, RNA and protein was determined by incorporation on the radioactive precursors
, RNA and protein was determined by incorporation on the radioactive precursors [3H]-thymidine, [3H]-uridine and [14C]-leucine (GE Healthcare, Amersham, UK). Briefly, 4×10 5 cells/ml have been cultured in 96-well round-bottom plates in a total volume of one hundred culture medium with or without the need of the PDE1 list indicated concentrations of CAUE. Following incubation for 4 h, [3H]-thymidine (37 MBq/ml), [3H]-uridine (37 MBq/ml) or [14C]-leucine (1.85 MBq/ml) wereCorrespondence to: Professor Syu-Ichi Kanno, Division ofClinical Pharmacotherapeutics, Tohoku Pharmaceutical University, 4-4-1 Komatsushima, Sendai, Miyagi 981-8558, Japan E-mail: [email protected] words: caffeic acid, telomerase reverse transcriptase,cytotoxicity, telomerase, NALM-TOMIZAWA et al: INHIBITION OF TELOMERASE BY CAFFEIC ACID UNDECYL ESTER IN NALM-6 CELLSadded, each corresponding to a total activity of 148 Bq, and incubated for an more 90 min. The cells were harvested on filter membranes utilizing a Labo Mash cell harvester (Futaba Health-related Inc., Tokyo, Japan). Subsequent to drying, the radioactivity of the material was measured by a LS-6500 liquid scintillation -counter (Beckman Coulter, Miami, FL, USA). Telomerase activity assay. Telomerase activity was measured employing a stretch PCR-based TeloChaser method (Toyobo Co., Ltd., Osaka, Japan), based on the manufacturer’s directions. Briefly, 4×105 cells have been lysed in 50 lysis reagent and incubated on ice for 20 min. Following centrifugation at 12,000 x g for 20 min, DNA products had been isolated and 26 cycles of PCR amplification had been performed at 95 for 30 sec, 68 for 30 sec and 72 for 45 sec. PCR items were electrophoresed on a ten polyacrylamide gel and stained with ethidium bromide. Pictures had been captured using the FLA3000G image analyzer (Fujifilm Corp., Tokyo, Japan). Western blotting. The effects of cellular signal transduction on hTERT protein expression by CAUE had been determined by western blotting (ten). Briefly, the cells have been incubated with all the indicated concentrations of CAUE, washed with phosphate-buffered saline (PBS) and lysed. Protein concentrations were measured making use of the BCATM protein assay kit (Thermo αvβ5 Species Fisher Scientific Inc., Rockford, IL, USA), based on the manufacturer’s directions. Samples of every protein (30 ) have been loaded onto 7.5 sodium dodecyl sulfate-polyacrylamide gels. Following electrophoresis, the protein was transferred to polyvinylidene difluoride membranes and blocked with Blocking One(Nacalai Tesque, Inc., Kyoto, Japan) for 1 h, before incubation with antibody overnight at four . The membranes have been then washed with wash buffer (PBS containing 0.05 Tween 20) and incubated with horseradish peroxidase-linked secondary antibody for 1 h. Subsequent to being washed with wash buffer, the protein levels had been analyzed by enhanced chemiluminescence making use of Pierce western blotting substrate (Thermo Fisher Scientific Inc.). Statistical analysis. Statistical evaluation was performed using a one-way evaluation of variance, followed by Williams’ a number of comparison test. P0.01 was regarded as to indicate a statistically substantial distinction. Results Effects of CAUE on DNA, RNA and protein synthesis. To investigate the cytotoxic mechanisms of CAUE, the kinetics of macromolecule synthesis were examined (Fig. 1) as well as the incorporation of radiolabeled substrates into DNA, RNA and protein was monitored. No effect was identified on CAUE at concentrations of 0.3 , nonetheless, CAUE showed important inhibition o.