Medical Radiology and Radiation Safety. 2024. Vol. 69. № 3
DOI:10.33266/1024-6177-2024-69-3-13-18
A.A. Osipov1, A.K. Chigasova1, 2, 3, E.I. Yashkina1, 2, M.A. Ignatov1, 2,
N.Yu. Vorobyеva1, 2, A.N. Osipov1, 2
Link Between Cellular Senescence and Changes in The Number and Size of Phosphorylated Histone H2ax Foci in Irradiated Human Fibroblasts
1 N.N. Semenov Federal Research Center for Chemical Physics, Moscow, Russia
2 A.I. Burnazyan Federal Medical Biophysical Center, Moscow, Russia
3 Institute of Biochemical Physics, Moscow, Russia
Contact person: N.Yu. Vorobyеva, e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it.
ABSTRACT
Purpose: To analyze the relationship between cellular aging and changes in the number and size of phosphorylated histone H2AX (γH2AX) foci in human fibroblasts and their descendants (up to 15 passages) after exposure to low and high doses of X-ray radiation.
Material and methods: The work was performed on a culture of human skin fibroblasts. Cells were irradiated in the exponential growth phase on an X-ray biological unit RUB RUST-M1 (Russia), equipped with two X-ray emitters, at a dose rate of 40 mGy/min (dose 100 mGy) or 850 mGy/min (doses 2000 and 5000 mGy) and temperature 4˚C. Immunocytochemical staining was used to assess the number and size of γH2AX foci and the proportion of proliferating cells using antibodies to γH2AX and Ki67 (a cell proliferation marker protein), respectively. To assess cellular senescence, the proportion of cells positive for senescence-associated β-galactosidase (CA-β-gal(+)) was analyzed. Statistical and mathematical analysis of the obtained data was carried out using the statistical software package Statistica 8.0 (StatSoft).
Results: The studies showed that irradiation of cultured human fibroblasts at a low dose (100 mGy) does not lead to statistically significant changes in the number and size of γH2AX foci, as well as the proportion of non-proliferating and senescent cells in the progenies of irradiated cells up to the 15th passage after irradiation. The phenomenon of aging-associated persistence of an increased number and size of γH2AX foci in passages of cells irradiated at a dose of 5000 mGy was discovered. Mathematical analysis of the relationship between changes in the proportion of CA-β-gal(+) cells, the number and size of γH2AX foci in populations of irradiated cells indicates that radiation-induced cellular aging is more associated with the size, rather than the number, of γH2AX foci.
Keywords: fibroblasts, X-ray radiation, γH2AX, senescence, proliferation, late radiation-induced effects
For citation: Osipov AA, Chigasova AK, Yashkina EI, Ignatov MA, Vorobyеva NYu, Osipov AN. Link Between Cellular Senescence and Changes in The Number and Size of Phosphorylated Histone H2ax Foci in Irradiated Human Fibroblasts. Medical Radiology and Radiation Safety. 2024;69(3):13–18. (In Russian). DOI:10.33266/1024-6177-2024-69-3-13-18
References
1. Dabin J, Mori M, Polo SE. The DNA Damage Response in the Chromatin Context: a Coordinated Process. Curr Opin Cell Biol. 2023;82:102176. doi: 10.1016/j.ceb.2023.102176.
2. Georgoulis A, Vorgias C, Chrousos G, Rogakou E. Genome Instability and γH2AX. International Journal of Molecular Sciences. 2017;18(9). doi: 10.3390/ijms18091979.
3. Shibata A, Jeggo PA. DNA Double-Strand Break Repair in a Cellular Context. Clin Oncol (R Coll Radiol). 2014;26(5):243-9. doi: 10.1016/j.clon.2014.02.004.
4. Rothkamm K, Barnard S, Moquet J, Ellender M, Rana Z, Burdak-Rothkamm S. DNA Damage Foci: Meaning and Significance. Environ Mol Mutagen. 2015;56(6):491-504. doi: 10.1002/em.21944.
5. Biswas H, Makinwa Y, Zou Y. Novel Cellular Functions of ATR for Therapeutic Targeting: Embryogenesis to Tumorigenesis. International Journal of Molecular Sciences. 2023;24(14). doi: 10.3390/ijms241411684.
