Biomedical subjects

C G Begley

Publications and source records attributed to C G Begley.

At least 109 records · Page 6Linked to original sources

PubMed1995

Adjuvant treatment of high-risk breast cancer using multicycle high-dose chemotherapy and filgrastim-mobilized peripheral blood progenitor cells.

Women with primary breast cancer associated with extensive axillary node involvement or large primary tumors have a very poor prognosis despite treatment with standard-dose adjuvant chemotherapy. In an attempt to improve the outlook of these patients, we investigated the safety and feasibility of delivering three cycles of high-dose epirubicin and cyclophosphamide supported with filgrastim-mobilized peripheral blood progenitor cells (PBPC). Fifteen previously untreated women, median age 50 (range, 30-58) years, with poor prognosis early stage breast cancer received filgrastim (12 microgram/kg daily for 6 days) prior to chemotherapy to mobilize progenitor cells. Patients were then given three cycles of epirubicin (200 mg/m2) and cyclophosphamide (4 g/m2) at planned 28-day intervals, each followed by infusion of one third of the PBPC collected and daily administration of filgrastim (5 microgram/kg s.c.). Three leukaphereses collected a median of 114.9 (range, 22.7-273.5) x 10(4) granulocyte-macrophage-colony-forming cells/kg body weight. Hemopoietic recovery was rapid after each cycle, and there was no correlation between the rate of recovery and the number of granulocyte-macrophage-colony-forming cells infused. There was a small but significant progressive delay in recovery from hematological and nonhematological toxicities across the three cycles. Left ventricular ejection fraction fell to below 50% in eight (53%) patients, but none developed congestive cardiac failure. Two patients did not complete three cycles because of insufficient PBPC for a third cycle (n = 1) and 2-mercaptoethane sodium sulfonate- related drug reaction during the second cycle (n = 1). There were no deaths during the study or during the follow-up period (median, 70 weeks; range, 50-85 weeks), and no late toxicities occurred. Therefore, we concluded that the delivery of multiple cycles of nonmyeloablative, dose-intensive chemotherapy supported by PBPC and filgrastim is safe, and may be widely applicable to a variety of common chemosensitive cancers with a poor prognosis. The efficacy of three cycles of high-dose epirubicin and cyclophosphamide is to be compared with standard-dose chemotherapy in a randomized trial in patients with high-risk, operable stage II and III breast cancer.

Adolescent↗

PubMed1994

Steel factor affects SCL expression during normal erythroid differentiation.

Steel factor is one of the growth factors that controls the proliferation and differentiation of hematopoietic cells and SCL, also known as Tcl-5 or Tal-1, is a transcription factor involved in erythropoiesis. In this report, we studied the role of SCL in the proliferation of human peripheral blood burst-forming unit-erythroid (BFU-E) and the effects of Steel factor on SCL expression in proliferating erythroid cells. BFU-E-derived colonies increase progressively in size, as determined by cell number, from day 7 to day 14 of culture, with the greatest increase in colony size (10-fold expansion) occurring between day 7 and day 10. SCL protein levels in BFU-E-derived cells were highest in day 7 cells and decreased progressively from day 7 to day 14 of culture, suggesting an association of SCL with erythroid proliferation. In contrast, SCL mRNA levels did not decrease significantly between day 7 and day 14 cells, suggesting that posttranscriptional mechanisms are largely responsible for the decrease in SCL protein observed. The role of SCL in Steel factor-induced erythroid proliferation was then examined. In BFU-E-derived colonies cultured with Steel factor, colony size was significantly increased compared to control. In day 7 and day 10 erythroid precursors cultured with Steel factor, SCL protein was increased significantly compared to control. The increase in SCL protein levels in early erythroid precursors stimulated with Steel factor suggests one mechanism through which Steel factor may enhance normal erythroid proliferation. SCL mRNA levels assessed by Northern blot in day 7 cells did not increase significantly in response to Steel factor stimulation, suggesting that posttranscriptional mechanisms may also be important in the increase in SCL protein observed in response to Steel.

