Figure 8. Age-associated liver cancer elevates gankyrin and eliminates C/EBPα.

(A) C/EBPα is hyperphosphorylated in livers of old mice. Upper panel: Western blotting analysis of C/EBPα in nuclear extracts isolated from livers of young mice (Y), from healthy sections adjacent to liver tumors of old mice (O), and from liver tumors of old mice (OT). Bottom panel: C/EBPα was IP with antibodies to total C/EBPα and probed with antibodies to ph-S193 isoform of C/EBPα. (B) Gankyrin is elevated in liver tumors of old mice. Western blotting of proteins isolated from control mice and tumor sections was performed with Abs shown on the left. (C and D) Levels of mRNA coding for cell-cycle proteins. RT-PCR was performed with RNA isolated from control and tumor sections of old livers. (E) Cdc2 is increased in liver tumors. Western blotting was performed with Abs to cdc2. The membrane was reprobed with Abs to β-actin. Cdc2-IP:kinase: cdc2 was IP and examined in a kinase assay with WT and S193A mutant C/EBPα. (F) Cdk4 is activated in liver tumors of old mice. Western blotting was performed with antibodies shown on the right. Cdk4 was IP, and gankyrin and cyclin D1 were examined in these IPs. (G) Kinase activity of cdk4 toward C/EBPα is increased in tumor sections of the livers. Upper image shows input of cdk4 used for the IP. Cdk4 IPs were examined in a kinase assay with WT and S193A-C/EBPα as substrates. (H) A hypothetical model for the molecular basis of liver cancer in old mice. Data shown are mean ± SD.