Cooperative Roles of Hepatocyte Growth Factor and Plasminogen Activator in Tubular Morphogenesis by Human Microvascular Endothelial Cells

Epidermal growth factor (EGF) or transforming growth factor-α (TGF-α) stimulated cell migration, chemotaxis, and the expression of tissue-type plasminogen activator (t-PA) in human omental microvascular endothelial (HOME) cells. Hepatocyte growth factor (HGF) stimulated cell proliferation, but had a negligible stimulatory effect on cell migration, the expression of t-PA and tube-like formation into collagen gel in HOME cells. Basic fibroblast growth factor stimulated cell proliferation, cell migration, tubulogenesis and the expression of urokinase-type plasminogen activator (u-PA) in bovine aortic endothelial (BAE) cells. HOME and BAE cells had both high- and low-affinity receptors for HGF. In BAE cells, u-PA activity and tube-like structures in collagen gel were induced in the presence of HGF alone. In contrast, in HOME cells, t-PA activity and tube-like structures were induced in the presence of TGF-a alone, but not in the presence of HGF alone. However, we observed a marked induction of tube formation by HOME cells when both t-PA and HGF were added simultaneously. In the model system for tumor angiogenesis, when HOME cells were co-cultured with a renal cancer cell line, KPK13, tube-like structures were induced in the presence of HGF: KPK13 cells expressed large amounts of t-PA mRNA. Our present study suggested that HGF in concert with active t-PA could be angiogenic in HOME cells.     

Role of Hepatocyte Growth Factor in Hemopoiesis

Hepatocyte growth factor (HGF) is a polypeptide that stimulates proliferation, motility, and morphogenesis of various cells, particularly epithelial cells. There is considerable evidence that HGF is a regulator in hemopoiesis not only in mice but also in humans. In mice, HGF and c-met (its receptor) mRNA are coexpressed in the fetal liver in the middle and late stages, when hemopoiesis is most active. HGF and c-met mRNA are also expressed in the stromal cells of both fetal liver and bone marrow. Human HGF (2 to 20 ng/ml) enhances colony-forming units in culture (CFU-C) counts and cobblestone colony counts in the long-term cultures of the fetal liver and bone marrow, although HGF has no effect on freshly isolated bone marrow or fetal liver cells in the CFU-C assay. However, when the bone marrow or fetal liver cells are cocultured with HGF in the presence of IL-3, CFU-C counts increase. In humans, it has also been shown that HGF in the presence of erythropoietin induces the formation of erythroid burst-forming unit (BFU-E) colonies from CD34⁺ cells purified from the bone marrow, peripheral blood, or cord blood. This review discusses the role of HGF as a regulator in hemopoiesis.     

Human Lung Cancer Cell Line Producing Hepatocyte Growth Factor/Scatter Factor

Hepatocyte growth factor (HGF)/scatter factor (SF) is a cytokine which is produced by mesenchymal cells and stimulates the motility of some epithelial cells, including cancer cells and vascular endothelial cells. Two human lung cancer cell lines, PC-1 and PC-13, were found to produce a protein which was indistinguishable from HGF/SF with regard to biological activities and immunological characteristics, although they were derived from epithelial cells. In general, highly aggressive cancer cells often show some mesenchymal characteristics, and production of HGF/SF by cancer cells is also considered as a phenomenon of acquisition of mesenchymal phenotype, which may be involved in cancer invasion and progression. These cell lines showed no apparent response to exogenous HGF/SF. In addition, no c-met proto-oncogene product was detectable in these cells by Western blot analysis. Although the function of HGF/SF produced by cancer cells, either autocrine or paracrine stimulation, remains to be studied, this is the first report to describe cancer cells producing HGF/SF.     

肝细胞生长因子及C-Met的表达与宫颈癌浸润和转移的关系

目的：研究肝细胞生长因子（HGF）及其受体C—Met在宫颈癌组织中的表达．探讨二者与宫颈癌浸润和转移的关系。方法：采用RT—PCR检测36例宫颈癌组织手术标本及31例正常宫颈组织标本中HGF mRNA和C—Met mRNA的表达，并进行相对定量研究。结果：宫颈癌组织中C—Met的阳性表达率显著高于正常宫颈组织：宫颈癌组织中，淋巴结转移组C—Met的表达水平高于未转移组；临床分期Ⅱ期组的表达水平高于IB期组；中、低分化组的表达水平高于高分化组。结论：宫颈癌组织中存在C—Met的高表达．肿瘤细胞丰富的C—Met受体可能是通过与旁分泌途径获得的HGF结合而在宫颈癌浸润和转移的过程中发挥重要作用。     

