epidermal-growth-factor and herbimycin

Epidermal growth factor (EGF) is known to play an important role in modulating renal transport functions. Thus, we investigated the effect of EGF on Ca(2+) uptake and its related signals in the primary cultured rabbit renal proximal tubule cells. EGF (50 ng/ml, 1 h) stimulated Ca(2+) uptake. Its effect was blocked by AG 1478 (an EGF receptor antagonist), genistein or herbimycin A (tyrosine kinase inhibitors). EGF increased intracellular cAMP level and SQ 22536 (an adenylate cyclase inhibitor), Rp-cAMP (a cAMP analogue), or PKI (a protein kinase A inhibitor) blocked the EGF-induced stimulation of Ca(2+) uptake. EGF-induced stimulation of Ca(2+) uptake was also blocked by neomycin or U-73122 (phospholipase C inhibitors), staurosporine, H-7, or bisindolylmaleimide I (protein kinase C inhibitors), nifedipine or methoxyverapamil (L-type Ca(2+) channel blockers). It increased IPs formation by 167 +/- 5% compare to control within 90 s. On the other hand, EGF increased [(3)H]-arachidonic acid release, which was significantly blocked by PKC inhibitors. In addition, PGE(2), one of cyclooxygenase metabolites, and 5,6-EET, one of cytochrome P-450 metabolites, increased Ca(2+) uptake. These results suggest that cAMP, PLC/PKC, and PLA(2) are involved in EGF-induced stimulation of Ca(2+) uptake.

Topics: Animals; Arachidonic Acid; Benzoquinones; Blotting, Western; Calcium; Calcium Radioisotopes; Cells, Cultured; Cyclic AMP; Dose-Response Relationship, Drug; Enzyme Inhibitors; Epidermal Growth Factor; Kidney Tubules, Proximal; Kinetics; Lactams, Macrocyclic; Male; Phospholipases A; Protein Kinase C; Quinones; Rabbits; Rifabutin

Suppression of protein breakdown occurs commonly in cell growth, but the pathways responsible for controlling proteolysis are poorly understood. Protein breakdown in NRK-52E renal epithelial cells treated with epidermal growth factor (EGF) and intracellular signaling inhibitors or dominant negative signaling molecules contained in an adenoviral vector were measured. The tyrosine kinase inhibitor, herbimycin A, eliminated the suppression of proteolysis induced by EGF. In contrast, the Src inhibitor, PP1, had no effect. Expression of dominant negative H-RasY57 blocked the ability of EGF to stimulate downstream targets of Ras and also reduced the ability of EGF to suppress proteolysis. Inhibiting MEK did not influence the ability of EGF to suppress proteolysis, but the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor, LY249002, stimulated basal proteolysis and completely eliminated the proteolytic response to EGF. Use of an adenovirus that expresses a dominant negative p85 subunit of class 1 PI 3-kinase completely blocked the ability of EGF to suppress proteolysis, whereas use of an adenovirus expressing a K227E constitutively active p110 subunit reproduced the reduction in protein breakdown. It was concluded that EGF suppresses proteolysis by a mechanism that involves Ras and class 1 PI 3-kinase.

Topics: Animals; Benzoquinones; Cell Line; Enzyme Inhibitors; Epidermal Growth Factor; Kidney Tubules; Lactams, Macrocyclic; LLC-PK1 Cells; Mitogen-Activated Protein Kinases; Peptide Hydrolases; Phosphatidylinositol 3-Kinases; Protein-Tyrosine Kinases; Quinones; ras Proteins; Rats; Rifabutin; Swine