Base Sequence↗

PubMed1994

Cloning of a murine IL-11 receptor alpha-chain; requirement for gp130 for high affinity binding and signal transduction.

An adult mouse liver cDNA library was screened with oligonucleotides corresponding to the conserved WSXWS motif of the haemopoietin receptor family. Using this method, cDNA clones encoding a novel receptor were isolated. The new receptor, named NR1, was most similar in sequence and predicted structure to the alpha-chain of the IL-6 receptor and mRNA was expressed in the 3T3-L1 pre-adipocytic cell line and in a range of primary tissues. Expression of NR1 in the factor-dependent haemopoietic cell line Ba/F3 resulted in the generation of low affinity receptors for IL-11 (Kd approximately 10 nM). The capacity to bind IL-11 with high affinity (Kd = 300-800 pM) appeared to require coexpression of both NR1 and gp130, the common subunit of the IL-6, leukaemia inhibitory factor (LIF), oncostatin M (OSM) and ciliary neurotrophic factor (CNTF) receptors. The expression of both NR1 and gp130 was also necessary for Ba/F3 cells to proliferate and M1 cells to undergo macrophage differentiation in response to IL-11.

3T3 Cells↗

PubMed1994

Structure of the gene encoding the murine SCL protein.

We have determined the molecular structure of the gene encoding the murine SCL protein (helix-loop-helix transcription factor). The gene consists of seven exons spanning approx. 20 kb. The intron/exon structure, coding region sequences and sequences present at the splice junctions were highly conserved between mouse and human. The 5' flanking sequence contains CCAAT and TATA consensus motifs with several putative binding sites for SP-1, AP-1 and GATA-1. Multiple mRNA transcripts were generated by alternate exon usage. The transcripts differed primarily in the 5' untranslated region (UTR), but potentially also encode a smaller SCL protein. Despite the high degree of conservation between species, the heptamer/nonamer signal sequences in the 5' region of the human SCL gene (the frequent site of SCL disruption in human leukemia) were poorly represented in the murine sequence. In keeping with this, structural abnormalities of murine SCL were uncommon in murine leukemias that express the SCL transcript.

Animals↗

PubMed1994

Human R1 subunit of ribonucleotide reductase (RRM1): 5' flanking region of the gene.

Ribonucleotide reductase is essential, in dividing cells, for the production of deoxyribonucleotides prior to DNA synthesis in S phase. Neither of its two subunits (R1 and R2) are detectable in quiescent cells. In cycling cells, RRM1 mRNA and R1 protein are present throughout the cell cycle. A fragment of the human cDNA was used to isolate a genomic clone that encompasses the 5' flanking region of human RRM1. Primer extension and PCR experiments were used to define six potential cap sites. The immediate upstream region does not have a TATA box and is not GC-rich. A 1.9-kb fragment (-1670 to +208) was able to direct transcription of a reporter gene in a transient expression system. Understanding the mechanisms regulating expression of this gene will provide insight into the processes involved in cell cycling and may be of particular importance in understanding the deregulated growth of transformed cells.

Amino Acid Sequence↗

PubMed1994

Synthesis of rabbit corneal epithelial glycocalyx in vitro.

