Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A human prostate cancer model was established by inoculating a prostate specific antigen (PSA)-producing LNCaP cell line with either prostate or bone fibroblasts. Alternatively, this human prostate cancer model can also be established by inoculating LNCaP cells with growth factor(s) (GFs) and extracellular matrix (ECM) immobilized on Gelfoam. The resulting LNCaP tumors were used to evaluate PSA production and excretion in athymic hosts. This model was also employed to examine the biochemical nature of mesenchymal cell-derived growth-promoting protein(s) and to assess the efficacy of potential chemotherapeutic agents. Because of the propensity of human prostate cancer to metastasize to the bone, this study defined a 1.0 M NaCl-eluted fraction, MS1, from the conditioned medium of a bone stromal cell line (MS) by heparin-affinity column chromatography. The growth-promoting activity was assayed both in vivo (e.g., tumor formation) and in vitro (e.g., soft agar colony formation). We found that the growth-promoting activity was trypsin- and heat-sensitive, and partially degraded by acid and dithiothreitol. Immunochemical studies indicated that the polyclonal antibody raised against MS1 blocked the growth-promoting effect elicited by the bone-conditioned media. This growth-promoting factor was found to be immunochemically dissimilar to KGF, HGF, and bFGF. However, addition of bFGF, HGF and NGF, but not aFGF, TGF beta, IGF1, IGF2, PDGF, EGF, TGF alpha and KGF, stimulated anchorage-independent growth of prostate cells, a condition closely parallel to tumor formation in vivo. We found that the MS1 fraction also contained fibronectin and tenascin but not laminin or collagen IV.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Human prostate cancer model: roles of growth factors and extracellular matrices. 128 80

The MET proto-oncogene encodes a transmembrane tyrosine kinase receptor for HGF (p190MET). In this work, p190MET was immunoprecipitated, allowed to phosphorylate in the presence of [gamma-32P]ATP, and digested with trypsin. A major phosphopeptide was purified by reverse phase chromatography. The phosphorylated tyrosine was identified as residue 1235 (Tyr1235) by Edman covalent radiosequencing. A synthetic peptide derived from the corresponding MET sequence was phosphorylated by p190MET in an in vitro assay and coeluted in reverse phase chromatography. Tyr1235 lies within the tyrosine kinase domain of p190MET, within a canonical tyrosine autophosphorylation site that shares homology with the corresponding region of the insulin, CSF-1 and platelet-derived growth factor receptors, and of p60src and p130gag-fps. The p190MET kinase is constitutively phosphorylated on tryosine in a gastric carcinoma cell line (GTL16), due to the amplification and overexpression of the MET gene. Metabolic labeling of GTL-16 cells with [32P]orthophosphate followed by immunoprecipitation and tryptic phosphopeptide mapping of p190MET showed that Tyr1235 is a major site of tyrosine phosphorylation in vivo as well. Since phosphorylation activates p190MET kinase, we propose a regulatory role for Tyr1235.
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PMID:Identification of the major autophosphorylation site of the Met/hepatocyte growth factor receptor tyrosine kinase. 165 90

Paracrine motogenic cytokines secreted by normal cells can stimulate metastatic cell invasion. For example, human fibroblasts secrete hepatocyte growth factor/scatter factor (HGF/SF), which stimulates paracrine migration of epithelial and certain carcinoma cells, and migration-stimulating factor (MSF), which stimulates autocrine migration of fibroblasts from certain breast carcinomas. We found that human peri-tumoral and lung fibroblasts secrete motility-stimulating activity for several recently established human sarcoma cell strains. Motility of lung metastasis-derived SYN-I sarcoma cells was preferentially stimulated by human lung and peri-tumoral fibroblast motility-stimulating factors (FMSFs). FMSFs were non-dialyzable, susceptible to trypsin and sensitive to dithiothreitol. Cycloheximide inhibited accumulation of FMSF activity in conditioned medium; however, addition of cycloheximide to the migration assay did not significantly affect motility-stimulating activity. Purified HGF/SF, rabbit anti-hHGF and RT-PCR analysis of peri-tumoral and lung fibroblast HGF/SF mRNA expression indicated that FMSF activity was unrelated to HGF/SF. Partial purification of FMSF by gel exclusion chromatography revealed several peaks of activity, suggesting multiple FMSF molecules or complexes. Since human soft tissue sarcomas have a distinctive hematogenous metastatic pattern (predominantly lung), FMSF may play a role in this process independent of HGF/SF.
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PMID:Human peri-tumoral and lung fibroblasts produce paracrine motility factors for recently established human sarcoma cell strains. 766 30

