Biomedical subjects

C G Begley

Publications and source records attributed to C G Begley.

At least 73 records · Page 4Linked to original sources

PubMed1997

Identification of a second murine interleukin-11 receptor alpha-chain gene (IL11Ra2) with a restricted pattern of expression.

The interleukin-11 receptor alpha-chain, a member of the hematopoietin receptor superfamily, forms, together with gp130, a functional high-affinity receptor complex for interleukin 11. We, and others, reported the cloning of the murine interleukin 11 receptor alpha-chain cDNA (IL11Ra) and recently described the structure of the IL11Ra locus. We also described the presence of a second IL11Ra-like locus in some mouse strains. In this study we report that the second locus, designated IL11Ra2, encodes an mRNA species. The transcript was 99% identical to the IL11Ra transcript in the coding and 3'-untranslated region, but had a different 5'-untranslated region. The complete genomic organization of the IL11Ra2 locus is presented, and the two loci are shown to be located on a 200-kb NaeI genomic fragment. Comparison of the expression pattern of the IL11Ra and IL11Ra2 genes using an RT-PCR restriction fragment length polymorphism strategy revealed that while the expression of IL11Ra was widespread, expression of IL11Ra2 was restricted to testis, lymph node, and thymus.

Amino Acid Sequence↗

PubMed1997

Expression and function of members of the cytokine receptor superfamily on breast cancer cells.

Receptors for the cytokines leukemia inhibitory factor (LIF), interleukin-6 (IL-6), oncostatin M (OSM), ciliary neurotrophic factor (CNTF) and interleukin-11 (IL-11) are members of the structurally conserved hemopoietin receptor superfamily. In addition, they all share the transmembrane signalling protein gp130. In this paper the expression and function of this family of receptors in breast cancer cells was examined. RT-PCR analyses demonstrated that gp130 was expressed in 12/12 breast cell lines and the specific receptor alpha-chains for IL-6, LIF, IL-11 and CNTF were expressed in the majority of these cell lines. This was in contrast to other hemopoietin receptors. Examination of 50 clinical samples of malignant breast tissue by RT-PCR showed a similar pattern of expression of gp130 associated receptors. Treatment of breast cancer cell lines with OSM resulted in changes in cellular morphology. Cellular proliferation was inhibited following exposure to OSM (3/4 cell lines), IL-11 (2/4 cell lines), and by IL-6 and LIF (1/4 cell lines). Cell surface binding of LIF and OSM was also documented. The expression of these receptors in 12/12 cell lines and greater than 95% of clinical samples suggests that these molecules may be important in regulating the growth of breast cells.

Base Sequence↗

PubMed1997

The structural basis of the biological actions of the GM-CSF receptor.

The receptor for granulocyte/macrophage colony-stimulating factor (GM-CSF) consists of a ligand-specific low-affinity binding chain (GM-CSFR alpha) and a second chain that is required for high-affinity binding and signal transduction. This second chain is shared by the ligand-specific alpha-chains for the interleukin 3 (IL-3) and IL-5 receptors and is therefore called beta common (beta c). In mice but not humans the IL-3 receptor can also use a closely related but IL-3-specific beta-chain (beta IL-3). In order to define the contributions of each chain to receptor signalling we generated mice in which either beta c or beta IL-3 expression was deleted. beta IL-3 null mice were phenotypically normal but displayed a decreased responsiveness to IL-3 in vitro. beta c null mice, on the other hand, were unresponsive to GM-CSF or IL-5 but still responded to IL-3. These data demonstrated that GM-CSF and IL-5 receptors can use only one beta-chain for signalling (beta c) while IL-3 can effectively use either beta-chain. The hierarchical basis of receptor transmodulation was shown to result from this differential usage of beta-chains. To define the regions required for different types of cell signalling, we constructed human beta c mutants with successive cytoplasmic truncation. By the use of appropriate biological read-out systems we found that the cytoplasmic region of the receptor has a modular design with distinct domains required for cell proliferation, cell survival, differentiation and growth suppression. Appropriate targeting of these domains and the signalling pathways they initiate may provide highly specific cell therapies in the future.

Animals↗

PubMed1997

The SCL/TAL1 gene: roles in normal and malignant haematopoiesis.

