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@INPROCEEDINGS{Dahmen:837949,
author = {Dahmen, Volker and Kriehuber, Ralf},
title = {{P}rocessing of {C}omplex {DNA} {L}esions in {M}ammalian
{C}ells {I}nduced by {I}-125 labeled {T}riplex-{F}orming
{O}ligonucleotides},
reportid = {FZJ-2017-06710},
year = {2017},
abstract = {Introduction: Triplex-forming oligonucleotides (TFOs) are
known for their ability to bind DNA in a sequence specific
manner and are therefore a promising tool to manipulate
genes or gene regulatory units. TFOs labeled with the
Auger-Electron-Emitter Iodine-125 can induce complex but
localized damage to the DNA. Using radionuclide-labeled TFO
the subsequent cellular damage was analyzed regarding
mutation frequency, mutation type and mutation
localization.Methods: The human squameous cell carcinoma
cell line SCL-II was used as the wildtype strain SCL-II WT
and the transgenic strain SCL-II p2RT. In the conducted
experiments the SCL-II WT strain was transiently transfected
with an in vitro pre-formed DNA-triplex of the p2RT vector
containing the target sequence and its specific TFO, the
I-125-TFO p2RT. The transgenic SCL-II p2RT strain carry the
stably integrated p2RT vector system harboring the specific
triplex target sequence for TFO-p2RT and was, therefore,
transfected with the I-125-TFO p2RT only. Efficient delivery
of vector + TFO or TFO only was ensured by electroporation
with the Nucleofector I system (Lonza GmbH, Basel). After
storage at -150°C for decay accumulation the samples were
analyzed for mutation frequency, type and localization using
blue/white screening and sequencing.Results: In the SCL-II
WT cell line an almost four fold increased mutation
frequency at the target region on the p2RT vector was found
when compared to the negative controls. In contrast, the
SCL-II p2RT transgenic strain did not show a significant
increase of the mutation frequency in comparison to the
negative controls. Sequencing revealed that most mutants
displayed large deletions of more than 100 bp located at the
TFO p2RT target site. Conclusions: The local complex DNA
damage induced by the decay of the TFO delivered Iodine-125
is likely to be accountable for the increased mutation rate
in the SCL-II WT cells. Since this increase could not be
detected in the SCL-II p2RT transgenic strain it can be
hypothesized that the site-specific triplex formation
between I-125-TFO p2RT and its target sequence is inhibited
to some extent in the cellular environment.},
month = {Sep},
date = {2017-09-17},
organization = {Joint Meeting of the European
Radiation Research Society and the
Society for Biological Radiation
Research, Essen (Germany), 17 Sep 2017
- 21 Sep 2017},
subtyp = {After Call},
cin = {S-US},
cid = {I:(DE-Juel1)S-US-20090406},
pnm = {899 - ohne Topic (POF3-899)},
pid = {G:(DE-HGF)POF3-899},
typ = {PUB:(DE-HGF)24},
url = {https://juser.fz-juelich.de/record/837949},
}