Chinese Hamster Ovary Transfectants Research Articles

A deep-well plate enabled automated high-throughput cell line development platform.

Cell line development (CLD) plays a crucial role in the manufacturing process development of therapeutic biologics. Most biologics are produced in Chinese hamster ovary (CHO) cell. Because of the nature of random transgene integration in CHO genome and CHO's inherent plasticity, stable CHO transfectants usually have a vast diversity in productivity, growth, and product quality. Thus, we often must resort to screening a large number of cell pools and clones to increase the probability of identifying the ideal production cell line, which is a very laborious and resource-demanding process. Here we have developed a deep-well plate (DWP) enabled high throughput (DEHT) CLD platform using 24-well DWP (24DWP), liquid handler, and other automation components. This platform has capabilities covering the key steps of CLD including cell passaging, clone imaging and expansion, and fed-batch production. We are the first to demonstrate the suitability of 24DWP for CLD by confirming minimal well-to-well and plate-to-plate variability and the absence of well-to-well cross contamination. We also demonstrated that growth, production, and product quality of 24DWP cultures were comparable to those of conventional shake flask cultures. The DEHT platform enables scientists to screen five times more cultures than the conventional CLD platform, thus significantly decreases the resources needed to identify an ideal production cell line for biologics manufacturing.

Model-based investigation of intracellular processes determining antibody Fc-glycosylation under mild hypothermia.

ABSTRACTDespite the positive effects of mild hypothermic conditions on monoclonal antibody (mAb) productivity (q mAb) during mammalian cell culture, the impact of reduced culture temperature on mAb Fc‐glycosylation and the mechanism behind changes in the glycan composition are not fully established. The lack of knowledge about the regulation of dynamic intracellular processes under mild hypothermia restricts bioprocess optimization. To address this issue, a mathematical model that quantitatively describes Chinese hamster ovary (CHO) cell behavior and metabolism, mAb synthesis and mAb N‐linked glycosylation profile before and after the induction of mild hypothermia is constructed. Results from this study show that the model is capable of representing experimental results well in all of the aspects mentioned above, including the N‐linked glycosylation profile of mAb produced under mild hypothermia. Most importantly, comparison between model simulation results for different culture temperatures suggests the reduced rates of nucleotide sugar donor production and galactosyltransferase (GalT) expression to be critical contributing factors that determine the variation in Fc‐glycan profiles between physiological and mild hypothermic conditions in stable CHO transfectants. This is then confirmed using experimental measurements of GalT expression levels, thereby closing the loop between the experimental and the computational system. The identification of bottlenecks within CHO cell metabolism under mild hypothermic conditions will aid bioprocess optimization, for example, by tailoring feeding strategies to improve NSD production, or manipulating the expression of specific glycosyltransferases through cell line engineering. Biotechnol. Bioeng. 2017;114: 1570–1582. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals Inc.

Generation and Characterization of Chimeric Antibodies against NS3, NS4, NS5, and Core Antigens of Hepatitis C Virus

Mouse-human chimeric antibodies (cAbs) against hepatitis C virus (HCV) core, NS3 (nonstructural), NS4, and NS5 antigens were developed as quality control (QC) reagents to replace the use of human sera/plasma for Abbott HCV immunoassays. The cAb retains the mouse monoclonal antibody (MAb) specificity and affinity but still reacts in the existing HCV assay format, which measures human anti-HCV immunoglobulin. Mouse heavy-chain (V(H)) and light-chain (V(L)) variable regions of anti-HCV core, NS3, NS4, and NS5 antigens were PCR amplified from hybridoma lines and then cloned with human IgG1 heavy-chain (C(H)) and light-chain (C(L)) constant regions, respectively. A single mammalian expression plasmid containing both heavy-chain and light-chain immunoglobulin genes was constructed and transfected into dihydrofolate reductase (DHFR)-deficient Chinese hamster ovary (CHO) cells. The transfected CHO cells were selected using hypoxanthine- and thymidine-free medium and screened by an enzyme immunoassay (EIA). The clone secreting the highest level of antibody was isolated from the CHO transfectants and further subcloned. Each cAb-expressing CHO cell line was weaned into serum-free medium, and the cAb was purified by protein A affinity chromatography. The levels of cAb production for the various CHO cell lines varied from 10 to 20 mg/liter. Purified anti-HCV cAbs were tested with Abbott HCV immunoassays and showed reactivity. Moreover, yeast surface display combined with alanine-scanning mutagenesis was used to map the epitope at the individual amino acid level. Our results suggest that these HCV cAbs are ideal controls, calibrators, and/or QC reagents for HCV assay standardization.