SCL (TAL1/TCL5) is a member of the helix-loop-helix family of transcription factors. Originally identified because of its involvement in a tumour-specific chromosomal translocation, overexpression of the SCL gene is the most common molecular abnormality found in human T cell leukaemia. Transgenic models have now formally demonstrated that overexpression of SCL within the T cell lineage is capable of causing malignant transformation. Gene targeting experiments have revealed that the SCL gene is crucial for the development of primitive haematopoiesis in the mouse and is also required for the generation of all adult haematopoietic lineages. Biochemical studies have indicated some of the proteins which interact with SCL and this has refined the hypotheses concerning the mechanisms by which SCL plays a role in leukaemogenesis and haematopoietic development.

Adaptor Proteins, Signal Transducing↗

PubMed1997

Mpl ligand (MGDF) alone and in combination with stem cell factor (SCF) promotes proliferation and survival of human megakaryocyte, erythroid and granulocyte/macrophage progenitors.

We examined cytokine-stimulated proliferation and survival of human megakaryocyte progenitor cells. We used a reliable, immunoenzymatic method of labeling CD41a-, CD42b-stained megakaryocytes in intact agar cultures to specifically identify and enumerate all megakaryocyte-containing colonies. We examined a previously defined population of cells enriched for megakaryocyte progenitors that coexpress CD34 and the megakaryocyte/platelet marker CD61. These CD34+61+ cells displayed clonogenicity of approximately 30% and contained myeloid, erythroid and megakaryocyte progenitor cells. With single CD34+61+ cells, megakaryocyte growth and development factor ([MGDF], also known as mpl-ligand or thrombopoietin) stimulated 9% of cells to complete their first cell division by day 2 (versus 21% with stem cell factor [SCF], or 13% with interleukin 3 alone). MGDF showed an additive effect with SCF and interleukin 3 to increase this number at least twofold. In purified CD34+61+ cells, MGDF stimulated the survival of megakaryocyte-colony forming cells (CFC) when addition of other proliferative factors was delayed for 5, 10 and 15 days (all p < 0.0001 versus saline control). MGDF also promoted survival of BFU-E and granulocyte-macrophage-CFC for at least 10 days (p < or = 0.0013 and p < or = 0.0362, respectively). SCF alone prolonged survival of CD34+61+ progenitor cells, however, MGDF + SCF was significantly more active. Whereas the action of MGDF on megakaryocyte-CFC was evident both in stimulating proliferation and survival, its ability to promote survival was between two- and fivefold greater than its action to stimulate proliferation. Thus MGDF alone, and in combination with SCF, was active in promoting the survival and proliferation of human progenitor cells of multiple hemopoietic lineages.

Antigens, CD↗

PubMed1997

Factors involved in leukaemogenesis and haemopoiesis.

This review describes the chromosomal abnormalities in T-cell acute lymphoblastic leukaemia (ALL) which result in the over-expression of the gene SCL, which encodes a helix-loop-helix transcription factor. Also described are how gene targeting studies have revealed a key role for SCL in normal haemopoiesis. Next, the BCR-ABL fusion protein, seen in chronic myeloid leukaemia (CML) and in some patients with ALL, is discussed. Finally, the involvement of members of the core-binding factor (CBF) gene family in leukaemogenesis are described. Members of this gene family are involved in the generation of fusion proteins as a result of t(8;21) and inv(16), the most common translocations associated with acute myeloid leukaemia (AML). They provide a useful model of the way in which aberrant transcriptional function, brought about through genetic alterations, can modify haemopoietic development.

Animals↗

PubMed1997

The gene for the human interleukin-11 receptor alpha chain locus is highly homologous to the murine gene and contains alternatively spliced first exons.

