Nuclear DNA, which is essential for the transmission of genetic information, is constantly damaged by external stresses and is subsequently repaired by the removal of the damaged region, followed by resynthesis of the excised region. Accumulation of DNA damage with failure of repair processes leads to fatal diseases such as cancer. Recent studies have suggested that intra- and extra-nuclear environments play essential roles in DNA damage. However, numerous questions regarding the role of the nuclear mechanical environment in DNA damage remain unanswered. In this study, we investigated the effects of cell confluency (cell crowding) on the morphology of cell nuclei, and cytoskeletal structures, and DNA damage in NIH3T3 skin fibroblasts and HeLa cervical cancer cells. Although nuclear downsizing was observed in both NIH3T3 and HeLa cells with cell crowding, intracellular mechanical changes in the two cell types displayed opposite tendencies. Cell crowding in NIH3T3 cells induced reinforcement of actin filament structures, cell stiffening, and nuclear downsizing, resulting in a significant decrease in endogenous DNA damage, whereas cell crowding in HeLa cells caused partial depolymerization of actin filaments and cell softening, inducing endogenous DNA damage. Ultraviolet (UV) radiation significantly increased DNA damage in NIH3T3; however, this response did not change with cell crowding. In contrast, UV radiation did not cause DNA damage in HeLa cells under either sparse or confluent conditions. These results suggested that cell crowding significantly influenced endogenous DNA damage in cells and was quite different in NIH3T3 and HeLa cells. However, cell crowding did not affect the UV-induced DNA damage in either cell type.
References
[1]
Paschke, R. (2011) Molecular Pathogenesis of Nodular Goiter. Langenbeck’s Archives of Surgery, 396, 1127-1136. https://doi.org/10.1007/s00423-011-0788-5
[2]
Someya, M., Hasegawa, T., Nakamura, A.J., Tsuchiya, T., Kitagawa, M., Gocho, T., et al. (2023) Prediction of Late Adverse Events in Pelvic Cancer Patients Receiving Definitive Radiotherapy Using Radiation-Induced γ-H2AX Foci Assay. Journal of Radiation Research, 64, 948-953. https://doi.org/10.1093/jrr/rrad079
[3]
Nakamura, A.J. (2021) Beyond Visualization of DNA Double-Strand Breaks after Radiation Exposure. International Journal of Radiation Biology, 98, 522-527. https://doi.org/10.1080/09553002.2021.1930268
[4]
Takata, H., Hanafusa, T., Mori, T., Shimura, M., Iida, Y., Ishikawa, K., et al. (2013) Chromatin Compaction Protects Genomic DNA from Radiation Damage. PLOS ONE, 8, e75622. https://doi.org/10.1371/journal.pone.0075622
[5]
Shah, P., Hobson, C.M., Cheng, S., Colville, M.J., Paszek, M.J., Superfine, R., et al. (2021) Nuclear Deformation Causes DNA Damage by Increasing Replication Stress. Current Biology, 31, 753-765.e6. https://doi.org/10.1016/j.cub.2020.11.037
[6]
Nagayama, K. and Fukuei, T. (2019) Cyclic Stretch-Induced Mechanical Stress to the Cell Nucleus Inhibits Ultraviolet Radiation-Induced DNA Damage. Biomechanics and Modeling in Mechanobiology, 19, 493-504. https://doi.org/10.1007/s10237-019-01224-3
[7]
Nagayama, K., Hamaji, Y., Sato, Y. and Matsumoto, T. (2015) Mechanical Trapping of the Nucleus on Micropillared Surfaces Inhibits the Proliferation of Vascular Smooth Muscle Cells but Not Cervical Cancer Hela Cells. Journal of Biomechanics, 48, 1796-1803. https://doi.org/10.1016/j.jbiomech.2015.05.004
[8]
Nagayama, K., Sagawa, C. and Sato, A. (2023) Deformation and Trapping of Cell Nucleus Using Micropillar Substrates Possibly Affect UV Radiation Resistance of DNA. Journal of Robotics and Mechatronics, 35, 1158-1164. https://doi.org/10.20965/jrm.2023.p1158
[9]
McBride, S.H. and Knothe Tate, M.L. (2008) Modulation of Stem Cell Shape and Fate A: The Role of Density and Seeding Protocol on Nucleus Shape and Gene Expression. Tissue Engineering Part A, 14, 1561-1572. https://doi.org/10.1089/ten.tea.2008.0112
[10]
Shah, B.S. and Chahine, N.O. (2018) Dynamic Hydrostatic Pressure Regulates Nucleus Pulposus Phenotypic Expression and Metabolism in a Cell Density-Dependent Manner. Journal of Biomechanical Engineering, 140, Article ID: 021003. https://doi.org/10.1115/1.4038758
