title: Optimization of the TRAP assay to evaluate specificity of telomerase inhibitors.
abstract: Telomerase inhibition represents a promising approach to anticancer treatment. In order to clarify the therapeutic potential of telomerase inhibitors we examined different substances (small molecule compounds BIBR1532 and BRACO19, as well as hTR antisense oligonucleotides 2'-O-methyl RNA and PNA) in A-549, MCF-7, and Calu-3 cell lines in a cell-free TRAP assay. We demonstrated that each of the tested agents inhibited telomerase in all used cell lines and that the antisense oligonucleotides represent the most potent inhibitors. Interestingly, upon evaluating the specificity of telomerase inhibitors we found out that not all agents acted specifically against telomerase. We observed that BRACO19 and PNA had an inhibitory effect also on PCR amplification of the TSR8 oligonucleotide which is provided in the TRAP(EZE) kit as a PCR control. By modifying the experimental protocol and using a different reverse primer we were able to enhance PNA selectivity, although the PCR inhibition of the TSR8 control template by BRACO19 could not be prevented. We propose an explanation for the lack of target specificity and suggest caution when testing putative telomerase inhibitors, as it appears that some of those substances may not affect specifically telomerase or telomeric G-rich sequences and thus can lead to the misinterpretation of experimental results.
authors.affiliation: Dr Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany. kamilla.piotrowska@ikp-stuttgart.de
authors.forename: Kamilla
authors.initials: K
authors.lastname: Piotrowska
authors.forename: Elke
authors.initials: E
authors.lastname: Kleideiter
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Mürdter
authors.forename: Sebastian
authors.initials: S
authors.lastname: Taetz
authors.forename: Christiane
authors.initials: C
authors.lastname: Baldes
authors.forename: Ulrich
authors.initials: U
authors.lastname: Schaefer
authors.forename: Claus-Michael
authors.initials: CM
authors.lastname: Lehr
authors.forename: Ulrich
authors.initials: U
authors.lastname: Klotz
mesh_terms: D000166:Acridines / Q000494:pharmacology*; D062365:Aminobenzoates / Q000494:pharmacology*; D001681:Biological Assay / Q000379:methods*; D045744:Cell Line, Tumor / Q000187:drug effects* / Q000201:enzymology; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D009281:Naphthalenes / Q000494:pharmacology*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D019098:Telomerase / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000235:genetics
chemical_list: D000166:Acridines; D062365:Aminobenzoates; C458523:BIBR 1532; D004791:Enzyme Inhibitors; D009281:Naphthalenes; D016376:Oligonucleotides, Antisense; D019098:Telomerase; C454064:BRACO-19