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Biomedical subjects

K Nabeshima

Publications and source records attributed to K Nabeshima.

At least 55 records · Page 3Linked to original sources
PubMed1997

Analysis of tissue factor and tissue factor pathway inhibitor expression in human colorectal carcinoma cell lines and metastatic sublines to the liver.

To investigate the expression of tissue factor (TF) and tissue factor pathway inhibitor (TFPI) in human colorectal carcinomas, Northern blot analysis was performed in a series of human colorectal carcinoma cell lines and in normal or tumoral colorectal tissues. Of 16 human colorectal carcinoma cell lines examined, most expressed TF mRNA, though the levels of expression varied significantly. Considerably higher expression was observed in the cell line CaR-1, while lines established from metastatic lesions tended to express abundant TF mRNA. By contrast, TFPI mRNA levels were low in these high TF-expressing cell lines. TFPI was expressed abundantly in WiDr and in a few other cell lines which expressed a very low level of TF mRNA. Immunocytochemically, both proteins were stained predominantly on the cell surface; however, diffuse cytoplasmic staining for TF also was observed in CaR-1 cells. In addition, the cell surface TF activity was significantly higher in CaR-1 cells than in WiDr cells, confirming the results of mRNA analysis. The level of TF mRNA in colorectal carcinoma tissue in vivo and its ratio to the normal counterpart also varied significantly among the cases. To search for a possible role of TF/TFPI in metastasis of colorectal carcinoma cells, the expression of these genes was compared between a rectal adenocarcinoma cell line, RCM-1, and its highly metastatic subline, RCM-1 L-10. Compared with the parent line, RCM-1 L-10 expressed 7.5-fold higher levels of TF mRNA, whereas TFPI expression was not altered significantly or even decreased slightly. The higher cellular TF activity was confirmed in the metastatic subline in comparison with the parent line.

Adenocarcinoma↗
PubMed1997

Expression of c-Met correlates with grade of malignancy in human astrocytic tumours: an immunohistochemical study.

AIMS: Recent studies suggest the involvement of hepatocyte growth factor/scatter factor (HGF/SF) in glioma cell invasion and tumour progression. We investigated the distribution and rate of tumour cells that express c-Met protein, which is the cell-surface receptor for HGF/SF, in astrocytic tumours. The type of cells that express c-Met in tumour tissues was also identified. METHODS AND RESULTS: c-Met expression was screened immunohistochemically in a total of 43 astrocytic tumours, including 14 low-grade astrocytomas (A), 13 anaplastic astrocytomas (AA) and 16 glioblastoma multiforme (GBM), c-Met reactivity was demonstrated predominantly in the cytoplasm of tumour cells. Bizarre large tumour cells tended to stain intensely. Higher c-Met expression levels (> or = 2+, more than 25% cells were positive) were noted in 21.4% of (A) vs. 53.8% in (AA) and 87.5% in (GBM) (P < 0.001), indicating a clear relationship between c-Met protein staining and higher grade astrocytic tumours. Moreover, c-Met immunoreactivity was also shown in tumour microvasculature, reactive astrocytes, and neurones in the cortex infiltrated by glioma cells. In 85.7% of cases containing infiltrated cortex, neurones were positive vs. no neurones in non-neoplastic regions (P < 0.002). CONCLUSIONS: This evidence suggests that c-Met expression in the brain could be associated with astrocytoma progression and also reactive process. Immunohistochemical determination of c-Met-expressing cell types helps to understand possible roles of c-Met in tumour tissues.

Adolescent↗
PubMed1997

Modulation of E-cadherin expression in TPA-induced cell motility: well-differentiated human adenocarcinoma cells move as coherent sheets associated with phosphorylation of E-cadherin-catenin complex.

