Search PubMed⌕ Search

Biomedical subjects

Y Yarden

Publications and source records attributed to Y Yarden.

At least 73 records · Page 4Linked to original sources
PubMed1996

Enhanced expression of heregulin in c-erb B-2 and c-Ha-ras transformed mouse and human mammary epithelial cells.

Heregulin beta 1 was found to stimulate the anchorage-dependent, serum-free growth of nontransformed human MCF-10A mammary epithelial cells. Unlike epidermal growth factor, transforming growth factor alpha, or amphiregulin, heregulin beta 1 was also able to induce the anchorage-independent growth of MCF-10A cells. In contrast, the anchorage-independent, serum-free growth of c-Ha-ras or c-erb B-2 transformed MCF-10A cells was unaffected by heregulin beta 1, whereas heregulin beta 1 was able to stimulate the anchorage-independent growth of these cells. c-Ha-ras or c-erb B-2 (c-neu) transformed MCF-10A or mouse NOG-8 mammary epithelial cells express elevated levels of 2.5, 5.0, 6.5, 6.8, and 8.5 kb heregulin mRNA transcripts and/or synthesize cell-associated 25, 29, 50, and 115 kDa isoforms of heregulin. Since the MCF-10A cells and transformants also express c-erb B-3, these data suggest that endogenous heregulin might function as an autocrine growth factor for Ha-ras or erb B-2 transformed mammary epithelial cells.

Animals↗
PubMed1996

Coupling of the c-Cbl protooncogene product to ErbB-1/EGF-receptor but not to other ErbB proteins.

The ErbB family of transmembrane tyrosine kinases includes the receptor for EGF (ErbB-1), two receptors for NDF/heregulin (ErbB-3 and ErbB-4) and an orphan receptor (ErbB-2). In order to examine the possibility that distinct signal transduction pathways are coupled to each ErbB protein, we examined the interaction of individual ligand-stimulated receptors with the c-Cbl protein, a protooncogene-encoded signaling molecule previously identified in hematopoietic cells. We report that c-Cbl undergoes rapid and sustained phosphorylation on tyrosine residues upon stimulation of fibroblast and epithelial cell lines with ligands of ErbB-1. By contrast, activation of either ErbB-3 or ErbB-4 by NDF did not affect tyrosine phosphorylation of c-Cbl. Likewise, activation of a chimeric ligand-stimulatable ErbB-2 by a heterologous ligand was ineffective. Despite rapidity of the EGF effect, we observed no association of c-Cbl with activated ErbB-1, implying that the interaction is indirect. Our in vitro experiments suggest that a candidate mediator of the interaction is the Grb-2/Ash adaptor protein, which is constitutively bound to c-Cbl. These results indicate that different ErbB proteins can couple to distinct signaling pathways, and therefore their physiological functions are probably non-redundant.

Animals↗
PubMed1996

Granulocyte-macrophage colony-stimulating factor stimulates JAK2 signaling pathway and rapidly activates p93fes, STAT1 p91, and STAT3 p92 in polymorphonuclear leukocytes.

Granulocyte-macrophage colony-stimulating factor (GM-CSF), supports proliferation, differentiation, and functional activation of hemopoietic cells by its interaction with a heterodimeric receptor. Although GM-CSF receptor is devoid of tyrosine kinase enzymatic activity, GM-CSF-induced peripheral blood polymorphonuclear leukocytes (PMN) functional activation is mediated by the phosphorylation of a large number of intracellular signaling molecules. We have previously shown that JAK2 becomes tyrosine-phosphorylated in response to GM-CSF in PMN. In the present study we demonstrate that also the signal transducers and activators of transcription (STAT) family members STAT1 p91 and STAT3 p92 and the product of the c-fps/fes protooncogene become tyrosine-phosphorylated upon GM-CSF stimulation and physically associated with both GM-CSF receptor beta common subunit and JAK2. Moreover GM-CSF was able to induce JAK2 and p93fes catalytic activity. We also demonstrate that the association of the GM-CSF receptor beta common subunit with JAK2 is ligand-dependent. Finally we demonstrate that GM-CSF induces a DNA-binding complex that contains both p91 and p92. These results identify a new signal transduction pathway activated by GM-CSF and provide a mechanism for rapid activation of gene expression in GM-CSF-stimulated PMN.

