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Gadd45 is a
p53-dependent, multifunctional protein. Our prior work has demonstrated on
important role for the tumor suppressor p53 in UV-dependent responses in
the skin. In order to determine if this control is mediated via a
downstream molecule, we investigated the role of GADD45 in the UVB response
using irradiated normal human and GADD45 deficient mouse keratinocytes. An
RNA protection assay and western blotting demonstrated that GADD45 was
induced by UVB. Up-regulation of GADD45 mRNA was observed 4 h after
irradiation and remained induced for 24 hrs at multiple chosen doses (100,
300 or 600 J/m2 UVB). The induction of Gadd45 protein was observed 8 h
after irradiation and it remained induced for 48 hrs, with the strongest
induction at 300 J/m2. Cell cycle kinetics after exposure to UV was
assessed using flow cytometry. G2/M and S phase arrest were both observed
however only G2M arrest was Gadd45 dependent. To establish a mechanism for
G2/M arrest, the activity of cdc2 cyclin kinase was assayed. After
immunoprecipitation of cell lysates from irradiated GADD45+/+ and GADD45-/-
cells with cdc2 antibody, the kinase activity was assessed via histone H1
phosphorylation. Whereas cdc2 kinase activity, thought to be G2 specific,
showed reduction in GADD45+/+ cells after UV exposure, there was no
reduction observed in GADD45-/- cells. Post-UV global genomic DNA repair
wasassessed using a slot-western technique with a thymine dimer specific
antibody.GADD45-/- cells showed slower nucleotide excision repair than
GADD45+/+ cells. And finally, the cytotoxic effect of UVB was assessed,
GADD45-/- cells showed higher UV sensitivity. Our results suggest that
GADD45 is, at least in part, involved in regulating the G2/M checkpoint
after UV exposure, by regulating cdc2 kinase. In addition, GADD45 enhances
global genomic repair and regulates cell death. GADD45 appears to be an
important factor in maintaining genomic integrity in keratinocytes after UV
exposure.
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Gadd45 is a
multifunctional protein that is regulated by p53 and that may play a role
in DNA repair and cell cycle checkpoints. p53 plays an important role in
regulating DNA repair and in the response to UVB in keratinocytes. This
study investigates the role of GADD45 in the response to UVB in normal and
GADD45-deficient mouse keratinocytes. RNase protection assays showed that
GADD45 mRNA increases after 4 h and remains elevated for 24 h in cells
irradiated at 100, 300 or 600 J/m2 UVB. The level of Gadd45
protein increases after 8 h and remains elevated for 48 h with maximal
induction at 300 J/m2. Flow cytometry indicates that normal
cells arrest at G2/M and S, but Gadd45-deficient cells only arrest at S
phase. cdc2 kinase activity in immunoprecipitates from normal and
GADD45-deficient cells decreases after irradiation in normal cells but not
in GADD45-deficient cells. GADD45-deficient cells also have a slower rate
of nucleotide excision repair and higher UV sensitivity than normal cells.
These results suggest that GADD45 may play a role in the G2/M checkpoint in
keratinocytes exposed to UV. GADD45 may also regulate cdc2 kinase activity
and play roles in global genomic DNA repair and apoptosis.
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