Oxidized extracellular DNA causes oxidative modifications, induces double-stranded DNA breaks and activates the anti-apoptotic response in human astrocytoma cells: possible contribution to drug/radiation resistance? - 11th International Symposium on Circulating Nucleic Acids in Plasma and Serum (CNAPS)

Ashot Nazaretyan ^1,3 Ekaterina Kozhina ¹ Anton Filev ^1,2 Vladimir Pisarev ² Marina Konkova ¹ Larisa Kameneva ¹ Elizaveta Ershova ^1,2 Galina Shmarina ¹ Elena Malinovskaya ¹ Alexander Khalansky ⁴ Natalya Veiko ¹ Svetlana Kostyuk ^1,2

¹Laboratory of Molecular Biology, Research Centre for Medical Genetics (RCMG), Moscow, Russia
²Laboratory of Molecular Mechanisms of Critical States, V. A. Negovsky Research Institute of General Reanimatology, Federal Research and Clinical Center of Intensive Care Medicine and Rehabilitology, Moscow, Russia
³Department of Biophysics, N. I. Pirogov Russian National Research Medical University, Moscow, Russia
⁴Laboratory of Neuromorphology, Research Institute of Human Morphology, Moscow, Russia

Astrocytoma belongs to the most deadly gliomas resistant to radiotherapy/chemotherapy. Clarification of molecular mehanisms contributing to tolerance of astrocytomas to drug therapies or radiation remains an urgent problem in medicine. We propose that anti-tumor therapy induces oxidation stress and cell death that results in liberation into intercellular space of oxidized extracellular DNA (ox-exDNA). The latter may serve as DAMP signal to activate the adaptive anti-apoptotic responses in tumor cells. To prove the hypothesis, we determined the DNA damage and anti-apoptotic transcriptome responses of human astrocytome cell line 1321NI cells to ox-exDNA fragments. Exposure of 1321NI cells to 50-100 ng/ml of ox-exDNA (500 molecules of 8-oxo-2`-deoxyguanosine per 10^6 nucleosides) for 30 min caused 2-4-fold increased levels of both 8-oxodG and double-strand DNA breaks in cells as determined by cytofluorimetry (P<0.01). RT-PCR studies revealed 3-5-fold increased expression of BRCA1 and BRCA2 genes responsible for the repair of DNA breaks following exposure of cells to ox-cfDNA (P <0.001). Later, in 3-5 hours after the exposure of 1321NI cells to ox-exDNA, the level of double-stranded DNA breaks returned to control value, whereas the expression of the anti-apoptotic BCL2 gene and protein demonstrated 2-3-fold increases as revealed by RT-PCR and flow cytofluorimetry up to 24 hrs after the exposure(P<0.01). Data demonstrate that in astrocytoma cells ox-exDNA triggers: (1) formation of double-strand breaks in cancer cells possesing potential to provoke genome rearrangements and accumulation of mutant clones, and (2) adaptive responses of transcriptome to enhance the expression of anti-apoptotic BCL2 gene/protein in the same tumor cells supporting the propensity of mutant cells to survive. We suggest that the combination of both ox-exDNA-triggered mechanisms synergistically drives generation of of tumor cells tolerance to cytotoxic therapies. The work was supported by the Ministry of Education and Science of Russian Federation and the RFBR grant 17-29-06017 ofi_m.