c-Src kinase activity is required for hepatocyte growth factor-induced motility and anchorage-independent growth of mammary carcinoma cells

Overexpression and amplification of hepatocyte growth factor (HGF) receptor (Met) have been detected in many types of human cancers, suggesting a critical role for Met in growth and development of malignant cells. However, the molecular mechanism by which Met contributes to tumorigenesis is not well known. The tyrosine kinase c-Src has been implicated as a modulator of cell proliferation, spreading, and migration; these functions are also regulated by Met. To explore whether c-Src kinase is involved in HGF-induced cell growth, a mouse mammary carcinoma cell line (SP1) that co-expresses HGF and Met and a nonmalignant epithelial cell line (Mv1Lu) that expresses Met but not HGF were used. In this study, we have shown that c-Src kinase activity is constitutively elevated in SP1 cells and is induced in response to HGF in Mv1Lu cells. In addition, c-Src kinase associates with Met following stimulation with HGF. The enhanced activity of c-Src kinase also correlates with its ability to associate with Met. Expression of a dominant negative double mutant of c-Src (SRC-RF), lacking both kinase activity (K295R) and a regulatory tyrosine residue (Y527F), in SP1 cells significantly reduced c-Src kinase activity and strongly blocked HGF-induced motility and colony growth in soft agar. In contrast, expression of the dominant negative c-Src mutant had no effect on HGF-induced cell proliferation on plastic. Taken together, our data strongly suggest that HGF-induced association of c-Src with Met and c-Src activation play a critical role in HGF-induced cell motility and anchorage-independent growth of mammary carcinomas and further support the notion that the presence of paracrine and autocrine HGF loops contributes significantly to the transformed phenotype of carcinoma cells.     

Possible roles of nonparenchymal cells in hepatocyte proliferation induced by lead nitrate and by tumor necrosis factor alpha

Several proteins from culture supernatants of Streptococcus sobrinus were able to bind avidly to Sephadex G-75. The proteins could be partially eluted from the Sephadex by low-molecular-weight alpha-1,6 glucan or fully eluted by 4 M guanidine hydrochloride. Elution profiles were complex, yielding proteins of 16, 45, 58 to 60, 90, 135, and 145 kDa, showing that the wild-type strain possessed multiple glucan-binding proteins. Two mutants of Streptococcus sobrinus incapable of aggregation by high-molecular-weight alpha-1,6 glucan were isolated. One mutant was spontaneous, from a cell suspension to which glucan had been added, whereas the other was induced by ethyl methanesulfonate. Both mutants were devoid of a 60-kDa protein, as shown by gel electrophoresis of culture supernatants and whole cells. Amino acid analysis showed that the 58- to 60-kDa protein and the 90-kDa protein were distinct, although both were N-terminally blocked. Both mutants retained their ability to adhere to glass in the presence of sucrose and to ferment mannitol and sorbitol. Both mutants retained their glucosytransferase activities, as shown by activity gels. Western blots (immunoblots), employing antibody against a glucan-binding protein of Streptococcus mutans, failed to reveal cross-reactivity with S. sobrinus proteins. The results show that even though S. sobrinus produces several proteins capable of binding alpha-1,6 glucans, the 60-kDa protein is probably the lectin needed for glucan-dependent cellular aggregation.     

Serum hepatocyte growth factor activity and hepatocyte proliferation in Long-Evans with a cinnamon-like coat color rats with hepatic lesions

We classified hepatic lesions spontaneously developed by Long-Evans with a cinnamon-like coat color (LEC) rats into the following four stages: Normal liver, acute hepatitis, chronic hepatitis, and hepatoma, by biochemical tests of the sera, and anatomical and histopathological examination of the livers. Hepatocyte growth factor (HGF) activity in the sera of LEC rats which developed acute hepatitis, chronic hepatitis, and hepatoma was higher than that of normal LEC rats. In particular, HGF activity in the sera of the LEC rats with acute hepatitis was about 70-fold that of normal LEC rats. However, primary cultured hepatocytes of LEC rats with hepatic lesions were hardly proliferated by stimulation with EGF and insulin in vitro or with increased HGF in vivo. These results suggest that the hepatocytes of LEC rats with hepatic lesions disorder the signal transduction of growth factors.     

