Investigation of electro-vascular phase-amplitude coupling during an auditory task.

Multimodal neuroimaging using electroencephalography (EEG) and functional near-infrared spectroscopy (fNIRS) provides complementary views of cortical processes, including those related to auditory processing. However, current multimodal approaches often overlook potential insights that can be gained from nonlinear interactions between electrical and hemodynamic signals. Here, we explore electro-vascular phase-amplitude coupling (PAC) between low-frequency hemodynamic and high-frequency electrical oscillations during an auditory task. We further apply a temporally embedded canonical correlation analysis (tCCA)-general linear model (GLM)-based correction approach to reduce the possible effect of systemic physiology on fNIRS recordings. Before correction, we observed significant PAC between fNIRS and broadband EEG in the frontal region (p ≪ 0.05), ß (p ≪ 0.05) and γ (p = 0.010) in the left temporal/temporoparietal (left auditory; LA) region, and γ (p = 0.032) in the right temporal/temporoparietal (right auditory; RA) region across the entire dataset. Significant differences in PAC across conditions (task versus silence) were observed in LA (p = 0.023) and RA (p = 0.049) γ sub-bands and in lower frequency (5-20 Hz) frontal activity (p = 0.005). After correction, significant fNIRS-γ-band PAC was observed in the frontal (p = 0.021) and LA (p = 0.025) regions, while fNIRS-α (p = 0.003) and fNIRS-ß (p = 0.041) PAC were observed in RA. Decreased frontal γ-band (p = 0.008) and increased ß-band (p ≪ 0.05) PAC were observed during the task. These outcomes represent the first characterization of electro-vascular PAC between fNIRS and EEG signals during an auditory task, providing insights into electro-vascular coupling in auditory processing.

Individual-specific characterization of event-related hemodynamic responses during an auditory task: An exploratory study.

Functional near-infrared spectroscopy (fNIRS) has been established as an informative modality for understanding the hemodynamic-metabolic correlates of cortical auditory processing. To date, such knowledge has shown broad clinical applications in the diagnosis, treatment, and intervention procedures in disorders affecting auditory processing; however, exploration of the hemodynamic response to auditory tasks is yet incomplete. This holds particularly true in the context of auditory event-related fNIRS experiments, where preliminary work has shown the presence of valid responses while leaving the need for more comprehensive explorations of the hemodynamic correlates of event-related auditory processing. In this study, we apply an individual-specific approach to characterize fNIRS-based hemodynamic changes during an auditory task in healthy adults. Oxygenated hemoglobin (HbO2) concentration change time courses were acquired from eight participants. Independent component analysis (ICA) was then applied to isolate individual-specific class discriminative spatial filters, which were then applied to HbO2 time courses to extract auditory-related hemodynamic features. While six of eight participants produced significant class discriminative features before ICA-based spatial filtering, the proposed method identified significant auditory hemodynamic features in all participants. Furthermore, ICA-based filtering improved correlation between trial labels and extracted features in every participant. For the first time, this study demonstrates hemodynamic features important in experiments exploring auditory processing as well as the utility of individual-specific ICA-based spatial filtering in fNIRS-based feature extraction techniques in auditory experiments. These outcomes provide insights for future studies exploring auditory hemodynamic characteristics and may eventually provide a baseline framework for better understanding auditory response dysfunctions in clinical populations.

Targeting tumor multicellular aggregation through IGPR-1 inhibits colon cancer growth and improves chemotherapy.

Adhesion to extracellular matrix (ECM) is crucially important for survival of normal epithelial cells as detachment from ECM triggers specific apoptosis known as anoikis. As tumor cells lose the requirement for anchorage to ECM, they rely on cell-cell adhesion 'multicellular aggregation' for survival. Multicellular aggregation of tumor cells also significantly determines the sensitivity of tumor cells to the cytotoxic effects of chemotherapeutics. In this report, we demonstrate that expression of immunoglobulin containing and proline-rich receptor-1 (IGPR-1) is upregulated in human primary colon cancer. Our study demonstrates that IGPR-1 promotes tumor multicellular aggregation, and interfering with its adhesive function inhibits multicellular aggregation and, increases cell death. IGPR-1 supports colon carcinoma tumor xenograft growth in mouse, and inhibiting its activity by shRNA or blocking antibody inhibits tumor growth. More importantly, IGPR-1 regulates sensitivity of tumor cells to the chemotherapeutic agent, doxorubicin/adriamycin by a mechanism that involves doxorubicin-induced AKT activation and phosphorylation of IGPR-1 at Ser220. Our findings offer novel insight into IGPR-1's role in colorectal tumor growth, tumor chemosensitivity, and as a possible novel anti-cancer target.

