Expression Of Tumor Suppressor P33～(ING1b) And Its Cooperation With P53 Gene In Hepatocellular Carcinoma

Tumor suppressor gene negatively control cell growth. Inactivation of its normal functions may cause tumorigenesis. p53, a tumor suppressor gene ,and its encoding protein act as a check point in cell cycle, inhibite the injured DNA to process mismatch repair and protect cells from malignant transformation . In primary hepatocellular carcinoma(HCC), inactivation of wild-type p53 is an important molecular event in tumorigenesis. One reason of p53 gene losts its normal function is gene mutation and another reason is p53- related gene network can not exert regulation as normally. As an important member of p53 protein family, tumor suppressor gene ING1 encoding protein P33~ING1b has a closely relation with wild-type p53 protein, both of them cooperate in regulation of many cell life activities. Therefore , to investigate the cooperating relation of p33~ING1b and p53 gene in tumorigenesis of liver cells is very significant in theory and practice.The expression of p33~ING1b and p53 proteins in 57 cases of primary hepatocellular carcinoma tissues (HCC)(including 4 cases of primary cholangiocarcinoma) and their matched surrounding tumor tissues were examined by immunohistochemistry(IHC). The infection rate of hepatitis B virus (HBV) in HCC tissues were also detected to analysize the correlation of HBV infection with the expression of p33~ING1b and p53 protein. The mutation rate of ING1b gene in HCC was examined by PCR-SSCP technique and DNA sequencing. The p33~ING1b cDNA fragment was cloned into PCDNA3 eukaryotic expression vector and the recombinant plasmid was transfected into hepatoma cell line SMMU7721 and the changes of SMMU7721 cell growth, apoptosis rate and clony efficiency in soft agar were analysized. To further observe the effects of p33~ING1b and p53 cooperation function on hepatoma cell biological behavior ,three hepatoma cell lines with different endogenous p53 gene expression status were chosed . After transfection of p33~ING1b and wild-type p53 cDNA into hepatoma cells, respectively ,the changes ofhepatoma cell apoptosis rate, G0/G1 arrest and the activation of p53 downstream gene p21WAF1 were analysized. The expression of p33~ING1b and p53 proteins in hepatoma cell lines after transfection were also detected using Western blot. The results show:1.The positive expression rate of p33~ING1b and p53 protein in 57 cases of HCC were 42.1% and 57.9% , respectively. Both of them had the same positive localization. The expression of p33~ING1b and p53 protein had a closely relation in HCC (P<0.01). In thirteen surrounding-tumor tissues (13/51) and two normal liver tissues(2/12), p33~ING1b protein present weak positive expression. But p53 protein exhibited negative expression in all surrounding tumor tissues and normal liver tissues. These results suggested p33~ING1b expression could have some other p53-independent pathway. The infection of HBV play an important role in influencing p53 normal activity in liver cell and the expression of HBV X protein had a significant association with p33~ING1b and p53 protein . HBV X protein might have an effect on the cooperation of p33~ING1b and p53.2. The results of PCR-SSCP and DNA sequencing demonstrated that ING1b gene did not have a fragmental frame shift or deletion in tumor and surrounding tumor tissues. Five cases of HCC present abnormal band shifting by SSCP, two of which had point mutation in ING1b gene exon 2. In all surrounding tumor liver tissues ING1b gene did not exhibite mutation. ING1b gene had a low mutation rate (7.1%) in HCC, which suggested that gene mutation was not the main reason that ING1b lost its tumor suppressing function in HCC.3. After transfection of p33~ING1b gene expression plasmid , the cell growth rate of SMMU7721 was decreased significantly (P<0.01). Cell apoptosis was enhanced and cloning efficiency in soft agar was lowered. These results showed that overexpression of p33~ING1b gene could effectively inhibit the growth of hepatoma cells and protect cells from malignant transformation . It suggested p33~ING1b eukaryo...