The effector loop and prenylation site of R-Ras are involved in the regulation of integrin function.

The closely related small GTP-binding proteins H-Ras and R-Ras have opposing effects on the regulation of integrin cell adhesion receptors. To gain insight into the properties of R-Ras with respect to the regulation of integrin function and interactions with downstream effectors we performed an analysis of R-Ras variants containing mutations in the effector binding domain and C-terminal prenylation site. We found that the activation of the downstream effector PI 3-kinase was sensitive to mutations in the effector binding domain, as was the binding to the effectors, Ral-GDS, Raf-1 and the novel effector Nore1. Furthermore, specific mutations in the effector binding loop and C-terminal prenylation motif impaired the ability of R-Ras to regulate integrin function in CHO cells. However, the ability of the R-Ras effector loop mutants to bind, and activate known effectors did not correlate with their ability to regulate integrin function. Thus, the known R-Ras effectors are not critical for regulating integrin activation, at least in CHO cells. Consequently, these studies provide insight into the structural basis of the interactions between R-Ras and its candidate effectors and suggest the existence of novel mechanisms through which this GTPase could regulate cell adhesion.

Phosphoinositide 3-kinase induces scattering and tubulogenesis in epithelial cells through a novel pathway.

Hepatocyte growth factor/scatter factor (HGF/SF) treatment of the Madin-Darby canine kidney epithelial cell line causes scattering of cells grown in monolayer culture and the formation of branching tubules by cells grown in collagen gels. HGF/SF causes prolonged activation of both the mitogen-activated protein (MAP) kinase extracellular signal-regulated kinase 2 (ERK2) and the phosphoinositide 3-OH kinase (PI 3-kinase) target protein kinase B (PKB)/Akt; inhibition of either the MAP kinase pathway by the MAP kinase/ERK kinase inhibitor PD98059 or the PI 3-kinase pathway by LY294002 blocks HGF/SF induction of scattering, although in morphologically distinct ways. Expression of constitutively activated PI 3-kinase, Ras, or R-Ras will cause scattering, but activated Raf will not, indicating that activation of the MAP kinase pathway is not sufficient for this response. Downstream of PI 3-kinase, activated PKB/Akt and Rac are both unable to induce scattering, implicating a novel pathway. Scattering induced by Ras or PI 3-kinase is sensitive to PD98059, as well as to LY294002, suggesting that basal MAP kinase activity is required, but not sufficient, for the scattering response. Induction of MDCK cell tubulogenesis in collagen gels by HGF/SF is inhibited by PD98059; expression of activated Ras and Raf causes disorganized growth in this system, but activated PI 3-kinase or R-Ras causes branching tubule formation similar to that seen with HGF/SF treatment. These data indicate that multiple signaling pathways acting downstream of Met and Ras are needed for these morphological effects; scattering is induced primarily by the PI 3-kinase pathway, which acts through effectors other than PKB/Akt or Rac and requires at least basal MAP kinase function. Elevated PI 3-kinase activity induces tubulogenesis, but total inhibition and excess activation of the MAP kinase pathway both oppose this effect.

PKB/Akt: connecting phosphoinositide 3-kinase to cell survival and beyond.

PKB/Akt is a serine/threonine kinase that contains a pleckstrin-homology (PH) domain and is activated in response to growth-factor treatment of cells by a mechanism involving phosphoinositide 3-OH kinase. PKB/Akt provides a survival signal that protects cells from apoptosis induced by various stresses, perhaps explaining its discovery as a retroviral oncogene and its amplification in many human tumours.

Role of phosphoinositide 3-OH kinase in cell transformation and control of the actin cytoskeleton by Ras.

The pathways by which mammalian Ras proteins induce cortical actin rearrangement and cause cellular transformation are investigated using partial loss of function mutants of Ras and activated and inhibitory forms of various postulated target enzymes for Ras. Efficient transformation by Ras requires activation of other direct effectors in addition to the MAP kinase kinase kinase Raf and is inhibited by inactivation of the PI 3-kinase pathway. Actin rearrangement correlates with the ability of Ras mutants to activate PI 3-kinase. Inhibition of PI 3-kinase activity blocks Ras induction of membrane ruffling, while activated PI 3-kinase is sufficient to induce membrane ruffling, acting through Rac. The ability of activated Ras to stimulate PI 3-kinase in addition to Raf is therefore important in Ras transformation of mammalian cells and essential in Ras-induced cytoskeletal reorganization.

