title: Quiescent keratocytes fail to repair MMC induced DNA damage leading to the long-term inhibition of myofibroblast differentiation and wound healing.
abstract: OBJECTIVE
  The purpose of this study was to determine the acute and long-term effects of mitomycin C (MMC) on quiescent rabbit corneal keratocytes regarding cell proliferation, myofibroblast differentiation and DNA repair.
  
  
  METHODS
  Quiescent keratocytes cultured in serum-free media were exposed to various concentrations of MMC and then treated with transforming growth factor-β (TGFβ). DNA damage was evaluated in both cultured keratocytes and live rabbit eyes following treatment with MMC. The long-term ability of quiescent keratocytes to repair MMC induced damage in vivo was evaluated in rabbits treated with MMC 2 months before 100 μm deep lamellar keratectomy (LK) injury.
  
  
  RESULTS
  MMC significantly blocked TGFβ-induced cell proliferation and myofibroblast differentiation in cultured quiescent keratocytes and altered the transcriptional regulation of macrophage chemotactic protein-1 (MCP-1) and alpha smooth muscle actin (αSMA). MMC also induced phosphorylation of the nuclear histone marker of DNA damage, γH2AX (a member of the H2A histone family), without induction of cell cycle entry or immediate DNA repair measured by Comet assay. In live rabbits, 0.2 mg/ml MMC significantly induced γH2AX nuclear immunostaining (p<0.05) throughout the cornea and corneas receiving 0.2 mg/ml MMC treatment 2 months before LK injury showed complete absence of any corneal scarring.
  
  
  CONCLUSIONS
  MMC induces DNA damage to quiescent corneal keratocytes, which remains unrepaired, resulting in abnormal cell replication and gene transcription that leads to long-term effects on corneal repair. Overall these findings suggest that there may be long-term and perhaps permanent consequences to the application of MMC as an anti-fibrotic therapy.
authors: Gavin Herbert Eye Institute, University of California-Irvine, CA, USA. jjester@uci.edu
authors: James V
authors: JV
authors: Jester
authors: Chyong Jy
authors: CJ
authors: Nien
authors: Vasilis
authors: V
authors: Vasiliou
authors: Donald J
authors: DJ
authors: Brown
mesh_terms: D000199:Actins / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000235:genetics / Q000378:metabolism; D002921:Cicatrix / Q000517:prevention & control; D020552:Comet Assay; D003315:Cornea / Q000187:drug effects* / Q000378:metabolism; D065306:Corneal Injuries; D060527:Corneal Keratocytes / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D016895:Culture Media, Serum-Free; D004249:DNA Damage; D004260:DNA Repair / Q000187:drug effects; D006657:Histones / Q000235:genetics / Q000378:metabolism; D016685:Mitomycin / Q000494:pharmacology*; D058628:Myofibroblasts / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D011817:Rabbits; D014158:Transcription, Genetic / Q000187:drug effects; D016212:Transforming Growth Factor beta / Q000494:pharmacology; D014945:Wound Healing / Q000187:drug effects*
chemical_list: D000199:Actins; D018932:Chemokine CCL2; D016895:Culture Media, Serum-Free; D006657:Histones; D016212:Transforming Growth Factor beta; D016685:Mitomycin