Hepatocyte Growth Factor/scatter Factor Research Articles

HGF/SF Activates Glycolysis and Oxidative Phosphorylation in DA3 Murine Mammary Cancer Cells

Hepatocyte growth factor/scatter factor (HGF/SF) is a paracrine growth factor which increases cellular motility and has also been implicated in tumor development and progression and in angiogenesis. Little is known about the metabolic alteration induced in cells following MetHGF/SF signal transduction. The hypothesis that HGF/ SF alters the energy metabolism of cancer cells was investigated in perfused DA3 murine mammary cancer cells by nuclear magnetic resonance (NMR) spectroscopy, oxygen and glucose consumption assays and confocal laser scanning microscopy (CLSM). 31P NMR demonstrated that HGF/SF induced remarkable alterations in phospholipid metabolites, enhanced the rate of glucose phosphorylation (P < .05). 13C NMR measurements, using [13C1] -glucose-enriched medium, showed that HGS/SF reduced the steady state levels of glucose and elevated those of lactate (P < .05). In addition, HGF/SF treatment increased oxygen consumption from 0.58±0.02 to 0.71±0.03 ymol/hour per milligram protein (P < .05). However, it decreased CO2 levels, attenuated pH decrease. The mechanisms of these unexpected effects were delineated by CLSM, using NAD(P)H fluorescence measurements, which showed that HGF/SF increased the oxidation of the mitochondrial NAD system. We propose that concomitant with induction of ruffling, HGF/SF enhances both the glycolytic and oxidative phosphorylation pathways of energy production.

HGF/SF-INDUCED SPREADING OF MDCK CELLS CORRELATES WITH DISAPPEARANCE OF BARMOTIN/7H6, A TIGHT JUNCTION-ASSOCIATED PROTEIN, FROM THE CELL MEMBRANE

Changes in expression of the two tight junction-associated proteins, barmotin/7H6 and ZO-1, as well as the adherence junction-associated protein, E-cadherin, were followed during hepatocyte growth factor/scatter factor (HGF/SC)-induced migration process of MDCK cells. Modulation of the HGF/SF-induced migration process by staurosporine, an inhibitor of protein kinase C (PKC), was also examined. Cell migration induced by HGF/SF consisted of two distinct phases, initial cell spreading between 2 and 9h after the start of treatment, and the scattering phase which started ∼12h after treatment. Both ZO-1 and E-cadherin were expressed at the cell–cell border of adherent cells in the scattering phase, whereas barmotin/7H6, a barrier function-related tight junction protein, was not seen during the early spreading phase. Confluent cultures of MDCK cells, which did not spread after HGF/SF treatment, were positive for barmotin/7H6 expression at cell–cell borders. Blocking PKC activation during HGF/SF treatment with staurosporine inhibited cell spreading, and the cells retained barmotin/7H6 expression until at 6h after HGF/SF treatment. The results indicate that disappearance of the tight junction protein, barmotin/7H6, is closely associated with cell spreading, with both barmotin/7H6 expression and cell spreading seemingly being regulated by PKC-mediated signaling.

Stimulation of DNA Synthesis and Cell Proliferation of Human Mammary Myoepithelial-like Cells by Hepatocyte Growth Factor/Scatter Factor Depends on Heparan Sulfate Proteoglycans and Sustained Phosphorylation of Mitogen-activated Protein Kinases p42/44

Hepatocyte growth factor/scatter factor (HGF/SF) is a heparan/dermatan sulfate-binding growth factor produced by stromal cells that acts as a paracrine effector on neighboring epithelia. HGF/SF stimulated DNA synthesis in human mammary (Huma) 109 myoepithelial-like cells grown on collagen I and fibronectin substrata but not when grown on plastic. Dual phosphorylation of mitogen-activated protein kinases (p42/44(MAPK)) was required for this stimulation of DNA synthesis. In Huma 109 cells cultured on plastic, HGF/SF stimulated a transient phosphorylation of p42/44(MAPK), which reached a maximum at 10 min after addition of the growth factor and returned to near basal levels after 20 min. In contrast, the phosphorylation of p42/44(MAPK) stimulated by HGF/SF in cells cultured on collagen I or fibronectin was sustained over 45 min. In Huma 109 cells deficient in sulfated glycosaminoglycans, HGF/SF failed to stimulate p42/44(MAPK) phosphorylation or DNA synthesis on any substratum, even when soluble heparan sulfate proteoglycans purified from the cells or from the culture medium were added. However, HGF/SF stimulated DNA synthesis and a sustained phosphorylation of p42/44(MAPK) in sulfated glycosaminoglycan-deficient Huma 109 cells plated on a substratum of medium HSPGs but not cell HSPGs. The HGF/SF-induced proliferation is thus highly dependent on heparan sulfate proteoglycans in myoepithelial-like cells.