Effect of hepatocyte growth factor on the proliferation of intrasplenically transplanted hepatocytes in rats

Expression of genes encoding insulin-like growth factor I (IGF-I), insulin-like growth factor II (IGF-II) and type I insulin-like growth factor receptor (IGFr) was measured in theca and granulosa cells from the ovary of the laying hen, using an RNase protection assay. Expression of genes encoding IGF-I and -II was confined to theca tissue and expression was not detected in granulosa cells. In contrast, expression of genes encoding IGFr in granulosa cells was significantly greater than that in theca tissue. The 98 base IGF-II probe was similar to a region of the second coding exon of chicken IGF-II and produced multiple RNase-protected RNA hybrids. Theca RNA from follicles at all stages of development produced RNase-protected hybrids of size 98, 96 and 90 bases; however, an additional band (66 bases) was also observed in theca RNA from small yellow follicles. The stage of follicular development during which maximum amounts of the 66 base RNase-protected fragment was detected correlates with the stage at which small follicles are selected for recruitment into the follicular hierarchy. The results provide evidence for the involvement of IGFs in the intraovarian control of ovarian function in a non-mammalian species, and highlight the importance of IGF-II in this process.     

Autocrine-paracrine effects of overexpression of hepatocyte growth factor gene on growth of endothelial cells

Although hepatocyte growth factor (HGF) is synthesized in vascular cells, it is not known whether locally synthesized HGF acts similarly to exogenously added HGF. Therefore, we transfected cultured cells with human HGF vector and examined the effects on growth of vascular cells. Endothelial cells (EC) transfected with HGF vector synthesized and secreted high levels of HGF, and also showed significantly higher number. Addition of conditioned medium from vascular smooth muscle cells (VSMC) or EC transfected with HGF vector to nontransfected EC resulted in a significant increase in cell number, which was abolished by anti-HGF antibody. Co-culture of HGF-transfected VSMC with EC showed that HGF released from VSMC or EC stimulated EC growth. These results demonstrate that endogenously produced HGF by transfection of human HGF vector can exert autocrine and paracrine stimulatory effects on EC growth, but not VSMC growth, suggesting the role of local HGF system in cardiovascular disease.     

Modulation of growth factor receptors on acute myeloblastic leukemia cells by retinoic acid

Interleukin-6 (IL-6) production and proliferative responses by draining lymph node cells were studied in mice exposed topically to a series of chemicals. Chemicals with the capacity to induce sensitisation, but not non-sensitisers, promoted both IL-6 production and lymph node cell proliferation ex vivo. The responses exhibited similar kinetics, were dependent upon the dose of topically applied allergen, and correlated significantly. We demonstrate that the main source of IL-6 within draining lymph nodes is not proliferating T lymphocytes. The induction of a strong IL-6 response, and the relationship of this to cellular proliferation indicate that production of this cytokine within the lymph node is closely associated with the induction of contact sensitivity in mice.     

Stimulation by histamine of TPA-dependent hepatocyte growth factor production in promyelocytic leukemia HL-60 cells

Hepatocyte growth factor (HGF) is most likely a physiological hepatotrophic factor that triggers regeneration of the injured liver. Histamine may also be important in the pathophysiology of the injured liver. Previously we showed that histamine production was increased in liver macrophages of mice injected with CCI4, a well-known hepatotoxin. Therefore, it is likely that the biological actions of histamine in repairing processes of the injured liver are mediated by HGF. This study was aimed at examining the effects of histamine on production of HGF using, as a model, the human promyelocytic leukemia cells, HL-60. 12-o-Tetradecanoylphorbol-13-acetate (TPA) markedly stimulated HGF production and release from the cells; the maximal amount of HGF was released at a concentration of 3 ng/ml of TPA. Histamine significantly stimulated the TPA-induced HGF production and release in these cells, depending on incubation time and its dose. These actions of histamine were abrogated by a H2 receptor antagonist, ranitidine.     

