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Biomedical subjects

G F Vande Woude

Publications and source records attributed to G F Vande Woude.

At least 19 recordsLinked to original sources
PubMed2006

Evidence that MIG-6 is a tumor-suppressor gene.

Mitogen-inducible gene 6 (MIG-6) is located in human chromosome 1p36, a locus frequently associated with human lung cancer. MIG-6 is a negative regulator of epidermal growth factor (EGF) signaling, and we show that Mig-6 - like EGF - is induced by hepatocyte growth factor/scatter factor (HGF/SF) in human lung cancer cell lines. Frequently, the receptors for both factors, EGFR and Met, are expressed in same lung cancer cell line, and MIG-6 is induced by both factors in a mitogen-activated protein kinase-dependent fashion. However, not all tumor lines express MIG-6 in response to either EGF or HGF/SF. In these cases, we find missense and nonsense mutations in the MIG-6 coding region, as well as evidence for MIG-6 transcriptional silencing. Moreover, germline disruption of Mig-6 in mice leads to the development of animals with epithelial hyperplasia, adenoma, and adenocarcinoma in organs like the lung, gallbladder, and bile duct. These data suggests that MIG-6 is a tumor-suppressor gene and is therefore a candidate gene for the frequent 1p36 genetic alterations found in lung cancer.

Adaptor Proteins, Signal Transducing↗
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

Inactivation of LRG-47 and IRG-47 reveals a family of interferon gamma-inducible genes with essential, pathogen-specific roles in resistance to infection.

The cytokine interferon (IFN)-gamma regulates immune clearance of parasitic, bacterial, and viral infections; however, the underlying mechanisms are poorly understood. Recently, a family of IFN-gamma-induced genes has been identified that encode 48-kD GTP-binding proteins that localize to the endoplasmic reticulum of cells. The prototype of this family, IGTP, has been shown to be required for host defense against acute infections with the protozoan parasite Toxoplasma gondii, but not for normal clearance of the bacterium Listeria monocytogenes and murine cytomegalovirus (MCMV). To determine whether other members of the gene family also play important roles in immune defense, we generated mice that lacked expression of the genes LRG-47 and IRG-47, and examined their responses to representative pathogens. After infection with T. gondii, LRG-47-deficient mice succumbed uniformly and rapidly during the acute phase of the infection; in contrast, IRG-47-deficient mice displayed only partially decreased resistance that was not manifested until the chronic phase. After infection with L. monocytogenes, LRG-47-deficient mice exhibited a profound loss of resistance, whereas IRG-47-deficient mice exhibited completely normal resistance. In addition, both strains displayed normal clearance of MCMV. Thus, LRG-47 and IRG-47 have vital, but distinct roles in immune defense against protozoan and bacterial infections.

Amino Acid Sequence↗
PubMed2001

Neutralizing monoclonal antibodies to hepatocyte growth factor/scatter factor (HGF/SF) display antitumor activity in animal models.

The hepatocyte growth factor (HGF/SF) receptor, Met, regulates mitogenesis, motility, and morphogenesis in a cell type-dependent fashion. Activation of Met via autocrine, paracrine, or mutational mechanisms can lead to tumorigenesis and metastasis and numerous studies have linked inappropriate expression of this ligand-receptor pair to most types of human solid tumors. To prepare mAbs to human HGF/SF, mice were immunized with native and denatured preparations of the ligand. Recloned mAbs were tested in vitro for blocking activity against scattering and branching morphogenesis. Our results show that no single mAb was capable of neutralizing the in vitro activity of HGF/SF, and that the ligand possesses a minimum of three epitopes that must be blocked to prevent Met tyrosine kinase activation. In vivo, the neutralizing mAb combination inhibited s.c. growth in athymic nu/nu mice of tumors dependent on an autocrine Met-HGF/SF loop. Importantly, growth of human glioblastoma multiforme xenografts expressing Met and HGF/SF were markedly reduced in the presence of HGF/SF-neutralizing mAbs. These results suggest interrupting autocrine and/or paracrine Met-HGF/SF signaling in tumors dependent on this pathway is a possible intervention strategy.

Animals↗
PubMed2001

Down-regulation of MET, the receptor for hepatocyte growth factor.

The ligand-dependent degradation of activated tyrosine kinase receptors provides a means by which mitogenic signalling can be attenuated. In many cell types the ligand-dependent degradation of the tyrosine kinase receptor Met is completely dependent on the activity of the 26S proteasome (Jeffers et al., 1997b). We now show that degradation also requires trafficking to late endosomal compartments and the activity of acid dependent proteases as determined by the effects of a dominant negative form of dynamin (K44A) and a vacuolar-ATPase inhibitor, concanamycin. We show that in the presence of the proteasome inhibitor lactacystin, Met fails to redistribute from the plasma membrane to intracellular compartments. This observation is most consistent with the interpretation that proteasome activity is required for Met internalization and only indirectly for its degradation.

