AP-2a Targets Methylation and AP-2a-downregulated SCC22B cells. This revealed a visible increase in protein expression in 3/5 of the genes (i.e. PTEN, MLH1, and RASSF1A) and no detectable change in RASGRF2 (Figure 1c). Not surprisingly, CDH1 showed a decrease in protein expression in response to AP-2a-downregulation, which correlates with previous findings that AP-2a is required for activation, not repression, of this gene (Figure 1c)[18]. AP-2a has previously been shown to regulate PTEN [19], but the findings of AP-2a regulation of MLH1 and RASSF1A are novel. AP-2a was also downregulated in additional HNSCC cell lines (SCC11B and SCC17as)(Figure 2a). The methylation status of the 3 genes with decreased methylation and increased expression in AP-2a-downregulated SCC22B cells (i.e. RASSF1A, MLH1, and PTEN) was analyzed. SCC17as displayed subtle decreases in PTEN and MLH1 methylation in the AP-2a-downregulated cells (Figure 2b and 2d), and both SCC11B and SCC17as revealed increased target gene expression after AP-2a downregulation (Figure 2c). AP-2a-targeted methylation was also investigated in malignancies other than HNSCC, and colon cancer was selected based on the association of MLH1 methylation [20,21,22]. AP-2a was downregulated in two colon cancer cell lines, HCT116 and HT29 (Figure S3a). The region analyzed for MLH1 showed insignificant methylation levels in the vector-only cancer cells for both HT29 and HCT116 (Figure S3b). However, both PTEN and RASSF1A demonstrated slightly higher levels of methylation, which were decreased in correlation with AP-2a downregulation in both HT29 and HCT116 (Figure S3c). AP-2a downregulation seems to be associated with decreased MSI Methylation of MLH1, a DNA repair gene, has been pre- viously shown to correlate with microsatellite instability in colon, gastric, and endometiral cancers [20,21,22]. Therefore, MSI was compared in SCC22B, SCC17as, SCC11B, HCT116, and HT29 cells with and without AP-2a downregulation. Interestingly, MSI primers yielded fewer products in both SCC22B and SCC17 as AP-2a-downregulated cells compared to the control cells (Figure 3a) (SCC22B = Bat25, D18S487, D17S250, D2S123, Bat40, and D18S56; SCC17as = Bat25, D18S487, D2S123, D18S67, and Bat40; D18S56 did not amplify in SCC17as). This Figure 2. Varied target methylation and expression following AP-2a downregulation in HNSCC cell lines SCC11B and SCC17as. a) Western blot analysis to confirm AP-2a downregulation in SCC11B and SCC17as. b) Methylation in 3 target genes was compared between SCC11B and SCC17as cells with and without AP-2a expression. Solid circles represent methylated CpGs; whereas, open circles represent unmethylated CpG sites. c) Western blot analysis was performed to analyze methylation’s affect on target expression. d) Graphical representation of the average methylation percentage in the 3 target genes analyzed. doi:10.1371/journal.pone.0006931.g002 PLoS ONE | www.plosone.org 3 September 2009 | Volume 4 | Issue 9 | e6931