Hemodialysis stimulates hepatocyte growth factor release

Studies were performed in 26 patients on regular dialysis treatment with cuprophane (CU), polymethylmetacrilate (PMMA) or cuprammonium (CAM) dialyzers. Controls were six patients with chronic renal failure but not on regular dialysis treatment (CRF) and six healthy subjects (N). Blood was collected at the start (T0), and at 15 (T15) and 240 (T240) minutes of dialysis to measure the serum hepatocyte growth factor (HGF) concentration and to study HGF production by peripheral blood mononuclear cells (PBMC) in vitro. The form of HGF (that is, inactive/monomeric, active/dimeric) present in the serum was analyzed by immunoblotting. In addition, the ability of serum to stimulate proliferation of tubular cells (HK-2) and HGF release by PBMC and fibroblasts (MRC-5) was investigated. At T0, serum HGF levels were identical to that of the controls. In patients treated with CU, serum HGF rose from 0.24 ng/ml at T0 to 7.44 ng/ml at T15, and remained high at T240. PBMC collected at T15 and T240 released significantly more HGF in vitro than those collected at T0. Serum at T15 stimulated proliferation of HK-2 cells and the release of HGF by PBMC and MRC-5 cells. The PMMA and CAM dialyzers had similar effects as the CU. These results indicate that dialysis induces a striking rise in serum HGF and a prompt circulation of factor(s) stimulating HGF release. Dialysis-activated PBMC release HGF.     

Immediate increase in circulating hepatocyte growth factor/scatter factor by heparin

Circulating levels of hepatocyte growth factor (HGF)/scatter factor have been recently found to be increased in the early phase of myocardial infarction, and it has been hypothesized that HGF plays a role in angiogenesis and collateral vessel growth. Heparin has also been shown to enhance angiogenesis and to improve collateral blood flow. This study was designed to study the effect of heparin on the release of HGF. In an experimental study, heparin was given to rats intravenously and plasma was collected for measurements of HGF by enzyme-linked immunosorbent assay. A dose-dependent increase in circulating HGF was measured with peak levels occurring 10 min after injection of 300 units/kg of heparin (15.4+/-2.0 ng/ml after v 0. 17+/-0.14 ng/ml before injection,P<0.0001). In a subsequent clinical study, 12 patients received 3000 units of heparin during cardiac catheterization. Circulating HGF increased steeply within 3 min of the injection. Comparable changes in plasma concentrations were measured in samples obtained from femoral vein (8.7+/-3.5 after v 0. 33+/-0.07 before injection P<0.05) or artery (10.5+/-3.2 ng/mlv 0. 27+/-0.05 P<0.01), pulmonary artery (9.1+/-2.0 ng/mlv 0.36+/-0.06 ng/ml,P=0.07 ) or right atrium (8.5+/-1.6 ng/mlv 0.42+/-0.11,P<0.01). This study suggests that heparin-induced effects such as the promotion of angiogenesis may be at least partly due to the release of HGF.     

Dual pathways of tubular morphogenesis of vascular endothelial cells by human glioma cells: vascular endothelial growth factor/basic fibroblast growth factor and interleukin-8

In this study, we examined whether human glioma cells are angiogenic in a model using human microvascular endothelial cells, and also which factor is responsible for the glioma-dependent angiogenesis. Tubular morphogenesis in type I collagen gel by human microvascular endothelial cells was stimulated in the presence of 10 and 100 ng/ml of vascular endothelial growth factor (VEGF), 10 ng/ml basic fibroblast growth factor (bFGF) and 10 ng/ml of interleukin-8 (IL-8). Tube formation of the microvascular endothelial cells was assayed in the glioma cell lines IN157 and IN301, co-cultured using the double chamber method. IN301 cells had much higher levels of VEGF, bFGF and transforming growth factor-beta mRNA than IN157 cells, whereas the two had similar levels of transforming growth factor-alpha mRNA. By contrast, IN157 cells had much higher levels of IL-8 mRNA than IN301 cells. IN301-dependent tubular morphogenesis was inhibited by anti-VEGF or anti-bFGF antibody, and the inhibition was almost complete when anti-VEGF and anti-bFGF antibodies were present. On the other hand, IN157-dependent tubular morphogenesis was inhibited by anti-IL-8 antibody, but not by anti-VEGF or anti-bFGF antibodies. These findings demonstrated dual paracrine controls of tumor angiogenesis by human glioma cells. One is mediated through VEGF and/or bFGF, and the other, through IL-8.     