Nek3 kinase regulates prolactin-mediated cytoskeletal reorganization and motility of breast cancer cells

Prolactin (PRL) stimulates the cytoskeletal re-organization and motility of breast cancer cells. During PRL receptor signaling, Vav2 becomes phosphorylated and activated, an event regulated by the serine/threonine kinase Nek3. Given the regulatory role of Vav2, the function of Nek3 in PRL-mediated motility and invasion was examined. Overexpression of Nek3 in Chinese hamster ovary transfectants potentiated cytoskeletal re-organization in response to PRL. In contrast, downregulation of Nek3 expression by small-interfering RNA (siRNA) attenuated PRL-mediated cytoskeletal reorganization, activation of GTPase Rac1, cell migration and invasion of T47D cells. In addition, PRL stimulation induced an interaction between Nek3 and paxillin and significantly increased paxillin serine phosphorylation, whereas Nek3 siRNA-transfected cells showed a marked reduction in paxillin phosphorylation. Analysis of breast tissue microarrays also demonstrated a significant up-regulation of Nek3 expression in malignant versus normal specimens. These data suggest that Nek3 contributes to PRL-mediated breast cancer motility through mechanisms involving Rac1 activation and paxillin phosphorylation.

A New Functional Role of the Fibrinogen RGD Motif as the Molecular Switch That Selectively Triggers Integrin αIIbβ3-dependent RhoA Activation during Cell Spreading

A number of RGD-type integrins rely on a synergistic site in addition to the canonical RGD site for ligand binding and signaling, although it is still unclear whether these two recognition sites function independently, synergistically, or competitively. Experimental evidence has suggested that fibrinogen binding to the RGD-type integrin alphaIIbbeta3 occurs exclusively through the synergistic gamma(400-411) sequence, thus questioning the functional role of the RGD recognition site. Here we have investigated the respective role of the fibrinogen gamma(400-411) sequence and the RGD motif in the molecular events leading to ligand-induced alphaIIbbeta3-dependent Chinese hamster ovary (CHO) cell or platelet spreading, by using intact fibrinogen and well characterized plasmin-generated fibrinogen fragments containing either the RGD motif (fragment C) or the gamma(400-411) sequence (fragment D), and CHO cells expressing resting wild type (alphaIIbbeta3wt), constitutively active (alphaIIbbeta3T562N), or non-functional (alphaIIbbeta3D119Y) receptors. Our data provide evidence that the gamma(400-411) site by itself is able to initiate alphaIIbbeta3 clustering and recruitment of intracellular proteins to early focal complexes, mediating cell attachment, FAK phosphorylation, and Rac1 activation, while the RGD motif subsequently acts as a molecular switch on the beta3 subunit to trigger cell spreading. More importantly, we show that the premier functional role of the RGD site is not to reinforce cell attachment but, rather, to imprint a conformational change on the beta3 subunit leading to maximal RhoA activation and actin cytoskeleton organization in CHO cells as well as in platelets. Finally, alphaIIbbeta3-dependent RhoA stimulation and cell spreading, but not cell attachment, are Src-dependent and phosphoinositide 3-kinase-independent and are inhibited by the Src antagonist PP2.

Novel Association of Vav2 and Nek3 Modulates Signaling through the Human Prolactin Receptor

Prolactin (PRL) receptor activation contributes to the progression and motility of human breast cancer. This event activates multimeric signaling pathways, including the activation of the Vav family of guanine nucleotide exchange factors. To detect novel proteins interacting with Vav, yeast two-hybrid analysis was performed and demonstrated an interaction between the serine/threonine NIMA (never in mitosis A)-related family kinase p56Nek3 and Vav1. The PRL-dependent interaction of Nek3 with Vav1 and Vav2 was confirmed by coimmunoprecipitation analysis. PRL stimulation of T47D cells induced Nek3 kinase activity and the interaction of Vav2/Nek3 with the PRL receptor. Increased Nek3 levels up-regulated Vav2 serine and tyrosine phosphorylation, whereas knockdown of Nek3 resulted in a reduction of Vav2 phosphorylation. Activation of guanosine triphosphatase Rac-1 in Chinese hamster ovary transfectants required both Nek3 and Vav2 and was inhibited by the overexpression of a kinase inactivating Nek3 mutant. However, overexpression of either Nek3 or kinase-inactive Nek3 had no effect on Vav2-potentiated signal transducer and activator of transcription 5-mediated gene expression. Overexpression of kinase inactive Nek3 in T47D cells led to a 50% increase in apoptosis vs. controls. These data suggest that the PRL-mediated activation of Nek3 contributes differentially to Vav2 signaling pathways involving Rac1 and signal transducer and activator of transcription 5 and implicates Nek3 during PRL-mediated actions in breast cancer.

