The normal erbB-2 product is an atypical receptor-like tyrosine kinase with constitutive activity in the absence of ligand

Overexpression of the erbB-2/neu gene is frequently detected in human cancers. When overexpressed in NIH 3T3 cells, the normal erbB-2 product, gp185erbB-2, displays potent transforming ability as well as constitutively elevated levels of tyrosine kinase activity in the absence of exogenously added ligand. To investigate the basis for its chronic activation we sought evidence of a ligand for gp185erbB-2 either in serum or produced by NIH 3T3 cells in an autocrine manner. We demonstrate that a putative ligand for gp185erbB-2 is not contained in serum. Chimeric molecules composed of the extracellular domain of gp185erbB-2 and the intracellular portion of the epidermal growth factor receptor (EGFR) did not show any transforming ability or constitutive autophosphorylation when they were expressed in NIH 3T3 cells. However, they were able to transduce a mitogenic signal when triggered by a monoclonal antibody directed against the extracellular domain of erbB-2. These results provide evidence against the idea that an erbB-2 ligand is produced by NIH 3T3 cells. Furthermore, we obtained direct evidence of the constitutive enzymative activity of gp185erbB-2 by demonstrating that the erbB-2 kinase remained active in a chimeric configuration with the extracellular domain of the EGFR, in the absence of any detectable ligand for the EGFR. Thus, under conditions of overexpression, the normal gp185erbB-2 is a constitutively active kinase able to transform NIH 3T3 cells in the absence of ligand.     

The carboxy-terminal domains of erbB-2 and epidermal growth factor receptor exert different regulatory effects on intrinsic receptor tyrosine kinase function and transforming activity.

The erbB-2 gene product, gp185erbB-2, displays a potent transforming effect when overexpressed in NIH 3T3 cells. In addition, it possesses constitutively high levels of tyrosine kinase activity in the absence of exogenously added ligand. In this study, we demonstrate that its carboxy-terminal domain exerts an enhancing effect on erbB-2 kinase and transforming activities. A premature termination mutant of the erbB-2 protein, lacking the entire carboxy-terminal domain (erbB-2 delta 1050), showed a 40-fold reduction in transforming ability and a lowered in vivo kinase activity for intracellular substrates. When the carboxy-terminal domain of erbB-2 was substituted for its analogous region in the epidermal growth factor receptor (EGFR) (EGFR/erbB-2COOH chimera), it conferred erbB-2-like properties to the EGFR, including transforming ability in the absence of epidermal growth factor, elevated constitutive autokinase activity in vivo and in vitro, and constitutive ability to phosphorylate phospholipase C-gamma. Conversely, a chimeric erbB-2 molecule bearing an EGFR carboxy-terminal domain (erbB-2/EGFRCOOH chimera) showed reduced transforming and kinase activity with respect to the wild-type erbB-2 and was only slightly more efficient than the erbB-2 delta 1050 mutant. Thus, we conclude that the carboxy-terminal domains of erbB-2 and EGFR exert different regulatory effects on receptor kinase function and biological activity. The up regulation of gp185erbB-2 enzymatic activity exerted by its carboxy-terminal domain can explain, at least in part, its constitutive level of kinase activity.     

A chimeric EGF-R-neu proto-oncogene allows EGF to regulate neu tyrosine kinase and cell transformation.

