title: Global identification of genes related to nutrient deficiency in intervertebral disc cells in an experimental nutrient deprivation model.
abstract: BACKGROUND
  Intervertebral disc degeneration is a significant cause of degenerative spinal diseases. Nucleus pulposus (NP) cells reportedly fail to survive in large degenerated discs with limited nutrient availability. Therefore, understanding the regulatory mechanism of the molecular response of NP cells to nutrient deprivation may reveal a new strategy to treat disc degeneration. This study aimed to identify genes related to nutrient deprivation in NP cells on a global scale in an experimental nutrient deprivation model.
  
  
  RESULTS
  Rat NP cells were subjected to serum starvation. Global gene expression was profiled by microarray analysis. Confirmation of the selected genes was obtained by real-time polymerase chain reaction array analysis. Western blotting was used to confirm the expression of selected genes. Functional interactions between p21(Cip1) and caspase 3 were examined. Finally, flow cytometric analyses of NP cells were performed. Microarray analysis revealed 2922 differentially expressed probe sets with ≥1.5-fold changes in expression. Serum starvation of NP cells significantly affected the expression of several genes involved in DNA damage checkpoints of the cell cycle, including Atm, Brca1, Cdc25, Gadd45, Hus1, Ppm1D, Rad 9, Tp53, and Cyclin D1. Both p27(Kip1) and p53 protein expression was upregulated in serum-starved cells. p21(Cip1) expression remained in NP cells transfected with short interfering RNA targeting caspase 3 (caspase 3 siRNA). Both G1 arrest and apoptosis induced by serum starvation were inhibited in cells transfected with caspase 3 siRNA.
  
  
  CONCLUSIONS
  Nutrient deprivation in NP cells results in the activation of a signaling response including DNA damage checkpoint genes regulating the cell cycle. These results provide novel possibilities to improve the success of intervertebral disc regenerative techniques.
authors.affiliation: Department of Advanced Medicine for Spine and Spinal Cord Disorders, Hokkaido University Graduate School of Medicine, Sapporo, Japan. hidekisudo@yahoo.co.jp
authors.forename: Hideki
authors.initials: H
authors.lastname: Sudo
authors.forename: Katsuhisa
authors.initials: K
authors.lastname: Yamada
authors.forename: Koji
authors.initials: K
authors.lastname: Iwasaki
authors.forename: Hideaki
authors.initials: H
authors.lastname: Higashi
authors.forename: Manabu
authors.initials: M
authors.lastname: Ito
authors.forename: Akio
authors.initials: A
authors.lastname: Minami
authors.forename: Norimasa
authors.initials: N
authors.lastname: Iwasaki
mesh_terms: D000818:Animals; D059447:Cell Cycle Checkpoints; D002478:Cells, Cultured; D004249:DNA Damage; D005786:Gene Expression Regulation; D007403:Intervertebral Disc / Q000378:metabolism* / Q000473:pathology; D055959:Intervertebral Disc Degeneration / Q000401:mortality* / Q000473:pathology; D008297:Male; D008954:Models, Biological; D051381:Rats; D017207:Rats, Sprague-Dawley