Increased Susceptibility to Apoptosis of Human Hepatocarcinoma Cells Transfected with Antisense N-Acetylglucosaminyltransferase V cDNA

The antisense cDNA of N-acetylglucosaminyltransferase V (GnT-V, EC 2.4.1.155) was constructed as pcDNA3/GnT-V-AS plasmid and transfected into 7721 cells, a human hepatocarcinoma cell line. The transfection was confirmed with Northern blot. By using HPLC and HRP-lectin staining, it was found that the...

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Bibliographic Details
Published in:Biochemical and biophysical research communications 1999-10, Vol.264 (2), p.509-517
Main Authors: Guo, Hua-Bei, Liu, Fei, Chen, Hui-Li
Format: Article
Language:English
Subjects:
all-trans-retinoic acid
antisense
Apoptosis
Blotting, Northern
Carcinoma, Hepatocellular
Cell Division
Cell Survival
DNA, Antisense - pharmacology
Flow Cytometry
Horseradish Peroxidase
human hepatocarcinoma cell line
Humans
In Situ Nick-End Labeling
Liver Neoplasms
N-acetylglucosaminyltransferase V (GnT-V)
N-Acetylglucosaminyltransferases - genetics
N-Acetylglucosaminyltransferases - metabolism
N-Acetylglucosaminyltransferases - pharmacology
Plasmids - genetics
Polysaccharides - chemistry
Polysaccharides - genetics
Transfection
Tretinoin - pharmacology
Tumor Cells, Cultured - drug effects
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Summary:The antisense cDNA of N-acetylglucosaminyltransferase V (GnT-V, EC 2.4.1.155) was constructed as pcDNA3/GnT-V-AS plasmid and transfected into 7721 cells, a human hepatocarcinoma cell line. The transfection was confirmed with Northern blot. By using HPLC and HRP-lectin staining, it was found that the cells transfected with pcDNA3/GnT-V-AS (GnT-V-AS/7721) expressed less GnT-V activity and β-1,6-GlcNAc branching in the cell glycoproteins compared with the cells mock-transfected with the vector pcDNA3 (pcDNA3/7721). The growth rate of GnT-V-AS/7721 was decreased in serum-containing medium, while the cell death was accelerated in serum-free medium. The GnT-V-AS/7721 cells were more susceptible to the apoptosis induced by ATRA than the mock-transfected cells. This was evidenced by the obvious appearance of a hypoploid sub-G1 fraction in the DNA histogram using FCM analysis, the more condensed new moon-type nuclei under morphological observation, and the more intensive TUNEL reaction for assaying the fragmented DNA. At the same time as GnT-V down-regulation by GnT-V-AS, an increase of another N-aceylglusaminyltransferase, GnT-III (EC 2.4.1.144), was observed, and the biological significance of this finding was discussed.
ISSN:0006-291X
1090-2104
DOI:10.1006/bbrc.1999.1303