Cytotoxic effects and specific gene expression alterations induced by I-125-labeled triplex-forming oligonucleotides

Abstract

Purpose: Triplex-forming oligonucleotides (TFO) bind to the DNA double helix in a sequence-specific manner. Therefore, TFO seem to be a suitable carrier for Auger electron emitters to damage exclusively targeted DNA sequences, e.g., in tumor cells. We studied the influence of I-125 labeled TFO with regard to cell survival and induction of DNA double-strand breaks (DSB) using TFO with different genomic targets and target numbers. Furthermore, the ability of TFO to alter the gene expression of targeted genes was examined.

Materials and methods: TFO were labeled with I-125 using the primer extension method. DNA triplex formation and sequence-specific DSB were demonstrated in vitro. Cell survival was analyzed by colony-forming assay and DNA damage was assessed by microscopic quantification of protein 53 binding protein 1 (53BP1) foci in the human squamous carcinoma cell line II (SCL-II). Quantitative real-time polymerase-chain-reaction (qRT-PCR) was performed to analyze gene expression alterations.

Results: The sequence-specific induction of a single DSB in a 1695 bp long DNA double stranded fragment was demonstrated in vitro. I-125-labeled TFO binding to single and multiple targets 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 is also in good accordance with the observed induction of DSB. Single gene targeting I-125-labeled TFO significantly decreased cell survival and altered gene expression in the targeted gene.

Conclusions: I-125-labeled TFO enable specific targeting of DNA in vitro as well as in a cellular environment and thus induce sequence-specific complex DNA lesions. Therefore I-125-labeled TFO might be a very useful tool for basic DNA repair research.