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C P Webb

Publications and source records attributed to C P Webb.

17 recordsLinked to original sources
PubMed2001

Suppression of Ras-mediated tumorigenicity and metastasis through inhibition of the Met receptor tyrosine kinase.

Mutations in the Ras family of GTP binding proteins represent one of the most frequently observed genetic alterations in human cancers. We and others have recently demonstrated that expression of Met, the tyrosine kinase receptor for hepatocyte growth factor/scatter factor (HGF/SF), is significantly up-regulated in Ras-transformed cells. Because HGF/SF-Met signaling is proposed to play a prominent role in tumor development and progression, we assessed the possible requirement for Met during Ras-mediated tumor growth and metastasis. To disrupt endogenous Met signaling, we constructed dominant-negative mutants of both human and murine Met and showed that these can inhibit HGF/SF-mediated Met signaling and cell invasion of ras-transformed cells in vitro. Moreover, ectopic expression of dominant-negative Met mutants reduced the s.c. tumor growth of ras-transformed cells and dramatically suppressed their ability to form lung metastases in vivo. Our data demonstrate that Met plays a prominent role during Ras-mediated tumor growth and metastasis, and further suggest that agents that inhibit HGF/SF-Met signaling may represent an important therapeutic avenue for the treatment of a variety of malignant tumors.

3T3 Cells↗
PubMed2001

Suppression of ras-mediated transformation and inhibition of tumor growth and angiogenesis by anthrax lethal factor, a proteolytic inhibitor of multiple MEK pathways.

Lethal factor is a protease, one component of Bacillus anthracis exotoxin, which cleaves many of the mitogen-activated protein kinase kinases (MEKs). Given the importance of MEK signaling in tumorigenesis, we assessed the effects of anthrax lethal toxin (LeTx) on tumor cells. LeTx was very effective in inhibiting mitogen-activated protein kinase activation in V12 H-ras-transformed NIH 3T3 cells. In vitro, treatment of transformed cells with LeTx caused them to revert to a nontransformed morphology, and inhibited their abilities to form colonies in soft agar and to invade Matrigel without markedly affecting cell proliferation. In vivo, LeTx inhibited growth of ras-transformed cells implanted in athymic nude mice (in some cases causing tumor regression) at concentrations that caused no apparent animal toxicity. Unexpectedly, LeTx also greatly decreased tumor neovascularization. These results demonstrate that LeTx potently inhibits ras-mediated tumor growth and is a potential antitumor therapeutic.

3T3 Cells↗
PubMed2001

Normal and malignant prostate epithelial cells differ in their response to hepatocyte growth factor/scatter factor.

Hepatocyte growth factor/scatter factor (HGF/SF) promotes the proliferation, differentiation, motility, and invasion of epithelial cells by binding to its cell surface receptor, the Met tyrosine kinase. In the prostate, Met is expressed predominantly by prostate epithelial cells (PrEC), whereas HGF/SF is synthesized by prostate stromal cells (PrSC). Met is also expressed in localized and metastatic prostate cancers. Our results show that PrECs in in vitro culture maintain expression of Met at a level comparable to DU145 cancer cell expression. HGF/SF secreted by PrSC stimulates tyrosine phosphorylation of the Met receptor. In normal PrEC, HGF/SF causes growth inhibition, sustained phosphorylation of mitogen-activated protein kinase, and increased CK18 expression consistent with cell differentiation. In contrast, HGF/SF significantly stimulates the proliferation of DU145 prostate cancer cells. HGF/SF in the conditioned medium of PrSC specifically induces migration of both normal and malignant prostate epithelial cells through MatriGel-coated Transwell filters. HGF/SF depletion reduces cell migration by approximately 50%. The response of PrEC is specific for HGF/SF since the other growth factors tested do not significantly affect growth or migration of PrECs. These results support the in vivo importance of the prostate stroma and specifically of HGF/SF as a unique stromal derived factor in the development and progression of prostate cancer.

Animals↗
PubMed2000

The geldanamycins are potent inhibitors of the hepatocyte growth factor/scatter factor-met-urokinase plasminogen activator-plasmin proteolytic network.

