[Effect of overexpression of glycosylphosphatidylinositol-specific phospholipase D on biological character of hepatocellular carcinoma cell line HepG2].
OBJECTIVE
To investigate the effect of overexpression of glycosylphosphatidyl-inositol-specific phospholipase D (GPI-PLD) on the biological character of hepatocellular carcinoma cell line HepG2.


METHODS
The GPI-PLD gene eukaryon expression vector pcDNA3.1(+)/ GPI-PLD was transiently transfected into HepG2 cell by lipid-media transfection. The untransfected HepG2 and HepG2 transfected with pcDNA3.1(+) were used as controls. After screening with G418, the single clone was obtained. The expression level of GPI-PLD mRNA in HepG2 was identified by reverse transcription polymerase chain reaction (RT-PCR). GPI-PLD activities were analyzed quantitatively by triton-X-114 partition with GPI anchored placental alkaline phosphatase (PLAP) as a substrate. Cell count was used to detect the proliferation of the 3 groups, and complement dependent cytotoxicity (CDC) effects were observed by the staining of trypan blue. Apoptosis cells were analyzed by flow cytometry. Carcinoembryonic antigen (CEA)was detected by enzyme linked immunosorbent assay (ELISA).


RESULTS
Compared with HepG2 and pcDNA3.1(+)/HepG2 cell, the levels of GPI-PLD activities and its mRNA from pcDNA3.1(+)/GPI-PLD/HepG2 were increased with almost 2 to 5 times,respectively. The GPI anchored PLAP and CEA released into the medium by GPI-PLD, and the rate of CDC killing on the cells were significantly increased. However, the proliferative capacity was obviously decreased, and the typical apoptosis cells were presented in positive clones and its apoptosis rates were increased significantly.


CONCLUSIONS
The stable cell line with overexpression of GPI-PLD has been constructed. The overexpression of GPI-PLD in these cells increases the sensitivity of these cells to CDC killing and impairs the proliferative capacity of cells, and promotes the apoptosis.
He
Wang-jiao
WJ
Department of Biochemistry, School of Biological Science and Technology, Central South University, Changsha, China.
Tang
Jian-hua
JH
Tan
Chao-chao
CC
Duan
Qiong
Q
Wang
Kai-jia
KJ
Zuo
Ke-qiang
KQ
Yuan
Xian-yu
XY
D017209:Apoptosis / Q000235:genetics*; D006528:Carcinoma, Hepatocellular / Q000235:genetics* / Q000473:pathology; D003167:Complement Activation / Q000235:genetics; D003602:Cytotoxicity, Immunologic / Q000235:genetics; D005057:Eukaryotic Cells / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics* / Q000473:pathology; D010739:Phospholipase D / Q000096:biosynthesis* / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection; D014407:Tumor Cells, Cultured; D015854:Up-Regulation
D012333:RNA, Messenger; D010739:Phospholipase D; C065017:glycoprotein phospholipase D