DNA Repair 8 (2009) 930–943 Contents lists available at ScienceDirect DNA Repair journal homepage: www.elsevier.com/locate/dnarepair High osmolality activates the G1 and G2 cell cycle checkpoints and affects the DNA integrity of nucleus pulposus intervertebral disc cells triggering an enhanced DNA repair response Eleni Mavrogonatou, Dimitris Kletsas ∗ Laboratory of Cell Proliferation and Ageing, Institute of Biology, National Centre for Scientific Research “Demokritos”, 153 10 Athens, Greece article info Article history: Received 21 January 2008 Received in revised form 11 May 2009 Accepted 15 May 2009 Available online 16 June 2009 Keywords: Nucleus pulposus Intervertebral disc Osmotic stress DNA damage p53 siRNA abstract Nucleus pulposus intervertebral disc cells experience a broad range of physicochemical stimuli in their native environment including osmotic fluctuations. Here we show that hyperosmotic treatment reduced nucleus pulposus cells’ proliferation by activating the G2 and G1 cell cycle checkpoints. p38 MAPK was found to participate in the manifestation of the G2 arrest under conditions of increased osmolality, since inhibition of its activity by SB203580 released the cells from G2 phase into mitosis. High osmolality resulted in the ATM-mediated phosphorylation of p53 on Ser15, the up-regulation of p21 WAF1 and the hypophosphorylation of the retinoblastoma protein in accordance to the observed G1 arrest. siRNA knock- ing down of p53 inhibited the expression of p21 WAF1 while maintaining the hyperphosphorylated form of the retinoblastoma protein and thus abrogated the G1 arrest observed under hyperosmotic conditions. Comet assay revealed that high osmolality provoked DNA damage to nucleus pulposus cells. Several previous reports have shown that renal cells become unable to sense and repair DNA damage under conditions of increased osmolality. On the contrary, nucleus pulposus cells residing within a hyperos- motic environment clearly preserved their ability to sense newly introduced DNA damage, as confirmed by the reactivation of p53 by ionizing radiation, retained the MRN complex in the nucleus and phos- phorylated H2A.X on Ser139. H2A.X phosphorylation was attenuated in cells persistently experiencing hyperosmotic stress which, combined with the concurrent reduction in comet tails’ length, indicated an active DNA repair machinery. Even more, when the DNA repair efficiency of nucleus pulposus cells was directly measured by a host cell reactivation of luciferase activity assay, it was found to be signifi- cantly increased under hyperosmotic pressure. Finally, p53 depletion of nucleus pulposus cells by siRNA enhanced and prolonged H2A.X phosphorylation, attributing to p53 a regulatory role in the DNA repair pathway induced by increased osmolality. © 2009 Elsevier B.V. All rights reserved. 1. Introduction Spine degenerative disorders, most of them leading to back pain, constitute a major public health problem with a great economic impact on the health care system and society [1,2]. Even though these disorders affect the quality of life of a large number of people, their exact etiology in many cases is still unidentified. Neverthe- less, low back pain has been clearly associated with intervertebral disc degeneration [3,4]. Intervertebral discs lie between the verte- bral bodies and consist of an outer layer of laminated fibres and a gelatinous core called annulus fibrosus and nucleus pulposus, respectively. The nucleus pulposus is the largest avascular struc- ture in the body thus conferring a very unusual environment for the cells that lie within it [5,6]. It has been shown to be a key ∗ Corresponding author. Tel.: +30 210 6503565; fax: +30 210 6511767. E-mail address: dkletsas@bio.demokritos.gr (D. Kletsas). player in very early disc degeneration since unfavorable conditions within this tissue seem to limit the density of its cells and their ability to respond to damage [6,7]. Despite their sparse popula- tion intervertebral disc cells are responsible for the maintenance of tissue homeostasis given that they regulate the delicate balance between extracellular matrix’s synthesis and degradation. Hence, it seems crucial to understand nucleus pulposus cells’ physiology as the preservation of a healthy nucleus would improve disc functions. One of the major stresses confronted by nucleus pulposus cells in their peculiar microenvironment is hyperosmotic stress. Hypertonicity (rising up to ∼500 mOsm/kg H 2 O in vivo) is the com- bined result of the extracellular matrix’s composition (containing increased amounts of aggrecan) and the mechanical forces the tis- sue is subjected to over a diurnal cycle [5]. The effects of high osmolality have been studied to an extent in the kidney, another physiologically relevant tissue to this stress [8]. Cells of the renal inner medulla are normally exposed to variable and often extreme hypertonicity as the result of the mechanism for concentrating the 1568-7864/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.dnarep.2009.05.005