Monoclonal Antireceptor Research Articles

C-reactive protein in the arterial intima: role of C-reactive protein receptor-dependent monocyte recruitment in atherogenesis.

Infiltration of monocytes into the arterial wall is an early cellular event in atherogenesis. Recent evidence shows that C-reactive protein (CRP) is deposited in the arterial intima at sites of atherogenesis. In this study, we demonstrate that CRP deposition precedes the appearance of monocytes in early atherosclerotic lesions. CRP is chemotactic for freshly isolated human blood monocytes. A specific CRP receptor is demonstrated on monocytes in vitro as well as in vivo, and blockage of the receptor by use of a monoclonal anti-receptor antibody completely abolishes CRP-induced chemotaxis. CRP may play a major role in the recruitment of monocytes during atherogenesis.

Human monocyte receptors involved in tumor necrosis factor responses to group B streptococcal products.

Several group B streptococcal products have been previously found to stimulate human monocytes to produce tumor necrosis factor alpha. In order to identify the receptors involved in these responses, monocytes were stimulated with purified group- or type-specific carbohydrates or lipoteichoic acid in the presence of anti-receptor monoclonal antibodies, soluble CD14, or lipopolysaccharide-binding protein. Results indicate that CD14 plays an important role in tumor necrosis factor alpha responses to all of the stimuli tested. Moreover, both CD14 and complement receptor type 3 may be involved in responses to the group-antigen.

Epidermal growth factor receptor tyrosine kinase as a target for anticancer therapy.

Increasing knowledge of the structure and function of the epidermal growth factor receptor (EGFR) subfamily of tyrosine kinases and of their role in the initiation and progression of various cancers has, in recent years, provided the impetus for a substantial research effort aimed at developing new anticancer therapies that target specific components of the EGFR signal transduction pathway. Selective compounds have been developed that target either the extracellular ligand-binding region of the EGFR or the intracellular tyrosine kinase region, resulting in interference with the signalling pathways that modulate mitogenic and other cancer-promoting responses (e.g. cell motility, cell adhesion, invasion and angiogenesis). Potential new anticancer agents that target the extracellular ligand-binding region of the receptor include a number of monoclonal antibodies, immunotoxins and ligand-binding cytotoxic agents. Agents that target the intracellular tyrosine kinase region include small molecule tyrosine kinase inhibitors (TKIs), which act by interfering with ATP binding to the receptor, and various other compounds that act at substrate-binding regions or downstream components of the signalling pathway. Currently, the most advanced of the newer therapies undergoing clinical development are antireceptor monoclonal antibodies (e.g. trastuzumab and cetuximab) and a number of small molecule EGFR-TKIs principally of the quinazoline and pyrazolo-pyrrolo-pyridopyrimidine inhibitor structural classes. The latter group of compounds offers several advantages in cancer chemotherapy, including the possibility of inhibiting specific deregulated pathways in cancer cells while having minimal effects on normal cell function. They also have favourable pharmacokinetic and pharmacodynamic properties and low toxicity, and some TKIs such as the reversible inhibitor ZD1839 ('Iressa') are now undergoing phase II to III clinical trials. In addition, the accumulation of evidence from laboratory studies strongly suggests that EGFR-selective TKIs will have synergistic effects with other antitumour agents or therapy such as cytostatic agents, conventional cytotoxic drugs and radiotherapy. As our knowledge of signal transduction pathways in cancer increases, it is hoped that further advances in this area will allow the therapeutic potential of these compounds as anticancer agents to be realised.

Functional Blockade of Tyrosine Kinase A in the Rat Basal Forebrain by a Novel Antagonistic Anti-Receptor Monoclonal Antibody

