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Biomedical subjects

J W Adamson

Publications and source records attributed to J W Adamson.

At least 19 recordsLinked to original sources
PubMed1992

Erythropoietin rapidly induces tyrosine phosphorylation in the human erythropoietin-dependent cell line, UT-7.

UT-7 is a human megakaryoblastic cell line capable of growing in interleukin-3, granulocyte-macrophage colony-stimulating factor, or erythropoietin (Epo) (Cancer Res 51:341, 1991). We used this cell line and a selected Epo-dependent subcell line (UT-7/Epo) to study the early signal transduction events induced by Epo. When UT-7 cells were exposed to Epo, tyrosine phosphorylation of several proteins (with molecular weight equivalent to that of p85, p110, and p145) was observed. Protein phosphorylation occurred in both a dose- and time-dependent manner. p85 showed a marked increase in phosphotyrosine content within 30 seconds; maximal phosphorylation was observed at 1 minute. Subsequently, tyrosine phosphorylation of p110 and p145 was observed, beginning at 1 minute and reaching plateau at 5 minutes. The degree of phosphorylation of these three proteins gradually decreased thereafter. In addition, in UT-7/Epo cells, Epo induced tyrosine phosphorylation of other proteins that were not observed in Epo-induced UT-7 cells. The concentration of Epo required to induce tyrosine phosphorylation was in the same range of concentration required to stimulate cell growth. Epo was also able to activate p21ras as measured by exchange of guanosine diphosphate for guanosine triphosphate. These data show that tyrosine phosphorylation and P21ras activation are early signals in the Epo-induced mitogenic pathway.

Erythropoietin↗
PubMed1992

Long-term generation of colony-forming cells in liquid culture of CD34+ cord blood cells in the presence of recombinant human stem cell factor.

Human cord blood was used as a source of progenitor and stem cells to evaluate the effect of recombinant human stem-cell factor (SCF) on colony formation and the generation of colony-forming cells (CFC) under highly defined, serum-deprived conditions. SCF interacted with a number of hematopoietic growth factors to stimulate colony growth and was particularly effective in stimulating the formation of mixed-cell colonies from CD34+ soybean agglutinin negative (SBA-) cells. In suspension culture of CD34+, SBA- cells, SCF alone was unable to maintain cell numbers or CFC but, in combination with interleukin-3 (IL-3), increased input numbers of cells by 10-fold and increased CFC of all kinds by nearly 20-fold. This included erythroid burst-forming cells (BFU-E), granulocyte/macrophage (GM) CFC, and mixed-cell CFC. In contrast, CD34- SBA- cells neither gave rise to CFC nor were maintained by combinations of growth factors including SCF. SCF interacted with erythropoietin (Epo) and granulocyte colony-stimulating factor (G-CSF) to maintain large numbers of cells as well as to generate a twofold to threefold increase in CFC in the case of Epo, and a 10-fold increase in CFC in the case of G-CSF. With Epo, the predominant CFC generated were BFU-E and erythroid CFC and many of the cells in suspension were erythroblasts. In contrast, SCF plus G-CSF resulted in large numbers of granulocytes at various stages of maturation and the CFC generated were almost exclusively granulocytic-CFC. IL-1 and IL-6, alone or in combination with SCF, showed little or no ability to increase cell numbers or generate CFC. In summary, SCF interacts with a variety of hematopoietic growth factors to promote colony formation, particularly mixed-cell colony formation, and also, in suspension culture, SCF interacts with IL-3, G-CSF, and Epo to generate large numbers of differentiated cells as well as a variety of CFC for up to 1 month.

Antigens, CD↗
PubMed1992

Expression of the interleukin-3 and granulocyte-macrophage colony-stimulating factor genes in Friend spleen focus-forming virus-induced erythroleukemia.

