GADD45 induction of a G ₂ /M cell cycle checkpoint

Abstract

G 1 ͞S and G 2 ͞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 G 2 ͞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 G 2 ͞M boundary with a phenotype of MPM2 immunopositivity, 4n DNA content and, in 15% of the cells, centrosome separation. The Gadd45-mediated G 2 ͞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 G 2 ͞M arrest in human fibroblasts, indicating that the mechanism of Gadd45-mediated G 2 ͞M checkpoint is at least in part through modulation of the activity of the G 2 -specific kinase, cyclin B1͞p34 cdc2 . Genetic and physiological evidence of a Gadd45-mediated G 2 ͞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 G 2 ͞M checkpoint after exposure to either ultraviolet radiation or methyl methanesulfonate but are still able to undergo G 2 arrest after ionizing radiation. Lymphocytes from gadd45-knockout mice (gadd45 ؊͞؊) also retained a G 2 ͞M checkpoint initiated by ionizing radiation and failed to arrest at G 2 ͞M after exposure to ultraviolet radiation. Therefore, the mammalian genome is protected by a multiplicity of G 2 ͞M checkpoints in response to specific types of DNA damage.Mammalian cells have evolved an intricate defense network to maintain genomic integrity by preventing the fixation of permanent damage from endogenous and exogenous mutagens. Cellcycle checkpoints, a major genomic surveillance mechanism, exist at the G 1 ͞S and G 2 ͞M transitions that are regulated in response to DNA damage (1). Defects in these steps may result in a mutator phenotype that is associated with tumorigenesis.Tumor suppressor gene product p53 is implicated to be one of the essential components of cell-cycle checkpoints (2-5). p53 is a transcription factor that up-regulates a number of important cell cycle-modulating genes, including p21 WAF1͞CIP1͞SDI1

Gadd45 in the response of hematopoietic cells to genotoxic stress

“…In addition, microinjecting a gadd45a expression vector into primary human fibroblasts arrested the cells at the G2/M boundary of the cell cycle (21). Given that gadd45a is a transcriptional target to p53 activation, these observations implicate Gadd45a in p53 mediated G2/M cell cycle arrest in response to certain genotoxic stress agents (21). The genomic instability observed in gadd45a null mice (22) may reflect perturbations in G2/M cell cycle progression &/or impaired DNA repair.…”

“…Cell cycle arrest-In human endothelial & fibroblast cells inhibiting endogenous Gadd45 expression by antisense gadd45 impaired the G2/M checkpoint following exposure to either UV radiation or MMS (2). In addition, microinjecting a gadd45a expression vector into primary human fibroblasts arrested the cells at the G2/M boundary of the cell cycle (21). Given that gadd45a is a transcriptional target to p53 activation, these observations implicate Gadd45a in p53 mediated G2/M cell cycle arrest in response to certain genotoxic stress agents (21).…”

Molecular Responses of Human Lung Epithelial Cells to the Toxicity of Copper Oxide Nanoparticles Inferred from Whole Genome Expression Analysis

“…GADD45B and GADD45G are members of the GADD45 family of proteins that are induced by genotoxic stresses and various apoptotic cytokines [11][12][13] and are involved in cell cycle arrest, [14][15][16][17][18][19] DNA repair, [20][21][22] cell survival, [22][23][24][25][26][27] and apoptosis. These functions are mediated by proliferating cell nuclear antigen (PCNA), cell division control 2 (CDC2), cyclin B1 (CCNB1), and cyclin-dependent kinase inhibitor 1A (CDKN1A; also known as p21), which are classified in the GO "cell cycle" category.…”

Hematopoietic cells from gadd45a-deficient and gadd45b-deficient mice exhibit impaired stress responses to acute stimulation with cytokines, myeloablation and inflammation

“…Overall, Gadd45a, Gadd45b and Gadd45g have been implicated in cell cycle arrest (Beadling et al, 1993;Hollander et al, 1999;Wang et al, 1999;Zhang et al, 1999), DNA repair (Smith et al, 1994;Vairapandi et al, 1996), apoptosis (Harkin et al, 1999;Vairapandi et al, 2000;Azam et al, 2001;Takekawa et al, 2002;Yoo et al, 2003), cell survival (Smith et al, 1996;De Smaele et al, 2001;Zazzeroni et al, 2003;Papa et al, 2004;Gupta et al, 2005), genomic stability (Hollander et al, 1999), inflammatory response and innate immunity (Lu et al, 2001;Yang et al, 2001;Lu et al 2004). Gadd45 proteins have been shown to stimulate the p38-c-Jun N-terminal kinase (JNK) mitogen-activated protein (MAP) kinase pathways in response to stress situations and thereby sensitize cells for growth arrest, apoptosis or survival (Takekawa and Saito, 1998;Harkin et al, 1999;De Smaele et al, 2001;Lu et al, 2001;Hildesheim et al, 2002;Yoo et al, 2003).…”