Oxidized extracellular DNA affects DNA damage and transcription of genes regulating DNA repair and apoptosis in human astrocytoma cells

A. S. Nazaretyan; E. M. Malinovskaya; A. D. Filev; E. S. Ershova; N. N. Veiko; V. M. Pisarev; A. S. Khalansky; L. V. Kameneva; V. Y. Tabakov; M. S. Konkova; S. V. Kostyuk

Oxidized extracellular DNA affects DNA damage and transcription of genes regulating DNA repair and apoptosis in human astrocytoma cells

A. S. Nazaretyan, E. M. Malinovskaya, A. D. Filev, E. S. Ershova, N. N. Veiko, V. M. Pisarev, A. S. Khalansky, L. V. Kameneva, V. Y. Tabakov, M. S. Konkova, S. V. Kostyuk

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Abstract

The effects of oxidized extracellular DNA (oecDNA) on genomic DNA damage and activation of transcription of genes regulating DNA repair and apoptosis in human astrocytoma 1321NI cells were studied. OecDNA fragments, 50-100 ng/ml, were added to 1321NI cells for 0.5-24 hours. The levels of oxidative damage and double stranded DNA breaks, BCL and BAX protein expression were determined using antibodies to 8-hydroxy-7,8-dihydroguanosine (8-ohdG), phosphorylated histone γH2AX, BCL2 and BAX proteins and flow cytofluorimetry. The levels of gene expression of BCL2, BAC, BRCA1, BRCA2, TBP (control) were evaluated by real-time PCR. The exposure of 1321NI cells to oecDNA resulted in a 2-4-fold increased levels of 8-ohdG (p <0.01) and 3-3.5-fold increases in double-strand breaks (p < 0.01), whereas the expression of damage repair genes BRCA1 and BRCA2 was enchanced by 3-5 times (p <0.001). In 5-24 hours after exposure to oecDNA cells, the ratio of BCL2/BAX gene expression increased by 3.5-5 times vs. unexposed cell cultures (p <0.001). Therefore, in astrocytoma cells, oecDNA induces both genome rearrangement and adaptive transcriptome reactions followed by activation of an anti-apoptotic path in malignant cells. It is suggested that oecDNA contributes to the accumulation in a residual tumor of mutant clones resistant to treatment thus provoking relapse.