Martha Brice

Analysis of human hemoglobin switching in MEL × human fetal erythroid cell hybrids

The switch from fetal to adult globin synthesis in man was studied using heterospecific cell hybrids between human fetal erythroblasts and mouse erythroleukemia cells. When erythroblasts from first trimester fetuses were used the hybrids expressed a fetal program of human globin expression. While in continuous culture, these hybrids switched from predominantly fetal to almost exclusively adult globin expression, providing direct evidence that switching can occur within a single cell lineage. Sequential studies of globin expression at a single cell level and subcloning experiments suggested that the switch reflects a progressive increase in the generation of beta + cells from gamma + cells. Hybrids formed with erythroblasts of second trimester fetuses switched faster than those produced with cells of first trimester fetuses. The findings suggest that the human gamma to beta switch is controlled by a developmental clock-like mechanism, which appears to be associated with chromosome 11.

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.

Developmental regulation of fetal to adult globin gene switching in human fetal erythroid × mouse erythroleukemia cell hybrids☆

Human fetal erythroid x murine erythroleukemia cell hybrids undergo human fetal (gamma) to adult (beta) globin gene switching in vitro under the control of a mechanism located on human chromosome 11. We investigated whether this mechanism acts in cis or in trans by preparing hybrid cells containing marked fragments of the gamma and beta genes known to switch in transgenic mice. In these cells the chromosomally introduced human globin locus undergoes the fetal to adult globin gene switch. In contrast, the marked globin gene fragments were expressed at all stages of hybrid development. These results suggest that either the mechanism of switching acts in cis or that sequences present in the chromosomal globin locus but missing from the transfected globin gene fragments mediate its action.

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.

Somatic-Cell Mutation Monitoring System Based on Human Hemoglobin Mutants

The system described in this chapter was developed as a means of detecting rare red cells, in genetically normal (HbA/HbA) individuals, that are heterozygous for an abnormal hemoglobin. It is assumed, first, that mutations arise spontaneously in human hemopoietic stem cells, as they do in gametal stem cells, and second, that somatic mutations of globin-chain genes do not diminish the viability of affected stem cells. The latter assumption is a reasonable one, since phenotypic expression of such mutations occurs very late in hemopoietic cell differentiation. It is expected that as a result of these stem cell mutations, lines of stem cells containing the mutant globin genes are established and produce erythrocytes heterozygous for structurally abnormal globin chains. Development of appropriate methods of screening blood samples should then permit detection and enumeration of red cells that contain an abnormal hemoglobin as a result of somatic mutation in a stem cell.

GM 58/8: a monoclonal antibody that identifies a new lineage-specific determinant expressed by myeloid progenitors (CFU-GM) and their progeny.

A cytotoxic (IgM) monoclonal antibody (McAb) raised against human erythroleukaemia cell line HEL appears specific for the myelomonocytic lineage. Indirect immunofluorescence and fluorescence‐activated cell sorter analyses of peripheral blood and bone marrow cells showed that this McAb (designated GM 58/8) reacts exclusively with more than 90% of the granulocytes, monocytes and myeloid precursors. The positive (fluorescent) fraction sorted from McAb‐treated bone marrow buffy coat cells showed a marked enrichment of myeloid precursors, while nonmyeloid cells were recovered in the negative fraction. GM 58/8 specifically inhibited the growth of 90‐100% of the committed myelomonocytic progenitors (CFU‐GM) in five complement‐mediated cytotoxicity experiments. GM 58/8 had virtually no effect on the erythroid progenitor (BFU‐E and CFU‐E) growth. Cell sorting experiments using this McAb resulted in recovery of 84% of CFU‐GM among the fluorescent fractions. Comparisons with other anti‐myelomonocytic McAbs reported so far suggest that GM 58/8 has more restricted reactivity and shows more efficient CFU‐GM cytotoxicity. Patterns of reactivity with normal cells and established lines indicate that the antigenic determinant identified by GM 58/8 is different from that recognized by previously described anti‐myelomonocytic McAbs. McAb GM 58/8 could be used to isolate or remove pure populations of progenitors and/or more differentiated cells of the myelomonocytic lineage for in vitro studies. This study also provides evidence that the HEL cell line, derived originally from an erythroleukaemia patient, expresses an antigenic determinant shared by the normal cells of myelomonocytic lineage.