Evidence for a functional hepatocyte growth factor receptor in human mesangial cells

In a previous study, mu-opioid receptor binding was decreased by chronic treatment of rats with a mu-opioid receptor-selective agonist [CH3Phe3, D-Pro4]morphiceptin (PL-017) [Tao, P.L., Lee, H.Y., Chang, L.R., Loh, H.H., 1990. Decrease in mu-opioid receptor binding capacity in rat brain after chronic PL-017 treatment. Brain Res. 526, 270-275]. However, there was a lack of correlation between the time course of receptor down-regulation and the loss of pharmacological effects of the drug. In the current study, we used immunohistochemistry to reinvestigate this issue. Male Sprague-Dawley rats were chronically treated with PL-017 i.c.v. for 1, 3 or 5 days, using an escalating dosage paradigm (0.75-6.0 microg), which resulted in a 1.4 to 32-fold increase in the AD50. Rat brains were removed, frozen, coronally sectioned (14 microm) and processed for mu-, delta- or kappa-opioid receptor immunohistochemistry by the avidin-biotin complex (ABC) method. Significant decreases in OP3 immunodensity were found in many brain regions which are enriched with OP3 after chronic treatment of PL-017. Time-dependent decreases in OP3 were detected and reached a plateau around 3 days of PL-017 treatment. No significant change in OP1 or OP2 immunodensity after chronic treatment with PL-017 was found. Our conclusion is that chronic treatment with PL-017 of rats selectively down-regulates mu-opioid receptors in the brain. This may be an important mechanism for PL-017 tolerance.     

Interaction of c-myc with transforming growth factor alpha and hepatocyte growth factor in hepatocarcinogenesis

Double transgenic mice bearing fusion genes consisting of mouse albumin enhancer/promoter-mouse c-myc cDNA and mouse metallothionein 1 promoter-human TGF-alpha cDNA were generated to investigate the interaction of these genes in hepatic oncogenesis and to provide a general paradigm for characterizing the interaction of nuclear oncogenes and growth factors in tumorigenesis. Coexpression of c-myc and TGF-alpha as transgenes in the mouse liver resulted in a tremendous acceleration of neoplastic development in this organ as compared to expression of either of these transgenes alone. The two distinct cellular reactions that occurred in the liver of the double transgenic mice prior to the appearance of liver tumors were dysplastic and apoptotic changes in the existing hepatocytes followed by emergence of multiple focal lesions composed of both hyperplastic and dysplastic cell populations. These observations suggest that the interaction of c-myc and TGF-alpha, during development of hepatic neoplasia contributes to the selection and expansion of the preneoplastic cell populations which consequently increases the probability of malignant conversion. These studies have now been extended to examine the interaction of hepatocyte growth factor (HGF) with c-myc during hepatocarcinogenesis in the transgenic mouse model. While sustained overexpression of c-myc in the liver leads to cancer, coexpression of HGF and c-myc in the liver delayed the appearance of preneoplastic lesions and prevented malignant conversion. Similarly, tumor promotion by phenobarbital was completely inhibited in the c-myc/HGF double transgenic mice whereas phenobarbital was an effective tumor promoter in the c-myc single transgenic mice. The results indicate that HGF may function as a tumor suppressor during early stages of liver carcinogenesis, and suggest the possibility of therapeutic application for this cytokine. Furthermore, we show for the first time that interaction of c-myc with HGF or TGF-alpha results in profoundly different outcomes of the neoplastic process in the liver.     

