Biomedical subjects

W T Chen

Publications and source records attributed to W T Chen.

At least 19 recordsLinked to original sources

PubMed1998

Activation of beta1 integrin signaling stimulates tyrosine phosphorylation of p190RhoGAP and membrane-protrusive activities at invadopodia.

The ligation of available alpha6beta1 integrin in adherent LOX melanoma cells by laminin G peptides and integrin stimulatory antibodies induced cell invasiveness, independent of adhesion activity of integrins that were pre-bound to extracellular matrix (Nakahara, H., Nomizu, M., Akiyama, S. K., Yamada, Y., Yeh, Y., and Chen, W.-T. (1996) J. Biol. Chem. 271, 27221-27224). Here, we show that this induced invasion involves an increase in tyrosine phosphorylation of a 190-kDa GTPase-activating protein for Rho family members (p190(RhoGAP); p190) and membrane-protrusive activities at invadopodia. This tyrosine phosphorylation does not occur when the adherent cells are treated with non-activating antibody against beta1 integrin, control laminin peptides, or tyrosine kinase inhibitors genistein and herbimycin A. Although p190 and F-actin co-distribute in all cell cortex extensions, tyrosine-phosphorylated proteins including p190 appear to associate with F-actin specifically in invadopodia. In addition, the localized matrix degradation and membrane-protrusive activities were blocked by treatment of LOX cells with tyrosine kinase inhibitors as well as microinjection of antibodies directed against p190 but not by non-perturbing antibodies or control buffers. We suggest that activation of the alpha6beta1 integrin signaling regulates the tyrosine phosphorylation state of p190 which in turn connects downstream signaling pathways through Rho family GTPases to actin cytoskeleton in invadopodia, thus promoting membrane-protrusive and degradative activities necessary for cell invasion.

Amino Acid Sequence↗

PubMed1997

Transmembrane/cytoplasmic domain-mediated membrane type 1-matrix metalloprotease docking to invadopodia is required for cell invasion.

The invasion of human malignant melanoma cells into the extracellular matrix (ECM) involves the accumulation of proteases at sites of ECM degradation where activation of matrix metalloproteases (MMP) occurs. Here, we show that when membrane type 1 MMP (MT-MMP) was overexpressed in RPMI7951 human melanoma cells, the cells made contact with the ECM, activated soluble and ECM-bound MMP-2, and degraded and invaded the ECM. Further experiments demonstrated the importance of localization of the MT-MMP to invadopodia. Overexpression of MT-MMP without invadopodial localization caused activation of soluble MMP-2, but did not facilitate ECM degradation or cell invasiveness. Up-regulation of endogenous MT-MMP with concanavalin A caused activation of MMP-2. However, concanavalin A treatment prevented invadopodial localization of MT-MMP and ECM degradation. Neither a truncated MT-MMP mutant lacking transmembrane (TM) and cytoplasmic domains (DeltaTMMT-MMP), nor a chimeric MT-MMP containing the interleukin 2 receptor alpha chain (IL-2R) TM and cytoplasmic domains (DeltaTMMT-MMP/TMIL-2R) were localized to invadopodia or exhibited ECM degradation. Furthermore, a chimera of the TM/cytoplasmic domain of MT-MMP (TMMT-MMP) with tissue inhibitor of MMP 1 (TIMP-1/TMMT-MMP) directed the TIMP-1 molecule to invadopodia. Thus, the MT-MMP TM/cytoplasmic domain mediates the spatial organization of MT-MMP into invadopodia and subsequent degradation of the ECM.

Amino Acid Sequence↗

PubMed1997

Molecular cloning of seprase: a serine integral membrane protease from human melanoma.

Seprase is a homodimeric 170 kDa integral membrane gelatinase whose expression correlates with the invasiveness of the human melanoma cell line LOX. Here, we report the molecular cloning of a cDNA that encodes the 97 kDa subunit of seprase. Its deduced amino acid sequence predicts a type II integral membrane protein with a cytoplasmic tail of 6 amino acids, followed by a transmembrane domain of 20 amino acids and an extracellular domain of 734 amino acids. The carboxyl terminus contains a putative catalytic region (approximately 200 amino acids) which is homologous (68% identity) to that of the nonclassical serine protease dipeptidyl peptidase IV (DPPIV). The conserved serine protease motif G-X-S-X-G is present as G-W-S-Y-G. However, sequence analysis of seprase cDNA from LOX and other cell lines strongly suggests that seprase and human fibroblast activation protein alpha (FAP alpha) are products of the same gene. We propose that seprase/FAP alpha and DPPIV represent a new subfamily of serine integral membrane proteases (SIMP).

