Role of p21(Waf-1) in regulating the G1 and G2/M checkpoints in ultraviolet-irradiated keratinocytes.

This study examines the role of p21(Waf-1) , a p53-dependent protein, in regulating mechanisms that protect keratinocytes against ultraviolet-B-induced cellular damage. Keratinocytes from p21(Waf-1) or p53-deficient mice were irradiated with ultraviolet B, and examined for DNA repair, cell cycle progression, and cell death. Both p21(Waf-1) -deficient and p53-deficient cells failed to maintain G2 arrest, and p21(Waf-1) -deficient cells, and to a lesser extent p53-deficient cells, also failed to undergo G1 arrest. After exposure to ultraviolet B, p53-deficient cells were more susceptible to cell death than wild-type cells. p21(Waf-1) -deficient cells did not undergo apoptotic cell death more often, however, but did have an increased frequency of nuclear abnormalities, suggesting mitotic catastrophe. TUNEL assay showed DNA fragmentation in the p53 +/+, p21(Waf-1) +/+, and p53 -/- cells, but not in p21(Waf-1) -/- cells. This result is consistent with the suggestion that p21(Waf-1) -deficient keratinocytes undergo mitotic cell death (catastrophe) after exposure to ultraviolet B irradiation in the system. Western analysis demonstrated that p21(Waf-1) expression was upregulated in p53-proficient and -deficient keratinocytes, supporting the notion that a p53-independent mechanism contributes to the response to ultraviolet B in keratinocytes. Finally, p21(Waf-1) -deficient cells had slightly less efficient nucleotide excision repair. In summary, this study suggests that p21(Waf-1) regulates the ultraviolet-B-induced G2/M checkpoint through p53, and the G1 checkpoint partially through p53. p21(Waf-1) does not significantly regulate DNA repair in ultraviolet-irradiated keratinocytes, however.

GADD45 regulates G2/M arrest, DNA repair, and cell death in keratinocytes following ultraviolet exposure.

GADD45 is a multifunctional protein that is regulated by p53. p53 plays an important role in regulating DNA repair and in the response to ultraviolet light in keratinocytes. This study investigates the role of GADD45 in the response to ultraviolet B. Cell cycle analysis demonstrated that wild-type and Gadd45-deficient cells have transient G2/M arrest, but only in the wild-type cells was arrest sustained. Cdc2 kinase activity in immunoprecipitates from normal and Gadd45-deficient cells decreases after irradiation in normal cells but not in Gadd45-deficient cells. An immunofluorescent study with Cdc2 antibody demonstrated diffuse cellular fluorescence before ultraviolet irradiation in both Gadd45-deficient and wild-type cells, but upon ultraviolet irradiation only Gadd45-proficient cells showed Cdc2 sequestration in the cytoplasm. Gadd45-deficient cells also have a slower rate of nucleotide excision repair. The lack of G2/M arrest coupled with reduced DNA repair leads to a higher ultraviolet sensitivity of Gadd45-deficient cells. These results reveal that GADD45 promotes G2/M arrest via nuclear export and kinase activity of Cdc2, increases global genomic DNA repair, and inhibits cell death in keratinocytes. Thus, GADD45 plays an important role in maintaining genomic integrity in ultraviolet-exposed skin.