Speaker
Mr
Volker Dahmen
(Radiation Biology Group, Department of Safety and Radiation Protection, Forschungszentrum Jülich, 52425 Jülich, Germany)
Description
Triplex-forming oligonucleotides (TFO) are able to bind DNA in a sequence specific manner and are a promising tool to manipulate genes or gene regulatory units in a cellular environment. TFO posses a therapeutic potential e.g. as a carrier molecule for Alpha- or Auger-Electron-Emitter (AEE) to target specific DNA sequences in tumour cells. We established a method for the effective labeling of TFO with the AEE Iodine-125 (I-125) and studied the influence of labeled TFO in SCL-II cells with regard to cell survival, appearance of DNA Double-Strand-Breaks (DSB) and the induction of cell cycle arrest.
The TFO employed in this study were two multi-binding TFO with several thousand binding sites each in the human genome and a single binding site TFO, specific for GAPDH. TFO labeling with I-125 was performed using the primer extension method. Cell survival and DNA DSB frequency in I-125-TFO transfected SCL-II cells were analyzed with the Colony-Forming-Assay and the 53BP1 Assay. Analysis of cell cycle was done after 7-AAD staining by flowcytometry.
I-125-labeled TFO were shown to induce a pronounced decrease in cell survival and an increase of DSB. TFO targeting multiple sites differing in the total target number showed a significant different cell killing per decay that was in good accordance with the observed induction of DSB per decay. Single gene targeting I-125-labeled TFO significantly decreased cell survival and induced DSB as well. All three investigated TFO induced a significant cell cycle arrest in G2/M phase 8 h post-transfection.
I-125-labeled TFO with a single binding site as well as TFO with multiple binding sites cause massive cell killing and increase substantially the DSB frequency in SCL-II cells. All investigated TFO induce a pronounced G2/M arrest at rather low numbers of accumulated decays. I-125-labeled TFO might be a very useful tool for basic DNA repair research.
Funded by the Bundesministerium für Bildung und Forschung (BMBF), Grant number 02NUK005A
Primary author
Mr
Volker Dahmen
(Radiation Biology Group, Department of Safety and Radiation Protection, Forschungszentrum Jülich, 52425 Jülich, Germany)
Co-author
Dr
Ralf Kriehuber
(Radiation Biology Group, Department of Safety and Radiation Protection, Forschungszentrum Jülich, 52425 Jülich, Germany)