Amino Acid Sequence↗
PubMed1996

ErbB-2 is a common auxiliary subunit of NDF and EGF receptors: implications for breast cancer.

Overexpression of the erbB-2 gene contributes to aggressive behavior of various human adenocarcinomas, including breast cancer, through an unknown molecular mechanism. The erbB-2-encoded protein is a member of the ErbB family of growth factor receptors, but no direct ligand of ErbB-2 has been reported. We show that in various cells ErbB-2 can form heterodimers with both EGF receptor (ErbB-1) and NDF receptors (ErbB-3 and ErbB-4), suggesting that it may affect the action of heterologous ligands without the involvement of a direct ErbB-2 ligand. This possibility was addressed in breast cancer cells through either overexpression of ErbB-2 or by blocking its delivery to the cell surface by means of an endoplasmic reticulum-trapped antibody. We report that ErbB-2 overexpression enhanced binding affinities to both EGF and NDF, through deceleration of ligand dissociation rates. Likewise, removal of ErbB-2 from the cell surface almost completely abolished ligand binding by accelerating dissociation of both growth factors. The kinetic effects resulted in enhancement and prolongation of the stimulation of two major cytoplasmic signaling pathways, namely: MAP kinase (ERK) and c-Jun kinase (SAPK), by either ligand. Our results imply that ErbB-2 is a pan-ErbB subunit of the high affinity heterodimeric receptors for NDF and EGF. Therefore, the oncogenic action of ErbB-2 in human cancers may be due to its ability to potentiate in trans growth factor signaling.

Adenocarcinoma↗
PubMed1996

Targeting of stealth liposomes to erbB-2 (Her/2) receptor: in vitro and in vivo studies.

Long-circulating (stealth) liposomes coated with polyethylene glycol (PEG), which show reduced uptake by the reticuloendothelial system (RES) and enhanced accumulation in tumours, were used for conjugation to monoclonal antibodies (MAbs) as a drug-targeting device. A MAb (N-12A5) directed against erbB-2 oncoprotein, a functional surface antigen, was used. Amplification and overexpression of the erbB-2 gene product, being unique to malignancy, confer onto this antibody-mediated therapy high tumour specificity. In vitro binding of [3H]cholesteryl ether ([3H]Chol ether) labelled anti-erbB-2 conjugated liposomes to N-87 cells (erbB-2-positive human gastric carcinoma) was compared with the binding of non-targeted liposomes and indicated a 16-fold increase in binding for the targeted liposomes. No difference in binding to OV1063 cells (erbB-2-negative human ovary carcinoma) was observed. These results indicate highly selective binding of antibody-targeted liposomes to erbB-2-overexpressing cells. Despite increased cell binding, doxorubicin (DOX) loaded in anti-erbB-2-conjugated liposomes did not cause increased in vitro cytotoxicity against N-87 cells, suggesting lack of liposome internalisation. In vivo, the critical factor needed to decrease the non-specific RES uptake and prolong the circulation time of antibody-conjugated liposomes is a low protein to phospholipid ratio ( < 60 micrograms mumol-1). Using these optimised liposome preparations loaded with DOX and by monitoring the drug levels and the [3H]Chol ether label, biodistribution studies in nude mice bearing subcutaneous implants of N-87 tumours were carried out. No significant differences in liver and spleen uptake between antibody-conjugated and plain liposomes were observed. Nevertheless, there was no enhancement of tumour liposome levels over plain liposomes. Both liposome preparations considerably enhanced DOX concentration in the tumour compared with free drug administration. Therapeutic experiments with N-87 tumour-bearing nude mice indicated that anti-tumour activity of targeted and non-targeted liposomes was similar, although both preparations had an increased therapeutic efficacy compared with the free drug. These studies suggest that efficacy is dependent on drug delivery to the tumour and that the rate-limiting factor of liposome accumulation in tumours is the liposome extravasation process, irrespective of liposome affinity or targeting to tumour cells.

