[Mutations of tumor suppressor gene PTEN mutations in hepatocellular carcinoma and its implications in tumor proliferation and apoptosis].
OBJECTIVE
To study mutations of tumor suppressor gene PTEN in human hepatocellular carcinomas and its effects on the proliferation and apoptosis of hepatocellular carcinoma cell line HHCC.


METHODS
(1) PCR-SSCP and sequence analysis were used to detect the mutations of the 5th and 8th exon of PTEN in 42 cases of human primary hepatocellular carcinoma. (2) Eukaryotic expression vectors of the wild-type (pEGFP-wt-PTEN) and the mutant type (pEGFP-PTEN, G129R) of PTEN were constructed. Lipofectamine 2000 mediated gene transfection was used to transfect hepatocellular carcinoma cell line HHCC, in which the PTEN protein is not expressed. Culture medium containing G418 was used to select stable transfectants. MTT colorimetry was used to analyze the proliferation ability of selected cell lines. Naive HHCC cells and HHCC cells transfected with empty vector (pEGFP-C1) served as controls. (3) TNF-alpha was used to induce apoptosis of selected cell clones.


RESULTS
(1) Point mutation involving the 5th exon of PTEN was detected in 4 of 42 primary hepatocellular carcinomas. (2) Compared with the control groups, the proliferation of hepatocellular carcinoma cells was significantly inhibited by the transfection of wild-type PTEN gene, while the transfection with mutant PTEN construct did not significantly change the proliferation. (3) The apoptosis indices of cells transfected with the wild-type and the mutant PTEN genes were 13.8% and 8.1% respectively. Compared with the control, the apoptosis index of HHCC cell transfected by the wild type PTEN was significantly lower (P < 0.05). There were no significant differences between HHCC cells transfected with mutated PTEN gene and the control (P > 0.05). The expression of internal 473-phosphorylated Akt of HHCC was weak, but was enhanced when the cells treated with TNF-alpha. However, it was down regulated by the wild type PTEN.


CONCLUSIONS
(1) First time report that PTEN mutations can be found in 9.5% human primary hepatocellular carcinomas. (2) The expression of the wild-type PTEN can suppress the proliferation of HHCC cells, and such suppression was lost when PTEN gene was mutated. (3) PTEN inhibition of the proliferation and the enhancement of apoptosis of hepatocellular carcinoma cells is likely related to a down-regulation of the TNF-alpha induced activation of protein kinase Akt pathway.
Guo
Shuang-ping
SP
Department of Pathology, Xijing Hospital of Fourth Military Medical University, Xi'an 710032, China. guoshp@fmmu.edu.cn
Wang
Li
L
Wang
Wen-liang
WL
Li
Qin-long
QL
Wang
Wen-yong
WY
Zhang
Jing
J
D017209:Apoptosis / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D001483:Base Sequence; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D004252:DNA Mutational Analysis; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation; D051059:PTEN Phosphohydrolase / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D014409:Tumor Necrosis Factor-alpha; D049452:Green Fluorescent Proteins; D051059:PTEN Phosphohydrolase; C494929:PTEN protein, human