HCV-core protein induces long-term transformation of NIH 3T3 cells.

IS Smirnova PhD^a, ND Aksenov PhD^a, MG Isaguliants PhD^b

^aInstitute of Cytology RAS, St. Petersburg, Russian Federation, ^bDepartment of Virology, Swedish Institute for Infectious Disease Control, SE 171 82, Stockholm, Sweden

AIM Hepatitis C virus (HCV) has been known to be a major causative agent of chronic hepatitis worldwide, which readily establishes chronic persistent infection that often leads to liver cirrhosis and hepatocellular carcinoma. Numerous studies have strongly suggested an oncogenic property of HCV-core protein. However the precise mechanism of cell transformation in the presence of HCV-core protein is not known. In the present report, we investigated the stages of cell transformation after HCV-core protein transfection. METHODS NIH 3T3 cells were transfected with plasmid PCMV-core, using lipofectamine. HCV-core protein expression was assessed with Western blotting and indirect fluorescent staining. P53 transactivation was estimated after transient transfection of the reporter plasmid pG13-luc containing p53-responsive wild type sites located before luciferase gene with the use of the luciferase assay. Cell cycle and cell ploidy was determined by flow cytometry. For DNA ploidy analysis mouse splenocytes were used as an internal standard. Anchorage independence was estimated using growth in soft agar and absence of contact inhibition by focus-forming morphology. RESULTS We found that expression of HCV-core protein leads to cell transformation. Shortly after transfection tetraploid cells were generated. Simultaneously doubling time increased, saturation density and colony formation efficiency, on contrary, decreased, cells were not able to form colonies in soft agar, focus-forming morphology was absent. Later tetraploid cells disappeared with DNA loss in comparison with NIH 3T3 cells (diploid index changed from 1.67 in control cells to 1.58 in HCV-core cells). Doubling time decreased, but saturation density and colony formation efficiency, on contrary, increased. The cells acquired some features of transformation: anchorage independence and absence of contact inhibition. During transformation p53 lost its transactivation function in comparison with control NIH 3T3 cells. CONCLUSIONS In HCV-core cell transformation even with the immortalized cells NIH 3T3 can be distinguished two stages: preneoplastic and neoplastic. Preneoplastic stage took around 50 passages. The fact that during this stage p53 lost its transactivation function suggests p53-dependent character of HCV-core transformation.

Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Nice, France; February 7 - 10, 2004; in poster session 791 (Viral infections).