| Background&Objective:Telomere 3'end of Eukaryotic chromosomes, which is composed of the G-rich DNA repeat sequences, with telomere-binding protein (telomere binding proteins, TBPS), like the metal shoelace knot of shoes, whoes main function is to maintain chromosome stability and to prevent the occurrence of degradation, rearrangements, terminal fusion and chromosome loss, such as enzymatic digestion. When normal cell is replicated, the telomere is shortened by 50-100 bp after each cell division. Because linear DNA can not be completely replicated with DNA polymerase, when the telomere is shorten to a certain extent, DNA is not stable any more and the double helix open, then the cells stop dividing and begin senescence or death. This is the normal cell's causes of their own genetic programming decisions of aging and death. Because the majority of malignant cells can activate telomerase and can fill up certain telomere shortage, telomere is not to decurtate after each cell division. The telomerase become an important identification of tumor cells between normal cells. It is has been shown that over 90% of malignant cells can activate telomerase. Telomerase therefore become a tumor diagnosis and the new drug target of anticancer. The smallest unit of telomerase which has activity is composed of hTERT and hTR. Telomerase reorganization activity required the correct assembly of these two parts. The combination of hTERT and hTR, as enzyme and substrate combine, which are reversible, in S phase of cell cycle, the tow parts are combined and show the telomerase activity, in the other phases, the combination separate and have no telomerase activity. Therefore, if the screening model is a combination of hTR-hTERT, we can greatly improve the specificity of screening. According to the theory, the telomerase yeast three-hybrid system is chosen to to discover specific telomerase inhibitors by our Lab. The high-throughput screening method related to yeast three-hybrid system is effective in screening samples that has potential telomerase inhibitory ability in the more than 2000 strains of actinomycetes fermentation broth. In the study, we only observe growth of the yeast strains which have been inserted combination of hTR-hTERT to screen specific telomerase inhibitors.Methods:1. To verify the yeast three-hybrid screening modelsThe high-throughput screening models which are held by our Lab are L40 ura3 /pHyblex/Zeo-MS2/hTR126-451/hTERT292-608aa,L40ura3/pHyblex/Zeo-MS2/hTR 240-450/hTERT292-608aa,L40ura3/pHyblex/Zeo-MS2/hTR(m)/hTERT292-952aa, which are inserted by plasmids which contain combination of hTR-hTERT based on L40 ura3/pHyblex/Zeo-MS2. The plasmids are possible missed if the yeast stains are held too long time or by a bad way. So the high-throughput screening models based on yeast three-hybrid system must be verified by yeast colony PCR,analysis of P-galactosidase reporter activity and so on before the screening.2. The sample libraries preparation of actinomycetes fermentation brothMore than 2000 strains of actinomycetes samples (fermented rice) are marked by different numbers. Each of the samples, which weigh 0.3g, were placed in 5ml penicillin bottles that are labeled, then infused 3ml 50% ethanol. Then put the penicillin bottles in the shaking table soaking for 24h at room temperature, and oscillating through the shaker filter collection of fluid filtration placed in the same label affixed penicillin bottle,4℃refrigerator spare.3. To determine appropriate screening conditionsAs mentioned above, the screening models used in the experiment is based on the yeast strain L40 ura3/pHyblex/Zeo-MS2 which are imported in the easy combination of hTERT and hTR fragments. While the yeast strain L40 ura3/ pHyblex/Zeo-MS2 is a kind of brewer's yeast, alcohol can inhibit its growth. Actinomycetes broth in the screening experiment was dissolved with the liquid 50% alcohol. So it is required to select the appropriate dose of actinomycetes fermentation broth in pre-screening regard to avoid the wrong effects of alcohol on the screen.On the condition that there is the appropriate dose of Actinomycetes broth, the best initial screening concentration of yeast. Because the yeast cell wall is thick, permeability is poor. It is needed to extend the incubation time in order to promote the drugs into cells. While the incubation time is longer, if the initial concentration of too much culture, yeast growth would be too early to enter the plateau is not conducive to screening, Therefore, pre-screening of the yeast need to select the appropriate concentration of initial training.4. High-throughput screening in Actinomyces broth sample It is first to discover specific telomerase inhibitors by high-throughput screening model of the yeast strains L40ura3/pHyblex/Zeo-MS2/hTRl 26-451/hTERT 292-608aa in the more than 2000 strains of actinomycetes fermentation broth, when the Initial appropriate concentration and the appropriate line of fermentation liquid dose is certain. In this study,96-well plates are used in high-through screening and each well contain 10μl sample and 190μl yeast liquid. The solvent control, blank control and negative control are also set. Then the yeast is incubated at 25℃for 48 hours in the thermostat oscillator. At the same time OD595nm of the yeast liquid were measured every 12h by using Multifunctional Microplate Analyzer. After all drug samples are tested, the screening models of yeast strains L40ura3/pHyblex/ Zeo-MS2/hTR240-450/hTERT292-608aa and L40ura3/pHyblex/Zeo-MS2/hTR(m) /hTERT292-952aa were used to screen the more than 2000 strains of actinomycetes fermentation broth again. The way is the same to the yeast strain L40ura3/pHyblex/ Zeo-MS2/hTR126-451/hTERT292-608aa.5. Calculation of inhibition rateBy using