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E Spooncer

Publications and source records attributed to E Spooncer.

At least 37 records · Page 2Linked to original sources
PubMed1992

Expression and downregulation of cytotoxic cell protease 1 or Granzyme 'B' transcripts during myeloid differentiation of interleukin-3-dependent murine stem cell lines.

Using the technique of differential cDNA library screening, we have molecularly cloned a gene that is highly expressed in an undifferentiated myeloid multipotent and growth factor-dependent stem cell line (FDCP-Mix) and that downregulates as these cells are induced to differentiate along monocytic, granulocytic, and erythroid cell lineages. Sequence analysis of this gene has shown homology with a previously cloned gene, cytotoxic cell protease 1 (CCP1 or Granzyme 'B'), that has been shown to be expressed only in thymocytes, activated T cells, a mast cell line, and peritoneal exudate leukocytes. In situ hybridization, Northern blot analysis, and nuclear run-off assay has confirmed that expression of CCP1 is restricted to the phenotypically primitive multipotent undifferentiated. FDCP-Mix cells that are undergoing self-renewal in the presence of growth factors such as interleukin-3.

Amino Acid Sequence↗
PubMed1992

The mouse M-lysozyme gene domain: identification of myeloid and differentiation specific DNasel hypersensitive sites and of a 3'-cis acting regulatory element.

The mouse M-lysozyme gene is exclusively expressed in myeloid cells of the blood system being progressively turned on upon cell differentiation. In this study the mechanism controlling this tissue- and differentiation stage-specific gene expression was analyzed at the level of chromatin structure. A complex pattern consisting of constitutive and differentiation dependent DNasel hypersensitive sites (HSs) was found in a set of various myeloid cell lines, representing different stages of maturity. The chromatin of a lymphoid cell line, which does not express the lysozyme gene, is completely insensitive to DNasel digestion. Chromatin analysis of two multipotent hematopoietic stem cell lines which can be differentiated in vitro to mature myeloid cells confirmed that these identified DNasel HSs are specific for distinct differentiation stages, rather than being a characteristic feature of the cell lines. Additionally, the stem cell studies revealed that the hypersensitivity of the chromatin domain is already established at the multipotent stage. DNA fragments spanning a cell type- and differentiation stage-specific cluster of HSs in the 3' region of the gene showed enhancer activity in all cell types tested. In the light of this lack of specificity, we suggest that cell type-specific modification of the chromatin structure in this region may play a role in determining the binding of a widespread transcription factor, and hence contribute to the time specificity of lysozyme M gene expression.

Animals↗
PubMed1992

Immunoglobulin heavy-chain and CD3 delta-chain gene enhancers are DNase I-hypersensitive in hemopoietic progenitor cells.

Multipotential interleukin 3-dependent non-immortalized murine hemopoietic progenitor cells have DNase I-hypersensitive sites in the immunoglobulin heavy-chain and CD3 delta enhancers and transcribe germ-line T-cell antigen receptor gamma-chain (TCR gamma), but not IgM or TCR beta, genes. Induction of myeloid differentiation in these cells clones down expression and/or transcriptional accessibility of the immunoglobulin heavy-chain and TCR gamma genes. The CD3 delta enhancer region remains DNase I-hypersensitive but closes down in B cells. In embryonic stem cells and pan-mesodermal cells, these genes or enhancer regions are neither expressed nor DNase I-hypersensitive. These data suggest that lineage potential may be programmed, at least in part, by alterations in the accessibility or conformation of regulatory regions of genes and that some promiscuity of gene expression and/or accessibility can precede lineage commitment and maturation in progenitor cells induced to self-renew by interleukin 3.

Animals↗
PubMed1992

Multilineage phenotypes of interleukin-3-dependent progenitor cells.

Interleukin-3 (IL-3)-dependent murine FDCP-mix cells have multilineage differentiation capacity; they are nonleukemic, have a normal karyotype, and are nonimmortalized. These cells coexpress on their cell surface the "early" B-lineage marker B220/CD45R and the myeloid marker Mac-1/iC3b receptor (CR3), transcribe germline T-cell receptor gamma genes, and express the macrophage lineage growth factor receptor gene c-fms as a predominant 8.4-kb transcript. They do not detectably express at the stable mRNA or protein level other lymphoid precursor cell genes including CD2, TdT, lambda 5, and BP1. Induction of granulocyte/macrophage differentiation in these cells closes down expression of the lymphoid genes and activates stable expression of genes specific to the myeloid lineage, including myeloperoxidase. Expression of the c-fms gene at the mRNA level is upregulated and the dominant stable transcript is now in the 4.1-kb form typical of the macrophage lineage. These data provide a plausible explanation for the coexpression of lymphoid and myeloid lineage markers on human leukemic cells of stem cell or progenitor cell origin and have implications for the programming of lineage potential in normal multipotential hematopoiteic progenitor cells.

Animals↗
PubMed1991

Expression of the GM-CSF gene after retroviral transfer in hematopoietic stem cell lines induces synchronous granulocyte-macrophage differentiation.