Animals↗
PubMed1996

A hierarchical network of interreceptor interactions determines signal transduction by Neu differentiation factor/neuregulin and epidermal growth factor.

The ErbB family includes four homologous transmembrane tyrosine kinases. Whereas ErbB-1 binds to the epidermal growth factor (EGF), both ErbB-3 and ErbB-4 bind to the Neu differentiation factors (NDFs, or neuregulins), and ErbB-2, the most oncogenic family member, is an orphan receptor whose function is still unknown. Because previous lines of evidence indicated the existence of interreceptor interactions, we used ectopic expression of individual ErbB proteins and their combinations to analyze the details of receptor cross talks. We show that 8 of 10 possible homo-and heterodimeric complexes of ErbB proteins can be hierarchically induced by ligand binding. Although ErbB-2 binds neither ligand, even in a heterodimeric receptor complex, it is the preferred heterodimer partner of the three other members, and it favors interaction with ErbB-3. Selective receptor overexpression in human tumor cells appears to bias the hierarchical relationships. The ordered network is reflected in receptor transphosphorylation, ErbB-2-mediated enhancement of ligand affinities, and remarkable potentiation of mitogenesis by a coexpressed ErbB-2. The observed superior ability of ErbB-2 to form heterodimers, in conjunction with its uniquely high basal tyrosine kinase activity, may explain why ErbB-2 overexpression is associated with poor prognosis.

Animals↗
PubMed1996

Type 1 receptor tyrosine kinases are differentially phosphorylated in mammary carcinoma and differentially associated with steroid receptors.

The neu/erbB-2/HER-2 proto-oncogene is amplified and/or overexpressed in up to 30% of mammary carcinomas and has been variably correlated with poor prognosis. The signaling activity of the encoded receptor tyrosine kinase is regulated by interactions with other type 1 receptors and their ligands. We have used a novel approach, phosphorylation-sensitive anti-Neu antibodies, to quantify signaling by Neu and epidermal growth factor receptor in a panel of frozen sections of mammary carcinoma specimens. We also determined the relationship of Neu, phosphorylated Neu (and epidermal growth factor receptor), and phosphotyrosine to the expression of Neu-related receptors (epidermal growth factor receptor, HER-3, and HER-4) and to prognostic factors (estrogen and progesterone receptor). We found that tyrosine phosphorylation of Neu (and hence signaling activity) is highly variable among mammary carcinomas. Neu and HER-4 were associated with divergent correlates, suggesting that they have profoundly different biological activities. These results have implications for etiology of mammary carcinoma for clinical evaluation of mammary carcinoma patients, and for development of Neu-targeted therapeutic strategies.

Biomarkers, Tumor↗
PubMed1995

Neu differentiation factor inhibits EGF binding. A model for trans-regulation within the ErbB family of receptor tyrosine kinases.

Neu differentiation factor (NDF, or heregulin) and epidermal growth factor (EGF) are structurally related proteins that bind to distinct members of the ErbB family of receptor tyrosine kinases. Here we show that NDF inhibits EGF binding in a cell type-specific manner. The inhibitory effect is distinct from previously characterized mechanisms that involve protein kinase C and receptor internalization because it occurred at 4 degrees C and displayed reversibility. The extent of inhibition correlated with both receptor saturation and affinity of different NDF isoforms, and it was abolished upon overexpression of either EGF receptor or ErbB-2. Binding kinetics and equilibrium analyses indicated that NDF reduced the affinity, rather than the number, of EGF receptors, through an acceleration of the rate of ligand dissociation and deceleration of the association rate. On the basis of co-immunoprecipitation of EGF and NDF receptors, we attribute the inhibitory effect to the formation of receptor heterodimers. According to this model, EGF binding to NDF-occupied heterodimers is partially blocked. This model of negative trans-regulation within the ErbB family is relevant to other subgroups of receptor tyrosine kinases and may have physiological implications.

