[Study of cooperation of tumor suppressor p33(ING1b) with p53 gene in hepatocellular carcinoma].
OBJECTIVE
To investigate the cooperation effect of p33(ING1b), coded protein of the novel tumor-suppressor gene ING1b, with p53 gene on the cell growth, cell cycle arrest, apoptosis and expression of the p53 downstream gene p21(WAF1/CIP1) in hepatocellular carcinoma.


METHODS
Recombinant plasmids pcDNA3-INGIb containing sense p33(ING1b) cDNA, and pCMV-wtp53 containing sense p53 gene, and were transfected into HepG2, PLC/PRF/5 and Hep3B hepatoma cell lines with different p53 endogenous expression status using lipofecTAMINE method. Recombinant plasmids pcDNA3-alphaING1b containing antisense p33(ING1b) cDNA, pCMV-alphawtp53 containing antisense p53 gene, and plain vector as control were transfected into the cells. Forty-eight hours after transfection, the apoptosis rates were detected and cell cycle arrest were analyzed by flow cytometry. The expression of p33(ING1b) and wtp53 proteins were detected by Western blot. The luciferase gene reporter plasmid drived by p21(WAF1/CIP1) promoter was also transfected into the cells to analyze the activation of p21(WAF1/CIP1) gene in hepatoma cells. Then 6% ethanol was added into the in DMEM culture medium and all of the above experiments were repeated.


RESULTS
After the p33(ING1b) gene was transfected, the apoptosis rate of HepG2 cells which express endogenous wtp53 was enhanced (22.53%), the number of cells arrested in G(0)/G(1) phase was increased (67.45%), and the activation of p21(WAF1/CIP1) promoter reached the highest level (13.08) (all P < 0.01). In the PLC/PRF/5 cells which express endogenous mutant p53, after combined p33(ING1b) and wtp53 gene transfection the percentage of cells arrested in G(0)/G(1) phase (78.16%) and the activation of p21(WAF1/CIP1) promoter (12.99) were higher than those in the experiment groups transfected with p33(ING1b) or wtp53 gene alone (both P < 0.01), however, the difference in apoptosis rate was not statistically significant (P > 0.05). After 6% ethanol treatment, apoptosis rate of PLC/PRF/5 cells transfected with combined p33(ING1b) and wtp53 gene was increased significantly (42.8%). The apoptosis rate and cell cycle arrest were not significantly different between the Hep3B cells with deletion of wtp53 which were transfected with p33(ING1b) and wtp53 gene and those of the control group (P > 0.05), however, the activity of the p21(WAF1/CIP1) promoter was activated in the combined transfectlion group (10.32, P < 0.01).


CONCLUSIONS
p33(ING1b) cooperate with wtp53 in the process of inhibiting hepatoma cell growth, inducing apoptosis, and activating p53 downstream gene p21(WAF1/CIP1), and after chemical inducing reagent treatment the cooperation between p33(ING1b) and wtp53 gene is enhanced.
Zhu
Zhi
Z
Department of Pathology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China.
Lin
Jing
J
Qu
Jian-hui
JH
Li
Fang-mei
FM
Ni
Can-rong
CR
Zhu
Ming-hua
MH
D017209:Apoptosis; D006528:Carcinoma, Hepatocellular / Q000235:genetics* / Q000473:pathology; D002453:Cell Cycle; D018797:Cell Cycle Proteins; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins; D016147:Genes, Tumor Suppressor; D016158:Genes, p53; D006801:Humans; D000076023:Inhibitor of Growth Protein 1; D047908:Intracellular Signaling Peptides and Proteins; D008113:Liver Neoplasms / Q000235:genetics* / Q000473:pathology; D009687:Nuclear Proteins; D011506:Proteins / Q000235:genetics*; D014162:Transfection; D025521:Tumor Suppressor Proteins
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C501503:ING1 protein, human; D000076023:Inhibitor of Growth Protein 1; D047908:Intracellular Signaling Peptides and Proteins; D009687:Nuclear Proteins; D011506:Proteins; D025521:Tumor Suppressor Proteins