The gene for the murine interleukin-11 receptor alpha chain (mIL-11R alpha) contains two loci (1 and 2), of which locus 2 is restricted to only some mouse strains. Two alternatively spliced exons (1a and 1b) encode the 5' untranslated region (5'UTR) of the murine locus 1. We have characterized the gene for the human interleukin-11 receptor alpha chain locus (hIL-11R alpha), examined its expression by Northern analysis and determined its chromosomal location by fluorescence in situ hybridization. The presence of exon(s) encoding the 5'UTR and mapping of transcription initiation sites was determined by reverse-transcriptase polymerase chain reaction and 5' rapid amplification of cDNA ends (5'RACE) techniques. The human locus spanned 10 kilobasepairs (kb) and consisted of 14 exons. Two alternatively spliced first exons (1a and 1b) encoding the 5'UTR were identified and shared 76 and 73% nucleotide identity with murine exons 1a and 1b. Multiple transcription start sites were demonstrated for human exon 1a. The promoter regions of both human exons 1a and 1b did not display a canonical TATA box. A predominant 1.8 kb transcript for the hIL-11R alpha was present in heart, brain, skeletal muscle, lymph nodes, thymus, appendix, pancreas and foetal liver. The hIL-11R alpha gene was localized to chromosome 9p13. In summary, the hIL-11R alpha gene was highly related to locus 1 of the murine gene and there was no evidence of a second hIL-11R alpha locus.

Alternative Splicing↗

PubMed1997

Establishment of multipotential and antigen presenting cell lines derived from myeloid leukemias in GM-CSF transgenic mice.

In this study neonatal mice expressing a GM-CSF transgene (GMT mice), and their normal littermate controls, were infected with Moloney murine leukemia virus (MoMLV) to examine in vivo tumorigenesis. By 200 days, all of the GMT mice had died whereas median survival had not been reached in the littermates (P < 0.0003). Thymomas developed in 32% of GMT mice and were more frequently CD4+CD8+ (83%) compared to the CD4+CD8- phenotype seen in 90% of thymomas developing in MoMLV-infected littermate mice. A primitive myeloid leukemia was induced in 21% of GMT mice, but none of the littermates. To characterize further the nature of the leukemic cells, a factor-dependent cell line (DGM36) was derived. DGM36 cells were tumorigenic, capable of differentiation to neutrophils, macrophages and eosinophils, and contained a partial deletion of chromosome 2. A subline arose spontaneously that was factor-independent and produced GM-CSF in an autocrine manner (IGM36 cells). Stimulation of the IGM36 cells with TNF alpha and IFNgamma resulted in increased expression of B7-1, class I MHC and class II MHC and consequent presentation of antigen in allogeneic MLRs. IGM36 cells thereby satisfy many of the criteria of dendritic cells and consequently may be used to examine antigen presentation by leukemic cells. This is the first report of primary myeloid leukemias arising in GMT mice and documents the derivation of a multipotential, autocrine leukemic cell line with dendritic cell characteristics.

Animals↗

PubMed1997

Multilineage mobilization of peripheral blood progenitor cells in humans following administration of PEG-rHuMGDF.

The most important physiological regulator of megakaryocytopoiesis is the ligand for the c-mpl receptor (thrombopoietin/megakaryocyte growth and development factor, MGDF). We examined the effect of pegylated-recombinant human MGDF (PEG-rHuMGDF): patients received PEG-rHuMGDF at doses of 0.03, 0.1, 0.3 or 1.0 microg/kg/d or placebo for 10d maximum in a double-blinded randomized study. There was a dose-dependent elevation in circulating platelet counts but no alteration in erythrocyte or total leucocyte counts. The number of bone marrow megakaryocytes was increased approximately 2-fold. The frequency of bone marrow progenitor cells was not altered. In contrast, both to the bone marrow results and to published pre-clinical data, there was a dose-dependent mobilization into the blood of progenitor cells of multiple cell lineages. Increased levels of Meg-CFC (maximum increase 30-fold), day 7 and day 14 GM-CFC and BFU-E were demonstrated at doses of 0.3 and 1.0 microg/kg/d PEG-rHuMGDF. At 0.1 microg/kg/d, mobilization of Meg-CFC alone occurred in two-thirds of patients. Maximum blood levels of progenitor cells occurred at day 12. Thus, administration of PEG-rHuMGDF to humans resulted in mobilization of progenitor cells of multiple lineages despite its 'lineage-specific' activity on mature cell development.

Adult↗

PubMed1997

Clinical studies with megakaryocyte growth and development factor (Mpl-ligand).