Breast Neoplasms↗
PubMed1995

Suppression and promotion of tumor growth by monoclonal antibodies to ErbB-2 differentially correlate with cellular uptake.

Amplification and overexpression of the erbB-2/neu protooncogene are frequently associated with aggressive clinical course of certain human adenocarcinomas, and therefore the encoded surface glycoprotein is considered a candidate target for immunotherapy. We previously generated a series of anti-ErbB-2 monoclonal antibodies (mAbs) that either accelerate or inhibit the tumorigenic growth of erbB-2-transformed murine fibroblasts. The present study extended this observation to a human tumor cell line grown as xenografts in athymic mice and addressed the biochemical differences between the two classes of mAbs. We show that the inhibitory effect is dominant in an antibody mixture, and it depends on antibody bivalency. By using radiolabeled mAbs we found that all of three tumor-inhibitory mAbs became rapidly inaccessible to acid treatment when incubated with tumor cells. However, a tumor-stimulatory mAb remained accessible to extracellular treatments, indicating that it did not undergo endocytosis. In addition, intracellular fragments of the inhibitory mAbs, but not of the stimulatory mAb, were observed. Electron microscopy of colloidal gold-antibody conjugates confirmed the absence of endocytosis of the stimulatory mAb but detected endocytic vesicles containing an inhibitory mAb. We conclude that acceleration of cell growth by ErbB-2 correlates with cell surface localization, whereas inhibition of tumor growth is associated with an intrinsic ability of anti-ErbB-2 mAbs to induce endocytosis. These conclusions are relevant to the selection of optimal mAbs for immunotherapy and may have implications for the mechanism of cellular transformation by an overexpressed erbB-2 gene.

3T3 Cells↗
PubMed1995

ErbB-3 mediates differential mitogenic effects of NDF/heregulin isoforms on mouse keratinocytes.

The family of Neu differentiation factors (NDFs, or heregulins) includes a dozen secreted glycoproteins, whose receptor binding domain displays two variants, alpha and beta, and they bind to two receptor tyrosine kinases, ErbB-3 and ErbB-4. Certain isoforms were reported to induce growth-arrest and differentiation of mammary tumor cells, while other breast cancer cell lines responded mitogenically. The present study addressed the biologic effects of various NDF isoforms on normal EGF-dependent epithelial cells, Balb/MK keratinocytes, that can undergo either proliferation or differentiation. We found that beta isoforms of NDF induced a mitogenic effect, that was significantly smaller than the maximal response to EGF. By contrast with NDF-beta, NDF-alpha isoforms exerted almost no mitogenic effect, but they were sufficient to maintain keratinocytes in culture. Consistent with their higher mitogenic potency, NDF-beta isoforms bound to Balb/MK cells with higher affinity (Kd = 2.2 nM) than alpha isoforms, however both groups shared their receptor, that we identified as ErbB-3. No transcript of ErbB-4 was detectable in the keratinocytes, but these cells express multiple NDF mRNAs and also ErbB-2. We conclude that different isoforms of NDF induce distinct growth regulatory effects on cultured keratinocytes, through direct interaction with ErbB-3.

Animals↗
PubMed1995

Roles for a cytoplasmic tyrosine and tyrosine kinase activity in the interactions of Neu receptors with coated pits.