The purpose of the present study was to investigate the involvement of cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), and tyrosine kinase on prostaglandin E2 (PGE2) production in human gingival fibroblasts stimulated by interleukin-1beta (IL-1beta) and/or epidermal growth factor (EGF). The cytokine IL-1beta and to a lesser extent EGF, enhanced COX-2 mRNA levels in gingival fibroblasts. Simultaneous treatment with EGF and IL-1beta resulted in enhanced COX-2 mRNA levels accompanied by a synergistic stimulation of PGE2 biosynthesis compared to the cells treated with IL-1beta or EGF alone. Neither IL-1beta EGF nor the combination of IL-1beta and EGF enhanced COX-1 mRNA levels in gingival fibroblasts. The tyrosine kinase inhibitors, Herbimycin A and PD 153035 hydrochloride, reduced COX-2 mRNA levels as well as PGE2 production induced by IL-1beta or the combination of IL-1beta and EGF whereas COX-1 mRNA levels were not affected. Furthermore, the COX-2 specific inhibitor, NS-398, abolished the PGE2 production induced by IL-1beta, EGF, or the combination. These results indicate that the synergy between IL-1beta and EGF on PGE2 production is due to an enhanced gene expression of COX-2 and that tyrosine kinase(s) are involved in the signal transduction of COX-2 in gingival fibroblasts.

Topics: Benzoquinones; Cells, Cultured; Child; Cyclooxygenase 1; Cyclooxygenase 2; Dinoprostone; Dose-Response Relationship, Drug; Drug Synergism; Epidermal Growth Factor; Fibroblasts; Gene Expression Regulation; Gingiva; Humans; Interleukin-1; Isoenzymes; Lactams, Macrocyclic; Membrane Proteins; Nitrobenzenes; Prostaglandin-Endoperoxide Synthases; Protein-Tyrosine Kinases; Quinazolines; Quinones; Rifabutin; RNA, Messenger; Sulfonamides; Time Factors

Hepatocyte growth factor/scatter factor (HGF/SF) contributes to the malignant progression of human gliomas. We investigated the effect of HGF/SF on matrix metalloproteinase-2 (MMP-2), membrane type 1 matrix metalloproteinase (MT1-MMP) and tissue inhibitors of metalloproteinases (TIMPs), expressions of c-Met/HGF receptor-positive human glioblastoma cells. Treatment of U251 human glioblastoma cells with HGF/SF resulted in enhanced secretion of MMP-2 with an increased level of the active form. This was accompanied by enhanced expression (2.5-fold) of mRNA specific for MMP-2. The stimulatory effect of HGF/SF on MMP-2 expression did not occur in the presence of herbimycin A, a protein tyrosine kinase inhibitor. MT1 -MMP, a cell-surface activator of proMMP-2, was also up-regulated by HGF/SF in a dose-dependent manner. By contrast, the level of TIMP- 1 mRNAs was not altered significantly and that of TIMP-2 was reduced mildly by the HGF/SF treatment, suggesting that HGF/SF may eventually modulate a balance between MMP-2 and TIMPs in favor of the proteinase activity in the glioma cell microenvironment. HGF/SF also stimulated MMP-2 expression of other glioblastoma cell lines. Since glioblastomas frequently co-express HGF/SF and its receptor, our results suggest that HGF/SF might contribute to the invasiveness of glioblastoma cells through autocrine induction of MMP-2 expression and activation.

Topics: Benzoquinones; Brain Neoplasms; Disease Progression; Enzyme Induction; Enzyme Inhibitors; Epidermal Growth Factor; Gelatinases; Gene Expression Regulation, Neoplastic; Glioblastoma; Glioma; Hepatocyte Growth Factor; Humans; Lactams, Macrocyclic; Matrix Metalloproteinase 2; Matrix Metalloproteinases, Membrane-Associated; Metalloendopeptidases; Neoplasm Invasiveness; Neoplasm Proteins; Protein-Tyrosine Kinases; Quinones; Recombinant Proteins; Rifabutin; RNA, Messenger; RNA, Neoplasm; Signal Transduction; Stimulation, Chemical; Tumor Cells, Cultured

