The Effect Of ELE On Telomerase Activity And Telomerase Catalytic Subunit HTERT In Hela Cell

IntroductionTelomerase is a ribonucleoprotein enzyme which is composed of RNA and protein. It can use its RNA as template to synthesize telomeric DNA sequences. It can remedy the loss of telomere after cellular mitosis, maintain telomere length and stabilize chromosome. Two proteins have been cloned;TEP1 and hTERT . Telomerase activity is found to be presents in most immortalized cells and over 85 -90% , but absent in most normal human somaic cells. Therefore, telomerase is regarded as a character of cancers.The regulation of telomerase activity can be expressed at various levels. It is important to regulate hTERT which is protein and can control telomerase activity. Telomerase activity can be inhibited by inhibiting telomerase catalytic sub-unit hTERT. The experiment indicates that tumor growth can be inhibited by inhibiting telomerase activity. The telomerase are related to tumor cell 's growth, proliferation, caducity and tumorigenesis. ELE is a medicament which can induce cell apoptosis. The ELE acts Hela cell by using different concentration , then we can observe the effect of ELE on telomerase activity of Hela cell and telomerase catalytic subunit hTERT. We can approach the relationship of telomerase activation and apoptosis in order to offer method of clinical tumor therapy.Materials and methods1. Materials1. 1 Cell;human cervical carcinoma cell (Hela cell line) comes from experiment center 3 department of china medical university.1.2 Reagent: ELE, RPMI 1640, Trizol,CHARPS,AEBSF come from Sigma co. of the USA;Erasol, PCR, RT -PCR reagent comes from Takara co.1.3 Implement;HitachiO5PR -22 centrifugal machine;tabletop ultracentri-fuge (Sorvall Biofuge Primor, German);THZ -95 tabletop constant temperature oscillation box jsuperclean bench2. Methods2. 1 Hela Cell anabiosis and culture.2. 2 Observe Cell structure2. 3 Prepare for telomerase of Hela cell: collect cell, schizolysis , centrifu-galization , pre - emergency .2. 4 Protein level assaying : determine protein level by using phenol reagent2.5 TRAP;set up TRAP - PCR system, 12% polyacrylamide gel elec -trophoresis ( PAGE) , argentation, to measure telomerase activation.2. 6 Extract RNA of Hela cell2. 7 RT - PCR: run RT - PCR reaction2. 8 RT - PCR product purification2.9 2% Agarose electrophoresis and EB staining2. 10 Tatistics disposalResultsThe effects on Hela cell proliferation with different con - centration : high concentration group(50 -300uJg/ml) ,Hela cell lost normal form, the number reduced;low concentration group(1 -50|xg/ml) , Hela cellar growth were inhibited gentle;there were no change compared with control group. The ELE act on Hela cell which is 200|jLg/ml. When it act 24h, the number reduced. When it act 48h, Hela cell lost normal form.During cell culture, the effects of ELE on telomerase activity are related to the effect concentration;compared with control group, telomerase activity begins to changed in the 50jxg/ml;been inhibited obviously with increase of concentration.During cell culture, the effects of ELE on telomerase activity are related tothe effect time;Telomerase activity begins to changed when acted 24h;was the weakest when ELE acted 48 h.During cell culture, the effects of ELE on telomerase catalytic subunit hTERT are related to the effect concentration. When ELE acted 48h. , telomerase catalytic subunit hTERT was more inhibited when concentration of ELE was higher.DiscussionThe ends of linear chromosomes are protected by telomeres that consist of double - stranded repetitive sequences complexed with specific telomere - binding proteins. It is important that telomerase can keep genome integrity and function stability. Telomerase is a specialized ribo - nucleoprotein polymerase that elongates the telomere 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 therefore provide a new strategy for anticancer therapy.ELE is a cell differentiation antileptic, which is essential component in cur-cumae . ELE can inhibite tumor cell proliferation and induce tumor cell apopto-sis. In this experiments, after different concentration of ELE acted Hela cell, different reduction of telomerase activity was found and telomerase catalytic sub-unit hTERT was inhibited. Under the 50jxg/ml,telomerase activity begins to reduce. With concentration higher,telomerase activity reduce obviously. The activation of telomerase is related to many regulation ways, in fact, the hTERT is the most important one. Telomerase catalytic subunit hTERT was more inhibited when concentration of ELE was higher. ELE can inhibit telomerase activation, maybe because ELE can directly inhibit catalytic subunit hTERT. Maybe ELE inhibit c - myc gene, by which indirectly inhibit catalytic subunit hTERT, reduce the activation of telomerase.In this experiment, we detect express change of catalytic subunit hTERT -Mrna which is related to the activation of telomerase. We find that the colour of electrophoresis strip of hTERT becomes weak . But the colour of electrophoresisstrip of ento - contrast is no change. The inhibition of telomerase activity probably promotes cell apoptosis. The telomerase maybe takes part in control of cell apoptosis. The research indicates that abnormal telomerase activity and control of cell apoptosis are related in tumor cell. Kyos research indicate that the level of express of telomerase catalytic subunit hTERT mRNA are positively related to telomerase active. After promoting cell apoptosis, the mechanism of telomerase changing probably one is that related gene can directly control apoptosis, another one is that maintaining telomerase activity depends on integrality of cell nucleus structure. Destroying integrality of cell nucleus structure can affect express of telomerase and telomerase activity.The activation of endogenous endonuclease disrupt cell DNA. It can affect express of telomerase and telomerase activity. In the apoptosis, the mechanism of express and telomerase activity changing need to be investigated deeply.Conclusion1. ELE reduces telomerase activity of Hela cell2. The effects of reducing telomerase activity are related to inhibiting telomerase catalytic subunit hTERT3. ELE inhibit telomerase catalytic subunit hTERT of Hela cell4. The effects of inhibition telomerase catalytic subunit hTERT are related to inducing cell apoptosis.