Different Endocytosis Pathways of the C5a Receptor and the N‐formyl Peptide Receptor

Two chemoattractant receptors, C5aR (the complement fragment C5a receptor) and FPR (the N-formyl peptide receptor), are involved in neutrophil activation at sites of inflammation. In this study, we found major differences in the intracellular trafficking of the receptors in transfected Chinese hamster ovary (CHO) cells. Western blot analysis showed that FPR was stable during a 3 h stimulation with ligand, but C5aR was reduced in quantity by 50%. Not all C5aR was targeted directly for degradation however; a small, but visible fraction of the receptor became re-phosphorylated upon subsequent addition of ligand, suggesting that some of the receptor had cycled to the cell surface. Light membrane fractions isolated from activated cells showed C5aR distribution at the bottom of a glycerol gradient, colocalizing with the main distribution of the late endosomal/lysosomal marker LAMP2, whereas FPR was found at the bottom of the gradient as well as in the middle of the gradient, where it cofractionated with the early/sorting endosomal marker Rab5. Using fluorescence microscopy, we observed ligand-dependent redistribution of C5aR-EGFP from the plasma membrane to LAMP2-positive compartments, whereas FPR-EGFP showed significant colocalization with the early/sorting endosomes. Analysis of endogenous C5aR and FPR in neutrophils revealed a pattern similar to the CHO transfectants: C5aR underwent degradation after prolonged ligand stimulation, while FPR did not. Finally, we confirmed the down-regulation of C5aR in a functional assay by showing reduced chemotaxis toward C5a in both CHO transfectants and neutrophils after preincubation with C5a. A similar decrease in FPR-mediated chemotaxis was not observed.

610. Elucidating the Mechanism(s) for Tumor Specificity of Attenuated Measles Virus

Attenuated Edmonston strain of measles virus (MV-Edm) is a potent oncolytic agent which inhibited the growth or caused complete remission of a variety of human tumor xenografts in mice. Interestingly, the virus is tumor selective, causing extensive cytopathic effects (cpe) of cell-to-cell fusion (syncytium) in tumor cells but minimal cytopathic damage or cell killing in non-transformed cells. The mechanism(s) underlying tumor specificity of MV-Edm is unknown and is the focus of this study. The oncolytic activity of MV-Edm is mediated through the CD46, a complement regulatory protein, and the measles hemagglutinin (H) and fusion (F) protein. The virus uses its H protein to attach to CD46 and uses F to mediate virus-cell entry or cell-to-cell fusion which results in syncytia formation. Hence, a differential in cpe between tumor cells and non-transformed cells could be due to a difference in receptor expression on the surfaces of these cells or due to a difference in H/F expression levels. Indeed, tumor cells are known to express higher levels of CD46 receptors compared to their normal counterparts. To define the role of CD46 receptor density in MV-Edm infection, a panel of Chinese hamster ovary (CHO) clones expressing a range of surface densities of CD46 was generated. Using these CHO transfectants, a recombinant MV-Edm expressing a green fluorescent protein (MV-eGFP) and an adenoviral vector expressing MV-H/F proteins, we established the correlation between CD46 receptor density, virus entry and cell fusion. Virus entry increased progressively with CD46 receptor density and was quite efficient even at relatively low receptor densities. In contrast, syncytium formation and cell killing was minimal at low CD46 densities but increased rapidly above a critical threshold in CD46 expression level, more typical of tumor cells. The median survival of mice bearing low, medium and high CD46 expressing CHO clones infected with oncolytic MV-Edm, and injected into the flank of nude mice confirmed that high CD46 receptor density is critical to the therapeutic potency of this oncolytic agent. This unique relationship between cytopathic effects and CD46 density, coupled with overexpression of CD46 on tumor cells makes MV-Edm an appealing targeted agent for virotherapy.