We previously presented a two-dimensional cell motility assay using L-10, a highly metastatic variant of the human rectal adenocarcinoma cell line RCM-1, as a motility model of tumor cells of epithelial origin. In this model, L-10 cells moved outward from the cell islands mainly as a localized coherent sheet of cells when stimulated with 12-O-tetradecanoylphorbol-13-acetate (TPA). Electronmicroscopic study of the migrating cell sheets revealed that wide intercellular gaps had developed at the lower portion of the cells, allowing them to extend leading lamellae, whereas close cell-cell contacts remained at the upper portion of the cells. In the present study, the mechanism involved in this localized modulation of cell-cell adhesion at the lower portion of the cells was investigated with special reference to E-cadherin expression. E-cadherin immunostaining, which was demonstrated using an anti-E-cadherin mAb, HECD-1, was decreased in migrating L-10 cell sheets. Apparently, however, E-cadherin was involved in the sheet formation of migrating cells because simultaneous or sequential treatment with TPA and HECD-1 inhibited sheet formation and caused scattering of migrating cells. With immunoelectron microscopic study, E-cadherin immunoreactivity was confined to the upper portion of migrating cells and lost at the lower portion. The level of E-cadherin and alpha-catenin expression was not altered by TPA treatment, although tyrosine phosphorylation of E-cadherin and catenins increased 1.6- to 1.9-fold. We propose that cells are released from cell-cell adhesion only at the lower portion of the cells via phosphorylation of the E-cadherin-catenin complex when stimulated with TPA. This change allows the cells to extend leading lamella and thus move together as coherent sheets (cohort migration).

Adenocarcinoma↗
PubMed1997

Modulation of matrix metalloproteinase-7 (matrilysin) secretion in coculture of human colon carcinoma cells with fibroblasts from orthotopic and ectopic organs.

Matrix metalloproteinase-7 (MMP-7) is a member of the family of matrix-degrading metalloproteinases that are believed to contribute to the complex process of cancer invasion and metastasis. The secretion level of MMP-7 as assayed by immunoblot analysis was low but distinct in the culture medium of a human colon carcinoma cell line, WIDr, whereas none of the fibroblasts secreted the detectable level of MMP-7. The coculture of WiDr with various human fibroblasts from orthotopic (colon) and ectopic (thyroid, brain, lung, and skin) organs significantly stimulated the secretion of MMP-7 compared with the cultures of individual cells. Reverse transcriptase-polymerase chain reaction analysis and RNA blot analysis suggested that this enhancement occurred at a pretranslational level. The extent of the stimulation was widely varied by the fibroblasts used and was dependent on the cellular ratios and density in the coculture. There may exist a tendency that fibroblasts of orthotopic origin stimulate more extensively than do those of ectopic origin. Moreover, in the coculture of high cell density, normal fibroblasts from the ectopic organs reduced the MMP-7 secretion. The stimulation of MMP-7 secretion may be partially mediated through soluble factor(s); however, direct cell-cell interactions would be required for maximum stimulation. The enhanced MMP-7 secretion was also observed in coculture of colon fibroblasts with other colorectal carcinoma cell lines such as RCM-1 and SW837, which secreted hardly detectable levels of MMP-7 in the individual culture. These results suggest that MMP-7 secretion by colon carcinoma cells is influenced by specific interactions between the carcinoma cells and host fibroblasts.

Cell Communication↗
PubMed1997

Establishment and characterization of a new human glioblastoma cell line (MGM-1) with highly motile phenotype.

A new cell line MGM-1 was established from a primary tumor of the left temporal lobe with histological diagnosis of glioblastoma multiforme, removed from a 64-year-old Japanese male. The patient died of recurrence and unusual extracranial metastases of the tumor 7 months after the surgery. The cultured MGM-1 cells are spindle or polygonal in shape. After serial passages, glial fibrillary acidic protein became negative immunocytochemically in vitro. The modal chromosome number was 61-64. Doubling time and soft agar colony forming efficiency were 42.9h and 0.4%, respectively (at 25th passage). MGM-1 is a highly motile cell line in vitro and its serum-free conditioned medium is chemotactic and chemokinetic for other glioma cells. Secretion of gelatinases (probably MMP-2/72-kDa type i.v. collagenase) and MMP-9/92-kDa type i.v. collagenase) and urokinase-type plasminogen activator were also investigated. MGM-1 would therefore be useful for studying the mechanisms regulating glioma-cell motility and invasion. The MGM-1 cell line has been propagated continuously by serial passages (more than 100 passages) during the past 4 years.