Friend spleen focus-forming virus (F-SFFV) is a replication-defective retrovirus that induces a multistage erythroleukemia in mice. In the first stage, expression of the SFFV envelope glycoprotein results in erythroid hyperplasia. Subsequently, the F-SFFV integrates near the Spi-1 gene and activates its expression, resulting in immortalized cells that represent a second stage in the disease process. We report here that media conditioned by erythroleukemia cell lines or leukemic spleen cells induced by the polycythemia-inducing strain of F-SFFV (F-SFFVp), but not medium conditioned by SFFVp-induced hyperplastic spleens, promote the proliferation of normal granulocyte-macrophage progenitor cells and of granulocyte-macrophage colony-stimulating factor (GM-CSF)- and/or interleukin-3 (IL-3)-dependent cell lines. The colony-stimulating activity of the conditioned media from four of five of the lines studied was neutralized by antibodies specific for IL-3 and/or GM-CSF, and IL-3 and GM-CSF-specific mRNA could be detected in the cells after amplification by the polymerase chain reaction. No rearrangements of the IL-3 or GM-CSF genes were observed by Southern blot analysis. However, as previously shown for SFFV-induced cell lines, the Spi-1 gene was expressed in all of these cells. Because the Spi-1 gene encodes a transcription factor whose cognate sequences are present in the promoter region of many hematopoietic growth factor genes, including IL-3 and GM-CSF, Spi-1 activation may be inducing the expression of these genes.

Animals↗
PubMed1992

Production of granulocyte colony-stimulating factor and granulocyte/macrophage-colony-stimulating factor after interleukin-1 stimulation of marrow stromal cell cultures from normal or aplastic anemia donors.

We have studied stromal cell function in naive or interleukin-1 (IL-1)-stimulated (100 pg/ml) long-term marrow cultures (LTC) from 12 normal donors and 21 patients with severe aplastic anemia (AA). Conditioned media (CM) from normal LTC contained levels of erythroid burst-promoting activity (BPA) and granulocyte/macrophage (GM) colony-stimulating activity (CSA) comparable to those previously described (Migliaccio et al., [1990] Blood, 75:305-312). The addition of IL-1 to these cultures increased the level of CSA and, specifically, of granulocyte colony-stimulating factor (G-CSF) released. Anti-GM-CSF antibody neutralized BPA and CSA in normal naive LTC CM but only the CSA in the CM from IL-1-stimulated LTC. Since the concentrations of GM-CSF, as detected with a specific immunoassay, did not increase after IL-1 treatment, these data suggest that IL-1-stimulated cultures contain an unidentified growth factor having BPA. CM from AA stromal cells contained levels of CSA comparable to those observed in normal stromal cell CM but had significantly lower levels of BPA. Neither anti-GM-CSF nor anti-IL-3 antibodies neutralized the BPA in AA stromal cell CM. This activity may be related to that found in the CM of IL-1-treated normal stromal cells. In nearly 50% of stromal cell cultures of AA patients, addition of IL-1 failed to increase the BPA, CSA, or G-CSF. The presence of an inhibitor in naive or IL-1-treated AA stromal cell CM was excluded by adding the CM to IL-3-stimulated cultures. These findings suggest that G-CSF and GM-CSF genes are differentially regulated in the marrow microenvironment. Furthermore, a marrow microenvironment, deficient in BPA production and, in some cases, unresponsive to IL-1 could contribute to marrow failure in some patients with AA.

Anemia, Aplastic↗
PubMed1992

A comparison of the responses to recombinant human erythropoietin in normal and uremic subjects.

The erythropoietic response to graded doses of recombinant human erythropoietin (epoetin alfa) was assessed in 24 hemodialysis patients by quantitative ferrokinetic studies, and measurement of the reticulocyte count and plasma levels of transferrin receptor protein. These responses were compared to those of 22 normal subjects. Epoetin alfa was given intravenously at 15, 50 or 150 U/kg every other day for four injections. Three patients with chronic renal failure were restudied after renal function was restored following renal transplantation. The results of these three different measurements of erythroid function showed that the acute response to recombinant human erythropoietin was similar in normal subjects and patients with renal failure. We conclude that chronic uremia does not alter the responsiveness to erythropoietin in vivo.

Anemia↗
PubMed1992

Effectiveness of recombinant human erythropoietin therapy in myelodysplastic syndromes.