We have exploited a new monoclonal antibody against the tyrosine kinase A (TrkA) nerve growth factor (NGF) receptor to block the NGF-TrkA interaction in the rat basal forebrain. The monoclonal antibody MNAC13 is a potent antagonist that prevents the binding of NGF to TrkA in a variety of systems. This antibody was used to study the maintenance of the cholinergic phenotype in the rat basal forebrain in vivo, by the implant of antibody-secreting cells. Basal forebrain cholinergic neurons (BFCNs) are greatly affected by the antibody treatment, both in terms of cell number and of cell soma size. When antibody-secreting cells are implanted at postnatal day 2 (P2), the effects observed at P8 are as severe as those obtained with anti-NGF antibodies and, interestingly, are observed also if anti-TrkA cells are implanted at P8, when anti-NGF antibodies, delivered by the same route, are no longer effective (). The effects induced by anti-TrkA, as those induced by anti-NGF, are reversible, but the time required for recovery and the critical period in the sensitivity of BFCNs to the functional inactivation of TrkA is twice as long than that observed when NGF is intercepted. These results demonstrate that BFCNs are more sensitive to the block of TrkA activation than they are to the block of NGF. The cloning of MNAC13 variable regions and their assembly into a functional polypeptide of reduced size (single chain Fv fragment) will allow its use in gene transfer applications.

Internalization and Recycling Pathways of the Thyrotropin Receptor

Scant information is available to date on the intracellular trafficking of the TSH receptor. In the present study we have used stably transfected L cells that express the TSH receptor, 125I-labeled TSH, and antireceptor antibodies as well as gold-conjugated antireceptor monoclonal antibodies and hormone. The latter allowed us to study, by electron microscopy, the cellular distribution and endocytosis of TSH receptor. The receptor was initially localized on the plasmalemma proper and in clathrin-coated pits but was excluded from smooth vesicles open to the cell surface. It was internalized through clathrin-coated vesicles. Constitutive endocytosis represented 10% of cell surface receptor molecules. Endocytosis was increased 3-fold by incubation with hormone. The majority of internalized receptor molecules (90%) was recycled to the cell surface, whereas the hormone was degraded in lysosomes. This recycling of receptor was inhibited by administration of monensin. Electron microscopic and confocal microscopic studies were repeated in primary cultures of human thyroid cells and showed a distribution, and endocytosis pathways, very similar to those observed in transfected L cells. A previous study has shown the LH receptor to be endocytosed in high proportion and to be degraded in lysosomes. Confocal microscopy and colocalization studies with transferrin receptor confirmed that the highly homologous LH and TSH receptors exhibit, when expressed in the same cells, very different cellular trafficking properties. The use of LH/TSH receptor chimeras showed that transmembrane-intracellular domains contain information orienting the protein toward recycling or degradative pathways. The extracellular domain seems to play a role in the extent of internalization. These observations should now allow the identification of the molecular signals involved.

Sequencing of antibodies.

Several antireceptor monoclonal antibodies (MAbs) have been described to compete with ligands for receptor binding. The possibility that structures derived from the hypervariable or complementarity determining regions (CDR) of such antibodies display similarity with those of the ligand binding site of receptors has been documented, thereby allowing the understanding of the structural basis of receptor-ligand interaction. Thus, the determination of the structure of these CDR regions can allow the identification of sequences responsible for the activity of the antibodies.

Anti-(epidermal growth factor) receptor monoclonal antibodies for the induction of antibody-dependent cell-mediated cytotoxicity against squamous cell carcinoma lines of the head and neck.

Squamous cell carcinomas of the head and neck (SCCHN) frequently display high levels of the epidermal growth factor receptor (EGFR). Since EGFR is expressed on the cell surface it may form a suitable target for anticancer therapy with anti-receptor monoclonal antibodies (mAb). Besides the interference with receptor/ligand interactions, binding of mAb to EGFR leads to immunoglobulin-coated tumour cells that may induce or enhance non-specific immune effector mechanisms like antibody-dependent cell-mediated cytotoxicity (ADCC). In established cell lines of SCCHN (UM-SCC 11B, 14C, 22B, and 8029 NA) we investigated the antitumour activity of allogeneic peripheral blood mononuclear cells (PBMC) in combination with rat (ICR 62), mouse (EMD 55900), and humanized (EMD 72000) anti-EGFR mAb. In addition, autologous PBMC were available for tumour line UD-SCC 4. The EGFR protein content of the tumour cell lines ranged between 170 fmol/mg protein and 8100 fmol/mg protein, and MCF-7 cells served as receptor-negative controls. PBMC activity against SCCHN targets was determined in 96-well microtitre-plate monolayer cultures by the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay after coincubation for 4 h, 24 h and 72 h at effector target ratios of 1:1, 5:1, 10:1 and 20:1. PBMC subpopulations were obtained by macrophage depletion (plastic adherence) or natural killer (NK) cell enrichment (magnetic bead negative selection). Prolonged time of exposure and increased effector:target ratios revealed marked antitumour activity of PBMC alone. This non-specific immune destruction was enhanced considerably by humanized and rat, but not mouse anti-EGFR mAb. Increased EGFR protein in tumour cells partly correlated with an intensification of ADCC but was accompanied by decreased primary PBMC cytotoxicity. The utilization of PBMC subpopulations suggested a mainly NK-cell-mediated ADCC, which appeared to benefit directly or indirectly, e.g. via the secretion of cytokines, from other PBMC components. In conclusion, humanized (EMD 72000) and rat (ICR 62) anti-EGFR mAb were able to generate strong antitumour ADCC in target monolayers of SCCHN.

