E. Gene Ontology Associated with the development and progression of can cer, silencing by hypermethylation often affects genes in important pathways. Therefore, we investigated whether http://www.selleckchem.com/products/Roscovitine.html our selected TOP3000 candidate genes are asso ciated with specific pathways or harbour related func tions. Multiple GO terms using Gene Ontology by GOstat and specific pathways using Ingenuity Pathway Analysis , were significantly over represented within the TOP3000 list when compared to all annotated human genes. These terms include regulation of transcription DNA dependent, transcription from RNA polymerase II pro moter, regulation of progression through cell cycle, posi tive regulation of programmed cell death as well as organ development and embryonic development.

Genes in these processes were reported to be often meth ylated during cancer progression. Genes responsible for development and differentiation are mainly silenced by methylation in normal tissues. On the other hand, in cancer tissues, genes responsible for cell cycle control and induction of apoptosis are often aberrantly expressed as many of these genes have tumour suppressor activity. DNA hypermethylation is one mechanism to regulate expression of tumour suppressor genes. The GO analysis provided additional strong indication that our highest ranking genes in the top list are signifi cantly enriched for methylated genes involved in cervical cancer tissue or cell lines. Validation of the 10 highest ranking candidate genes using COBRA In order to validate whether the highest ranking genes rep resent markers that are functionally hypermethylated in cervical cancer, we performed COBRA on bisulfite treated DNA of 10 cervical cancers and 5 normal cervices.

For this analysis, we focused on those first 10 genes from the high est ranking probe list that represent a known gene contain a CpG island surrounding the TSS are located on any chromosome except chromosome X are expressed in less than 15 carcinomas, in order to identify markers to be methylated in 60% of cer vical cancers BSP was used to amplify the CpG islands of these candi date genes using bisulfite treated DNA and COBRA to determine the methylation status. CCNA1 was included as a positive control for the highest listed, reported cervical cancer specific methyl ation gene promoter. COBRA of CCNA1 revealed that 6 of 10 carcinomas are methylated at the restriction enzyme sites.

Sequence analysis of the BSP products of these 10 carcinomas revealed that in 6 carcinomas the promoter is hypermeth ylated in good agreement with the COBRA results. Table Cilengitide 3 summarizes the methylation status of the 10 high est ranking genes in 10 cervical cancer and 5 normal cer vices using COBRA. One gene could not be analyzed for methylation as no specific BSP prod ucts could be amplified using several selleck chem Trichostatin A combinations of primer pairs.