Manjusri Das

Binding of an antagonistic monoclonal antibody to an intact and fragmented EGF-receptor polypeptide☆

A murine monoclonal antibody (No. 425) raised against human A431 carcinoma cells specifically immunoprecipitates the 170,000 molecular weight epidermal growth factor (EGF)-receptor from extracts of A431 cells as well as from extracts of human placenta and cultured fibroblasts, but does not recognize the murine receptor. Binding to the external domain of the human EGF-receptor was indicated by indirect immunofluorescent staining of fixed nonpermeable cells. The antibody binds to both glyco- and aglycoreceptor forms, indicating that the epitope is a part of the polypeptide chain. Binding of the antibody to the receptor is conformation dependent; i.e., denatured receptors lacking EGF-binding activity are not recognized by the antibody. The results of antibody binding studies indicate that the epitope is closely linked to the EGF binding active site, and is common to both high- and low-affinity EGF-receptors. Interaction of this epitope with the antibody inhibits EGF binding and bioactivity, and triggers receptor down-regulation, but does not generate EGFlike kinase-stimulatory or mitogenic responses either in vitro or in vivo. The antibody was tested for its ability to bind to domain-sized fragments of the 170-kDa EGF-receptor. It can recognize both the proteolytically generated 110-kDa EGF binding peptide, and a soluble 100-kDa EGF-receptor secreted by A431 cells. This indicates that the epitope recognized this antibody retains its conformation after proteolytic separation of the EGF binding domain from the rest of the receptor molecule.

Epidermal growth factor: mechanisms of action.

Publisher Summary Epidermal growth factor (EGF), a single chain polypeptide, belongs to a new class of cytomodulatory factors that are hormone-like in their biological potencies. EGF-like polypeptides may be produced during tumorigenesis by the reactivation of genes that are expressed only during development of the embryo. The EGF-receptor system has been an important stimulus to the development of ideas on the mechanism of receptor-mediated ligand endocytosis. The EGF receptor is a multifunctional, multidomain protein, whose inherent kinase function is activated after EGF interacts with its external binding site. EGF stimulates DNA synthesis/cell division in a variety of cell types. The EGF-receptor system has been an important stimulus to the development of new ideas on receptor action and mitogenesis. In addition, it has provided some new insights into the vital processes that occur during embryogenesis and development, and during tumorigenic transformation in animals.

Effect of methylamine on internalization, processing and biological activation of epidermal growth factor receptor

Epidermal growth factor (EGF), a mitogenic polypeptide of Mr 6045 interacts with responsive cells through high affinity surface receptors [ 11. The membrane receptor for EGF is a polypeptide of &fr -184 000 [2,3], and interaction of EGF with this surface receptor has been shown to lead to the intracellular generation of an activator of DNA replication [4]. As with many other polypeptide ligands, binding of EGF to the surface receptor has been shown to be followed by endocytic uptake of the hormonereceptor complex leading to subsequent proteolytic degradation of both EGF and receptor within lysosomes [3,5,6]. Our earlier studies had indicated that the phenomenon of EGF-induced receptor internalization may play an essential role in the induction of the message necessary for the mitogenic response [3,4]. We are therefore interested in using various inhibitory compounds which block one or more of the steps in the cascade of cellular reactions that follow hormone-receptor binding, and in studying their effect on the induction of the mitogenic message. There have been reports on the inhibitory effects of various amines on the biological fate of hormones bound to target cells [5,7,8]. Here we report the effect of methylamine (MA) on the interaction of EGF with murine 3T3 cells. We find that the crucial event in hormone-cell interaction which is blocked by MA is not the initial endocytic event, but rather the later intracellular proteolytic processing of hormone and receptor. We also report some of the effects of MA on EGF-induced stimulatory responses,

Insulin degradation in 3T3-L1 adipocytes: role of the endocytic lysosomal pathway.

