The Effect Of Basic Amino Acid On The Cell Proliferation And The Telomerase Activity In Hela

IntroductionTelomerase, a ribonucleoprotein enzyme, which RNA component acts as template for elongating and maintaining telomeric DNA through adding TTAGGG repeats to the end of eukaryocyte chromosome, playing a crucial role in the development of cellular immortality and carcinogenesis. The main components of human telomerase containing: hTR,hTP1,hTERT. Among these components, hTERT is crucial agent for telomerase activity. Telomerase activity has been examined in most immortalized cells and cancer cells, and has been considered as a criterion for clinical diagnosis and prognosi. Recent study show there are many pathways of controlling telomerase activity.Basic amino acid exist in organis widely, including of arginine,lysine and histidine. Amino acid is the basic unit of protein,participating a few of metabolisms in organis. We found asp,arg ect. amino acid ,especially L - Arg, could change telomerase activity, influence cellular growth in force - experiment, but there is no article to report it at present. The study purpose to observe the effect of basic amino acid on telomerase activity and offer evidence of the relationship between amino acid and malignant tumor.Materials and Methods1. Materials1. 1 Cell: Hela cell line comes from experiment center 3 department of China Medical University.Reagent: L - Arg,Lys,His,MTT,CHARPS, AEBSF comes from SIGMACo. of the U. S. A; PCR reagent comes from TAKARA Co.. 2. Methods2.1 Cell culture: Hela cell line were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum containing lOOu/ml penicillin and 100 jxg/ml streptomycin in a 5% C02incubator with 100% humidity at 37 X..2.2 Cell proliferation determine: with MTT method.2. 3 Extract telomerase of Hela cell; washed Hela cell extract once in phosphate - buffered saline, resuspended in ice cold lysis buffer; centrifuge for 30 min and store at -70 T!.2.4 TRAP: set up TRAP -PCR system 50 |xl,amplificated 30 cycles.2.5 PCR product purification2.6 PAGE & silver staining2.7 Statistics disposal; t -test with SPSS softwareResults1. The effects on Hela cell proliferation with different concentration; high concentration group (50 ~ lOOmM) ,Hela cell lost normal form;middle concentration group (5 ~25mM) , Hela cellular growth were inhibited obviously; low concentration group ( < lmM), there were no change compared with control group.2. MTT chromatometry check cell proliferational activity;when L — arg concentration ( <5mM) , promote Hela cellular growth gently; when L — arg concentration ( >5mM) , with increase of concentration and time, inhibit Hela cellular growth obviously.3. During cell culture, the effects of L - arg on telomerase activity are related to the effect concentration;compare with the control group, telomerase activity had no change in the lower group ( < lmM) ; been promoted obviously in the mid - group (5 ~ 25mM) ;been inhibited obviously in the higher group(50 ~ lOOmM).4. The effects of L - Arg on telomerase activity are related to the effect time:telomerase activity was almost twice over the control group when L - Arg acted 24h; was weaker when L - Arg acted 48h; was nearly as same as the control group when L - Arg acted 96h.5. Telomerase activity of Hela cell extract treated with basic amino acid had no significant difference from the control group.6. The effects of lysine and histidine on telomerase activity were not significant compared to the control group.DiscussionTelomerase is a specialized ribonucleoprotein 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. However,people is not quite clear yet the regulation mechanism of telomerase activity.Some paper report the handicap of the function of tumor cell mitochondria lead to ATP generate deficiently and increase glycolysis, so depress the results of MTT. Another explanation believe accumulation of the metabolite of amino acid, lead to the metabolite stack in cell, so depress the proliferation of Hela cell.Arginine degradate to omithine,the latter decarboxyl to form poly amines. Ectogenesis arginine could inhibit cell growth through inhibiting synthesis poly-amines of tumor cell. Polyamines are believe 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. Some study show that polyamines could accelerate the synthesis of DNA, ectogenesis polyamines could reverse the inhibition of DNA synthesis. The activation of telomerase is related to many regulation ways,in fact,the hTERT is the most important one. Indeed, the 200 — bp proximal region, designated as the hTERT core promoter,contains the basao transcriptional activity of hTERT. Perhaps polyamines binding to the proximal region of the hTERT promoter and activating transcription of the hTERT is responsible for this effect. However, we ha-...