title: Induction of p21Cip1-mediated G2/M arrest in H2O2-treated lens epithelial cells.
abstract: OBJECTIVE
  Oxidative damage is one of the major factors associated with the formation of age-related cataract and with senescence of various cell types. Although the effects of oxidative stress are complex, we focused on whether oxidative damage affects control of the cell cycle in lens epithelial cells.
  
  
  METHODS
  BrdU labeling and FACS analysis were used to investigate the effect of H2O2 on the cell cycle of HLE B-3 cells. In addition, western and Northern blot analysis were performed to assess the expression of cell cycle regulatory proteins and transfection with siRNA was used to knock out expression of p21Cip1. The activation of MAPK family members by oxidative stress was assessed using antibodies to detect the activated forms. To confirm the effect of H2O2 on an ex vivo model, its effect on cultures of the lenses of 3-week-old SD rats were examined. The localization and expression of PCNA and p21Cip1 in the rat lenses were analyzed by immunohistochemistry.
  
  
  RESULTS
  FACS analysis showed that H2O2 treatment induced G2/M phase arrest of HLE B-3 cells. p21Cip1 was strongly induced by H2O2, whereas expression of other cell cycle genes was unchanged. Attenuation of p21Cip1 expression using siRNA reduced the H2O2 induced G2/M arrest. Furthermore, JNK and ERK were activated by H2O2 and their specific inhibitors SP600125 (for JNK) and U0126 (for ERK1/2) prevented p21Cip1 expression and blocked cell cycle arrest. H2O2 treatment of a rat lens organ culture also caused an increase in p21Cip1. However, H2O2 treatment lowered the levels of p27Kip1, cdc2, and PCNA in the rat lens culture, unlike in the HLE B-3 cells.
  
  
  CONCLUSIONS
  The accumulation of p21Cip1 in lenses exposed to oxidative stress may play a role as a defensive mediator of oxidative damage, an indicator for senescence or aging, or an inducer for the formation of cataract. This finding links oxidative stress with p21Cip1-mediated control of the cell cycle in lens epithelial cells.
authors.affiliation: Korea Eye Tissue and Gene Bank, College of Medicine, The Catholic University of Korea, Seoul, Korea. youngsm@catholic.ac.kr
authors.forename: Young
authors.initials: Y
authors.lastname: Seomun
authors.forename: Jong-Tak
authors.initials: JT
authors.lastname: Kim
authors.forename: Hae-Sook
authors.initials: HS
authors.lastname: Kim
authors.forename: Ji-Young
authors.initials: JY
authors.lastname: Park
authors.forename: Choun-Ki
authors.initials: CK
authors.lastname: Joo
mesh_terms: D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D016203:CDC2 Protein Kinase / Q000378:metabolism; D002386:Cataract / Q000139:chemically induced / Q000378:metabolism; D018929:Cell Culture Techniques; D002455:Cell Division / Q000187:drug effects*; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000096:biosynthesis*; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005434:Flow Cytometry; D016195:G2 Phase / Q000187:drug effects*; D006801:Humans; D006861:Hydrogen Peroxide / Q000633:toxicity*; D007150:Immunohistochemistry; D048031:JNK Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D007908:Lens, Crystalline / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000037:antagonists & inhibitors / Q000378:metabolism; D009924:Organ Culture Techniques; D018384:Oxidative Stress / Q000187:drug effects; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D018809:Proliferating Cell Nuclear Antigen; D034741:RNA, Small Interfering; D050760:Cyclin-Dependent Kinase Inhibitor p27; D006861:Hydrogen Peroxide; D016203:CDC2 Protein Kinase; D048031:JNK Mitogen-Activated Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3