Red Cell ICAM-4 Is a Novel Ligand for Platelet-activated αIIbβ3 Integrin

ICAM-4 (LW blood group glycoprotein) is an erythroid-specific membrane component that belongs to the family of intercellular adhesion molecules and interacts in vitro with different members of the integrin family, suggesting a potential role in adhesion or cell interaction events, including hemostasis and thrombosis. To evaluate the capacity of ICAM-4 to interact with platelets, we have immobilized red blood cells (RBCs), platelets, and ICAM-Fc fusion proteins to a plastic surface and analyzed their interaction in cell adhesion assays with RBCs and platelets from normal individuals and patients, as well as with cell transfectants expressing the alpha(IIb)beta(3) integrin. The platelet fibrinogen receptor alpha(IIb)beta(3) (platelet GPIIb-IIIa) in a high affinity state following GRGDSP peptide activation was identified for the first time as the receptor for RBC ICAM-4. The specificity of the interaction was demonstrated by showing that: (i) activated platelets adhered less efficiently to immobilized ICAM-4-negative than to ICAM-4-positive RBCs, (ii) monoclonal antibodies specific for the beta(3)-chain alone and for a complex-specific epitope of the alpha(IIb)beta(3) integrin, and specific for ICAM-4 to a lesser extent, inhibited platelet adhesion, whereas monoclonal antibodies to GPIb, CD36, and CD47 did not, (iii) activated platelets from two unrelated type-I glanzmann's thrombasthenia patients did not bind to coated ICAM-4. Further support to RBC-platelet interaction was provided by showing that dithiothreitol-activated alpha(IIb)beta(3)-Chinese hamster ovary transfectants strongly adhere to coated ICAM-4-Fc protein but not to ICAM-1-Fc and was inhibitable by specific antibodies. Deletion of individual Ig domains of ICAM-4 and inhibition by synthetic peptides showed that the alpha(IIb)beta(3) integrin binding site encompassed the first and second Ig domains and that the G65-V74 sequence of domain D1 might play a role in this interaction. Although normal RBCs are considered passively entrapped in fibrin polymers during thrombus, these studies identify ICAM-4 as the first RBC protein ligand of platelets that may have relevant physiological significance.

A novel and functional interaction between cyclophilin A and prolactin receptor.

Precedent data have revealed that peptidyl isomerases can modulate the function of cell-surface receptors, but no such interactions have been previously shown for the members of the cytokine receptor superfamily. We demonstrate here that a functional interaction occurs between the prolactin receptor (PRLR) and peptidyl prolyl cis/trans isomerase cyclophilin A (CypA). CypA was co-immunoprecipitated with the PRLR in vivo from the breast epithelial cell line T47D and Chinese hamster ovary transfectants overexpressing transfected human PRLR. In addition, in vitro binding assays demonstrated a direct interaction of CypA with the PRLR, in the presence or absence of cyclosporine. Co-immunoprecipitation studies also showed an association of CypA with Jak2. Functional analysis revealed that overexpression of CypA inhibited PRL-induced Rac activation, while simultaneously prolonging Jak2 phosphorylation. These proximal actions had profound downstream effects: CypA overexpression significantly enhanced the basal and PRL-stimulated expression from a beta-casein reporter construct. Hence, the interaction between CypA and the PRLR plays a differential regulatory role in the various signaling pathways leading from the PRLR.

Cloning and expression of the ovine CD40 molecule and the inhibition of the mixed lymphocyte reaction by the ovine CD40 e–EGFP fusion protein