Brain Neoplasms↗
PubMed1996

Stimulation of gelatinase B and tissue inhibitors of metalloproteinase (TIMP) production in co-culture of human osteosarcoma cells and human fibroblasts: gelatinase B production was stimulated via up-regulation of fibroblast growth factor (FGF) receptor.

Co-cultures of human osteosarcoma Takase (OST) cells with various human fibroblasts derived from surgical specimens stimulated production of gelatinase B (92-kDa type-IV collagenase, MMP-9), tissue inhibitors of metalloproteinase (TIMP)-1, and TIMP-2 when compared to cultures of individual cells. The maximum stimulation of gelatinase-B production occurred at a cellular ratio of 1:1. Conditioned media from several fibroblast cultures stimulated OST cells to produce gelatinase B, TIMP-1 and TIMP-2, but not vice versa. Among various recombinant growth factors or cytokines, tumor necrosis factor (TNF)-alpha stimulated gelatinase-B production in cultures of OST cells alone, while recombinant basic fibroblast growth factor (bFGF) stimulated gelatinase-B production in co-cultures of OST cells with skin fibroblasts but not in individual cultures of each cell type. In the co-cultures, gelatinase-B production was inhibited by anti-bFGF monoclonal antibody (MAb), but not by anti TNF-alpha MAb. This co-culture-specific stimulation of gelatinase-B production by bFGF was associated with increased expression of the FGF receptor in the co-culture.

Animals↗
PubMed1996

rHuEPO enhances the production of plasminogen activator inhibitor-1 in cultured endothelial cells.

The direct effects of recombinant human erythropoietin(rHuEPO) on coagulation and fibrinolysis factors were evaluated in a cultured endothelial cell (EC) system. Confluent quiescent ECs were incubated with or without 5.0 U/ml rHuEPO for 1, 6, and 18 hours, and supernatant concentrations of plasminogen activator inhibitor-1 (PAI-1): antigen (Ag), tissue plasminogen activator and thrombomodulin, and supernatant activities of tissue factor pathway inhibitor and von Willebrand factor were measured. The results showed that only PAI-1 levels were increased by the presence of rHuEPO. In order to assess the effect of rHuEPO on PAI-1 production by EC more precisely, confluent ECs were incubated with various doses of rHuEPO (0, 1.0, 2.5, 5.0, 10.0 U/ml) for 1, 6, 12, and 18 hours, and PAI-1:Ag concentrations in the supernatants of media were measured. PAI-1:Ag in the supernatants were increased by the presence of rHuEPO at all incubation times (P < 0.01) and the increase in PAI-1:Ag was dependent on rHuEPO concentration. The increases in PAI-1:Ag by 5.0 U/ml rHuEPO were comparable to those by 0.1 U/ml tumor necrosis factor-alpha, 1.0 microgram/ml lipopolysaccharide, and 0.5 U/ml thrombin. The increase in PAI-1:Ag by rHuEPO was suppressed by pre-incubation with 10 micrograms/ml cycloheximide (P < 0.01) or 0.2 microgram/ml actinomycin D (P < 0.01). These results indicate that rHuEPO directly stimulates PAI-1 production in cultured EC via de novo protein and RNA syntheses.

Cells, Cultured↗
PubMed1996

Dissection of fission yeast microtubule associating protein p93Dis1: regions implicated in regulated localization and microtubule interaction.

