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1.
3.
Int Immunol ; 11(12): 1935-44, 1999 Dec.
Article in English | MEDLINE | ID: mdl-10590259

ABSTRACT

mAb against human IL-5 inhibit pulmonary eosinophilia, tissue damage and airway hyper-reactivity in allergic animal models. Sch 55700 is a humanized, neutralizing anti-IL-5 antibody. To better understand the molecular mechanism by which Sch 55700 blocks IL-5 bioactivity, we have mapped its epitope by scanning IL-5 with synthetic peptides. Those peptides containing a region, ERRRV, corresponding to amino acids 89-93 of IL-5 specifically interact with both Sch 55700 and its parental rat IgG, 39D10. Among the five residues of this region, all three arginine residues were particularly critical for interaction of these peptides with Sch 55700. We further characterized this region by alanine scanning using site-directed mutagenesis. Examination of COS-expressed IL-5 mutants by Western blot showed that single mutations of E(89), R(90), R(91) or R(92) to alanine caused a loss of IL-5 binding to both Sch 55700 and 39D10. We further demonstrated in surface plasmon resonance studies using a BIAcore biosenosor that E(89), R(90) or R(91) are involved in the interaction between IL-5 and its receptor alpha subunit. Based upon the findings here and previously reported structures of the IL-5 and 39D10 variable region, we propose a model suggesting that the molecular interactions between the IL-5 and Sch 55700 mainly involve several ion pair interactions. We conclude that Sch 55700 occupies a region, ERRR, on IL-5 that is essential for its interaction with the receptor and thereby blocks IL-5 bioactivity.


Subject(s)
Antibodies, Monoclonal/immunology , Epitope Mapping , Interleukin-5/antagonists & inhibitors , Animals , Base Sequence , Binding Sites , COS Cells , Humans , Interleukin-5/chemistry , Interleukin-5/metabolism , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Rats , Receptors, Interleukin/metabolism , Receptors, Interleukin-5
4.
Anal Biochem ; 268(1): 134-42, 1999 Mar 01.
Article in English | MEDLINE | ID: mdl-10036172

ABSTRACT

The high-affinity receptor for human interleukin-5 (hIL-5) is composed of alpha and beta subunits. A baculovirus expression system was established in Sf9 cells capable of expressing hIL-5 receptor alpha and beta subunits simultaneously. By using wheat germ agglutinin (WGA)-coated scintillation proximity assay (SPA) beads to capture 125I-labeled hIL-5-bound Sf9 cells, a SPA was developed and used to measure hIL-5 high-affinity binding. The hIL-5 receptors expressed in the Sf9 cells represented a single class of high-affinity binding sites with a dissociation constant (Kd) of 0. 24 nM and a density of 2.95 x 10(5) sites/cell. This is the first study in which the high-affinity Kd value similar to that for hIL-5 binding to human eosinophils was achieved using a recombinant expression system. The SPA compared favorably with the filter binding assay with regard to various binding parameters. We also found that several lectins, when coated on SPA beads, were even more effective than WGA-coated SPA beads for capturing the insect cells. We conclude that the baculovirus expression system was highly efficient in producing the high-affinity hIL-5 receptors and that the SPA was a simple and sensitive assay that could be readily adapted into a high-throughput screening format. The SPA described here could be a prototype for binding assays for other multimeric receptors.


Subject(s)
Cloning, Molecular/methods , Interleukin-5/metabolism , Receptors, Interleukin/genetics , Scintillation Counting , Animals , Baculoviridae , Cells, Cultured , Genetic Vectors , Humans , Insecta , Lectins/metabolism , Protein Binding , Receptors, Interleukin/metabolism , Receptors, Interleukin-5 , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Transfection
5.
J Immunol ; 159(11): 5412-21, 1997 Dec 01.
Article in English | MEDLINE | ID: mdl-9548481

ABSTRACT

In addition to a previously characterized promoter (P1), we now show the existence of a second promoter for the human IL-5Ralpha gene. Initially, a genomic region (P2) 5' upstream of human IL-5Ralpha exon 2 was cloned by an inverted PCR. The transcriptional start site was then mapped to a deoxycytidine (C) residue within P2 by analyzing cellular mRNA with both the 5' rapid amplification of cDNA end-PCR and S1 nuclease protection assays. Transfection of eosinophilic HL-60 cells with reporter gene constructs in which either P1 or P2 was linked to the bacterial chloramphenicol acetyltransferase (CAT) gene resulted in CAT expression; little or no CAT expression occurred in other myeloid and nonmyeloid cell lines. Deletion studies showed that a 66-bp region, ranging from -31 to +35, was sufficient to promote CAT expression in eosinophilic HL-60 cells. Analysis of linker-scanning mutants identified a novel 6-bp element (5' CTAATT 3') spanning -19 to -14 that was essential for P2 promoter activity. In electrophoretic mobility shift assays, a P2 region from -31 to +1 containing the unique 6-bp element, when used as a probe, formed a complex with a protein(s) that was found only in the eosinophilic cell line. This binding activity was lost upon replacement of the 6-bp element with a 6-bp linker, suggesting that this element likely serves as the binding site for an eosinophilic HL-60 cell-specific transcription factor(s). Together, these data suggest an important role for P2 promoter in the regulation of eosinophil-specific expression of the human IL-5 receptor alpha gene.