Prostate-specific antigen (PSA), a M(r) 34,000 serine protease, is recognized as a useful marker for the detection and prognosis of patients with prostate cancer. Although serum PSA is an excellent prognostic indicator, an increasing number of factors were found to regulate the PSA expression of prostatic cancer cells, which include androgenic steroids, the growth factors (GFs) and the extracellular matrix. The purpose of this study is to define a novel protein factor that may be responsible for regulating PSA expression by androgen-independent (AI) human prostate cancer cells. We have established a LNCaP subline (C4) from a parental LNCaP tumor grown in a castrated host. The C4 subline overexpressed PSA mRNA and protein. Serum-free conditioned medium (CM) isolated from the C4 subline is able to stimulate PSA gene expression in parental LNCaP cells in a concentration-dependent manner. This autocrine PSA-inducing activity was found to be organ specific because CMs from other fibroblast cell lines (such as bone, prostate, kidney, and lung fibroblasts) and the CMs from several prostatic carcinoma cell lines (such as parental LNCaP, PC-3, DU-145) and a bladder transitional carcinoma cell line (WH) fail to exhibit similar activity. The activity of the CM from the C4 subline cannot be substituted by GFs such as TGF-alpha, TGF-beta, bFGF, HGF, KGF, or NGF; neuropeptide (bombesin/GRP); secondary messenger analogue (dibutyryl cAMP); beta 2-adrenergic agonist (isoproterenol); or alpha 1-adrenergic agonist (phenylephrine), indicating that the factor(s) may be a novel prostate-specific autocrine factor (PSAF). Both androgen and PSAF exhibit an additive effect on up-regulating PSA gene expression, suggesting that the signal transduction pathway elicited by PSAF may differ from that mediated by the androgen receptor. Further characterization of PSAF by heat, acid, and trypsin digestion revealed that the PSAF may be a protein factor with a unique amino acid composition. These observations suggest that a novel autocrine pathway mediated by PSAF may be responsible for the overexpression of PSA mRNA and protein in a human prostatic cancer cell line. The potential clinical significance of this factor will be discussed.
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PMID:Autocrine regulation of prostate-specific antigen gene expression in a human prostatic cancer (LNCaP) subline. 768 49

Epithelial-mesenchymal interactions mediate aspects of normal lung growth and development and are important in the restoration of normal alveolar architecture after lung injury. To determine if fibroblasts are a source of soluble growth factors for alveolar type II cells, we investigated the effect of fibroblast-conditioned medium (CM) on alveolar type II cell DNA synthesis. Serum-free CM from confluent adult human lung fibroblasts was concentrated fivefold by lyophilization. Type II cells were isolated from adult rats by elastase dissociation and incubated with [3H]thymidine and varying dilutions of concentrated CM and serum from day 1 to 3 of culture. Stimulation of type II cell DNA synthesis by fibroblast-CM was maximal after 48 h of conditioning and required the presence of serum. The activity of the CM was eliminated by boiling and by treatment with trypsin, pepsin, or dithiothreitol and was additive with saturating concentrations of acidic fibroblast growth factor, epidermal growth factor, and insulin. The growth factor activity bound to heparin-Sepharose and was eluted with 0.6 and 1.0 M NaCl. Neutralizing antibody studies demonstrated that the primary mitogens isolated in the 0.6 and 1.0 M NaCl fractions were keratinocyte growth factor (KGF, fibroblast growth factor 7) and hepatocyte growth factor/scatter factor (HGF/SF), respectively. HGF/SF was demonstrated in the crude CM and KGF was detected in the 0.6 M NaCl eluent by immunoblotting. Northern blot analysis confirmed that the lung fibroblasts expressed both KGF and HGF/SF transcripts. Human recombinant KGF and HGF/SF induced a concentration- and serum-dependent increase in rat alveolar type II cell DNA synthesis. We conclude that adult human lung fibroblasts produce at least two soluble heparin-binding growth factors, KGF and HGF/SF, which promote DNA synthesis and proliferation of rat alveolar type II cells in primary culture. KGF and HGF/SF may be important stimuli for alveolar type II cell proliferation during lung growth and after lung injury.
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PMID:Keratinocyte growth factor and hepatocyte growth factor/scatter factor are heparin-binding growth factors for alveolar type II cells in fibroblast-conditioned medium. 768 69