Mutational analysis of the virus and monoclonal antibody binding sites in MHVR, the cellular receptor of the murine coronavirus mouse hepatitis virus strain A59.

The primary cellular receptor for mouse hepatitis virus (MHV), a murine coronavirus, is MHVR (also referred to as Bgp1a or C-CAM), a transmembrane glycoprotein with four immunoglobulin-like domains in the murine biliary glycoprotein (Bgp) subfamily of the carcinoembryonic antigen (CEA) family. Other murine glycoproteins in the Bgp subfamily, including Bgp1b and Bgp2, also can serve as MHV receptors when transfected into MHV-resistant cells. Previous studies have shown that the 108-amino-acid N-terminal domain of MHVR is essential for virus receptor activity and is the binding site for monoclonal antibody (MAb) CC1, an antireceptor MAb that blocks MHV infection in vivo and in vitro. To further elucidate the regions of MHVR required for virus receptor activity and MAb CC1 binding, we constructed chimeras between MHVR and other members of the CEA family and tested them for MHV strain A59 (MHV-A59) receptor activity and MAb CC1 binding activity. In addition, we used site-directed mutagenesis to introduce selected amino acid changes into the N-terminal domains of MHVR and these chimeras and tested the abilities of these mutant glycoproteins to bind MAb CC1 and to function as MHV receptors. Several recombinant glycoproteins exhibited virus receptor activity but did not bind MAb CC1, indicating that the virus and MAb binding sites on the N-terminal domain of MHVR are not identical. Analysis of the recombinant glycoproteins showed that a short region of MHVR, between amino acids 34 and 52, is critical for MHV-A59 receptor activity. Additional regions of the N-terminal variable domain and the constant domains, however, greatly affected receptor activity. Thus, the molecular context in which the amino acids critical for MHV-A59 receptor activity are found profoundly influences the virus receptor activity of the glycoprotein.

Receptor Associated Phosphorylation Following Monoclonal Antibody or Synthetic Peptide Binding to Nonspecific Cytotoxic Cells

We have previously shown that crosslinkage of a receptor protein on catfish nonspecific cytotoxic cells (NCC) with anti-receptor monoclonal antibody or with a synthetic peptide activates cytotoxicity and initiates signalling responses. Receptor linked signalling was associated with the production of increased levels of expression of 50-60 and 20-30 kDa phosphoproteins determined by immunoprecipitation with anti-phosphoserine and anti-phosphotyrosine mabs. These proteins are components of a macromolecular protein complex (>200 kDa) determined by reducing and nonreducing SDS-PAGE. The calcium ionophore A23187 treatment produced the same pattern of phosphoprotein expression as peptide or mab. Maximum phosphoserine expression occurred at 15′-30′ post-mab binding. We now show that synthetic peptide or mab treatment initiated the same serine and tyrosine phosphorylation profiles. The PKC specific inhibitor MDL 29,152 produced 50% inhibition of NCC lysis of IM-9 target cells, and completely inhibited serine phosphorylation of peptide activated cells but had no effect on tyrosine phosphorylation of the phosphointermediates. Genistein pretreatment of NCC inhibited cytotoxicity and tyrosine phosphorylation. Sequential immunprecipitation of the phosphointermediate demonstrated that the phosphorylated serine and tyrosine residues were on the same 50-60 kDa protein. These data indicate that both proximal and distal signalling events required for NCC activation may be associated with ATPase phosphorylation.