The neu oncogene, characterized by Weinberg and colleagues, is a transforming gene found in ethylnitrosourea-induced rat neuro/glioblastomas; its human proto-oncogene homologue has been termed erbB2 or HER2 because of its close homology with the epidermal growth factor receptor (EGF-R) gene (c-erbB1). Expression of the rat neu oncogene is sufficient for transformation of mouse NIH 3T3 fibroblasts in culture and for the development of mammary carcinomas in transgenic mice, but the neu proto-oncogene has not been associated with cell transformation. We constructed a vector for expression of a chimeric cDNA and hybrid protein consisting of the EGF-R extracellular, transmembrane and protein kinase C-substrate domains linked to the intracellular tyrosine kinase and carboxyl terminal domain of the rat neu cDNA. Upon transfection with the construct, NIH 3T3 cells gave rise to EGF-R antigen-positive cell clones with varying amounts of specific EGF binding. Immunofluorescence and immunoprecipitation using neu- and EGF-receptor specific antibodies demonstrated a correctly oriented and positioned chimeric EGF-R-neu protein of the expected apparent mol. wt on the surface of these cells. EGF or TGF alpha induced tyrosine phosphorylation of the chimeric receptor protein, stimulated DNA synthesis of EGF-R-neu expressing cells and led to a transformed cell morphology and growth in soft agar. In contrast, the neu proto-oncogene did not show kinase activity or transforming properties when expressed at similar levels in NIH 3T3 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Multiple independent activations of the neu oncogene by a point mutation altering the transmembrane domain of p185

The neu oncogene, which is frequently activated in neuro- and glioblastomas of BDIX rats, was originally identified in the NIH 3T3 focus-forming assay. cDNA clones of the normal and transforming alleles of neu have been isolated. When these clones are inserted into the expression vector pSV2, they direct the synthesis of p185, the neu gene product. The transforming cDNA clone yields foci when transfected onto a NIH 3T3 monolayer, but the normal cDNA does not. The construction of in vitro recombinants between the normal and transforming cDNAs has allowed the determination of the mutation responsible for the activation of the neu proto-oncogene. A single point mutation changes a valine in the transmembrane domain of the predicted protein product insert to a glutamic acid. The DNAs from four independent cell lines containing activated neu oncogenes contain the identical mutation at this position.     

The role of autophosphorylation in modulation of erbB-2 transforming function

The product of the erbB-2 gene is a 185-kD receptor-like glycoprotein. erbB-2 gp185 displays constitutive tyrosine kinase activity and transforms NIH 3T3 cells when expressed 100-fold over the normal levels. We have analyzed the role of tyrosine kinase function and of receptor autophosphorylation in the regulation of erbB-2 biological activity. Abolition of erbB-2 gp185 tyrosine kinase function resulted in complete loss of its transforming activity and the absence of in vivo tyrosine phosphorylation. The steady-state content of phosphotyrosine in erbB-2 gp185 was found to be solely dependent on receptor autophosphorylation and to be dependent on the specific enzymatic activity of the erbB-2 protein. The major sites of erbB-2 autophosphorylation were shown to be in its COOH-terminal domain. Biological analysis of erbB-2 mutants containing either individual or multiple Tyr----Phe substitutions at the potential sites of autophosphorylation revealed that autophosphorylation upregulates erbB-2 gp185 transforming activity. Autophosphorylation did not modulate receptor turnover. A Tyr----Phe substitution of erbB-2 Tyr-877 homologous to pp60c-src Tyr-416 did not alter erbB-2 biological and biochemical properties, thus excluding the possibility that phosphorylation of this residue, located in the kinase domain, modulates erbB-2 gp185 catalytic function. Hence, autophosphorylation of tyrosine residues localized in its COOH terminus appears to be required for optimal coupling of erbB-2 gp185 with its mitogenic pathway.     

The erbB-2 mitogenic signaling pathway: tyrosine phosphorylation of phospholipase C-gamma and GTPase-activating protein does not correlate with erbB-2 mitogenic potency.