The haemopoietic growth factor mpl-ligand (also known as thrombopoietin, and Megakaryocyte Growth and Development Factor [MGDF] is a recently cloned and characterized megakaryocyte-active factor. Administration of MGDF to humans was associated with a dose-related increase in the platelet count with maximum levels observed between days 12-18. Platelets had normal appearance and functioned normally in assays of platelet aggregation and ATP-release. There was no evidence of platelet activation as assessed by platelet surface markers. The earliest detectable effect of MGDF was an increase in early (reticulated) platelets by day 3-4. MGDF also hastened recovery from thrombocytopenia when given after myelosuppressive chemotherapy. MGDF caused mobilisation into the blood of progenitor cells of multiple haemopoietic lineages, and was synergistic with G-CSF in this action after chemotherapy. MGDF was well tolerated with no adverse effects directly attributable to its administration. Further clinical evaluation is on-going.

Blood Platelets↗

PubMed1996

Retroviral transduction of human progenitor cells: use of granulocyte colony-stimulating factor plus stem cell factor to mobilize progenitor cells in vivo and stimulation by Flt3/Flk-2 ligand in vitro.

The clinical application of gene transfer is hindered by the availability of the multipotential stem cells and the difficulty in obtaining efficient retroviral transduction. To assess potential means by which gene transfer into human hemopoietic stem cells might be enhanced, the retroviral transduction efficiency of human bone marrow cells (BM) or peripheral blood progenitor cells (PBPC) was compared at multiple time points after in vivo administration of granulocyte colony-stimulating factor (G-CSF). This was further compared with the transduction efficiency of cells mobilized with G-CSF plus stem cell factor (SCF) in a cohort of patients randomized to receive either one or two growth factors and with normal BM function. Using the LNL6 retrovirus, retroviral transduction efficiencies of up to 19% were observed for both PBPC and BM (n = 26 patients). There was at least a 100-fold increase in PBPC with G-CSF alone and a further 30-fold increase in the total number of progenitor cells available for retroviral transduction using the combination of SCF plus G-CSF. However, pretreatment of patients with G-CSF with or without SCF did not enhance the retroviral infectability of growth factor-mobilized progenitor cells. The effect of the growth factor, Flk-2/Flt3 ligand (FL), was also examined with respect to retroviral transduction efficiency of human progenitor cells. FL plus IL-3 in vitro increased the retroviral transduction efficiency up to eightfold compared with results observed using other combinations of cytokines tested (P < .001). These findings have clinical implications both for increasing the number of target cells for in vivo gene-marking/gene-therapy studies and improving the efficiency of gene transfer.

Cell Cycle↗

PubMed1996

Thrombopoietic effects of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) in patients with advanced cancer.

BACKGROUND: Pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) is a potent stimulator of megakaryocyte colony formation and platelet production. It is likely to be useful in the management of severe thrombocytopenia. To determine its clinical activity and safety, we gave it to patients with advanced cancer before chemotherapy. METHODS: Patients were randomly assigned to receive either PEG-rHuMGDF or placebo in a three to one ratio. PEG-rHuMGDF was given at a dose of 0.03, 0.1, 0.3, or 1.0 microgram/kg body weight. The study drug or placebo were administered daily by subcutaneous injection for up to 10 days or until a target platelet count was reached. FINDINGS: 17 patients, median age 59 years, received either PEG-rHuMGDF (13 patients) or placebo (four patients). PEG-rHuMGDF produced a dose-dependent increase in platelet counts. Patients given placebo. 0.03, and 0.1 microgram/kg of PEG-rHuMGDF had median increases in platelet counts of 16%, 12%, and 39%. Those receiving 0.3 and 1.0 microgram/kg of PEG-rHuMGDF had an increase in blood platelets of between 51% and 584%. Platelets rose from day 6 of PEG-rHuMGDF administration and continued to rise after stopping the drug. The platelet count peaked between days 12 and 18 and remained above 450 x 10(9)/L for up to 21 days. There were no alterations in white-blood-cell count or haematocrit, and low toxicity. Platelets taken from patients during PEG-rHuMGDF administration and at the time of peak platelet count were morphologically and functionally normal. INTERPRETATION: The potency with which PEG-rHuMGDF stimulates platelet production and its low toxicity indicate that this is likely to be a useful agent for the management of thrombocytopenia.