[11]
Doolin, M.T., Smith, I.M. and Stroka, K.M. (2021) Fibroblast to Myofibroblast Transition Is Enhanced by Increased Cell Density. Molecular Biology of the Cell, 32, ar41. https://doi.org/10.1091/mbc.e20-08-0536
[12]
Vadla, R., Chatterjee, N. and Haldar, D. (2020) Cellular Environment Controls the Dynamics of Histone H3 Lysine 56 Acetylation in Response to DNA Damage in Mammalian Cells. Journal of Biosciences, 45, Article No. 19. https://doi.org/10.1007/s12038-019-9986-z
[13]
Oram, S.W., Liu, X.X., Lee, T., Chan, W. and Lau, Y.C. (2006) TSPY Potentiates Cell Proliferation and Tumorigenesis by Promoting Cell Cycle Progression in Hela and NIH3T3 Cells. BMC Cancer, 6, Article No. 154. https://doi.org/10.1186/1471-2407-6-154
[14]
Mattos dos Santos, P.C., Feuser, P.E., Cardoso, P.B., Steiner, B.T., Córneo, E.d.S., Scussel, R., et al. (2018) Evaluation of in Vitro Cytotoxicity of Superparamagnetic Poly(Thioether-Ester) Nanoparticles on Erythrocytes, Non-Tumor (NIH3T3), Tumor (HeLa) Cells and Hyperthermia Studies. Journal of Biomaterials Science, Polymer Edition, 29, 1935-1948. https://doi.org/10.1080/09205063.2018.1564134
[15]
Paddillaya, N., Ingale, K., Gaikwad, C., Saini, D.K., Pullarkat, P., Kondaiah, P., et al. (2022) Cell Adhesion Strength and Tractions Are Mechano-Diagnostic Features of Cellular Invasiveness. Soft Matter, 18, 4378-4388. https://doi.org/10.1039/d2sm00015f
[16]
An, J., Huang, Y., Xu, Q., Zhou, L., Shang, Z., Huang, B., et al. (2010) DNA-PKcs Plays a Dominant Role in the Regulation of H2AX Phosphorylation in Response to DNA Damage and Cell Cycle Progression. BMC Molecular Biology, 11, Article No. 18. https://doi.org/10.1186/1471-2199-11-18
[17]
Lu, L., Oswald, S.J., Ngu, H. and Yin, F.C. (2008) Mechanical Properties of Actin Stress Fibers in Living Cells. Biophysical Journal, 95, 6060-6071. https://doi.org/10.1529/biophysj.108.133462
[18]
Harris, A.R., Daeden, A. and Charras, G.T. (2014) Formation of Adherens Junctions Leads to the Emergence of a Tissue-Level Tension in Epithelial Monolayers. Journal of Cell Science, 127, 2507-2517. https://doi.org/10.1242/jcs.142349
[19]
Leontieva, O.V., Demidenko, Z.N. and Blagosklonny, M.V. (2014) Contact Inhibition and High Cell Density Deactivate the Mammalian Target of Rapamycin Pathway, Thus Suppressing the Senescence Program. PProceedings of the National Academy of Sciences of the United States of America, 111, 8832-8837. https://doi.org/10.1073/pnas.1405723111
[20]
Pavel, M., Renna, M., Park, S.J., Menzies, F.M., Ricketts, T., Füllgrabe, J., et al. (2018) Contact Inhibition Controls Cell Survival and Proliferation via YAP/TAZ-Autophagy Axis. Nature Communications, 9, Article No. 2961. https://doi.org/10.1038/s41467-018-05388-x
[21]
Zhao, B., Wei, X., Li, W., Udan, R.S., Yang, Q., Kim, J., et al. (2007) Inactivation of YAP Oncoprotein by the Hippo Pathway Is Involved in Cell Contact Inhibition and Tissue Growth Control. Genes & Development, 21, 2747-2761. https://doi.org/10.1101/gad.1602907
[22]
Sun, N., Kamarajan, P., Huang, H. and Chao, C.C (2002) Restoration of UV Sensitivity in UV-Resistant Hela Cells by Antisense-Mediated Depletion of Damaged DNA‐binding Protein 2 (DDB2). FEBS Letters, 512, 168-172. https://doi.org/10.1016/s0014-5793(02)02250-0
[23]
Ambrosio, S., Di Palo, G., Napolitano, G., Amente, S., Dellino, G.I., Faretta, M., et al. (2015) Cell Cycle-Dependent Resolution of DNA Double-Strand Breaks. Oncotarget, 7, 4949-4960. https://doi.org/10.18632/oncotarget.6644
[24]
Besaratinia, A., Yoon, J., Schroeder, C., Bradforth, S.E., Cockburn, M. and Pfeifer, G.P. (2011) Wavelength Dependence of Ultraviolet Radiation-Induced DNA Damage as Determined by Laser Irradiation Suggests That Cyclobutane Pyrimidine Dimers Are the Principal DNA Lesions Produced by Terrestrial Sunlight. The FASEB Journal, 25, 3079-3091. https://doi.org/10.1096/fj.11-187336
[25]
Knips, A. and Zacharias, M. (2017) Both DNA Global Deformation and Repair Enzyme Contacts Mediate Flipping of Thymine Dimer Damage. Scientific Reports, 7, Article No. 41324. https://doi.org/10.1038/srep41324