Possible involvement of p21/waf1 in the growth inhibition of HepG2 cells induced by hepatocyte growth factor

Patients with malignant tumors of the head and neck often have immune defects. Higher serum immunoglobulin (Ig)A levels were reported in this group of patients. We investigated whether IgA-anti-Fab- or IgA-anti-F(ab')2 autoantibodies, which have been shown to correlate with severe dysfunction of the immune system, also appear in patients with head and neck cancer. Sera of 110 patients with squamous cell carcinoma (SCCHN), eight patients with adenoid cystic carcinoma, and 57 healthy control subjects were tested by enzyme-linked immunosorbent assay for IgA-anti-Fab autoantibody activity. Patients with head and neck cancer showed a higher IgA-anti-Fab activity (optical density (OD) = 399; n = 118) than did healthy control subjects (OD = 84; n = 57; p < 0.0001). An association between stage of disease and IgA-anti-Fab activity could be established in patients with SCCHN. Patients with stage IV disease had a significantly higher IgA-anti-Fab activity (OD = 538; n = 51) than had patients with stage I disease (OD = 283; n = 18; p < 0.05). Patients with stage II (OD = 293; n = 13) or stage III (OD = 379; n = 28) disease had intermediate activity. Also a higher IgA-anti-Fab activity than in healthy control subjects could be shown in the eight patients with adenoid cystic carcinoma (OD = 314; n = 8; p < 0.01). The highest IgA-anti-Fab activity was observed in eight patients with SCCHN who died within 6 months after testing (OD = 1004; n = 8), suggesting an association between autoimmunity and final desintegration of physiologic body functions. The occurrence of IgA-anti-Fab/IgA-anti-F(ab')2 autoantibodies might be interpreted as an aspect of immune deficiency in patients with malignant tumors of the head and neck.     

Epidermal growth factor receptor-mediated motility in fibroblasts

Cell motility is induced by many growth factors acting through cognate receptors with intrinsic tyrosine kinase activity (RPTK). However, most of the links between receptor activation and the biophysical processes of cell motility remain undeciphered. We have focused on the mechanisms by which the EGF receptor (EGFR) actuates fibroblast cell motility in an attempt to define this integrated process in one system. Our working model is that divergent, but interconnected pathways lead to the biophysical processes necessary for cell motility: cytoskeleton reorganization, membrane extension, formation of new adhesions to substratum, cell contraction, and release of adhesions at the rear. We postulate that for any given growth factor some of the pathways/processes will be actively signaled and rate-limiting, while others will be permissive due to background low-level activation. Certain couplings have been defined, such as PLCgamma and actin modifying proteins being involved in cytoskeletal reorganization and lamellipod extension and MEK being implicated in detachment from substratum. Others are suggested by complementary investigations in integrin-mediated motility, including rac in membrane protrusion, rho in new adhesions, myosin II motors in contraction, and calpain in detachment, but have yet to be placed in growth factor-induced motility. Our model postulates that many biochemical pathways will be shared between chemokinetic and haptokinetic motility but that select pathways will be activated only during RPTK-enhanced motility.     

Modulation of the insulin-like growth factor system by chronic alcohol feeding

Insulin-like growth factor (IGF)-I is a potent anabolic agent that plays an important role in regulating muscle protein balance. Alterations in one or more of the various components of the IGF system may be in part responsible for the muscle wasting that accompanies chronic alcohol consumption. The purpose of the present study was to characterize changes in the growth hormone-IGF axis produced by chronic alcohol consumption in rats. After 8 weeks of alcohol feeding, the IGF-I concentration was decreased in plasma (31%) as well as in the liver and skeletal muscle (40-50%), compared with pair-fed control animals. In addition, alcohol consumption decreased IGF-I mRNA abundance in liver and muscle (approximately 50%). IGF-I content in duodenum and kidney, however, was not altered by alcohol feeding. Concomitantly, the relative concentration of IGF binding protein (IGFBP)-1 was increased in plasma, liver, and muscle of alcohol-fed rats, compared with control values. In contrast, no changes in the plasma concentrations of IGFBP-2, -3, or -4 were detected in alcohol-fed rats at this time point Previous studies have indicated that elevations in glucocorticoids or decreases in insulin or growth hormone might be responsible for the decrease in IGF-I and/or the increase in IGFBP-1 in other catabolic conditions. However, there was no difference in the plasma concentrations of these hormones between alcohol-fed and control animals in this study. These data indicate that chronic alcohol feeding in rats decreases IGF-I and increases IGFBP-1 in the circulation and in skeletal muscle and that these changes appear to be independent of changes in classical hormonal regulators of the IGF system. The observed alterations in the IGF system are consistent with a reduction in the anabolic actions of IGF-I induced by chronic alcohol consumption.     

