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Assessment of DNA damage produced by 125I-triplex-forming oligonucleotides in cells
Olga A Sedelnikova1, Irina V Panyutin, Ronald D Neumann
1NIH/NMD, 10 Center Dr., Room 4D45, Bethesda, MD 20892, USA.
Purpose:
Triplex-forming oligodeoxyribonucleotides (TFOs) bind specifically to their target sequences by forming hydrogen bonds within the major groove of the target duplex. When labeled with Auger-electron-emitting radioisotopes, TFOs are able to damage the target gene in a process named antigene radiotherapy. We compared radiotoxicity and the amount of DNA damage produced within cultured cells by two 125I-labeled TFOs, one with a single target in the genome and another with multiple targets.
Materials And Methods:
Radiotoxicity was measured by clonogenic assay while DNA damage was assessed by the number of histone gamma-H2AX foci formed at the sites of DNA double strand breaks (DSBs).
Results:
The TFO with multiple nuclear targets was 1.7 fold more radiotoxic and produced on average 1.9 fold more gamma-H2AX foci per cell than the TFO with a single target.
Conclusion:
Since the two methods gave comparable results, measuring the number of gamma-H2AX foci per decay may be a useful procedure for the assessment of cytotoxic effects and the intranuclear localization of radionuclides when they produce DSBs.
Insights
Triplex-forming oligodeoxyribonucleotides (TFOs) with multiple DNA targets showed higher radiotoxicity and DNA damage compared to single-target TFOs. Measuring gamma-H2AX foci is a reliable method for assessing antigene radiotherapy effects.
Area of Science:
- Molecular Biology
- Radiochemistry
- Genetics
Background:
- Triplex-forming oligodeoxyribonucleotides (TFOs) bind specific DNA sequences via major groove hydrogen bonding.
- Antigene radiotherapy utilizes Auger-electron-emitting radioisotope-labeled TFOs to induce targeted gene damage.
- Assessing the efficacy of TFO-based therapies requires quantifying both radiotoxicity and DNA damage.
Purpose of the Study:
- To compare the radiotoxicity and DNA damage induced by two 125I-labeled TFOs with distinct genomic target profiles (single vs. multiple targets).
- To evaluate the correlation between cellular radiotoxicity and DNA double-strand break (DSB) formation in the context of antigene radiotherapy.
Main Methods:
- Radiotoxicity was assessed using clonogenic assays.
- DNA damage was quantified by counting gamma-H2AX foci, markers of DNA double-strand breaks (DSBs).
- Two 125I-labeled TFOs, one targeting a single gene and the other multiple genes, were used in cultured cells.
Main Results:
- The TFO with multiple nuclear targets exhibited 1.7-fold higher radiotoxicity compared to the single-target TFO.
- Cells treated with the multi-target TFO showed an average of 1.9-fold more gamma-H2AX foci per cell.
- Both methods (clonogenic assay and gamma-H2AX foci counting) yielded comparable results regarding TFO efficacy.
Conclusions:
- The number of gamma-H2AX foci per decay can serve as a valuable method for assessing cytotoxic effects of radionuclides.
- This method aids in evaluating the intranuclear localization and efficacy of radionuclides used in antigene radiotherapy.
- TFOs with multiple targets demonstrate enhanced potential for antigene radiotherapy due to increased radiotoxicity and DNA damage induction.