A method to study the synthesis and cellular processing of epithelial apical membrane glycoproteins in the rabbit cornea was developed. Fluorescent derivatives of wheat germ agglutinin (WGA; alpha-N-acetylglucosamine and sialic acid hapten affinities), succinylated WGA (alpha-N-acetylglucosamine hapten affinity) and concavalin A (Con A; D-mannose and D-glucose hapten affinity) were reacted with the corneal surface and the extent of binding attained was measured by en face, microscope-aided fluorophotometry. Minimal binding of succinylated WGA and a large reduction in WGA binding following neuraminidase treatment demonstrated that the attachment of WGA to the corneal surface occurred via sialic acid residues, i.e. via structures associated with terminal glycosylation. Corneas were treated with digitonin to induce the exfoliation of the outer squamous-like cell layers. The time-dependent changes in lectin binding density at the apical surface of the newly exposed intrastratal cells were then determined. Binding densities for WGA and Con A at the time of exfoliation of the digitonin-devitalized squamous cell layers (< 2 hr post-devitalization) were similar to the densities measured at the surface of untreated corneas. Over the subsequent 18-20 hr, the WGA and Con A binding increased by 2.63 +/- 0.24 and 3.0 +/- 0.68 (+/- S.D., n = 4) fold, respectively. The effect of inhibitors of transcription (actinomycin D, alpha-amanitin), translation (cycloheximide), core glycosylation of polypeptides (tunicamycin), endoplasmic reticulum glucosidases (deoxinojirimycin) and Golgi mannosidase (swainsonine) indicated that the increases were underpinned by new glycoprotein synthesis driven by a stable, pre-existing mRNA pool. Retinoic acid (2 microM) inhibited the increase in WGA binding by 55 +/- 6% (n = 4) but did not affect the Con A density increase suggesting that this agent either, modifies the terminal glycosylation pattern of apical membrane proteins and/or inhibits the synthesis of proteins bearing sialic acid. Actinomycin D or alpha-aminitin reverted the retinoic acid action, indicating that the retinoid effect is mediated by induced gene expression.

Animals↗

PubMed1994

Analysis of cerbB2 expression using a panel of 6 commercially available antibodies.

Results are presented of a study comparing cerbB2 (neu or Her2) expression as assessed immunohistochemically in breast neoplasia using a panel of 6 commercially available antibodies. The antibodies were examined utilizing conventional formalin fixed paraffin embedded tissue, and compared with molecular analysis of gene amplification. The aim was to determine the practical utility of each antibody, assessing ease of use, specific and non-specific staining characteristics, and expense, thus allowing a specific recommendation as to antibody of choice for immunohistochemical assessment of cerbB2 expression. Reassuringly, amongst the 38 breast lesions (36 carcinomas, 2 fibroadenomas) subjected to immunohistochemically (IHC) with the panel of 6 antibodies (Ab), no gross discrepancy of staining pattern was seen. Of the 38 cases, 10 were positive (26%), where at least one Ab demonstrated clear cytoplasmic membrane staining. Of a total of 45 breast lesions (43 carcinomas, 2 fibroadenomas), including all those examined by IHC, the total number of cases showing cerbB2 amplification by DNA analysis was 14 (31%). Using the DNA amplification as a base line for comparison, one Ab (No. 4) was found to stain 6 of the 14 cases of breast carcinoma that were assessed as showing amplification at the DNA level. Four Abs (1,3,5,6) stained 5 of these cases. However, Abs 3,4 and 6 displayed artefactual cytoplasmic staining (in the absence of membrane staining) that precluded the practical use of these reagents. Therefore, based on additional considerations of cost and ease of use, Ab No. 1 was finally chosen for recommendation from the 6 Ab panel.

Adult↗

PubMed1994

Two cases of de novo precursor B-cell acute lymphoblastic leukemia with t(14;18), but without cytogenetic evidence of an associated Burkitt's or Burkitt's variant translocation.

This study reports two cases of acute lymphoblastic leukemia of precursor B-cell type. The cases were associated with a t(14;18), but no evidence of a Burkitt's-type translocation was found. There was also no evidence of a preexisting follicular lymphoma. This cytogenetic variety of acute lymphoblastic leukemia has not been reported previously.

Adolescent↗

PubMed1994

A study of granular lymphoproliferative disorders including a CD3 negative case with a rearrangement of the T-cell receptor locus.

