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H Yasumitsu

Publications and source records attributed to H Yasumitsu.

At least 37 records · Page 2Linked to original sources
PubMed1996

Specific accumulation of tumor-derived adhesion factor in tumor blood vessels and in capillary tube-like structures of cultured vascular endothelial cells.

Tumor-derived adhesion factor (TAF) was previously identified as a cell adhesion molecule secreted by human bladder carcinoma cell line EJ-1. To elucidate the physiological function of TAF, we examined its distribution in human normal and tumor tissues. Immunochemical staining with an anti-TAF monoclonal antibody showed that TAF was specifically accumulated in small blood vessels and capillaries within and adjacent to tumor nests, but not in those in normal tissues. Tumor blood vessel-specific staining of TAF was observed in various human cancers, such as esophagus, brain, lung, and stomach cancers. Double immunofluorescent staining showed apparent colocalization of TAF and type IV collagen in the vascular basement membrane. In vitro experiments demonstrated that TAF preferentially bound to type IV collagen among various extracellular matrix components tested. In cell culture experiments, TAF promoted adhesion of human umbilical vein endothelial cells to type IV collagen substrate and induced their morphological change. Furthermore, when the endothelial cells were induced to form capillary tube-like structures by type I collagen, TAF and type IV collagen were exclusively detected on the tubular structures. The capillary tube formation in vitro was prevented by heparin, which inhibited the binding of TAF to the endothelial cells. These results strongly suggest that TAF contributes to the organization of new capillary vessels in tumor tissues by modulating the interaction of endothelial cells with type IV collagen.

Cell Adhesion↗
PubMed1996

Cloning of the cDNA encoding mouse PP5/TFPI-2 and mapping of the gene to chromosome 6.

Placental protein 5 (PP5)/tissue factor pathway inhibitor-2 (TFPI-2) is a new homologue of TFPI, which contains three tandemly repeated Kunitz-type proteinase inhibitory (KPI) domains and potently inhibits the extrinsic blood coagulation cascade. In this study, mouse PP5/TFPI-2 cDNA was cloned using a human PP5/TFPI2 cDNA fragment as a probe. The characteristic three KPI domains with short spacer sequences and a basic amino acid stretch in the carboxyl-terminal region present in human PP5/TFPI-2 were well conserved in mouse PP5/TFPI-2. In general, the P1 reactive site residues of active KPI domains are basic amino acids. However, the putative P1 residues of the first, second, and third KPI domains were glutamine, aspartic acid, and serine, respectively. Mouse PP5/TFPI-2 mRNA was highly expressed in developing placenta as in humans. Adult liver and kidney also contained a significant amount of its transcripts. The mouse PP5/TFPI-2 gene was found to be located in the R-positive A2 band by the direct R-banding FISH and identified at 2.7 cM proximal to D6Mit 1 by interspecific backcross analysis.

Amino Acid Sequence↗
PubMed1996

Purification and identification of a novel and four known serine proteinase inhibitors secreted by human glioblastoma cells.

Our previous studies have shown that some human cancer cell lines produce pancreatic trypsinogen, plasminogen, and tissue-type kallikrein. To understand the regulatory mechanism of these proteinases, serine proteinase inhibitors secreted by human glioblastoma cell line T98G were analyzed by gelatin reverse zymography with trypsin. The serum-free conditioned medium of T98G cells showed more than ten trypsin inhibitor bands ranging from 16 to 150 kDa in the reverse zymography. Major trypsin inhibitors were purified by trypsin-affinity chromatography. Analysis of their N-terminal amino acid sequences demonstrated that the purified inhibitors were identical to the secreted forms of amyloid protein precursors (APPs), tissue factor pathway inhibitor (TFPI), placental protein 5 (PP5)/TFPI-2, and secretory leukocyte proteinase inhibitor (SLPI). In addition, a novel 25-kDa trypsin-binding protein, tentatively named p25TI, was identified. p25TI showed weak inhibitory activity against trypsin in reverse zymography as compared with the other inhibitors. The secretion of multiple forms of serine proteinase inhibitors by human cancer cells raises the possibility that they might be involved in the abnormal growth of cancer cells.

Amino Acid Sequence↗
PubMed1996

Differential expression of laminin-5/ladsin subunits in human tissues and cancer cell lines and their induction by tumor promoter and growth factors.

