β-1,6-N-acetylglucosaminyltransferase V (GnT-V) (EC 2.4.1.155) that catalyzes β-1,6 branching in asparagine-linked oligosaccharides is activated on viral or oncogenic transformation and is associated with tumor metastasis. To study the molecular mechanisms involved in regulation of expression of the GnT-V gene, we cloned cDNA and genomic DNA for the enzyme (Saito, H., Nishikawa, A., Gu, J., Ihara, Y., Soejima, Y., Sekiya, C., Niikawa, N., and Taniguchi, N.(1994) Biochem. Biophys. Res. Commun. 198, 318-327). We found that transforming growth factor β (TGFβ) specifically induced GnT-V expression in mouse melanoma cells. The activity of GnT-V was increased 24 h after the addition of TGFβ and remained at high levels up to 72 h. Northern blot analysis showed that the mRNA levels of GnT-V were consistent with the increased activity. To further investigate the nature of the induction, mRNA stability and transcriptional activity were assayed. The enhancement of the GnT-V mRNA expression resulted from prolonged mRNA stability, not from increased transcription. Consequently, elevated mRNA levels were observed even 72 h after the addition of TGFβ. Lectin blot analysis involving leukoagglutinin showed newly synthesized β-1,6 branching structures in the sugar chains of a protein of approximately 130 kDa at 48 h after TGFβ treatment. These results suggested that TGFβ caused changes in the sugar chains of proteins in melanoma cells by up-regulating GnT-V expression. β-1,6-N-acetylglucosaminyltransferase V (GnT-V) (EC 2.4.1.155) that catalyzes β-1,6 branching in asparagine-linked oligosaccharides is activated on viral or oncogenic transformation and is associated with tumor metastasis. To study the molecular mechanisms involved in regulation of expression of the GnT-V gene, we cloned cDNA and genomic DNA for the enzyme (Saito, H., Nishikawa, A., Gu, J., Ihara, Y., Soejima, Y., Sekiya, C., Niikawa, N., and Taniguchi, N.(1994) Biochem. Biophys. Res. Commun. 198, 318-327). We found that transforming growth factor β (TGFβ) specifically induced GnT-V expression in mouse melanoma cells. The activity of GnT-V was increased 24 h after the addition of TGFβ and remained at high levels up to 72 h. Northern blot analysis showed that the mRNA levels of GnT-V were consistent with the increased activity. To further investigate the nature of the induction, mRNA stability and transcriptional activity were assayed. The enhancement of the GnT-V mRNA expression resulted from prolonged mRNA stability, not from increased transcription. Consequently, elevated mRNA levels were observed even 72 h after the addition of TGFβ. Lectin blot analysis involving leukoagglutinin showed newly synthesized β-1,6 branching structures in the sugar chains of a protein of approximately 130 kDa at 48 h after TGFβ treatment. These results suggested that TGFβ caused changes in the sugar chains of proteins in melanoma cells by up-regulating GnT-V expression.
Abstract Oligosaccharides are some of the most important factors in the posttranslational modification of proteins and lipids. It is well known that oligosaccharide structures change during malignant transformation. Fucose is a type of constituent of oligosaccharides, and is notably associated with cancer and inflammation. Fucosylation is catalyzed by several kinds of fucosyltransferases, the GDP-fucose synthetic enzymes, and GDP-fucose transporter. Recently, we found a mutation of the GMDS gene, which involved in the synthesis of GDP-fucose, in a colon cancer cell line, HCT116, leading to a virtually complete deficiency of fucosylation. Down-regulation of cellular fucosylation rendered the cells resistant to tumor necrosis factor-related apotosis-inducing ligand (TRAIL) -induced apoptosis, resulting in the escape from natural killer (NK) cell-mediated tumor immune surveillance. Thus, GMDS and fucosylation are thought to be regulatory factors for TRAIL-induced apoptosis and tumor immune surveillance. In the present study, we searched mutation of the GMDS gene in human cancer cell lines and various kinds of cancer tissues. The aberrant transcripts with deletions of exons 2-4 and 5-7 were detected in the cDNA of LS174T and NCI-H716 cells, respectively. Since these mutations were heterozygous, the deficiency of fucosylation was not observed. In a gastric choriocarcinoma cell line, SCH, only short transcript with deletion of exons 2-4 was detected. This cell line showed a marked decrease in the fucosylation level. PCR and subsequent direct sequencing analyses using 100 human colon cancer tissues identified mutations in two cases. One of the two was c.739 C>T (p.R247X) in exon7, and the other was c.345+5 G>A (IVS4+5) in intron 4. Furthermore, RT-PCR analysis identified aberrant transcripts in 5 of 10 ovarian cancer tissues. All mutant GMDS genes found in this study had no enzymatic activity. Subsequently, the level of fucosylation in many colon cancer tissues was investigated by immunohistochemical analysis using two types of fucosylated oligosaccharide-specific lectin. The decrease of fucosylation was observed in certain cases. Especially, core-fucosylation was decreased in colon cancer with lymph node metastasis. These results suggest that cancer progression in certain patients with colon cancer is dependent on de-fucosylation. Thus, fucosylation is an attractive target for cancer diagnosis and therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 800.