The clinical, and laboratory features of 9 patients presenting with chronic proliferations of large granular lymphocytes (LGL) are described. The median patient age was 61 years (33-80) and median patient follow up was 3.5 years (28 mo-10 years) with all patients surviving. Clinical features and blood and bone marrow findings are documented. Immunophenotypic analysis showed lymphocytes from 4 patients were CD3 negative and 5 were CD3 positive with natural killer associated cell surface antigens expressed in both these groups. Analysis of the T-cell receptor (TCR) loci revealed a clonal rearrangement in 4 samples including one CD3 negative sample. Clonality did not correlate with immunophenotype or clinical or haematological features. We conclude that patients with persistent LGL have a wide diversity of cell surface marker expression and that whilst some patients with CD3 negative LGL proliferations have cells which are most likely of natural killer (NK) cell origin, in others TCR rearrangements can be demonstrated suggesting these cells are possibly of T-cell, not NK cell, origin.

Adult↗

PubMed1994

Combined chemotherapy and granulocyte colony-stimulating factor (G-CSF) mobilise large numbers of peripheral blood progenitor cells in pretreated patients.

The aim of this study was to examine the effect of G-CSF given after salvage chemotherapy on the mobilisation of peripheral blood progenitor cells (PBPC) in pretreated patients. Seven patients with relapsed or refractory non-Hodgkin's lymphoma (NHL) were treated with methotrexate, cyclophosphamide, cytarabine, etoposide and dexamethasone. G-CSF was given at a dose of 3.8-7.2 micrograms/kg (1-2 ampoules) daily by subcutaneous injection from the onset of neutropenia (< 1.0 x 10(9)/L). A median of 3 leukaphereses was performed when the white cell count was recovering. The median number of granulocyte-macrophage colony-forming cells (GM-CFC) collected was 99 x 10(4)/kg per leucapheresis (range 19-800) or 260 x 10(4)/kg in total per patient (110-1800). Six patients underwent myeloablative chemotherapy with PBPC rescue. No autologous bone marrow or growth factors post-PBPC infusion were administered. The median duration of severe neutropenia (< 0.5 x 10(9)/L) was 8.5 days (range 5-10) and to recovery of neutrophils post-PBPC infusion was 11.5 days (10-15). Severe thrombocytopenia (< 20 x 10(9)/L) was present for 4 days (range 1-5) and the median number of days post-infusion to platelet-transfusion independence was 9 (6-12). In conclusion, G-CSF combined with chemotherapy mobilised large numbers of PBPC for subsequent autotransplantation in pretreated patients with NHL. A single leukapheresis procedure may be sufficient following this protocol.

Adult↗

PubMed1994

Phase II study of autologous filgrastim (G-CSF)-mobilized peripheral blood progenitor cells to restore hemopoiesis after high-dose chemotherapy for lymphoid malignancies.

The hemopoietic growth factor filgrastim (r-metHu G-CSF) stimulates granulopoiesis after autologous BMT and can also be used as a peripheral blood progenitor cell (PBPC)-mobilizing agent. Rapid platelet recovery follows the addition of filgrastim-mobilized PBPC to autologous BMT. We have now studied 29 adults with malignant lymphoma, Hodgkin's disease or ALL to assess the ability of filgrastim-mobilized PBPC to rapidly and durably restore hemopoiesis without bone marrow (BM) infusion. Patients with a high yield of PBPC from three leukaphereses, defined as > 30 x 10(4)/kg GM-CFC, were eligible for PBPC transplant without BM. Patients with a low yield of GM-CFC received both PBPC and BM infusion. After filgrastim therapy 12 or 24 micrograms/kg/day by continuous sc infusion for 6 or 7 days, a high yield was obtained in 11 of 29 patients. Kinetics of recovery of both the platelet and neutrophil counts were more rapid in the high yield group than in the low yield group. The platelet count recovered to > 20 x 10(9)/l at a median of 9 days, to > 50 x 10(9)/l at 11 days and the neutrophil count to > 0.5 x 10(9)/l at 9 days in the high yield group compared with 12 days, 37 days and 10 days, respectively, in the low yield group (p = 0.028, p < 0.001 and p = 0.027). Fewer platelet transfusions were required in the high yield group (median 11 vs 29.5 units, p = 0.021).(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗

PubMed1994

The SCL transcription factor and differential regulation of macrophage differentiation by LIF, OSM and IL-6.