R-Ras can activate the phosphoinositide 3-kinase but not the MAP kinase arm of the Ras effector pathways.

BACKGROUND: The small GTPase R-Ras displays a less potent transforming activity than the closely related Ras oncogene products. Although R-Ras has been reported to interact with c-Raf1 and Ral-GDS in vitro, the pathways by which it exerts its effects on cellular proliferation are not known. RESULTS: Both Ras and R-Ras interact with phosphoinositide (PI) 3-kinase in vitro, and induce elevation of the levels of PI 3-kinase lipid products in intact cells. Unlike Ras, R-Ras does not activate Raf or mitogen-activated protein (MAP) kinase in cells. In co-transfection assays, the serine/threonine protein kinase PKB (or Akt) is effectively stimulated by R-Ras, Ras, mutants of Ras that activate PI 3-kinase but not other effectors, and activated forms of PI 3-kinase. Ras and R-Ras stimulate PKB/Akt through a non-autocrine mechanism that involves PI 3-kinase. The constitutive activation of PI 3-kinase alone is sufficient to activate PKB/Akt, but not the MAP kinase ERK or the stress-activated protein kinase, Jun N-terminal kinase. Transformation assays in fibroblasts suggest that PKB/Akt and Raf are part of distinct oncogenic signalling pathways. CONCLUSIONS: Both the Raf-MAP kinase and PI 3-kinase-PKB/Akt pathways are activated by Ras, but only the PI 3-kinase-PKB/Akt pathway is activated by R-Ras. PI 3-kinase, and downstream targets such as PKB/Akt, are likely to be essential mediators of transformation induced by R-Ras. PI 3-kinase, as well as Raf, is thus implicated also in Ras transformation.

Growth, differentiation and survival of HC11 mammary epithelial cells: diverse effects of receptor tyrosine kinase-activating peptide growth factors.

The HC11 mouse mammary epithelial cells are a useful in vitro model of mammary cell differentiation. When treated with the lactogenic hormones mix dexamethasone, insulin and prolactin (DIP) these cells synthesize the milk protein beta-casein. HC11 cells express receptor tyrosine kinases (RTK) of various subclasses. Here we present an analysis of the effect of their stimulation on growth, differentiation and survival. Growth conditions are an important part in the HC11 cell differentiation program. In order to respond optimally to DIP, cells must be grown to confluency in medium containing epidermal growth factor (EGF) plus insulin, at which stage the cells are defined as competent. During the growth phase all the peptide factors rested in this study: EGF, fibroblast growth factor (FGF)-2, insulin, IGF-I, platelet-derived growth factor (PDGF) and stem cell factor (SCF), stimulated MAP kinase (ERK2) activity and-DNA synthesis. However, not all factors were equivalent in promoting competency. Only FGF-2 replaced EGF during growth, while IGF-1 or SCF were able to substitute for insulin. PDGF replaced neither EGF nor insulin and was ineffective as a competence factor. The only peptide which could substitute for insulin in the lactogenic DIP mix and induce beta-casein synthesis was IGF-1, albeit at a high concentration. Competent cultures of HC11 cells maintained in serum-free medium in the presence of only dexamethasone and prolactin undergo apoptosis, which is prevented by the addition of either insulin, IGF-1, FGF-2, or EGF, but not PDGF or SCF. We conclude that in HC11 cells all peptide factors induce DNA synthesis but have distinct effects on differentiation and survival in HC11 cells.

Phosphatidylinositol 3' kinase: one of the effectors of Ras.

Ras proteins are proto-oncogene products that are critical components of signalling pathways leading from cell surface receptors to control of cellular proliferation, morphology and differentiation. the ability of Ras to activate the MAP kinase pathway through interaction with the serine/threonine kinase Raf is now well established. However, recent work has shown that Ras can also interact directly with the catalytic subunit of phosphatidylinositol 3' kinase and is involved in control of the lipid kinase in intact cells. A model is presented in which both tyrosine phosphoprotein interaction with the regulatory p85 subunit and Ras. GTP interaction with the catalytic p110 subunit is required to achieve optimal activation of phosphatidylinositol 3'kinase in response to extracellular stimuli. The ability of Ras to regulate phosphatidylinositol 3' kinase may be important both in Ras control of cellular morphology through the actin cytoskeleton and also in Ras control of DNA synthesis.

NDF/heregulin activates MAP kinase and p70/p85 S6 kinase during proliferation or differentiation of mammary epithelial cells.