The CD40 molecule is a member of the tumour necrosis factor receptor (TNFR)-like supergene family and plays a major role as a co-stimulatory molecule in the activation of T cells in response to antigens presented by dendritic cells. In this study, reverse transcription–PCR cloning was used to derive the sequence encoding ovine CD40. The ovine CD40 sequence demonstrated a similarity of 97, 76 and 64% with the bovine, human and murine sequences, respectively, at the nucleic acid level. The cysteine residues characteristic of the TNFR family and N-linked glycosylation sites are conserved. Furthermore, RNA analysis confirmed expression of CD40 mRNA in both ovine dendritic cells from lymphatic drainage and dermal fibroblasts in culture. In addition, cDNA encompassing the extracellular region of ovine CD40 (CD40 e) was fused ‘in-frame’ with the enhanced green fluorescent protein (EGFP) to generate a fusion protein upon the transfection of Chinese hamster ovary (CHO) cells. Immunoprecipitation with an anti-GFP monoclonal antibody of a 78 kD a protein from conditioned medium of CHO transfectants confirmed that the CD40 e–EGFP was secreted in the supernatant. All experiments were controlled with a pEGFP-N1 vector-blank construct. Moreover, the biological activity of ovine CD40 e–EGFP was demonstrated by its ability to inhibit a two-way mixed lymphocyte reaction. Thus these observations confirm that ovine CD40 blockade inhibits co-stimulation mediated by CD40–CD40L (CD154) interactions as has been reported in murine and human studies.

Feedback Inhibition of Amidophosphoribosyltransferase Regulates the Rate of Cell Growth via Purine Nucleotide, DNA, and Protein Syntheses

To clarify the contributions of amidophosphoribosyltransferase (ATase) and its feedback regulation to the rates of purine de novo synthesis, DNA synthesis, protein synthesis, and cell growth, mutated human ATase (mhATase) resistant to feedback inhibition by purine ribonucleotides was engineered by site-directed mutagenesis and expressed in CHO ade (-)A cells (an ATase-deficient cell line of Chinese hamster ovary fibroblasts) and in transgenic mice (mhATase-Tg mice). In Chinese hamster ovary transfectants with mhATase, the following parameters were examined: ATase activity and its subunit structure, the metabolic rates of de novo and salvage pathways, DNA and protein synthesis rates, and the rate of cell growth. In mhATase-Tg mice, ATase activity in the liver and spleen, the metabolic rate of the de novo pathway in the liver, serum uric acid concentration, urinary excretion of purine derivatives, and T lymphocyte proliferation by phytohemagglutinin were examined. We concluded the following. 1) ATase and its feedback inhibition regulate not only the rate of purine de novo synthesis but also DNA and protein synthesis rates and the rate of cell growth in cultured fibroblasts. 2) Suppression of the de novo pathway by the salvage pathway is mainly due to the feedback inhibition of ATase by purine ribonucleotides produced via the salvage pathway, whereas the suppression of the salvage pathway by the de novo pathway is due to consumption of 5-phosphoribosyl 1-pyrophosphate by the de novo pathway. 3) The feedback inhibition of ATase is more important for the regulation of the de novo pathway than that of 5-phosphoribosyl 1-pyrophosphate synthetase. 4) ATase superactivity leads to hyperuricemia and an increased bromodeoxyuridine incorporation in T lymphocytes stimulated by phytohemagglutinin.

Growth Hormone (GH)-induced Dimerization Inhibits Phorbol Ester-stimulated GH Receptor Proteolysis

Growth hormone (GH) initiates its cellular action by properly dimerizing GH receptor (GHR). A substantial fraction of circulating GH is complexed with a high-affinity GH-binding protein (GHBP) that in many species can be generated by GHR proteolysis and shedding of the receptor's ligand-binding extracellular domain. We previously showed that this proteolysis 1) can be acutely promoted by the phorbol ester phorbol 12-myristate 13-acetate (PMA), 2) requires a metalloprotease activity, 3) generates both shed GHBP and a membrane-associated GHR transmembrane/cytoplasmic domain remnant, and 4) results in down-regulation of GHR abundance and GH signaling. Using cell culture model systems, we now explore the effects of GH treatment on inducible GHR proteolysis and GHBP shedding. In human IM-9 lymphocytes, which endogenously express GHRs, and in Chinese hamster ovary cells heterologously expressing wild-type or cytoplasmic domain internal deletion mutant rabbit GHRs, brief exposure to GH inhibited PMA-induced GHR proteolysis (receptor loss and remnant accumulation) by 60-93%. PMA-induced shedding of GHBP from Chinese hamster ovary transfectants was also inhibited by 70% in the presence of GH. The capacity of GH to inhibit inducible GHR cleavage did not rely on JAK2-dependent GH signaling, as evidenced by its continued protection in JAK2-deficient gamma2A rabbit GHR cells. The GH concentration dependence for inhibition of PMA-induced GHR proteolysis paralleled that for its promotion of receptor dimerization (as monitored by formation of GHR disulfide linkage). Unlike GH, the GH antagonist, G120K, which binds to but fails to properly dimerize GHRs, alone did not protect against PMA-induced GHR proteolysis; G120K did, however, antagonize the protective effect of GH. Our data suggest that GH inhibits PMA-induced GHR proteolysis and GHBP shedding by inducing GHR dimerization and that this effect does not appear to be related to GH site 1 binding, GHR internalization, or GHR signaling. The implications of these findings with regard to GH signaling and GHR down-regulation are discussed.