The Met receptor tyrosine kinase and its ligand, hepatocyte growth factor/scatter factor (HGF/SF), have been implicated in human tumor development and metastasis. HGF/SF induces the expression of urokinase plasminogen activator (uPA) and the uPA receptor (uPAR), important mediators of cell invasion and metastasis. We have developed a cell-based assay to screen for inhibitors of this signaling system using the induction of endogenous uPA and uPAR and the subsequent conversion of plasminogen to plasmin as the biological end point. Assay validation was established using a neutralizing antiserum to HGF/SF and a uPA inhibitor (B428), as well as inhibitors of the MKK-MAPK1/2 pathway, shown previously to be important in the induction of uPA and uPAR. Using this assay, we found several classes of molecules that exhibited inhibition of HGF/SF-dependent plasmin activation. However, we discovered that certain members of the geldanamycin family of anisamycin antibiotics are potent inhibitors of HGF/SF-mediated plasmin activation, displaying inhibitory properties at femtomolar concentrations and nine orders of magnitude below their growth inhibitory concentrations. At nanomolar concentrations, the geldanamycins down-regulate Met protein expression, inhibit HGF/SF-mediated cell motility and invasion, and also revert the phenotype of both autocrine HGF/SF-Met transformed cells as well as those transformed by Met proteins with activating mutations. Thus, the geldanamycins may have important therapeutic potential for the treatment of cancers in which Met activity contributes to the invasive/metastatic phenotype.

3T3 Cells↗
PubMed1998

The mutationally activated Met receptor mediates motility and metastasis.

Mutations in Met have been identified in human papillary renal carcinomas. We have shown previously that these mutations deregulate the enzymatic activity of Met and that NIH 3T3 cells expressing mutationally activated Met are transformed in vitro and are tumorigenic in vivo. In the present investigation, we find that mutant Met induces the motility of Madin-Darby canine kidney cells in vitro and experimental metastasis of NIH 3T3 cells in vivo, and that the Ras-Raf-MEK-ERK signaling pathway, which has been implicated previously in cellular motility and metastasis, is constitutively activated by the Met mutants. We also report that transgenic mice harboring mutationally activated Met develop metastatic mammary carcinoma. These data confirm the tumorigenic activity of mutant Met molecules and demonstrate their ability to induce the metastatic phenotype.

3T3 Cells↗
PubMed1998

Evidence for a role of Met-HGF/SF during Ras-mediated tumorigenesis/metastasis.

Aberrations in Met-hepatocyte growth factor/scatter factor (HGF/SF) signaling have been implicated in the acquisition of tumorigenic and metastatic phenotypes. Here we show that murine NIH3T3 and C127 cells transformed by the Ras oncogene overexpress the Met receptor, resulting in enhanced HGF/SF-mediated responses in vitro including invasion through basement membrane. Accompanying the increase in Met in ras-transformed NIH3T3 cells, there is a decrease in endogenous HGF/SF expression as previously observed in cells exogenously overexpressing Met. However, subcutaneously grown tumors and experimental lung metastases derived from these cells express significantly higher levels of endogenous HGF/SF together with high levels of Met. These results suggest Met-HGF/SF signaling enhances tumor growth and metastasis of Ras-transformed NIH3T3 cells.

3T3 Cells↗
PubMed1998

Decreased fibronectin expression in Met/HGF-mediated tumorigenesis.

The tyrosine kinase receptor Met and its ligand, hepatocyte growth factor (HGF)/scatter factor are involved in the etiology and progression of a number of human cancers. Coexpression of Met and HGF in mesenchymal cells increases the tumorigenic and metastatic potential of the cells. In the studies described here, we used differential display screening to identify changes in gene expression that are initiated by Met/HGF, and that may lead to these phenotypes. We learned that Met/HGF signaling resulted in greatly decreased fibronectin mRNA production in three different human and mouse tumor cell lines; these decreases in fibronectin mRNA were paralleled by decreases in fibronectin protein. We also found a progressive decrease in fibronectin in tumor explants and metastases derived from the Met/HGF transformed cells. The absence of fibronectin expression is a frequent cancer phenotype; our results indicate that decreases in fibronectin correlate with, but are not essential for, MetHGF/SF-mediated tumorigenesis.