Hyphal wall protein 1 gene: A potential marker for the identification of different Candida species and phylogenetic analysis.

BACKGROUND AND PURPOSE: Hyphal wall protein 1 (HWP1) is an important adhesin which usually is expressed on the germ tube and hyphal surface produced by different Candida species. The hyphal wall protein-coding gene (HWP1) was evaluated as a novel identification and phylogenetic marker in Candida tropicalis, C. orthopsilosis, C. parapsilosis and C. glabrata. MATERIALS AND METHODS: Initially, four specific primer pairs were designed, and the target was amplified and finally sequenced. A total of 77 Candida isolates from four different species were included in the study. Consensus sequences were used for the evaluation of phylogenetic tree using the CLC Genome Workbench, GENEIOUS, and MEGA softwares and the levels of nucleotide and amino acid polymorphism were assessed. RESULTS: According to the results, the specific amplified fragments of HWP1 gene were useful for the differentiation of four species. Intra-species variation was observed only in C. tropicalis with two DNA types. The phylogenetic tree of Candida species based on the HWP1 gene showed consistency in topology with those inferred from other gene sequences. CONCLUSION: We found that HWP1 gene was an excellent marker for the identification of non-albicansCandida species as well as the phylogenetic analysis of the most clinically significant Candida species.

A prospective study of a modified pin-in-plaster technique for treatment of fractures of the distal radius.

OBJECTIVES: There are various pin-in-plaster methods for treating fractures of the distal radius. The purpose of this study is to introduce a modified technique of 'pin in plaster'. METHODS: Fifty-four patients with fractures of the distal radius were followed for one year post-operatively. Patients were excluded if they had type B fractures according to AO classification, multiple injuries or pathological fractures, and were treated more than seven days after injury. Range of movement and functional results were evaluated at three and six months and one and two years post-operatively. Radiographic parameters including radial inclination, tilt, and height, were measured pre- and post-operatively. RESULTS: The average radial tilt was 10.6° of volar flexion and radial height was 10.2 mm at the sixth month post-operatively. Three cases of pin tract infection were recorded, all of which were treated successfully with oral antibiotics. There were no cases of pin loosening. A total of 73 patients underwent surgery, and three cases of radial nerve irritation were recorded at the time of cast removal. All radial nerve palsies resolved at the six-month follow-up. There were no cases of median nerve compression or carpal tunnel syndrome, and no cases of tendon injury. CONCLUSION: Our modified technique is effective to restore anatomic congruity and maintain reduction in fractures of the distal radius. Cite this article: Bone Joint Res 2015;4:176-180.

In vitro activity of econazole in comparison with three common antifungal agents against clinical Candida strains isolated from superficial infections.

BACKGROUND AND PURPOSE: Candida species are the most common organisms involved in superficial fungal infections, worldwide. Although econazole is among the most frequently used topical formulations for the treatment of candidiasis, no information is available regarding the susceptibility profiles of Candida species in Iran. MATERIALS AND METHODS: In vitro susceptibility of 100 clinical Candida isolates belonging to 6 species from superficial candidiasis of Iran towards to econazole was compared with three other common antifungal agents including itraconazole, fluconazole, and miconazole. Minimum inhibitory concentrations (MICs) values were analyzed according to the Clinical and Laboratory Standards Institute (CLSI) M38-A3 document. All isolates were previously identified to the species level, using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) on ITS region. RESULTS: The MIC of econazole, itraconazole, miconazole, and fluconazole were within the range of 0.016-16, 0.032-16, 0.016-16, and 0.25-64 µg/ml, respectively. In general, econazole and miconazole were more active against Candida isolates, compared to the other two agents. CONCLUSION: The present study demonstrated that for Candida albicans isolates, miconazole and econazole had the best effect, but in non-albicans Candida species, itraconazole and miconazole displayed more activity than other antifungal agents.