The erbB-2 gene product, gp185erbB-2, unlike the structurally related epidermal growth factor (EGF) receptor (EGFR), exhibits constitutive kinase and transforming activity. We used a chimeric EGFR/erbB-2 expression vector to compare the mitogenic signaling pathway of the erbB-2 kinase with that of the EGFR, at similar levels of expression, in response to EGF stimulation. The EGFR/erbB-2 chimera was significantly more active in inducing DNA synthesis than the EGFR when either was expressed in NIH 3T3 cells. Analysis of biochemical pathways implicated in signal transduction by growth factor receptors indicated that both phospholipase C type gamma (PLC-gamma) and the p21ras GTPase-activating protein (GAP) are substrates for the erbB-2 kinase in NIH 3T3 fibroblasts. However, under conditions in which activation of the erbB-2 kinase induced DNA synthesis at least fivefold more efficiently than the EGFR, the levels of erbB-2- or EGFR-induced tyrosine phosphorylation of PLC-gamma and GAP were comparable. In addition, the stoichiometry of tyrosine phosphorylation of these putative substrates by erbB-2 appeared to be at least an order of magnitude lower than that induced by platelet-derived growth factor receptors at comparable levels of mitogenic potency. Thus, our results indicate that differences in tyrosine phosphorylation of PLC-gamma and GAP do not account for the differences in mitogenic activity of the erbB-2 kinase compared with either the EGFR or platelet-derived growth factor receptor in NIH 3T3 fibroblasts.     

Differential processing and turnover of the oncogenically activated neu/erb B2 gene product and its normal cellular counterpart

In nontransformed DHFR/G-8 cells (NIH 3T3 cells transfected with normal rat neu gene), the normal neu gene product was initially synthesized as a 170-kDa protein bearing endoglycosidase H-sensitive oligosaccharide chains and was then processed to a 175-kDa mature form with endoglycosidase H-resistant, endoglycosidase F-sensitive oligosaccharide chains. Most of this 175-kDa mature form appeared on the cell surface 2 h following synthesis and showed a half-life of approximately 3 h. In the presence of a growth factor(s) partially purified from bovine kidney, the half-life of this 175-kDa normal neu gene product was shortened to less than 30 min. In B104-1-1 cells (NIH 3T3 cells transfected with neu gene activated oncogenically by a point mutation that changes a valine residue to a glutamic acid residue in the putative transmembrane region), the oncogenically activated neu gene product was also synthesized as a 170-kDa precursor with endoglycosidase H-sensitive oligosaccharide chains. However, this 170-kDa precursor diminished very fast and was only partially processed to a 185-kDa mature form which exhibited a half-life of less than 30 min. The 185-kDa activated neu gene product possessed an unidentified post-translational modification in addition to N-linked oligosaccharide chains. Both the precursor and mature forms of the mutationally activated neu gene product showed increased tyrosine-specific phosphorylation as compared with those of their normal counterparts in DHFR/G-8 cells. The mutationally activated neu gene product in B104-1-1 cells shared several features which have been reported previously for the ligand-activated platelet-derived growth factor receptor in v-sis- or c-sis-transformed cells. These properties include: 1) accelerated turnover of the precursor and mature forms compared with the rates of turnover of its normal counterparts, 2) insensitivity of this rapid turnover to lysosomotropic amines, and 3) increased in vivo tyrosine-specific phosphorylation of both the precursor and mature forms. These findings suggest that the mutationally activated neu gene product may transform the cells by mimicking ligand-induced activation.     

Synergistic interaction of p185c-neu and the EGF receptor leads to transformation of rodent fibroblasts

The protein product of the rodent neu oncogene, p185neu, is a tyrosine kinase with structural similarity to the epidermal growth factor receptor (EGFR). Transfection and subsequent overexpression of the human p185c-erbB-2 protein transforms NIH 3T3 cells in vitro. However, NIH 3T3 cells are not transformed by overexpressed rodent p185c-neu. NIH 3T3 transfectants overexpressing EGF receptors are not transformed unless incompletely transformed. Several groups have recently demonstrated EGF-induced, EGFR-mediated phosphorylation of p185c-neu. During efforts to characterize the interaction of p185c-neu with EGFR further, we created cell lines that simultaneously overexpress both p185c-neu and EGFR and observed that these cells become transformed. These observations demonstrate that two distinct, overexpressed tyrosine kinases can act synergistically to transform NIH 3T3 cells, thus identifying a novel mechanism that can lead to transformation.     

