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B Nakamoto

Publications and source records attributed to B Nakamoto.

At least 37 records · Page 2Linked to original sources
PubMed1990

Dynamics of erythropoietin receptor expression on erythropoietin-responsive murine cell lines.

We examined erythropoietin receptor expression in two murine cell lines, B6SUtA and DA-1, that respond to erythropoietin in different ways. While B6SUtA cells undergo erythroid differentiation with limited proliferation after addition of erythropoietin, DA-1 cells show only a proliferative response. Equilibrium binding experiments with 125I-erythropoietin revealed that both B6SUtA and DA-1 cells express a single class of erythropoietin receptors. In the absence of erythropoietin, B6SUtA cells exhibited 145 receptors per cell with a dissociation constant (kd) of 380 pmol/L. Six days after induction with erythropoietin, the B6SUtA cells displayed 310 receptors per cell without a change in binding affinity; exposure to erythropoietin also increased cellular hemoglobin content. DA-1 cells adapted to erythropoietin-dependent growth over a period of months and exhibited a progressive increase in erythropoietin receptor expression, from 85 per cell (kd = 540) to 550 per cell (kd = 530), although the cells remained uniformly benzidine-negative. We interpret the data with B6SUtA cells to indicate that early erythroid differentiation stages are attended by an increase in erythropoietin receptor display, coordinate with the initiation of expression of erythroid-specific genes. In contrast, the results with DA-1 cells are most compatible with clonal selection as the mechanism underlying enhanced receptor expression. Thus, display of the erythropoietin receptor is dynamic and can be modulated during the course of erythropoietin-induced differentiation.

Cell Differentiation↗
PubMed1988

Erythropoietin changes the globin program of an interleukin 3-dependent multipotential cell line.

B6SUtA is a factor-dependent murine cell line of adult origin displaying the functional properties of a multipotent hematopoietic stem cell. We analyzed the globin programs of B6SUtA cells undergoing erythroid differentiation in both suspension and clonal cultures. In the absence of added erythropoietin, a small number of hemoglobinized cells were present, and these expressed predominantly embryonic globin. Addition of erythropoietin increased the number and maturation of hemoglobinized cells and led to a preferential augmentation of adult globin. Analysis of individual B6SUtA erythroid bursts showed that embryonic and adult globin can be expressed in cells derived from a single progenitor. Furthermore, by studying globin expression in cultured cells from mouse embryos, we found that the globin programs of B6SUtA cells are similar to those of erythroid progenitors at the period of transition from yolk sac to fetal liver erythropoiesis. Since B6SUtA cells are derived from adult bone marrow and they have the capacity to express embryonic globin, we speculate that the globin locus is not irreversibly modified during development and that adult cells at early stages of erythroid differentiation can transiently express ontogenetically primitive globin programs.

Animals↗
PubMed1988

Surface antigenic profile and globin phenotype of two new human erythroleukemia lines: characterization and interpretations.

Detailed characterization of the composite phenotype of two newly established erythroleukemia lines (OCIM1, OCIM2) shows that these lines share many of their erythroid markers (ie, surface antigens and globin program) as well as several of their nonerythroid properties (myeloid/monocytic/megakaryocytic) with the two known erythroleukemia lines (K562, HEL). In addition, each displays novel and instructive features. We argue that the surface and globin phenotype of all erythroleukemia lines is nonrandom and that it may be of physiologic relevance; it could represent the most prevalent phenotype of cells transformed by leukemia in vivo, and it raises the possibility that cells with similar potentials exist transiently during normal hematopoietic differentiation before their irreversible commitment to a single lineage. As such, these cells demonstrate a greater phenotypic adaptability in vitro than do their single lineage-committed counterparts since they can differentiate toward more than one lineage.

Antigens, Surface↗
PubMed1987

Constitutive and inducible secretion of platelet-derived growth factor analogs by human leukemic cell lines coexpressing erythroid and megakaryocytic markers.

We have examined the constitutive and inducible secretion of platelet-derived growth factor (PDGF)-like proteins in a variety of human hemopoietic cell lines. The highest levels of secreted protein were noted in four human erythroleukemia lines which, in addition to erythroid lineage markers, express one or more megakaryocytic lineage markers. Induction of these lines by 12-O-tetradecanoylphorbol-13-acetate enhanced the expression of megakaryocytic markers and increased secretion of PDGF-like proteins several fold. In concert with these changes, there was significant induction of c-sis/PDGF-B messenger RNA (mRNA) expression in all lines, whereas one line showed significant concurrent induction of PDGF-A mRNA expression. Whether PDGF-like secretion is part of the stem cell-like phenotype displayed by these lines or is secondary to their leukemic transformation remains to be determined. Nevertheless, these lines provide new cellular models for studying the expression and function of PDGF analogs in hemopoietic cells.