Dose-Response Relationship, Drug↗

PubMed1996

Administration of pegylated recombinant human megakaryocyte growth and development factor to humans stimulates the production of functional platelets that show no evidence of in vivo activation.

This report describes the effect of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) on platelet production and platelet function in humans. Subjects with advanced solid tumors received PEG-rHuMGDF daily for up to 10 days. There was no increase in circulating platelet count at doses of 0.03 or 0.1 microgram/kg/d by day 12 of study. At doses of 0.3 and 1.0 microgram/kg/d there was a threefold median increase (maximum 10-fold) in platelet count by day 16. The platelets produced in vivo in response to PEG-rHuMGDF showed unchanged aggregation and adenosine triphosphate (ATP)-release responses in in vitro assays. Tests included aggregation and release of ATP in response to adenosine diphosphate (ADP) (10, 5, 2.5, and 1.25 mumol/L), collagen (2 micrograms/mL), thrombin-receptor agonist peptide (TRAP, 10 mumol/L) and ristocetin (1.5 mg/mL). Administration of aspirin to an individual with platelet count of 1,771 x 10(3)/L resulted in the typical aspirin-induced ablation of the normal aggregation and ATP-release response to stimulation with arachidonic acid (0.5 mg/mL), collagen, and ADP (2.5 and 1.25 mumol/L). There was no change in the expression of the platelet-surface activation marker CD62P (P-selectin) nor induction of the fibrinogen binding site on glycoprotein IIb/IIIa as reported by the monoclonal antibody, D3GP3. An elevation of reticulated platelets was evident after 3 days of treatment with PEG-rHuMGDF and preceded the increase in circulating platelet count by 5 to 8 days; this reflected the production of new platelets in response to PEG-rHuMGDF. At later time points, the mean platelet volume (MPV) decreased in a manner inversely proportional to the platelet count. Levels of plasma glycocalicin, a measure of platelet turnover, rose 3 days after the initial increase in the peripheral platelet count. The level of plasma glycocalicin was proportional to the total platelet mass, suggesting that platelets generated in response to PEG-rHuMGDF were not more actively destroyed. Thus, the administration of PEG-rHuMGDF, to humans, increased the circulating platelet count and resulted in fully functional platelets, which showed no detectable increase in reactivity nor alteration in activation status.

Cohort Studies↗

PubMed1996

Five new mutations in the uroporphyrinogen decarboxylase gene identified in families with cutaneous porphyria.

We describe five new mutations in the uroporphyrinogen decarboxylase (UROD) gene. All mutations were observed in conjunction with decreased erythrocyte UROD and clinical familial porphyria cutanea tarda (fPCT), (four families) or hepatoerythropoietic porphyria (HEP), (one family). The fPCT mutations included three point mutations that resulted in amino acid substitutions: a lysine to glutamine at amino acid position 253 (exon 7); a glycine to arginine at position 318 (exon 10); an isoleucine to threonine at position 334 (exon 10). The lysine to glutamine at amino acid position 253 was found in conjunction with a single C nucleotide deletion in exon 8 on the same allele of the UROD gene in the same family. This deletion resulted in a shift in the reading frame and the introduction of a premature stop codon 8 amino acids downstream. In the fourth family, a 31-bp deletion (nucleotides 828-858: exon 8) of the coding region, resulted in a frameshift and the introduction of a stop codon 19 amino acids downstream. A point mutation was observed in an individual diagnosed with HEP, resulting in an alanine to glycine change at amino acid position 80 and was present on both alleles. All mutations were confirmed in at least one other family member. The impact of these mutations on the function of the UROD protein was examined using in vitro protein expression and with activity assessed using pentacarboxylic acid porphyrinogen I as a substrate for UROD. Although three mutations reduced UROD activity to < 15% of normal, one resulted in a UROD protein with 50% functional activity and the other had near normal activity. These results indicate that many different genetic lesions of the UROD gene are associated with fPCT.

Family↗

PubMed1996

Molecular cloning of a novel human gene (D11S4896E) at chromosomal region 11p15.5.

