Over-expression of hepatocyte growth factor/scatter factor (HGF/SF) and the HGF/SF receptor (cMET) are associated with a high risk of metastasis and recurrence for children and young adults with papillary thyroid carcinoma

OBJECTIVE: The study determined if hepatocyte growth factor/scatter factor (HGF/SF) or the HGF/SF receptor (cMET) might be important for metastasis in thyroid cancer. DESIGN: We examined HGF/SF and cMET expression by immunohistochemistry in a retrospective group of benign and malignant thyroid lesions from children and young adults, and correlated the intensity of expression with clinical outcome. PATIENTS: Patients included 42 children and young adults with papillary thyroid carcinomas (PTC), seven with follicular thyroid carcinomas (FTC), two with medullary thyroid carcinomas (MTC), 14 with benign thyroid disorders, and two with normal thyroids. MEASUREMENTS: Expression of cMET was graded from 0 (absent) to 4 (intense); and HGF/SF expression was graded from 0 (absent-minimal) to 3 (diffuse and intense). RESULTS: cMET staining was greater in PTC (mean intensity 2.3 +/- 0.4 vs. 0.8 +/- 0.2, P < 0.005) and FTC (2.4 +/- 0.6 vs. 0.8 +/- 0.2, P = 0.04) than benign lesions (0.8 +/- 0.2) or normal thyroids (0.4 +/- 0.5). PTC with intense cMET staining had shorter disease free survival (P = 0.05) and increased HGF/SF staining (r = 0.39, P = 0.017). HGF/SF correlated with the extent of disease at diagnosis (r = 0.33, P = 0.049). Patients with PTC were stratified into quartiles based on combined cMET and HGF/SF staining. Those with intense cMET and HGF/SF staining were younger (P = 0.05), and had reduced disease free survival (P = 0.03). CONCLUSIONS: We conclude that increased cMET and HGF/SF expression is associated with a high risk for metastasis and recurrence in children and young adults with papillary thyroid carcinoma.     

Nitric oxide modulates hepatocyte growth factor/scatter factor-induced angiogenesis

Objective: There is limited knowledge about potential therapeutic targets in Hepatocyte growth factor/scatter factor (HGF)-induced pathophysiological angiogenesis. Recent candidates have included phosphatidylinositol-3-kinase, which is an upstream activator for endothelial nitric oxide (NO) synthase (NOS III). The current study is the first to evaluate the possible involvement of NOS-NO cascade in HGF-induced angiogenesis. Methods and results: NOS III inhibitors blocked the HGF-induced functional neovascularization in vivo, as quantified using vessel counts, ¹³³Xe-clearance, and immunohistology. This was reversed by L-arginine. Western blot analysis of HGF-treated cells also revealed a temporal increase in HGF-induced phosphorylation. In a deconstructional approach, HGF induced the proliferation and chemokinesis of human endothelial cells. These phenotypic effects were inhibited by NOS inhibitors, L-NAME and L-NIO, and the NO scavenger, carboxy PTIO, but unaltered by 1400W, a NOS II inhibitor. This inhibition was reversed by spermine NONOate, a NO donor, which independently exerted a biphasic effect on endothelial cell proliferation. The modulation of NO did not alter HGF-induced chemoinvasion of endothelial cells, while spermine-NONOate destabilized HGF-induced tubulogenesis, suggesting that a single assay is not sufficient for predicting the final phenotypic outcome on angiogenesis. Conclusions: The study is the first to demonstrate that the NOS III nitric oxide is a key signal cascade in HGF-induced angiogenesis, and represents a promising target for the clinical management of pathological conditions characterized by overt HGF signaling.     