A Metabonomic Study of the Effect of Methanol Extract of Ginger on Raji Cells Using (1)HNMR Spectroscopy.

Cancer is currently a major international health problem. The development of resistance to chemotherapy has resulted in the search for herbal drugs. Ginger is a medicinal plant with several clinical applications. Metabolomics is a simultaneous detection of all the metabolites by use of (1)HNMR or mass spectroscopy and interpretation by modeling software. The purpose of this study was to detect the altered metabolites of Raji cells in the presence of ginger extract in vitro. Cells were cultured in the presence and absence of methanolic ginger extract in RPMI medium. IC50 determined by MTT and lipophilic and hydrophilic extracts were prepared from control and treated groups which were analyzed by (1)HNMR. The IC50 was 1000 µg/mL. Modeling of spectra was carried out on the two groups using OSC-PLS with MATLAB software and the main metabolites detected. Further analysis was carried out using MetaboAnalyst database. The main metabolic pathways affected by the ginger extract were detected. Ginger extract was seen to effect the protein biosynthesis, amino acid, and carbohydrate metabolism and had a strong cytotoxic effect on Raji cells in vitro.

c-Cbl inhibits angiogenesis and tumor growth by suppressing activation of PLCγ1.

Angiogenesis is regulated by highly coordinated function of various proteins with pro- and anti-angiogenic functions. Among the many cytoplasmic signaling proteins that are activated by VEGFR-2, activation of PLCγ1 is considered to have a pivotal role in angiogenic signaling. In previous study we have identified c-Cbl as a negative regulator of PLCγ1 in endothelial cells, the biochemical and biological significance of c-Cbl, however, in angiogenesis in vivo and molecular mechanisms involved were remained elusive. In this study, we report that genetic inactivation of c-Cbl in mice results in enhanced tumor angiogenesis and retinal neovascularization. Endothelial cells derived from c-Cbl null mice displayed elevated cell proliferation and tube formation in response to VEGF stimulation. Loss of c-Cbl also resulted in robust activation of PLCγ1 and increased intracellular calcium release. c-Cbl-dependent ubiquitination selectively inhibited tyrosine phosphorylation of PLCγ1 and mostly refrained from ubiquitin-mediated degradation. Hence, we propose c-Cbl as an angiogenic suppressor protein where upon activation it uniquely modulates PLCγ1 activation by ubiquitination and subsequently inhibits VEGF-driven angiogenesis.

Identification of tyrosine residues in vascular endothelial growth factor receptor-2/FLK-1 involved in activation of phosphatidylinositol 3-kinase and cell proliferation.

Activation of vascular endothelial growth factor receptor-2 (VEGFR-2) plays a critical role in vasculogenesis and angiogenesis. However, the mechanism by which VEGFR-2 activation elicits these cellular events is not fully understood. We recently constructed a chimeric receptor containing the extracellular domain of human CSF-1R/c-fms, fused with the entire transmembrane and cytoplasmic domains of murine VEGFR-2 (Rahimi, N., Dayanir, V., and Lashkari, K. (2000) J. Biol. Chem. 275, 16986-16992). In this study we used VEGFR-2 chimera (herein named CKR) to elucidate the signal transduction relay of VEGFR-2 in porcine aortic endothelial (PAE) cells. Mutation of tyrosines 799 and 1173 individually on CKR resulted in partial loss of CKR's ability to stimulate cell growth. Double mutation of these sites caused total loss of CKR's ability to stimulate cell growth. Interestingly, mutation of these sites had no effect on the ability of CKR to stimulate cell migration. Further analysis revealed that tyrosines 799 and 1173 are docking sites for p85 of phosphatidylinositol 3-kinase (PI3K). Pretreatment of cells with wortmannin, an inhibitor of PI3K, and rapamycin, a potent inhibitor of S6 kinase, abrogated CKR-mediated cell growth. However, expression of a dominant negative form of ras (N(17)ras) and inhibition of the mitogen-activated protein kinase (MAPK) pathway by PD98059 did not attenuate CKR-stimulated cell growth. Altogether, these results demonstrate that activation of VEGFR-2 results in activation of PI3K and that activation of PI3K/S6kinase pathway, but not Ras/MAPK, is responsible for VEGFR-2-mediated cell growth.

