Abstract LB-102: The p21Cip1/Waf1 cyclin-dependent kinase inhibitor is required for the activation of the FA-BRCA pathway

Fanconi anemia (FA) is a rare recessive disorder characterized in part by pronounced cancer susceptibility. The FA proteins, and the protein products of the major breast cancer susceptibility genes BRCA1 and BRCA2, function cooperatively in the FA-BRCA pathway, to repair damaged DNA and to prevent cellular transformation. A major step in the activation of the FA-BRCA pathway is the mono-ubiquitination of the FANCD2 and FANCI proteins, targeting these proteins to discrete chromatin-associated nuclear foci. Importantly, the regulation of FANCD2 ( and FANCI ) mono-ubiquitination remains poorly understood. To gain further insight into this important post-translational modification step, here we have examined the roles of p53 tumor suppressor as well its downstream target p21 Ci p 1/Waf1 in the regulation of the mono-ubiquitination of FANCD2. In this study we have used the isogenic HCT116 p53 +/+ , p53 −/− , p21 +/+ , and p21 −/− cell lines, to determine that p21, and not p53, plays a key role in the regulation of the mono-ubiquitination of FANCD2 following exposure to DNA damaging agents. For example, robust FANCD2 mono-ubiquitination is observed in HCT116 p21 +/+ cells 2 h following exposure to 20 J/m 2 UV irradiation, while this effect is markedly attenuated in p21 −/− cells. Using immunofluorescence microscopy we also demonstrate that p21 is necessary for the efficient recruitment of FANCD2 to nuclear foci following exposure to UV irradiation. Furthermore, preliminary protein-protein interaction analyses in COS-7 cells demonstrate that FLAG-p21 and 6xHis/V5-FANCD2 physically interact. Interestingly, despite a failure to efficiently activate the mono-ubiquitination of FANCD2 following exposure to DNA damaging agents, p21 −/− cells are not hypersensitive to the cytotoxic effects of the DNA crosslinking agent mitomycin C (MMC). However, elevated levels of distinct chromosome aberrations, including telomere associations and dicentric chromosomes, are observed in p21 −/− cells compared with p21 +/+ cells following exposure to MMC. Taken together, our results demonstrate an important upstream role for the p21 cyclin-dependent-kinase inhibitor in the activation of the FA-BRCA pathway. A greater understanding of the regulation of this important tumor suppressor pathway will lead to improved diagnostic and therapeutic approaches to FA and enhance our understanding of the elevated cancer susceptibility of FA patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-102.