Essential role of HGF (hepatocyte growth factor) in blood formation in Xenopus

In this study, we investigated the role of hepatocyte growth factor (HGF) in blood formation during Xenopus development. First, we examined the gene expression of HGF and its receptor, c-met, by whole-mount in situ hybridization during development. Strong signals of HGF as well as c-met were detected early in the developing ventral mesoderm, which later gives rise to the ventral blood island. Furthermore, to study the role of HGF, we blocked the HGF signaling pathway in Xenopus embryos by using truncated c-met lacking the tyrosine kinase domain. Injection of truncated c-met mRNA resulted in a marked decrease in the number of circulating blood cells. Similar results were obtained using morpholino antisense HGF oligonucleotides. Moreover, we also analyzed the expression of several early primitive blood markers in the blood island of these embryos. RNA in situ analysis revealed a significant reduction (or absence) of stem cell leukemia (SCL), -globin, and GATA-1 expression, but not GATA-2 expression. In contrast, no significant difference was observed in the levels of expression of early definitive blood markers, SCL, GATA-2, and GATA-3 in the dorsolateral plate, as analyzed by in situ hybridization. Overall, the present study demonstrated that HGF is necessary for primitive hematopoiesis by regulating the expression of SCL. (Blood. 2004;103:3320-3325)      

Elevated levels of osteoprotegerin (OPG) and hepatocyte growth factor (HGF) in rheumatoid arthritis

Rheumatic diseases are often associated with changes in bone metabolism. Excessive production and release of cytokines and other growth factors due to inflammation, e.g. tumor necrosis factor-alpha (TNF-alpha), receptor activator of NF-kappaB ligand (RANKL), interleukins such as IL-1 and IL-6, may cause alterations in bone homeostasis leading to bone degradation. Other components such as osteoprotegerin (OPG) and possibly the ligand-receptor pair hepatocyte growth factor (HGF) and c-met may counteract this destruction, we have measured the levels of OPG, and HGF c-met, in serum, synovial fluid (SF), and cartilage from patients with rheumatoid arthritis (RA) and other arthritides. We found a) elevated levels of both OPG and HGF in SF from RA patients relative to arthritides of other causes, b) increased levels of both OPG and HGF in SF from seropositive RA patients (RA+) compared to seronegative RA patients (RA-), c) elevated levels or both OPG and HGF in serum from RA patients compared to healthy controls, d) no correlation between severity of inflammation and levels of OPG or HGF, and e) presence of HGF c-met in both cartilage and synovial tissue. The most significant elevations of OPG and HGF were found in patients with RA, the rheumatic disease most frequently associated with the development of secondary osteoporosis.     

Expression of hepatocyte growth factor (HGF) and HGF receptor (c-met) proteins in liver diseases: an immunohistochemical study

BACKGROUND: Hepatocyte growth factor (HGF) is a potent mitogen for hepatocytes in vivo as well as in vitro. Serum levels of HGF vary in liver diseases, reflecting liver damage and dysfunction. However there are no studies reporting expression of HGF and HGF receptor (c-met protein) simultaneously in various liver diseases. METHODS: To clarify the clinical significance of HGF/c-met protein expression in liver diseases, liver tissues from 62 patients consisting of 7 with acute hepatitis (AH), 20 with chronic hepatitis (CH), 9 with liver cirrhosis (LC) and 26 with hepatocellular carcinoma (HCC) were immunohistochemically examined. RESULTS: Intense staining of HGF was observed in patients from AH, CH and LC, although no immunoreactivity was seen in HCC. The expression of c-met protein was higher in patients with HCC and AH than in those with CH (p < 0.05). A correlation of immunoreactivity between HGF and c-met protein was not observed expect in patients with LC (p < 0.01). The extent of c-met expression had no correlation with differentiation of HCC, tumour size, presence of portal invasion, or serum AFP levels. CONCLUSION: The results of the present study suggest that HGF plays an important role in human liver diseases, mostly in a manner independent of c-met protein expression.     

Production and characterization of immortalized rat hepatocytes secreting hepatocyte growth factor/scatter factor