The SCL gene is a member of the helix-loop-helix family of transcription factors. First identified because of its involvement in a rare chromosome translocation in human T cell acute lymphoblastic leukemia, it is now recognized to be involved in up to 25% of T cell leukemias. Normally within the hemopoietic system the gene is expressed in progenitor cells, erythroid cells, mast cells and megakaryocytes. During macrophage differentiation the level of SCL mRNA and protein becomes undetectable. To examine this further, SCL was over expressed in murine M1 cells. This resulted in perturbation of macrophage differentiation induced by leukemia inhibitory factor (LIF) and oncostatin-M (OSM) but not interleukin (IL)-6. Moreover the perturbation of LIF-induced differentiation applied to some components of macrophage differentiation but not others. This suggests that signaling through the gp130 homodimer (as occurs with IL-6) does not utilize an SCL-inhibitable pathway. In contrast the LIF receptor/gp130 heterodimer does utilize an SCL-inhibitable pathway for some elements of macrophage differentiation (e.g., lysozyme induction) but not others (e.g., M-CSF receptor induction).

Animals↗

PubMed1994

Hereditary protein C deficiency associated with mutations in exon IX of the protein C gene.

This report describes five families with symptomatic hereditary protein C deficiency. Using a polymerase chain reaction (PCR)-based method, the entire coding sequence and intron-exon boundaries of the protein C gene was amplified from genomic DNA. In each family a single point mutation in the protein C gene was identified. Two unrelated families were found to share the same mutation, while the other three had different mutations. In the first two families with type I protein C deficiency the normal cytosine residue at nucleotide position 8551 in the protein C gene was replaced by thymidine leading to substitution of the normal proline residue at amino acid position 279 by leucine. In the third family with type I deficiency a previously undescribed mutation was identified. In this family the guanosine residue at position 8559 was replaced by adenosine (glycine 282 substituted by serine). In the fourth family, also with type I deficiency, guanosine 8589 was replaced by adenosine (glycine 292 substituted by serine). The fifth family had type II deficiency and in affected members cytosine 8769 was replaced by thymidine (arginine 352 substituted by tryptophan). All these mutations lead to amino acid substitutions in the serine protease domain of the mature protein. All were able to be confirmed by restriction enzyme analysis of PCR-derived DNA. In addition the novel mutation at nucleotide position 8559 was also demonstrable using single strand conformation polymorphism (SSCP) analysis of PCR-derived DNA. These mutations were likely examples of deamination of methylated cytosine occurring in cytosine-phosphate-guanosine (CpG) dinucleotide sequences. These findings confirm the genetic heterogeneity of hereditary protein C deficiency in these families.

5-Methylcytosine↗

PubMed1994

Complex pattern of alternative splicing in the normal uroporphyrinogen decarboxylase gene: implications for diagnosis of familial porphyria cutanea tarda.

We describe multiple alternative transcripts of uroporphyrinogen decarboxylase mRNA in normal individuals and patients with familial porphyria cutanea tarda. mRNA was reverse-transcribed, subjected to the polymerase chain reaction, and analyzed for nucleotide sequence. Seven different transcripts were characterized, and a cryptic splice acceptor site was identified in intron 1. In all mRNAs the exons abutted at previously defined exon boundaries. Characterization of the splice junctions in the genomic DNA showed that splice donor and acceptor sequences complied with the consensus sequences for these sites except for the splice acceptor sequences of exons 3 and 10. THese deviations were present in two normal individuals and one patient with familial porphyria cutanea tarda and were thus unable to explain the multiple aberrant uroporphyrinogen decarboxylase transcripts. We conclude that apparent deletions observed in transcripts derived from the uroporphyrinogen decarboxylase gene in patients with familial porphyria cutanea tarda should be interpreted with caution.

Alternative Splicing↗

PubMed1994

No evidence for GM-CSF receptor alpha chain gene mutation in AML-M2 leukemias which have lost a sex chromosome.