Hepatocyte growth factor/scatter factor (HGF/SF), a fibroblast-derived mediator of epithelial and endothelial growth and motility, is regulated by factors present in media conditioned by breast tumor cell lines. Both inhibitory and stimulatory effects were observed dependent on culture conditions. The present work shows that breast tumor cell conditioned medium contains a latent HGF/SF inhibitory activity, which can be activated by a variety of treatments known to activate latent transforming growth factor-beta. Using blocking antibodies and other criteria, we show that transforming growth factor-beta present in epithelial cell conditioned medium is primarily responsible for mediating the down-regulation of fibroblast HGF/SF. Epithelial cell conditioned medium also contains a trypsin-sensitive and heat-stable stimulatory activity. Stromal cell density but not proliferation rate markedly alters HGF/SF expression. These results indicate that the expression of at least one epithelial morphogen, HGF/SF, is interdependently regulated by mesenchymal condensation and by factors released by neighboring epithelial and carcinoma cells.
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PMID:Tumor-stroma interactions and stromal cell density regulate hepatocyte growth factor protein levels: a role for transforming growth factor-beta activation. 772 Jun 42

A number of cytokines have been reported to increase the cell motility (scatter) of cohesive cell colonies. We report here a novel scattering factor produced by human monocytes. Media from both stimulated and non-stimulated human monocytes or the monocytic cell line, U937, increased the cell motility of 2 human colon-cancer cell lines, HT115 and HT29, but not the canine epithelial cell line MDCK. Motility was assayed by cell dissociation from carrier beads and colony scattering. HT115 cells were strongly scattered by the conditioned media but not by interleukins IL-1, 2, 4, 6, 8, 10, TNF alpha, TGF beta, EGF, GM-CSF, IGF-I, PDGF, interferon-gamma. This factor is distinct from hepatocyte growth factor/scatter factor (HGF/SF), since the activity was not blocked by anti-HGF/SF antibody. The activity was reduced by treatment with acid, heat, trypsin and dithiothreitol; this, together with gel filtration, suggests that the factor is a protein (MW 40 to 60 kDa). This new factor, which is secreted from monocytes and the monocytic cell line, U937, has the ability to increase the motility of cancer cells and may be important in controlling the behaviour of tumours in vivo.
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PMID:Monocyte-conditioned media possess a novel factor which increases motility of cancer cells. 842 96

Macrophage-stimulating protein (MSP) and hepatocyte growth factor/scatter factor (HGF/SF) are plasminogen-related growth and motility factors that interact with cell-surface protein tyrosine kinase receptors. Each one is a heterodimeric protein comprising a disulfide-linked alpha chain and a serine protease-like beta chain. Despite structural similarities between MSP and HGF, the primary receptor binding site is located on the alpha chain of HGF/SF but on the beta chain of MSP. To obtain insight into the structural basis for MSP beta chain binding, beta chain structure was modeled from coordinates of an existing model of the HGF beta chain. The model revealed that the region corresponding to the S1 specificity pocket in trypsin is filled by the Asn(682)/Glu(648) interacting pair, leaving a shallow cavity for possible beta chain interaction with the receptor. Mutants in this region were created, and their binding characteristics were determined. A double mutation of Asn(682)/Glu(648) caused diminished binding of the beta chain to the MSP receptor, and a single mutation of neighboring Arg(683) completely abolished binding. Thus, this region of the molecule is critical for binding. We also found that at equimolar concentrations of free alpha and beta chains, alpha chain binding to receptor was detectable, at levels considerably lower than beta chain binding. The EC(50) values determined by quantitative enzyme-linked immunosorbent assay are 0.25 and 16.9 nM for beta and alpha chain, respectively. The data suggest that MSP has two independent binding sites with high and low affinities located in beta and alpha chain, respectively, and that the two sites together mediate receptor dimerization and subsequent activation.
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PMID:Interaction of macrophage-stimulating protein with its receptor. Residues critical for beta chain binding and evidence for independent alpha chain binding. 1051 76