Unique epithelial cell production of hepatocyte growth factor/scatter factor by putative precancerous intestinal metaplasias and associated "intestinal-type" biliary cancer chemically induced in rat liver.

Recently, we observed that Met, the receptor for hepatocyte growth factor/scatter factor (HGF/SF), is overexpressed in epithelial cells of both early-appearing intestinal metaplastic glands in precancerous hepatic cholangiofibrotic tissue and neoplastic glands in later developed intestinal-type of cholangiocarcinoma originated from the furan rat model of cholangiocarcinogenesis when compared with normal and hyperplastic intrahepatic biliary epithelia. We now show that HGF/SF is also aberrantly expressed in a manner closely paralleling that of its receptor in the neoplastic epithelial cells of furan-induced rat cholangiocarcinomas and in a majority of metaplastic epithelial cells within earlier formed precancerous hepatic cholangiofibrotic tissue. Using in situ hybridization and reverse transcription-polymerase chain reaction (RT-PCR), we further showed specific expression of HGF/SF messenger RNA (mRNA) in a novel rat cholangiocarcinoma epithelial cell line overexpressing Met. This cholangiocarcinoma cell line, termed C611B, was established from tumorigenic cells isolated from a furan-induced transplantable tumor. Moreover, we detected by in situ hybridization strong expression of HGF/SF mRNA transcripts in the cancerous epithelial glands of cholangiocarcinoma developed in recipient rats after in vivo cell transplantation of C611B cells. In contrast, mRNA transcripts and protein immunoreactivity for this cytokine were not detected in hepatocytes and biliary epithelial cells in adult normal rat liver nor in rat hyperplastic intrahepatic biliary epithelium. Our results clearly show that HGF/SF becomes aberrantly expressed in cholangiocarcinoma epithelium and in putative precancerous intestinal metaplastic epithelium induced in the liver of furan-treated rats.

Immunoreactivity for cadherins, HGF/SF, met, and erbB-2 in pleural malignant mesotheliomas.

To assess the immunoreactivity of malignant mesotheliomas for N- and E-cadherins, hepatocyte growth factor/scatter factor (HGF/SF) and the tyrosine kinase receptors, met and erbB-2. Pleural malignant mesotheliomas were stained using a standard indirect immunoperoxidase method applied to paraffin sections. Malignant mesotheliomas were immunoreactive for N-cadherin (26/29; 90%), met (29/29; 100%) and erbB-2 (28/29; 97%). Focal immunoreactivity was present for E-cadherin in epithelioid or mixed tumours (14/25; 56%), and for HGF/SF (9/24; 38%). Expression of N-cadherin supports the diagnosis of malignant mesothelioma and use of appropriate antibodies would be a useful addition to a diagnostic antibody panel. Focal staining for E-cadherin does not exclude mesothelioma. Signalling pathways mediated via met and erbB-2 may play a role in the growth and spread of malignant mesotheliomas.

Requirement of Regulated Activation of Ras for Response of MDCK Cells to Hepatocyte Growth Factor/Scatter Factor

Hepatocyte growth factor/scatter factor (HGF/SF) induces cell scattering, migration, and branching tubule formation of MDCK cells. To examine the role of the Ras protein in the HGF/SF-induced responses, we constructed MDCK cell clones expressing either inducible dominant-negative Ras or constitutively activated Ras and analyzed their effects on responses of cells to HGF/SF. Induced expression of dominant-negative Ras prevented cell dissociation required for cell scattering, migration, and cystic formation as well as branching morphology required for branching tubule formation. Constitutively activated Ras induced cell dissociation, but not a scattered fibroblastic morphology even in the presence of HGF/SF. MDCK cells expressing constitutively activated Ras migrated at a level similar to that of wild-type MDCK cells stimulated by HGF/SF. MDCK cells expressing constitutively activated Ras showed disorganized growth in three-dimensional culture and did not form the branching tubule structures. These results indicate that activation of the Ras protein is essential for the cell scattering, migration, and branching tubule formation of MDCK cells induced by HGF/SF, and a properly regulated activation is required for some stages of the HGF/SF-induced responses of MDCK cells.