6. Bushmanov A, Vorobyeva N, Molodtsova D, Osipov AN. Utilization of DNA Double-Strand Breaks for Biodosimetry of Ionizing Radiation Exposure. Environmental Advances. 2022;8. doi: 10.1016/j.envadv.2022.100207.
7. Merighi A, Gionchiglia N, Granato A, Lossi L. The Phosphorylated Form of the Histone H2AX (γH2AX) in the Brain from Embryonic Life to Old Age. Molecules. 2021;26(23). doi: 10.3390/molecules26237198.
8. Palla V-V, Karaolanis G, Katafigiotis I, Anastasiou I, Patapis P, Dimitroulis D, et al. gamma-H2AX: Can It Be Established as a Classical Cancer Prognostic Factor? Tumor Biology. 2017;39(3). doi: 10.1177/1010428317695931.
9. Marcotte R, Lacelle C, Wang E. Senescent Fibroblasts Resist Apoptosis by Downregulating Caspase-3. Mech Ageing Dev. 2004;125(10-11):777-83. doi: 10.1016/j.mad.2004.07.007.
10. Neumaier T, Swenson J, Pham C, Polyzos A, Lo AT, Yang P, et al. Evidence for Formation of DNA Repair Centers and Dose-Response Nonlinearity in Human Cells. Proceedings of the National Academy of Sciences. 2011;109(2):443-8. doi: 10.1073/pnas.1117849108.
11. Belov O, Chigasova A, Pustovalova M, Osipov A, Eremin P, Vorobyeva N, et al. Dose-Dependent Shift in Relative Contribution of Homologous Recombination to DNA Repair after Low-LET Ionizing Radiation Exposure: Empirical Evidence and Numerical Simulation. Current Issues in Molecular Biology. 2023;45(9):7352-73. doi: 10.3390/cimb45090465.
12. Vaurijoux A, Voisin P, Freneau A, Barquinero JF, Gruel G. Transmission of Persistent Ionizing Radiation-Induced Foci Through Cell Division in Human Primary Cells. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis. 2017;797-799:15-25. doi: 10.1016/j.mrfmmm.2017.03.003.
13. Stewart RD. Two-Lesion Kinetic Model of Double-Strand Break Rejoining and Cell Killing. Radiation Research. 2001;156(4):365-78. doi: 10.1667/0033-7587(2001)156[0365:tlkmod]2.0.co;2.
14. Miller I, Min M, Yang C, Tian C, Gookin S, Carter D, et al. Ki67 is a Graded Rather than a Binary Marker of Proliferation versus Quiescence. Cell Rep. 2018;24(5):1105-12 e5. doi: 10.1016/j.celrep.2018.06.110.
15. Sobecki M, Mrouj K, Camasses A, Parisis N, Nicolas E, Lleres D, et al. The cell proliferation antigen Ki-67 organises heterochromatin. Elife. 2016;5:e13722. doi: 10.7554/eLife.13722.
16. Sobecki M, Mrouj K, Colinge J, Gerbe F, Jay P, Krasinska L, et al. Cell-Cycle Regulation Accounts for Variability in Ki-67 Expression Levels. Cancer Res. 2017;77(10):2722-34. doi: 10.1158/0008-5472.CAN-16-0707.
17. Maier AB, Westendorp RG, D VANH. Beta-Galactosidase Activity as a Biomarker of Replicative Senescence during the Course of Human Fibroblast Cultures. Annals of the New York Academy of Sciences. 2007;1100:323-32. doi: 10.1196/annals.1395.035.
18. Osipov A, Chigasova A, Yashkina E, Ignatov M, Fedotov Y, Molodtsova D, et al. Residual Foci of DNA Damage Response Proteins in Relation to Cellular Senescence and Autophagy in X-Ray Irradiated Fibroblasts. Cells. 2023;12(8). doi: 10.3390/cells12081209.
PDF (RUS) Full-text article (in Russian)
Conflict of interest. The authors declare no conflict of interest.
Financing. The work was carried out with the support of the RSF (project No. 23-14-00078).
Contribution. Article was prepared with equal participation of the authors.
Article received: 20.01.2024. Accepted for publication: 27.02.2024.