Abstract Cancer treatment with antibodies (Abs) is one of the most successful therapeutic strategies for obtaining high selectivity. In this study, α‐gal–Ab conjugates were developed that dramatically increased cellular cytotoxicity by recruiting natural Abs through the interaction between α‐gal and anti‐gal Abs. The potency of the α‐gal–Ab conjugates depended on the amount of α‐gal conjugated to the antibody: the larger the amount of α‐gal introduced, the higher the level of cytotoxicity observed. The conjugation of antibodies with an α‐gal dendrimer allowed the introduction of large amounts of α‐gal to the Ab, without loss of affinity for the target cell. The method described here will enable the re‐development of Abs to improve their potency.
Expression of cell-cycle modulators at the G1 -S boundary, retinoblastoma gene product (pRb), p21, p16, p27, p53, cyclin D1 , and cyclin E was investigated with 104 hepatocellular carcinomas (HCC), as well as 90 of their adjacent noncancerous lesions and 9 normal liver control specimens. The labeling indices (LI) of pRb, p21, p16, and p27 were higher in HCC lesions than in the adjacent noncancerous lesions and normal controls. Especially, p27 LI in noncancerous lesions was significantly higher than that in normal livers (P = .011). Aberrant p53 expression and cyclin D1 and E overexpression were observed exclusively in HCC lesions. pRb was positive in 85.6% of the HCC cases and was not related to any clinicopathological parameters. The p21 LI was generally low (average, 5.5 ± 9.8). Although a negative regulator, p21 LI was higher in cases with intrahepatic metastasis (P = .0359). The p16 LI was significantly decreased (P = .0121) in cases with advanced stage. p27 LI was significantly decreased in cases with portal invasion (P = .0409), poor differentiation (P < .0001), larger size (P = .0421), and intrahepatic metastasis (P = .0878, borderline significance). On the other hand, aberrant p53 expression showed positive relationships with poor differentiation (P = .0004) and Ki-67 LI (P = .0047). Cyclin D1 overexpression was found in 32.6% of the cases and occurred more frequently in those with high Ki-67 LI (P = .0032), pRb expression (P = .0202), poor differentiation (P = .0612, borderline significance), and intrahepatic metastasis (P = .0675, borderline significance). Cyclin E was overexpressed in 35.5% and had positive relationships with Ki-67 LI (P = .0269) and stage (P = .0125). In univariate analysis, cases with p27 LI < 50 (P = .0004), cyclin D1 overexpression (P = .0041), and cyclin E overexpression (P = .0572, borderline significance) showed poorer outcomes for disease-free survival (DFS). In multivariate analysis, p27 expression could be recognized as an independent prognostic marker for DFS. These findings suggest that in HCC: 1) p27 is active against HCC progression in early phases and, possibly, hepatocarcinogenesis as a negative regulator and can be a novel prognostic marker for DFS; and 2) cyclin D1 predominantly works for cell-cycle progression at the G1 -S boundary
Elastography is currently used clinically to diagnose the degree of liver stiffness. We sought to develop a shear-wave elastography (SWE) measurement method using ultrasound in mice and to compare its results with those of other noninvasive tests for liver fibrosis. We divided male mice into three groups (normal (G1), liver fibrosis (G2), and fatty liver (G3)). We measured mouse liver SWE values and compared them with T1rho and T2 values from magnetic resonance imaging results. We also compared the SWE values with the expression levels of a serum liver fibrosis biomarker (Mac-2-binding protein (M2BP)) and hepatic genes. SWE values significantly increased over time in G2 but did not change in G3. T1rho values in G2 and G3 were significantly increased compared with those in G1. T2 values in G2 did not increase compared with those in group 1. T2 values in G3 significantly increased compared with those in groups 1 and 2. In G2, SWE values significantly and positively correlated with T1rho values. SWE values significantly correlated with serum M2BP levels in G2 but did not correlate with inflammatory gene expression. We could measure SWE values to assess the degree of liver fibrosis in mouse models of liver disease.
