.1. Of Tbp And Tlp On Htert Transcription Adjust The Effect And Mechanism Of 2.dna Methylation In Cervical Cancer Diagnosis

Human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase in human, has been identified the rate-limiting factor in telomerase activity, but its detailed mechanism of transcription regulation remains unclear. Many investigations are focusing on the precise mechanism of hTERT transcription regulation. The transcription of genes encoding functional proteins in cells has been identified to be dependent on activity of RNA polymerase II. However, RNA polymerase II does not directly bind to promoter as elucidated so far but mediated by cellular proteins. The proteins for initiating transcription include two major proteins: TBP (TATA-binding protein) and TLP (TBP-like protein). In this investigation, TBP and TLP were expressed and purificated. The interaction of TBP with hTERT promoter was identified, but the interaction of TLP with hTERT promoter was not identified. The in vivo interaction between TBP and hTERT promoter was identified by chromatin immunoprecipitation (ChIP) assay. Transfection of TBP into HeLa cells presented marked transactivation of hTERT promoter. These results suggested that TBP protein might be a transcription regulator and take part in activation of hTERT. Objectives: Gene promoter hypermethylation is an important mechanism involved in carcinogenesis. Several gene promoters are known to be hypermethylated in cervical cancer. However, not much is known about hypermethylation in cervical cancer precursor lesions. In order to obtain more insight in the course of hypermethylation throughout cervical carcinogenesis, and to allow selection and adjustment of a marker panel for the detection of cervical cancer and high grade (H)SIL in scrapings, the hypermethylation status of nine gene promoters, previously reported to be hypermethylated in cervical cancer, was analyzed using quantitative methylation specific PCR (QMSP).Methods: Paraffin embedded tissue from normal cervix (n=20), LSIL (n=20), HSIL (n=20), adenocarcinomas (AC) (n=20) and squamous cell cervical cancers (SCC) (n=40) was retrieved together with 55 corresponding cervical scrapings and DNA was extracted. Promoter methylation analysis was performed using QMSP for DAPK, CALCA, ESR1, APC, RAR-β2, TFPI2, SPARC, CCNA1 and CADM1.Results: The level of methylation of all gene promoters was increased with severity of the underlying lesion. The number of samples methylated of most gene promoters was increased as well with the severity of the lesion, except for CALCA, ESR1 and RAR-β2. DAPK and CADM1 became more prominently methylated in HSIL lesions compared to LSIL, while CCNA1, TFPI2 and ESR1 became mainly methylated in cervical cancers. Methylation ratios determined in the scrapings were reflecting the methylation status of the underlying lesion. Furthermore, in cervical scrapings, CCNA1 was most frequently hypermethylated in cervical cancer cases (80%) and in HSIL (56%), while only 25% of LSIL showed hypermethylation.Conclusion: The number of samples methylated and the level of methylation was increased during carcinogenesis in paraffin embedded tissue as well as in corresponding cervical scrapings. DAPK and CADM1 gene promoter methylation were the best gene promoters to distinguish LSIL vs HSIL lesions, which might be important for treatment of SIL lesions.