Neu differentiation factors (NDF) are a novel family of polypeptide factors which activate sub-class I tyrosine kinase receptors. In all mammary epithelial cells analysed in this study, NDF activates the same signalling pathways while it induces different, cell-specific biological effects. In AU565 cells which are growth inhibited, as well as in T47D or HC11 cells which proliferate in response to NDF, the MAP kinase isoforms p44ERK1 and p42ERK2 and the p70/p85 S6 kinase are activated. NDF stimulates tyrosine phosphorylation and the in vitro kinase activity of ErbB-2. When PKC is activated by TPA, NDF is no longer able to activate ErbB-2 in T47D cells, leading to a blockage of cell proliferation. Activation of ErbB-2 by point mutation, or by monoclonal antibodies, also stimulates both the MAPK and the p70/p85 S6 kinase pathways. The same monoclonal antibodies can induce AU565 cell differentiation. In summary, during growth or differentiation of mammary epithelial cells, NDF stimulates several independent signalling pathways which can also be triggered by ErbB-2 stimulation alone. PKC activation blocks the biological effect induced by NDF through negative modulation of ErbB-2.

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.

EGF receptor and p185erbB-2-specific single-chain antibody toxins differ in their cell-killing activity on tumor cells expressing both receptor proteins.

Many human tumors over-express erbB-2 and EGF receptors. The membrane localization of these receptor tyrosine kinases make them appropriate targets for directed tumor therapy. We have used recombinant DNA technology to produce single-chain antibody exotoxin A (scFv-ETA) fusion proteins which specifically bind the erbB-2 and EGF receptors. The scFv portion is composed of the heavy- and light-chain variable domains of monoclonal antibodies which recognize the extracellular portion of each receptor. We have previously described the anti-tumor activity of the bacterially produced scFv(FRP5)-ETA directed to the erbB-2 receptor. In this paper we describe the characteristics of scFv(225)-ETA, a protein which binds the EGF receptor. The bacterially produced recombinant protein binds to the receptor with high affinity and inhibits the in vitro growth of the EGF receptor over-expressing tumor cell lines A431 and MDA-MB468. Combination treatment with scFv-(FRP5)-ETA and scFv(225)-ETA led to an additive inhibitory effect on the in vitro growth of A431 cells. SKBR3 cells expressing low levels of EGF receptor but high levels of p185erbB-2 were not affected by scFv(225)-ETA treatment but were sensitive to scFv(FRP5)-ETA. Stimulation of SKBR3 cells and HCII RI#11 mouse mammary epithelial cells expressing the human erbB-2 with EGF led to an increase in scFv(FRP5)-ETA activity, showing that the EGF-induced activation of erbB-2 can potentiate the action of the erbB-2-directed toxin. Treatment of athymic nude mice with scFv(FRP5)-ETA and the combination of both scFv-ETA proteins led to the transient arrest of growth of established A431 tumors. scFv(225)-ETA treatment alone was the most effective, leading to tumor shrinkage during the course of treatment, whereas treatment with the parental monoclonal antibody 225 led to retarded tumor growth.

Protein kinase C and mammary cell differentiation: involvement of protein kinase C alpha in the induction of beta-casein expression.

Treatment of HC11 mouse mammary epithelial cells with the lactogenic hormones dexamethasone, insulin, and prolactin (DIP) leads to cellular differentiation and production of the milk protein beta-casein. The following experimental evidence suggests the involvement of protein kinase C (PKC) in DIP induced signal transduction. Down-regulation of PKC by 12-O-tetradecanoylphorbol-13-acetate or addition of CGP 41251, a selective inhibitor of PKC, inhibited beta-casein protein expression induced by DIP in HC11 cells. This inhibition occurs at the level of transcription, since the DIP mediated activation of a beta-casein promoter-luciferase reporter construct or of mammary gland specific factor (MGF), an essential transcription factor for beta-casein promoter activity, was also inhibited by CGP 41251. Inhibition or down-regulation of PKC reduced the activation of MGF by prolactin as well. PKC-alpha, the only conventional PKC isoform expressed in HC11 cells, is most likely involved in the DIP induced beta-casein expression. (a) Only PKC-alpha and PKC-epsilon are down-regulated by 12-O-tetradecanoylphorbol-13-acetate whereas PKC-delta and PKC-zeta are not. (b) Of the PKC isoforms expressed in HC11 cells, CGP 41251 inhibits PKC-alpha more potently than PKC-delta, PKC-epsilon, and PKC-zeta. The IC50 for the inhibition of beta-casein synthesis, MGF activation, and beta-casein promoter activity by CGP 41251 correlated well with the IC50 of PKC-alpha inhibition. (c) Finally, only PKC-alpha translocated to membrane fractions after DIP or prolactin treatment. Taken together, these data indicate that PKC-alpha plays an important role in the signaling pathway activated by prolactin during beta-casein induction.