3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase is the major rate-limiting enzyme in sterol and non-sterol isoprenoid synthesis. Isoprenoids are involved in the mechanisms of cell proliferation and transformation leading notably to crucial post-translational maturation of small G-proteins of the Ras superfamily. HMG-CoA reductase is among the most highly regulated enzymes. It is controlled by several feedback regulation mechanisms induced by sterol and non-sterol metabolites. The present results show that tyrosine kinase activity is also involved in the regulation of HMG-CoA reductase activity in the human breast cancer cell line SKBR-3. Incubation of SKBR-3 cells with the tyrosine kinase inhibitor, herbimycin A, induces a concentration-dependent reduction of HMG-CoA reductase activity with an IC50 of 80nM. The inhibition of HMG-CoA reductase activity by herbimycin A is also time-dependent. A similar effect of herbimycin A was obtained on the steady-state level of the HMG-CoA reductase protein. The effect of herbimycin A is probably specific as it abolished the stimulation of reductase activity by epidermal growth factor. To elucidate the molecular basis of the inhibition of HMG-CoA reductase activity and protein level by herbimycin A, we performed experiments to study the metabolic turnover of this enzyme using [35S]methionine and [35]cysteine. Herbimycin A (1 microM) did not have any significant effect on the rate of HMG-CoA reductase protein degradation but did affect its rate of synthesis and mRNA levels. The decrease in protein synthesis rate correlates with the lower reductase protein level but is more pronounced than the decrease in mRNA levels. Taken together, the results reveal a novel pathway of regulation of HMG-CoA reductase expression and activity by cellular tyrosine kinase activities.

Topics: Adenocarcinoma; Benzoquinones; Breast Neoplasms; Down-Regulation; Enzyme Inhibitors; Epidermal Growth Factor; Female; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Hydroxymethylglutaryl CoA Reductases; Lactams, Macrocyclic; Protein-Tyrosine Kinases; Quinones; Rifabutin; RNA, Messenger

Prostaglandin H2 synthase (PGHS)-1 and PGHS-2 expression was examined in primary cultures of human amnion cells, an in vitro model of amnion tissue. Epidermal growth factor (EGF), the protein kinase C (PKC) activating phorbol ester TPA, and the protein phosphatase inhibitor, okadaic acid (OA), stimulated PGHS activity and the level of PGHS-2 mRNA, but did not affect the level of PGHS-1 mRNA. In situ hybridization suggested that the same population of cells responded to EGF, TPA and OA. Okadaic acid promoted PGHS activity independently of PKC. EGF stimulated the activity of extracellular signal-regulated protein kinase (Erk) and N-terminal c-Jun kinase (Jnk). OA increased Jnk activity but had no effect on Erk activity, while TPA had no influence on either Erk or Jnk activity. PD098059, a selective inhibitor of the Erk-activating kinase MEK, blocked the stimulation of PGHS expression by EGF, but did not decrease stimulation in response to OA. Herbimycin A, a tyrosine kinase inhibitor, suppressed the stimulation of PGHS activity and PGHS-2 mRNA abundance by all three stimulants, and blocked signalling via the Erk and Jnk mitogen-activated protein kinase pathways. Thus, growth factor stimulation, PKC activation and protein phosphatase inhibition induced the expression of PGHS-2 in primary amnion cells by distinct regulatory mechanisms involving tyrosine kinase(s). Tyrosine kinase inhibitors may constitute a new category of PGHS-2 inhibitors that act by blocking the expression of the enzyme.

Topics: Amnion; Benzoquinones; Calcium-Calmodulin-Dependent Protein Kinases; Cells, Cultured; Enzyme Activation; Enzyme Inhibitors; Epidermal Growth Factor; Flavonoids; Gene Expression Regulation; Humans; In Situ Hybridization; JNK Mitogen-Activated Protein Kinases; Lactams, Macrocyclic; Mitogen-Activated Protein Kinases; Okadaic Acid; Prostaglandin-Endoperoxide Synthases; Protein Kinase C; Protein-Tyrosine Kinases; Quinones; Rifabutin; RNA, Messenger; Tetradecanoylphorbol Acetate