Possible role of hepatocyte growth factor/scatter factor and activin A produced by the target organ in liver metastasis

The molecular mechanism of organ-specific metastasis to the liver remains largely unknown. However, it is conceivable that paracrine growth factors produced by a target organ induce migration and proliferation of malignant cells to that organ, and this is the cause of organ-specific metastasis. In this study, we investigated the effect of hepatocyte growth factor/scatter factor (HGF/SF) and activin A, which are known to be produced by the liver, on the motility and growth of liver-metastatic cell line FBJ-LL. HGF/SF and activin A induced motility synergistically, but they did not affect the proliferation of FBJ-LL cells. Expression of the HGF/SF receptor, the c-met gene, and the activin-receptor type IA, type IB, and type IIA genes in FBJ-LL cells was detected by reverse transcription polymerase chain reaction. These findings suggest that both HGF/SF and activin A promote organ-specific metastasis to the liver by induction of migration through their specific receptors on liver-metastatic cells.

Mechanism of Regulation of HGF/SF Gene Expression in Fibroblasts by TGF-β1

The effect of transforming growth factor β 1 (TGF-β1) on levels of hepatocyte growth factor/scatter factor (HGF/SF) gene transcripts was investigated in the human lung embryonic fibroblast cell line, MRC-5. TGF-β1 markedly reduced the expression of the 6.0-kb and 3.0-kb HGF/SF mRNA, which encode full-length HGF/SF, but it had little effect on the expression of the alternatively spliced 1.5-kb mRNA, which encodes NK2, a competitive HGF/SF antagonist. Using actinomycin D to block RNA synthesis, it was observed that TGF-β1 had little effect on the stability of the 1.5-kb NK2 mRNA but increased the rate of degradation of the 6.0- and 3.0-kb HGF/SF mRNA transcripts by a mechanism that was dependent on new protein synthesis. TGF-β1 minimally increased rather than reduced HGF/SF promoter activity in cells transiently transfected with chloramphenicol acetyltransferase (CAT) reporter genes driven by HGF/SF gene 5′-flanking sequences. In MRC-5 cells, TGF-β1 modulates HGF/SF gene transcripts at the posttranscriptional level in order to favour expression of the 1.5-kb mRNA that encodes the truncated protein NK2.

Signaling of Hepatocyte Growth Factor/Scatter Factor (HGF) to the Small GTPase Rap1 via the Large Docking Protein Gab1 and the Adapter Protein CRKL

Hepatocyte growth factor (HGF; scatter factor) is a multipotent protein with mitogenic, motogenic, and developmental functions. Upon activation, the HGF-receptor c-Met binds and phosphorylates the multisite docking protein Gab1. Besides binding motifs for phosphatidylinositol 3-kinase and Grb2, Gab 1 contains multiple Tyr-X-X-Pro (YXXP) motifs which, when phosphorylated, are potential binding sites for the adapter proteins c-Crk and Crk-like (CRKL). Stimulation of human embryonic kidney cells (HEK293) with HGF leads to Gab1 association with CRKL. The Gab1-CRKL interaction requires both, the SH2 domain of CRKL and the region containing the YXXP motifs in Gab1. CRKL binds via its first SH3 domain to several downstream signal transducers, including C3G an activator of the small GTPase Rap1. Indeed, Rap1 was rapidly activated after HGF stimulation of HEK293 cells. Rap1 activation through HGF was suppressed through transfection of a truncated C3G protein which only contains the SH3-binding motifs of C3G. Transfection of nonmutated Gab1 led to a strong increase of Rap1.GTP in the absence of HGF. In contrast, transfection of the GabDeltaYXXP mutant abolished the elevation of Rap1.GTP by HGF. A replating assay indicated that HGF decreases the adhesion of HEK293 cells. The results presented here delineate a novel signaling pathway from HGF to the GTPase Rap1 which depends on the interaction of the adapter protein CRKL with the exchange factor C3G and could be linked to cell migration.

Cortisol inhibits hepatocyte growth factor/scatter factor expression and induces c-met transcripts in osteoblasts.