We previously reported a new laminin variant containing laminin gamma 2 (or B2t) chain, ladsin, which exerted prominent cell-scattering, cell-adhesion, and cell-migration activities. In the present study, this laminin was further characterized, and gene expression of its three subunits in various human tissues and cancer cell lines was examined by Northern blotting. cDNA cloning of the largest subunit of ladsin and partial amino acid sequencing of its beta (or B1) subunit revealed that ladsin was identical to laminin-5 (kalinin/epiligrin/ nicein). Among various human tissues, placenta, lung, and fetal kidney expressed high levels of mRNAs for the three subunits of laminin-5 (laminin alpha 3EPA, beta 3, and gamma 2 chains). Most gastric and squamous carcinoma cell lines constitutively expressed all of the three subunit mRNAs, while other types of carcinoma cell lines expressed one or two of them. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and epidermal growth factor (EGF) strongly enhanced the gene expression of the three subunits, increasing 2 to 8-fold the secretion of laminin-5 from carcinoma cells into culture medium. However, TPA treatment did not increase the secretion of laminin beta 1 chain, a subunit of laminins-1, -3, and -6. The unique properties and inducibility by TPA and EGF of laminin-5 suggest that it is associated with growth and migration of cancer cells.

Blotting, Northern↗
PubMed1996

Synergistic growth stimulation of mouse fibroblasts by tumor-derived adhesion factor with insulin-like growth factors and insulin.

Tumor-derived adhesion factor (TAF) has been purified as a major secretory protein with cell-adhesion activity from culture medium conditioned by human bladder carcinoma cells (K. Akaogi et al., Biochem. Biophys. Res. Commun., 198: 1046-1053, 1994). TAF is closely related or identical to a putative protein encoded by the mac25 gene (M. Murphy et al., Cell Growth & Differ., 4: 715-722, 1993) and prostacyclin-stimulating factor (T. Yamauchi et al., Biochem. J., 303: 591-598, 1994). TAF has a characteristic cysteine cluster in its NH2-terminal sequence, which is conserved in the NH2 termini of insulin-like growth factor-binding proteins. In the present study, we examined the interaction of TAF with insulin-like growth factors (IGFs) and insulin. Although in a ligand blotting assay TAF bound neither IGFs nor insulin, immunoprecipitation assay showed weak interaction of TAF with IGF-I, IGF-II, and insulin. TAF by itself stimulated the growth of BALB/c3T3 cells on the usual plastic substrates and significantly enhanced IGF- or insulin-mediated cell growth. Moreover, marked potentiation of the mitogenic activities of IGFs and insulin by TAF was found in the culture on type IV collagen substrates. The synergistic growth stimulation was completely blocked by heparin. These results suggest that TAF may regulate the growth of fibroblastic cells in collaboration with IGFs and insulin.

3T3 Cells↗
PubMed1995

Trypsinogen expression in human ovarian carcinomas.

Increased secretion of matrix metalloproteinases and serine proteinases is well known to be associated with cancer invasion and metastasis. We aimed to elucidate the implication of trypsin, a serine proteinase and a representative digestive enzyme in invasion and metastasis of human carcinomas. Northern blot, RT-PCR and Western blot analyses and immunohistochemical studies were performed to detect and analyze trypsinogen expression in 5 ovarian carcinoma cell lines and 10 human ovarian carcinoma tissues using a DNA probe for trypsinogen I, and monoclonal and polyclonal antibodies to human trypsin I. Among the 5 ovarian carcinoma cell lines, only the MCAS (mucinous cystadenocarcinoma) cell line showed a high level of trypsinogen production and mRNA expression by Western and Northern blot analyses, respectively. However, Southern blot analysis of RT-PCR products could detect considerable levels of trypsinogen mRNA in all ovarian cancer cell lines. In Northern analysis of ovarian cancer tissues, all advanced cancer samples showed trypsinogen gene expression. Serous cystadenocarcinomas exhibited particularly high levels of gene expression. Immunohistochemical staining also detected trypsin in ovarian carcinoma tissues. In contrast, normal ovaries and tumors with low malignant potential did not show trypsinogen expression. Our results demonstrate the extra-pancreatic production and distribution of trypsinogen in human ovarian carcinomas.

Adenocarcinoma↗
PubMed1995

Marked induction of gelatinases, especially type B, in host fibroblasts by human ovarian cancer cells in athymic mice.