A novel gene (GOK) has been cloned from human chromosome region 11p15.5 that is believed to contain a gene or genes associated with a number of pediatric malignancies, including Wilms tumor. A 4-kb cDNA has been cloned and it encodes a predicted protein of approximately 84 kDa that could be translated in vitro. Computer analysis predicted that the protein had a signal peptide and may contain a transmembrane helix. Restriction mapping by pulsed-field electrophoresis indicates that GOK is located 1.7 kb telomeric of RRM1, and both genes are transcribed in the same direction. GOK displays high evolutionary conservation: cloning and partial sequencing of a mouse genomic clone revealed 90% identity with the human gene at both the nucleotide and the predicted amino acid levels.

Amino Acid Sequence↗

PubMed1996

The scl gene product is required for the generation of all hematopoietic lineages in the adult mouse.

Homozygosity for a null mutation in the scl gene causes mid-gestational embryonic lethality in the mouse due to failure of development of primitive hematopoiesis. Whilst this observation established the role of the scl gene product in primitive hematopoiesis, the death of the scl null embryos precluded analysis of the role of scl in later hematopoietic development. To address this question, we created embryonic stem cell lines with a homozygous null mutation of the scl gene (scl-/-) and used these lines to derive chimeric mice. Analysis of the chimeric mice demonstrates that the scl-/- embryonic stem cells make a substantial contribution to all non-hematopoietic tissues but do not contribute to any hematopoietic lineage. These observations reveal a crucial role for the scl gene product in definitive hematopoiesis. In addition, in vitro differentiation assays with scl-/- embryonic stem cells showed that the scl gene product was also required for formation of hematopoietic cells in this system.

Animals↗

PubMed1996

Structural analysis of the gene encoding the murine interleukin-11 receptor alpha-chain and a related locus.

In this study the gene for the murine interleukin-11 receptor alpha chain (IL-11Ralpha) has been characterized. The gene spans 9 kilobase pairs of DNA, and the organization of its 14 exons conforms to the pattern observed for other members of the hematopoietin receptor family. Analysis of the 5' end of the cDNA using 5' RACE showed that the first two exons, designated exons 1a and 1b, are spliced to form alternate transcripts. Transcripts initiating from exon 1b were not found in adult tissues but were present in embryonic stem cells. S1 nuclease and 5' rapid amplification of cDNA ends assays demonstrated multiple major and minor sites of transcription initiation for each exon. The putative promoter regions of both exons lacked TATA boxes, although potential recognition sites for several transcription factors including Sp1, AP1, and AP2 were present. A comparison of the murine and human IL-11Ralpha revealed that the 5' sequence upstream of the major site of transcription initiation site for exon 1b is highly conserved. Northern analysis showed that IL-11Ralpha is expressed in many adult murine tissues. A second IL-11Ralpha-like locus containing a sequence homologous to exons 2-13 was also identified.

Alternative Splicing↗

PubMed1996

Functional inactivation in mice of the gene for the interleukin-3 (IL-3)-specific receptor beta-chain: implications for IL-3 function and the mechanism of receptor transmodulation in hematopoietic cells.

The receptors for granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 and -5 (IL-3, IL-5) share a common signaling subunit (beta c). However, in the mouse, IL-3 can also use an alternative IL-3-specific receptor beta-chain (beta IL-3). To assess the relative contributions of beta c and beta IL-3 to IL-3 receptor formation and function, mice were generated in which the beta IL-3 gene was functionally inactivated by replacement of exons 9-13 with a neomycin resistance cassette. Bone marrow cells from these mice displayed a lower affinity IL-3 receptor than normal and were hyporesponsive to IL-3, but the mice displayed no obvious hematopoietic abnormalities. The data suggested that beta c and beta IL-3 are normally coexpressed on IL-3-responsive cells and have identical qualitative signaling capacities. Receptor transmodulation studies on bone marrow cells from wild-type, beta c -/-, and beta IL-3 -/- mice showed that the previously described hierarchical pattern of transmodulation was dependent on the relative numbers of both beta IL-3 and beta c receptor chains and also provided evidence for an unexpected interaction between beta c chains and G-CSF and M-CSF receptors.

Animals↗