Hepatocyte growth factor/scatter factor (HGF/SF) is expressed by osteoblasts and has important effects on repair and bone remodeling. Because glucocorticoids regulate these two functions, we tested the effects of cortisol on the expression of HGF/SF and c-met, the protooncogene encoding the HGF/SF receptor, in cultures of osteoblast-enriched cells from 22-day fetal rat calvariae (Ob cells). Cortisol decreased HGF/SF mRNA levels and diminished the induction of HGF/SF transcripts by fibroblast growth factor-2 (FGF-2) and platelet-derived growth factor BB (PDGF BB). Cortisol also decreased FGF-2 and PDGF BB-induced HGF/SF mRNA and polypeptide levels in MC3T3 cells. In contrast, cortisol enhanced the expression of c-met transcripts in Ob cells. Cortisol did not modify the half-life of HGF/SF or of c-met mRNA in transcriptionally arrested cells, and it increased the rate of transcription of c-met. In conclusion, cortisol decreases HGF/SF transcripts in Ob cells and enhances c-met expression transcriptionally. The effects of cortisol on HGF/SF could be relevant to its inhibitory actions on bone formation and repair.

Nk4, a new HGF/SF variant, is an antagonist to the influence of HGF/SF on the motility and invasion of colon cancer cells

Hepatocyte Growth Factor/Scatter Factor (HGF/SF) is a heterodimeric molecule that plays a key role in the regulation of migration, invasion and angiogenesis in cancer, via activation of its receptor, c-met. HGF/SF is composed of an alpha-chain, containing the N-terminal hairpin domain and 4 kringle domains, plus the serine protease-like beta-chain. We have examined here the properties of NK4, an HGF variant containing the N-terminal hairpin plus the 4 kringle domains, on tumour cell proliferation, dissociation and invasion using human colorectal cancer cells (HT115). The expression of HGF/SF and its receptor was also examined by RT-PCR and Western blotting. Analysis revealed NK4 to be an HGF/SF antagonist that, at a wide range of concentrations, did not exert any biological effects of its own. HT115 cells were shown to express the HGF/SF receptor mRNA and protein. HGF/SF-induced receptor tyrosine phosphorylation was suppressed, in a dose-dependent manner, upon addition of NK4, whereas the addition of NK4 alone caused no phosphorylation. Tumour cell motility was induced by HGF and inhibited by NK4. Furthermore, HGF/SF induced the invasion of cells through Matrigel basement membrane components, and again this induced invasion was suppressed by NK4. Our results show that the ability of HGF/SF to stimulate tumour cell motility and invasion, properties required for metastatic spread, can be inhibited by NK4. Thus, NK4 may have an important role in the control of cancer metastasis.

HGF is an autocrine growth factor for skeletal muscle satellite cells in vitro.

Muscle satellite cell activation following injury is essential for muscle repair, and hepatocyte growth factor/scatter factor (HGF) was the first growth factor shown to be able to stimulate activation and early division of adult satellite cells in culture and in muscle tissue. In addition, HGF was shown to be present in uninjured and injured skeletal muscle. Experiments in this report demonstrate that cultured satellite cells also synthesize and secrete HGF. Reverse transcription-polymerase chain reaction (RT-PCR) was used to demonstrate the presence of HGF mRNA in cultured adult satellite cells as early as 12 h from the time of plating. Message content was detectable at early times in culture and appeared to increase between 36 and 48 h. HGF protein expression was demonstrated during this time period by immunofluorescence localization; HGF was localized to mononucleated cells and multinucleated myotubes. HGF message was not detectable in muscle-derived fibroblast clones, and fibroblast-like cells in satellite cell cultures were negative for HGF by immunofluorescence analysis. Furthermore, Western blot analysis revealed the presence of HGF in satellite cell culture conditioned medium, associated with the cell surface and inside cells. Finally, the addition of neutralizing HGF antibodies during the proliferation phase in culture (42-90 h) significantly reduced cell proliferation. These experiments indicate that HGF is expressed by cultured satellite cells and that endogenous HGF from satellite cells can act in an autocrine fashion. Because HGF plays a central role in satellite cell activation, it is likely that direct administration of HGF into damaged muscle may represent a potentially useful approach for stimulating muscle repair. This approach may also be useful in enhancing the efficiency of myoblast transplantation in vivo.

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.

Hepatocyte growth factor/scatter factor (HGF/SF) increases cutaneous wound healing in the diabetes mouse

HGF/SF is a heparin binding polypeptide that stimulates cell proliferation, motility and morphogenesis via activation of it's receptor c-Met tyrosine kinase. It has also been shown that HGF/SF is a potent angiogenic factor in a sponge implant model in rats. To evaluate the potential HGF/SF has to heal chronic cutaneous wounds we have topically applied recombinant murine HGF/SF to full thickness excisional injuries using the diabetes mouse model. The diabetes mouse is the result of a spontaneous mutation in an inbred mouse strain. Homozygote animals (db/db) are obese, hyperglycemic and exhibit impaired cutaneous wound healing compared to their wildtype littermates.