Protein kinase activators as well as several neuropeptides are able to increase the GnRH-binding capacity of cultured adenohypophyseal cells. To determine whether such up-regulation of GnRH-binding sites can be achieved by a substance(s) endogenous to the pituitary, binding experiments were performed after exposure of cells to increasing amounts of medium conditioned by incubation with primary cultures of adenohypophyseal cells for 4 days. Addition of the conditioned medium elicited a 50% increase in GnRH binding. Characterization of the agent(s) responsible for the effect was attempted by submitting the conditioned medium to molecular sieve filtration, adding or immunoprecipitating endogenous substances, and comparing the susceptibilities of the responses to various inhibitors of transduction processes. Fractionation of the medium indicated that active molecules were of a proteic nature, with M(r) ranging from 5,000-10,000. Among major endogenous moieties corresponding to these criteria [epidermal] growth factor (EGF), transforming growth factor-alpha, and insulin-like growth factors I and II), only the first two exhibited properties similar to those of the conditioned medium. EGF stimulated binding with an EC50 of 3.6 +/- 0.8 pM. Immunoprecipitation of EGF, but not transforming growth factor-alpha, inactivated the conditioned medium. The effects of both conditioned medium and EGF were inhibited by herbimycin, a tyrosine kinase inhibitor; U73122, a phospholipase C inhibitor; and prior desensitization of protein kinase C. In contrast, both were insensitive to pertussis toxin pretreatment. In parallel, EGF did not increase LH secretion by itself, but potentiated its response to GnRH in a concentration range of 1 pM to 1 nM, resulting in a shift of the curve toward lower values of GnRH. It is concluded that EGF is able to control the accessibility of binding sites to GnRH and to potentiate the responsiveness of gonadotropes to the decapeptide.

Topics: Animals; Benzoquinones; Cells, Cultured; Chromatography, Gel; Culture Media, Conditioned; Enzyme Inhibitors; Epidermal Growth Factor; Gonadotropin-Releasing Hormone; Immunosorbent Techniques; Lactams, Macrocyclic; Luteinizing Hormone; Male; Pituitary Gland, Anterior; Protein-Tyrosine Kinases; Quinones; Rats; Receptors, LHRH; Rifabutin; Transforming Growth Factor alpha; Type C Phospholipases

Endothelin 1 (ET-1) is produced in ovarian cancer cell lines and has been shown to act through ET(A) receptors as an autocrine growth factor to promote tumor cell proliferation in vitro. In OVCA 433 cells, the efficacy of ET-1 as a stimulus of [3H]thymidine incorporation was equivalent to that of epidermal growth factor. ET-1 also stimulated the rapid expression of c-fos, an action mediated by ET(A) receptors. The mitogenic action of ET-1 was not mediated by a pertussis toxin-sensitive G protein. An analysis of the effects of inhibition and depletion of protein kinase C (PKC) on mitogenic responses demonstrated that PKC was necessary but not sufficient for maximal stimulation by ET-1. In quiescent OVCA 433 cells, ET-1-induced stimulation of [3H]thymidine incorporation was prevented by two structurally distinct inhibitors of tyrosine kinase, herbimycin A and genistein. These results indicate that both PKC and protein tyrosine kinase participate in ET-1-stimulated mitogenic signaling. ET-1 rapidly stimulated tyrosine phosphorylation of several cellular proteins, among which p125FAK and p42 mitogen-activated protein kinase were identified. The additivity between the potent mitogenic actions of ET-1 and epidermal growth factor is consistent with the independence of their signal transduction pathways in ovarian cancer cells. These findings also indicate that intracellular signaling between the ET(A) receptor and a yet unidentified tyrosine kinase is involved in the mitogenic response to ET-1.

Topics: Benzoquinones; Blotting, Northern; Calcium-Calmodulin-Dependent Protein Kinases; Cell Adhesion Molecules; Cell Division; DNA, Neoplasm; Endothelin-1; Enzyme Inhibitors; Epidermal Growth Factor; Female; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Gene Expression Regulation; Genes, fos; Genistein; Humans; Immunoblotting; Indoles; Isoflavones; Lactams, Macrocyclic; Maleimides; Ovarian Neoplasms; Pertussis Toxin; Phosphorylation; Protein Kinase C; Protein-Tyrosine Kinases; Quinones; Rifabutin; Signal Transduction; Tumor Cells, Cultured; Virulence Factors, Bordetella