BACKGROUND: Fission yeast microtubule associating protein (MAP) p93Dis1 functions for sister chromatid separation: dis1 mutants fail to separate chromosomes, while the spindle elongates but without cyclin destruction. p93Dis1 localizes along microtubules in interphase cytoplasm, but shifts to the spindle pole body (SPB) and spindle microtubules upon the entry into mitosis. In this study, regions of p93Dis1 were dissected to examine their role. RESULTS: Nitrocellulose filter blotting shows that recombinant Dis1 binds to bovine brain microtubules in vitro. A basic central region rich in S, T and P is essential for this association. However, the whole p93Dis1 with N- and C-termini containing a conserved repeat motif and heptad repeats, respectively, is necessary for normal microtubule association in vivo. The N-truncated region also binds to microtubules but only to the portions near the SPBs. Overproduction phenotypes indicate that p93Dis1 greatly affects spindle formation and cell morphogenesis. The central region is essential but, by itself, not sufficient for generating such effects. CONCLUSIONS: We propose that p93Dis1 consists of three regions which carry distinct properties for localization: the N-region for cell cycle dependent localization, the central region for direct microtubule association, and the C-region for SPB and nuclear localization. The essential role of p93Dis1 is carried out in the C-region, while the N-region acts as a regulator.

Amino Acid Sequence↗
PubMed1996

The regulatory subunits of fission yeast protein phosphatase 2A (PP2A) affect cell morphogenesis, cell wall synthesis and cytokinesis.

BACKGROUND: Protein phosphatase 2A (PP2A) holoenzymes have a trimeric structure, consisting of a catalytic subunit C and two regulatory subunits A (PR65) and B (PR55). In fission yeast the C subunits, being 80% identical to their mammalian counterparts, are essential for viability and negatively regulate the entry into mitosis. Genetic analyses in budding yeast and Drosophila show that the regulatory subunits are implicated in chromosome segregation, cell morphogenesis and/or cytokinesis. RESULTS: We isolated fission yeast genes paa1+ and pab1+ encoding the regulatory subunits PR65 and PR55, respectively. Gene disruption showed that the paa1+ gene was essential for viability while pab1+ was not required at 26-33 degrees C. Microtubule and actin distributions were anomalous in gene disrupted delta paa1 cells which were incapable of forming a polarized cell shape. Gene disrupted delta pab1 cells were pear- or round-shaped, and lost the polar distributions of actin and microtubules. In addition, delta pab1 cells were defective in cell wall synthesis and sporulation at permissive temperatures. At restrictive temperatures, delta pab1 cells showed an osmoremedial temperature-sensitive phenotype and delayed in cytokinesis. However, chromosome segregation was normal. CONCLUSION: Fission yeast PP2A regulatory subunit plays a critical role in cell morphogenesis, probably through regulation of the cytoskeletal network and cell wall synthesis.

Amino Acid Sequence↗
PubMed1996

Aberrant mitosis in fission yeast mutants defective in fatty acid synthetase and acetyl CoA carboxylase.

Two fission yeast temperature-sensitive mutants, cut6 and lsd1, show a defect in nuclear division. The daughter nuclei differ dramatically in size (the phenotype designated lsd, large and small daughter). Fluorescence in situ hybridization (FISH) revealed that sister chromatids were separated in the lsd cells, but appeared highly compact in one of the two daughter nuclei. EM showed asymmetric nuclear elongation followed by unequal separation of nonchromosomal nuclear structures in these mutant nuclei. The small nuclei lacked electron-dense nuclear materials and contained highly compacted chromatin. The cut6+ and lsd1+ genes are essential for viability and encode, respectively, acetyl CoA carboxylase and fatty acid synthetase, the key enzymes for fatty acid synthesis. Gene disruption of lsd1+ led to the lsd phenotype. Palmitate in medium fully suppressed the phenotypes of lsd1. Cerulenin, an inhibitor for fatty acid synthesis, produced the lsd phenotype in wild type. The drug caused cell inviability during mitosis but not during the G2-arrest induced by the cdc25 mutation. A reduced level of fatty acid thus led to impaired separation of non-chromosomal nuclear components. We propose that fatty acid is directly or indirectly required for separating the mother nucleus into two equal daughters.

