UTF1 and Cervical Carcinogenesis Figure 1. Hierarchical cluster analysis of methylation levels of genes involved in stem cell physiology. Gene methylation was analyzed by Illumina microarray in DNA from microdissected normal ectocervix (Ecto1 to 4) and cervical squamous cell carcinoma (SCC1 to 5). doi:10.1371/journal.pone.0042704.g001 Effect of 5-aza-deoxycytidine on cell proliferation and viability To assess a potential link between UTF1 promoter methylation and its expression, cervical cell lines were treated with 5-aza. We first analyzed the impact of 5-aza on cell proliferation and viability. As shown on Figure 6A, 5-aza induced a decrease in HeLa and CaSki cells proliferation (p,0.01 and p,0.001 vs untreated cells, respectively), whereas it had no significant impact on SiHa and A431 cells (p = NS). Cell viability in response to 5-aza was assessed with MTT test. Similar results were obtained, with a decrease of cell viability observed only in HeLa and CaSki cells (Figure 6B). Figure 2. UTF1 promoter methylation increases during cervical carcinogenesis. A) Mean methylation value for each CpG analysed by direct bisulfite pyrosequencing. Position of each CpG is indicated (TSS = +1). B) CpG methylation profile in each sample (cut-off value of 20%). Each line represents one sample. Ecto, normal ectocervix; LSIL, low grade squamous intraepithelial lesion; HSIL, high grade squamous intraepithelial lesion; SCC, squamous cell carcinoma; white circle, hypomethylated CpG; black circle, hypermethylated CpG. The CpG in red was the one analyzed by microarray. doi:10.1371/journal.pone.0042704.g002 Figure 3. UTF1 promoter methylation analysis by direct bisulfite pyrosequencing in white blood cells. Mean methylation value for each CpG analysed by direct bisulfite pyrosequencing in DNA from white blood cells. Position of each CpG is indicated (TSS = +1). The mean methylation values for HSIL and SCC in LBC samples were added as a comparison. WBC, white blood cells. doi:10.1371/journal.pone.0042704.g003 PLoS ONE | www.plosone.org 5 August 2012 | Volume 7 | Issue 8 | e42704