The high basal level of prolactin (PRL) gene expression in rat pituitary GH3 cells is maintained through the spontaneous activity of voltage-sensitive calcium channels (VSCCs). This can be observed experimentally by addition of 0.5 mM CaCl2 to GH3 cells cultured in a low calcium, serum-free medium. CaCl2 specifically induces PRL gene expression and this induction is inhibited by VSCC blockers. PRL gene expression is also stimulated by several hormones and growth factors. In the present study, we examined the effects of tyrosine kinase inhibitors on the ability of CaCl2, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) and thryrotropin-releasing hormone (TRH) to increase PRL mRNA levels. Of several PTK inhibitors used, one PTK inhibitor, herbimycin A, specifically inhibited the CaCl2-induced increase in cytoplasmic and nuclear prolactin (PRL) mRNA without affecting cell viability, cell-cell and cell-matrix adhesion, or the expression of several other genes. The effects of herbimycin A were reversible. In cells pretreated with herbimycin A, PRL mRNA levels were reduced by 69 +/- 12% (P < 0.001; n = 4). Western blot analysis using anti-phosphotyrosine antibody revealed a decrease of 91 +/- 1% (P < 0.001; n = 4) in the phosphotyrosine content of proteins in the molecular weight range of 130-160 kDa. After changing the medium back to SFM plus 0.5 mM CaCl2, levels of PRL mRNA increased over a period of several hours, and this increase was accompanied by the tyrosine phosphorylation of two or more proteins in the approximate size range of 130-160 kDa. Herbimycin A also inhibited PRL gene expression in the independently-derived 235-1 lactotrope cell line and lowered the tyrosine specific phosphorylation of protein(s) in a similar size range. Herbimycin A inhibited the ability of bFGF, EGF and TRH to stimulate PRL gene expression in GH3 cells. Again, in cells pretreated with herbimycin A, bFGF induced a reappearance of tyrosine-specific phosphorylation, followed by a reappearance of PRL mRNA. These findings provide evidence for a role for at least one PTK which is necessary for basal and stimulated PRL gene expression.

Topics: Animals; Benzoquinones; Calcium Chloride; Epidermal Growth Factor; Fibroblast Growth Factor 2; Gene Expression; Lactams, Macrocyclic; Phosphorylation; Pituitary Neoplasms; Prolactin; Protein-Tyrosine Kinases; Quinones; Rats; Rifabutin; RNA, Messenger; Thyrotropin-Releasing Hormone; Tumor Cells, Cultured

Topics: Amnion; Benzoquinones; Calcium-Calmodulin-Dependent Protein Kinases; Cells, Cultured; Cycloheximide; Cyclooxygenase 2; Enzyme Induction; Epidermal Growth Factor; Female; Humans; Isoenzymes; JNK Mitogen-Activated Protein Kinases; Lactams, Macrocyclic; Membrane Proteins; Mitogen-Activated Protein Kinases; Okadaic Acid; Pregnancy; Prostaglandin-Endoperoxide Synthases; Protein-Tyrosine Kinases; Quinones; Rifabutin; RNA, Messenger; Tetradecanoylphorbol Acetate; Transcription, Genetic

Breast cancers frequently over-express a number of growth factor receptors. In addition, elevated src family kinase activity is present in a percentage of these neoplasms and has been implicated in signal transduction in these cells. Therefore, inhibiting tyrosine kinase activity is a potential approach for treating these tumors. Utilizing the SKBR3 and MCF-7 breast cancer cell lines, we evaluated the effects of broadly targeting growth factor receptor and cytoplasmic tyrosine kinases with tyrosine kinase inhibitors (herbimycin A and genistein) to inhibit proliferation. We also evaluated these inhibitor's effects on proteins that regulate ras function, which is a convergence point for signaling through both src family kinases and a number of growth factor receptors with tyrosine kinase activity (e.g., epidermal growth factor and erbB-2 receptors). We specifically evaluated whether these compounds affected 2 recently discovered proteins involved in controlling ras function: Shc, which is tyrosine-phosphorylated by src and activated growth factor receptors, and Grb-2, which mediates signal transduction from activated growth factor receptors through ras. We evaluated their effects on tyrosine phosphorylation of Shc, binding of Grb-2 to Shc and MAP kinase activity. Both cell lines were inhibited in a dose-dependent manner by each compound. This was accompanied by decreased Shc tyrosine phosphorylation, Shc's association with Grb-2 and MAP kinase activity. Thus, tyrosine kinase inhibitors can inhibit proliferation of breast cancer cells, accompanied by inhibition of signal transduction steps potentially mediated through ras. Tyrosine kinase inhibitors might, therefore, be useful for the treatment of breast cancer.