Amino Acid Sequence↗

PubMed1997

Identification of the 170-kDa melanoma membrane-bound gelatinase (seprase) as a serine integral membrane protease.

The 170-kDa membrane-bound gelatinase, seprase, is a cell surface protease, the expression of which correlates with the invasive phenotype of human melanoma and carcinoma cells. We have isolated seprase from cell membranes and shed vesicles of LOX human melanoma cells. The active enzyme is a dimer of N-glycosylated 97-kDa subunits. Sequence analysis of three internal proteolytic fragments of the 97-kDa polypeptide revealed up to 87.5% identity to the 95-kDa fibroblast activation protein alpha (FAPalpha), the function of which is unknown. Thus, we used reverse transcription-polymerase chain reaction to generate a 2.4-kilobase cDNA from LOX mRNA with FAPalpha primers. COS-7 cells transfected with this cDNA expressed a 170-kDa gelatinase that is recognized by monoclonal antibodies directed against seprase. Sequence analysis also showed similarities to the 110-kDa subunit of dipeptidyl peptidase IV (DPPIV). Like DPPIV, the gelatinase activity of seprase was completely blocked by serine-protease inhibitors, including diisopropyl fluorophosphate. Seprase could be affinity-labeled by [3H]diisopropyl fluorophosphate, but the proteolytically inactive 97-kDa subunit could not, confirming the existence of a serine protease active site on the dimeric form. Proteolytic activity is lost upon dissociation into its 97-kDa subunit following treatment with acid, heat, or cysteine and histidine-modifying agents. We conclude that seprase, FAPalpha, and DPPIV are related serine integral membrane proteases and that seprase is similar to DPPIV, the proteolytic activities of which are dependent upon subunit association.

Amino Acid Sequence↗

PubMed1996

A mechanism for regulation of melanoma invasion. Ligation of alpha6beta1 integrin by laminin G peptides.

Invasion of LOX human melanoma cells involves extracellular matrix (ECM) degradation and formation of cell surface invadopodia. Here we show that the ligation of alpha6beta1 by two peptides derived from the COOH-terminal globular domain of laminin-1 alpha1 chain (laminin G peptides), designated AG-10 (NPWHSIYITRFG) and AG-32 (TWYKIAFQRNRK), and antibodies against alpha6 and beta1 integrins promoted invasiveness. AG-10 and AG-32 inhibited cell adhesion on laminin, and the antibodies blocked cell adhesion on immobilized AG-10 and AG-32, suggesting that the peptides interact primarily with alpha6beta1 integrin. These soluble peptides and integrin antibodies induced invasiveness by causing an 2-3-fold increase in ECM degradation and invadopodial activity independently of adhesion activity of integrins that were prebound to ECM. The induced ECM degradation and invasion was associated with an increased surface expression of the 170-kDa membrane-bound gelatinase, seprase, as well as its intense localization at invadopodia but not at focal adhesions. However, the total expression levels of seprase, gelatinase A and beta1 integrins were not altered. We suggest that laminin G peptides act on the alpha6beta1 integrin signaling of invasion by stimulating invadopodial activities, which is distinct from their direct effects on cell adhesion on immobilized ECM.

Amino Acid Sequence↗

PubMed1996

Blood pressure-lowering effect of fish oil is independent of thromboxane A2 level in spontaneously hypertensive rats.

Whether the alterations in the synthesis of thromboxane A2 (TXA2) is the direct mechanism underlying the blood pressure-lowering effect of fish oil was investigated in this study. Six groups of 11 male spontaneously hypertensive rats were fed semipurified diets containing corn or fish oils and graded levels (50, 5000 or 15,000 ppm) of dietary vitamin E for 8 weeks. Plasma TXA2, assayed by RIA, was significantly greater in the corn oil group than in the fish oil group (P < 0.05). Compared to 50 ppm dietary vitamin E, 5000 and 15 000 ppm dietary vitamin E, respectively, significantly decreased plasma TXA2 (P < 0.05). Systolic, mean or diastolic blood pressure, evaluated by the tail cuff method, were significantly higher in the corn oil group than in the fish oil group (P < 0.05). However, vitamin E had no effect on blood pressure. No relationship between TXA2 and blood pressure was found. Experimental results indicated that the alterations in the synthesis of TXA2 were not the direct antihypertensive effect of fish oil.

Animals↗

PubMed1996

Proteases associated with invadopodia, and their role in degradation of extracellular matrix.