Animals↗
PubMed1998

Signaling pathways in Ras-mediated tumorigenicity and metastasis.

The effector domain mutants of oncogenic Ras, V12S35 Ras, V12G37 Ras, and V12C40 Ras were tested for their abilities to mediate tumorigenic and metastatic phenotypes in athymic nude mice when expressed in NIH 3T3 fibroblasts. All mutants displayed comparable tumorigenic properties, but only the mutant that activates the Raf-mitogen-activated protein kinase kinase (MEK)-extracellular regulated kinase (ERK) 1/2 pathway, V12S35 Ras, induced tumors in the experimental metastasis assay. Furthermore, direct activation of the MEK-ERK1/2 pathway in NIH 3T3 cells by mos or a constitutively active form of MEK was sufficient to induce metastasis whereas R-Ras, which fails to activate the ERK1/2 pathway, is tumorigenic but nonmetastatic. The subcutaneous tumors and lung metastases derived from V12S35 Ras-transformed NIH 3T3 cells expressed higher levels of activated ERK1/2 in culture when compared with the parental cellular pool before injection, indicating that selection for cells with higher levels of activated ERK1/2 occurred during tumor growth and metastasis. By contrast, cells explanted from V12G37-Ras or V12C40-Ras-induced tumors did not show changes in the level of ERK1/2 activation when compared with the parental cells. When tumor-explanted cell lines derived from each of the effector domain mutants were passaged one additional time in vivo, all mediated rapid tumor growth, but, again, only cells derived from V12S35 Ras-tumors formed numerous metastatic lesions within the lung. These results show that the metastatic properties of the Ras effector domain mutants segregate, and that, whereas Ras-mediated tumorigenicity can arise independently of ERK1/2 activation, experimental metastasis appears to require constitutive activation of the ERK1/2 pathway.

3T3 Cells↗
PubMed1998

Proteolytic inactivation of MAP-kinase-kinase by anthrax lethal factor.

Anthrax lethal toxin, produced by the bacterium Bacillus anthracis, is the major cause of death in animals infected with anthrax. One component of this toxin, lethal factor (LF), is suspected to be a metalloprotease, but no physiological substrates have been identified. Here it is shown that LF is a protease that cleaves the amino terminus of mitogen-activated protein kinase kinases 1 and 2 (MAPKK1 and MAPKK2) and that this cleavage inactivates MAPKK1 and inhibits the MAPK signal transduction pathway. The identification of a cleavage site for LF may facilitate the development of LF inhibitors.

Animals↗
PubMed1997

Activating mutations for the met tyrosine kinase receptor in human cancer.

Recently, mutations in the Met tyrosine kinase receptor have been identified in both hereditary and sporadic forms of papillary renal carcinoma. We have introduced the corresponding mutations into the met cDNA and examined the effect of each mutation in biochemical and biological assays. We find that the Met mutants exhibit increased levels of tyrosine phosphorylation and enhanced kinase activity toward an exogenous substrate when compared with wild-type Met. Moreover, NIH 3T3 cells expressing mutant Met molecules form foci in vitro and are tumorigenic in nude mice. Enzymatic and biological differences were evident among the various mutants examined, and the somatic mutations were generally more active than those of germ-line origin. A strong correlation between the enzymatic and biological activity of the mutants was observed, indicating that tumorigenesis by Met is quantitatively related to its level of activation. These results demonstrate that the Met mutants originally identified in human papillary renal carcinoma are oncogenic and thus are likely to play a determinant role in this disease, and these results raise the possibility that activating Met mutations also may contribute to other human malignancies.

3T3 Cells↗
PubMed1996

Involvement of the NMDA receptor in a non-cholinergic action of acetylcholinesterase in guinea-pig substantia nigra pars compacta neurons.

