due to inhibition of gamma gene silencing rather than to induction of gamma gene transcription. Since in other cellular systems, glucocorticoids antagonize the action of butyrate, the effect of dexamethasone on the gamma-to-beta switch in HFE x MEL hybrids was examined. Dexamethasone strikingly accelerated the gamma-to-beta switch, and its effect was irreversible. The effects of dexamethasone and butyrate on the gamma-to-beta switch of the HFE x MEL hybrids appear to be codominant. These results indicate that steroids can have a direct effect on globin gene switching in erythroid cells.

MAPPING OF ANTIGENIC SITES ON HUMAN HAEMOGLOBIN BY MEANS OF MONOCLONAL ANTIBODIES AND HAEMOGLOBIN VARIANTS

Monoclonal antibodies specific for human globin chains have been prepared and the following strategy has been applied in delimiting the antigenic sites involved in antibody binding. The structural sites of the human globin subunit that might be recognised by the monoclonal antibody were deduced from comparisons of the primary structures of mammalian globin chains that did or did not react with the antibody. The involvement of individual residues at these specific sites was subsequently tested by reacting the antibody with abnormal human haemoglobins in which there was either a substitution or a deletion of one of the residues in question. The primary structural site recognised by monoclonal antibody HuHb beta 3-2(an antibody that reacts with the adult haemoglobins from man and macaque monkey, but not with those from baboon and mouse) includes the aspartic acid residue at position 52 of the beta-globin subunit.

The asynchrony of γ- and β-chain synthesis during human erythroid cell maturation: III. γ- and β-mRNA in immature and mature erythroid clones

Abstract Globin chain synthesis and the accumulation of globin mRNAs were examined in immature and mature erythroid clones produced by circulating erythroid progenitors (burst forming units) of the neonate. Immature clones were composed of early erythroblasts containing little hemoglobin while the mature clones appeared at later days in culture and were composed of well hemoglobinized erythroblasts. There was a strikingly higher γ γ + β chain synthetic ratio in the nonhemoglobinized clones compared to their fully hemoglobinized counterparts. RNA was prepared from immature and from mature colonies and α-, β-, and γ-mRNA content was measured by solution hybridization to α-, β-, and γ-[32P]DNA probes. The probes were sense strand coding globin sequences prepared from chimeric plasmids by isolation of nick-translated [32P]DNA which annealed to cord blood or adult reticulocyte globin mRNA. In three experiments, the γ γ + β mRNA ratios in immature bursts were 0.58, 0.55, and 0.70 while ratios in the mature counterparts were 0.21, 0.38, and 0.45. There was excellent agreement between globin chain synthetic ratios and globin mRNA ratios suggesting that the globin mRNA species present in immature and mature erythroblasts are translated with similar efficiencies. These data suggest that the maturational modulation of non-α-globin chain synthesis is not under translational control. The absolute mRNA values (molecules per cell) consistently showed similar amounts of γ-mRNA per cell in both the immature and the mature cell populations. The most striking effect of maturation was the increase in the amount of β-mRNA per cell (and the concomitant increase in α-mRNA). These data, together with previously published results on globin chain biosynthesis suggest that the maturation related decline in γ/γ + β chain ratios is due to asynchrony in γ- and β-mRNA accumulation. Apparently γ-mRNA accumulates early (at or near the onset of globin synthesis) and the accumulation of β-mRNA follows. This asynchrony in mRNA accumulation results in a decline in the relative synthesis of fetal hemoglobin during maturation.

Greek (Aγ) variant of hereditary persistence of fetal haemoglobin: globin gene organization and studies of expression of fetal haemoglobins in clonal erythroid cultures

Individuals heterozygous for the Greek (Aγ) variant of hereditary persistence of fetal haemoglobin (HPFH) synthesize Hb F whose γ‐globin chains are predominantly of the Aγ type. DNA obtained from Greek HPFH heterozygotes was used to test for abnormalities in the organization of non α‐globin genes. In addition, γ‐ and β‐globin expression was studied in BFUe cultures. Restriction endonuclease mapping showed that the γGγ, δ and β genes in cis to the Greek HPFH determinant are intact. Overproduction of γ‐globin chains synthesis was observed in the BFUe cultures. A significant portion of the γ chain synthesis was of the Gγ type, suggesting that the Gγ genes cis and trans to the HPFH chromosome are active in culture.

Toward a system for detecting somatic-cell mutations. V. Preparation of fluorescent antibodies to hemoglobin Hasharon, a human α-chain variant

Antibodies against the abnormal human hemoglobin, Hb Hasharon (alpha 47 Asp leads to His), were raised in horse and purified by absorption against Sepharose 4B to which normal hemoglobins or Hb Hasharon were bound. The purified, non-precipitating antibodies were tested for specificity against normal hemoglobins and Hb Hasharon by immunodiffusion in the presence of anti-horse IgG, and by exposing mixtures of normal and Hb Hasharon-containing red cells to the antibodies after conjugation of the latter with fluorescein isothiocyanate. The ease with which antibodies specific for different variant hemoglobins have been prepared, and their potential for identifying individual erythrocytes that contain these hemoglobins by virtue of somatic mutation, underscore their value as aids to detection and analysis of mutational events in human subjects.