Mammalian cells have evolved an intricate defense network to maintain genomic fidelity by preventing the fixation of permanent damage from genotoxic stress. Cell cycle checkpoint, a major genomic surveillance mechanism, is governed by a series of control systems and their inactivation may result in dramatic consequences on genomic stability and therapeutic sensitivity. In contrast to G1 checkpoint, the control of mammalian G2-M cell cycle checkpoint after DNA damage is poorly understood. We have previously reported that expression of GADD45, a p53-regulated and stress-inducible gene, is able to suppress cell growth although the mechanism by which GADD45-induced growth suppression remains unclear. Recent findings have demonstrated that over-expression of GADD45 in normal fibroblast via a microinjection approach caused cells to arrest in an early mitotic phase. The evidence includes that following microinjection with GADD45 expression vectors, the cells displayed a completely rounded shape, positive staining with the mitotic- specific marker (MPM2, Ab), a 4N amount of DNA and a single centrosome Also in agreement with these results, an increased level of GADD45 protein expression after transient transfection was shown to result in a higher G2/M fraction in human cells. These results raise the possibility that GADD45 may participate in the control of G2-M arrest in response to DNA damage. Therefore, the long- term objective described in this application will focus on three key issues: (1). To determine the role of GADD45 in the G2-M cell cycle checkpoint after genotoxic stress. (2) To define the molecular and biochemical mechanism(s) by which GADD45 plays a role in the G2-M checkpoint. Our hypothesis are as follows: (1). Cells with disrupted endogenous GADD45 will exhibit a perturbed G2-M delay following DNA damage. (2). In order to activate the machinery of G2-M transition, Gadd45 protein may target the Cdc2/cyclin B1 and Cdc2/cyclin A complexes, which """"""""drive"""""""" cells from G2 to mitosis. (3). The capability of GADD45 on the control of G2-M checkpoint will contribute to the GADD45-induced growth suppression. Experimental techniques will include flow cytometric analysis, mitotic index assay, Cdc2 kinase assay, immunoblotting, construction of recombinant Gadd45 deletion proteins, cell survival assay and microinjection. The studies proposed in this application would define a new pathway controlling G2-M cell cycle checkpoint after certain DNA damage as well as determine the mechanism(s) by which GADD45 exerts its role in the negative control of cell growth. In addition, these studies would be helpful in the elucidation of how G2-M checkpoint affects therapeutic sensitivity and might provide the insights into the development of novel anti-cancer agents.
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| Jin, Shunqian; Tong, Tong; Fan, Wenhong et al. (2002) GADD45-induced cell cycle G2-M arrest associates with altered subcellular distribution of cyclin B1 and is independent of p38 kinase activity. Oncogene 21:8696-704 |
| Jin, S; Antinore, M J; Lung, F D et al. (2000) The GADD45 inhibition of Cdc2 kinase correlates with GADD45-mediated growth suppression. J Biol Chem 275:16602-8 |