In an attempt to determine predictors of response to recombinant human erythropoietin (r-HuEPO) therapy in 20 patients with various subtypes of myelodysplastic syndrome (MDS), plasma concentrations of transferrin receptor protein were measured before and after 4 doses of r-HuEPO. An r-HuEPO dosage of 150 U/kg was administered subcutaneously 3 times weekly and increased to 300 U/kg in patients who failed to raise plasma concentrations of transferrin receptor protein by at least one third. Ten (50%) patients had an effective clinical response to therapy by reducing (greater than 50%) or eliminating transfusion requirements, or by showing an improvement in haematocrit of greater than or equal to 6 percentage points. Changes in plasma transferrin receptor protein concentrations failed to predict which patients would eventually respond to r-HuEPO therapy. A subset of MDS patients demonstrated a delayed response to therapy in order to achieve a satisfactory clinical outcome. Precise predictors of response, either laboratory or clinical, remain to be determined. Continued research is warranted in this group of patients in order to specifically target r-HuEPO therapy. It is, however, likely that r-HuEPO therapy will have an effective and important role in this subset of MDS patients.

Anemia↗
PubMed1991

Response to erythropoietin in erythroid subclones of the factor-dependent cell line 32D is determined by translocation of the erythropoietin receptor to the cell surface.

Regulation of the expression of the erythropoietin (Epo) receptor (EpoR) gene is under the control of transcriptional regulatory factor GATA-1. GATA-1 is expressed widely among the nonerythroid, factor-dependent subclones of the interleukin 3-dependent mouse cell line 32D. Consequently, to determine whether GATA-1 and EpoR gene expression are linked even in nonerythroid cells, we have studied the correlation of GATA-1 expression with expression and function of EpoR in these cell lines. EpoR mRNA (by RNase protection analysis) and EpoR protein (by specific antibody immunoprecipitation of metabolically labeled EpoR protein) were detectable not only in 32D and 32D Epo (an Epo-dependent subclone) but also in 32D GM, a subclone dependent for growth on granulocyte/macrophage colony-stimulating factor. EpoR mRNA also was detectable by PCR in 32D G, a subclone dependent for growth on granulocyte colony-stimulating factor. However, only 32D Epo cells bound 125I-labeled Epo and expressed EpoR protein on the cell surface, as determined by immunoprecipitation of surface-labeled proteins. These results indicate that, in these factor-dependent cell lines, the major regulatory step determining the erythroid-specific response to Epo is the efficiency of EpoR protein translocation to the cell surface. Mechanisms that could affect lineage-specific translocation are the presence of a chaperone protein, erythroid-specific editing of EpoR mRNA, or altered processing of the EpoR protein to the cell surface. In this model, lineage-restricted responses to growth factors such as Epo are determined not by expression of the genes for growth factor receptors but, rather, by appropriate processing of the receptor protein.

Animals↗
PubMed1991

Stem cell factor induces proliferation and differentiation of highly enriched murine hematopoietic cells.

Recombinant rat stem cell factor (SCF) was studied for its ability to stimulate the growth of murine hematopoietic progenitor cells and to generate colony-forming cells (CFC) from highly enriched populations of hematopoietic cells. In serum-deprived cultures, SCF alone stimulated few colonies but interacted with a number of other hematopoietic growth factors, particularly interleukin 3, to promote colony formation. The most marked effect was on the generation of mixed-cell colonies. Hematopoietic cells were sorted into wheat-germ agglutinin-negative, monocyte-depleted, rhodamine 123 (Rh123)-bright or Rh123-dull cells. Historically, Rh123-bright cells are capable of short-term (less than 1 mo) marrow engraftment, whereas among Rh123-dull cells are cells capable of long-term marrow engraftment. Enriched cells (2.5 x 10(3) were placed into serum-deprived liquid cultures with various hematopoietic growth factors. Initially, the Rh123-bright and Rh123-dull cells had few CFC but, in the presence of interleukin 3 and SCF, Rh123-bright cells gave rise to greater than 15,000 granulocyte/macrophage CFC, greater than 1500 erythroid burst-forming cells, and greater than 700 mixed-cell CFC by day 5. In contrast, Rh123-dull cells proliferated only in the presence of interleukin 3 and SCF, but total cell numbers rose to a peak of 18,000 by day 21, and one-third of the cells were CFC. Thus, SCF, in combination with other growth factors, can generate large numbers of CFC from pre-CFC and appears to act earlier than hematopoietic growth factors described to date.