Evidence is accumulating that a soluble, secretory form of acetylcholinesterase may have novel, non-cholinergic functions in certain brain regions, such as the substantia nigra. In this study, application of human recombinant acetylcholinesterase (rhAChE) to pars compacta neurons in the rostral substantia nigra resulted in a sustained hyperpolarization that was not only mimicked by application of N-methyl-D-aspartate (NMDA) but also blocked by the NMDA receptor antagonists MK8O1 and 2-amino-5-phosphonopentanoic acid. Neither the rhAChE- nor the NMDA-induced hyperpolarization was seen when the calcium chelator BAPTA was injected into the neuron; hence the effect is mediated by accumulation of intracellular calcium. This intracellular calcium appears sufficient to compromise neuronal metabolism since the rhAChE-induced hyperpolarization was reversed by application of the K-ATP channel antagonist tolbutamide. Butyrylcholinesterase, a protein of similar molecular weight to acetylcholinesterase, which also hydrolyses acetylcholine, had no effect whatsoever. The results suggest that, independent of its normal catalytic function, acetylcholinesterase can act via the NMDA receptor complex to enhance calcium entry into nigral neurons and jeopardize cell metabolism. This non-classical action of acetylcholinesterase might thus be an important factor in the mechanisms underlying parkinsonian neurodegeneration.

2-Amino-5-phosphonovalerate↗
PubMed1996

C-Met signalling in an HGF/SF-insensitive variant MDCK cell line with constitutive motile/invasive behaviour.

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.

Animals↗
PubMed1992

Non-cholinergic effects of acetylcholinesterase in the substantia nigra: a possible role for an ATP-sensitive potassium channel.

Within the substantia nigra acetylcholinesterase has non-cholinergic actions that can be demonstrated at both behavioural and cellular levels: the aim of this study was, thus, to explore, in the in vitro guinea pig substantia nigra, the ionic mechanisms which mediate these non-classical phenomena. Acetylcholinesterase had a reversible hyperpolarizing action, via an opening of potassium channels, on a selective population of nigral neurons. These neurons could be identified by an ability to generate bursts of action potentials and by a sensitivity to either amphetamine or to a reduction of glucose in the perfusing medium. The acetylcholinesterase-induced hyperpolarization could not be attributed to a contaminant in the exogenous solution, since a highly purified preparation was even more potent. Furthermore, enzymatic action of any kind could be eliminated as boiled acetylcholinesterase was equally efficacious. The effect of acetylcholinesterase was not subject to tachyphylaxis and was resistant to blockade of potassium channels with tetraethylammonium: since both these phenomena are features of the D2 autoreceptor for dopamine within the substantia nigra, it seems unlikely that acetylcholinesterase is operating on the same target as dendritically released local dopamine. On the other hand, the actions of acetylcholinesterase were enhanced by low glucose and blocked by the sulfonylurea, tolbutamide. These results strongly suggest that acetylcholinesterase can exert a nonenzymatic action and that this action, in the substantia nigra, is mediated by an ATP-sensitive potassium channel.

Acetylcholinesterase↗
PubMedDate not supplied

Genes that regulate metastasis and angiogenesis.

Genetic instability and an accumulation of genetic and epigenetic changes during tumor progression lead to an increasingly aggressive and treatment-resistant phenotype, and ultimately metastasis. In recent years it has become well established that angiogenesis, the process by which new vasculature is formed from pre-existing vessels, is an essential component to primary tumor growth and distant metastasis. A greater understanding of the complex multitude of factors involved in tumor angiogenesis and metastasis is fundamental to the development of potential therapeutics to treat malignant disease. As highlighted throughout this review, angiogenesis and metastasis share many common cellular and molecular features. We will briefly discuss the pertinent genes involved in the regulation of angiogenesis and metastasis.

Angiogenesis Inhibitors↗
PubMedDate not supplied

Hepatocyte growth factor expression in human cancer and therapy with specific inhibitors.

Hepatocyte growth factor/Scatter Factor (HGF/SF) mediated stimulation of the Met receptor tyrosine kinase results in pleiotropic cellular effects including proliferation, morphogenesis, motility and invasion. In vivo, HGF/SF-Met activation has been shown to participate in tumorigenesis, angiogenesis and metastasis. Coupled with accumulating evidence that aberrant HGF/SF-Met expression is frequently observed in a variety of human tumors, often in association with progressive disease, these data present HGF/SF-Met as an attractive target for therapeutic intervention in human cancer. In this review, we will present the most compelling evidence suggesting a key role for HGF/SF-Met signaling in tumorigenesis, and discuss several possible therapeutic strategies.

Animals↗