Abstract Insulin bound to 3T3-L1 adipocytes at 12 °C rapidly becomes processed to higher and lower molecular weight components at 37 °C. A part of this insulin processing (degradation) appears to have no role in the expression of its biological effects on hexose and amino acid transport. Degradation is strongly (~70%) inhibited by bacitracin, and very weakly inhibited (~5%) by methylamine, monodansylcadavarine, and bromophenacylbromide. All the other compounds tested for inhibition—azide, dinitrophenol (inhibitors of energy-dependent endocytosis), chloroquine (a lysosomotropic agent), chlorpromazine, phenylglyoxal (reported inhibitors of macromolecular internalization)—inhibited degradation partially to about the same extent (~20%), suggesting that the endocytic lysosomal pathway accounts for only a fifth of insulin degradation in 3T3-L1 adipocytes.

Aberrant energy metabolism in a variant epidermal growth factor receptor-negative fibroblastic cell line.

A variant 3T3 cell line has been isolated [l] lacking mitogenic response to epidermal growth factor [2,3]. This non-responder variant (NR-6) lacked epidermal growth factor (EGF) receptors, but retained the ability to respond to other mitogens, and displayed growth control in culture [ 11. NR-6 cells have been used as specificity controls in identification of the EGF receptor [4-61 on 3T3 cells and for studies on insertion of exogenous hormone receptors into receptornegative mutant cells [7]. We were interested in further character~ing this variant cell line, and examined whether the lack of EGF receptors is accompanied by other losses as well. Such studies may provide new insights into receptor genetics and the regulatory mechanisms involved in receptor biosynthesis. Here we report our preliminary findings on the energy metabo~sm characteristics of NR-6 cells. We find that compared with the parent 3T3 cells, the variant NR-6 cells are extremely deficient in cytochrome c oxidase, a key enzyme in the oxidative phosphorylation pathway, but this deficiency is compensated for by an exceptionally high rate of aerobic glycolysis. This aberrant energy metabolism does not appear to be associated with any transformed growth characteristics.

EGF stimulates the processing and export of a secreted form of EGF receptor

Human A431 cells express a 100 kDa EGF‐receptor homolog that contains only the external domain. The kinetics of its maturation and export are slow and comparable to those of the transmembrane receptor. Here we demonstrate that exogenously added EGF stimulates post‐translational processing and export of this receptor through a pathway that is insensitive to inhibitors of protein synthesis. The results suggest that EGF may influence one or more of the rate determining steps that control receptor export from endoplasmic reticulum. This may constitute one of the path‐ways by which EGP regulates the expression of its receptor.

A hepatic membrane-associated factor stimulates nuclear DNA synthesis in cultured fibroblastic cells

Abstract Nuclear DNA replication in cultured mouse fibroblasts is stimulated by isolated hepatic plasma membranes in a time- and concentration-dependent manner. The plasmalemmal activity is susceptible to trypsin treatment, and to treatment with protein modifying agents, N-ethylmaleimide, N-bromosuccinimide, and 2-hydroxy-5-nitro-benzylbromide.

Cell- and tissue-specific expression of a 34,000-molecular-weight peptide growth factor in humans.

A 34,000-dalton peptide growth factor that we originally identified in human placental trophoblasts and in certain carcinomas was shown to be expressed in several normal human tissues. A highly specific antibody to the trophoblast-derived growth factor was used in an immunoperoxidase staining technique to identify the immunoreactive peptide in tissue sections. Immunoreactivity was seen in the adrenal cortex, Leydig cells of the testes, and follicular cells of the thyroid. In addition, strong staining was seen in the ducts and terminal ductules of the pancreas, in glandular epithelium of the prostate, in the chief cells of the stomach, and in the columnar epithelium of the trachea and bronchus of the lung. Certain tissues were negative for the peptide, including the adrenal medulla, liver, esophagus, small intestine, colon, bladder, lymph node, spleen, bone marrow, and thymus. Thus, the expression of the peptide depends on cell lineage and the state of differentiation; tissues of hematopoietic lineage are devoid of the 34,000-dalton peptide, whereas some of the major hormone-secreting tissues that are under pituitary control show the highest immunoreactivity.