Animals↗
PubMed1991

Effects of recombinant human stem cell factor (SCF) on the growth of human progenitor cells in vitro.

We have studied the effect of recombinant human Stem Cell Factor (SCF) on the growth of human peripheral blood, bone marrow, and cord blood progenitor cells in semisolid medium. While SCF alone had little colony-stimulating activity under fetal bovine serum (FBS)-deprived culture conditions, SCF synergized with erythropoietin (Epo), granulocyte/macrophage colony-stimulating factor (GM-CSF), and interleukin 3 (IL-3) to stimulate colony growth. Colony morphology was determined by the late-acting growth factor added along with SCF. Of all the combinations of growth factors, SCF plus IL-3 and Epo resulted in the largest number of mixed-cell colonies--a larger number than observed with IL-3 and Epo alone even in FBS-supplemented cultures. These results suggest that SCF is a growth factor that more specifically targets early progenitor cells (mixed-cell colony-forming cells) and has the capacity to synergize with a wide variety of other hematopoietic growth factors to cause the proliferation and differentiation of committed progenitor cells. Our studies indicate that SCF may be the earliest acting growth factor described to date.

Bone Marrow Cells↗
PubMed1990

Muramyl dipeptide induces production of hemopoietic growth factors in vivo by a mechanism independent of tumor necrosis factor.

Monocyte products including TNF and IL-1 can stimulate hemopoietic growth factor production in vitro and in vivo. Endotoxin-resistant C3H/HeJ mice have a cellular defect in the regulation of TNF production and provide a model in which to examine the relative importance of TNF and IL-1. To assess the relative role of TNF and IL-1, we injected the synthetic bacterial cell wall derivative muramyl dipeptide (MDP) or LPS into C3H/HeJ mice. Both MDP and LPS stimulated hemopoietic growth factor production in a dose-dependent manner. Northern blot analysis of splenic and lung mRNA revealed that MDP treatment induced granulocyte-macrophage CSF and macrophage-CSF transcripts, whereas LPS treatment induced macrophage-CSF transcripts. Messenger RNA for granulocyte-CSF, IL-3, IL-4, and IL-5 was not detected in control or treated mice. MDP treatment induced IL-1 mRNA but not TNF mRNA, and TNF bioactivity was not detected in the serum of MDP-treated mice. In contrast, LPS treatment induced TNF production. These results identify the hemopoietic growth factors induced by MDP and LPS in vivo, and suggest that MDP stimulates growth factor production by a mechanism independent of TNF.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1990

Comparative analysis of hematopoietic growth factors released by stromal cells from normal donors or transplanted patients.

We compared the erythroid burst-promoting activity (BPA) and colony-stimulating activity (CSA) released under serum-deprived conditions by stromal cells derived from nine normal subjects and from nine patients after bone marrow transplantation. BPA and CSA were defined according to the capacity of the conditioned media (CM) to stimulate formation of erythroid bursts and granulocyte/macrophage (GM) colonies in serum-deprived cultures of nonadherent marrow cells. Six patients (group A) failed to establish or maintain successful allografts during the study. The remaining three (group B) did not experience problems with engraftment. CM from all stromal cell cultures contained detectable levels of BPA. Preincubation of the CM with an anti-GM colony-stimulating factor (GM-CSF) monoclonal antibody (MoAb), but not with a rabbit anti-interleukin-3 (IL-3) serum, reduced BPA by an average of 94%. CM from normal and group B stromal cell cultures contained detectable CSA, and the levels correlated with the amounts of granulocyte-CSF (G-CSF) detected by a specific bioassay. G-CSF was not detectable in medium conditioned by stromal cells from transplanted patients with poor marrow function. These results indicate that CM from stromal cells from normal subjects and transplanted patients with good marrow function contain both GM-CSF and G-CSF, while CM from stromal cells from transplanted patients with poor marrow function contain detectable levels of GM-CSF only. The reduced capacity of these stromal cells to produce G-CSF is associated with a reduced capacity of the CM to sustain GM colony formation and may be associated with the inability of these patients to sustain their neutrophil counts in vivo.

Biological Assay↗
PubMed1979

Acute nonlymphocytic leukemia: expression in cells restricted to granulocytic and monocytic differentiation.

