Vero cell heparin-binding epidermal growth factor-like growth factor (HB-EGF) is synthesized as a 20-to 30-kDa membrane-anchored HB-EGF precursor (proHB-EGF). Localization and processing of proHB-EGF, both constitutive and 12-O-tetradecanoylphorbol 13-acetate (TPA)-inducible, was examined in Vero cells overexpressing recombinant HB-EGF (Vero H cells). Flow cytometry and fluorescence immunostaining demonstrated that Vero cell proHB-EGF is cell surface-associated and localized at the interface of cell to cell contact. Cell surface biotinylation and immunoprecipitation detected a 20-to 30-kDa heterogeneous proHB-EGF species. Vero H cell surface proHB-EGF turned over constitutively with a half-life of 1.5 h. Some of the 20-to 30-kDa cell surface-associated proHB-EGF was processed and a 14-kDa species of bioactive HB-EGF was released slowly, but most of the proHB-EGF was internalized, displaying a diffuse immunofluorescent staining pattern and accumulation of proHB-EGF in endosomes. Addition of TPA induced a rapid processing of proHB-EGF at a Pro148-Val149 site with a half-life of 7 min. The TPA effect was abrogated by the protein kinase C inhibitors, staurosporine and H7. Kinetic analysis showed that loss of cell surface proHB-EGF is maximal at 30 min after addition of TPA and that proHB-EGF is resynthesized and the initial cell surface levels are regained within 12-24 h. Loss of cell surface proHB-EGF was concomitant with appearance of 14-and 19-kDa soluble HB-EGF species in conditioned medium. Vero H cell-associated proHB-EGF is a juxtacrine growth factor for EP170.7 cells in coculture. Processing of proHB-EGF resulted in loss of juxtacrine activity and a simultaneous increase in soluble HB-EGF paracrine mitogenic activity. It was concluded that processing regulates HB-EGF bioactivity by converting it from a cellsurface juxtacrine growth factor to a processed, released soluble paracrine growth factor.
SummaryIn the present study. we analysed human choriocarcinoma cell lines for abnormalities in the tumour-suppressor gene p53 by Southern blotting, Northern blotting, non-radioisotopic single-stranded conformational polymorphism (SSCP) and complementary DNA sequencing. In all cell lines (Bewo, GCH-1, GCH-2. SCH, JAR, JEG-3. NUC-1 and HCCM-5), no p53 gene abnormality was detected by using Southern blotting. p53 mRNA of the expected size was detected in all cell lines tested by Northern blotting. SSCP analysis revealed abnormalities of p53 cDNA in the SCH cell line. Sequencing analysis of the entire coding region of the p53 gene revealed that both alleles were expressed in the JEG-3 cell line, and one of the alleles contained a point mutation (G to T) in codon 167 (Gln to His). In the NUC-1 cell line both alleles were point mutated. One allele had a point mutation (A to T) that resulted in a codon 17 change (Glu to Asp), and another had a point mutation (A to T) that caused a codon 24 change (Lys to Asn). In the SCH cell line, AGG was inserted between codon 249 and 250; this insertion resulted in an abnormal structure of the p53 protein. In three out of eight human choriocarcinoma cell lines, a p53 gene abnormality was detected. Therefore our data demonstrate that p53 gene abnormalities are associated with choriocarcinoma cell lines.