Growth suppression of normal mammary epithelial cells by wild-type p53.

p53 mutations are frequent in human breast cancer. In order to understand the role of p53 in the context of the accumulation of mutations in breast cancer, a model of non transformed mammary cells was sought. The HC11 cells are immortalized, non transformed rodent mammary epithelial cells which synthesize milk proteins following stimulation with lactogenic hormones. p53 protein was readily detected in HC11 protein extracts with the PAb421 antibody. Two mutations were identified in the p53 cDNA from HC11 cells: a missense mutation at codon 138, substituting Trp for Cys, and a microdeletion, codon 123 to 130, of exon 5. The latter results from an intronic mutation of the splice acceptor site at the intron 4/exon 5 junction. The mutations affect separate p53 alleles, and no wt allele was found. Wt p53 was introduced into HC11 cells by means of a retroviral vector, under the control of a Cd(++)-inducible promoter. In the presence of CdSO4 a dramatic growth inhibition was observed. A temperature-sensitive mutant p53 gene was also transfected into HC11 cells. This resulted in a marked inhibition of cells growth at 32 degrees C, when the p53 is in the wt conformation, while no effect was observed at 37 degrees C, when the mutant conformation is predominant. wt p53-mediated inhibition of monolayer growth does not involve induction of programmed cell death and does not activate de novo synthesis of differentiation-specific milk proteins. We conclude that mutations in the p53 gene likely played a role in their immortalization. The HC11 cells provide a model for assessing the cooperative action of other mutations in mammary tumorigenesis.

Drosophila evolution challenges postulated redundancy in the E(spl) gene complex.

The Enhancer of split [E(spl)] gene complex belongs to the class of neurogenic loci, which, in a concerted action, govern neurogenesis in Drosophila. Two genetically distinct functions, vital and neurogenic, reside within the complex defined by lethal mutations in the l(3) gro gene and by the typical neurogenic phenotype of deletions, respectively. Such deletions always affect several of the many embryonically active genes in the region, which cannot be mutated separately to lethality. Seven of these genes are extremely similar at the transcription and sequence level sharing the basic helix-loop-helix (bHLH) motif of transcriptional regulators. While these E(spl) bHLH genes seem to be required collectively for neurogenesis, they are nonessential individually, suggesting functional redundancy of the encoded gene products. No specific functions could yet be ascribed to any of the other genes located within the complex. One might expect these apparently dispensable genes, as well as the supposedly redundant bHLH genes, to be under little evolutionary constraint and, thus, to evolve most rapidly. However, we find the entire E(spl) gene complex highly conserved during Drosophila evolution, indicating that all the genes as well as their organization are of functional importance.

Monoclonal antibodies against the extracellular domain of the erbB-2 receptor function as partial ligand agonists.

In this paper we describe the isolation and characterization of four monoclonal antibodies (FRP5, FSP16, FWP51, and FSP77) which specifically recognize the human erbB-2 protein. All of the antibodies recognize epitopes on the extracellular domain of the receptor protein. FRP5 and FSP16 compete with one another for binding while FWP51 and FSP77 each recognize a different epitope. The effects of the antibodies on the erbB-2 receptor protein have been analyzed. Two different erbB-2-expressing cell lines, SKBR3 breast tumor cells and HC11 R111 cells, were examined. The SKBR3 cells express approximately 1 x 10(6) molecules of the erbB-2 protein/cell; HC11 R111 cells, a clone of mouse mammary epithelial cells derived by transfection of a human erbB-2 expression plasmid, contain 10-fold less erbB-2 protein than the SKBR3 cells. Treatment of the two cell lines with FRP5, FSP16, and FWP51 led to a rapid increase in the phosphotyrosine content of the erbB-2 protein. Three of the antibodies, FRP5, FSP16, and FSP77, stimulated the turnover of the erbB-2 protein. Binding of the antibodies did not stimulate DNA synthesis in HC11 R111 cells. Thus, the erbB-2-specific monoclonal antibodies behave as partial ligand agonists. The antibodies were examined for their effects upon the growth of SKBR3 and HC11 R111 cells. The growth of SKBR3 cells was inhibited by 90% following long term treatment of the cells with FSP77.