Topics: Adaptor Proteins, Signal Transducing; Benzoquinones; Breast Neoplasms; Cell Division; Enzyme Inhibitors; Epidermal Growth Factor; Female; Genistein; GRB2 Adaptor Protein; Humans; Isoflavones; Lactams, Macrocyclic; Protein-Tyrosine Kinases; Proteins; Proto-Oncogene Proteins p21(ras); Quinones; Rifabutin; Signal Transduction; Tumor Cells, Cultured

Mice carrying homozygous disruption of the c-src proto-oncogene (Src-/-) develop osteopetrosis due to an impaired ability of osteoclasts to adhere to the bone surface and/or to form bone-resorbing ruffled border. It has also been reported that osteopontin (OPN), a secreted phosphoprotein, mediates osteoclast adherence to the bone matrix. We report here that cells from Src-/- mice, both in vitro and in vivo, express OPN mRNA and protein at a significantly reduced level as compared to cells from Src+/- and +/+ animals, suggesting a potential role for the proto-oncogene c-src in the regulation of OPN gene expression. Our data also show that OPN gene expression can be induced by treatment of SR-/- cells with epidermal growth factor (EGF) and 12-O-tetradecanoyl phorbol-13-acetate (TPA). Results obtained from studies using inhibitors of receptor tyrosine kinases (RTKs) and protein kinase C (PKC) suggest that PKC and RTK are positioned in a pathway with PKC as the downstream effector for the EGF-induced OPN gene expression in SRC-/- cells, and that pp60c-src and EGF may regulate OPN gene expression through a common signalling pathway. Furthermore, contrary to published reports, our study shows that EGF-mediated cell signalling does not require functional interaction between the EGF-receptor and pp60c-src.

Topics: 3T3 Cells; Animals; Base Sequence; Benzoquinones; Bone and Bones; Cell Adhesion; Cell Line; DNA Primers; DNA Replication; Epidermal Growth Factor; ErbB Receptors; Gene Expression; Homozygote; Lactams, Macrocyclic; Mice; Microscopy, Electron; Molecular Sequence Data; Osteopetrosis; Osteopontin; Protein Kinase C; Proto-Oncogene Proteins pp60(c-src); Quinones; Rifabutin; Sialoglycoproteins; Tetradecanoylphorbol Acetate

Interleukin-6 (IL-6) is a multifunctional cytokine that is produced not only by a variety of normal cells but also by cancer cells. IL-6 produced by cancer cells stimulates the proliferation of these cancer cells in an autocrine/ paracrine manner and causes paraneoplastic syndromes including hypercalcemia, cachexia, and leukocytosis. We have reported previously that a human oral squamous cancer associated with hypercalcemia produces large amounts of IL-6, that animals bearing this cancer exhibit elevated levels of plasma IL-6, and that neutralizing antibodies to human IL-6 reverse hypercalcemia in tumor-bearing animals, indicating an important role of IL-6 in the hypercalcemia in this model. Because these cancer cells overexpress epidermal growth factor receptors (EGFR) with intrinsic tyrosine kinase (TK) activity similar to many other squamous cancers, we examined the effects of herbimycin A, a tyrosine kinase inhibitor, on IL-6 production and hypercalcemia in animals bearing this cancer to develop a new approach to treat the hypercalcemia associated with malignancy. Intraperitoneal administration (once a day for 2 days) of herbimycin A to cancer-bearing hypercalcemic mice reduced the plasma levels of human IL-6 and impaired the hypercalcemia. During 2-day treatment with herbimycin A, no changes were observed in tumor size. Of interest, plasma levels of mouse, but not human, soluble IL-6 receptors were also elevated. However, herbimycin A showed no effects on plasma levels of mouse soluble IL-6 receptors. Herbimycin A suppressed the tyrosine autophosphorylation of EGFR and IL-6 mRNA expression and production, all of which were stimulated by EGF. The data raise the possibility that TK inhibitors may be potential mechanism-based therapeutic agents for the treatment of hypercalcemia associated with squamous cancers which overexpress EGFR.