血清肝细胞生长因子水平与乳腺癌转移的关系

目的:研究血清肝细胞生长因子(hepatocytegrowthfactory,HGF)与乳腺癌转移的关系。方法:应用双抗体夹心ELISA方法检测80例术后复发转移乳腺癌患者,50例术后无复发乳腺癌患者和20例献血员的血清肝细胞生长因子水平。结果:术后复发转移组血清HGF水平(0.90±0.52ng/ml)显著高于无复发组(0.38±0.30ng/ml;P<0.01)和正常对照组(0.22±0.36ng/ml;P<0.01)。肝转移组患者血清HGF水平(1.25±0.57ng/ml)显著高于其它转移组(0.78±0.48ng/ml;P<0.01)。多处转移组血清HGF水平(1.10±0.66ng/ml)显著高于其它转移组(0.78±0.48ng/ml;P<0.05)。结论:血清HGF水平与乳腺癌肝转移和多处转移发生密切相关。     

Acquisition of Invasive Phenotype in Gallbladder Cancer Cells via Mutual Interaction of Stromal Fibroblasts and Cancer Cells as Mediated by Hepatocyte Growth Factor

Growth and motility of carcinoma cells are regulated through their interactions with host stromal cells, i. e., tumor-stromal interactions. Hepatocyte growth factor (HGF), a ligand for c-Met tyrosine kinase, is a stromal-derived regulator of growth, motility, and morphogenesis. HGF stimulated proliferation and motility of GB-d1 gallbladder carcinoma cells from a patient with gallbladder cancer. HGF induced in vitro invasion of GB-d1 cells into a collagen gel matrix, and this potent, invasive effect was not seen with epidermal growth factor, transforming growth factor-β1, basic fibroblast growth factor, or platelet-derived growth factor. Although GB-d1 did not produce HGF, the cells did produce a factor which enhances HGF production in human skin fibroblasts, and this factor proved to be interleuldn-1β (IL-1β). When GB-d1 cells were co-cultured with fibroblasts such that a collagen gel matrix was layered between the GB-d1 cells and fibroblasts, GB-d1 cells invaded the gel, but invasion of the cells in the co-culture system was inhibited by antibodies against HGF and partially inhibited by antibodies against IL-1β. Thus, GB-d1 cell-derived IL-1β stimulates HGF production in stromal fibroblasts and HGF up-regulated in the fibroblasts induces invasion of GB-d1 cells. The looped interaction of carcinoma cells and stromal fibroblasts mediated by HGF and a HGF-inducer such as IL-1β may be one mechanism which would explain the acquisition of malignant phenotype through tumor-stromal interactions.     

Immunohistochemical Localization of Hepatocyte Growth Factor Protein in Pancreas Islet A-Cells of Man and Rats

Hepatocyte growth factor (HGF), a potent mitogen for adult rat hepatocytes in primary culture, has previously been shown to be primarily expressed in the nonparenchymal cells of the liver. Using polyclonal antisera against human and rat HGFs we studied the tissue distribution of HGF immunohistochemically and found the most intense staining in the pancreas islet cells in both man (autopsy cases) and the rat. Differential localization of 4 pancreas islet hormones, glucagon, insulin, somatostatin and pancreatic polypeptide, revealed HGF to be preferentially expressed within the glucagon-positive cells. The results indicate that HGF is primarily produced or stored in A-cells and may act as a growth factor in a paracrine and an endocrine fashion, like various other hormones.     

Establishment of a Hepatocyte Cell Line Producing Growth-promoting Factors for Liver-colonizing Tumor Cells

A hepatocyte-derived cell line designated MLE-15A2 was established from a primary culture of mouse hepatocytes. The MLE-15A2 cells appeared to retain the basic nature of hepatocytes in that they showed morphology of an epithelial cell type and secreted albumin into the culture medium. These cells were grown on collagen-coated plates and could be easily expanded to a large-scale culture. Therefore, MLE-15A2 cells may provide a more useful model for studying liver microenvironments than primary cultures of hepatocytes. We found that conditioned media from MLE-15A2 cells, as well as from primary cultures of hepatocytes, promoted the proliferation of highly liver-colonizing colon 26 NL-17 cells better than the poorly liver-colonizing colon 26 NL-4 cells. Moreover, the conditioned media stimulated the growth of some human colon cancer cell lines. These results indicate that MLE-15A2 cells secrete growth factors that selectively stimulate certain tumor cell types. Hepatocyte-derived growth factors may regulate selective survival and colonization of tumor cells in the process of liver metastasis. The growth-promoting activity was unaffected by dialysis, was stable at 80°C for 30 min and was bound to a heparin-Sepharose column. The major activity was eluted from the column with 0.7–0.75 M NaCl, and some minor activities eluted with lower concentrations of NaCl. These results suggest that the active components are heterogeneous heparin-binding proteins with lower affinity to heparin than platelet-derived and fibroblast growth factors.     

