γH2AX, 53BP1 and Rad51 protein foci changes in mesenchymal stem cells during prolonged X-ray irradiation

Abstract

// Anastasia Tsvetkova 1 , Ivan V. Ozerov 2, 3 , Margarita Pustovalova 2, 4 , Anna Grekhova 2, 4, 5 , Petr Eremin 6 , Natalia Vorobyeva 2, 3 , Ilya Eremin 6 , Andrey Pulin 6 , Vadim Zorin 6, 7 , Pavel Kopnin 8 , Sergey Leonov 9 , Alex Zhavoronkov 3 , Dmitry Klokov 10 and Andreyan N. Osipov 2, 3, 4, 9 1 Lomonosov Moscow State University, Moscow 119991, Russia 2 State Research Center - Burnasyan Federal Medical Biophysical Center of Federal Medical Biological Agency (SRC-FMBC), Moscow 123098, Russia 3 Insilico Medicine, Inc, ETC, Johns Hopkins University, Baltimore, Maryland 21218, USA 4 Semenov Institute of Chemical Physics, Russian Academy of Sciences, Moscow 119991, Russia 5 Emanuel Institute for Biochemical Physics, Russian Academy of Sciences, Moscow 119991, Russia 6 Federal State Budgetary Institution “Central Clinical Hospital with Outpatient Health Center” of The Business Administration for The President of The Russian Federation, Moscow 121359, Russia 7 PJSC Human Stem Cells Institute, Moscow 119333, Russia 8 N.N. Blokhin Cancer Research Center, Moscow 115478, Russia 9 Moscow Institute of Physics and Technology, Moscow 141700, Russia 10 Canadian Nuclear Laboratories, Chalk River, Ontario K0J1P0, Canada Correspondence to: Andreyan N. Osipov, email: andreyan.osipov@gmail.com Keywords: DNA double strand breaks, DNA repair, mesenchymal stem cells, X-rays, continuous irradiation Received: December 02, 2016      Accepted: June 20, 2017      Published: July 12, 2017 ABSTRACT At high exposure levels ionizing radiation is a carcinogen. Little is known about how human stem cells, which are known to contribute to tumorigenesis, respond to prolonged radiation exposures. We studied formation of DNA double strand breaks, accessed as γH2AX and 53BP1 foci, in human mesenchymal stem cells (MSCs) exposed to either acute (5400 mGy/h) or prolonged (270 mGy/h) X-irradiation. We show a linear γH2AX and 53BP1 dose response for acute exposures. In contrast, prolonged exposure resulted in a dose-response curve that had an initial linear portion followed by a plateau. Analysis of Rad51 foci, as a marker of homologous recombination, in cells exposed to prolonged irradiation revealed a threshold in a dose response. Using Ki67 as a marker of proliferating cells, we show no difference in the γH2AX distribution in proliferating vs. quiescent cells. However, Rad51 foci were found almost exclusively in proliferating cells. Concurrent increases in the fraction of S/G2 cells were detected in cells exposed to prolonged irradiation by scoring CENPF-positive cells. Our data suggest that prolonged exposure of MSCs to ionizing radiation leads to cell cycle redistribution and associated activation of homologous recombination. Also, proliferation status may significantly affect the biological outcome, since homologous repair is not activated in resting MSCs.