Cell Differentiation↗
PubMed1987

Analysis of the erythroid phenotype of HEL cells: clonal variation and the effect of inducers.

The erythroid phenotype of HEL cells, before and after the addition of a variety of inducers, was assessed at the cellular and biochemical level. Among 14 inducers used, delta-aminolevulinic acid (delta-ALA) was identified as the most optimal inducer of heme and globin synthesis in HEL cells. The relative synthesis of globin chains produced by HEL cells, mainly gamma and alpha chains with traces of epsilon and zeta chains, was not influenced by the majority of the inducers used. However, delta-ALA and bromodeoxyuridine did increase the relative synthesis of alpha and epsilon chains respectively. Subcloning experiments revealed heterogeneity in the constitutive expression of alpha globin; however, the latter was inducible in all clones by either hemin or delta-ALA. One rare clone of HEL cells was found to produce, in contrast to parental cells, significant amounts of epsilon globin. This clone differed from K562 cells by the absence of any zeta globin expression, thus demonstrating the independent regulation of the two embryonic chains, epsilon and zeta. Changes in the expression of several surface markers specific for erythroid cells were found to accompany the globin accumulation in these cells, and some of these changes appeared to be inducer specific. Thus, the unique globin and nonglobin phenotypic properties of HEL cells and their subclones make them valuable cellular models complementary to the existing K562 cells for studying regulatory aspects of erythroid-specific proteins.

Antigens, Surface↗
PubMed1986

The modulation of Hb F synthesis in adult erythroid progenitor (burst-forming unit) cultures reflects changes in gamma-globin gene transcription and chromatin structure.

Fetal hemoglobin production can be reactivated in vivo in adult persons with various hemoglobinopathies and other hemopoietic disorders, and in cultures of adult erythroid progenitors. We show that the activation of Hb F in adult cells is transcriptional in nature and is accompanied by the appearance of DNase I-hypersensitive sites and undermethylation of Hpa II sites 5' to the gamma-globin genes. Production of Hb F in culture is strongly modulated by the environment, and the repression of Hb F synthesis by specific culture conditions has been reported. By nuclear runoff, chromatin, and methylation analyses, we show that this inhibition of Hb F production in vitro is at the level of transcription with the concomitant loss of characteristic gamma hypersensitive sites and methylation of gamma Hpa II sites. These data indicate, first, that the organization of globin chromatin of adult cells that produce fetal hemoglobin resembles that of fetal erythroid cells and, second, that this organization switches from a fetal to an adult pattern in response to changes in the environment of the erythroid cells.

Cells, Cultured↗
PubMed1986

Fetal to adult hemopoietic cell transplantation in humans: insights into hemoglobin switching.

A 2-year-old boy with refractory acute leukemia (ALL) was transplanted with liver cells from twin fetuses of an 18-gestational-week age. Regeneration of hemopoietic cells was evident during the second week following transplantation when a cellular, predominantly erythroid, marrow was present. Studies of bone marrow and peripheral blood cells obtained 21 days posttransplant showed that bone marrow and peripheral blood BFU-E-derived erythroblasts displayed typical fetal patterns of globin chain synthesis (gamma/gamma + beta ratios: 0.87 to 0.98). In addition, all of the individually analyzed erythroid clones displayed a fetal type of globin program, suggesting that the presence of rare, partially switched clones was unlikely. Additional evidence supported the fetal phenotype of these progenitors. The il expression of culture-derived erythroblasts was typical for fetal erythroid cells. As in fetal cells, fetal sheep serum influenced neither the globin nor the il phenotypes, and the growth characteristics were as those observed in fetal liver cultures. That these fetal progenitors matured in vivo and produced cells with a fetal program was shown by the pattern of globin biosynthesis in bone marrow cells and peripheral blood reticulocytes (gamma/gamma + beta ratios: 0.85 to 0.95) at days 14 and 21 posttransplantation. These results indicate that the transplanted fetal cells, in spite of their proliferation and differentiation in the environment of the recipient, continued to express during the early posttransplantation period fetal patterns of globin, surface antigenic determinants, and growth and response to environmental modulation. The observations in this patient support the notion that hemoglobin switching is primarily controlled by a mechanism intrinsic to the stem cell.

Bone Marrow↗
PubMed1983

Direct evidence for interaction between human erythroid progenitor cells and a hemoglobin switching activity present in fetal sheep serum.