Two human ovarian adenocarcinoma cell lines, MCAS-3 and OVISE-3 were found to secrete little of any type of gelatinase in tissue culture. However, when these cell lines were implanted subcutaneously into nude mice the cyst fluids from the resultant tumors contained gelatinase A and/or B. The enzyme activities, especially of gelatinase B, were much higher in the malignant MCAS-3 tumors than in those of the less malignant OVISE-3 tumor cells. To elucidate the origin of gelatinase B in cyst fluids of the MCAS-3 tumors, murine skin fibroblasts (MSF) were isolated from a subcutaneous tumor in a nude mouse and tested for their proteinase secretion in culture. MSF cells, which secreted some gelatinase A and gelatinase B, were induced to secrete high levels of both enzymes, especially gelatinase B, by co-cultivation with MCAS-3 cells. In addition, gelatinase A activity was induced by incubation of MSF cells with the conditioned medium of either MCAS-3 or OVISE-3 cells, whereas gelatinase B was induced only with that of MCAS-3. Although cytokines or growth factors such as IL-1 beta, TGF-beta 1, TNF-alpha or EGF stimulated the secretion of gelatinases A and B from MSF cells, their effects on gelatinase B activity were far less than that of the MCAS-3 conditioned medium. These results indicate that the major part of gelatinase B activity in the cyst fluids of the ovarian tumors is secreted by host interstitial cells stimulated by tumor-derived humoral factors. Similar tumor cell-host cell interactions may be important in the production of various proteinases in other tumor types.

Animals↗
PubMed1995

Characterization of matrix-degrading proteinases and their inhibitors secreted by human gynecological carcinoma cells.

Matrix-degrading proteinases secreted by tumor cells play crucial roles in tumor cell invasion and metastasis. Serum-free conditioned media of 7 human gynecological carcinoma cell lines were examined for proteinases and their inhibitors by using gelatin zymography, reverse zymography and immunoblotting. All of three ovarian adenocarcinoma cell lines secreted urokinase-type plasminogen activator. Among them, a mucinous cystadenocarcinoma cell line also secreted tissue-type plasminogen activator, plasmin-like enzyme and trypsinogen. On the other hand, two ovarian undifferentiated carcinoma cell lines mainly secreted glatinase A or B. A choriocarcinoma cell line secreted multiple metalloproteinases in the highest amount, whereas an endometrial adenocarcinoma cell line (HEC-1) derived from an early clinical stage hardly secreted any gelatinolytic enzyme. The five high proteinases producers hardly secreted the corresponding inhibitors, such as tissue inhibitor of metalloproteinases (TIMP)-1, -2 or plasminogen activator inhibitor-1. In contrast to these highly malignant cell lines, a poor proteinase producer, HEC-1, secreted a large amount of TIMPs. Therefore, an enhanced proteolytic tendency appears to be associated with gynecological cancer cells established from highly malignant tumors.

Adenocarcinoma, Clear Cell↗
PubMed1995

[TIMPs (tissue inhibitors of metalloproteinases): their species, functions and effects on tumor metastasis].

TIMPs (tissue inhibitors of metalloproteinases) belong to a family composed of three members: TIMP-1, -2 and -3. They possess a high degree similarity in their structure including 12 conserved cystenyl residues at the same relative positions. Recently, they have been shown to inhibit not only proteolytic activities of metalloproteinases but also tumor cells metastasis in vitro and in vivo. In this review, we summarize their structure information, inhibitory mechanism of metalloproteinases, TIMPs-like secretory inhibitors, other biological functions of TIMPs such as growth-regulating activity, their chromosomal localizations and related diseases. We also mention their inhibitory effects on tumor cells metastasis in vitro and in vivo, and their clinical applications.

Glycoproteins↗
PubMed1994

Identification of one- and two-chain forms of trypsinogen 1 produced by a human gastric adenocarcinoma cell line.

It has previously been reported that two kinds of human gastric adenocarcinoma cell lines (STKM-1 and MKN28) secrete a trypsin-like enzyme. In this study, four molecular forms of the enzyme (26, 25, 24 and 23 kDa on non-reducing SDS/PAGE) were purified from the serum-free conditioned medium of STKM-1 cells. Analysis of N-terminal amino acid sequences showed that the 26 kDa protein was a two-chain form of trypsinogen 1 which had been produced by proteolytic cleavage of the Arg107-Val108 bond of trypsinogen 1, and the 24 kDa protein was the one-chain form of trypsinogen 1. The 25 and 23 kDa proteins were the activated forms of the two-chain and one-chain trypsinogen 1 respectively. Isoelectric focusing gave pI values of 6.3 and 6.6 for the 26 kDa two-chain form and the 24 kDa one-chain form of trypsinogen 1 respectively. Comparison of the proteolytic activities indicated that the one-chain trypsin 1 had amidolytic activity about four times higher than that of the two-chain enzyme.