BACKGROUND/AIMS: Hepatocyte transplantation is a recently attractive field in the treatment of liver failure and enzyme-deficient diseases. However, procurement of sufficient quantities of hepatocytes is almost impossible. We attempted to create a hepatocyte cell line that could be used for hepatocyte transplantation. METHODOLOGY: L2A2 is a conditionally immortalized rat hepatocyte cell line produced by transfection of temperature-sensitive simian virus T antigen to the hepatocytes in the Lewis rat. Hepatocyte Growth Factor/Scatter Factor (HGF/SF)-secreting L2A2 cells, designated as SF-21, was produced by transfecting human HGF/SF cDNA into L2A2 cells. RESULTS: This cell line was able to produce HGF/SF at the rate of 5-10 ng/10(6) cells/24 hrs, and the recombinant HGF/SF was of the expected size and was functionally active in that it could scatter Madin-Darby canine kidney cells. The SF-21 cells grew faster than its parental cell clone, and survived and proliferated at 37 degrees C in vitro. Also, the SF-21 cells were able to survive and proliferate when transplanted into the spleen of syngeneic rat, and expressed glucose-6-phosphatase. CONCLUSIONS: These HGF/SF-secreting hepatocytes can be used as a model system to test a feasibility of using genetically engineered hepatocyte cell line for hepatocyte transplantation in the rat.     

Serum levels of the angiogenic cytokines basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) in multiple myeloma

Angiogenesis is a crucial process in growth and progression of cancer and there is growing evidence that neovascularisation is important in hematological malignancies. Since an increased angiogenic potential has been identified in multiple myeloma, we simultaneously measured circulating serum levels of the cytokines bFGF, VEGF, HGF and IL-6 by ELISA in 67 patients with multiple myeloma or monoclonal gammopathies of undetermined significance (MGUS) and in 20 controls. Median values of bFGF were 4.7 pg/ml in healthy volunteers, 6.2 in MGUS, 6.3 in myeloma stage I, 13.4 in stage II and 21.7 in stage III. Myeloma patients had significantly higher bFGF serum levels than controls (p<0.001). Pretreatment bFGF levels differed significantly in the Salmon and Durie stages I-III (p=0.02) and were significantly elevated in stage II-III compared to stage I myeloma (p=0.02). In patients responding to chemotherapy according to the CLMTF criteria, a significant decrease in serum bFGF, VEGF and HGF levels occurred (median pretreatment values for bFGF 23.9 pg/ml, post-treatment 6.5 pg/ml; p<0.001, for VEGF 223 pg/ml versus 105 pg/ml; p=0.02 and for HGF 1429 pg/ml versus 1077 pg/ml; p=0.02, respectively). In 11 patients who did not achieve a remission, there was no significant decrease in bFGF, VEGF and HGF levels. These data show that myeloma in stages II and III is associated with an increase in serum bFGF concentrations and give the first report that effective chemo-therapy is accompanied by a significant decrease in the angiogenic factors bFGF, VEGF and HGF, while no decrease of these factors could be found in nonresponders.     

离散因子/肝细胞生长因子cDNA转染对肝癌SMMC7721细胞生长和转移的影响

目的 通过离散因子／肝细胞生长因子（scatter factor/Hepatocyte factor,SF/HGF）cDNA转染肝癌SMMC7721细胞来探讨SF／HGF对肝癌生长和转移的影响。方法 用脂质体法进行基因转染,以ELISA和western blot检测SF／HGF及其受体c-met的表达,通过生长曲线、划良实验比较转染前后细胞的增长状况及细胞的运动能力。以转染前后细胞分别接种裸鼠,观察移植的生长及转移情况。结果 转染后细胞表达SF／HGF的量为694pg/ml,其受体c-met量未见明显变化;转染后细胞增殖明显加快,运动能力增强,并伴明显的形态学变化。初步动物实验显示转染后细胞生长较快,瘤组织中有瘤栓形成,并在肺内发现转移灶。结论 SF／HGF在肝癌细胞中的高表达能促进肝癌细胞生长、侵袭及转移。     

Neutralizing and nonneutralizing monoclonal antibodies to the human granulocyte-macrophage colony-stimulating factor receptor alpha-chain

