| IntroductionPolyamines are recognized as cell growth factors in relation to cell proliferation, differentiation, regeneration and malignant transformation. Polyamines play an important role in the growth of normal cells like vascular endothelial cells and also exert various effects on the proliferation and metastasis of malignant cells. The recent studies on the biosynthesis have clearly elucidated its mechanism at the gene levels, which reflects to the development of the inhibitors of the polyamine biosynthesis. The measurement of the polyamine contents in biological fluids including urine and blood has been shown to be useful as the tumor marker.Telomere is a specific structure of eukaryon linear chromosome, which is composed of a simple overlapping sequences with G and protein. The DNA sequences in human and various animals are (TTAGGG)n. To certain extent telomere shortens with cell dividing, cells will stop dividing and enter senecence ever die. So telomere plays a key role in senscence and tumor.Telomerase is a specilized ribonucleoprotein polymerase containing an integral RNA with a short template element that directs the donovo synthesis of telomeric repeats at chromosome ends. Telomerase is repressed in normal human somatic cells but is activated during development and upon neoplasia. Three subunits comprising the humantelomerase complex have recently been identified. Human telomerase RNA component (hTR) functions as a template for telomere elongation by telomerase. HTRT is a rate - limiting determinant of the enzymatic activity of human telomerase.Experimental Materials and Methods1. MaterialsCAOV3 cell line conies from the Molecular biology laboratory of China Medical University. Tumor tissue comes from the second clinical hospital of China Medical University.2. Methods2. 1. Cell culture: CAOV3 cell were maintained as monolayer cultures growing in RPMI 1640 containing 10% Nu - serum.2.2. Cell growth curve:with MIT method2.3. Extract telomerase of CAOV3 cell2. 4. Extract telomerase of tumor tissue; Tumor tissue were washed once in phosphate - buffered saline, resuspended in ice cold lysis buffer; centrifuged for 30min and stored at - 70C.2.5. Detect telomerase activity with TRAP - silver staining assay.2. 5. 1. PCR amplification :The reaction mixture was subjected to PCR cycles at 941 for 30 seconds, 50C for 30 seconds, and 72C for 90 seconds, then 10 min for the final step.2.5. 2. 12% polyacrylamide non - denaturing gels electrophore-sis.2. 5. 3. Silver staining2.6. Analysis the result with gel analysis systemExperimental results1. Telomerase activity of gastric carcinoma tissue treatment with polyamine has no significant difference from the control group.2. Telomerase activity of CAOV3 cell extraction treatment with polyamine has no significant difference from the control group.3. During cell growth, increase exogenous polyamine concentration result in increased rate of cell proliferation.4. Telomerase activity of CAOV3 cell line treatment with polyamine significant increased compared to the control group.DiscussionSome paper reported that Inhibition of polyamine biosynthesis could result in the proliferation inhibition and apoptosis induction of Hep - 2 cells, which was associated with suppressed telomerase activity. It is suggest that inactivation of telomerase would be one of the important molecular events in reversion of tumor malignant phenotype via inhibition of polyamine biosynthesis. Polyamines are believed to exert their effects by binding to negatively charged macromolecules such as DNA, RNA, and proteins to influence the chromatin structure and sequence - specific DNA - or RNA - protein interactions, which alter the rate of gene transcription and the stabilization of mRNA and proteins. Telomerase activity is tightly regulated during development and oncogenesis. The hTERT promoter is a GC - rich, TATA - less promoter. Deletion analyses in reporter assays show that the proximal region of the hTERT promo... |
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