three kinds of telomerase inhibitor screening model to discover specific telomerase inhibitors in more than 2000 strains of actinomycetes fermentation broth samples, all the data of screening in 96-well plates in processing are calculated by the following formula:Inhibition rate (%)= (Solvent control OD595nm—Sample OD595nm)/(Solvent control OD595nm-Blank control OD595 nm)×100%6.Re-screeningAfter the calculation of antibacterial efficiency for the screening results, all the samples whose rate are more than 45% are regarded the highly active actinomycetes fermentation broth samples. Then the active actinomycetes fermentation broth samples are re-screened.Since Actinomyces broth is natural medicine resource, it has been reported that a number of actinomycetes broth is with cell toxicity in the literature. It may be also toxic to the yeast. In order to prevent the toxicity to yeast of actinomyces broth samples cause growth inhibition to screening models, rather than through the use of screening mechanisms to inhibit yeast models growth,then to produce false positive results. So the control yeast strain is established in re-screening of this experiment. The control yeast strain is L40ura3/pHyblex/Zeo-MS2 (with the YPD yeast culture).Control yeast strain L40ura3/pHyblex/Zeo-MS2 is cultured under the same conditions to the three models yeast. The rate of calculations in re-screening is same to the screening models.7 Evaluating inhibition efficiency of the positive samples:Culture K562 leukemia cells at 37℃,5% CO2, and the culture medium is with penicillin-streptomycin.14 active actinomycete fermentation samples were filtered by 0.22μl anotopr. When cells grow well, pipe cells to 48-well cell plate and each well is added 10μl acitive actinomycete fermentation. Set solvent control sample and the control cells.In the process of cultivation of K562 cell, the growth status is observed every 12 hours and compared with the control cells.Result:1. The verification of the yeast three-hybrid screening modelsAfter verifying by yeast colony PCR and analysis of P-galactosidase reporter activity, the results show that the three kinds of telomerase inhibitor yeast three-hybrid screening models preserved by our lab are right and the plasmids in them are no loss occur, which can be used to re-screen.2. To try to find out the best screening conditionsBy screening initial concentration of yeast culture models in experiment, the results show that when the absorbance of 595nm (OD595nm) is 0.04, the yeast concentration is the best initial screening concentration.Under this condition, the yeast continued to grow within 48 hours and does not appear plateau. When the initial concentration of yeast models in liquid culture is 0.04 for OD595nm, the alcohol concentration is 2.5%which has little effect on yeast growth. Therefore, liquid culture of 2.5% alcohol concentration and yeast initial screening concentration of 0.04 for OD595nm are the best screening conditions. 3. The primary screening resultsThrough high-throughput screening telomerase inhibitors and the comparison results of the inhibitory efficiency in more than 2000 strains of actinomycetes fermentation broth in 96-well plates, the samples whose inhibitory efficiency are over 0.45 are regarded as positive samples. In the primary screening,21 strains actinomycetes fermentation broth was regarded as telomerase inhibitors, which can be used for the re-screening. These strains are marked as 100-05,148-14,133-05,144-03,GD17,59-27,51-27,160-11,51-2,155-20,37-7,157-05,37-5,51-10,58-09,H41-50,11-11,73-8,40-08,GD-17,179-02,72-5,47-12,H120-20,H102-4,GD18,12-01.4. The results of secondary screeningRe-screen the screening results of the primary screening results which contain 21 strains actinomycetes broth and in the double screenings, there are a total of 14 strains actinomycetes broth regarded positive samples. These strains are marked as 100-05,148-14,59-27,160-11,51-2,155-20,157-05,37-5,58-09,H41-50,GD-17,H120-20,GD18,12-01.5. The evaluation for the positive samples regarded the telomerase inhibitionAfter Leukemia K562 cells and 10μl positive actinomycete fermentation samples were incubated together for 12 hours, observe the cells compared with solvent control and blank control cells. The active samples have inhibitory effect on cell growth compared with solvent control wells. The amount of cells is less and the cells state is poor in sample wells. The liquid medium has more cell debris. K562 cells which were incubated with the positive samples were observed after 24 hours. Compared with solvent control wells, the amount of cells is significantly less and the cells are in worse condition. The liquid medium has a large number of cell debris. From this we can infer that the positive samples may have strong inhibition of telomerase activity. Conclusion:It is the first time to use telomerase yeast three-hybrid screening model for high-throughput screening telomerase inhibitors. This study shows that high-throughput screening method related to yeast three-hybrid system is simple and stable in discovering telomerase inhibitors, and 14 new antitumor compounds were identified, which lays the foundation in the development of new anti-tumor agents.The innovations in the study:1. A use of RNA three-hybrid theory, for the first time using the two core components of human telomerase that are hTERT and hTR as the only variable is whether the telomerase-specific inhibitor of high-throughput screening model for high-throughput screening anticancer drugs;2. It is the first time to screen telomerase inhibitors in the natural products-fermentation broth of actinomycetes, which has opened up find a new way in order to search new anti-tumor agents. |
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