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RUNX1(AML1) gene is a common participant in chromosomal translocations found in patients with “treatment-related” leukemia. This disease arises as a consequence of anti-cancer therapy with DNA-topoisomerase inhibitors (i.g. etoposide). Chromosomal rearrangements in these cases occur after the erroneous repair of etoposide-induced double-stranded DNA breaks (DSBs) within RUNX1 gene. We previously showed that after etoposide treatment of human cultured T-lymphocytes (Jurkat) the ends of DSB become mobile and tend to leave the inherent chromosome territory. The molecular mechanisms of these events may be unraveled by using the fluorescent microscopy followed by data processing. We treated cultured human T-lymphocytes (Jurkat) with etoposide and inhibitors of Rad51 and Mre11 (B02 and mirin, correspondingly), which participate in double-stranded DNA breaks repair. Besides, we treated the cells with etoposide in combination with aclarubicin, a topoisomerase II catalytic inhibitor. In all cases cells were fixed with formaldehyde after the treatment. 3D-FISH was performed with RUNX1 dual color break-apart probe and whole chromosome-21 painting probe. Images obtained with confocal microscope were processed in a computer software with a view to detect the signals and measure the distances between them. Etoposide promotes the DSBs within RUNX1 gene with measurable frequency. Addition of aclarubicin to etoposide-treated cells significantly decrease the amount of DSBs, supporting the role of DNA-topoisomerase II in the induction of such breaks. Parts of broken RUNX1 are located outside their chromosome territory more frequently than non-broken ones. Inhibition of strand exchange activity of Rad51 by B02 decreases the amount of etoposide-induced DSBs. Inhibition of exonuclease activity of Mre11 by mirin does not affect the number of etoposide-induced DNA disruption. The usage of Rad51 or Mre11 inhibitors leads to a decline in proportion of broken RUNX1 gene outside the chromosome 21 territory, suggesting the participation of these proteins in the DSBs ends mobility.