A human breast cancer model for the study of telomerase inhibitors based on a new biotinylated-primer extension assay

Telomerase is an RNA-dependent polymerase that synthesizes telomeric DNA (T TAGGG)(n) repeats. The overall goal of our work was to establish human canc er models that can be used to design clinical trials with telomerase inhibi tors. The objectives of this study were (1) to set up a human breast cancer system that allows evaluation of the effects of telomerase inhibitors in c ultured cells using a non-amplified telomerase assay and (2) to test this s ystem using two drugs (cisplatin and TMPyP4) that affect the telomerase exp ression in breast cancer cells in culture. We first compared the telomerase activity in a variety of human breast cancer cell lines to that of other t umour types using a new biotinylated-primer extension assay. Our method, ba sed on a non-amplified primer extension assay shows the direct incorporatio n of P-32-labelled nucleotides induced by telomerase on human telomeric pri mers. The P-32-dGTP labelled telomerase-extended 5'-biotinylated (TTAGGG)(3 ) primer can subsequently be separated using streptavidin-coated magnetic b eads. As compared to other non-amplified method, we showed that this proced ure improved the characterization and the quantification of the banding pat tern resulting from telomerase extension by reducing the radioactive backgr ound. Using this method, we observed that telomerase activity varies marked ly in a panel of 39 human cancer cell lines. For example, MCF7 breast cance r cells in culture showed intermediate telomerase activity corresponding to 33.8 +/- 3.4% of that of the HeLa cells (reference cell line). Similarly, the telomere length varied with each cell line (average: 6.24 +/- 6.16). No correlation between the level of telomerase and telomere length was observ ed, suggesting that a high processivity is not required to maintain telomer es and that, in some cell lines, another mechanism of telomere elongation c an maintain telomere length. From this study, we selected MCF7 and MX1 mode ls that showed reproducible telomerase activity and a relatively limited te lomere length for the testing of potential telomere-telomerase interacting agents. Using cisplatin and a new porphyrin-derived compound TMPyP4, we sho wed that our model was able to detect a down-regulation of the telomerase a ctivity in MCF7 cells in culture and in a human MX1 tumour xenografts, Base d on these results, a breast cancer model for evaluating telomerase and tel omere interactive agents is proposed.