The regulation of normal melanocyte proliferation.

In vitro, normal human melanocytes require synergistic mitogens, in addition to the common growth factors present in serum, in order to proliferate. The peptide growth factors that confer stimulation are fibroblast growth factors (such as bFGF/FGF2), hepatocyte growth factor/scatter factor (HGF/SF), mast/stem cell factor (M/SCF), endothelins (such as ET-1) and melanotropin (MSH). The proper function of these factors and their cognate receptors is likely to be important in vivo, as all five ligands are produced in the skin, and disruption of their normal function, by elimination due to deletions or mutations, or overproduction due to ectopic expression, disrupts the normal distribution of melanocytes. The synergistic growth factors activate intracellular signal transduction cascades and maintain the intermediate effectors at optimal levels and duration required for nuclear translocation and modification of transcription factors. The consequent induction of immediate-early response genes, such as cyclins, and subsequent activation of cyclin-dependent kinases (CDK4, CDK6 and CDK2) inactivates the retinoblastoma family of proteins (pRb, p107 and p130, together termed pocket proteins), and releases their suppressive association with E2F transcription factors. Molecular events that disrupt this tight control of pocket proteins and cause their inactivation, increase E2F transcriptional activity and confer autonomous growth on melanocytes.

Intravenous Injection of an Adenovirus Encoding Hepatocyte Growth Factor Results in Liver Growth and Has a Protective Effect Against Apoptosis

Hepatocyte growth factor/scatter factor (HGF/SF) is a pleiotropic cytokine with mitogenic, motogenic and morphogenic effects for a wide variety of cells. Previous studies have reported that the in vivo infusion in normal, untreated mice of recombinant HGF results in low levels of DNA synthesis and liver proliferation. In this study, we examined whether liver regeneration could be obtained by the in vivo injection of a recombinant adenoviral vector encoding human HGF (Ad.CMV.rhHGF) in normal, intact mice. C57BL/6 mice were infused intravenously with doses increasing from 1 to 4 x 1011 particles of the recombinant human HGF (rhHGF) adenoviral vector or with a control virus encoding Escherichia coli beta-galactosidase (Ad.CMV.lacZ). At day 5, mice were sacrificed and evaluated for the presence of hepatocyte mitogenesis and liver regeneration (5-bromo-2'-deoxyuridine (BrdU) assays and liver weight determination) and for the presence of liver damage (serum alanine amino-transferase (ALT) measurements and TUNEL assays). In vivo administration of rhHGF stimulated DNA synthesis of hepatocytes and liver weight in a dose-dependent fashion. The maximal effect was seen after the infusion of 3 x 1011 particles which resulted at day 5 in >130% increase in relative liver mass with little cytopathic effect. In contrast, administration of the lacZ adenoviral vector caused little hepatocyte replication, but induced high levels of serum ALT (approximately 3 times higher than the rhHGF vector) and significant apoptotic cell death. This study shows that a single injection of Ad.CMV.rhHGF alone is able to induce in vivo and in a very short period of time, robust DNA synthesis and liver proliferation in normal mice without liver injury or partial hepatectomy. This recombinant adenoviral vector has a lower toxicity than the control lacZ adenovirus. This suggests that HGF may have a protective effect against adenovirus-induced pathology.

Hepatocyte growth-factor-scatter factor can stimulate post-operative tumor-cell proliferation in childhood hepatoblastoma

Rapid growth of residual tumor after partial hepatectomy has been observed during the period of liver regeneration in children with malignant embryonal hepatoblastoma. The aim of this study was to elucidate the role of hepatocyte growth-factor-scatter factor (HGF-SF) in this phenomenon. Markedly increased serum levels of HGF-SF up to 15 ng/ml were found in 13/18 patients after liver resection and in 6/16 patients with regressive tumors after chemotherapy, in comparison with 15 patients with non-pre-treated hepatoblastoma and 20 healthy children of the same age group. In the tumors, epithelial tumor cells highly expressed the HGF-SF receptor c-met, as shown by immunohistochemistry and m-RNA RT-PCR. The hepatoblastoma cell lines HepT1, HepT3 and HUH6 reacted with significantly increased proliferation to rhHGF-SF in these concentrations (1-15 ng/ml). In the tumors, HGF-SF was found to be expressed in the stromal fibroblasts. In culture, hepatoblastoma cells (HepT3, HUH6) stimulated secretion of the factor by human fibroblasts, indicating the paracrine fashion of intratumoral HGF-SF production. Cultured hepatoblastoma cells ceased to proliferate at 20-50 ng/ml HGF-SF, and they underwent cell death at ≥100 ng/ml. In contrast, the hepatocellular-carcinoma cell line HepG2 decreased growth under HGF-SF in a dose-dependent manner. We conclude that post-operatively secreted and intratumorally produced HGF-SF can function as a growth factor for hepatoblastoma, while the same agent has a cytostatic effect in unphysiologically high concentrations. Int. J. Cancer 85:151–159, 2000. ©2000 Wiley-Liss, Inc.

