scholarly journals β1-4N-acetylgalactosaminyltransferase can synthesize both asialoglycosphingolipid GM2 and glycosphingolipid GM2in vitro and in vivo: isolation and characterization of a β1-4N-acetylgalactosaminyltransferase cDNA clone from rat ascites hepatoma cell line AH7974F

1994 ◽  
Vol 303 (3) ◽  
pp. 957-965 ◽  
Author(s):  
K I-P J Hidari ◽  
S Ichikawa ◽  
K Furukawa ◽  
M Yamasaki ◽  
Y Hirabayashi
Keyword(s):  
Nucleotide Sequence ◽  
Cell Line ◽  
Cdna Clone ◽  
Hepatoma Cell Line ◽  
Rat Tissues ◽  
Ascites Hepatoma ◽  
Terminal Residue ◽  
Isolation And Characterization ◽  
Rat Ascites Hepatoma

We have cloned a cDNA encoding beta 1-4N-acetylgalactosaminyltransferase (EC 2.4.1.92) (GalNAc-T) from rat ascites hepatoma of the free-cell type AH7974F. The cell line only expressed asialo-series glycosphingolipids (GSLs) including asialo-GM2 [Taki, T., Hirabayashi, Y., Ishiwata, Y., Matsumoto, M., and Kojima, K. (1979) Biochim. Biophys. Acta 572, 113-120]. The cDNA, pGNA56, was isolated by screening AH7974F cDNA library in lambda gt10 with a probe. The probe was obtained from AH7974F cDNA by PCR using primers with the nucleotide sequence of the human GalNAc-T cDNA. The amino acid sequence deduced from the nucleotide sequence of pGNA56 exhibited 88% similarity to the human GalNAc-T sequence. The enzyme was a typical type II membrane protein, which consisted of a short N-terminal residue, a transmembrane region, and a long C-terminal residue, including the catalytic domain. The substrate specificity of rat GalNAc-T was determined using homogenates from cells into which the cDNA clone was transfected. The enzyme catalysed not only the formation of GM2 and GD2 from GM3 and GD3 respectively, but also asialo-GM2 from CDH. It also acted on GSL substrates, including GM1b, sialylparagloboside and GD1 alpha. On the other hand, the enzyme did not transfer GalNAc to soluble substrates such as glycoproteins and oligosaccharide. The GSL compositional and immunocytochemical analyses of stable transfectants obtained by transfection of the cDNA showed simultaneous expression of asialo-GM2 and GM2 on the plasma membrane. Therefore, we concluded that the formation of asialo-GM2, GM2 and GD2 was catalysed by the single GalNAc-T. Northern-blot hybridization showed that the GalNAc-T mRNA was strongly expressed in rat brain, testis, and spleen. The gene was also expressed in rat normal liver to a lesser extent. We found the GSLs in asialo- and alpha-pathways such as asialo-GM1 and GD1 alpha in the rat tissues by using a sensitive t.l.c.-immunostaining method. These observations also supported our conclusion that the single GalNAc-T synthesizes asialo-GM2, GM2 and GD2 in vivo.

scholarly journals Isolation and Characterization of Subpopulations of Rat Ascites Hepatoma Cell Line of AH109A with Different Metastatic Potentials

2003 ◽  
Vol 43 (1-3) ◽  
pp. 27-32
Author(s):  
Yutaka Miura ◽  
Miyako Ariga ◽  
Maiko Miyauchi ◽  
Katsuhiko Arai ◽  
Kazumi Yagasaki
Keyword(s):  
Cell Line ◽  
Hepatoma Cell ◽  
Hepatoma Cell Line ◽  
Ascites Hepatoma ◽  
Isolation And Characterization ◽  
Rat Ascites Hepatoma

In vivo cisplatin resistance of rat ascites hepatoma AH66

1996 ◽  
Vol 108 (2) ◽  
pp. 153-156 ◽  
Author(s):  
Tomoyoshi Minamino ◽  
Masaaki Nomura ◽  
Mitsuo Tamai ◽  
Shuzo Moritani ◽  
Tohru Ohshima ◽  
...  
Keyword(s):  
Cisplatin Resistance ◽  
Ascites Hepatoma ◽  
Rat Ascites Hepatoma

Non-genotoxic hepatocarcinogenesis in vitro: the FaO hepatoma line responds to peroxisome proliferators and retains the ability to undergo apoptosis