Metastasizing cancer cells invade the extracellular matrix using plasma membrane protrusions (invadopodia) that contact and dissolve the matrix. Evidence suggests that membrane-associated proteases, 170-kD gelatinase (seprase) and Gelatinase A, exert their mechanisms of action on invadopodia. Potential roles that other metallo- and serine-types of membrane proteases, including membrane-type matrix metalloprotease, meprin, dipeptidyl peptidase IV, fibroblast activation protein alpha and guanidinobenzoatase, play in the cell surface proteolysis are also discussed. It is proposed that formation of a structurally and functionally linked protease complex on invadopodia allows the invasion of cancer cells into the extracellular matrix.

Animals↗

PubMed1996

Clinical response of tuberculous pericarditis to medical treatment: a retrospective survey.

BACKGROUND: The prevalence of tuberculosis has declined with advanced antituberculous chemotherapy. However, the occurrence of subsequent constrictive pericarditis in tuberculosis has not reduced. Clinical progress of tuberculous pericarditis was investigated in patients receiving antituberculous chemotherapy. METHODS: Thirteen patients with tuberculous pericarditis (11 men and 2 women aged 14 to 86 years [mean 60.0 +/- 17.0]), treated initially with antituberculous medications were analyzed. All patients underwent pericardiocentesis on admission following echocardiography. RESULTS: Dyspnea was the most common clinical pictures. Bloody effusion fluids were noted in 10 patients. Moreover, the lymphocytic fluids were present in 78% of all patients. The neutrophilic fluids tended to have a bloody color, and there was progress to constrictive pericarditis even for those on anti-tuberculous chemotherapy. Five patients with subsequent constrictive pericarditis received pericardiectomy within 3.5 months of admission. Three of them received pericardiectomy later, despite pericardial window procedure. CONCLUSIONS: Follow-up by echocardiogram is necessary within three months after commencing medical treatment because of the high incidence of progression constrictive pericarditis despite aggressive medical treatment. Pericardiectomy seems to be the only solution to the catastrophic outcome of constrictive pericarditis.

Adolescent↗

PubMed1996

Heterogeneous clinical characteristics of identical twins concordant for systemic lupus erythematosus: a report of one pair of twins.

Lupus is a mysterious disease without definite etiology and pathogenesis. The heterogeneous clinical manifestation of systemic lupus erythematosus (SLE) increases the delay and difficulty of diagnosis and the complexity of pathogenesis. Even in twins with identical genome and similar environmental milieu, the incidence of concordance for SLE is not high. Identical twins, concordant for SLE with discordance to disease onset and expression, are reported. Besides genetics, multiple factors are responsible for the pathogenesis of SLE.

Adult↗

PubMed1995

The value of ultrasound measurement of gallbladder wall thickness in predicting laparoscopic operability prior to cholecystectomy.

We prospectively analysed 51 consecutive cases who underwent laparoscopic cholecystectomy from June 1992 to February 1993. There were 35 cases of chronic cholecystitis and 16 cases of acute cholecystitis. All underwent pre-operative ultrasonography, complete blood cell count, liver function test and endoscopic retrograde cholangiopancreatography. Of those 44 had post-operative ultrasound within the first 2 d and again on the seventh day. In 35 cases of chronic cholecystitis, 31 of 32 cases with a pre-operative gallbladder (GB) wall thickness of less than 6 mm were successfully resected laparoscopically. All three cases with a GB wall thicker than 6 mm were converted to open cholecystectomy. In acute cholecystitis, the wall thickness of the laparoscopic cholecystectomy group ranged from 2 to 9 mm (average 4 mm) and the wall thickness of the conversion group was 4-7 mm (average 6 mm). Post-operative fluid accumulation was noted in 28 (63.6%) cases. There was no correlation between post-operative pyrexia, duration of post-operative pain, clinical complications and the presence of fluid accumulation in the GB fossa. However, of four cases with increasing fluid on the seventh day, three developed complications. We conclude that ultrasonography is valuable in chronic cholecystitis for selecting cases for laparoscopic cholecystectomy.

Adult↗

PubMed1994

A potential marker protease of invasiveness, seprase, is localized on invadopodia of human malignant melanoma cells.