The function of the spike protein of mouse hepatitis virus strain A59 can be studied on virus-like particles: cleavage is not required for infectivity.

The spike protein (S) of the murine coronavirus mouse hepatitis virus strain A59 (MHV-A59) induces both virus-to-cell fusion during infection and syncytium formation. Thus far, only syncytium formation could be studied after transient expression of S. We have recently described a system in which viral infectivity is mimicked by using virus-like particles (VLPs) and reporter defective-interfering (DI) RNAs (E. C. W. Bos, W. Luytjes, H. Van der Meulen, H. K. Koerten, and W. J. M. Spaan, Virology 218:52-60, 1996). Production of VLPs of MHV-A59 was shown to be dependent on the expression of M and E. We now show in several ways that the infectivity of VLPs is dependent on S. Infectivity was lost when spikeless VLPs were produced. Infectivity was blocked upon treatment of the VLPs with MHV-A59-neutralizing anti-S monoclonal antibody (MAb) A2.3 but not with nonneutralizing anti-S MAb A1.4. When the target cells were incubated with antireceptor MAb CC1, which blocks MHV-A59 infection, VLPs did not infect the target cells. Thus, S-mediated VLP infectivity resembles MHV-A59 infectivity. The system can be used to identify domains in S that are essential for infectivity. As a first application, we investigated the requirements of cleavage of S for the infectivity of MHV-A59. We inserted three mutant S proteins that were previously shown to be uncleaved (E. C. W. Bos, L. Heijnen, W. Luytjes, and W. J. M. Spaan, Virology 214:453-463, 1995) into the VLPs. Here we show that cleavage of the spike protein of MHV-A59 is not required for infectivity.

Expression of insulin receptors and of 60-kDa receptor substrate in rat mature and immature enterocytes.

The mechanism(s) by which rat immature enterocytes exhibit increased responsiveness to insulin before weaning is unknown. Therefore, we have analyzed the distribution, ontogeny, and molecular properties of insulin receptors (IR) and of related substrates in immature and mature enterocytes. IR were studied by radioligand binding assays, cross-linking labeling, immunohistochemistry, and in vitro phosphorylated substrates by immunoprecipitation. Regardless of age, 125I-insulin binding to IR was five times higher in crypt cells than in villus cells and two times higher in the ileum than in the jejunum. Binding capacity to villus cells from sucklings (day 14) exceeded three times that of older animals (day 30 and day 60). Scatchard analysis of equilibrium binding data confirmed an age-related decrease in low- and high-affinity receptor classes without change in affinity constants. In concordance, both alpha- and beta-IR subunits were more abundant in immature than in mature membranes. In vitro, insulin elicited the phosphorylation of three membrane proteins (96, 60 and 42 kDa), whose signals were virtually inhibited by preincubating membranes with antireceptor monoclonal antibodies. By immunoprecipitation, the 60-kDa signal was rapidly detected as a tyrosine-phosphorylated protein, expressed in mature and immature membranes, and identified as a receptor substrate phosphorylated in vitro by the IR tyrosine kinase. In conclusion, 1) increased responsiveness of rat immature enterocytes to insulin could be related to high membrane concentrations of IR and 2) normal rat enterocytes express a 60-kDa phosphotyrosine protein identified as a direct substrate of the IR tyrosine kinase.

Cell-surface perturbations of the epidermal growth factor and vascular endothelial growth factor receptors by phosphorothioate oligodeoxynucleotides.

Antisense oligodeoxynucleotides offer potential as therapeutic agents to inhibit gene expression. Recent evidence indicates that oligodeoxynucleotides designed to target specific nucleic acid sequences can interact nonspecifically with proteins. This report describes the interactive capabilities of phosphorothioate oligodeoxynucleotides of defined sequence and length with two essential protein tyrosine receptors, flk-1 and epidermal growth factor receptor (EGFR), and their effects on receptor signaling in a transfected and tumor cell line, respectively. Phosphorothioate oligodeoxynucleotides bound to the cell surface, as demonstrated by fluorescence-activated cell-sorter analyses (FACS), and perturbed receptor activation in the presence and absence of cognate ligands, EGF (EGFR) and vascular endothelial growth factor (flk-1), in phosphorylation assays. Certain phosphorothioate oligodeoxynucleotides interacted relatively selectively with flk-1 and partially blocked the binding of specific anti-receptor monoclonal antibodies to target sites. They stimulated EGFR phosphorylation in the absence of EGF but antagonized ligand-mediated activation of EGFR and flk-1. In vivo studies showed that a nonspecific phosphorothioate oligodeoxynucleotide suppressed the growth of glioblastoma in a mouse model of tumorigenesis. These results emphasize the capacity of phosphorothioate oligodeoxynucleotides to interact with cells in a sequence-selective nonantisense manner, while associating with cellular membrane proteins in ways that can inhibit cellular metabolic activities.

