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


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.
Radiation Biology Unit, Department of Safety and Radiation Protection, Forschungszentrum Jülich GmbH, Jülich, Germany.
Volker
V
Dahmen
Ralf
R
Kriehuber
D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D053903:DNA Breaks, Double-Stranded / Q000528:radiation effects; D005786:Gene Expression Regulation / Q000528:radiation effects*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D007457:Iodine Radioisotopes / Q000378:metabolism; D007624:KB Cells; D009838:Oligodeoxyribonucleotides / Q000378:metabolism*; D059467:Transcriptome / Q000528:radiation effects*; D000071857:Tumor Suppressor p53-Binding Protein 1
D047908:Intracellular Signaling Peptides and Proteins; D007457:Iodine Radioisotopes; D009838:Oligodeoxyribonucleotides; C498301:TP53BP1 protein, human; D000071857:Tumor Suppressor p53-Binding Protein 1; C051802:triplex DNA; D004247:DNA