Seprase, a large, gelatin-degrading membrane-protease complex, is expressed at the invasive front of malignant melanoma cells on invadopodia, and its surface expression contributes to the invasive phenotype. An in vitro assay was used to determine the matrix-degrading activity of four malignant human melanoma cell lines. The lines differ in matrix-degrading activity with LOX > RPM17951 > A375 > SKMEL28. The seprase and Gelatinase A activities of these cell lines were also investigated. Seprase and active gelatinase A are found in cell membranes of LOX and RPM17951 cells but not those of SKMEL28 cells. Experiments using anti-seprase monoclonal antibodies in conjunction with a cell fractionation technique indicate that seprase consists of M(r) 97,000 polypeptides and is enriched on the ventral membrane of LOX in contact with planar extracellular matrix substratum. Confocal microscopy further substantiates our biochemical findings that seprase, as well as Gelatinase A, is localized on invadopodia membranes with a 6-fold increase of seprase and 4-fold increase of Gelatinase A intensity over the level expressed on dorsal membranes. In addition, LOX cells expressing higher levels of seprase at the cell surface, as selected by fluorescence-activated cell sorting, are significantly more degradative than LOX cells with lower seprase expression. Taken together, our data show a concordance between seprase and Gelatinase A expression on the cell surface at invadopodia and the matrix-degrading activity of human malignant melanoma cells. Seprase and major secreted proteases may act in concert to degrade components of the extracellular matrix during invasion.

Biomarkers, Tumor↗

PubMed1994

Expression of tau exon 8 in different species.

A synthetic peptide corresponding to a region encoded by tau gene exon 8 was used to raise an antibody. The antibody, E8, was used to probe normal tau from different species and abnormal tau proteins from subjects with Alzheimer's disease. Immunoblotting and enzyme-linked immunosorbent assays demonstrated that only bovine tau reacted with the E8 antibody. The E8 immunoreactive tau isoform was estimated to represent less than 2% of the bovine tau. Our results indicate that bovine tau is unique in containing isoforms positive with E8, and that PHF formation does not require the presence of PHF-tau with exon 8.

Amino Acid Sequence↗

PubMed1994

Invadopodia promote proteolysis of a wide variety of extracellular matrix proteins.

Chicken embryo fibroblasts (CEF) transformed by Rous sarcoma virus invade the extracellular matrix (ECM) using plasma membrane protrusions, termed invadopodia, that contact and dissolve the matrix. Normal cells neither form invadopodia nor degrade the ECM. Here we show that cells expressing invadopodia degrade and enter into a fibronectin-rich matrix produced by normal fibroblasts. Within 6 h after seeding onto the matrix, the invasive cells create an area devoid of matrix fibrils surrounding the cell body. Proteolysis mediates this matrix clearing because sevenfold more radiolabeled matrix is released into the growth media by the transformed cells relative to the normal cells. In addition to this assembled matrix, transformed cells were grown on thin layers of purified ECM proteins, revealing that invadopodia can degrade fibronectin, collagen type I, collagen type IV, and laminin. A 160 kDa protease that is extracted from transformed cells by Triton X-114 partitions into the detergent phase and is prominent in ventral plasma membranes that contact the ECM suggesting that it is a membrane associated protease.

Animals↗

PubMed1994

Membrane proteases as potential diagnostic and therapeutic targets for breast malignancy.

Metastasizing cancer cells can invade the extracellular matrix using plasma membrane protrusions, termed invadopodia, that contact and dissolve the matrix. Various membrane associated proteases localized on the invadopodial membranes are responsible for the extracellular matrix degradation. Work from our laboratory shows that secreted proteases including Gelatinase A, and high molecular weight integral membrane proteases are associated with cell surface invadopodia. Three cell types, including chicken embryonic cells transformed by Rous sarcoma virus, human malignant melanoma cell line LOX, and human breast carcinoma cell line MDA-MB-231, retain the invasive phenotype in vitro, express invadopodia, degrade and enter into a fibronectin-rich collagenous matrix. We suggest that invadopodium-associated proteases are ideal targets for the diagnosis and treatment of cancer as their presence in association with primary tumors may signal increased metastatic potential. An approach toward the development of new prognostic markers for breast malignancy involved production of monoclonal antibodies directed against membrane proteases in a mixture of glycoproteins. Double immunofluorescent technique using a known invadopodium marker is designed to select specific monoclonal antibodies colocalizing at the invasion front, on invadopodia of cancer cells. Membrane protease accessibility at the cell surface can therefore be exploited for therapeutic advances by the development of specific antibodies and inhibitors that block their activities, and by the use of monoclonal antibodies to target cytotoxic molecules to micrometastases. Also, this same accessibility may potentially be used to detect surface proteases on micrometastases or to detect components shed by micrometastases in serum.

Antigens, Neoplasm↗

PubMed1993

Binding and localization of M(r) 72,000 matrix metalloproteinase at cell surface invadopodia.