An activity that induces Hb F to Hb A switching in human cells is present in fetal sheep serum. To test directly the role of cell-to-environment interactions in hemoglobin switching and to define the level of erythroid cell differentiation at which this activity operates, colony transfer experiments were done. Clones grown in the presence of switching activity-containing medium (fetal sheep serum) or control medium (fetal calf serum) were transferred, at the 16- to 30-cell stage, to either fetal sheep serum or fetal calf serum plates and Hb F synthesis was determined in the fully mature erythroid bursts. Fetal calf serum-to-fetal calf serum transfers produced colonies with the high Hb F levels characteristic of undisturbed fetal calf serum-grown clones. Fetal sheep serum-to-fetal calf serum transfers resulted in significant decrease in Hb F synthesis, revealing an interaction between hemoglobin switching activity and cells at an early stage of progenitor cell development. The reduction of Hb F synthesis in fetal calf serum-to-fetal sheep serum transfers indicated that hemoglobin switching activity interacts with cells at later stages of progenitor cell development. Maximal decrease in Hb F synthesis was observed in fetal sheep serum-to-fetal sheep serum transfers, indicating that optimal effects on Hb switching are obtained when the environment that induces Hb switching is present throughout the development of progenitor cells. By splitting single early clones into two parts and transferring them to either a fetal sheep serum or a fetal calf serum environment, these interactions were further demonstrated in the progeny of a single erythroid burst-forming unit. Since all clone transfers were done on cell-free plates, the results of fetal calf serum-to-fetal sheep serum and of fetal sheep serum-to-fetal sheep serum transfers indicated that the switching activity does not require helper cells for its action. These studies show directly that (i) Hb F synthesis is controlled at the level of progenitors and (ii) it involves interactions between progenitor cells and their environment.

Animals↗
PubMed1983

Hemoglobin switching activity.

We describe observations that suggest the existence of an activity that induces the forward HbF-to-HbA switch. Studies in normal adult BFU-E cultures, in cultures of mutant BFU-E, and in cultures of cells from the neonatal and fetal stages of development reveal that the cells from various stages of ontogeny respond differently to this activity. Experiments using transfers of erythroid clones indicate that the hemoglobin switching activity acts on progenitors and that the interaction is of a direct nature. Our findings that the fetal BFU-E fail to respond whereas the neonatal and adult BFU-E do respond to the switching activity leads us to suggest that hemoglobin switching during ontogeny is controlled by a combined intrinsic/interactive mechanism. We propose that during ontogeny a locus becomes activated, the product of which (a receptor?) allows the cells to respond to their environment. Fetal cells do not switch because they cannot interact with their environment. The appearance of cellular receptors in erythroid progenitors after birth allows the progenitor cells to interact with their environment, and HbF-to-HbA switching ensues.

Animals↗
PubMed1983

Human erythroleukemia cell line (HEL) undergoes a drastic macrophage-like shift with TPA.

We investigated the effect of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on the human erythroleukemia cell line, HEL, and found that TPA addition (10(-6)-10(-8) M) to HEL cell cultures induces morphological, functional, and biochemical changes in HEL cells that are characteristic for macrophage-like cells. Apart from the drastic changes in morphology, the cells greatly enhance their phagocytic ability and acquire receptors for binding and degradation of chemically modified lipoproteins. At the biochemical level, a newly synthesized 85K glycoprotein is observed, and the cells are unresponsive to inducers of globin synthesis. Comparative observations with K562 cells indicate that TPA inhibits, as in HEL cells, spontaneous and induced globin synthesis, but induces minimal macrophage-like properties in these cells. The results with HEL cells are interpreted to indicate that TPA uncovers a latent monocyte-like phenotype in these cells.

Acid Phosphatase↗
PubMed1983

Carbohydrate antigen profiles of human erythroleukemia cell lines HEL and K562.

The expression of major carbohydrate antigens carried by polylactosaminyl chains in human erythroleukemia cell lines, K562 and HEL, was investigated by applying monoclonal antibodies recognizing specific carbohydrate determinants. The two cell lines showed common differences in their glycolipid compositions: (1) the presence of significant amounts of ganglio-series glycolipids, which are absent in normal erythrocytes; and (2) a remarkable reduction in the amount of globo-series glycolipids, which are the major glycolipids in normal human erythrocytes. A variety of differences were also detected in the carbohydrate antigens carried by lacto-series glycolipids and glycoproteins having related carbohydrate chains. K562 cells were i+H-X+, with a minor population of I+ cells. HEL cells were I-i+H+X-. The presence of the I+ population in K562 cells is particularly noteworthy, since I-antigen is characteristic of adult mature erythrocytes and is absent in most human leukemic cell lines. Several clones showing I+, I+/-i+/-, or I-i+ specificities were isolated from K562 cells by cloning in either methylcellulose media or limiting dilution, and I+ and I- cells were sorted by FACS fluorometer. HEL cells and these K562 clones provide a useful experimental model for studying the biologic significance and enzymatic control in expression of cell surface polylactosamines.