Cloned hst gene from normal human leukocyte DNA transforms NIH3T3 cells

The hst gene was originally identified as a transforming gene in DNAs from stomach cancers and a noncancerous portion of stomach mucosa by transfection assays using NIH3T3 cells (1,2). Subsequently, the hst gene obtained directly from leukocyte DNA of a leukemia patient was sequenced (3,4). Here, cosmid clones containing the hst gene were isolated directly from normal human leukocyte DNA and from T361-2nd-1 cells, a secondary transformant of NIH3T3 cells induced by transfection of DNA from a stomach cancer. All clones containing the hst gene from these different sources transformed NIH3T3 cells with similar efficiency. Restriction map of the hst gene from normal leukocyte DNA was identical with that from leukocyte DNA of a leukemia patient, while the hst gene from T361-2nd-1 cells was rearranged at the 168th nucleotide upstream of the TATA box.     

PC3 overexpression affects the pattern of cell division of rat cortical precursors

The PC3 gene is transiently expressed during neurogenesis in precursor cells of the telencephalic ventricular/subventricular zone, and is rapidly downregulated before cell migration and differentiation. It is thought to have a role in controlling cell proliferation, but its precise function is not known. Here we present evidence that PC3, when overexpressed in vitro by retroviral-mediated gene transfer, acts by interfering with the normal pattern of cell division. Firstly, we report evidence that PC3 overexpression reduces the rate of cell proliferation in both NIH 3T3 cells and embryonic precursor cells from the rat cerebral cortex. Secondly, when studying the pattern of BrdU dilution in clones of cortical precursors, we observe that clones transduced with PC3 show an asymmetric pattern of BrdU dilution more frequently than clones transduced with a control vector. We discuss the hypothesis that the higher number of PC3 transduced clones showing an asymmetric pattern of BrdU dilution may be due to an increase in asymmetric cell divisions.     

Down-modulation of an oncogene protein product and reversion of the transformed phenotype by monoclonal antibodies

Exposure of neu-oncogene-transformed NIH 3T3 cells to monoclonal antibodies reactive with the neu gene product, p185, results in the rapid and reversible loss of both cell-surface and total cellular p185. Although not directly cytotoxic, monoclonal anti-p185 antibody treatment causes neu-transformed NIH 3T3 cells to revert to a nontransformed phenotype, as determined by anchorage-independent growth. Isotype matched control antibodies of an unrelated specificity do not affect p185 levels or colony formation in soft agar by neu-transformed NIH 3T3 cells. Soft agar colony formation by NIH 3T3 cells transformed by ras oncogenes is not affected by anti-p185 antibody treatment. Anchorage-independent growth of cells from the ethylnitrosourea-induced rat neuroblastoma line in which neu was originally detected by DNA transfection is also inhibited in the presence of anti-p185 monoclonal antibodies. Collectively, these results suggest that p185 is required to maintain transformation induced by the neu oncogene.     

Overexpression of the human EGF receptor confers an EGF-dependent transformed phenotype to NIH 3T3 cells

The epidermal growth factor receptor (EGFR) gene is frequently amplified and/or overexpressed in human malignancies. To investigate the biological effects of its overexpression, we constructed a eukaryotic vector containing human EGFR cDNA. Introduction of this construct led to reconstitution of functional EGF receptors in NR6 mutant cells, which are normally devoid of this receptor. Transfection of NIH 3T3 resulted in no significant alterations in growth properties. However, EGF addition led to the formation of densely growing transformed foci in liquid culture and colonies in semisolid medium. NIH 3T3-EGFR clonal lines, which expressed the EGF at 500- to 1000-fold levels over control NIH 3T3 cells, demonstrated a marked increase in DNA synthesis in response to EGF. Thus EGF receptor overexpression appears to amplify normal EGF signal transduction. Finally, high levels of EGFR expression, which conferred a transformed phenotype to NIH 3T3 cells in the presence of ligand, were demonstrated in representative human tumor cell lines that contained amplified copies of the EGFR gene.     

