GADD45 PLAYS A ROLE IN P53-DEPENDENT G2/M CHECKPOINT CONTROL

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.