The neu proto-oncogene product, p185neu (HER2, c-ErbB-2), encodes a cell-surface tyrosine kinase receptor with high oncogenic potential, which correlates with increased tyrosine kinase activity and a rapid receptor internalization rate. To investigate the interactions and signal(s) leading to the endocytosis of Neu receptors, we employed lateral mobility and internalization studies. Fluorescence photobleaching recovery measurements revealed that activation of Neu receptors (induced by mutation or by agonistic antibodies) markedly reduced their mobile fractions. To elucidate the signals involved, other mutants, all carrying a constitutively dimerizing oncogenic mutation, were analyzed. A kinase-negative mutant and a mutant lacking all cytoplasmic tyrosine phosphorylation consensus sequences exhibited high mobile fractions, similar to nonactivated Neu. Retention of a single tyrosine autophosphorylation site (Tyr-1253) out of the five known such sites was sufficient to immobilize a large fraction of the receptor. For all mutants, internalization correlated with receptor immobilization and was blocked by treatments that interfere with coated pit structure, indicating that the immobilization is due to interactions with coated pits. This was supported by the coimmunoprecipitation of alpha-adaptin only with the constitutively activated Neu mutants. We conclude that activated Neu receptors become stably associated with coated pits via plasma membrane adaptor complexes (AP-2). Efficient Neu receptor endocytosis requires activation, a functional kinase domain, and at least one tyrosine autophosphorylation site.

3T3 Cells↗
PubMed1995

The fourth immunoglobulin domain of the stem cell factor receptor couples ligand binding to signal transduction.

Receptor dimerization is ubiquitous to the action of all receptor tyrosine kinases, and in the case of dimeric ligands, such as the stem cell factor (SCF), it was attributed to ligand bivalency. However, by using a dimerization-inhibitory monoclonal antibody to the SCF receptor, we confined a putative dimerization site to the nonstandard fourth immunoglobulin-like domain of the receptor. Deletion of this domain not only abolished ligand-induced dimerization and completely inhibited signal transduction, but also provided insights into the mechanism of the coupling of ligand binding to dimer formation. These results identify an intrinsic receptor dimerization site and suggest that similar sites may exist in other receptors.

Animals↗
PubMed1995

Activation of a transfected FGFR-1 receptor in Madin-Darby epithelial cells results in a reversible loss of epithelial properties.

Basic fibroblast growth factor (bFGF) is a potent mitogen for a wide variety of cell types derived from mesoderm and neuroectoderm. The activity of bFGF is mediated by several types of closely related receptors belonging to the tyrosine-kinase family of receptors. We have found that Madin-Darby epithelial cells (MDCK) do not seem to produce bFGF or bFGF receptors. High level expression of human bFGF cDNA in these cells did not produce any mitogenic or morphological effects. Expression of the mouse-derived cDNA encoding FGF receptor-1 (FGFR-1) in MDCK cells resulted in the acquisition of a fibroblast-like morphology when the transfected cells were cultured at low density in the presence of 0.6% fetal calf serum and 20 ng/ml bFGF. Acidic fibroblast growth factor (aFGF) also induced these morphological changes but not keratinocyte growth factor. The morphological effect was not accompanied by increased bFGF-induced cell proliferation and did not result in the loss of epithelial cell markers such as cytokeratins. However, the morphological transition was accompanied by changes in the intracellular distribution of actin. In spite of these changes the transfected cells formed monolayers even in the presence of bFGF. Coexpression of bFGF and FGFR-1 in the MDCK cells resulted in similar morphological effects that were not dependent upon exogenous bFGF. These morphological effects were mimicked by exposure of MDCK cells to either orthovanadate or phorbol ester. Parental and FGFR-1-expressing MDCK cells formed monolayers that displayed high electrical resistance. Incubation of monolayers of FGFR-1-transfected cells with bFGF resulted in the loss of trans-epithelial resistance. Monolayers of parental MDCK cells did not lose their trans-epithelial resistance in response to bFGF, although exposure to phorbol ester did result in the loss of their trans-epithelial resistance, indicating that the effects on the trans-epithelial resistance are mediated by protein kinase C activation. Interestingly, orthovanadate did not cause a loss of transepithelial resistance, suggesting that the loss of trans-epithelial resistance is separable from the morphological transition.

