GADD45 induction of a G2/M cell cycle checkpoint.
G1/S and G2/M cell cycle checkpoints maintain genomic stability in eukaryotes in response to genotoxic stress. We report here both genetic and functional evidence of a Gadd45-mediated G2/M checkpoint in human and murine cells. Increased expression of Gadd45 via microinjection of an expression vector into primary human fibroblasts arrests the cells at the G2/M boundary with a phenotype of MPM2 immunopositivity, 4n DNA content and, in 15% of the cells, centrosome separation. The Gadd45-mediated G2/M arrest depends on wild-type p53, because no arrest was observed either in p53-null Li-Fraumeni fibroblasts or in normal fibroblasts coexpressed with p53 mutants. Increased expression of cyclin B1 and Cdc25C inhibited the Gadd45-mediated G2/M arrest in human fibroblasts, indicating that the mechanism of Gadd45-mediated G2/M checkpoint is at least in part through modulation of the activity of the G2-specific kinase, cyclin B1/p34(cdc2). Genetic and physiological evidence of a Gadd45-mediated G2/M checkpoint was obtained by using GADD45-deficient human or murine cells. Human cells with endogenous Gadd45 expression reduced by antisense GADD45 expression have an impaired G2/M checkpoint after exposure to either ultraviolet radiation or methyl methanesulfonate but are still able to undergo G2 arrest after ionizing radiation. Lymphocytes from gadd45-knockout mice (gadd45 -/-) also retained a G2/M checkpoint initiated by ionizing radiation and failed to arrest at G2/M after exposure to ultraviolet radiation. Therefore, the mammalian genome is protected by a multiplicity of G2/M checkpoints in response to specific types of DNA damage.
Wang
X W
XW
Laboratory of Human Carcinogenesis, Division of Basic Science, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Zhan
Q
Q
Coursen
J D
JD
Khan
M A
MA
Kontny
H U
HU
Yu
L
L
Hollander
M C
MC
O'Connor
P M
PM
Fornace
A J
AJ
Harris
C C
CC
D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins; D002453:Cell Cycle / Q000502:physiology*; D018797:Cell Cycle Proteins; D002460:Cell Line; D003110:Colonic Neoplasms; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000235:genetics; D004249:DNA Damage; D004268:DNA-Binding Proteins; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology; D016195:G2 Phase; D016158:Genes, p53; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008214:Lymphocytes / Q000166:cytology* / Q000502:physiology; D051379:Mice; D008815:Mice, Inbred Strains; D008938:Mitosis; D017346:Protein Serine-Threonine Kinases; D011506:Proteins / Q000235:genetics / Q000502:physiology*; D011994:Recombinant Proteins / Q000378:metabolism; D013154:Spleen / Q000166:cytology; D014162:Transfection; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins; D000097642:GADD45 Proteins
D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D017346:Protein Serine-Threonine Kinases; D011506:Proteins; D011994:Recombinant Proteins; D025521:Tumor Suppressor Proteins; D000097642:GADD45 Proteins; C493956:CDKN1A protein, human; C493957:Cdkn1a protein, mouse; C576324:ATM protein, human; C576325:Atm protein, mouse