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GADD45 induction of a G2/M cell cycle checkpoint
X W Wang1, Q Zhan, J D Coursen
1Laboratory of Human Carcinogenesis, Division of Basic Science, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Summary
Growth arrest and DNA-damage-inducible 45 (Gadd45) mediates a G2/M cell cycle checkpoint in human and murine cells. This Gadd45-mediated checkpoint is essential for genomic stability following genotoxic stress.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- Cell cycle checkpoints are crucial for maintaining genomic stability in response to DNA damage.
- The G2/M checkpoint prevents cells with damaged DNA from entering mitosis.
- Growth arrest and DNA-damage-inducible 45 (Gadd45) proteins are involved in cellular responses to stress.
Purpose of the Study:
- To investigate the role of Gadd45 in the G2/M cell cycle checkpoint.
- To elucidate the mechanism of Gadd45-mediated G2/M arrest.
- To determine the dependence of the Gadd45 checkpoint on p53 and its interaction with other cell cycle regulators.
Main Methods:
- Microinjection of Gadd45 expression vectors into human fibroblasts.
- Assessment of cell cycle arrest using MPM2 antibody, DNA content analysis, and centrosome separation.
- Analysis of Gadd45-mediated arrest in p53-null cells and cells expressing p53 mutants.
- Investigation of the effect of cyclin B1 and Cdc25C on Gadd45-induced arrest.
- Utilizing antisense GADD45 expression in human cells and gadd45-knockout mice to assess checkpoint function after DNA damage.
Main Results:
- Increased Gadd45 expression induced G2/M arrest in human fibroblasts, characterized by MPM2 positivity, 4n DNA content, and centrosome separation.
- The Gadd45-mediated G2/M arrest was dependent on wild-type p53.
- Overexpression of cyclin B1 and Cdc25C inhibited the Gadd45-mediated G2/M arrest.
- Reduced Gadd45 expression impaired the G2/M checkpoint in response to UV and methyl methanesulfonate, but not ionizing radiation.
- Gadd45-deficient cells showed impaired G2/M arrest after UV exposure but retained checkpoint function after ionizing radiation.
Conclusions:
- Gadd45 plays a significant role in mediating the G2/M cell cycle checkpoint in mammalian cells.
- The Gadd45-mediated G2/M checkpoint is partially regulated by modulating cyclin B1/p34(cdc2) activity and is dependent on p53.
- Mammalian cells possess multiple G2/M checkpoints that respond to specific types of DNA damage, highlighting a complex network for maintaining genomic integrity.