DNA Damage Produced by ~(125)I-Triplex-Forming Oligonucleotides as a Measure of Their Succesful Delivery into Cell Nuclei

摘要

Decay of an Auger-electron-emitting radioisotope can knock out a targeted gene by producing DNA strand breaks within its sequence. For deliv-ery of Auger emitters to genomic targets we used triplex-forming oligonucle-otides (TFOs) that bind specifically to their target sequences by forming hydrogen bonds within the major groove of the target duplex. We named this approach antigene radiotherapy. In our previous studies, we demonstrated that 1251-labeled TFOs targeted against the human MDRI gene produced sequence-specific double strand breaks (DSBs) within this gene in live cultured cells. We also found that conjugation of TFO with nuclear localization signal peptide significantly increased the efficiency of targeting. To screen the wide variety of possible TFO modifications a sensitive and robust assay of DNA damage produced by such ~(125)I-TFOs would be highly desirable. Recently we showed a direct correspondence between the number of decays of ~(125)I incorpo-rated into DNA as ~(125)I-UdR and the number of histone γ-H2AX foci per cell revealed by staining with γ-H2AX antibodies. The technique is 100-fold more sensitive than other DSB-detection methods, thus it is possible to detect as few as an average of 0.5 DSBs per cell in a population of cultured cells. Here we ap-plied this method to evaluate the intracellular DNA damage produced by two ~(125)I-TFOs, the first targeted to the single-copy HPRT gene (~(125)I-TFO-HPRT) and second to a multicopy repeated sequence (GA)_n that occurs almost 7000 times in the human genome (~(125)I-TFO-GA). DNA damage produced by ~(125)ITFO was assessed by staining the cells with γ-H2AX antibody followed by either direct counting γ-H2AX foci or by measuring the γ-H2AX signal using flow cytometry. Both methods produced quantitatively close results; ~(125)I-TFOGA with multiple nuclear targets produced on average 1.93 times more γ- H2AX foci per cell and generated 1.96 times increase in γ-H2AX antibody staining signal than ~(125)I-TFO-HPRT with a single target. The γ-H2AX-based assay requires considerably less time and effort than the direct measurement of DSB by Southern hybridization applied previously. Therefore, we believe that γ-H2AX-based measurement of DNA damage could be useful for evaluation and cellular DNA accessibility by ~(125)I-labeled DNA targeting agents.