Naslov
Promoter methylation in the PTCH gene in cervical epithelial cancer and ovarian cancer tissue as studied by eight novel Pyrosequencing assays

Autori
Lof-Ohlin, Zarah ; Levanat, Sonja ; Sabol, Maja ; Sorbe, Bengt ; Nilsson, Torbjorn K.

Izvornik
International journal of oncology (1019-6439) 38 (2010), 3; 685-692

Vrsta, podvrsta i kategorija rada
Radovi u časopisima, članak, znanstveni

Ključne riječi
CpG sites; DNA methylation; Hedgehog pathway; Pyrosequencing technology

Sažetak
DNA methylation status in the CpG sites of promoter regions in cancer-related genes, such as PTCH, has traditionally been investigated using either dye-terminator sequencing or methylation-specific PCR. We aimed to study the PTCH gene promoter methylation in gynecological cancers, with a method that gives a quantitative measure of the methylation status of the promoter region of the studied gene, and for this purpose, we designed novel Pyrosequencing based assays. Bisulfite-treated genomic DNA (bsDNA)was amplified by standard PCR and applied to novel Pyrosequencing assays, in order to measure the methylated fraction(%) at each CpG site of the PTCH gene promoter. We analyzed 22 squamous cell cervical cancer tissue specimens (11 with good and 11 with poor outcomes after radiotherapy)and 5 ovarian cancer tissue specimens matched with 5 normal ovarian tissue specimens. Six optimized PCR protocols which generated 8 Pyrosequencing assays covering 63 CpG sites in the promoter regions 1 and 2 as well as the previously unanalyzed promoter region 3 in the PTCH gene were developed. The 27 tumor tissue specimens and 5 normal tissues did not show any methylation within any of the 63 CpG sites. Our data suggest that methylation of the PTCH promoter is not a high-prevalence feature of squamous cell cervical cancer or ovarian cancer, but Pyrosequencing assays are a good method for studying promoter methylation.

Izvorni jezik
Engleski

Znanstvena područja
Temeljne medicinske znanosti

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