A Point Mutation Thr799Met on the 2 Integrin Leads to the Formation of New Human Platelet Alloantigen Sita and Affects Collagen-Induced Aggregation

A new platelet-specific alloantigen, termed Sita, was identified in a severe case of neonatal alloimmune thrombocytopenia. The Sita alloantigen is of low frequency (1/400) in the German population. Immunochemical studies demonstrated that the Sita epitopes reside on platelet glycoprotein (GP) Ia. Nucleotide sequence analysis of GPIa cDNA derived from Sita-positive platelets showed C2531→T2531 point mutation, resulting in Thr799Met dimorphism. Analysis of genomic DNA from 22 Sita-negative normal individuals showed that the Thr799 is encoded by ACG2532 (90.9%) or ACA2532 (9.1%). To establish a DNA typing technique, we elucidated the organization of the GPIa gene adjacent to the polymorphic bases. The introns (421 bp and 1.2 kb) encompass a 142-bp exon with the 2 polymorphic bases 2531 and 2532. Polymerase chain reaction-restriction fragment length polymorphism analysis on DNA derived from 100 donors using the restriction enzyme MaeIII showed that the Met799 form of GPIa is restricted to Sita (+) phenotype. Analysis of stable Chinese hamster ovary transfectants expressing allele-specific recombinant forms of GPIa showed that anti-Sita exclusively reacted with the Glu505Met799, but not with the Glu505Thr799 and the Lys505Thr799 isoforms. In contrast, anti-Bra (HPA-5b) only recognized the Lys505Thr799 form, whereas anti-Brb(HPA-5a) reacted with both Glu505Thr799 and Glu505Met799 isoforms. These results demonstrated that the Met799 is responsible for formation of the Sita alloantigenic determinants, whereas amino acid 505 (Lys or Glu) specifically controls the expression of Bra and Brb epitopes, respectively. Platelet aggregation responses of Sita (+) individuals were diminished in response to collagen, indicating that the Thr799Met mutation affects the function of the GPIa/IIa complex.

A Point Mutation Thr799Met on the α2 Integrin Leads to the Formation of New Human Platelet Alloantigen Sita and Affects Collagen-Induced Aggregation

AbstractA new platelet-specific alloantigen, termed Sita, was identified in a severe case of neonatal alloimmune thrombocytopenia. The Sita alloantigen is of low frequency (1/400) in the German population. Immunochemical studies demonstrated that the Sita epitopes reside on platelet glycoprotein (GP) Ia. Nucleotide sequence analysis of GPIa cDNA derived from Sita-positive platelets showed C2531→T2531 point mutation, resulting in Thr799Met dimorphism. Analysis of genomic DNA from 22 Sita-negative normal individuals showed that the Thr799 is encoded by ACG2532 (90.9%) or ACA2532 (9.1%). To establish a DNA typing technique, we elucidated the organization of the GPIa gene adjacent to the polymorphic bases. The introns (421 bp and 1.2 kb) encompass a 142-bp exon with the 2 polymorphic bases 2531 and 2532. Polymerase chain reaction-restriction fragment length polymorphism analysis on DNA derived from 100 donors using the restriction enzyme MaeIII showed that the Met799 form of GPIa is restricted to Sita (+) phenotype. Analysis of stable Chinese hamster ovary transfectants expressing allele-specific recombinant forms of GPIa showed that anti-Sita exclusively reacted with the Glu505Met799, but not with the Glu505Thr799 and the Lys505Thr799 isoforms. In contrast, anti-Bra (HPA-5b) only recognized the Lys505Thr799 form, whereas anti-Brb(HPA-5a) reacted with both Glu505Thr799 and Glu505Met799 isoforms. These results demonstrated that the Met799 is responsible for formation of the Sita alloantigenic determinants, whereas amino acid 505 (Lys or Glu) specifically controls the expression of Bra and Brb epitopes, respectively. Platelet aggregation responses of Sita (+) individuals were diminished in response to collagen, indicating that the Thr799Met mutation affects the function of the GPIa/IIa complex.

