Biological impact of the COOH-terminal 40 amino acid deletions of hepatitis B virus X protein in hepatocellular carcinoma cells

Abstract

To investigate the biological impact of the wild type hepatitis B virus X protein (HBx) and HBx3'-40, an engineered deletion mutant of HBx lacking the last 40 C-terminal amino acids and a transcriptional transactivator on hepatoma cells. Human hepatocellular cells of the lines Huh7 and SMMC-7721 were transfected with HBx3'-40 or HBx constructs. The integration of the plasmid DNA and the expression of HBx3'-40 and HBx were confirmed by Neo gene PCR and Western blotting respectively. The growth curves of different cells were obtained by MTT method. Plate clone formation test to calculate the clone formation rate. Flow cytometry (FCM), and Chloramphenicol acetyl transferase (CAT)-ELISA. Male BALB/c mice were divided into 3 groups to be inoculated subcutaneously with plasmids pcDNA3HBx, pcDNA3HBx3'-40, and blank plasmas pcDNA3 as controls. The mice were killed 15 and 30 days after and the tumors were taken out to undergo measurement and immunohistochemistry. Growth curve showed that after transfection the HBx3'-40 group cells grew faster than HBx and pcDNA3 transfected group cells. FCM analysis showed that HBx3'-40 transfection enhanced the progression of G(1) --> S phase in Huh7 cell cycle and HBx did not show such impact. The clone formation rates of pcDNA3HBx3'-40 group was 12.2% +/- 1.4%, significantly higher than those of the pcDNA3HBx, blank vector, and control groups (6.3% +/- 0.7%, 4.1% +/- 0.9%, and 6.1% +/- 1.1% respectively, all P < 0.05) in the Huh7 cells. The situation was the similar in the SMMC-7721 cells: 83% +/- 6% vs.49% +/- 8%, 27% +/- 3%, and 51% +/- 5% respectively (all P < 0.05). The apoptotic rates of SMMC-7721 cells under no serum condition transfected with HBx and HBx3'-40, especially the former, were 12.57% and 9.15%, significantly higher than that of the pcDNA3HBx group. The CAT expression of the pcDNA3HBx-transfected SMMC-7721 cells was 2.913 and 3.652 times those of the pcDNA3HBx3'-40 group and pcDNA3 group. The tumor weights of the mice inoculated with pcDNA3HBx3'-40, pcDNA3HBx, and blank vector-transfected Huh7 cells were 1.2 g +/- 0.17 g, 0.55 g +/- 0.12 g, and 0.48 g +/- 0.15 g respectively. The tumor weights of the mice inoculated with pcDNA3HBx3'-40, pcDNA3HBx, and blank vector-transfected SMMC-7721 cells showed similar features. HBx3'-40 protein significantly promote the proliferation of hepatocellular carcinoma cells and is involved in cell apoptosis and invasion, thus supporting the hypothesis that HBx mutants play a key role in hepatocarcinogenesis by modifying the biological functions of HBx. Besides, C-terminal mutants of HBx makes p53 tumor suppressor gene lose its cancerostatic function.