Topics: Animals; Antibiotics, Antineoplastic; Antigens, CD; Benzoquinones; Carcinoma, Squamous Cell; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Humans; Hypercalcemia; Interleukin-6; Lactams, Macrocyclic; Male; Mice; Mice, Nude; Neoplasm Transplantation; Protein-Tyrosine Kinases; Quinones; Receptors, Interleukin; Receptors, Interleukin-6; Rifabutin; Solubility; Tumor Cells, Cultured

The Met protein is a receptor tyrosine kinase for hepatocyte growth factor/scatter factor (HGF/SF), a multifunctional growth factor with mitogenic, motogenic and morphogenic properties. A morphologically altered variant of the MDCK cell line, MDCK-1, spontaneously exhibits a number of features associated with a partial HGF/SF-Met induced phenotype (less adhesive colonies in culture, enhanced invasion and motility, nascent tubule formation), but paradoxically does not respond to HGF/SF treatment. Although the overall cell surface expression and distribution of Met were found to be similar in parental MDCK cells and the MDCK-1 cell line, p145met autophosphorylation (+/ HGF/SF) was significantly reduced in MDCK-1 cells in vitro and in vivo when compared with parental MDCK cells. In contrast, EGF induced cell proliferation and EGF receptor autophosphorylation to similar levels in both cell lines. The basal levels of protein tyrosine phosphorylation were higher in MDCK-1 cells when compared with parental MDCK cells, including that of two prominent proteins with molecular masses of approximately 185 kDa and 220 kDa. Moreover, both p185 and p220 are present and tyrosine phosphorylated in Met immunoprecipitates from MDCK-1 cells (+/-HGF/SF), but not parental MDCK cells. In addition, Met immunocomplexes from MDCK-1 cells exhibited an approximately 3-fold increased tyrosine kinase activity in vitro when compared with MDCK cells, correlating with the higher basal levels of total phosphotyrosine. Treatment of MDCK-1 cells with the tyrosine kinase inhibitor herbimycin A reverted the cell phenotype to a more MDCK-like morphology in culture, with a concomitant reduction in the tyrosine phosphorylation predominantly of p220. Taken together these data suggest that aberrations in Met activity and associated signalling render MDCK-1 cells insensitive to HGF/SF, and may also mediate alterations in MDCK-1 cell behaviour.

Topics: Animals; Benzoquinones; Cell Line; Cell Movement; Cell Size; Dogs; Drug Resistance; Enzyme Inhibitors; Epidermal Growth Factor; Hepatocyte Growth Factor; Lactams, Macrocyclic; Phenotype; Phosphorylation; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-met; Quinones; Receptor Protein-Tyrosine Kinases; Rifabutin; Signal Transduction; Tyrosine

Epidermal growth factor (EGF) mediates many pleiotrophic biological effects, one of which is alteration of cellular morphology. In the present study, we examine the possibility that this alteration in cell morphology is caused in part by the dysfunction of cadherin-mediated cell-cell adhesion using the human oesophageal cancer cell line TE-2R, which expresses E-cadherin and EGF receptor. In the presence of EGF, TE-2R changed its shape from round to fibroblastic and its colony formation from compact to sparse. Vanadate, a tyrosine phosphatase inhibitor, further potentiated the EGF response, whereas herbimycin A, a tyrosine kinase inhibitor, interfered with it. Moreover, EGF enabled the cells to invade in organotypic raft culture. These phenomena were accompanied not by decreased expression of the E-cadherin molecule but by a change in its localisation from the lateral adhesion site to the whole cell surface. Both alpha- and beta-catenin, cadherin-binding proteins, were also expressed at the same level throughout these morphological changes. Finally, we examined tyrosine phosphorylation of E-cadherin and alpha- and beta-catenin, and observed tyrosine phosphorylation of beta-catenin induced by EGF. These results suggest that EGF counteracts E-cadherin-mediated junctional assembly through phosphorylation of beta-catenin and modulates tumour cell behaviour to a more aggressive phenotype.

