UTF1 and Cervical Carcinogenesis Figure 7. Effect of 5-aza on UTF1 promoter methylation and expression in epithelial cancer cell lines. A) BP analysis of UTF1 methylation status in cancer cell lines derived from the anogenital area with (5-AZA) or without (UN) 5-AZA for 72 hrs. B) Quantitative RT-PCR of cancer cell lines derived from the anogenital sphere with (5-AZA) or without (UN) 5-AZA for 72 hrs. Results are representative of 3 independent experiments. **, p,0.01 vs UN (Mann-Whitney U Test). doi:10.1371/journal.pone.0042704.g007 Discussion Cervical cancer development can take decades. Therefore adequate diagnosis and treatment of preneoplastic lesions might decrease its rate of development [2,3]. It is well established that DNA methylation patterns could be used to improve cancer diagnosis and/or prognosis [34]. However, in spite of clinical research progress, there is still no reliable epigenetic biomarker for cervical cancer diagnosis [35]. Currently, methylation specific PCR (MSP) is the gold standard to identify CpG hypermethyla- tion. However it can lead to false positive results and the methylation status of only one CpG can be assessed in each PCR reaction [34]. Reports have already evaluated promoter methyl- ation by MSP in Papanicolaou smears, which contain a mixed cell population, thus confirming the usefulness of these samples for the identification of putative epigenetic biomarkers [36,37]. Other commercialized techniques, such as Methylight or quantitative MSP, were also used to identify promoter methylation profile in DNA from liquid-based cytological (LBC) samples. These tech- niques allowed to discriminate between normal cytological samples and HSIL [38,39]. In this study we performed direct bisulfite pyrosequencing (BP) which was reported to give accurate and reproducible results for up to 20 CpG within up 120 nucleotides [34,40]. The data obtained in this study were far below these limits since the pyrosequenced fragment was of small size (50 bp) and contained only 8 CpG, thus reducing the risk for loss of sensitivity or specificity [34]. Direct BP of LBC samples has already been used to assess methylation status of HPV DNA in cervical lesions. The authors were able to demonstrate that CpG methylation status within the PLoS ONE | www.plosone.org 8 August 2012 | Volume 7 | Issue 8 | e42704