Acetyl-CoA Carboxylase↗
PubMed1996

Feasibility of archival non-buffered formalin-fixed and paraffin-embedded tissues for PCR amplification: an analysis of resected gastric carcinoma.

Although several factors affecting the sensitivity of polymerase chain reaction (PCR) amplification from formalin-fixed tissues have been investigated mostly by experiments, the feasibility of archival formalin-fixed, paraffin-embedded tissue samples stored in pathology departments for PCR amplification has rarely been examined directly. Thus, the feasibility of 74 archival unbuffered 10% formalin-fixed, paraffin-embedded tissues for PCR amplification with primers producing a 190 b.p. DNA segment of p53 exon 5 was investigated. Fixation time was the critical factor influencing the sensitivity of PCR amplification. All (6/6) of the samples fixed for only 1 day, 44% (7/16) of the samples fixed for 2-3 days and 14% (4/28) of the samples fixed for 4-6 days showed successful amplification, while no amplification was obtained for the samples fixed for 7 days or more. The peak size of DNA extracted from the archival tissues decreased as the fixation time became longer. Experiments using xenografted tumor tissues fixed for various times showed longer permissible fixation time; up to 9 days of fixation, decreasing amounts of PCR products were obtained while no amplification was obtained for the samples fixed for 12 days or more. The time in paraffin seemed to be a minor factor for PCR amplification since all of the 1 day fixation samples, including those that had been embedded for up to 5 years, resulted in efficient amplification. The size of the amplified DNA segments, however, could be another factor influencing the sensitivity of amplification because even the 1 day fixation samples showed less amplification of 345 b.p. DNA compared with those of 167 and 262 b.p. DNA. Additionally, a point mutation was detected in the amplified p53 products from archival tissues using a non-isotopic method, temperature gradient gel electrophoresis. In conclusion, archival tissue samples that had been fixed immediately for only up to 1 day were constantly available for PCR amplification of approximately 200 b.p. DNA segments, suggesting that surgical specimens should be subjected to cutting and paraffin embedding just after 1 day or less fixation for subsequent use in PCR amplification.

Animals↗
PubMed1995

p93dis1, which is required for sister chromatid separation, is a novel microtubule and spindle pole body-associating protein phosphorylated at the Cdc2 target sites.

Fission yeast cold-sensitive (cs) dis1 mutants are defective in sister chromatid separation. The dis1+ gene was isolated by chromosome walking. The null mutant showed the same phenotype as that of cs mutants. The dis1+ gene product was identified as a novel 93-kD protein, and its localization was determined by use of anti-dis1 antibodies and green fluorescent protein (GFP) tagged to the carboxyl end of p93dis1. The tagged p93dis1 in living cells localizes along cytoplasmic microtubule arrays in interphase and the elongating anaphase spindle in mitosis, but association with the short metaphase spindle microtubules is strikingly reduced. In the spindle, the tagged p93dis1 is enriched at the spindle pole bodies (SPBs). Time-lapse video images of single cells support the localization shift of p93dis1 to the SPBs in metaphase and spindle microtubules in anaphase. The carboxy-terminal fragment, which is essential for Dis1 function, accumulates around the mitotic SPB. We propose that these localization shifts of p93dis1 in mitosis facilitates sister chromatid separation by affecting SPB and anaphase spindle function.

Amino Acid Sequence↗
PubMed1995

The human tumor cell-derived collagenase stimulatory factor (renamed EMMPRIN) is a member of the immunoglobulin superfamily.

Tumor cell-derived collagenase stimulatory factor, renamed extracellular matrix metalloproteinase inducer (EMMPRIN), is a M(r) approximately 58,000 glycoprotein which is located on the outer surface of human tumor cells and which interacts with fibroblasts to stimulate expression of several matrix metalloproteinases in the fibroblasts. In this study, we have used several approaches to isolate a complementary DNA encoding EMMPRIN. Several peptide sequences obtained from the isolated M(r) 58,000 glycoprotein are found in the translated complementary DNA clone, verifying its identity. Computer database searches indicate that EMMPRIN is a member of the immunoglobulin superfamily and that the deduced amino acid sequence of EMMPRIN is identical to that recently reported for human basigin and M6 antigen, molecules of previously undetermined biological function.