Degradation (turnover) of collagenous matrix occurs on the surface of specialized membrane extensions termed "invadopodia," which are sites of cell invasion into the extracellular matrix. Here we show the localization of the M(r) 72,000 type IV collagenase of the matrix metalloproteinase family at invadopodia. When added exogenously, latent M(r) 72,000 collagenase binds to invadopodia of chicken embryo fibroblasts transformed by Rous sarcoma virus, whereupon the bound collagenase loses its propeptide. The collagenase binds to a component contained within the detergent extract of transformed cells, and increased levels of the active M(r) 62,000 form of the collagenase are seen here. Such an association is not detected in the detergent extract derived from normal cells. Using a recently developed cell fractionation procedure to collect cell surfaces enriched in invadopodia, we show that the M(r) 72,000 collagenase associates with the invadopodial fraction and active forms of the enzyme become immobilized on the collagenous surface. Thus, invadopodia direct intense localized degradation of the extracellular matrix by concentrating active membrane-associated collagenases at sites of cellular invasion.

Amino Acid Sequence↗

PubMed1993

Protease production by cultured microglia: substrate gel analysis and immobilized matrix degradation.

The production of collagen-degrading proteases by cultured neonatal rat microglia was examined using an immobilized fibronectin-gelatin matrix coupled to a fluorescent marker and by substrate gel analysis. When microglia were plated onto the surface of the matrix and incubated under resting (nonstimulated) conditions, a small but visible amount of immobilized matrix was degraded. Treatment with lipopolysaccharide (LPS) or interleukin-1 (IL-1) significantly increased the number of microglia demonstrating substrate degradation. Substrate-SDS polyacrylamide gel electrophoresis of samples of supernatants from untreated cultured microglia indicated the presence of a 72 and a 92 kD metalloproteinase with characteristics corresponding to collagenases. Supernatants from untreated astrocyte cultures were shown to have primarily a 72 kD metalloproteinase. Proteinase activity increased on stimulation of the microglia with LPS and IL-1 in a dose-dependent fashion. These results indicate that cultured microglia release active proteases capable of degrading the extracellular matrix in a localized region. The production of proteases by activated microglia may have important physiological and pathophysiological consequences within the restricted extracellular matrix of the CNS.

Animals↗

PubMed1993

Nuclear proteins of the bovine esophageal epithelium. I. Monoclonal antibody W2 specifically reacts with condensed nuclei of differentiated superficial cells.

Cells from three layers of the bovine esophageal epithelium, representing different stages of differentiation, were dissociated and separated by Percoll gradient centrifugation into fractions of small, medium and large sizes. A majority of the large cells possessed condensed nuclei, a characteristic feature of terminal differentiation of the superficial epithelium. The small cells resembled the proliferate cells of the basal layer. In vitro culture of the esophageal epithelial cells resulted in proliferation of the small cells, colony formation, and, in some cases, differentiation into cells with condensed nuclei. Nuclei, or nuclear subfractions derived from cells of the different layers, were used as immunogens for the generation of hybridomas secreting monoclonal antibodies that bound specifically to different regions of the esophageal tissue. One such antibody, designated W2, labeled the condensed nuclei from the superficial layer of stratified esophageal and corneal epithelia in situ, as well as the large cells from esophageal culture in vitro. Thus, the expression of the W2 antigen may be associated with the process of nuclear condensation during epithelial differentiation. Immunoisolation of the target antigen of W2 from extracts of large cells of the bovine esophagus yielded a band of M(r) approximately 33,000 on nonreducing polyacrylamide gels. This band dissociated into two polypeptides, of M(r) approximately 22,000 and approximately 11,000, upon treatment with dithiothreitol. Amino acid sequence analysis of the larger polypeptide showed extensive homology to a group of small calcium-binding proteins, including two helix-turn-helix motifs designated as the EF-hand, characteristic of the configuration of the metal-ion coordinating ligands of the calcium-binding site. Similarly, the sequence at the amino terminus of the polypeptide of approximately 11,000 indicated that it was the light chain counterpart of the same calcium-binding protein complex.

Amino Acid Sequence↗

PubMed1993

Proteases of cell adhesion proteins in cancer.

Neoplastic cells elaborate a vast array of proteases that enable them to proteolytically digest underlying adhesion molecules. In doing this, the tumor cell disrupts the adhesive interactions holding it in place so it is free to degrade and migrate through the basement membrane and interstitial stroma resulting in invasion and ultimately metastasis. Invasive cells elaborate specialized membrane protrusions, invadopodia, that actively degrade the underlying substratum. Evidence indicates that integral membrane proteases and receptors for secreted proteases are present on these surface protrusions. All the major classes of secreted proteases are reported to associate with the plasma membrane. Interactions between proteases occurring at the plasma membrane may result in proenzyme activation. It is possible that various proteases in close proximity to each other on the plasma membrane could interact in a proteolytic cascade resulting in in vivo activation, and the subsequent degradation of adhesion proteins.

Animals↗