Urokinase and the Urokinase Receptor: Association With In Vitro Invasiveness of Human Bladder Cancer Cell Lines

The plasminogen activators urokinase and tissue plasminogen activator are enzymes that degrade proteins in tissue basement membranes and the extracellular matrix (a biomolecular complex surrounding individual cells in tissues that serves as a barrier between the cells and the vascular and lymph systems). The action of these enzymes allows tumor cells to escape their local environment and metastasize. Plasminogen activator activity can be influenced by the urokinase receptor, which is expressed on the surface of cells, and by the plasminogen activator inhibitors 1 and 2. Because bladder tumors differ in their propensity to invade local areas and distant sites, we studied the expression of both plasminogen activators, the two plasminogen activator inhibitors, and the urokinase receptor in four human bladder cancer cell lines (RT4, 253J, EJ, and T24) to see if there was an association between the expression of these proteins and tumor cell invasiveness in vitro. The expression of urokinase, tissue plasminogen activator, and the two inhibitors was measured by enzyme-linked immunosorbent assays of serum-free, cell-conditioned media (i.e., culture fluids). Cell surface expression of the urokinase receptor was assayed by flow cytometry, using an anti-receptor monoclonal antibody (Mab3936). The invasive capacity of untreated cells and of cells exposed to exogenous, high-molecular-weight urokinase was analyzed by use of Matrigel invasion chambers. The four bladder cancer cell lines demonstrated differential expression of both plasminogen activators and both inhibitors; three of the cell lines (T24, EJ, and 253J) expressed the urokinase receptor. The four cell lines differed in their invasive potential in vitro. Neither expression of tissue plasminogen activator nor production of the inhibitors appeared to influence Matrigel invasion. EJ cells and 253J cells produced the highest levels of urokinase and demonstrated the greatest propensity for invasion; T24 cells, which produced only small amounts of urokinase, exhibited a low invasive potential. Pretreatment of T24 cells with exogenous high-molecular-weight urokinase markedly increased their invasiveness. Similar pretreatment of EJ and 253J cells increased their invasiveness as well. RT4 cells, which lacked urokinase receptor expression but produced moderate amounts of urokinase, were not invasive and did not become so after exposure to exogenous high-molecular-weight urokinase. Binding of Mab3936 to urokinase receptors inhibited Matrigel invasion. To our knowledge, this is the first study demonstrating that bladder tumor cells express the urokinase receptor and that both receptor expression and urokinase expression are required for bladder tumor cell invasion in vitro.

Clinical coxsackievirus B isolates differ from laboratory strains in their interaction with two cell surface receptors.

Coxsackie B viruses interact with two putative cell surface receptor molecules. Experiments with prototype laboratory strains suggest that all 6 coxsackie B serotypes interact with a 46-kDa protein recognized by the monoclonal antibody RmcB, whereas CB1, CB3, and CB5 may also bind to decay accelerating factor. Antireceptor monoclonal antibodies were used to study interactions between low-passage clinical coxsackie B virus isolates and the two receptors. In contrast to observations made with single prototype strains, these data indicate that receptor use by clinical isolates is not strictly related to serotype and that even prototype strains with different passage histories may differ in receptor use. Within a given serotype, variation exists in the capacity of individual virus isolates to bind to specific receptors, and variants with altered receptor specificity may arise during infection in humans and in tissue culture.