Sixty-five percent of acute myeloid leukemias subtype M2 (AML-M2), displaying a translocation between chromosomes 8 and 21 (t8;21) have also lost one or other copy of the sex chromosomes (XO). This finding has led to the hypothesis that a recessive oncogene may be present in the DNA common to both sex chromosomes, that is the pseudoautosomal region. The alpha chain of the receptor for the hemopoietic growth factor GM-CSF has recently been mapped to the human pseudoautosomal region and, given the role of this molecule in the control of normal hemopoiesis, it is thought to be a good candidate gene. This paper examines the structure, expression, and sequence of the GM-CSF receptor alpha chain gene (GMR alpha) in three primary AML-M2,XO samples and one AML-M2,XO cell line. Results of this work show no gross rearrangement or loss of the remaining allele of GMR alpha, detectable expression of its transcript and protein, and no changes at the nucleotide sequence level. Thus, we find no evidence to support a role for the GM-CSF receptor alpha chain molecule in the development of AML-M2,XO leukemias.

Base Sequence↗

PubMed1994

Constitutive expression of the putative transcription factor SCL associated with proviral insertion in the myeloid leukemic cell line WEHI-3BD-.

The murine myeloid leukemia WEHI-3B contains an intracisternal A-particle insertion in a growth factor gene (IL-3) and a homeobox gene (Hox 2.4). Southern blot analysis of the differentiation unresponsive subline WEHI-3BD- demonstrated a rearrangement of the SCL gene, a gene that encodes a protein of the helix-loop-helix class of transcription factors. Northern blot analysis demonstrated an abnormal SCL transcript in the WEHI-3BD- cells, although SCL expression was not detected in the parental cells. The rearrangement was cloned and confirmed as an insertion of an intracisternal A-particle in the 3' untranslated region of the SCL gene. Although SCL is implicated in up to 25% of human T-cell acute lymphoblastic leukemia, this is the first example of SCL involvement in a myeloid leukemia.

Animals↗

PubMed1994

The effects of soft contact lens disinfection solutions on rabbit corneal epithelium.

This investigation compared the effects of three commercial soft contact lens solutions on the rabbit corneal epithelium. Fifteen rabbits wore lenses soaked in ReNu multi-purpose disinfecting solution, Opti-Free rinsing, disinfecting and storage solution, and neutralized AOSEPT for a period of 30 minutes. Control eyes wore lenses soaked in unpreserved saline for the same time period. Corneas were photographed by scanning electron microscopy and graded in random locations across the corneal surface. Slides of these photographs were projected at a magnification of x3000 and graded according to two scales by an observer unaware of the treatment used. The results showed a significant treatment effect, with all three experimental solutions scoring higher than controls. When the three experimental solutions were compared, corneas exposed to ReNu multi-purpose disinfecting solution showed a significantly increased effect by contingency table analysis of data from the adapted Burstein's scale.

Animals↗

PubMed1994

The SCL protein displays cell-specific heterogeneity in size.

SCL protein production was examined in a variety of hemopoietic cell lines by immunoblotting using specific polyclonal antisera. SCL protein was detected in erythroid, megakaryocyte, mast and early myeloid cell lines, as well as in several lymphoid leukemia cell lines which are known to harbor SCL gene rearrangements. In most cell lines, proteins of molecular weight 49 and 44 kDa were found, however two myeloid cell lines expressed only lower molecular weight species of 24 and 22 kDa. This size discrepancy appeared to be due to cell-specific translational regulation, since overexpression of a retrovirally transfected SCL gene yielded the higher molecular weight forms in most cell lines (GP+E-86, AT2.5, M1) but only the 22 kDa form in the myeloid cell line, WEHI-3B/D+. Overexpression of full-length SCL protein in the lymphoid cell lines, SupT1 and Raji, did not alter cell phenotype and there was no evidence for autoregulation of SCL transcription. The restricted pattern of SCL protein synthesis is consistent with the restricted expression of SCL mRNA documented previously. In addition, the present results indicate that SCL protein size was determined by regulation of translation in a cell-specific manner.

Amino Acid Sequence↗