Receptor chimeras indicate that the vascular endothelial growth factor receptor-1 (VEGFR-1) modulates mitogenic activity of VEGFR-2 in endothelial cells.

Vascular endothelial growth factor (VEGF) provokes angiogenesis in vivo and stimulates growth and differentiation of endothelial cells in vitro. Although VEGF receptor-1 (VEGFR-1) and VEGFR-2 are known to be high affinity receptors for VEGF, it is not clear which of the VEGFRs are responsible for the transmission of the diverse biological responses of VEGF. For this purpose we have constructed a chimeric receptor for VEGFR-1 (CTR) and VEGFR-2 (CKR) in which the extracellular domain of each receptor was replaced with the extracellular domain of human colony-stimulating factor-1 receptor (CSF-1R), and these receptors were expressed in pig aortic endothelial (PAE) cells. We show that CKR individually expressed in PAE cells is readily tyrosine-phosphorylated in vivo, autophosphorylated in vitro, and stimulates cell proliferation in a CSF-1-dependent manner. In contrast, CTR individually expressed in PAE cells showed no significant in vivo, in vitro tyrosine phosphorylation and cell growth in response to CSF-1 stimulation. The kinase activity of CKR was essential for its biological activity, since mutation of lysine 866 to arginine abolished its in vivo, in vitro tyrosine phosphorylation and mitogenic signals. Remarkably, activation of CTR repressed CKR-mediated mitogen-activate protein kinase activation and cell proliferation. Similar effects were observed for VEGFR-2 co-expressed with VEGFR-1. Collectively, these findings demonstrate that VEGFR-2 activation plays a positive role in angiogenesis by promoting endothelial cell proliferation. In contrast, activation of VEGFR-1 plays a stationary role in angiogenesis by antagonizing VEGFR-2 responses.

Vascular endothelial growth factor and hepatocyte growth factor levels are differentially elevated in patients with advanced retinopathy of prematurity.

Although the roles of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) in angiogenesis are well described, the putative roles of these factors in retinopathy of prematurity (ROP) remain unknown. We evaluated VEGF and HGF protein levels in subretinal fluid of eyes with ROP, and expression of their corresponding receptors in retrolental membranes associated with stage 5 ROP. We examined subretinal fluid samples from eyes using rhegmatogenous retinal detachment as a control. VEGF and HGF were differentially elevated in eyes with ROP. In Stage 5 ROP (n = 22), the mean VEGF and HGF levels were 14.77 +/- 14.01 ng/ml and 16.56 +/- 9.62 ng/ml, respectively. Interestingly, in patients with active stage 4 ROP, mean VEGF levels were highly elevated (44.16 +/- 18.72 ng/ml), whereas mean HGF levels remained very low (4.77 +/- 2.50 ng/ml). Next, we investigated in vivo expression of VEGF receptor-2 and HGF receptor in retrolental membranes from 16 patients with stage 5 ROP. Both VEGF receptor-2 and HGF receptor proteins were detected mainly in posterior portions of the membrane as well as in vessel walls and along the retinal interface where angiogenesis was active. These findings together suggest that VEGF and HGF play important roles in the pathogenesis of ROP.

Cooperative effect of hepatocyte growth factor and fibronectin in anchorage-independent survival of mammary carcinoma cells: requirement for phosphatidylinositol 3-kinase activity.

Anchorage-independent survival and growth are critical characteristics of malignant cells. We showed previously that the addition of exogenous hepatocyte growth factor (HGF) and the presence of fibronectin fibrils stimulate anchorage-independent colony growth of a murine mammary carcinoma, SP1, which expresses both HGF and HGF receptor (Met; R. Saulnier et al., Exp. Cell Res., 222: 360-369, 1996). We now show that tyrosine phosphorylation of Met in carcinoma cells is augmented by cell adhesion and spreading on fibronectin substratum. In contrast, detached serum-starved cells exhibit reduced tyrosine phosphorylation of Met and undergo apoptotic cell death within 18-24 h. Under these conditions, the addition of HGF stimulates tyrosine phosphorylation of Met and restores survival of carcinoma cells. Soluble fibronectin also stimulates cell survival and shows a cooperative survival response with HGF but does not affect tyrosine phosphorylation of Met; these results indicate that fibronectin acts via a pathway independent of Met in detached cells. We demonstrated previously that inhibition of phosphatidylinositol (PI) 3-kinase activity blocks HGF-induced DNA synthesis of carcinoma cells (N. Rahimi et al., J. Biol. Chem., 271: 24850-24855, 1996). We now show in detached cells a cooperative effect of HGF and FN in the activation of PI 3-kinase and on the phosphorylation of PKB/Akt at serine 473. PI 3-kinase activity is also required for the HGF- and fibronectin-induced survival responses, as well as anchorage-independent colony growth. However, c-Src kinase or MEK1/2 activities are not required for the cell survival effect. Together, these results demonstrate that the PI 3-kinase/Akt pathway is a key effector of the HGF- and fibronectin-induced survival response of breast carcinoma cells under detached conditions and corroborate an interaction between integrin and HGF/ Met signalling pathways in the development of invasive breast cancer.