We investigated the extent to which NO participates in the developmental competence (oocyte maturation, fertilization and embryo development to blastocyst) using an in vitro culture system adding sodium nitroprusside (SNP), NO donor, and NOS inhibitor (N‐omega‐nitro‐L‐arginine methyl ester, L‐NAME). We also assessed the effects of NO/NOS system on blastocyst implantation using an in vitro trophoblast outgrowth assay. The treatment of low concentrations of SNP (10−7 M) significantly stimulated meiotic maturation to metaphase II stages in cumulus enclosed oocytes. In contrast, 10−3 and 10−5 M L‐NAME demonstrated a significant suppression in resumption of meiosis. This inhibition was reversed by the addition of SNP. No development beyond the four‐cell stage was observed by the addition of high concentration of SNP (10−3 M). Inhibition of embryo development, especially the conversion of morulae to blastocysts, was also observed in the treatment of lower doses of SNP (10−5 and 10−7 M). Similarly, inhibition of NO by NOS inhibitor resulted in the dose‐dependent inhibition of embryo development and hatching rates, but the concomitant addition of SNP with L‐NAME reversed the inhibitory effect by each SNP or L‐NAME treatment. Furthermore, low concentration of SNP (10−7 M) but not high concentration of SNP (10−3 M) significantly stimulated trophoblast outgrowth, whereas the addition of L‐NAME suppressed the spreading of blastocysts in a dose‐dependent manner. These results suggest that NO may have crucial roles in oocyte maturation and embryogenesis including the process of implantation. The observed differences in required amount of NO and the sensitivity to cytotoxicity of NO in each developmental stage embryos may also suggest that NO/NOS system is tightly regulated in developmental stage specific manner. Mol. Reprod. Dev. 58:262–268, 2001. © 2001 Wiley‐Liss, Inc.
a1,6-Fucosyltransferase (Fut8), an enzyme that catalyzes the introduction of α1,6 core fucose to the innermost N-acetylglucosamine residue of the N-glycan, has been implicated in the development, immune system, and tumorigenesis. We found that α1,6-fucosyltransferase and E-cadherin expression levels are significantly elevated in primary colorectal cancer samples. Interestingly, low molecular weight population of E-cadherin appeared as well as normal sized E-cadherin in cancer samples. To investigate the correlation between α1,6-fucosyltransferase and E-cadherin expression, we introduced α1,6-fucosyltransferase in WiDr human colon carcinoma cells. It was revealed that the low molecular weight population of E-cadherin was significantly increased in α1,6-fucosyltransferase-transfected WiDr cells in dense culture, which resulted in an enhancement in cell-cell adhesion. The transfection of mutated a1,6-fucosyltransferase with no enzymatic activity had no effect on E-cadherin expression, indicating that core fucosylation is involved in the phenomena. In α1,6-fucosyltransferase knock down mouse pancreatic acinar cell carcinoma TGP49 cells, the expression of E-cadherin and E-cadherin dependent cellcell adhesion was decreased. The introduction of α1,6-fucosyltransferase into kidney epithelial cells from α1,6-fucosyltransferase -/-mice restored the expression of E-cadherin and E-cadherin-dependent cell-cell adhesion. Based on the results of lectin blotting, peptide Nglycosidase F treatment, and pulse-chase studies, it was demonstrated that the low molecular weight population of E-cadherin contains peptide N-glycosidase F insensitive sugar chains, and the turnover rate of E-cadherin was reduced in α1,6-Fucosyltransferase transfectants. Thus, it was suggested that core fucosylation regulates the processing of oligosaccharides and turnover of E-cadherin. These results suggest a possible role of core fucosylation in the regulation of cell-cell adhesion in cancer. (Cancer Sci 2009; 100: 888-895) I t is generally accepted that glycosylation affects many properties of glycoproteins, including their conformation, flexibility, and hydrophilicity. As a result, it regulates protein sorting, stability, and protein-protein interactions.(1-5) N-Glycans have a common core structure, and their branching patterns are determined by glycosyltransferases.(6,7) Fut8 is an enzyme that catalyzes the introduction of α1,6 core fucose on the asparagine-branched N-acetylglucosamine residue of the chitobiose unit of complextype N-glycans. (8,9) Fut8 has been investigated especially in terms of oncogenesis, since the α1,6-fucosylation of α-fetoprotein is a well-known marker of hepatocellular carcinoma. (10) In previous studies, our group reported that Fut8 expression is markedly enhanced in several types of cancer cell lines (11) rat hepatoma tissues (12) and in ovarian serous adenocarcinoma cells.E-cadherin is a 120 kDa type I membrane protein, which belongs to the class of calcium-dependent cell adhesion molecules. (14) It mediates cell-cell adhesi...
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