Effects of DNA Demethylation on Human Intervertebral Disk Cells: Pilot study

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Introduction The intervertebral disk (IVD) cell distribution depends on the developmental stage of the human disk.1 DNA methylation has been shown to be essential to mammalian development,2,3 and aberrant methylation patterns are a common feature of many kinds of age-related diseases.3 We hypothesize that the methylation status of the promoter region of the genomic DNA affects cell proliferation, progression, and alterations in the gene expression of IVD cells. Materials and Methods IVD tissues were obtained from donors undergoing anterior lumbar discectomy procedures with Institutional Review Board approval. The tissues were transported to the laboratory, minced and immediately underwent cell isolation by sequential enzyme digestion. The digested cells were centrifuged and seeded in culture dishes, supplemented with complete medium (DMEM/F12) containing 10% fetal bovine serum (FBS), 1% L-glutamine, and 1% antibiotic. The demethylation agent 5-azacytidine (5-aza-C)4 was used to inhibit DNA methylation in IVD cells. The cells were then plated and treated with 5-aza-C followed by cell proliferation assay by MMT assay, cell progression by fluorescent cell sorting, and analyzed for gene regulation changes by real time PCR. Results Our results demonstrate that 5-azacytidine treatment inhibits cell proliferation in both dose-dependent and time-dependent manners. Cell cycle analysis revealed that 5-azacytidine (5 µM) treatment significantly increased the percentage of cells within the sub-G1 phase, suggestive of DNA fragmentation and apoptotic cell death. 5Aza-C (5 µM) treatment also increases the percentage of cells in S phase and G2/M phase indicating cell arrest. Gene expression analysis following 5-azaC treatment revealed that the MMP-1, MMP-13, IL-1b, and IL-6 were upregulated, whereas the anabolic gene aggrecan was downregulated. Conclusion The experimentally-induced loss of DNA methylation results in the inhibition of cell proliferation and an increase in apoptotic cell death. Furthermore, demethylation results in both increased gene transcription of catabolic and inflammatory genes as well as a downregulation of aggrecan, thus confirming a cause-and-effect relationship between DNA demethylation and transcription of these genes in IVD cells. DNA demethylation has been previously shown to inhibit cell proliferation5 and induce apoptotic cell death5,6 in different cell types. Our results suggest that demethylation of IVD cells may be one of the influential causes for IVD degeneration. Further studies are required to confirm our findings and to better understand the effects of aberrant DNA methylation on IVD biology and disease pathophysiology. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared Guehring et al. European Spine Journal 2010;19(1):113–121 Takebayashi et al. Molecular and Cellular Biology 2007;27(23):8243–8258 Christensen et al. PLoS Genetics 2009;5(8) Quddus et al. Journal of Clinical Investigation 1993;92:38–53 Nilsson et al. Journal of Endocrinology 2005;186(1):241–249 Ho et al. Cell Biology International 2006;30(3):288–294