Release of the Thrombin Receptor (PAR-1) N-terminus from the Surface of Human Platelets Activated by Thrombin

Platelet activation by thrombin is at least partially mediated by a G-protein-coupled receptor whose extended N-terminus is cleaved by thrombin. Theoretically, this should release a small fragment containing the original receptor N-terminus. However, the fate of this fragment is unknown, as is its biological role, if any. To begin to examine these issues, we have prepared monoclonal anti-receptor antibodies whose epitopes lie entirely N-terminal to the thrombin cleavage site. By flow cytometry and fluorescence microscopy of human platelets and megakaryoblastic CHRF-288 cells, these antibodies were found to recognize intact receptors and receptors activated by the agonist peptide, SFLLRN, but not receptors whose N-terminus had been cleaved by thrombin or cathepsin G. Incubating CHRF-288 cells with thrombin released pre-bound antibody from the cell surface. An assay based upon the antibodies was able to detect a fragment containing the original receptor N-terminus in the supernate of thrombin-treated human platelets. The concentration of the fragment obtained with platelets from 15 normal donors was 4.8 +/- 0.9 pmol per 10(9) platelets (mean +/- sem), which is similar to the value expected if all of the thrombin receptors present on human platelets have been cleaved. Taken together, these results demonstrate that 1) following receptor cleavage a fragment containing the original N-terminus of the receptor is released from the platelet surface, 2) based upon epitope mapping, this fragment is at least 15-20 residues long, 3) it is possible to quantitate the receptor fragment in the supernates of cells exposed to thrombin, and 4) the results of the quantitation suggest that on platelets all of the receptors have been cleaved and 100% of the fragment is present in the cell supernate. Depending on it survival time, measurements of the receptor fragment in blood or urine samples may eventually prove to be a useful marker for thrombin receptor activation in vivo.

Two primitive neuroectodermal tumor cell lines require an activated insulin-like growth factor I receptor for growth in vitro.

To determine the expression of the insulin-like growth factors (IGFs) and the IGF-I receptor in primitive neuroectodermal tumor cell lines and to assess the importance of these proteins in the growth of cell lines in vitro. Ribonucleic acid blotting and reverse transcriptase-polymerase chain reaction were used for detection of IGF and IGF-I expression. Ribonucleic acid blotting was used for detection of up-regulation of c-fos in the presence of exogenous growth factor. Immunoprecipitation was used to demonstrate autophosphorylation of the receptor in the presence of exogenous growth factor. Ligand binding analysis was used to determine the binding affinity of the receptor and the number of receptors per cell. Growth of curves in the presence of monoclonal antibody that blocks binding of ligand to receptor was measured to determine the requirement for an activated receptor during growth. Expression of IGF-II was identified in one cell line. No expression of IGF-I was seen in any cell line. Expression of IGF-I receptor was detected in all three cell lines. Immunoprecipitation experiments demonstrated autophosphorylation of the receptor after addition of IGF-I to growing cells. Ligand binding analysis revealed 9.2 x 10(4) and 4 x 10(4) receptors per cell in the Daoy and PFSK cell lines, respectively. Addition of either IGF alone or in combination to serum-starved cells was not able to restore growth of the cell lines. A blocking monoclonal antireceptor antibody decreased growth of Daoy and PFSK cells in a dose-dependent fashion. Complete arrest of growth occurred at 1 microgram/ml antibody in both cell lines. The IGF-I receptor is expressed by primitive neuroectodermal tumor cell lines in vitro. An activated receptor is important for cell proliferation in vitro. Additional work will establish the importance of these findings for tumors in vivo.

Epidermal Growth Factor Promotes a Cardiomyoblastic Phenotype in Human Fetal Cardiac Myocytes

Peptide growth factors likely play an important role in cardiac development, but growth factors which inhibit or prevent differentiation in cardiac myocytes are largely unknown. Using immunocytochemistry, Western and Northern blotting, and RNase protection assays, we demonstrate that epidermal growth factor (EGF) significantly inhibits differentiation and promotes proliferation in cultured human fetal ventricular cardiac myocyte cell lines. In enriched cell lines and in a pure myocyte cell strain, EGF inhibited increases in immunoreactive sarcomeric actin and sarcomeric myosin heavy chain (SMHC) normally seen after serum withdrawal. In the pure myocyte strain, EGF induced a cardiomyoblastic phenotype; i.e., it caused a complete loss of detectable sarcomeric proteins in the majority of cells; it was also mitogenic. EGF inhibited expression of cardiac α-actin and SMHC mRNAs, but inhibition of SMHC expression was predominantly of the β-MHC isoform. Removal of EGF was followed by reexpression of sarcomeric proteins. Blocking the EGF receptor (EGFR) with monoclonal anti-receptor antibody completely abolished the dedifferentiating effects of EGF and also significantly reduced the mitogenic effect of the peptide. The results indicate that activation of the EGFR both inhibits differentiation and promotes proliferation of human fetal ventricular myocytesin vitro.These findings suggest an important role for EGF in human cardiac differentiation and development.

