GADD45 is a ubiquitously expressed mammalian gene that is induced by DNA damage and certain other stresses. Like another p53-regulated gene, p21WAF1/CIP1, whose product binds to cyclin-dependent kinases (Cdk's) and proliferating cell nuclear antigen (PCNA), GADD45 has been associated with growth suppression. Gadd45 was found to bind to PCNA, a normal component of Cdk complexes and a protein involved in DNA replication and repair. Gadd45 stimulated DNA excision repair in vitro and inhibited entry of cells into S phase. These results establish GADD45 as a link between the p53-dependent cell cycle checkpoint and DNA repair.
Exposure of mammalian cells to ionizing radiation (IR) induces a complex array of cellular responses including cell cycle arrest and͞or apoptosis. IR-induced G 1 arrest has been shown to depend on the presence of the tumor suppressor p53, which acts as a transcriptional activator of several genes. p53 also plays a role in the induction of apoptosis in response to DNA damage, and this pathway can be activated by both transcription-dependent and -independent mechanisms. Here we report the identification of a novel transcript whose expression is induced in response to IR in a p53-dependent manner, and that shows homology to the type 2C protein phosphatases. We have named this novel gene, wip1. In vitro, recombinant Wip1 displayed characteristics of a type 2C phosphatase, including Mg 2؉ dependence and relative insensitivity to okadaic acid. Studies performed in several cell lines revealed that wip1 accumulation following IR correlates with the presence of wild-type p53. The accumulation of wip1 mRNA following IR was rapid and transient, and the protein was localized to the nucleus. Similar to waf1, ectopic expression of wip1 in human cells suppressed colony formation. These results suggest that Wip1 might contribute to growth inhibitory pathways activated in response to DNA damage in a p53-dependent manner.
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
We have studied several aspects of DNA damage formation and repair in human ovarian cancer cell lines which have become resistant to cisplatin through continued exposure to the anticancer drug. The Cells in culture were treated with cisplatin, and the two main DNA lesions formed, intrastrand adducts and interstrand cross-links, were quantitated before and after repair incubation. This quantitation was done for total genomic lesions and at the level of individual genes. In the overall genome, the initial frequency of both cisplatin lesions assayed was higher in the parental than in the derivative resistant cell lines. Nonetheless, the total genomic repair of each of these lesions was not increased in the resistant cells. These differences in initial lesion frequency between parental and resistant cell lines were not observed at the gene level. Resistant and parental cells had similar initial frequencies of intrastrand adducts and interstrand cross-links in the dihydrofolate reductase (DHFR) gene and in several other genes after cisplatin treatment of the cells. There was no increase in the repair efficiency of intrastrand adducts in the DHFR gene in resistant cell lines compared with the parental partners. However, a marked and consistent repair difference between parental and resistant cells was observed for the gene-specific repair of cisplatin interstrand cross-links. DNA interstrand cross-links were removed from three genes, the DHFR, multidrug resistance (MDR1), and b-globin genes, much more efficiently in the resistant cell lines than in the parental cell lines. Our findings suggest that acquired cellular resistance to cisplatin may be associated with increased gene-specific DNA repair efficiency of a specific lesion, the interstrand cross-link.The metal coordination complex cisplatin has been widely used in the treatment of ovarian, testicular, head and neck, bladder, and small-cell lung carcinomas (19). DNA is thought to be the most critical target for the antitumor action of cisplatin, although the compound can also react with other biologically important molecules such as RNA and proteins (25, 29). The major lesions produced by cisplatin are DNA intrastrand adducts (IA) between adjacent purines GpG or ApG. Minor lesions include IA between two guanine residues separated by one nucleotide residue (GpXpG), interstrand cross-links (ICL) between two adjacent guanine residues on opposite DNA strands, and DNA-protein crosslinks (13,27). The lesions of most therapeutic significance are thought to be IA and ICL.Platinum-based chemotherapy is the standard treatment in the initial therapy of advanced-stage ovarian cancer. Although up to 80% of patients will have an early favorable response to therapy, less than 20% of individuals will experience long-term disease-free survival (33). As a result of primary or acquired resistance, most ovarian cancer patients will eventually relapse with cisplatin-resistant disease (5). Attempts to overcome drug resistance are central to both clinical and basic molecular research ...
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