1993 ◽  
Vol 104 (2) ◽  
pp. 307-315 ◽  
Author(s):  
A.C. Bayly ◽  
N.J. French ◽  
C. Dive ◽  
R.A. Roberts
Keyword(s):  
Cell Death ◽  
Cell Line ◽  
Cell Lines ◽  
Hepatoma Cell ◽  
Hepatoma Cell Line ◽  
Peroxisome Proliferator ◽  
Peroxisome Proliferators ◽  
Hepatoma Cell Lines

A range of hepatoma cell lines (RH1, HTC, FaO, 7800C1 and MH1C1), has been studied with the aim of establishing an in vitro model to investigate the molecular mechanisms of hepatocarcinogenicity induced by the peroxisome proliferator class of non-genotoxic carcinogens. In view of speculation that peroxisome proliferators suppress hepatocyte apoptosis in vivo, we have placed particular emphasis on evaluating whether hepatoma cell lines retain the ability to undergo apoptotic cell death. Expression of the liver-specific differentiation marker albumin and the peroxisome proliferator-activated receptor (PPAR) was highest in the Reuber hepatoma cell line, FaO. This cell line also demonstrated the most marked response to the peroxisome proliferator nafenopin with a 2.2-fold induction of the microsomal enzyme cytochrome p450IVA1. This response was found to display intercellular heterogeneity by immunocytochemistry. Thus, the FaO cell line maintained characteristics of hepatocytes, both in vivo and in vitro, in terms of expression of constitutive and inducible markers. However, none of the cell lines tested mirrored the hyperplastic response of hepatocytes to nafenopin, since no increase in cell growth kinetics was observed on addition of nafenopin to the growth medium. The mode of cell death in confluent FaO cultures was characterised as apoptosis, by fluorescence microscopy and agarose gel electrophoresis of extracted DNA. Cells detaching from confluent FaO cultures exhibited chromatin condensation and DNA fragmentation patterns characteristic of cels undergoing apoptotic death.Interestingly, no apoptosis was seen in monolayer cells, suggesting that apoptosis in vitro is associated with cell shrinkage and detachment similar to that documented for the liver in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Development of an interleukin (IL) 6 receptor antagonist that inhibits IL-6-dependent growth of human myeloma cells.

1994 ◽  
Vol 180 (6) ◽  
pp. 2395-2400 ◽  
Author(s):  
F D de Hon ◽  
M Ehlers ◽  
S Rose-John ◽  
S B Ebeling ◽  
H K Bos ◽  
...  
Keyword(s):  
Cell Line ◽  
Inflammatory Diseases ◽  
Hepatoma Cell ◽  
Myeloma Cell ◽  
Oncostatin M ◽  
Mutant Protein ◽  
Hepatoma Cell Line ◽  
Wild Type ◽  
Human Hepatoma Cell

The pleiotropic cytokine interleukin 6 (IL-6) plays a role in the pathogenesis of various diseases, such as multiple myeloma, autoimmune and inflammatory diseases and osteoporosis. Therefore, specific inhibitors of IL-6 may have clinical applications. We previously succeeded in developing receptor antagonists of IL-6 that antagonized wild-type IL-6 activity on the human Epstein-Barr virus (EBV)-transformed B cell line CESS and the human hepatoma cell line HepG2. However, these proteins still had agonistic activity on the human myeloma cell line XG-1. We here report the construction of a novel mutant protein of IL-6 in which two different mutations are combined that individually disrupt the association of the IL-6/IL-6 receptor (R) alpha complex with the signaltransducing "beta" chain, gp130, but leave the binding of IL-6 to IL-6R alpha intact. The resulting mutant protein (with substitutions of residues Gln160 to Glu, Thr163 to Pro, and replacement of human residues Lys42-Ala57 with the corresponding residues of mouse IL-6) was inactive on XG-1 cells and weakly antagonized wild-type IL-6 activity on these cells. By introducing two additional substitutions (Phe171Leu, Ser177Arg), the affinity of the mutant protein for IL-6R alpha was increased fivefold, rendering it capable of completely inhibiting wild-type IL-6 activity on XG-1 cells. Moreover, this mutant also antagonized the activity of IL-6, but not that of leukemia inhibitory factor, oncostatin M, or GM-CSF on the human erythroleukemia cell line TF-1, demonstrating its specificity for IL-6. These data demonstrate the feasibility of developing specific IL-6R antagonists. The availability of such antagonists may offer an approach to specifically inhibit IL-6 activity in vivo.

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