A role for cadherin-5 in regulation of vascular endothelial growth factor receptor 2 activity in endothelial cells.

FLK-1/vascular endothelial growth factor receptor 2 (VEGFR-2) is one of the receptors for VEGF. In this study we examined the effect of cell density on activation of VEGFR-2. VEGF induces only very slight tyrosine phosphorylation of VEGFR-2 in confluent (95-100% confluent) pig aortic endothelial (PAE) cells. In contrast, robust VEGF-dependent tyrosine phosphorylation of VEGFR-2 was observed in cells plated in sparse culture conditions (60-65% confluent). A similar cell density-dependent phenomenon was observed in different endothelial cells but not in NIH-3T3 fibroblast cells expressing VEGFR-2. Stimulating cells with high concentrations of VEGF or replacing the extracellular domain of VEGFR-2 with that of the colony-stimulating factor 1 receptor did not alleviate the sensitivity of VEGFR-2 to cell density, indicating that the confluent cells were probably not secreting an antagonist to VEGF. Furthermore, in PAE cells, ectopically introduced platelet-derived growth factor alpha receptor could be activated at both high and low cell density conditions, indicating that the density effect was not universal for all receptor tyrosine kinases expressed in endothelial cells. In addition to lowering the density of cells, removing divalent cations from the medium of confluent cells potentiated VEGFR-2 phosphorylation in response to VEGF. These findings suggested that cell-cell contact may be playing a role in regulating the activation of VEGFR-2. To this end, pretreatment of confluent PAE cells with a neutralizing anti-cadherin-5 antibody potentiated the response of VEGFR-2 to VEGF. Our data demonstrate that endothelial cell density plays a critical role in regulating VEGFR-2 activity, and that the underlying mechanism appears to involve cadherin-5.

Hepatocyte growth factor receptor in human RPE cells: implications in proliferative vitreoretinopathy.

PURPOSE: To determine whether hepatocyte growth factor (HGF) receptor (HGFR) is expressed in retinal pigment epithelial (RPE) cells and to test whether RPE cells are responsive to HGF. To evaluate expression of HGFR in human donor eyes and in several epiretinal membranes associated with proliferative vitreoretinopathy and idiopathic epiretinal membranes. METHODS: HGF-dependent migration and proliferation in primary and simian virus (SV) 40-transformed human RPE cells was studied using a Boyden chamber and [3H]thymidine uptake, respectively. The expression and tyrosine phosphorylation of HGFR protein was evaluated in RPE cells by immunoprecipitation and western blot analysis. Expression of HGFR in human donor eyes and in several epiretinal membranes associated with proliferative vitreoretinopathy (PVR) and idiopathic epiretinal membranes was analyzed by immunohistochemistry. RESULTS: HGFR was expressed in RPE cells and was tyrosine-phosphorylated in response to HGF. Whereas HGF was a potent motogen for RPE cells, it induced only a modest, dose-dependent uptake of [3H]thymidine. Evaluation of human donor eyes showed that the RPE monolayer was the major cell type that was strongly positive for HGFR. HGFR was uniformly and readily detected in the cellular component of epiretinal membranes associated with PVR, whereas little or no HGFR was found in idiopathic epiretinal membranes. CONCLUSIONS: HGFR is expressed in cultured RPE cells, in the RPE monolayer in human donor eyes, and in epiretinal membranes obtained from patients with PVR. Furthermore, HGF is a potent chemoattractant for cultured human RPE cells. These observations suggest a role for HGF and HGFR in normal function of RPE cells and in RPE-related disease such as PVR.