[49] Spontaneous transfer of exogenous epidermal growth factor receptors into receptor-negative mutant cells

Publisher Summary This chapter focuses on the spontaneous transfer of exogenous epidermal growth factor (EGF) receptors into receptor-negative mutant cells. Receptor-EGF interaction produces a variety of rapid events, including activation of a receptor-associated protein and receptor clustering and endocytosis. One or more of these events leads to the intracellular generation of macromolecular activator(s) of DNA replication. The mechanism of receptor action is examined with a variant cell line NR-6 derived from mouse 3T3 that can neither bind nor biologically respond to EGF. Development of a procedure for receptor transfer is an important step toward elucidation of the mechanism of receptor action and understanding the mode of integration of receptor proteins in the membrane. The chapter describes some of the properties of transfer process. EGF receptor is transferred from donor receptor-enriched membranes to recipient receptorless cells in the absence of added fusogenic agents. The cells are assayed for transferred receptor activity by 125 I-labeled EGF binding, or by measuring EGF-induced stimulation of [ 3 H]thymidine incorporation into DNA.

Abstract 531: Human γδ T cells limit breast tumor growth partly by down regulating cell survival-related molecules and up regulating apoptosis-related molecules in tumor cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: Innate immune system has been known to play an important role in inhibiting the malignant transformation, tumor progression and invasion. However, the mechanistic basis remains ambiguous. Despite polyclonality of human γδ T cells, Vγ2Vα2 T cell subset, major subset of peripheral γδ T cells, uniformly recognize non-peptide antigens and provide the first line of defense towards many microbial infections. This γδ T cell subset was also shown to recognize and limit the growth of the various tumors such as myeloma, lymphoma, pancreatic tumors and ovarian tumors. The differential recognition of the tumor cells by the Vγ2Vα2 T cells depends on the expression levels of certain surface molecules on the tumor cells. Methods: To determine innate immune response of γδ T cells towards breast tumor cells, human peripheral γδ T cells were isolated and expanded in vitro using aminobisphosphonate, risedronate and co-cultured with breast tumor cells to assess tumor cell viability and proliferation using MTT assays and to determine the alteration in molecular levels in tumor cells using western blots. Cell morphology was recorded under microscope and flowcytometric analysis was performed to evaluate tumor surface molecules. Xenograft of the human breast tumor cells and human γδ T cells were performed in immunocompromised NOD/SCID mice. Immunohistochemistry was performed to evaluate status of the tumor tissue morphology, angiogenesis and apoptosis. Western blot was also performed to evaluate key signaling molecules. Results: Breast cancer cell death was associated with the surface expression levels of MICA/B and ICAM-1. Molecular signaling analysis demonstrated that cell survivability or immune escape from γδ T cells was associated with the higher expression levels of cell survival-related molecules such as Akt, Erk1/2 and Bcl2 and concomitant down regulation of apoptosis-related molecules, PARP and cleaved Caspase 3, and down regulation of tumor suppressor genes, PTEN and P53. However, opposite molecular signaling was observed in susceptible cell lines after co-culture with γδ T cells. In vivo, anti-neoplastic effects of γδ T cells were also documented, where tumor growth was inhibited due to the disruption of microvasculature, induction of tumor suppressor genes p53, downregulation of survival signals, and a strong induction of apoptotic molecules. Conclusions: Our current study reveals that γδ T cells limit the in vitro growth of breast cancer cell lines as SKBR7 (HER2+), T47D (ER+) and MDA-MB-231 (ER-) by inhibiting their survival and inducing apoptosis, except BR-CA-MZ01 (PR+) cells. In vivo xenograft also confirms the anti-neoplastic effect of γδ T cells towards breast tumor cells. These findings reveal that complex molecular signaling mechanisms are involved in γδ T cell-mediated anti-neoplastic effects other than cytotoxicity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 531. doi:1538-7445.AM2012-531