Acetylcysteine↗
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

Anti-apoptotic signaling by hepatocyte growth factor/Met via the phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase pathways.

Hepatocyte growth factor (HGF) is a ligand of the receptor tyrosine kinase encoded by the c-Met protooncogene. HGF/Met signaling has multifunctional effects on various cell types. We sought to determine the role of HGF/Met in apoptosis and identify signal transducers involved in this process. In experiments with human SK-LMS-1 leiomyosarcoma cells, we show that the Akt kinase is activated by HGF in a time- and dose-dependent manner by phosphatidylinositol 3-kinase (PI3-kinase). Akt is also activated by active tumorigenic forms of Met, i.e., ligand-independent Tpr-Met, a truncated and constitutively dimerized form of Met, and a mutationally activated version of Met corresponding to that found in human hereditary papillary renal carcinoma. In NIH 3T3 cells transfected with wild-type Met, HGF inhibits apoptosis induced by serum starvation and UV irradiation. HGF-induced survival correlates with Akt activity and is inhibited by the specific PI3-kinase inhibitor LY294002, indicating that HGF inhibits cell death through the PI3-kinase/Akt signal transduction pathway. Furthermore, transiently transfected Tpr-Met activates Akt (both Akt1 and Akt2) and protects cells from apoptosis. Mitogen-activated protein kinase (MAPK) also is activated by HGF and rescues cells from apoptosis, although the cytoprotective effect is less marked than for PI3-kinase/Akt. Blocking MAPK with the specific MAPK kinase inhibitor PD098059 impairs the ability of HGF to promote cell survival. Similar results were obtained with NIH 3T3 cells expressing the fusion protein Trk-Met and stimulated with nerve growth factor, the Trk ligand. These results demonstrate that HGF/Met is capable of protecting cells from apoptosis by using both PI3-kinase/Akt and, to a lesser extent, MAPK pathways.

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

Met receptor tyrosine kinase: enhanced signaling through adapter proteins.

The Met receptor tyrosine kinase is the prototypic member of a small subfamily of growth factor receptors that when activated induce mitogenic, motogenic, and morphogenic cellular responses. The ligand for Met is hepatocyte growth factor/scatter factor (HGF/SF) and while normal HGF/SF-Met signaling is required for embryonic development, abnormal Met signaling has been strongly implicated in tumorigenesis, particularly in the development of invasive and metastatic phenotypes. Following ligand binding and autophosphorylation, Met transmits intercellular signals using a unique multisubstrate docking site present within the C-terminal end of the receptor. The multisubstrate docking site mediates the binding of several adapter proteins such as Grb2, SHC, Crk/CRKL, and the large adapter protein Gab1. These adapter proteins in turn recruit several signal transducing proteins to form an intricate signaling complex. Analysis of how these adapter proteins bind to the Met receptor and what signal transducers they recruit have led to more substantial models of HGF/SF-Met signal transduction and have uncovered new potential pathways that may be involved into Met mediated tumor cell invasion and metastasis.

Animals↗
PubMed2000

A novel germ line juxtamembrane Met mutation in human gastric cancer.

Activating mutations in the Met receptor tyrosine kinase, both germline and somatic, have been identified in human papillary renal cancer. Here we report a novel germline missense Met mutation, P1009S, in a patient with primary gastric cancer. The dosage of the mutant Met DNA was elevated in the tumor when compared to its matched normal DNA. Therefore, as with hereditary renal papillary cancer, the mutant Met allele may also be selectively duplicated in the tumor. Different from previously reported Met mutations, which occur in the tyrosine kinase domain, this missense mutation is located at the juxtamembrane domain, and is not constitutively activated. However, following treatment with HGF/SF, the P1009S mutant Met protein, expressed in NIH3T3 cells, displays increased and persistent tyrosine phosphorylation compared to the wild-type Met. Importantly, these cells also form colonies in soft agar, and are highly tumorigenic in athymic nude mice. A second nucleotide change in this region of Met, T1010I, was found in a breast cancer biopsy and a large cell lung cancer cell line. Although this previously reported 'polymorphism' did not stimulate NIH3T3 cell growth in soft agar, it was more active than the wild-type Met in the athymic nude mice tumorigenesis assay, suggesting that it may have effects on tumorigenesis. Met has been shown to be highly expressed in human gastric carcinoma cell lines, and our results raise the possibility that activating missense Met mutations could contribute to tumorigenesis of gastric cancer.

3T3 Cells↗
PubMed2000

Hepatocyte growth factor-stimulated invasiveness of monocytes.