Subject(s)
Promoter Regions, Genetic , Receptors, Interleukin/genetics , Base Sequence , Cloning, Molecular , Eosinophils/chemistry , HL-60 Cells , Humans , Molecular Sequence Data , Mutagenesis , Polymerase Chain Reaction , Receptors, Interleukin-5
6.
Eur J Cancer ; 29A(16): 2279-83, 1993.
Article in English | MEDLINE | ID: mdl-7509165

ABSTRACT

Cells from patients with acute myeloid leukaemia (AML) or chronic myeloid leukaemia (CML) were separated into CD34-enriched and CD34-depleted subpopulations. The clonogenic capacities of these two subpopulations were then compared to each other and to the original unseparated cell population. In every study, the CD34-enriched subpopulation demonstrated a substantial increase in clonogenicity in vitro in comparison with the original cell population, while the reverse was the case for the CD34-depleted subpopulations. For reasons not clear at present, the enrichment for clonogenic cells far exceeded the enrichment for cells expressing the CD34 antigen. Additionally, the clonogenic potential was found to be unrelated to the level of myc expression in the various cell populations.


Subject(s)
Antigens, CD/analysis , Leukemia, Myeloid/pathology , Antigens, CD34 , Cell Division , Cell Separation , Gene Expression , Genes, myc , Humans , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/immunology , Leukemia, Myeloid/genetics , Leukemia, Myeloid/immunology , Tumor Stem Cell Assay
7.
Leuk Res ; 16(10): 1003-11, 1992 Oct.
Article in English | MEDLINE | ID: mdl-1405703

ABSTRACT

Monoclonal antibodies and flow cytometry were used to detect the expression of c-myc and c-myb in the bone marrow (BM) and peripheral blood (PB) cells of patients with acute myelogenous leukemia (AML). The expression of neither gene was correlated with the percent blast cells in the BM or PB nor was there a correlation between c-myc and c-myb expression. A wide range of expression of each gene was found within each FAB type of AML. Patients who had a high proportion of leukemia cells expressing c-myb were less likely to respond to remission induction therapy than patients in whom a low proportion of cells expressed c-myb. This association appears to reflect an inverse relationship between the proportion of cells expressing c-myb and the sensitivity of leukemia cells to the killing effects of chemotherapy in vivo. Treatment outcome was unrelated to c-myc expression.


Subject(s)
Leukemia, Myeloid, Acute/genetics , Proto-Oncogene Proteins c-myc/analysis , Proto-Oncogene Proteins/analysis , Bone Marrow/ultrastructure , Flow Cytometry , Gene Expression , Humans , Proto-Oncogene Proteins c-myb
8.
Anticancer Res ; 12(1): 171-5, 1992.
Article in English | MEDLINE | ID: mdl-1567165

ABSTRACT

Sections of normal colon (n = 14), hyperplastic polyps (n = 31) ulcerative colitis (n = 97) and tubular adenomas (n = 40) were examined by immunohistochemistry for the expression of the c-myc proto-oncogene product in order to assess its potential diagnostic value in predicting the malignant potential of these lesions. We compared the degree of epithelial abnormality in these colorectal specimens with the extent of immunoperoxidase staining for c-myc oncoprotein, we found that high c-myc protein expression correlated with the degree of epithelial alteration in ulcerative colitis and tubular adenoma groups. Weakly positive staining was found in 10 out of 14 normal colon samples and 28 out of 31 hyperplastic polyps. High tissue expression of c-myc protein, when combined with histologic dysplasia, may prove to be an additional factor in the evaluation of malignant potential in ulcerative colitis specimens and adenomas.


Subject(s)
Adenoma/chemistry , Colitis, Ulcerative/metabolism , Colonic Polyps/chemistry , Colorectal Neoplasms/chemistry , Precancerous Conditions/chemistry , Proto-Oncogene Proteins c-myc/analysis , Antibodies, Monoclonal/immunology , Colon/chemistry , Humans , Proto-Oncogene Mas , Proto-Oncogene Proteins c-myc/immunology
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