Multipotent murine stem cell lines (FDC-Pmix) depend on IL-3 for self-renewal and proliferation and can be induced to differentiate into multiple hematopoietic lineages. Single FDC-Pmix cells infected with retroviral vectors expressing GM-CSF are induced to differentiate into granulocytes and macrophages. This results in a complete loss of clonogenic cells if IL-3 is not exogenously supplied; however, multipotent variants can be selected that do not terminally differentiate if cells are kept in the presence of IL-3. Unidirectional and synchronous granulocyte and macrophage differentiation accompanied with loss of self-renewal capacity is induced when IL-3 is removed. Our data indicate that activation of the GM-CSF receptor induces differentiation of stem cells by an instructive mechanism that can be blocked by the activated IL-3 receptor. A model of how receptors can induce proliferation and cell-specific differentiation by two separate pathways is discussed.

Animals↗
PubMed1991

Interleukin-3-dependent hematopoietic stem cell lines capable of osteoclast formation in vitro.

Recently we reported that the osteoclast originates from the pluripotent hematopoietic stem cell. However, a detailed analysis of the progenitor and precursor stages of the osteoclast lineage is hard to perform with primary cultures of stem cells. In the present investigation interleukin-3 (IL-3)-dependent multipotent hematopoietic stem cell lines (FDCP-mix), which have many characteristics in common with freshly isolated hematopoietic stem cell lines (FDCP-mix), which have many characteristics in common with freshly isolated hematopoietic stem cells, were assayed for their osteoclast formation capacity. FDCP-mix cell lines A4, C2GM, and 15S were cocultured with periosteum-free 17-day-old fetal metatarsal bones. The effects of culture time, medium composition, and addition of WEHI-3b-conditioned medium (an unpurified IL-3 preparation) on osteoclast formation were studied. 15S cells never differentiated into osteoclasts. Both A4 and C2GM cells were able to generate osteoclasts. Osteoclast formation was visualized by staining for tartrate-resistant acid phosphatase activity and confirmed by 45Ca release assays and electron microscopic studies. Medium supplemented with fetal calf serum clearly supported osteoclast formation from A4 cells better than medium supplemented with cock serum. The difference between fetal calf serum and horse serum is generally less pronounced. C2GM cells formed osteoclasts more readily and, generally, earlier than A4 under all culture conditions. WEHI-3b-conditioned medium addition increased the numbers of osteoclasts and their resorption activity. The coculture of stripped metatarsal bones with FDCP-mix cell lines therefore offers a model system with many possibilities for the study of osteoclastogenesis and its regulation.

Acid Phosphatase↗
PubMed1991

Retroviral vectors related to the myeloproliferative sarcoma virus allow efficient expression in hematopoietic stem and precursor cell lines, but retroviral infection is reduced in more primitive cells.

Retroviral vectors are considered to be the most suited vehicles for somatic gene therapy with hematopoietic stem cells as targets. Retrovirus-mediated gene transfer into differentiation-restricted hematopoietic precursor (FDC-P1, FDC-P2) and multipotent progenitor (stem) cell lines (FDC-Pmix) is inefficient. Two cellular restrictions are involved. One is specific for stem but not precursor cells and is at the level of transcription. Due to a unique property of the transcriptional control region of the myeloproliferative sarcoma virus (MPSV), vectors derived from MPSV are not affected by this block. The second restriction occurs before proviral DNA synthesis and integration. This inhibition of effective viral infection depends on the state of differentiation, being more pronounced in multipotent clonogenic blast cells. This block to retroviral infection affects all retroviral vectors tested.

Animals↗
PubMed1990

The role of growth factors in self-renewal and differentiation of haemopoietic stem cells.

Haemopoietic stem cells in vivo proliferate and develop in association with stromal cells of the bone marrow. Proliferation and differentiation of haemopoietic stem cells also occurs in vitro, either in association with stromal cells or in response to soluble growth factors. Many of the growth factors that promote growth and development of haemopoietic cells in vitro have now been molecularly cloned and purified to homogeneity and various techniques have been described that allow enrichment (to near homogeneity) of multipotential stem cells. This in turn, has facilitated studies at the mechanistic level regarding the role of such growth factors in self-renewal and differentiation of stem cells and their relevance in stromal-cell mediated haemopoiesis. Our studies have shown that at least some multipotential cells express receptors for most, if not all, of the haemopoietic cell growth factors already characterized and that to elicit a response, several growth factors often need to be present at the same time. Furthermore, lineage development reflects the stimuli to which the cells are exposed, that is, some stimuli promote differentiation and development of multipotential cells into multiple cell lineages, whereas others promote development of multipotential cell into only one cell lineage. We suggest that, in the bone marrow environment, the stromal cells produce or sequester different types of growth factors, leading to the formation of microenvironments that direct cells along certain lineages. Furthermore, a model system has been used to show the possibility that the self-renewal probability of multipotential cells can also be modulated by the range and concentrations of growth factors present in the environment. This suggests that discrete microenvironments, preferentially promoting self-renewal rather than differentiation of multipotential cells, may also be provided by marrow stromal cells and sequestered growth factors.

