Study On The Effects Of Telomerase Activity Inhibition On The Cell Proliferation And Apoptosis In Human Multiple Myeloma And The Underlying Meehanisms

Part1Study on the effects of telomerase activity inhibition on the Cell Proliferation and apoptosis in human multiple myelomaObjective:Observation of telomerase inhibitor3-azido-2,3inhibited, dideoxythymidine (AZT) on telomerase activity in RPMI8226.Then (TA) to observe its effects on cell proliferation and apoptosis in humanmultiple myeloma cell line, TA inhibition. Change detection of tumor suppressor gene expression level, the relationship between the expression level of change when cell proliferation and inhibition of telomerase activity of AZT.Methords:Human multiple myeloma cell line RPMI8226DMEM culture medium of1-5×106cells/ml.exponentially cell growth were divided into four groups:(1) control:no AZT;(2) azt100um treatment group:100μm;(3):azt200um group200micron AZT treatment (4); azt400um group:treatment with400well AZT telomerase activity. Using telomerasere repeated amplification protocol (TRAP) assessment. By clone formation assay methods were inhibitory effect evaluation of AZT on the proliferation of human multiple myeloma cell line RPMI8226, PCNA,immunocytochemicalstaini-ng,immunohistochemical staining for p53. Using flow cytometry, TUNEL is induced by the comet assay of AZT on apoptosis of human multiple myeloma cell line RPMI8226cells.Results:The results show that theTRAP, the telomerase activity in three groups were lower than that of control group, especially in AZT400um group (P<0.05). The results showed that the telomerase activity of AZT inhibited RPMI8226cell, inhibitory effect was dose dependent. Cloning experiments show that, compared with control group, the therapeutic cloning formation rate is reduced, the AZT group than in400um was significantly lower than that of other groups, and the difference was significant (P <0.05). Three groups PCNA labeling index decreased, while the p53labeling index increased in these groups, and the difference is significant (P<0.05). These results confirm, inhibition of telomerase AZT can inhibit the proliferation of RPMI8226cell line.TUNEL detection, apoptosis index in AZT group was higher than control group, there was significant difference of AZT400um AL was the highest (P<0.01), the result is similar to the comet assay. From the flow cytometry, we can see that the treatment group the apoptosis peak there, apoptosis rate is low. Flow cytometry concluded, AZT can induce cell apoptosis, inhibit telomerase activity.Conclusions:Telomerase abnormal reaction causes inhibition of multiple myeloma prolifation and apoptosis. AZT can inhibit the telomerase activity of RPMI8226cells, and inhibitory dose dependently. Part2Telomerase activity inhibition targets and downregulates C-myc、P14ARFObjective:The purpose of this study was to explore the downstream mechanisms of cell proliferation and apoptosis of RPMI8226cells, telomerase activity decreased. Before C-myc and P14ARF induced cell senescence. The purpose of this study is to explore and prospect of AZT in the treatment of multiple myeloma, and put forward by theory and experiment on telomerase as a target for the treatment of multiple myeloma.Methords:P14ARFgene was amplified by RT-PCR, then we detect changes in four groups, P14ARFmRNA c-myc gene. C-myc protein, P14ARF protein by Western blotting was detected in the four groups.Results:Compared with the control group, c-myc, P14ARF mRNA relative gray and a decrease in AZT concentration significantly (P<0.01). The expression level of c-myc, P14ARFmRNA positive rate of colon formation and proliferation index correlation (r=-0.994-0.990, P<0.01) and negatively with apoptosis index. And p53LI (R=0.996-0.999, P<0.01). Conclusions:1. Concentration by the inhibitory effect of AZT on telomerase activity of c-myc, P14ARFmRNA expression level and the level directly related to downregulation of AZT.2. When telomerase activity was inhibited the effect of prolifation AZT on the induction of cell, cell senescence.3. P14ARFmRNA and c-myc expression levels were positively correlated, negative junction formation rate of negative p53LI and the degree of apoptosis and PCNA.4. These results indicate that activation of telomerase, the P14ARFmRNA expression is regulated by the tmeans c-myc, directly or indirectly, leading to improved myeloma.AZT proliferation and apoptosis inhibition can effectively prevent the telomerase activation, AZT has the therapeutic effect by inhibiting and inducing apoptosis of tumor cell proliferation.5. The basic theory and experiment about the target by telomerase to the treatment of multiple myeloma to be presented.