| IntroductionTelomerase is a ribonucleoprotein enzyme which is composed of UNA and protein. It can synthesize telomeric DNA sequences using its RNA as template. It can remedy the loss of telomere after cellular mitosis, maintain telomere length and stabilize chromosome. Telomerase activity was found to be absent in most normal human somatic cells but present in most immortalized cells and over 90% of cancer cells. In cancer cells activation of telomerase plays a critical role in uncontrol proliferation of cells. Therefore, telomerase has been considered as a new target for cancer therapy.The regulation of telomerase activity can be expressed at various levels. It has been testified by experiment that inhibition of telomerase activity can inhibit growth of cancer cells, so telomerase is closely related with tumor cell growth, proliferation , senescence and carcinogenesis. Malate is a promoting agent of ox-idative metabolization which can stimulate aerobic oxidation and accelerate mitochondria respiration. While tumor tissue acquire energy mainly by anaerobic fermentation and aerobic oxidation is not beneficial for growth and deterioration of cancer cells. Gamma - Aminobutyric Acid( GABA) is the major inhibitory neu-rotransmitter in the brain and has many beneficial functions for human body. Recent study has shown that some amino - acid neurotransmitters play roles in stimulation or inhibition of cancer cell proliferation in vitro. Hela cells (human cervical cancer) are common cells for telomerase study. Our aim was to investigate the effect of malate and GABA on telomerase activity of Hela cells and ex-plore the relationship of telomerase activity change with growth, metabolization and deterioration of cancer cells to provide new tactics for treatment of malignant tumor.Material and Method1. Material:1.1 Cell lines: Hela cell, regular culture.1.2 Reagents and equipment: malate, GABA, and AEBSF were purchased from Hua - Mei company. PCR kit, from Takara company in Japan. All other chemicals were of reagent grade.C02 incubator, Eppendorf microcentrifugal machine, Sorvall centrifugal machine ( Germany) , HRSP0520 PCR instrument ( Hybaid, UK) , super clean bench,DYY-4 electrophoresis apparatus, etc.2. Method;2. 1 Culture of Hela cells and determination of cell number.2.2 Treatment with various concentration of malate and GABA respectively for different time.2.3 Preparation of Hela cell telomerase2.4 Determination of protein content2. 5 The activity of telomerase was examined by the method of TRAP, poly-acrylamide gel electrophoresis and silver staining. 2.6 Statistic treatment with SPSS soft.Results1. The effect of malate on telomerase activity of Hela cells After treatment with malate at various (ljxmol/L - 0. lmol/L) concentration for 48h, we examined Hela cells for changes in telomerase activity. The results show that malate can decrease telomerase activity of treated cells even at relatively low doses (1 |xmol/L). The maximum telomerase down - regulation is obtained at a concentration of 10|xmol/L. A time course experiment of telomer-ase activity in Hela cells treated with lOjxmol/L malate revealed decreased level as early as 8h after treatment beginning and down to approximately 11% as contrast to the control level at 48 h. Afterwards there are minor changes of telomerase activity, but all lower than contrast group.2. The effect of GABA on telomerase activity of Hela cells After treatment with GABA at various (ljxmol/L -0. lmol/L) concentration for 48h, we examined Hela cells for changes in telomerase activity. The results show that GABA can decrease telomerase activity of treated cells even at relatively low doses (1 jxmol/L). The maximum telomerase down - regulation is obtained at a concentration of lmmol/L. A time course experiment of telomerase activity in Hela cells treated with lmmol/L GABA revealed decreased level as early as 8h after treatment beginning and down to approximately 13% as contrast to the control level at 48 h. Afterwards there are minor changes of telomerase activity, but all lower than contrast group.DiscussionTelomerase is a kind of RNA - dependent DNA polymerase that elongates the telomeres of chromosome to compensate for losses that occur with each round of DNA replication. Hela cells (human cervical cancer cells) , being a kind of malignant tumor cells, telomerase of which are activated to maintain the length of telomere and prevent cell death. The activity of telomerase can be used as an index of measuring malignant degree of tumor. However, it is not very clear a-bout regulation mechanism of telomerase activity.Malate is an important organic acid being produced during human me-tabolization and middle metabolite of tricarboxylic acid cycle ( TAC). Supplement of malate can promote aerobic metabolization of tissue and accelerate mitochondria respiration. While tumor tissue acquire energy mainly by anaerobic fermentation even under the condition with adequate oxygen. Aerobic oxidation is not beneficial for growth and deterioration of tumor cells. Therefore malate is promoting agent that can inhibit growth of tumor cells. In this report, we investigate the effect of malate on telomerase activity of Hela cells. As is shown above,treated with malate at various concentrations, telomerase activity in Hela cells was decreased to different extent. Among which the maximum telomerase down - regulation was obtained at a concentration of lOjxmol/L after malate-treatment for 48 h. The decrease of telomerase activity may be related to promotion of aerobic metabolization of tumor cells by malate.The gamma - aminobutyric acid (GABA) is the major and the best known neurotransmitter with inhibitory properties in central nervous system . It has man-y important functions in Central Nervous System (CNS) and peripheral region. Recent study has shown that some amino - acid neurotransmitters play roles in stimulation or inhibition of cancer cell proliferation in vitro. In this report, we investigate the effect of GABA on telomerase activity of Hela cells. As is shown above, treated with GABA at various concentrations, telomerase activity in Hela cells was decreased to different extent. Among which the maximum telomerase down - regulation was obtained at a concentration of 1 mmol/L after GABA treatment for 48h. Activation of telomerase in cancer cells plays a critical role in un-control proliferation of cells. Therefore the decrease of telomerase activity may be related to inhibition of Hela cells proliferation by GABA. Research data overseas show that GABA can inhibit growth of gastric carcinoma of rat, which is concerned with GABA inhibiting proliferation of mucosa cells of gastric sinus .In the peripheral region, as in CNS GAB As function is known to be mediated by GABAAand GABAB receptors. GABAAreceptor is GABA - gate chloride channel which can be regulated by cAMP, Ca2+and cell volume. In cervical cancer, cell swelling was shown to activate ATP - dependent Cl~ currents, while Volume - regulated anion channels (VRAC) have been linked with cell cycle progression in cervical cancer. During cell cycle progression, cells undergo a significant increase in size (especially at the Gj/S transition) that perturbs cell volume homeostasis. Meanwhile during cell cycle progression, telomerase activity has been noted as highly been activated in S phase. The down - regulation of GABA on telomerase activity of Hela cells may be realized by decreasing the percentage of cells in S phase.In summary, we observed down - regulation of telomerase activity in Hela cells after exposure to malate and GABA. This down - regulation may be related...
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