Previously, we found that hepatocyte growth factor receptor (c-Met)-and alpha-fetoprotein (AFP)-expressing cells were present in adult rat bone marrow, and that these cells also expressed hematopoietic stem cell markers, such as CD34, Thy-1, and c-Kit. When bone marrow cells were cultured in a hepatocyte growth medium (HGM) with HGF and EGF, colonies composed of polygonal cells resembling mature hepatocytes appeared by 2 weeks and grew very slowly because of overgrowth of stromal cells. At days 34-41, 2-mm2 sheets of hepatocyte-like cells were cut out of their colonies by scratching with an injection needle under observation with a phase contrast microscope, transferred into wells of 24-well plates, and cultured in the HGM medium in the presence or absence of HGF and EGF. When cells reached confluence, cells were detached with trypsin and EDTA and transferred step by step into bigger culture vessels. Thus, hepatocyte-like cells were expanded 1000-fold during less than 4 months. These cells were immunocytochemically stained for albumin and also for AFP and the hematopoietic stem cell markers described above, showing characteristics of oval cells. By RT-PCR, we detected mRNAs of tryptophan-2,3-dioxygenase and tyrosine aminotransferase, markers of hepatocytes at a terminal differentiation stage. The present culture system may be useful for supply of hepatocyte resources for cell transplantation therapy.
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PMID:Propagation of adult rat bone marrow-derived hepatocyte-like cells by serial passages in vitro. 1546 80

Hepsin is a membrane-anchored, trypsin-like serine protease with prominent expression in the human liver and tumours of the prostate and ovaries. To better understand the biological functions of hepsin, we identified macromolecular substrates employing a tetrapeptide PS-SCL (positional scanning-synthetic combinatorial library) screen that rapidly determines the P1-P4 substrate specificity. Hepsin exhibited strong preference at the P1 position for arginine over lysine, and favoured threonine, leucine or asparagine at the P2, glutamine or lysine at the P3, and proline or lysine at the P4 position. The relative activity of hepsin toward individual AMC (7-amino-4-methylcoumarin)-tetrapeptides was generally consistent with the overall peptide profiling results derived from the PC-SCL screen. The most active tetrapeptide substrate Ac (acetyl)-KQLR-AMC matched with the activation cleavage site of the hepatocyte growth factor precursor sc-HGF (single-chain HGF), KQLR downward arrowVVNG (where downward arrow denotes the cleavage site), as identified by a database analysis of trypsin-like precursors. X-ray crystallographic studies with KQLR chloromethylketone showed that the KQLR peptide fits well into the substrate-binding cleft of hepsin. This hepsin-processed HGF induced c-Met receptor tyrosine phosphorylation in SKOV-3 ovarian cancer cells, indicating that the hepsin-cleaved HGF is biologically active. Activation cleavage site mutants of sc-HGF with predicted non-preferred sequences, DPGR downward arrowVVNG or KQLQ downward arrowVVNG, were not processed, illustrating that the P4-P1 residues can be important determinants for substrate specificity. In addition to finding macromolecular hepsin substrates, the extracellular inhibitors of the HGF activator, HAI-1 and HAI-2, were potent inhibitors of hepsin activity (IC50 4+/-0.2 nM and 12+/-0.5 nM respectively). Together, our findings suggest that the HGF precursor is a potential in vivo substrate for hepsin in tumours, where hepsin expression is dysregulated and may influence tumorigenesis through inappropriate activation and/or regulation of HGF receptor (c-Met) functions.
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PMID:Hepatocyte growth factor is a preferred in vitro substrate for human hepsin, a membrane-anchored serine protease implicated in prostate and ovarian cancers. 1583 37


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