Two patients with acute nonlymphocytic leukemia who were heterozygous for the X-chromosome-linked enzyme glucose-6-phosphate dehydrogenase were studied to determine the number and type of cells in which the disease arises. Both type A and B isoenzymes were found in normal tissues, but the myeloblasts showed only one enzyme type, indicating that at the time of study, the disease had a clonal origin. The observation in one patient that erythroid cells did not arise from this clone contrasts with conclusions reached in patients previously studied with chromosomal markers. The results suggest that in this patient, the leukemic clone suppressed expression of normal granulopoiesis but did not inhibit erythroid differentiation from normal progenitors. They suggest also that the disease is heterogeneous. In some patients, the disease is expressed in cells with differentiation restricted to the granulocyte-macrophage pathway; in others, it involves stem cells that also differentiate into erythrocytes. This heterogeneity may reflect differences in causation and could have prognostic importance.

Acute Disease↗
PubMed1979

Cellular origins of the fetal-haemoglobin-containing cells of normal adults.

The origin of the small population of adult red cells which contain Hb F (F-cells) has been studied in a clonal disorder of haemopoiesis, polycythaemia rubra vera (P.R.V.). In eleven patients who had not received cytotoxic therapy F-cells comprised less than 0.1% to 11.9% of the circulating red cells, compared with 0.34% to 4.6% in 21 haematologically normal controls. Two additional patients were glucose-6-phosphate-dehydrogenase heterozygotes in whom the clonal nature of the P.R.V. could be demonstrated directly; they had F-cell values of 2.1% and 8.3%. These observations indicate that F-cells arise from the same population of stem cells as other adult cells and not from a separate stem-cell pool.

Aged↗
PubMed1979

Patterns of globin chain synthesis in erythroid colonies grown from sheep marrow of different developmental stages.

Erythroid colonies were grown from fetal sheep bone marrow at different stages of development and from adult marrow. Colony numbers increased with erythropoietin concentration, but fetal erythroid cells were more sensitive to the hormone than adult cells. Haemoglobin synthesis was characterized in the colonies and compared to that in control marrow incubations. No beta chain synthesis was detectable in marrow incubations or erythroid colonies from fetal marrow at 87-96 d gestation. Incubations of 115-120 d marrow shoed 5% beta chain synthesis while erythroid colonies synthesised up to 20% beta chains. Incubations and erythroid colonies from 125 d marrows showed about 27% beta chain synthesis, despite a similar increase in colony numbers with erythropoietin as the other marrows. Later in gestation, beta chain synthesis was lower in erythroid colonies than in marrow incubations and declined further in erythroid colonies as erythropoietin concentration increased. Adult marrow showed no detectable gamma chain synthesis but erythroid colonies produced up to 12% gamma chains, the identity of which was confirmed by peptide mapping. All changes in globin synthesis in vitro were correlated with colony numbers. In adult marrow, the maximal level of gamma chain production was inversely related to the cloning efficiency of the marrow samples studied. Potential uses of this model for studying globin gene expression are discussed.

Animals↗
PubMed1979

Modulation of in vitro erythropoiesis. Studies with euthyroid and hypothyroid dogs.

The interactions of adrenergic agonists and thyroid hormones on the growth of erythroid colony-forming units were studied in cultures of dog marrow before and after the establishment of hypothyroidism. Erythroid colony growth in cultures form euthyroid dogs was enhanced by isoproterenol and other adrenergic agonists having beta 2-receptor specificity. With hypothyroidism, however, this responsiveness was lost, and sensitivity to alpha-agonists, such as phenylephrine and norepinephrine, was acquired. This alteration in receptor specificity appeared to be dependent upon thyroid hormone and was rapidly reversible. Preincubation of marrow cells from hypothyroid animals with thyroid hormone resulted in the reappearance of responsiveness to beta-adrenergic agonists and the loss of sensitivity to alpha-agonists. These findings are in agreement with previous suggestions that beta-adrenergic receptor activity is modulated by thyroid hormone levels and demonstrate that the specificity of adrenergic modulations of erythropoiesis in culture may accurately reflect the thyroid status of the intact animal.

Adenylyl Cyclases↗