Co-transfection of normal NIH/3T3 DNA and retroval LTR sequences: a novel strategy for the detection of potential c-onc genes.

Morphologically transformed, tumorigenic cell lines were obtained after co-transfecting normal NIH/3T3 DNA and cloned 3'-long terminal repeat sequences of Moloney leukemia virus (Mo-LTR) onto NIH/3T3 recipient cells. In four such cell lines the malignant phenotype was found to be associated with single and specific Mo-LTR integration sites that were retained after serial passages through NIH/3T3 and rat 208F cells, indicating that Mo-LTR sequences are linked to the activated oncogenes. In one of these clones the activated transforming gene was identified as c-raf, the cellular homologue of a recently described retroviral oncogene. This finding not only demonstrates that the mouse c-raf gene can be activated to exhibit an oncogenic potential but also that the approach chosen in this study is suitable for the detection of potential c-onc genes. In contrast to this clone, the activated transforming genes in other cell lines appear to be different from 19 previously isolated v-onc and c-onc genes. These results demonstrate the potential of the established transformation system for the detection and isolation of previously unidentified c-onc genes.     

Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent growth potential, and the ability to form tumors in experimental animals¹. In NIH 3T3 cells, the Ras signal transduction pathway is known to trigger many of these events, what is known as Ras transformation. The introduction of an overexpressed gene in NIH 3T3 cells may promote morphological transformation and loss of contact inhibition, which can help determine the oncogenic potential of that gene of interest. An assay that provides a straightforward method to assess one aspect of the transforming potential of an oncogene is the Focus Formation Assay (FFA)². When NIH 3T3 cells divide normally in culture, they do so until they reach a confluent monolayer. However, in the presence of an overexpressed oncogene, these cells can begin to grow in dense, multilayered foci¹ that can be visualized and quantified by crystal violet or Hema 3 staining. In this article we describe the FFA protocol with retroviral transduction of the gene of interest into NIH 3T3 cells, and how to quantify the number of foci through staining. Retroviral transduction offers a more efficient method of gene delivery than transfection, and the use of an ecotropic murine retrovirus provides a biosafety control when working with potential human oncogenes.     

Different structural alterations upregulate in vitro tyrosine kinase activity and transforming potency of the erbB-2 gene.

Compared with normal erbB-2 gp185, mutant erbB-2 proteins generated by mutations either in the transmembrane domain or by NH2-terminal deletion are able to transform NIH 3T3 cells at a 10- to 100-fold greater efficiency. Mutant proteins of both classes show increased tyrosine kinase activity, suggesting that an abnormal level of receptor-associated tyrosine kinase activity is a major determinant of erbB-2 oncogenic potential.     

Constitutively activated neu oncoprotein tyrosine kinase interferes with growth factor-induced signals for gene activation.

The neu receptor oncoprotein tyrosine kinase, capable of transforming cultured fibroblasts and causing mammary carcinomas in transgenic mice, carries a point mutation in its transmembrane domain and shows a constitutive tyrosine kinase activity. We analyzed the neu tyrosine kinase and its substrates in transfected NIH 3T3 fibroblasts by phosphotyrosine immunoblotting. Tyrosine phosphorylated proteins were similar but not identical in epidermal growth factor (EGF)-stimulated cells expressing the human EGF receptor (EGFR) or a chimeric EGFR/neu receptor but differed from phosphotyrosyl proteins constitutively expressed in neu oncogene-transformed cells. The neu oncoprotein in the latter cells was phosphorylated in tyrosine in a ligand-independent manner and had a shortened half-life in comparison with the normal neu protein. Tumor promoter pretreatment inhibited ligand-induced receptor tyrosine phosphorylation and decreased tyrosine phosphorylated neu oncoprotein. Prolonged pretreatment with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) also prevented the induction of immediate early growth factor-regulated genes in response to neu activation. Expression of the neu oncogene but not the protooncogene in NIH 3T3 cells was associated with enhanced levels of the jun and fos oncoproteins and loss of serum growth factor induction of immediate early mRNA responses. The constitutively activated neu oncoprotein tyrosine kinase thus deregulates cellular genomic responses to growth factors.     