Adenocarcinoma↗
PubMed1994

Cell adhesion activity of a 30-kDa major secreted protein from human bladder carcinoma cells.

Human bladder carcinoma cell line EJ-1 secreted a protein of 30 kDa as a major component. This protein was purified from the serum-free conditioned medium of EJ-1. Its N-terminal amino acid sequence was identical to that of fibroblast-derived endothelial cell growth factor (f-ECGF), but there were differences in their amino acid compositions and isoelectric points. The 30-kDa protein showed no effect on the growth of bovine capillary endothelial cells, but it promoted the attachment and spreading of rat liver cell line BRL and human umbilical vein endothelial cell line ECV-304. These results suggest that the 30-kDa protein, tentatively named "tumor-derived adhesion factor (TAF)", may be related with the aberrant cell adhesion of cancer cells.

Amino Acid Sequence↗
PubMed1994

cDNA cloning and mRNA expression of a serine proteinase inhibitor secreted by cancer cells: identification as placental protein 5 and tissue factor pathway inhibitor-2.

A serine proteinase inhibitor was purified from conditioned medium of the human glioblastoma cell line T98G. Analysis of its partial amino acid sequences indicated that this protein was identical to placental protein 5 (PP5), a placenta-derived glycoprotein with serine proteinase inhibitor activity, the amino acid sequence of which had been partially determined. cDNA cloning of PP5 demonstrated that it belonged to the Kunitz-type serine proteinase inhibitor family, having three putative Kunitz-type inhibitor domains, and that it was identical to a recently reported inhibitor, tissue factor pathway inhibitor-2 (TFPI-2) [Sprecher et al. (1994) Proc. Natl. Acad. Sci. USA 91, 3353-3357]. PP5/TFPI-2 transcripts were highly abundant in the full-term placenta and widely expressed in various adult human tissues, such as the liver, skeletal muscle, heart, kidney, and pancreas. Several ovarian carcinoma cells as well as T98G also contained significant amounts of the transcripts.

Amino Acid Sequence↗
PubMed1993

A large cell-adhesive scatter factor secreted by human gastric carcinoma cells.

Human gastric carcinoma cell line STKM-1 secretes a large protein that induces scattering of a rat liver epithelial cell line (BRL) into disconnected individual cells in monolayer culture. This cell-scattering factor was purified from serum-free conditioned medium of STKM-1 cells and found to be composed of three disulfide-linked subunits of 140, 150, and 160 kDa. The 140-kDa peptide contains an amino acid sequence homologous to that of the laminin B2t chain. The native protein has an apparent molecular mass of > 1000 kDa and a pI of 5.0. In addition to the cell-scattering activity, the purified protein stimulates attachment of BRL cells to substrate and their migration. Similar effects have been observed toward various cell lines, including nontumorigenic epithelial, endothelial, and fibroblastic cell lines and human cancer cell lines. Similar cell-scattering activity was secreted by human squamous carcinoma and gastric carcinoma cell lines and nontumorigenic epithelial and endothelial cell lines. These results indicate that the protein, named "ladsin," is probably an extracellular matrix protein that regulates cell-cell and cell-substrate interactions and cell migration.

Animals↗
PubMed1993

Purification and characterization of a two-chain form of tissue inhibitor of metalloproteinases (TIMP) type 2 and a low molecular weight TIMP-like protein.

Multiple forms of metalloproteinase inhibitors were found in the serum-free conditioned medium of the EJ-1 human bladder carcinoma cell line by reverse zymography assay with gelatinase A as the indicator enzyme. Two novel forms of inhibitor with apparent molecular masses of 18 and 22 kDa on nonreducing SDS-polyacrylamide gel electrophoresis (PAGE), together with tissue inhibitor of metalloproteinases (TIMP) and TIMP-2, were purified from the conditioned medium by a series of chromatographic steps. Structural analysis showed that the 18-kDa inhibitor is a two-chain form of TIMP-2 (tc-TIMP-2) produced by proteolytic processing, and the 22-kDa inhibitor may be a partially glycosylated form of TIMP. The purified tc-TIMP-2 was separated into a 17-kDa peptide and a small peptide of about 2.5 kDa by reducing SDS-PAGE and into four isoforms with pI 7.6, 7.3, 7.2, and 6.8 by isoelectric focusing. tc-TIMP-2 has essentially the same inhibitory activity as TIMP-2 toward gelatinase A, collagenase, stromelysin, and matrilysin. Unlike TIMP-2, however, tc-TIMP-2 does not bind to the latent precursor fo gelatinase A. Similar two-chain forms of TIMP-2 were produced by its partial digestion with trypsin or less effectively with plasmin. These results suggest that proteolytic processing of TIMP-2 plays a role in the regulation of gelatinase A activity in the extracellular matrix.

