TY - JOUR

T1 - Unrepairable analogous of nucleotide excision repair substrates as a potential anti-cancer drugs

AU - Evdokimov, Alexey N.

AU - Dol-Gova, Evgeniya V.

AU - Popov, Alexey A.

AU - Petruse-Va, Irina O.

AU - Bogachev, Sergey S.

AU - Lavrik, Olga I.

N1 - Publisher Copyright: © 2019, National Academy of Sciences of UkraineAll rights reserved.

PY - 2019/1/1

Y1 - 2019/1/1

N2 - In the previous studies we have demonstrated that DNA with the bulky Fap-dC derivative is a hardly repairable substrate for the cellular NER system [1]. Such type of compounds could be of particular interest as possible selective inhibitors of the NER system, considerably reducing the potency of DNA repair due to competitive immobilization of protein factors involved in this process. Tumor-initiating stem cells (TISCs) which are capable to internalize exogenous DNA [2, 3] could be the potential target for such synthetic analogues of NER substrates. In the current work the process of DNA internalization was considered as an approach to deliver the model DNAs into TISCs in order to reduce the reparative potential of cancer cells. Methods: Enzymatic DNA synthesis, PCR, RT-PCR, NER-competent cell extract preparation, in vitro NER assay, lab animals breeding, fluorescence microscopy, laser scanning microscopy. Results: 756 bp PCR product containing bulky photoactivable dC adducts inhibits the nucleotide excision repair system. The Krebs-2 ascites cells were shown to natively internalize such modified DNA. The basic parameters for this DNA probe internalization by the murine Krebs-2 tumor cells were characterized. Upon internalization, the fragments of DNA undergo partial and non-uniform hydrolysis of 3′ ends followed by ligation into a ring. The degree of hydrolysis, assessed by sequencing several clones with the insertion of specific PCR product, was 30-60 nucleotides. Conclusions: Results of the current investigation suggest the possibility to use Fap-dC-modified DNA fragments for further analysis of both membrane-associated and intracellular factors mediating the internalization of eDNA by Krebs-2 cells. Capability of Fap-dC-DNA to impair the NER process presumes their possible applicability in antitumor therapy.

AB - In the previous studies we have demonstrated that DNA with the bulky Fap-dC derivative is a hardly repairable substrate for the cellular NER system [1]. Such type of compounds could be of particular interest as possible selective inhibitors of the NER system, considerably reducing the potency of DNA repair due to competitive immobilization of protein factors involved in this process. Tumor-initiating stem cells (TISCs) which are capable to internalize exogenous DNA [2, 3] could be the potential target for such synthetic analogues of NER substrates. In the current work the process of DNA internalization was considered as an approach to deliver the model DNAs into TISCs in order to reduce the reparative potential of cancer cells. Methods: Enzymatic DNA synthesis, PCR, RT-PCR, NER-competent cell extract preparation, in vitro NER assay, lab animals breeding, fluorescence microscopy, laser scanning microscopy. Results: 756 bp PCR product containing bulky photoactivable dC adducts inhibits the nucleotide excision repair system. The Krebs-2 ascites cells were shown to natively internalize such modified DNA. The basic parameters for this DNA probe internalization by the murine Krebs-2 tumor cells were characterized. Upon internalization, the fragments of DNA undergo partial and non-uniform hydrolysis of 3′ ends followed by ligation into a ring. The degree of hydrolysis, assessed by sequencing several clones with the insertion of specific PCR product, was 30-60 nucleotides. Conclusions: Results of the current investigation suggest the possibility to use Fap-dC-modified DNA fragments for further analysis of both membrane-associated and intracellular factors mediating the internalization of eDNA by Krebs-2 cells. Capability of Fap-dC-DNA to impair the NER process presumes their possible applicability in antitumor therapy.

UR - http://www.scopus.com/inward/record.url?scp=85073366431&partnerID=8YFLogxK

U2 - 10.7124/bc.0009B8

DO - 10.7124/bc.0009B8

M3 - Article

AN - SCOPUS:85073366431

VL - 35

SP - 180

EP - 181

JO - Biopolymers and Cell

JF - Biopolymers and Cell

SN - 0233-7657

IS - 3

ER -