Amino Acid Sequence↗
PubMed1995

Reverse-zymographic analysis of protease nexin-II/amyloid beta protein precursor of human carcinoma cell lines, with special reference to the grade of differentiation and metastatic phenotype.

Trypsin inhibitors in serum-free conditioned media (SFCM) of various human carcinoma cell lines were analyzed by reverse zymography. Most of the cells secreted high-molecular-weight trypsin inhibitors (HMTI) larger than 100 kDa. The cell lines of colorectal carcinoma origin had a tendency to secrete HMTI whose molecular weight was a little higher than that of the other cell lines. Analysis of SFCM of subclones with different histological differentiation and metastatic/invasive potentials derived from a single pancreatic carcinoma cell line SUIT-2 showed that the HMTI activity in SFCM was correlated to the degree of histological differentiation in vivo and tended to be inversely correlated to their metastatic/invasive capabilities. Immunoblotting analysis revealed that these HMTI were protease nexin-II/amyloid beta protein precursors (PN-II/APP). Semi-quantificative reverse-transcriptase/polymerase-chain reaction study for PN-II/APP mRNAs suggested that the differences in PN-II/APP activities in SFCM between the subclones might be post-transcriptional or post-secretional events. In addition, SFCM of a highly metastatic subclone contained 43-kDa protein which reacted to anti-APP monoclonal antibody (MAb) suggesting that the subclone may have APP-degrading activity.

Adenocarcinoma↗
PubMed1995

Ultrastructural study of TPA-induced cell motility: human well-differentiated rectal adenocarcinoma cells move as coherent sheets via localized modulation of cell-cell adhesion.

We previously found that 12-O-tetradecanoylphorbol-13-acetate (TPA)-enhanced invasion of Matrigel was associated with augmentation of cell motility but not with metalloproteinase activity in a highly metastatic variant (L-10) of human rectal adenocarcinoma cell line RCM-1. In a two-dimensional cell motility assay, TPA induced active L-10 cell locomotion with characteristic morphology; the cells moved outwards from the cell islands mainly as a localized coherent sheet of cells. The leading cells showed locomotor morphologies with fan-shaped leading lamellae while the following cells had cell contacts on all sides and appeared to lack leading lamellae. In the present ultrastructural study, the following cells frequently showed tapering cytoplasmic protrusions and leading lamella-like processes underlapping a preceding cell, indicating that the locomotion mechanism is almost the same for both the leading and following cells. For this type of locomotion as a coherent sheet we propose that localized modulation of cell-cell adhesion was induced such that wide intercellular gaps occurred at the lower portion of the cells to allow the cells to extend the tapering cytoplasmic processes and leading lamellae while close cell-cell contacts remained at the upper portion of the cells. These TPA-induced changes took place predominantly in the cells at the periphery of the cell islands, while the cells in the middle of the cell islands maintained close cell-cell contacts including complex interdigitation all around the cells, suggesting the modulation of TPA action by cell-cell interaction. Additionally, consistent with the evidence for junctional complexes between the cells moving outwards, the Lucifer-yellow dye transfer studies showed some, limited cell-cell coupling, suggesting the presence of at least some gap junctional intercellular communication in the moving cell sheets.

Actin Cytoskeleton↗
PubMed1995

Esophagoaortic fistula caused by esophageal tuberculosis: report of a case.

We report herein the case of a 73-year-old woman who was urgently admitted to hospital with severe hematemesis. An emergency endoscopy revealed a protruding lesion 36 cm from the incisors; however, respiratory insufficiency precluded surgery and despite aggressive medical treatment, the patient's respiratory status continued to deteriorate, leading to death on the 36th hospital day. An autopsy revealed the source of the hemorrhage to be a fistula connecting the esophagus and the descending aorta. Histopathologic studies showed that this fistula was caused by esophageal tuberculosis.

Aged↗