Processing and juxtacrine activity of membrane‐anchored betacellulin

Betacellulin (BTC) was originally isolated as a secreted growth factor from a mouse pancreatic β-tumor cell line, whereas the cDNA sequence predicts that BTC is synthesized as a larger transmembrane protein. In the present study, we have characterized the membrane-anchored forms of BTC, using Chinese hamster ovary (CHO) cells, mouse fibroblast A9 cells, and a human breast cancer cell line MCF-7, all of which were stably transfected with human BTC cDNA. A9 and MCF-7 transfectants produced membrane-anchored BTC isoforms of 21, 25, 29, and 40 kDa on the cell surface, as well as a secreted BTC isoform. CHO transfectants secreted little BTC but accumulated the membrane-anchored isoforms. The cleavage of the membrane-anchored forms to release a secreted from of BTC was not enhanced by biological mediators such as a phorbol ester, which stimulates the cleavage of other membrane-anchored growth factors. The membrane-anchored forms of BTC expressed on the transfected cells induced the insulin production and/or promoted the growth in subclones of AR42J rat pancreatic cells. These results suggest that the membrane-anchored BTC can function as a juxtacrine factor in regulating the growth and differentiation of pancreatic endocrine cells. J. Cell. Biochem. 72:423–434, 1999. © 1999 Wiley-Liss, Inc.

Processing and juxtacrine activity of membrane-anchored betacellulin

Betacellulin (BTC) was originally isolated as a secreted growth factor from a mouse pancreatic beta-tumor cell line, whereas the cDNA sequence predicts that BTC is synthesized as a larger transmembrane protein. In the present study, we have characterized the membrane-anchored forms of BTC, using Chinese hamster ovary (CHO) cells, mouse fibroblast A9 cells, and a human breast cancer cell line MCF-7, all of which were stably transfected with human BTC cDNA. A9 and MCF-7 transfectants produced membrane-anchored BTC isoforms of 21, 25, 29, and 40 kDa on the cell surface, as well as a secreted BTC isoform. CHO transfectants secreted little BTC but accumulated the membrane-anchored isoforms. The cleavage of the membrane-anchored forms to release a secreted from of BTC was not enhanced by biological mediators such as a phorbol ester, which stimulates the cleavage of other membrane-anchored growth factors. The membrane-anchored forms of BTC expressed on the transfected cells induced the insulin production and/or promoted the growth in subclones of AR42J rat pancreatic cells. These results suggest that the membrane-anchored BTC can function as a juxtacrine factor in regulating the growth and differentiation of pancreatic endocrine cells.

Integrin ?3?1 expressed by human colon cancer cells is a major carrier of oncodevelopmental carbohydrate epitopes

Oncodevelopmental carbohydrate epitopes are a common feature of human colorectal carcinoma, yet their biological significance remains unclear. We have shown previously that monoclonal antibody (MAb) 3A7, which recognizes a determinant on type 2 chain blood group A and B oligosaccharides, detects oncodevelopmental changes in azoxymethane-induced rat colon tumors and some human colon cancer cell lines. (Laferté S et al. [19951 J. Cell. Biochem. 57:101-119). In this study, we set out to purify gp140, the major glycoprotein carrier of the 3A7 epitope expressed by human colon cancer cells, as a first step towards elucidating the contribution of the 3A7 epitope and its major glycoprotein carrier to colon cancer progression. To this end, gp140 was purified from HT29 cells and used for the preparation of polypeptide-specific monoclonal antibodies. Five monoclonal antibodies (7A8, 7B11, 8C7, 8H7, and 11D4) immunoprecipitated a 3A7-immunoreactive glycoprotein complex of 140 kDa which was subsequently identified by partial protein sequencing as alpha3beta1 integrin. Flow cytometric analysis of Chinese hamster ovary (CHO) cells expressing different human integrin chains revealed that MAbs 7A8 and 7B11 detect the alpha3 integrin subunit whereas MAbs 8C7 and 8H7 detect the beta1 integrin subunit. MAb 11D4, which did not bind to any of the CHO transfectants, detected type 2 chain blood group A determinant. Flow cytometric analysis of a panel of human colon carcinoma cell lines obtained from blood group A, AB, or B individuals revealed a direct correlation between cell-surface expression of the 3A7 epitope and alpha3 integrin subunit, suggesting that alpha3beta1 integrin is a preferred target of the 3A7 epitope in colon cancer cells. Using lectins and glycosidases to examine further the carbohydrate structure of alpha3beta1 integrin, we demonstrated that it is a sialoglycoprotein containing both N- and O-linked oligosaccharides. In addition, both alpha3 and beta1 integrin subunits express beta1-6 branched Asn-linked oligosaccharides and short poly-N-acetyllactosamine units (Galbeta1-4GlcNAc-R; n < or = 3), glycans previously implicated in cancer metastasis.Thus, alpha3beta1 integrin expressed by human colon carcinoma cells is a major carrier of oncodevelopmental carbohydrate epitopes whose presence may modulate tumor cell adhesion, migration, and/or invasion.