Animals↗
PubMed1990

Haemopoietic colony stimulating factors promote cell survival by suppressing apoptosis.

The survival, differentiation, proliferation and development of haemopoietic precursor cells and the functional activity of mature blood cells are all influenced by colony stimulating factors (CSFs). As haemopoietic cells rapidly die in the absence of appropriate CSF, the promotion of cell survival mediated by CSFs, or growth factors, is fundamental to all the other effects exerted by these factors. This enhancement of cell survival is distinct from the stimulation of proliferation. Here we show that the death of haemopoietic precursor cells on withdrawal of the relevant CSF. is due to active cell death, or apoptosis, indicating that CSFs promote cell survival by suppression of the process of apoptosis. The existence of a positive control mechanism regulating precursor cell survival has important implications both for the regulation of normal haemopoiesis and for tumorigenesis.

Animals↗
PubMed1990

Stromal cells in haemopoiesis.

Stromal cells of the bone marrow can provide the growth-promoting and differentiation-inducing molecules which are necessary for haemopoiesis. While the nature of these stimuli is largely unknown, the development of haemopoietic cells in association with stromal cells requires intimate cell contact. Molecules of the extracellular matrix, such as heparan sulphate, are able to bind growth factors and in this way the stromal cells may form microenvironmental niches which preferentially promote development of multipotent and committed cells along discrete lineages. Cells from some patients with acute and chronic myeloid and lymphoid leukaemias are defective in their ability to interact with stromal cells and consequently cannot survive in stromal cell-mediated long-term marrow cultures. We have exploited this phenomenon to obtain normal haemopoietic cells from patients with leukaemia, and to use these cells for successful autografting in patients with acute and chronic myeloid leukaemias.

Animals↗
PubMed1989

Cellular interactions between 3T3 cells and interleukin-3-dependent multipotent haemopoietic cells: a model system for stromal-cell-mediated haemopoiesis.

With the aid of a multipotent stem cell line (FDCP-mix cells) co-cultured with either normal or irradiated Swiss 3T3, cellular interactions between stromal cells and haemopoietic stem cells were studied by electron microscopy and time-lapse video microscopy. When cultured in the presence of interleukin 3 (IL-3) but in the absence of stromal cells, the FDCP-mix cells have a characteristic blast morphology. In the absence of IL-3, the cells die unless they are co-cultured with marrow stromal cells or 3T3 cells. In the latter case, they attach, proliferate, and differentiate on both normal and irradiated Swiss 3T3 cell layers without the addition of extrinsic growth factor (IL-3). At the initial attachment sites of these two cell lines, cellular recognition seemed to be mediated by the formation of microvillus cytoplasmic projections and extracellular matrix. These areas may well be the sites of plasma-membrane-bound signalling/adhesional molecules between the interacting cells.

Animals↗
PubMed1989

Aberrant expression of the multi-CSF gene in hematopoietic precursor and stem cell lines initiates leukemogenic progression.

Tumorigenesis of hemopoietic cells and acquisition of factor independence as a consequence of aberrant growth factor release are closely correlated. In previous work we were able to dissect two stages leading to growth factor autonomy of cells: the first step requires the secretion of the constitutively expressed CSF gene product and extracellular interaction with its cognate receptor. This requirement for external stimulation is abrogated by a second step. We were interested in characterizing the parameters that influence the conversion from nonautonomous to autonomous growth properties of hematopoietic precursor cells. The frequency with which this alteration occurs varies and correlates with the level of growth factor production. However, a significant increase of CSF production accompanying the progression to autonomy could not be detected. We thus conclude that there is no direct link between level of CSF production and acquisition of true autonomy but an indirect influence enhancing the frequency of genetic alteration(s) that lead to growth autonomy. Lang et al. have suggested that the acquisition of autonomous growth occurs due to internal receptor-ligand interaction. Indeed, Keating and Williams have claimed that PDGF may react with an intracellular PDGF receptor resulting in autocrine stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Rates of mutation to growth factor autonomy and tumorigenicity differ in hematopoietic stem and precursor cells expressing the multilineage colony-stimulating factor gene.

At least two separate but interdependent events are required to attain autonomous growth as a consequence of ectopic expression of the multilineage colony-stimulating factor gene in hematopoietic progenitor cells. The rate at which the second event occurs is more than 3 orders of magnitude higher in precursor cell lines (FDC-P1 or FDC-P2) than in stem cell lines (FDC-Pmix). Autonomous, but not density-dependent, growth is tightly coupled to tumorigenicity in precursor cells; however, neither growth-factor-independent nor autonomously growing stem cell lines are tumorigenic.

Animals↗
PubMed1989

Evaluation of a mouse Y chromosome probe for assessing marrow transplantation.

The multicopy mouse Y chromosome DNA probe 80Y/B (1) has been used to probe genomic DNA isolated from male and female bone marrow cells, mixed together in known ratios. It was found that in a mixture with a ratio of 1 male:200 female marrow cells the male cells could readily be detected by this method. The potential use of this technique for assessing repopulation kinetics of marrow transplantation is discussed.

Animals↗