Hepatocyte growth factor (HGF) is a pluripotent cytokine with mitogenic, motogenic, and morphogenic activity for mainly epithelial and endothelial target cells. We previously demonstrated that the specific HGF receptor, MET, is induced in stimulated peripheral blood monocytes. In this study, we analyzed the functional consequences of MET activation in primary cultures of peripheral blood monocytes from healthy donors. After stimulation of MET-expressing monocytes with recombinant HGF, the gene-expression profile of peripheral blood mononuclear cells and monocytes was significantly modulated, especially with regard to genes involved in cell movement. After stimulation of primary cultured monocytes with HGF, invasion assays showed a significantly increased matrigel invasion rate that was completely abolished by neutralizing antibodies to HGF. The HGF-activated invasiveness and the altered gene-expression profile suggest a proinflammatory role for HGF stimulation of monocytes and support the hypothesis that the HGF/MET signaling system plays an important part in the activation of the nonspecific cellular inflammatory response. (Blood. 2000;95:3964-3969)

Cell Division↗
PubMed2000

Dominant negative Met reduces tumorigenicity-metastasis and increases tubule formation in mammary cells.

Activation of the Met tyrosine kinase growth factor receptor by its ligand HGF/SF has been shown to increase in vitro invasiveness in epithelial cell lines. To study the effect of Met-HGF/SF signaling in breast cancer cells, we transfected met, hgf/sf and dominant negative (DN) forms of met into the poorly differentiated metastatic murine mammary adenocarcinoma cell line DA3. These cells express moderate levels of endogenous Met, which is rapidly phosphorylated in response to HGF/SF treatment. Met+hgf/sf transfection results in significantly increased tumorigenic and metastatic activity in vivo accompanied by reduced tubule formation. DA3 cells transfected with DN forms of Met (DN-DA3) exhibit reduced Met phosphorylation following exposure to HGF/SF. Furthermore, as compared to the parental cells, the DN-DA3 cells exhibit diminished in vitro scattering and invasiveness, while in vivo they display greatly reduced tumorigenicity and spontaneous metastasis. Tumors emanating from DN-DA3 cells injected to BALB/C mice are highly differentiated and display extensive tubule formation. These results suggest that Met-HGF/SF signaling is a determining factor in the delicate balance between differentiation/tubule formation and tumorigenicity-metastasis. Oncogene (2000) 19, 2386 - 2397

Adenocarcinoma↗
PubMed2000

Regulation of P311 expression by Met-hepatocyte growth factor/scatter factor and the ubiquitin/proteasome system.

P311 is a mouse cDNA originally identified for its high expression in late-stage embryonic brain and adult cerebellum, hippocampus, and olfactory bulb. The protein product of P311, however, had not been identified previously, and its function remains unknown. We report here that P311 expression is regulated at multiple levels by pathways that control cellular transformation. P311 mRNA expression was decreased sharply in both neural and smooth muscle cells when the cells were transformed by coexpression of the oncogenic tyrosine kinase receptor Met and its ligand hepatocyte growth factor/scatter factor. The P311 mRNA was found to encode an 8-kDa polypeptide that was subject to rapid degradation by the lactacystin-sensitive ubiquitin/proteasome system and an unidentified metalloprotease, resulting in a protein half-life of about 5 min. These data suggest that P311 expression is dramatically decreased by several pathways that regulate cellular growth.

Acetylcysteine↗
PubMed2000

Pathogen-specific loss of host resistance in mice lacking the IFN-gamma-inducible gene IGTP.

Interferon-gamma (IFN-gamma) is critical for defense against pathogens, but the molecules that mediate its antimicrobial responses are largely unknown. IGTP is the prototype for a family of IFN-gamma-regulated genes that encode 48-kDa GTP-binding proteins that localize to the endoplasmic reticulum. We have generated IGTP-deficient mice and found that, despite normal immune cell development and normal clearance of Listeria monocytogenes and cytomegalovirus infections, the mice displayed a profound loss of host resistance to acute infections of the protozoan parasite Toxoplasma gondii. By contrast, IFN-gamma receptor-deficient mice have increased susceptibility to all three pathogens. Thus, IGTP defines an IFN-gamma-regulated pathway with a specialized role in antimicrobial resistance.

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↗
PubMed1999

Ras oncogene-induced sensitization to 1-beta-D-arabinofuranosylcytosine.

Human tumor cells containing ras oncogenes display enhanced sensitivity to 1-beta-D-arabinofuranosylcytosine (Ara-C) and other deoxycytidine analogues (H-M. Koo, et al., Cancer Res., 56: 5211-5216, 1996). Human tumor cell lines with or without a ras oncogene as well as a pair of isogenic cell lines with one containing an activated ras oncogene were used to study the basis for differential sensitivity. We found that human tumor cells containing ras oncogenes upon entry into the S phase of the cell cycle underwent apoptosis in response to Ara-C treatment. By contrast, human tumor cells harboring wild-type ras alleles were only delayed in the S phase when exposed to Ara-C. Thus, the ras oncogene specifically renders human cells more sensitive to Ara-C by preventing S-phase arrest. This may occur by the ras oncogene compromising an S-phase checkpoint.

Antimetabolites, Antineoplastic↗