Actins↗
PubMed1995

Neu differentiation factor is a neuron-glia signal and regulates survival, proliferation, and maturation of rat Schwann cell precursors.

We show that beta forms of Neu differentiation factor (NDF), homologous to acetylcholine receptor-inducing activity, glial growth factor, and heregulin, prevent apoptotic death and stimulate DNA synthesis of the E14 Schwann cell precursor, an early cell in the rat Schwann cell lineage. When precursors are exposed to NDF in defined medium, they generate Schwann cells without the requirement for DNA synthesis and with a time course that is similar to that with which Schwann cells appear in embryonic nerves in vivo. Furthermore, a neuronal signal that also mediates precursor survival and maturation is blocked by the extracellular domain of the ErbB4 NDF receptor, a protein that specifically blocks the action of NDFs. These observations provide important evidence that NDF is one of the hitherto elusive neuron-glia signaling molecules long proposed to regulate development in the Schwann cell lineage.

Animals↗
PubMed1995

Neu differentiation factor activation of ErbB-3 and ErbB-4 is cell specific and displays a differential requirement for ErbB-2.

Neu differentiation factor (NDF)-induced signaling involves the activation of members of the ErbB family of receptor tyrosine kinases. Although ectopic expression of recombinant ErbB receptors has yielded valuable insight into their signaling properties, the biological function and in vivo interplay of these receptors are still poorly understood. We addressed this issue by studying NDF signaling in various human cell lines expressing moderate levels of all known ErbB receptors. NDF-induced phosphorylation of ErbB-2 and ErbB-3 was found in the breast epithelial cell line MCF10A, the breast tumor cell lines T47D and MCF7, and the ovarian tumor cell line OVCAR3. Despite similar expression levels, NDF-induced phosphorylation of ErbB-4 was cell specific and only detected in T47D and OVCAR3 cells. Blocking cell surface expression of ErbB-2 by intracellular expression of a single-chain antibody revealed that in these two cell lines, ErbB-2 significantly enhanced phosphorylation of ErbB-4. Efficient NDF-induced phosphorylation of ErbB-3 was strictly ErbB-2 dependent in the breast tumor cell lines T47D and MCF7, while it was largely ErbB-2 independent in MCF10A and OVCAR3 cells. Consequently, NDF-stimulated intracellular signaling and induction of a biological response displayed a cell-specific requirement for ErbB-2. Thus, while ErbB-2 cooperates with NDF receptors in the breast tumor cell lines, ErbB-2 independent mechanisms seem to prevail in other cellular contexts.

Breast↗
PubMed1995

Neu differentiation factor/heregulin modulates growth and differentiation of HC11 mammary epithelial cells.

The HC11 mouse mammary epithelial cell line has proven to be a valuable in vitro model to study the roles of peptide factors and hormones involved in the growth and differentiation of mammary cells. Treatment of HC11 cells with the lactogenic hormones, dexamethasone, insulin, and PRL (DIP), leads to cellular differentiation and production of the milk protein beta-casein. We have analyzed the effects of Neu differentiation factor (NDF)/heregulin, a newly described activating ligand for erbB-2 and other members of the epidermal growth factor (EGF) receptor family, on cell growth and the expression of milk proteins in HC11 cells. In these cells, NDF induces tyrosine phosphorylation of erbB-2 and erbB-3. Both NDF and EGF stimulate HC11 cell proliferation and promote the responsiveness of HC11 cells to lactogenic hormones. NDF induces the expression of a 22-kilodalton milk protein. This protein is up-regulated by other factors, including dexamethasone, EGF, and basic fibroblast growth factor, and is controlled in a manner distinct from that of beta-casein. Like EGF, NDF inhibits the DIP-induced expression of beta-casein at the level of transcription. The inhibition is due to the negative effect of NDF on the activation of mammary gland factor (MGF/Stat5), a member of the Stat family of transcription factors, which is essential for beta-casein gene expression.

Animals↗