Induction of p21Cip1-mediated G2/M arrest in H2O2-treated lens epithelial cells

PURPOSE: 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 p21(Cip1). 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 p21(Cip1) in the rat lenses were analyzed by immunohistochemistry. RESULTS: FACS analysis showed that H2O2 treatment induced G(2)/M phase arrest of HLE B-3 cells. p21(Cip1) was strongly induced by H2O2, whereas expression of other cell cycle genes was unchanged. Attenuation of p21(Cip1) expression using siRNA reduced the H2O2 induced G(2)/M arrest. Furthermore, JNK and ERK were activated by H2O2 and their specific inhibitors SP600125 (for JNK) and U0126 (for ERK1/2) prevented p21(Cip1) expression and blocked cell cycle arrest. H2O2 treatment of a rat lens organ culture also caused an increase in p21(Cip1). However, H2O2 treatment lowered the levels of p27(Kip1), cdc2, and PCNA in the rat lens culture, unlike in the HLE B-3 cells. CONCLUSIONS: The accumulation of p21(Cip1) 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 p21(Cip1)-mediated control of the cell cycle in lens epithelial cells.