Transforming Growth Factor-β and Hepatocyte Growth Factor Produced by Gastric Fibroblasts Stimulate the Invasiveness of Scirrhous Gastric Cancer Cells

Scirrhous gastric carcinoma is characterized by cancer cells that infiltrate rapidly in the stroma with extensive growth of fibroblasts. In the present study, we examined the effect of gastric fibroblasts on the invasiveness of a Scirrhous gastric cancer cell line, OCUM-2D, using an invasion assay. Gastric fibroblast-derived conditioned medium (CM) significantly stimulated the invasiveness of OCUM-2D cells, as did transforming growth factor- β (TGF- β ) and hepatocyte growth factor (HGF). The stimulating activity of gastric fibroblast-derived CM was inhibited significantly by anti-TGF- β neutralizing antibody or anti-HGF neutralizing antibody. TGF- β and HGF were detected in the gastric fibroblast-derived CM, and TGF- β receptor and C-met (HGF receptor) were expressed on OCUM-2D cells. Thus, TGF- β and HGF produced by gastric fibroblasts appear to affect the invasiveness of scirrhous gastric cancer cells. TGF- β was also detected in the conditioned medium derived from OCUM-2D cells, though HGF was not. TGF- β appears to affect the invasiveness of OCUM-2D cells in both paracrine and autocrine fashions.     

HGF和其受体c-met在NSCLC组织中的表达及其与VEGF的关系

目的检测肝细胞生长因子（HGF）及其受体（c-met）和血管生长因子（VEGF）在非小细胞肺癌（NSCLC）中的表达，分析其与NSCLC临床病理参数的关系及HGF和VEGF相关关系。方法应用免疫组织化学方法SABC法对68例NSCLC组织中的HGF、c-met和VEGF进行检测。结果68例NSCLC组织中HGF、c-met和VEGF表达率分别为45．6％（31／68）、51．5％（35／68）和41．8％（28／68）。HGF的表达与组织类型、分化程度无关（P〉0．05）。c-met的表达与组织类型、分化程度相关（P〈0．01，P〈0．005）。VEGF的表达与组织类型无关（P〉0．05）而与分化程度相关（P〈0．05）。HGF的表达与c-met的表达呈明显正相关（P=0．000，γ=0．471），HGF的表达与VEGF的表达亦呈明显正相关（P=0．000，7=0．662）。结论HGF、c-met及VEGF的增强表达与NSCLC发展演进和肿瘤血管生成有密切关系。     

Hepatocyte Growth Factor Enhancement of Preneoplastic Hepatic Foci Development in Rats Treated with Diethylnitrosamine and N-Ethyl-N-hydroxyethylnitrosamine

Effects of hepatocyte growth factor were investigated in a two-stage rat liver carcinogenesis protocol. Male F344 rats were first treated with diethylnitrosamine (200 mg/kg, i.p.) and then, starting two weeks later, with N-ethyl-N-hydroxyethylnitrosamine (EHEN) for 6 weeks at a dose of 0.01% in drinking water. Hepatocyte growth factor, which was injected i.v. at a dose of 200 μg/kg body weight one (at week 3) or two times (at weeks 3 and 4) during EHEN administration, significantly increased the development of preneoplastic glutathione S-transferase placental form-positive foci. Although the observed effects of hepatocyte growth factor were weaker than that of the two-thirds partial hepatectomy (PH) performed at week 3, the present results suggest that the enhancing effects of PH performed during the promotion stage may be largely mediated through induction of hepatocyte growth factor.     

