A major focus of this research group is the study of the cellular responses to genotoxic and related stresses in mammalian cells. This has included the cloning and characterization of a variety of DNA-damage-inducible (DDI) genes, including the gadd genes, and elucidation of the regulatory mechanisms controlling their expression. Cell-cycle checkpoint activation and growth inhibition are universal responses to genotoxic stress. We have found that the 5 gadd (growth-arrest and DNA-damage inducible) genes to be coordinately induced by cellular exposure to many DNA-damaging agents and certain other stresses that trigger growth arrest (Mol. Cell. Biol. 1989; 9:4196). Evidence for such responses have been found in all mammalian cells examined to date and indicate that this is a well-conserved stress response(s). In the case of Gadd45a, this was the first cellular gene found to be regulated by the key tumor suppressor p53 via a pathway that is activated by ionizing radiation (and many other stresses). This pathway also involves ATM, the gene defective in ataxia telangiectasia (Mol. Cell. Biol. 1991; 11:1009; Cell 1992; 71:587). Responses to ionizing radiation have been characterized in a variety of human tumor lines including the regulation of key cell death genes like BAX, BCL2, BCL-X, KILLER/DR5 , TRID/TRAIL-R3 (Oncogene 1998; 17:3287). This laboratory has shown that a functional and physical interaction between p53 and WT1 plays a central role in the regulation of such genes after certain stresses, such as UV radiation (Mol. Cell. Biol. 1998; 18:2768). Interestingly, the same promoter control element, which binds to WT1, has been found to be suppressed by c-Myc, a growth-stimulatory signaling protein (Oncogene 1998; 17:2149). Moreover, deletion of c-Myc has been found to markedly upregulate the Gadd45a growth-arrest gene. Both c-myc and Brca1 are implicated in breast cancer, and have been shown to contribute to the regulation of the Gadd45a gene. We have also shown that the p38 kinases of the MAP kinase pathway have an important role in activation of p53 after certain stresses (EMBO J. 1999; 18:6845). We have gone on to show that p38 plays a central role in G2 checkpoint activation (Nature 2001; 411:102), regulation of the key cell cycle Cdc25 proteins (Nature Cell Biology 2003; 5:545), and tumor suppression (Nature Genetics 2002; 31:210; 2004; 36:343). We have applied a functional genomics approach to the study of DDI genes. Using cDNA microarray hybridization, a very complex pattern of responses has been found in various human cells which is dependent on p53 status, apoptotic potential, and a variety of other control factors (Oncogene 1999; 18:3666; Oncogene 2003; 22:5828; Cancer Research 2004; 64: 6368). Characterization of such responses in tumor cells will be used to elucidate the status of signal transduction pathways and may have predictive value in treatment planning.A second major focus is the characterization of the products encoded by particular stress genes with emphasis on p53-regulated genes. This project involves both a genetic and biochemical approach. Targeted disruption of the Gadd45a gene has been carried out in mice and characterization of these mice is currently underway. Studies are being expanded to other engineered mice including strains with disruption of p53, cip1waf1 (Cdkna1), Gadd45b, Gadd45g, Gadd34, and other selected genes. Defects in important parameters, such as genomic stability, growth control, resistance to carcinogenesis, and DNA repair, have been found in Gadd45a-/- mice (Nature Genetics 1999; 23:176) (Mol. Cell. Biol. 2003; 23,3859). From analysis of these various knockout strains, cip1/waf1, gadd45, and other effector genes contribute to the phenotype of p53-/- mice. Targeted disruption of related DDI genes is currently underway. Using a biochemical approach, this laboratory has already demonstrated interactions between Gadd45a with p38, PCNA, p21Cip1/Waf1, Cdc2, and core histone proteins, and evidence for roles in signal transduction, immune regulation, DNA repair and cell cycle control. The goal of these studies is to contribute to the understanding of the function of these and related DDI genes, and their potential as targets for cancer therapy in the future.More details on this project and a complete bibliography can be found at http://rex.nci.nih.gov/RESEARCH/basic/lbc/fornace.htm
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