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.

Autocrine secretion of TGF-beta 1 and TGF-beta 2 by pre-adipocytes and adipocytes: a potent negative regulator of adipocyte differentiation and proliferation of mammary carcinoma cells.

We have developed an in vitro system to examine the influence of adipocytes, a major mammary stromal cell type, on the growth of a murine mammary carcinoma, SP1. Previously, we have shown that 3T3-L1 adipocytes release a mitogenic factor, hepatocyte growth factor, which strongly stimulates proliferation of SP1 cells. We now show that 3T3-L1 pre-adipocytes secrete active inhibitory molecules which inhibit DNA synthesis in SP1 cells. In addition, latent inhibitory activity is present in conditioned media (CM) from both pre-adipocytes and adipocytes, and is activated following acid treatment. CM also inhibited DNA synthesis in Mv1Lu wild type epithelial cells, but not DR27 mutant epithelial cells which lack TGF-beta type II receptor. Inhibitory activity of CMs was partially abrogated by neutralizing anti-TGF-beta1 and anti-TGF-beta2 antibodies, and was removed following ultrafiltration through membranes of 10,000 Mr but not 30,000 Mr pore size. These results show that the inhibitory effect on DNA synthesis is mediated by TGF-beta1-like and TGF-beta2-like molecules. In addition, acid-treated CM as well as purified TGF-beta inhibited differentiation of pre-adipocytes. Untreated pre-adipocyte CM, but not mature adipocyte CM, spontaneously inhibited adipocyte differentiation. Together, these findings indicate that pre-adipocytes spontaneously activate their own secreted TGF-beta, whereas mature adipocytes do not, and suggest that activation of TGF-beta has a potent negative regulatory effect on adipocyte differentiation and tumor growth. Thus, TGF-beta may be an important modulator of tumor growth and adipocyte differentiation via both paracrine and autocrine mechanisms. These findings emphasize the importance of adipocyte-tumor interactions in the regulation of tumor microenvironment.

Phosphatidylinositol 3-kinase activity is required for hepatocyte growth factor-induced mitogenic signals in epithelial cells.

Phosphatidylinositol (PI) 3-kinase is an important enzyme implicated in growth factor-stimulated intracellular signaling. In this study we have shown that hepatocyte growth factor (HGF) induces a rapid tyrosine phosphorylation of PI 3-kinase and association with HGF receptor/Met in Mv1Lu epithelial cells. Murine mammary carcinoma (SP1) cells, which co-express HGF and HGF receptor/Met, showed sustained phosphorylation of PI 3-kinase. Wortmannin, a potent inhibitor of PI 3-kinase, inhibited HGF-induced PI 3-kinase activity, proliferation of Mv1Lu cells, and spontaneous growth of SP1 cells in a dose-, and time-dependent manner. Transfection of a dominant negative mutant p85 (Deltap85) subunit of PI 3-kinase into SP1 cells strongly inhibited HGF-stimulated proliferation and PI 3-kinase activity. However, wortmannin did not influence HGF-induced c-Jun expression. Furthermore, HGF stimulated S6 kinase activity, but its activity was not required for HGF-induced proliferation. Overall, these results suggest that HGF-induced PI 3-kinase activity is important for the mitogenic action of HGF in epithelial cells and further demonstrate that expression of c-Jun is not influenced by inhibition of PI 3-kinase activity.

Hepatocyte growth factor (HGF) is a copper-binding protein: a facile probe for purification of HGF by immobilized Cu(II)-affinity chromatography.

Hepatocyte growth factor (HGF) is a multifunctional protein expressed in a variety of cell types and tissues. Here we describe a novel one-step method to separate and identify HGF, based on a unique interaction between HGF and Cu(II). Conditioned medium (CM) from mouse 3T3-L1 adipocytes which contains HGF or purified human recombinant HGF was used for analysis. Mouse 3T3-L1 adipocyte CM was applied to a Cu(II)-affinity column and rinsed with equilibration buffer. HGF was then eluted with 10 mM imidazole. Fractions eluted from the column were analyzed by SDS-PAGE. Analysis by silver staining revealed an 85kDa protein. Further analysis by Western blotting with polyclonal anti-HGF IgG demonstrated that this protein corresponded to HGF. Human recombinant HGF, when applied to a Cu(II)-affinity column, showed a stronger affinity to Cu(II) than did mouse HGF. Human recombinant HGF was not eluted from the Cu(II) column with either 10 or 20 mM imidazole; however, it was readily eluted with 40 mM imidazole. The percentages of recovery of both human and mouse HGF were greater than 90%. Both mouse HGF and human recombinant HGF eluted from the Cu(II)-affinity column retained their biological activity as measured by HGF-induced cell proliferation of Mv1Lu cells. Our findings provide the first evidence that HGF is a copper-binding protein and that a Cu(II)-affinity column can be used for efficient one-step purification of biologically active HGF.