Immunohistochemical Detection of Hepatocyte Growth Factor/Scatter Factor in Human Cancerous and Inflammatory Lesions of Various Organs

Hepatocyte growth factor (HGF)/scatter factor (SF) is a multifunctional factor considered to be potentially involved in tissue regeneration, wound healing, embryogenesis, angiogenesis and cancer invasion. Here we examined immunohistochemically the distribution of HGF/SF in human tissues, including cancerous and inflammatory tissues, using anti-HGF antibody. HGF/SF accumulation was clearly detected in the extracellular matrix, particularly along the basement membrane, in cancerous and inflammatory tissues, but only a little was detected in normal tissues. HGF/SF is well known to have a strong affinity for heparin in vitro , and from the results of our immunohistochemical assay, we considered that HGF/SF was bound to heparin or heparan sulfate of the extracellular matrix and basement membrane. HGF/SF was well localized in cancerous and inflammatory lesions of human lung, liver and pancreas, and in apparently normal tissues of kidney, adrenal gland and pancreas obtained at autopsy. In lung, HGF/SF was localized along the basement membranes of cancer cell nests, in the extracellular matrix of the cancer cell surface, cancer stroma and tissues invaded by cancer, and the basement membranes of bronchial epithelium and capillary vessels in inflammatory stroma. Since HGF/SF makes some cancer cells more invasive in vitro , the accumulation of HGF/SF in cancerous tissue suggests that the invasiveness of some cancer cells may be increased by HGF/SF in vivo .     

Expression of Ly-6D on the Surface of Normal and Neoplastic Mammary Epithelial Cells of the Mouse

Rat were immunized with mouse mammary epithelial cells and monoclonal antibodies (MAbs) were obtained to identify antigens stably expressed on the surface of both normal and neoplastic mammary epithelial cells of the mouse. Examination of the reactivities of the MAbs by immunofluorescence staining of tissue sections showed that one of the antibodies, MAb 2A2, reacted with luminal epithelium of the mammary gland and spontaneous mammary carcinomas of SHN mice. Further examination of the tissue lysates by western blot analysis revealed that MAb 2A2 reacts with a 17-kDa antigen expressed in normal mammary epithelial cells and mammary carcinoma cells. The antigen recognized by MAb 2A2 was absent in the lysates of liver, lung, salivary gland, kidney, small intestine, ovary and uterus. After immunoaffinity purification of the MAb 2A2- recognized antigen and determination of its N-terminal amino acid sequence, we identified the antigen as Ly-6D, also known as ThB, which belongs to a family of glycosyl-phosphatidylinositol-anchored cell surface proteins. Northern blot analysis further demonstrated that Ly-6D mRNA is expressed in the mammary gland. Based on these observations we concluded that Ly-6D is stably expressed on the surface of both normal and neoplastic mammary epithelial cells of the mouse. Ly-6D will serve as a useful epithelial cell surface marker for the study of mammary gland development, as well as for breast cancer research.     

Expression of Type II and Type XI Collagens in Canine Mammary Mixed Tumors and Demonstration of Collagen Production by Tumor Cells in Collagen Gel Culture

The development of cartilaginous collagen types, II and XI, in canine mammary mixed tumors was studied biochemically and immunohlstochemically. In mixed tumor, an alcian blue-positive myxomatous region appeared in the stroma, where round-shaped proliferating myoepithelial cells were scattered. Type II collagen was distributed in metaplastic cartilage matrix, while type XI was located only in the pericellular region, where proliferating cells were positively stained with anti-actin and anti-keratin antibodies. The accumulation of collagen types II and XI in the tumor mass was confirmed by sodium do decyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting of the extract of the lesion using type-specific antibodies to collagen types II and XI. Tumor cells isolated from metaplastic tumor mass expressed both collagen types II and XI and myoepithelial types of cytoskeleton in gel culture, in which an alcian blue-positive substance became detectable in the pericellular region on day 3 and type II and type XI collagens on day 5. This may be a useful model for studying chondrocyte-type gene expression during tumorigenesis
.     

血清肝细胞生长因子水平与胃癌关系的研究

目的 探讨胃癌患者血清肝细胞生长因子(hepatocyte growth factor,HGF)水平变化的临床意义.方法 采用酶联免疫吸附实验法检测60例胃癌患者血清HGF水平,其中40例行胃癌根治术,12例因有远处转移而未手术,8例为胃癌术后复发,同时选取15例良性胃病患者以及12例门诊健康体检者作为对照,分别检测其血清HGF的水平,并进行比较分析.结果 胃癌组血清HGF水平高于良性胃病组和正常对照组(P＜0.05);胃癌术后复发组血清HGF水平明显高于良性胃病组和正常对照组(P＜0.01);40例胃癌术前组血清HGF水平明显高于胃癌术后组(P＜0.01);胃癌复发组血清HGF水平明显高于胃癌手术组和非手术组(P＜0.01).结论 HGF在胃癌的发生发展过程中可能起重要作用,血清HGF水平的检测可成为胃癌病情程度监测和预后判断的客观指标.     