A panel of monoclonal antibodies was raised against the low-affinity human granulocyte-macrophage colony-stimulating factor (hGM-CSF) receptor alpha-chain expressed as recombinant protein on murine FDC-P1 cells. All the selected antibodies were of the IgG2A isotype and bound to protein A. They each recognized both native and recombinant receptors by indirect surface immunofluorescence and by immunoprecipitation. Several of the antibodies also recognized presumably denatured receptors as detected by immunoblotting of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three different epitopes on the extracellular domain of the GM-CSF receptor alpha-chain were defined by these antibodies, and two of the epitopes did not appear to be involved in binding hGM-CSF or in interactions with the beta-chain of the GM-CSF receptor that are required for high-affinity binding of GM-CSF. On the other hand, the epitope recognized by antibody 2B7-17-A appeared to be critically involved in the binding of GM-CSF because this antibody completely abrogated both high- and low- affinity binding of GM-CSF to native and recombinant receptors. Antibody 2B7-17-A had a relatively high affinity for the GM-CSF receptor alpha-chain (kd = 3 nmol/L) and slow dissociation kinetics (kd = 0.002 min-1). These properties made the 2B7-17-A antibody a potent inhibitor of hGM-CSF biologic action in several different bioassays, with a half-maximal inhibitory dose of about 6 nmol/L (1 microgram/mL). This antibody could prove useful in alleviating any pathologic states mediated by excess GM-CSF levels and in defining the domains of the GM- CSF receptor required for ligand binding.     

Multiple myeloma cells catalyze hepatocyte growth factor (HGF) activation by secreting the serine protease HGF-activator

Multiple myeloma (MM) is a common hematologic neoplasm consisting of malignant plasma cells, which expand in the bone marrow. A potential key signal in the evolution of MM is hepatocyte growth factor (HGF), which acts as a potent paracrine and/or autocrine growth factor and survival factor for MM cells. Proteolytic conversion of HGF into its active form is a critical limiting step in HGF/MET signaling. Here, we show that malignant MM plasma cells convert HGF into its active form and secrete HGF-activator (HGFA), a serine protease specific for HGF activation. By using serine protease inhibitors and neutralizing antibodies, we demonstrate that HGFA produced by the MM cells is responsible for their ability to catalyze HGF activation. We, therefore, suggest that autocatalyzation of HGF conversion by MM cells is an important step in HGF/MET-induced myeloma growth and survival, which may have implications for the management of this incurable form of cancer.     

The protooncogene c-sea encodes a transmembrane protein-tyrosine kinase related to the Met/hepatocyte growth factor/scatter factor receptor.

c-sea is the cellular homologue of the avian erythroblastosis virus S13-encoded oncogene v-sea. We have isolated and determined the nucleotide sequence of overlapping chicken cDNAs that encode the putative c-sea protooncogene product. The predicted reading frame encoded a 1404-aa polypeptide that had the structure of a receptor-like protein-tyrosine kinase and exhibited the highest degree of sequence similarity with the Met/hepatocyte growth factor/scatter factor receptor. Analysis of steady-state RNA expression revealed that c-sea mRNA levels were elevated approximately 5-fold in chicken embryo cells transformed by activated variants of the src nonreceptor protein-tyrosine kinase gene but not in cells transformed by the nuclear oncogenes v-myc or v-rel. A survey of c-sea expression in a variety of chicken tissues indicated that the highest levels of mRNA were located in peripheral white blood cell populations and in the intestine.     

Transfer of motogenic and invasive response to scatter factor/hepatocyte growth factor by transfection of human MET protooncogene.

The MET protooncogene encodes p190MET, a tyrosine kinase which is the receptor for a molecule known as scatter factor or hepatocyte growth factor (SF/HGF). This molecule has different biological activities, including stimulation of cell motility, promotion of matrix invasion and, in some cells, mitogenesis. We have cloned the full-length MET cDNA and transfected it into NIH 3T3 fibroblasts. Stable transfectants expressed the p190MET receptor together with two previously described truncated forms of 140 and 130 kDa lacking the tyrosine kinase domain. All three forms bound radiolabeled SF/HGF. The factor stimulated tyrosine kinase activity of the transfected p190MET and induced changes in cell shape, migration in Boyden chambers, and invasion of collagen matrices in vitro. The motile and invasive phenotype was transient and strictly dependent on the presence of SF/HGF. The factor did not stimulate either cell growth or thymidine incorporation in transfected cells, while it promoted colony formation in soft agar in the presence of 5% fetal calf serum. These data show that, in the presence of its ligand, the MET receptor expressed in fibroblasts induces cells to pursue a motogenic-invasive rather than a proliferative program.