| IntroductionTelomerase, a ribonucleoprotein enzyme, elongates and maintains telomeric DNA through the addition of TTAGGG repeats and plays a critical role in the development of cellular immortality and car-cinogenesis. telomerase activity has been examined in most immortalized cells and cancer cells, and has been considered as a new target for cancer therapy.Recent reports showed that telomerase activity in some cancer cells could been significantly down - regulated after exposure to some DNA - damaging agents, which may result from the disruption of telomeric structure or other indirect effects following cell cycle arrest or apoptosis. MMS, a kind of DNA - damaging agents, predominately methylates ring nitrogens of pyrine , leads to DNA double - strand breaks Npoint mutation, and so on . Therefore, MMS has the potential ability to damage telomeric structure and cause apoptosis of cancer cells. Our aim was to investigate the influence of MMS on telomerase activity of CAOV3 cells and observe whether MMS can induce the ap-optosis of CAOV3 cells.MethodsCAOV3 cells were cultured in 10ml of RPMI 1640 medium supplemented with 10% fetal brovine serum containing 100u/ml penicillin and 100 g/ml streptomycin in a 5% CO2 incubator with 100% humidity at 371. Concentrations of MMS determined by MTT test were: 2mmol/L, Immol/L, 0. Immol/L and 0. Olmmol/L. The number of cells alive was counted by trypan blue exclusion method, percentage of viable cells = [ the number of viable cells/ ( the number of viable cells + the number of dead cells) ] 100% . Apoptosis and cycles of cells were observed by flow cytometric analysis. The activity of telom-erase was examined by the method of TRAP, poly aery lamide gel elec-trophoresis and silver staining. The significance of differences in mean values was determined by chi square test.ResultsIn order to observe the differences of the percentages of viable cells between MMS groups and control groups after exposure to MMS for 120h, we conducted trypan blue exclusion method to count the number of viable cells in each group. The percentages of viable cells were 93. 8% ( control group) , 93. 3% (0. Olmmol/L MMS) , 93. 5% (0. Immol/L MMS) ,and 92. 7% ( Immol/L MMS) respectively, no significant difference was observed among all groups ( P > 0. 05 ) . Flow cytometric analysis revealed that the cells had been mainly arrested at S phase after MMS treatment for 24 and 48 h and no sub - G,fraction was observed . Compared with that of control group, the telom-erase activity of CAOV3 cells after MMS treatment was increased. After treatment with MMS at various ( 0. 01 -2mmol/L) concentrations for 24h, we examined CAOV3 cells for changes in telomerase activity. MMS significantly enhanced telomerase activity of treated cells even at relatively low doses. The maximum telomerase up - regulation was obtained at a concentration of Immol/L. A time course experiment of telomerase activity in CAOV3 cells treated with Immol/L MMS revealed elevated levels as early as lOh after treatment beginning and increased up to approximately twofold the control level at 24h. After 48h, telomerase activity decreased gradually and almost returned to the pretreat-ment level at 96h. When MMS was added directly to CAOV3 cell extracts immediately before primer addition, telomerase activity remained unchanged. Thus, a direct influence of MMS on TRAP assay does not seem to be involved in telomerase activation observed after drug treatment.DiscussionTelomerase is a specialized ribonucleoprotein polymerase that e-longates the telomeres of chromosomes to compensate for losses that occur with each round of DNA replication. Unlimited proliferation in tumor cell requires this enzyme to maintain chromosomal stability. Inhibition of telomerase could therefor provide a new strategy for antican-cer therapy. Recent study suggestes that many DNA - damaging a-gents can inhibit the telomerase activity of cancer cells, and considerable evidence shows that apoptosis plays a key role in cancer cell death induced by some...
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