Protein-protein interactions in receptor activation and intracellular signalling.

We review here signalling complexes that we have defined using X-ray analysis in our laboratory. They include growth factors and their receptors: nerve growth factor (NGF) and its hetero-hexameric 7S NGF storage complex, hepatocyte growth factor/scatter factor (HGF/SF) NK1 dimers and fibroblast growth factor (FGF1) in complex with its receptor (FGFR2) ectodomain and heparin. We also review our recent structural studies on intracellular signalling complexes, focusing on phosducin transducin GPry, CK2 protein kinase and its complexes, and the cyclin D-dependent kinase, Cdk6, bound to the cell cycle inhibitor p19INK4d. Comparing the structures of these complexes with others we show that the surface area buried in signalling interactions does not always give a good indication of the strength of the interactions. We show that conformational changes are often important in complexes with intermediate buried surface areas of 1500 to 2000 A2, such as Cdk6INK4 interactions. Some interactions involve recognition of continuous epitopes, where there is no necessity for a tertiary structure and very often the binding conformation is induced during the process of interaction, for example phosducin binding to the betagamma subunits (Gtbetagamma) of the heterotrimeric G protein transducin.

Hepatocyte growth-factor-scatter factor can stimulate post-operative tumor-cell proliferation in childhood hepatoblastoma.

Rapid growth of residual tumor after partial hepatectomy has been observed during the period of liver regeneration in children with malignant embryonal hepatoblastoma. The aim of this study was to elucidate the role of hepatocyte growth-factor-scatter factor (HGF-SF) in this phenomenon. Markedly increased serum levels of HGF-SF up to 15 ng/ml were found in 13/18 patients after liver resection and in 6/16 patients with regressive tumors after chemotherapy, in comparison with 15 patients with non-pre-treated hepatoblastoma and 20 healthy children of the same age group. In the tumors, epithelial tumor cells highly expressed the HGF-SF receptor c-met, as shown by immunohistochemistry and m-RNA RT-PCR. The hepatoblastoma cell lines HepT1, HepT3 and HUH6 reacted with significantly increased proliferation to rhHGF-SF in these concentrations (1-15 ng/ml). In the tumors, HGF-SF was found to be expressed in the stromal fibroblasts. In culture, hepatoblastoma cells (HepT3, HUH6) stimulated secretion of the factor by human fibroblasts, indicating the paracrine fashion of intratumoral HGF-SF production. Cultured hepatoblastoma cells ceased to proliferate at 20-50 ng/ml HGF-SF, and they underwent cell death at >/=100 ng/ml. In contrast, the hepatocellular-carcinoma cell line HepG2 decreased growth under HGF-SF in a dose-dependent manner. We conclude that post-operatively secreted and intratumorally produced HGF-SF can function as a growth factor for hepatoblastoma, while the same agent has a cytostatic effect in unphysiologically high concentrations.

Absence of mutations in the kinase domain of the Met gene and frequent expression of Met and HGF/SF protein in primary gastric carcinomas.

Hepatocyte growth factor/scatter factor (HGF/SF) and its receptor, Met, are known to play important roles in tumor cell migration, invasion, and metastasis. We analyzed the expression of these genes and the mutations in the kinase domain of the Met gene in 43 gastric carcinomas. Of the 43 cases, the Met and HGF/SF protein were expressed in 29 (67%) and 22 (51%), respectively. All of the cases with HGF/SF immunopositivity also expressed Met. Of 22 cases with HGF/SF immunopositivity, 13 (59%) expressed HGF/SF in tumor cells as well as fibroblasts. We detected no aberrant single-strand conformational polymorphism patterns, suggesting that there are no genetic alterations in the kinase domain of the Met gene. These results indicate that HGF/SF-mediated autocrine and/or paracrine stimulation and overexpression rather than structural alteration of its receptor may contribute to the development and progression of gastric carcinoma, and that expression of Met and HGF/SF may confer a growth advantage to neoplastic cells.