Effect of mutations at the residues R25, D27, P69, and N70 of B95a-MCP on receptor activities for the measles viruses Nagahata wild-type strain and CAM vaccine strain.

Human membrane cofactor protein (MCP, CD46) is a regulator of complement activation and also serves as a receptor for measles virus (MV). We recently isolated an MCP homolog from B95a, an Epstein-Barr virus-transformed marmoset B-lymphoblastoid cell line, which is 76% identical to human-MCP. B95a-MCP acts as an MV receptor for CAM, a vaccine strain of MV, but not for Nagahata, wild-type MV strain. The four residues in human-MCP (Asp27, Lys29, Arg69, and Asp70) are reportedly MV binding sites, and these are changed in B95a-MCP (Glu27, Asp29, Pro69, and Asn70). In the present study, we constructed B95a-MCP mutants by replacing the four residues with those in human-MCP, and tested whether the Chinese hamster ovary (CHO) transfectants expressing B95a-MCP mutants become susceptible to the Nagahata strain. The CHO transfectants expressing B95a-MCP mutants formed syncytium with the CAM strain but not with the Nagahata strain. The binding of the hemagglutinin (H) of MV with B95a-MCP mutants was observed with the CAM strain but not with the Nagahata strain. These results suggest that the failure of B95a-MCP as the MV receptor for the Nagahata strain is not due simply to the natural mutations at these four residues. The critical residues for MV binding in an MCP molecule seem to differ depending upon the structure of the MV H protein.

The Use of Allele-Specific Recombinant Fcγ Receptor IIIb Antigens for the Detection of Granulocyte Antibodies

AbstractThe Fcγ receptor IIIb (FcγRIIIb) for the Fc domain of IgG is expressed exclusively on neutrophils. The FcγRIIIb bears allotypic polymorphisms referred to as NA1, NA2, and SH, which are known for their frequent involvement in alloimmune and autoimmune neutropenias as well as in transfusion reactions. The bactericidal capacity of isolated neutrophils is easily activatable, and activation results in self-desintegration, thus preventing storage of neutrophils. As a result, only freshly isolated granulocytes can be used for antibody screening, often making it impossible to use typed panel cells. To provide a readily available source of typed panel cells, we therefore established stable mammalian cells expressing recombinant NA1, NA2, and SH antigens. We isolated mRNA from typed neutrophils and then transcribed it in cDNA. The cDNA that codes for the different forms of the FcγRIIIb was amplified by polymerase chain reaction and was subsequently subcloned into the mammalian expression vector pcDNA3. Chinese hamster ovary (CHO) cells were transfected with allele-specific constructs, and stable cell lines expressing FcγRIIIb were selected by flow cytometry. Because human sera show high background fluorescence with transfectants in flow cytometry, the monoclonal antibody–specific isolation of granulocyte antigens (MAIGA) assay was performed. By MAIGA assay, we tested 14 well-characterized human alloantibodies directed against the antigens NA1, NA2, and SH; 5 FcγRIIIb-specific isoantibodies; and 12 FcγRIIIb-reactive autoantibodies. Except one NA1- and one SH-specific alloantibody, all other antibodies could be identified by the use of CHO transfectants. In contrast to neutrophils, fixed CHO cells can be stored at 4°C for at least 4 weeks or stored frozen for a longer period. This longer shelf life of the transfected CHO cells compared with isolated neutrophils will simplify the detection of the clinically most important FcγRIIIb-reactive alloantibodies and autoantibodies.