2 were detectable in all analyzed cell lines (see Supporting Table 1). However, these data do not sufficiently explain the distinct decrease of AKAP12 protein expression observed in this study. As a possible suppressive mechanism, we investigated the DNA methylation of promoter-related CpG islands of both AKAP12 isoforms in NL, CL, DN, and HCC samples by quantitative MassARRAY analysis (Fig. 1). In tumor samples, hypermethylation was detected

in the AKAP12α promoter (Fig. 5A,B), but not in the AKAP12β promoter (Fig. 5C,D). Methylation analysis of the AKAP12β promoter showed a methylation value higher than 10% only for one HCC sample. For the AKAP12α promoter, a mean methylation of 12% for NL and CL, 15% for DN, and of 30% for Proteasome inhibitor HCC was observed. As high amounts of fibroblasts, infiltrating immune cells, and other nonparenchymal cells may inflict on the genuine methylation status of hepatocytes in CL, microdissection of

hepatocytes was performed. However, the methylation values of CL specimens after microdissection (18%) did not significantly differ from undissected samples of the same tissues. Elevated PD0325901 DNA methylation levels of the AKAP12α promoter were also detected in HCC cell lines. This analysis revealed DNA methylation of 96% (AKN1), 42% (HuH7), 41% (Hep3B), 24% (HepG2), and 20% (PLC/PRF/5) (Fig. 5A,B). Methylation analysis of the AKAP12β promoter in cell lines showed methylation values lower than 1% (Fig. 5C,D). Hypermethylation of the AKAP12α promoter was confirmed by an independent method (combined bisulfite restriction check details analysis; see Supporting Fig. 2). To verify the functional relationship between promoter hypermethylation and loss of AKAP12 gene expression, methylation and mRNA expression levels of both isoforms were compared before and after treatment with 5-aza-dC in cell lines AKN1, HepG2, and HuH7. Isoform-specific mRNA expression of AKAP12 differed between untreated hepatic cell lines and PHH but confirmed protein data (Fig. 3B; Fig. 6A). The 5-aza-dC treatment resulted in a decrease in AKAP12α promoter methylation in the highly methylated AKN1

cell line (Fig. 6B), accompanied by a strong increase in AKAP12α mRNA expression (Fig. 6C), demonstrating a relationship between AKAP12α expression and methylation of its promoter. In HepG2 and HuH7 cells, which showed lower methylation levels than AKN1, demethylation as well as re-expression of 5-aza-dC was moderate. Similarly, only a marginal increase in expression was detected for isoform β with its unmethylated promoter (Fig. 6B,C). Data were confirmed in two independent experiments (see Supporting Table 6). Although we have demonstrated that silencing of AKAP12 is associated with DNA hypermethylation in HCC, promoter methylation does not explain the loss of expression in earlier stages of hepatocarcinogenesis. Thus, we postulated that a posttranscriptional mechanism may cause silencing in CL and DN.