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 cells transfected with pcDNA3/GnT-V-AS (GnT-V-AS/7721) expressed less GnT-V activity and beta-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-G(1) 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.
Guo
H B
HB
Ministry of Health, Shanghai Medical University, Shanghai, 200032, China.
Liu
F
F
Chen
H L
HL
D017209:Apoptosis; D015152:Blotting, Northern; D006528:Carcinoma, Hepatocellular; D002455:Cell Division; D002470:Cell Survival; D016373:DNA, Antisense / Q000494:pharmacology*; D005434:Flow Cytometry; D006735:Horseradish Peroxidase; D006801:Humans; D020287:In Situ Nick-End Labeling; D008113:Liver Neoplasms; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D010957:Plasmids / Q000235:genetics; D011134:Polysaccharides / Q000737:chemistry / Q000235:genetics; D014162:Transfection; D014212:Tretinoin / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000187:drug effects
D016373:DNA, Antisense; D011134:Polysaccharides; D014212:Tretinoin; D006735:Horseradish Peroxidase; D017351:N-Acetylglucosaminyltransferases; C052254:alpha-1,6-mannosylglycoprotein beta 1,6-N-acetylglucosaminyltransferase