The Effect Of Estrogen And Progesterone On The Telomerase Activity Of Ovarian Cancer CAOV3 Cells Line

Telomerase is a celler reverse transcriptase which catalyzes the synthesis and extension of telomeric DNA. Telomerase activation is not observed in normal tissues of somatic origin but in almost 90% of human cancer, with the result the telomere shorten progressively with cell division and thus is a critical step for multistep carcinogenesis. The factors and mechanism involved in telomerase regulation are not well understood.Ovary is the main organ to synthesize and excrete estrogen and progesterone, which can affect its physiological change periodically. Ovary tumor is one of the tumors of the women to see frenquently. Sex steroid hormones involve in the tight regulation of telomerase expression and activity in ovary malignaint tumor. In order to find the evident that sex steroid hormones may be good candidates as physiological regulator of telomerase activity, we detected the telomerase activity of treated cells and issues by TRAP - Sliver Stain method to investigate the relationship between 17β-Estrogen, progesterone and the expression levels of telomerase activity in ovarian cancer cells and tissue and observed the morphological and functional changes of the cells. In thisstudy, we provide evidence for a role of estrogens and progesterone in the activation of the telomerase. Those results may help elucidate the mechanisms of hormonal control of telomerase activity and aid understanding of the roles of sex steroids in cellular senescence and aging as well as estrogen - induced carcinogenesis.Material and methodMaterial:Cell lines and Tissue; CAOV3 cells were purchased from Cell Biology department in China Medical University and were grown in RPMI -1640 with 10% FCS in the presence of 5% CO2 at 37℃; Ovary tumor samples were obtained from surgery of the Main Army Hospital in Shen Yang with histological confirmed Ovary Tumor. Chemical: 17β - Estrogen, Charps and Aebsf was purchased from Sigma company in USA. Progesterone was purchased from Xianju Pharmaceutical Factory in China. PCR kit, from Takara company in Japan. All other chemicals were of reagent grade.Method:1. MTT Assay: For determining the influence of the medium containing 17β -Estrogen or progesterone on cells growth, CAOV3 were treated with them for 48h and then determined in MTT Assay.2. Preparation of crude cell extracts; The cultured cells were treated with different medium containing 17β - E2 and/or progesterone of different concentration. After 12h,24h,48h,72h, the treated cells were collected and the extracts from CAOV3 was prepared by the Chaps - lysis method. Protein concentration were determined by folin method and the rest extracts were conserved in -70℃ quickly.3. Preparation of crude tiss ue extracts: The tumor samples were cut, whetted, and prepared by the Chaps -lysis method. Protein concentration were determined by folin method. The rest extracts were conserved in -70℃ quickly. The extracts were treated with 17β -Estrogen in different concentration, then purified by ethanol and reserved in -20℃.4. TRAP Assay: The reaction mixture contained the total of 50 ul, run in 30 cycles and stopped at 72℃ for 10 min.5. Purification of the amplified PCR product; To purify the PCR product of cells and tissue of ovary tumor with hydroxybenzene and chloroform: isoamyl alcohol ( 24:1).6. Electrophoresis and Sliver - Staining:In order to detect the te-lomeric repeats, PCR products were electrophoresed on a 12% polyac-rylamide gel with 0. 5M x TBE eletrophoretic buffer. Fixation, staining, and deoxidization were performed respectively and the reaction was stopped by 5% acetic acid until we have got a good show. The gel was dried and taken photographs.Finally, we contrasted the experimental groups with the control by SPSS soft.Results1. CAOV3 were treated with the medium containing 17)3 - Estrogen or progesterone for 48h and then determined in MTT Assay. We found that the growth of cells bloom obviously after treated by 10nM estrogen and subsequently decrease. Progesterone...