Promoter CpG Islands Methylation Of Candidate Tumor Suppressor Genes On 11q22-q25 In Cervical Cancer

Background:Cervical cancer is one of the most frequent gynecologic tumors. Human papilloma virus(HPV) infection is now recognized as a key cause of cervical cancer. However, because only a minority of HPV-infected women develops cervical cancer, HPV infection alone is insufficient to induce cervical cancer. Researches have indicated that additional genetic changes such as oncogene activation, tumor suppressor gene (TSG) inactivation should be involved in the development of cervical cancer.TSG plays a key role in regulating cell growth and differentiation. TSG inactivation in cells causes appearance of tumorigenic phenotype. Gene mutation, promoter CpG island methylation and Loss of heterozygosity(LOH) are believed to be three mechanisms of TSGinactivation according to Knudson's hypothesis of biallelic gene inactivation. The regions of frequent LOH in human tumors indicate the existence of TSG.LOH in the chromosomal regions llq was frequently observed in human ovarian, breast, colorectal and cervical cancers. Although reports of the regions and frequency of LOH on llq maybe slightly differ among various types of tumors, 11 q22-q25 has been considered as the common region of LOH with high frequency. Many candidate TSG were identified in this region, such as ATM, PPP2R1B, OPCML,POU2F3 and CUL-5. However, of these three genes, no mutation(POU2F3,CUL-5) or low frequency of mutaion(ATM, PPP2RlB,OPCML)was found, while high frequency of promoter CpG island methylation were detected in human tumors. The association of the candidate TSG with human cervical cancer has not been reported up to date.To evaluate effect of candidate TSGs, OPCML, POU2F3 and CUL-5 on Ilq22-q25, on the development of cervical cancer, in this study, we determined CpG island methylation of three candidate TSGs and OPCML mRNA expression by Methylation-specific PCR(MSP) and Fluorescence Quantitative RT-PCR(FQ-RT-PCR) respectively.Material and methods:Sixteen normal cervical tissues , thirty-six cervical cancer tissues were collected for detection, in department of Gynecologic Oncology, Women's Hospital,Zhejiang University,between 2004-2005.Samples were collected before chemotherapy and radiotherapy and were stored frozen at -70 "C.The median age were 51.5 years(range,38-65years) in normal cervical tissues and 46.5 years(range,28-65years) in cervical cancer tissues. Of the 36 cervical cancer tissues, there were 30 of squamous cell carcinomas, 1 of adenocarcinoma and 5 of adenosquamous carcinomas. All samples were reviewed by the same experienced pathologist.Hela cell lines was purchased from American Type Culture Collection(ATCC).The promoter CpG island methylation of OPCML,POU2F3 and CUL-5 was determined by MSP. OPCML mRNA relative expression was evaluated by FQ-RT-PCR.Thermocycle threshold(Ct) values for OPCML and GAPDH transcripts were measured and OPCML mRNA relative expression was standardized by GAPDH.Results:1. Of the 36 cervical cancer tissues analysed in this study, we found 23(63.9%) displayed promoter CpG island methylation of OPCML,but no methylaion was detected in normal cervical tissues. Amplification with methylation-specific primer sets only also indicated OPCML methylation in Hela cells.2. NO methylation was detected in normal cervical tissues, cervical cancer tissues and Hela cells of POU2F3 and CUL-5 promoter CpG island, except one cancer sample of Cul-5.3. OPCML transcripts were detected in normal cervical tissues. Cervical cancer tissues and Hela cells displayed diminished expression of OPCML mRNA compared to normal tissues ( t = -2.506,P=0.017) .OPCML mRNA relative abundance in Cervical cancer tissues without OPCML promoter methylation was close to normal tissues (f=-0.393,P=0.700) . OPCML mRNA expression was reduced significantly in cervical cancer tissues with OPCML promoter methylation compared to normal tissues(f = -6.311,P=0.000) and cancer tissues without OPCML promoter methylation(/ = -3.038,P=0.006).Two cervical cancer tissues displayed>2/3 reduced OPCML mRNA expression (relative to normal tissues) without OPCMLpromoter methylation. Conversely, two cervical cancer tissues were found to harbor methylated OPCML promoter exhibited only modest reduction in OPCML mRNA abundance.Conclusions:1. Promoter CpG island of OPCML was hypermethylated and expression of OPCML mRNA was reduced, suggesting that OPCML promoter methylation is an important mechanism of gene inactivation. OPCML maybe partly contribute to the development of cervical cancer and is a cervical cancer-associated candidate TSG2. NO methylation was detected in cervical cancer tissues of POU2F3 and CUL-5 promoter CpG island.So the association of POU2F3 and CUL-5 with the development of cervical cancer need to be determined.