Identification of a hepatocyte growth factor autocrine loop in a murine mammary carcinoma.

Constitutive activation of growth factor receptors through autocrine/paracrine mechanisms occurs frequently in human cancers and is thought to play an important role in carcinogenesis. We have demonstrated previously that hepatocyte growth factor (HGF) is a potent mitogenic factor for murine mammary carcinoma (SP1) cells in vitro. We report here an autocrine HGF loop in SP1 cells. HGF receptor/Met is expressed in SP1 cells and is constitutively tyrosine phosphorylated. The phosphorylation of HGF receptor/Met is inhibited when cells are exposed to suramin or anti-HGF IgG. This finding suggests that constitutive tyrosine phosphorylation of HGF receptor/Met is sustained by an extracellular factor, most likely HGF. Using Northern blot and Western blot analysis, we detected expression of a 6-kb HGF mRNA in SP1 cells and a M(r) 85,000 HGF protein in SP1-conditioned medium, respectively. In vitro translation of mRNA from SP1 cells and metabolic labeling confirmed expression and synthesis of HGF by SP1 cells. SP1 cells also invade through Matrigel-coated transwell membranes in an in vitro invasion assay, and invasion of these cells was inhibited by neutralizing anti-HGF IgG. In addition, SP1-conditioned medium induced scatter activity of Madin-Darby canine kidney epithelial cells, and this activity was inhibited by neutralizing anti-HGF IgG. We have also shown that several signaling molecules including phosphatidylinositol 3-kinase, Src, focal adhesion kinase, and phospholipase C-gamma in SP1 cells are constitutively tyrosine phosphorylated, suggesting that coexpression of HGF and HGF receptor/Met may in part contribute to sustained tyrosine phosphorylation of these cytoplasmic proteins in SP1 cells. Our observations in the SP1 model suggest that HGF contributes to growth and invasive phenotypes of mammary carcinomas via both paracrine and autocrine mechanisms.

Fibronectin fibrils and growth factors stimulate anchorage-independent growth of a murine mammary carcinoma.

Stromal cells are important regulators of mammary carcinoma growth and metastasis. We have previously shown that a 3T3-L1 adipocyte cell line secretes hepatocyte growth factor (HGF), which stimulates proliferation of a murine mammary carcinoma (SP1) in monolayer cultures (DNA Cell Biol. 13, 1189-1897, 1994). We now examine the role of growth factors and the extracellular matrix protein fibronectin in stimulation of anchorage-independent growth of SP1 cells. Purified transforming growth factor-beta (TGF-beta) stimulated significant colony growth in soft agar cultures, whereas HGF had a lesser effect. Analysis by confocal microscopy revealed that carcinoma cell colonies contained extracellular microfibrils composed of fibronectin. Partial depletion of fibronectin from 7% FBS/agar cultures reduced the number of colonies; colony growth could be recovered by adding back exogenous fibronectin. Addition of the 70-kDa N-terminal fragment of fibronectin, which inhibits fibronectin fibril formation, reduced growth of SP1 cell colonies, but an 85-kDa fragment containing the cell binding domain did not inhibit colony growth. These findings indicate that deposition of extracellular fibronectin fibrils is necessary, but not sufficient, for anchorage-independent growth of SP1 mammary carcinoma cells; growth factors are also required. SP1 cells had less fibronectin mRNA and secreted less fibronectin protein under anchorage-independent conditions than under anchorage-dependent conditions, as determined by Northern blotting and immunoprecipitation analysis. Thus, both growth factors (HGF and TGF-beta) and fibronectin may be important regulators of paracrine stimulation by stromal cells of anchorage-independent growth of mammary carcinoma cells.