Topics: alpha Catenin; Benzoquinones; beta Catenin; Cadherins; Carcinoma, Squamous Cell; Cell Adhesion; Cell Aggregation; Cell Line; Culture Techniques; Cytoskeletal Proteins; Epidermal Growth Factor; ErbB Receptors; Esophageal Neoplasms; Fibroblasts; Fluorescent Antibody Technique; Gels; Humans; Lactams, Macrocyclic; Neoplasm Invasiveness; Neoplasm Proteins; Phenotype; Phosphorylation; Phosphotyrosine; Protein Processing, Post-Translational; Quinones; Rifabutin; Signal Transduction; Trans-Activators; Tumor Cells, Cultured; Tumor Stem Cell Assay; Tyrosine; Vanadates

We examined effect of the tyrosine kinase inhibitor herbimycin A on A431 human epidermoid carcinoma cells which over-express epidermal growth factor (EGF) receptor. Herbimycin A inhibited the autophosphorylation of EGF-stimulated receptors in intact cells both time- and dose-dependently. The inhibition was found to be due to a decrease in the level of expression of the receptor amount, because herbimycin A equally decreased the receptor quantity and EGF-stimulated receptor kinase activity in intact cells, but did not exhibit a direct inhibitory effect on EGF receptor kinase activity in vitro. The decrease of the level of EGF receptor was also confirmed by 125I-EGF binding to herbimycin A-treated cells, and Scatchard analysis showed that the decrease in the receptor number occurred in the major population of the low-affinity binding ones, whereas the number with high-affinity binding was unaffected. Interestingly, although the proliferation of A431 cells was inhibited by EGF, herbimycin A converted EGF into a stimulatory ligand for cell growth, as determined by both cell number and DNA synthesis. These findings indicated that herbimycin A decreased the level of expression of EGF receptor by a mechanism other than inactivation of the receptor kinase and reversed the transformed phenotype of A431 cells to a normal one in the proliferative response to EGF.

Topics: Benzoquinones; Carcinoma, Squamous Cell; Cell Division; Epidermal Growth Factor; ErbB Receptors; Humans; Lactams, Macrocyclic; Ligands; Phosphorylation; Protein-Tyrosine Kinases; Quinones; Rifabutin; Tumor Cells, Cultured

In resting DDT1MF-2 smooth muscle cells, the cytosolic free Ca2+ concentration ([Ca2+]c) was higher than the free Ca2+ concentration in the nucleus ([Ca2+]n). However, this nucleocytosolic [Ca2+] gradient was reversed by Ca2+ agonists like ATP or, as is shown here, by the epidermal growth factor (EGF). The ATP-induced reversal of the nucleocytosolic [Ca2+] gradient was blocked by stimulation of protein kinase C with phorbol 12-myristate 13-acetate or with the diacylglycerol kinase inhibitor R59949, or by inhibition of the Ser/Thr-specific protein phosphatases-1 and -2A with okadaic acid or calyculin A. Moreover, the magnitude of the ATP-induced reversal of the [Ca2+] gradient diminished during prolonged culture of the cells. The EGF-induced [Ca2+] rise in the cytosol and nucleus was blocked by okadaic acid and by the tyrosine kinase inhibitors herbimycin A and psi-tectorigenin. Our data suggest that the nucleocytosolic [Ca2+] gradient is modulated by (de)phosphorylation processes catalyzed by tyrosine protein kinases, by protein kinase C, and by Ser/Thr protein phosphatases-1 and -2A.

Topics: Adenosine Triphosphate; Animals; Benzoquinones; Calcium; Cell Line; Cell Nucleus; Colforsin; Cricetinae; Cytosol; Diacylglycerol Kinase; Epidermal Growth Factor; Ethers, Cyclic; Homeostasis; Kinetics; Lactams, Macrocyclic; Leiomyosarcoma; Marine Toxins; Mesocricetus; Models, Biological; Muscle, Smooth; Okadaic Acid; Oxazoles; Phosphoproteins; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Piperidines; Protein Kinase C; Protein Kinases; Protein Tyrosine Phosphatases; Quinazolines; Quinazolinones; Quinones; Rifabutin; Tetradecanoylphorbol Acetate; Time Factors; Tumor Cells, Cultured