Basic fibroblast growth factor promotes the proliferation of human megakaryocyte progenitor cells

Basic fibroblast growth factor (bFGF), a multifunctional growth factor produced by bone marrow stromal cells, is known to be a potent modulator of hematopoiesis. Because bFGF is present in both human megakaryocytes (MKs) and platelets, we have hypothesized that this growth factor might affect human megakaryocytopoiesis. To test this hypothesis, either low density bone marrow (BM) cells (LDBM), a human BM subpopulation (CD34+ DR+) enriched for the colony-forming unit megakaryocyte (CFU-MK) or a BM subpopulation (CD34+ DR-) enriched for the more primitive burst-forming unit megakaryocyte (BFU-MK) were assayed in the presence of this growth factor. The effect of bFGF on MK colony formation differed according to the cell population assayed. bFGF alone had on MK colony-stimulating activity (MK-CSA) when either CD34+ DR+ or CD34+ DR- BM cells were cloned, but exhibited MK-CSA equivalent to that of interleukin-3 (IL-3) when LDBM cells were used as the target cell population. The MK-CSA of bFGF was inhibited by the addition of neutralizing antisera to either IL-3 and/or granulocyte-macrophage colony-stimulating factor (GM-CSF) but not IL-6. The addition of excess amounts of either IL-3 or GM-CSF to cultures containing bFGF plus anti-IL-3 or anti-GM-CSF reversed the inhibition by the corresponding antisera. The addition of bFGF and IL-3 to assays containing CD34+ DR+ or CD34+ DR- cells increased the size of both CFU-MK- and BFU-MK-derived colonies, respectively, when compared with assays containing IL-3 alone. This increase in MK colony size mediated by bFGF was not affected by addition of either an anti-GM-CSF or anti-IL-6 neutralizing antisera. When LDBM cells were assayed, bFGF alone increased CFU-MK-derived colony size when compared with control values. However, this potentiation of MK colony size by bFGF could be reversed by the addition of either anti-IL-3 or anti-GM-CSF but not anti-IL-6 antisera. In addition, the effects of bFGF and IL-3 on the size of MK colonies cloned from LDBM were not additive. These results suggest that bFGF affects human megakaryocytopoiesis by directly promoting MK progenitor cell proliferation and stimulating BM accessory cells to release growth factor(s) with MK-CSA, such as IL-3 and GM-CSF. We conclude that bFGF, likely produced by cellular components of the BM microenvironment, plays an important role in the control of human megakaryocytopoiesis.     

Circular ruffle formation and closure lead to macropinocytosis in hepatocyte growth factor/scatter factor-treated cells

Treatment with hepatocyte growth factor/scatter factor (HGF/SF) rapidly induced the formation of conspicuous circular ruffles on the apical surfaces of two kidney cell lines, MDCK and PtK2. The ruffles were found to contain significant amounts of F-actin and myosin as judged by immunofluorescence microscopy. Time-lapse photomicroscopy demonstrated that the ruffles constrict, closing over, and were followed by the formation of phase bright structures. That these structures were macropinocytotic vesicles was confirmed using fluorescein isothiocyanate (FITC)-dextran as a marker for fluid uptake. It is hypothesized that the constriction of the ruffles followed by membrane fusion causes the vesicles to form. Treatment with suramin blocked both circular ruffle formation and scattering, suggesting that ligand binding was the causal agent for ruffle formation. The drugs amiloride and SITS (4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid) also completely inhibited ruffle formation, suggesting that ion transport was an early consequence of HGF/SF binding and that these transport effects had a major role in the cytoskeletal changes leading to circular ruffle formation. The appearance of macropinocytotic vesicles was also blocked by amiloride treatment. Surprisingly though, subsequent scattering was not blocked by amiloride treatment, although suramin and SITS both entirely inhibited scattering.