Estrogen Receptor Immunocytochemistry in Endometrial Carcinoma: A Prognostic Marker for Survival

Only a few parameters such as tumor grade and stage are of value in prognosticating disease course in endometrial carcinoma. Biochemical steroid hormone receptor assays could also be useful but are difficult to perform and interpret. Immunocytochemical assay (ICA) might be the method of choice for detecting endometrial receptors. Methods: Frozen tissue from 78 cases of endometrial adenocarcinoma was examined for the presence of estrogen (ER) and progesterone receptors (PgR) with specific monoclonal anti-receptor antibodies and the peroxidase–antiperoxidase method. In over 60 cases, frozen tissue was also assayed for ER and PgR by biochemical means. Results: Fifty-five (71%) of the endometrial carcinomas were ERICA-positive and 55 (71%) PgRICA-positive. Although both ERICA and PgRICA correlated significantly with biochemical ER and PgR only ERICA was predictive of survival. A woman with a negative ERICA was 4 times more likely to die of her disease than if she were ERICA-positive (P= 0.009; mean follow-up, 37.5 months). Three cases ERICA-positive and PgRICA-negative survived while 3 others ERICA-negative and PgRICA-positive died. Conclusion: ERICA, a technique easy to perform and interpret at the community hospital level, appears to provide prognostic information independent of tumor stage and grade. Such information might be of value in planning postoperative therapies for women with endometrial cancer.

Potent human p140-TrkA agonists derived from an anti-receptor monoclonal antibody

Monoclonal antibody (mAb) 5C3 directed against human p140 TrkA is a structural and functional mimic of nerve growth factor (NGF) and an artificial receptor agonist. mAb 5C3 binds in the NGF-docking site and, like NGF, it promotes TrkA internalization, TrkA and phosphatidylinositol-3 kinase tyrosine phosphorylation, and increased transformation of TrkA-expressing fibroblasts. More important, mAb 5C3 protects human TrkA-expressing cells from apoptotic death in serum-free media. Interestingly, agonistic activity is observed with monomeric F(ab) 5C3 fragments. mAb 5C3 (Kd approximately 2 nM) was used to study features of ligand binding by TrkA and the distribution of TrkA protein in normal human brain.

Cytotoxicity of KDEL-terminated ricin toxins correlates with distribution of the KDEL receptor in the Golgi.

DNAs encoding ricin toxin A chain (RTA), with or without a C-terminal endoplasmic reticulum retention signal KDEL, were subcloned into pGEX2T bacterial expression plasmid. After transformation of JM105 E. coli cells and induction with isopropylthio-beta-galactoside (IPTG), fusion proteins were bound to an immobilized glutathione matrix and recombinant ricin A chains released with thrombin. Both recombinant wild-type RTA and RTA with KDEL had immunological reactivity and catalytic activity indistinguishable from plant RTA. The bacterial RTA products reassociated with plant ricin B chain (RTB) similarly to plant RTA. Cell cytotoxicities were measured on seven cell lines for each A-chain and heterodimer. Although KDEL sequences enhanced cytotoxicity in most cases, significant variability was observed. In each case, addition of KDEL enhanced A-chain cytotoxicity more than holotoxin cytotoxicity. Three cell lines showed reduced KDEL enhancement of both RTA and ricin cytotoxicity. The concentration of KDEL receptor was examined on each cell line by immunofluorescence microscopy with an antireceptor monoclonal antibody. Differences in sensitivity to KDEL-containing toxins correlated with altered distribution of KDEL receptor between endoplasmic reticulum (ER) and Golgi compartments.