ABSTRACT
Mutations of the tumor-suppressor gene p53 have been found in 30-50% cases of hepatocellular carcinoma (HCC). In this study, E1-negative adenoviral vector encoding wild-type p53 under the control of the human cytomegalovirus promoter (AdCMV-p53w) was constructed to evaluate its therapeutic efficacy against tumor nodules developing after injection of HuH7 cell lines in ten nude mice. When each nodule had reached 10 mm in perpendicular diameter, 1.5 x 10(8) pfu of AdCMV-p53w per session was injected intratumorally as follows: In group I (n=3), five sessions were injected every other day. In group II (n=3), only one session. Group III (n=4) as negative controls. The mice were sacrificed at 28 days post AdCMV-p53w injection. Tumor growth was significantly suppressed and delayed in group I and II compared to group III as compared by tumor volume at the end of observation. These results suggest that AdCMV-p53w may not only be effective in treating HCCs expressing mutant p53, but also useful as a local injectable gene therapy.
Subject(s)
Humans , Mice , Adenoviruses, Human , Animals , Apoptosis , Carcinoma, Hepatocellular/therapy , Carcinoma, Hepatocellular/pathology , Genetic Therapy/methods , Genetic Vectors , Liver Neoplasms/therapy , Liver Neoplasms/pathology , Mice, Nude , Neoplasm Transplantation , Tumor Suppressor Protein p53/genetics , Tumor Cells, CulturedABSTRACT
The truncated E1192-283 and E2384-649 genes of hepatitis C virus (HCV) linked to the gene for glutathione 5-transferase (GST) were constructed and their expressions were analyzed. The GST-E1192-283 fusion gene overexpressed the fusion protein in E. coli as a soluble form, while the GST-E1192-383 plasmid did not express expected fusion protein. The purified GST-E1192-283 fusion protein was efficiently cleaved by thrombin. More than 90% pure, HCV E1192-283 protein was obtained by GST-agarose chromatography. The truncated GST-E2384-649 fusion gene expressed the fusion protein mainly as an insoluble form, whereas the GST-E2384-740 did not express the fusion protein. The truncated GST-E1 182-283 and GST-E2384-649 fusion proteins reacted specifically with an HCV patient serum. In addition, mice immunized with either the purified E1192-283 or GST-E2384-649 proteins generated specific antibodies to each antigen. The results suggested that hydrophobic carboxyl portions of the E1 and E2 proteins might affect expression levels as well as the solubility of each fusion protein in bacteria. Also, the truncated E1 protein with Tyr-192 to Ser-283 contained antigenic epitope(s) which could be specifically recognized by an HCV patient serum.