The Dynamics of Tumor Cords in an Irradiated Mouse Mammary Carcinoma with a Large Hypoxic Cell Component

The tumor cord model represents a histologically based framework for interpretation of radiobiological phenomena, particularly the resistance to radiation conferred by absence of oxygen. For the mammary carcinoma T50/80 grown in B6D2F1 male mice, average oxygenation was poor, based on tumor growth delay after irradiation. There was no improvement in radiobiological oxygenation for several days after a high dose of radiation. This was consistent with events in the cords of the tumor, where although up to 20% of all cells became pyknotic by 8 hr, the cords did not shrink for at least 2 days. The cellular kinetics of populations of intact and dead cells, adjacent to and remote from the capillaries of the cords, were examined for up to 60 hr after irradiation and it was found that: (i) before treatment, average LI (adjacent) was 13% and LI (remote) was 2%, (ii) after irradiation, cells expressed pyknosis after passing through the S phase of the cell cycle, so that (iii) at early intervals there was a larger proportional rise in pyknotic cells in the adjacent than the remote zone. However, (iv) at later intervals there was always a higher proportion of dead cells in the remote zone.     

肝细胞生长因子及其受体c-met在卵巢癌细胞中的表达

 目的 探讨肝细胞生长因子（HGF）及其受体c-met蛋白表达与卵巢肿瘤临床病理特征间的关系。方法 采用免疫组织化学SABC法对不同病理类型的卵巢肿瘤组织中HGF／c-met蛋白的表达进行定位，定量分析研究。结果 在卵巢癌组织中，HGF阳性细胞呈黄色或棕黄色着色，且在上皮细胞中的表达较强；c-met在上皮细胞强着色，间质细胞则着色较弱。HGF和c-met在卵巢癌组织中表达较交界性肿瘤和良性肿瘤组织有统计学意义（P＜0.05）。手术病理分期Ⅲ ～ Ⅳ 期HGF和c-met蛋白表达也较Ⅰ～Ⅱ期显著升高（P＜0.05）。病理分级中、高分化与中分化、低分化间存在明显差异（P＜0.05）。结论 卵巢肿瘤组织中存在HGF和c-met蛋白的表达且两者存在相关关系，它们与卵巢癌的发生、侵袭和转移密切相关。     

Mitogenic Properties of Insulin-Like Growth Factors I and II, Insulin-like Growth Factor Binding Protein-3 and Epidermal Growth Factor on Human Breast Stromal Cells in Primary Culture

Insulin-like growth factors I and II (IGF-I and IGF-II) are growth factors implicated in both normal mammary gland development and breast cancer. We have previously reported on the effects of components of the IGF system on breast epithelial cells. Since data suggests that stromal-epithelial interactions play a crucial role in breast cancer, we have now investigated the mitogenic properties of IGF-I, IGF-II, insulin-like growth factor binding protein-3 (IGFBP-3) and epidermal growth factor (EGF) on human breast stromal cells in primary culture. We show that, under serum-free conditions, stromal cells are stimulated to grow in response to IGF-I and IGF-II in a dose-dependent manner. IGF-I and EGF, a potent stimulator of human breast epithelial cell growth in primary culture and also associated with breast cancer, appear to stimulate stromal cell growth in a synergistic manner. IGFBP-3 does not inhibit the stimulation of growth by IGF-I, or IGF-I plus EGF. However, IGFBP-3 does inhibit the stimulation of growth by IGF-II. In contrast to our previous results with human breast epithelial cells, IGFBP-3 does not have an IGF-independent inhibitory effect on stromal cell growth. This study is the first to address the effects of IGF-I, IGF-II and IGFBP-3 alone and in combination with EGF on human breast stromal cell growth in primary culture. Characterizing the role of the IGF system in both normal breast epithelial cells and stromal cells will aid in our understanding of the mechanisms behind the role of the IGF system in breast cancer.     

肝细胞生长因子和血管内皮生长因子的生物学特性研究进展

肝细胞生长因子(hepatocyte growth factor,HGF)与血管内皮生长因子(vascular endothelialgrowth factor,VEGF)在肿瘤细胞生长、浸润、转移过程中起重要的调节作用。近些年来,对这些生长因子及其受体的研究,无论是在基础方面,还是在临床病理学方面都取得了很大进展,为抗肿瘤治疗开辟了广阔的前景。对HGF与VEGF的特征及其生物学特性,以及目前研究的重要进展做一综述。