Intrinsic factors influencing the maintenance of contractile embryonic heart cells in vitro: I. The heart muscle conditioned medium effect

Abstract Dissociated heart muscle cells from 7-day chicken embryos were cultured at low density in a conditioned medium prepared by incubating heart muscle fragments in a chemically defined medium. Higher proportions of contracting cells were seen in conditioned medium than in either chemically defined or serum containing media in both short term (24 h) and long term (36 days) cultures. Conditioned medium also maintained contractile activity for longer periods (26% at 36 days) than did serum containing medium (0% at 28 days). Improved survival and spreading on glass were also a feature of culture in heart conditioned medium. Two factors appear to be present in the conditioned medium which affect contraction and spreading independently. These factors were operative even at dilutions of 1:600 and arise during the conditioning process by some mechanism other than cell death.

Intrinsic factors influencing the maintenance of contractile embryonic heart cells in vitro. II. Biochemical analysis of heart muscle conditioned medium.

Abstract Results are presented concerning the nature of the active factors in heart conditioned medium which enhance spreading and contractile activity in low density cultures of 7-day embryonic heart cells. Aliquots of conditioned medium were either analysed directly, or pretreated in a variety of ways and then assayed for activity on cultured heart cells. The contractile factor was found to be non-dialysable, fairly heat-stable, inactivated by both trypsin and hyaluronidase, unaffected by neuraminidase, and could be removed from the medium by centrifugation at 200 000 RCF. The factor which enhances spreading of cultured heart cells was found to be non-dialysable, heat-stable, inactivated by trypsin, unaffected by hyaluronidase or neuraminidase, and was still active after centrifugation at 200 000 RCF. Pooled lots of heart conditioned medium contained approx. 40 μg/ml protein, 20 μg bound hexose sugars per 100 μg protein, 1.3 μg bound lipid per 100 μg protein, and glucosamine and galactosamine. DNA, RNA, and hydroxyproline were not detected. The results suggest that the contractile factor is a protein-carbohydrate complex of the proteoglycan type and that the spreading factor contains protein.

Cellular regulation of fetal hemoglobin synthesis in man: Investigation of γ and β mRNA accumulation in clonal erythroid cultures initiated from erythroid progenitors derived from fetuses, neonates, and adult individuals☆

The accumulation of globin mRNA in erythroid cells during human development was investigated in clonal cultures of fetal, newborn, and adult origin erythroid progenitors and in control uncultured erythroblasts and reticulocytes. The relative concentrations of α, β, and γ mRNA sequences were measured by hybridization with purified α, β, and γ[3H]cDNA probes. We found that the amount of γ mRNA is characteristic of the developmental stage from which the erythroid progenitor cells are obtained. In colonies from fetal liver cells, 89% of the non-α mRNA is γ mRNA. Colonies derived from neonatal (cord blood) cells produce an average of 55% γ mRNA, while colonies generated from either adult bone marrow or progenitors circulating in the peripheral blood of the adult individual produce γ mRNA ranging from 15 to 38% of the non-α sequences. When compared with erythroid cells (erythroblasts or reticulocytes) from the same tissues, the fetal liver colonies produce identical proportions of γ mRNA as liver cells in vivo. Colonies from circulating erythroid progenitors from the perinatal (switchover) period accumulate somewhat less γ mRNA (55%) than do circulating reticulocytes in vivo (64%). The concentration of γ mRNA (15 to 38%) in colonies of adult marrow or peripheral blood origin is substantially higher than the trace amounts (<1%) found in mature erythroid cells in vivo. The amounts of β and γ globin chain synthesis from fetal liver, cord blood, and adult peripheral blood are proportional to β and γ mRNA (r = 0.97) and thus indicate that selective translation of β and γ mRNA is not a significant factor in the switch from Hb F to Hb A production. (However, both neonatal reticulocytes in vivo and erythroid colonies in vitro contain excess non-α mRNA but exhibit balanced globin chain synthesis; translational control mechanisms apparently prevent excess synthesis of total non-α chains.) The expression of stage-specific patterns of γ and β mRNA in erythroid colonies suggests that the erythroid progenitor cells in vivo possess the potential for unique programs of γ and β mRNA accumulation before they actually enter the pathway to terminal maturation.