Amino Acid Sequence↗
PubMed1993

Vitronectin secretion by hepatic and non-hepatic human cancer cells.

Thirty-eight human cancer cell lines and subclones derived from 12 different organs were screened for vitronectin secretion in their culture media. By immunoblotting analysis we detected high secretion by three out of five hepatoma cell lines tested but no secretion by the others. In addition, significant secretion was observed in seven non-hepatic cancer cell lines and subclones derived from the cervix, lung, and pancreas. These vitronectin-secreting cells included PLC/PRF/5, HuH-6 #5, HuH-7, HeLa S3, HeLa.P3 #2, #3, #6, #8, A549, and MIAPaCa-2. The results were further confirmed by quantitative analysis using sandwich enzyme-linked immunoassay, and activity analysis of cell attachment promotion on Western blotted filters.

Blotting, Western↗
PubMed1993

Synthetic peptide-generated monoclonal antibodies to transforming growth factor-beta 1.

Using a synthetic peptide with the amino acid sequence corresponding to residues 78-109 of human TGF-beta 1 (A78/109 peptide) as an immunogen, we generated and characterized monoclonal antibodies (Mabs) against transforming growth factor-beta 1 (TGF-beta 1). Spleen cells from a mouse immunized with carrier-free A78/109 peptide were fused with a myeloma cell line, P3U1. Two hybridoma cell lines were selected with A78/109 peptide used as the antigen, and the Mabs were designated as 3H10 and 4C10. Mab 4C10 recognized TGF-beta 1 only in immunoblotting analysis, but not in ELISA. In contrast, Mab 3H10 recognized it in not only ELISA but also immunoblotting analysis. Neither Mab was capable of recognizing both porcine TGF-beta 2 and chicken TGF-beta 3, and, therefore, both Mabs were TGF-beta 1 specific. From the results of epitope-mapping analysis, both Mab 3H10 and Mab 4C10 recognized the 78-87 portion of TGF-beta 1. The epitope recognized by Mab 4C10 was mapped specifically to amino acids 85-87. These results suggest that the region within 78-87 is exposed in the dimeric TGF-beta 1 and is one of the antigenic determinants in this molecule.

Amino Acid Sequence↗
PubMed1993

Characterization of cell-adhesive proteins secreted by cell lines growing in protein- and lipid-free synthetic medium: mouse L.P3 cells secrete a procollagen molecule with potent cell-attachment activity.

We have previously shown that mouse L.P3 cells secrete fibronectin and a novel protein, gelatin-non-binding and heparin-binding cell-adhesive protein (GNCP). Here, we screened and characterized cell-adhesive proteins in the conditioned media (CM's) of a series of cell lines growing in a protein- and lipid-free synthetic medium (P3 cell lines). Although cell-attachment activity of the CM's ranged from undetectable to highly significant, fractionation with affinity columns revealed the presence of significant cell-attachment activity in all CM's. Using cell-attachment assay and immunoassay on blotted filters, various cell-adhesive proteins were detected in the CM's, and most of them were identified as fibronectin, laminin, vitronectin, and collagen. GNCP-like proteins were detected in the CM's of HeLa.P3, JTC-16.P3, L.P3, and JTC-12.P3. There was no relationship between the origin of the cell lines and the cell-adhesive proteins secreted. GNCP purified from L.P3-CM was separated into 120-, 140-, 150-, and 160-kDa proteins on SDS-PAGE, which were judged to be a type of mouse type I procollagen from the following results: (1) they were digested by collagenase, (2) pepsin treatment converted the 150- and 160-kDa proteins into 120- and 140-kDa proteins, (3) they were recognized by anti-type I collagen antiserum, and (4) amino-terminal sequence of the pepsin-digested 140-kDa protein had significant homology with type I collagen. GNCP showed half-maximum activity of cell attachment at 0.03 micrograms/ml, indicating that GNCP was a cell-adhesive protein with an extremely high specific activity compared to other known cell-adhesive proteins.

Amino Acid Sequence↗