GADD45 PLAYS A ROLE IN P53-DEPENDENT G2/M CHECKPOINT CONTROL
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Date
1996-11
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Department
Hood College Biology
Program
Biomedical and Environmental Science
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Abstract
The G1/S and G2/M cell cycle checkpoints are important for
maintaining eukaryotic genomic stability in response to genotoxic stress.
The p53 tumor suppressor protein plays a central role in regulating both Gl/S
and G2/M checkpoints and also regulates programmed cell death (apoptosis)
in response to DNA damage. It is thought that p53 mediates these
pathways by acting as a transcription factor to transactivate downstream
target genes and by directly interacting with cellular proteins. While p53
regulation of the G1/S checkpoint and apoptosis have been extensively
studied, the mechanism of the p53-mediated G2/M checkpoint is poorly
understood. We report here that Gadd45, a member of the growth arrest
and DNA damage-inducible (GADD) gene family and a downstream target for
p53 transactivation, plays an important role in G2/M checkpoint regulation.
Overexpression of Gadd45 via microinjection of an expression vector into
primary normal human fibroblasts resulted in cell cycle arrest primarily at the
G2/M boundary, characterized by mitotic morphology, tetraploid DNA
content, and expression of mitotic epitopes. These arrested cells have not
formed a mitotic spindle and contain a single centrosome. The arrest is
dependent on wild-type p53, since no arrest was observed in either Li-
Fraumeni fibroblast cells that have homozygous loss of p53, or in normal
fibroblasts when Gadd45 was co-expressed with dominant negative p53
mutants. Flow cytometry analysis confirmed that Gadd45 induced a p53-
dependent G2/M arrest in a colon cancer cell line. Furthermore, cells with
diminished endogenous Gadd45 failed to arrest at G2/M following ultraviolet
light radiation or methyl methanesulfonate exposure. However, these cells
still retained a functional G2/M checkpoint following ionizing radiation.
These results indicate that Gadd45 plays an essential role in controlling at
least one of the G2/M checkpoints in response to DNA damage.