ABO Blood-Group System↗
PubMed1983

The surface antigen profile of HEL cells.

The surface antigen profile of HEL cells was probed with a panel of monoclonal antibodies that have been associated with various hemopoietic lineages. Although the list of antibodies tested is by no means complete, the following characteristic picture emerges. a. Affiliation of HEL cells with erythroid lineage has been secured by other than the globin criteria. In this respect differences with K562 cells have been mostly of a quantitative nature, with only a few exceptions. One notable exception is the presence of group H determinant in HEL cells. b. There is a strong expression in HEL cells of determinants present in the platelet/megakaryocytic series. K562 cells express only selective determinants for this lineage. c. In contrast to K562 cells, there is a strong expression in HEL cells of monocyte-associated determinants. Corroborating this are the cytochemical properties of the HEL cells [11] and their induction by TPA (12-0-tetradecanoyl-phorbol-13-acetate) to a macrophage-like phenotype [41]. d. Granulocyte-specific antigens have not been found in HEL cells, although shared antigenic determinants of granulocytes with other cells can be expressed. In contrast, K562 cells display several granulocyte-specific antigens. e. No T-cell-associated antigens are found in HEL cells, although some B-cell-associated determinants can be detected. The interpretation of the composite phenotype in HEL cells is, as yet, unclear. One fact stands out, however; the only two erythroid-associated lines available today also display a multilineage-affiliated phenotype that seems to have no parallel with the other cell lines thus far described.

Antigens, Surface↗
PubMed1982

Hemoglobin switching in culture: evidence for a humoral factor that induces switching in adult and neonatal but not fetal erythroid cells.

An erythropoietic activity that exerts a profound effect on fetal Hb synthesis is present in fetal sheep sera and it attains a peak concentration at the end of the second to the middle of the third trimester of fetal life. The activity consistently inhibits the increased synthesis of fetal Hb in cultures of burst-forming units (BFUes) from normal adults. In cultures of BFUes from homozygous beta+-thalassemias the activity produces a striking decline in gamma chain synthesis, a decline in G gamma/A gamma chain synthesis ratio, and an increase in delta/gamma and alpha/non-alpha ratios--i.e., findings suggesting a genuine gamma-to-beta switch. The activity accelerates Hb F-to-Hb A switching in neonatal BFUe cultures but it has no effect on fetal Hb synthesis in cultures of BFUe obtained from human fetuses. These findings provide direct evidence that (a) humoral factors play a role in the regulation of the switch from fetal to adult Hb formation, and (b) progenitor cells from various stages of ontogeny respond differently to these factors. The results are compatible with the hypothesis that Hb switching during development is mediated through a change in a developmental program which controls the responsiveness of progenitor cells to "switching" activities in their environment.

Animals↗
PubMed1981

Globin synthesis in erythroid bursts that mature sequentially in culture. I. Studies in cultures of adult peripheral blood BFU-Es.

To study whether the culture time at which the burst populations mature influences the expression of fetal hemoglobin in bursts, we measured hemoglobin synthesis in cohorts of fully hemoglobinized erythroid bursts maturing sequentially in cultures of adult peripheral blood BFU-Es. In 13 of 15 experiments, a decline in gamma/gamma + beta ratio was noted as the culture time advanced. On the average, erythroid bursts that mature during the third culture week showed lower levels of fetal Hb synthesis compared to bursts that are already mature in the second culture week. The decline of gamma/gamma + beta ratio with culture time was also noted in erythroid bursts composed of immature erythroblasts. The enhanced HbF formation in peripheral blood BFU-E cultures is thus most pronounced among the bursts that become hemoglobinized early, and there is a tendency for normalization of HbF synthesis in bursts that mature in late culture days. These results can be interpreted by several alternatives, including the possibility that the expression of high HbF levels in the early days of adult BFU-E cultures is a reflection of premature commitment to terminal differentiation of progenitors that possess an active HbF program. The present data indicate that the variation of HbF synthesis with culture time should be taken into consideration when the influence of various culture conditions of HbF synthesis is studied in BFU-E cultures.

Adult↗
PubMed1979

Hb F production of endogenous colonies of polycythemia vera.

Fetal hemoglobin was studied in endogenous colonies produced in plasma clot and methylcellulose cultures of circulating progenitors from patients with polycythemia vera (PV). Analysis of globin chain synthesis showed that gamma chains constituted from 13% to 42% of the non-alpha chains produced in cultured cells, whereas from 27% to over 50% of the endogenous colonies contained Hb F, as indicated by the fluorescent antibody probe. Since the endogenous colonies in PV cultures originate from the abnormal PV clone, the findings provide direct evidence that a single pluripotent stem cell can have committed progeny that differ in their expressions of the Hb F production program.

Cells, Cultured↗