过表达Gankyrin基因细胞模型的建立及其成瘤性分析

目的：Gankyrin作为一个新的癌基因,在细胞周期调控和肿瘤发生过程中有重要功能。建立Gankyrin过表达的细胞和动物模型,以便进一步研究其在肿瘤形成过程中的作用机制。方法：采用逆转录病毒感染细胞的方法构建Gankyrin稳定表达的细胞株,采用Western-blot检测Gankyrin的表达,采用软琼脂和裸鼠成瘤实验验证该细胞株的恶性转化效应。结果：构建了Gankyrin稳定过表达的NIH3T3细胞株,且该细胞株具有成瘤性。结论：采用逆转录病毒感染细胞的方法可以有效建立Gankyrin转化细胞株,为进一步研究Gankyrin的作用机制及其在肿瘤形成中的功能提供了基础。     

Loss of the amino-terminal helix-loop-helix domain of the vav proto-oncogene activates its transforming potential.

vav, a novel human oncogene, was originally generated in vitro by replacement of its normal 5' coding sequences with sequences from pSV2neo DNA, cotransfected as a selectable marker (S. Katzav, D. Martin-Zanca, and M. Barbacid, EMBO J. 8:2283-2290, 1989). The vav proto-oncogene is normally expressed in cells of hematopoietic origin. To determine whether the 5' rearrangement of vav or its ectopic expression in NIH 3T3 cells contributes to its transforming potential, we isolated murine and human proto-vav cDNA clones as well as human genomic clones corresponding to the 5' end of the gene. Normal proto-vav was poorly transforming in NIH 3T3 cells, whereas truncation of its 5' end greatly enhanced its transforming activity. The relative failure of full-length proto-vav cDNA clones to transform NIH 3T3 cells indicates that the transforming activity of vav is not simply due to ectopic expression. Analysis of the predicted amino terminus of the vav proto-oncogene shows that it contains a helix-loop-helix domain and a leucine zipper motif similar to that of myc family proteins, though it lacks a basic region that is usually found adjacent to helix-loop-helix domains. Loss of the helix-loop-helix domain of proto-vav, either by truncation or by rearrangement with pSV2neo sequences, activates its oncogenic potential.     

HER2 cytoplasmic domain generates normal mitogenic and transforming signals in a chimeric receptor.

We have investigated the biological function of an unidentified human growth factor, the ligand of the putative HER2 receptor, by characterizing the signalling properties of its receptor. HER2 (or c-erbB-2), the human homolog of the rat neu proto-oncogene, encodes a transmembrane glycoprotein of the tyrosine kinase family that appears to play an important role in human breast carcinoma. Since a potential ligand for HER2 has not yet been identified, it has been difficult to analyze the biochemical properties and biological function of this cell surface protein. For this reason, we replaced the HER2 extracellular domain with the closely related ligand binding domain sequences of the epidermal growth factor (EGF) receptor, and examined the ligand-induced biological signalling potential of this chimeric HER1-2 protein. This HER1-2 receptor is targetted to the cell surface of transfected NIH 3T3 cells, forms high and low affinity binding sites, and generates normal mitogenic and cell transforming signals upon interaction with EGF or TGF alpha. The constitutive activation of wild-type HER2 in transfected NIH 3T3 cells suggests the possibility that these cells synthesize the as yet unidentified HER2 ligand and activate HER2 by an autocrine mechanism.