transgenic mouse model of hepatocellular carcinoma that shows hepatotropic expression of HBx gene (36). gene expression by E2F1. Besides, HBx impaired the APCCdh1-dependent protein degradation pathway and conferred intracellular stability to CDC6 protein. Increase in CDC6 levels correlated with increase in CDC6 occupancy on the -globin origin of replication, suggesting increment in origin licensing and re-replication. In conclusion, our findings strongly suggest a novel role for CDC6 in abetting the oncogenic sabotage carried out by HBx and support the paradigm that pre-replicative complex proteins have a role in oncogenic transformation. gene is transcriptionally regulated in a Azaperone cell cycle- and E2F1-dependent manner in mammalian cells (1C3), (4), and plants (5). In early G1 phase of the cell cycle, CDC6, along with Cdt1 (chromatin licensing and DNA replication factor 1), forms a pre-replication complex with a six-subunit origin recognition complex on the origins of replication followed by recruitment of the minichromosome maintenance complex 2C7 helicases. These molecular events lead to licensing of origins, from which DNA synthesis begins at the onset of S-phase (6, 7). CDC6 protein must be rendered incompetent for its function after origin firing to ensure prevention of replication reinitiation. In accordance, the cellular levels of Azaperone CDC6 are under tight control of post-translational modifications, gene via E2F1-Rb axis and stabilization of CDC6 protein via post-translational modifications and impairment of APCCdh1 proteolytic machinery. Further, we show that increased CDC6 levels correlated with its specific enrichment on the -globin origin of replication that seems to favor origin licensing, re-replication, and unscheduled entry of cells into S-phase. EXPERIMENTAL PROCEDURES Cell Culture and DNA Transfections Maintenance of human hepatoma Huh7, HepG2, HEK293 (ATCC CRL-1573), and HepG2.2.15 cells was described elsewhere (30). Immortalized human hepatocyte (IHH) cell line and HepG2.2.15 cells were maintained in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS) at 37 C in a humidified atmosphere with 5% CO2. Transfection was carried out in a 60-mm culture dish (0.6 106 cells) with 2.0 g of the indicated plasmids by Lipofectamine (Invitrogen) according to the manufacturer’s instructions. EGFP-N1 (0.5 g) was used in each experiment as a transfection control. For reporter assays, 0.2 g of Luciferase reporter plasmids was co-transfected with 0.5C1 g of indicated expression plasmids. Recombinants, Reagents, and Antibodies Construction of the eukaryotic expression vectors for wild-type HBx (X0) and its deletion mutant (X9) was as described previously (31). pEGFP-N1 was procured from Clontech. Luciferase reporter constructs CDC6-WT, CDC6-SM1, pGL3-E2F1-WT, and pGL3-E2F1-mut were kindly provided by Kristian Helin (1, 32). The expression vectors for wild-type E2F1 (E2F1-WT) and its transactivation Mouse Monoclonal to C-Myc tag defective mutant E2F1-(1C374) were kind gifts from Xin Lu (33). The chemical inhibitors used and their working concentrations were: CDK2 inhibitor II (10 m for 6 h) from Calbiochem and thymidine (2 mm for 10C12 h) from Sigma-Aldrich. The luciferase assay system was procured from Promega. Dulbecco’s modified Eagle’s medium was obtained from Invitrogen. Antibodies were obtained from the following sources: CDC6, phospho-CDC6 Ser-54, E2F1, Rb, phospho-Rb Ser-807, p27Kip1, phospho-p27Kip1, and GAPDH were from Santa Cruz Biotechnology, and Cdh1 was from Abcam. The production of monoclonal antibody against HBx has been reported earlier (34). The polyclonal antiserum against HBx was raised in rabbit using recombinant HBx produced in (34). Luciferase Assay Luciferase assay was performed according to the manufacturer’s instructions (Promega). The relative luciferase activities were measured after normalizing each sample with protein amount, and transfection efficiency. Double Thymidine Block 48 h after transfection, cells were incubated in complete medium containing 2 mm thymidine for 10C12 h following which cells were washed twice with 1 PBS and incubated in complete medium for 12 h. Subsequently, for second block, cells were again incubated in complete medium containing 2 mm thymidine Azaperone for 10C12 h. Later, cells were washed twice with 1 PBS and induced to enter S-phase by incubation in complete medium. Harvesting of cells was carried out at the indicated time points. Flow Cytometry (FACS, Fluorescence-Activated Cell Sorting) Huh7 and IHH cells 24 h after transfection were starved for 72 h and.