IntroductionFanconi anemia (FA) is a genetic disease characterized by genomic instability and cancer predisposition. [1][2][3] Patients with FA have a pro-oxidant state that is associated with overproduction or impaired detoxification of reactive oxygen species (ROS). [4][5][6] As a consequence, cells from FA patients demonstrate hypersensitivity to ambient oxygen and increased chromosomal aberrations. 7-9 FA oxidant hypersensitivity has been documented in many studies using primary and immortalized cell cultures as well as ex vivo materials from patients. [4][5][6][7][8][9][10][11] Significant evidence also suggests that excessive apoptosis of hematopoietic stem/progenitor cells induced by oxidative stress may be a critical factor in the pathogenesis of bone marrow failure and cancer progression in FA. 5,12 Mammalian forkhead members of the class O (FOXO) transcription factors, including FOXO1, FOXO3a, FOXO4, and FOXO6, are implicated in the regulation of diverse physiologic processes, including cell-cycle arrest, apoptosis, DNA repair, stress resistance, and metabolism. 13,14 Among these FOXO proteins, FOXO3a functions as a major regulator of oxidative stress. 14,15 In this study, we identified a novel oxidative stress response pathway that converges Fanconi anemia complementation group D2 (FANCD2) and FOXO3a. Methods Cell culture and treatmentsHuman lymphoblast cell lines JY (normal), HSC72 (FA-A), HSC536 (FA-C), and PD20 (FA-D2) were cultured in 10% fetal bovine serum RPMI 1640 medium. HeLa cells were cultured in Dulbecco modified Eagle medium containing 10% fetal bovine serum. Cells were treated with H 2 O 2 (0.5mM for 6 hours), ionizing radiation (IR; 5 Gy), or mitomycin C (MMC; 0.5M for 18 hours). ConstructsThe retroviral vectors encoding human pMMP-Puro, pMMP-wt-FANCD2, and pMMP-K561R-FANCD2 were generously provided by Dr Alan D'Andrea (Harvard Medical School, Boston, MA). The cDNA encoding the human FOXO3a was from Addgene Plasmid 8360 (Addgene). The retroviral vectors MIEG3, MIEG3-FANCA, and MIEG3-FANCC have been described elsewhere. 16 The Flag-tagged FOXO3a was generated by polymerase chain reaction (PCR) using primers 5Ј-GGG GGA TCC ACC ATG GAT GGA CTA CAA GGA CGA TGA CGA TAA ACC-3Ј (forward) and 5Ј-CCT CTA GAT CAG CCT GGC ACC CAG CTC TGA GAT G-3Ј (reverse), and subcloned into the BamHI-XbaI of Lenti-X mammalian inducible vector (Clontech). ROS productionCells were incubated with CM-H 2 DCFDA (Invitrogen) in the dark for 15 minutes at 37°C. After washing, cells were analyzed by flow cytometry using a FACSCanto (BD Biosciences). Data were analyzed using the CellQuest program (BD Biosciences). 17 Real-time PCRTotal RNA was prepared with RNeasy kit (QIAGEN) following the manufacturer's procedures. After treatment with RNase-free DNase, RNA was reverse transcribed using Superscript II reverse transcriptase (Invitrogen). Real-time PCR was performed on an ABI PRISM 7700 sequence detection system (Applied Biosystems) with SYBR green PCR master mix (Applied Biosystems), according to the manufacturer's instr...
1089 Poster Board I-111 Fanconi anemia (FA) is a human genomic instability syndrome that is uniquely sensitive to oxidative stress. Members of the FA protein family are involved in repair of genetic damage caused by DNA cross-linkers. The molecular pathway in which the FA proteins function in oxidative stress response has not been defined. Here we report functional interaction between the FA protein FANCD2 and the forkhead transcription factor FOXO3a in response to oxidative stress. FOXO3a was colocalized to FANCD2 foci in cells subjected to oxidative stress. The FANCD2-FOXO3a complex was not detected in cells deficient for the FA core complex component FANCA, but could be restored after complementation with a functional FANCA. Consistent with this, a non-monoubiquitinated FANCD2 mutant failed to bind FOXO3a. While both DNA cross-linker mitomycin C and ionizing radiation induced monoubiquitination of FANCD2, neither was able to induce the association of FANCD2 and FOXO3a. This indicates that the FOXO3a-FANCD2 interaction is oxidative stress specific. Overexpression of FOXO3a reduced abnormal accumulation of reactive oxygen species, enhanced cellular resistance to oxidative stress, and increased antioxidant gene expression in corrected but not mutant FA-D2 cells. The novel oxidative stress response pathway converging FANCD2 and FOXO3a identified in this study is likely to contribute to cellular anti-oxidant defense. Disclosures No relevant conflicts of interest to declare.
3098 Poster Board III-35 In a subset of AML with a normal karyotype, a frame-shift mutation in the extreme C-terminal of the nucleophosmin (NPM) gene results in the creation of a nuclear export signal, generating a mutant NPM protein (NPMc) that is permanently dislocated in the cytoplasm. In the present study, we have analyzed the interaction between NPMc and a cytoplasmic subcomplex of Fanconi anemia (FA) proteins. Sequence analysis of bone marrow samples from 46 FA patients shows that NPMc mutations were excluded from FA genome. NPMc was degraded more rapidly in AML bone marrow cells from FA patients (t1/2 < 30 min) than in AML cell line HL60 (t1/2 > 90 min). Further analysis revealed that inducible knockdown of FANCA or FANCC in leukemic OCI/AML3 cells carrying the NPMc mutation induced degradation of the cytoplasmic NPMc protein. Forced localization of FANCC to the nucleus also caused rapid NPMc degradation. We also show that NPMc degradation was mediated by the proteasome and that correction of mutant lymphoblasts from FA-A or FA-C patients with a functional FANCA or FANCC protein prevented NPMc ubiquitination and consequently degradation. Moreover, we demonstrate that the cytoplasmic FANCA and FANCC interacted with NPMc in the cytosolic fractions of normal human lymphoblaststic cells and that the acidic domains of NPM were required for the cytoplasmic FA-NPMc complex formation. Finally, using patient-derived FANCC mutant, a nuclearized FANCC and a NOG/SGM3 xenotansplant model, we present evidence that the cytoplasmic FANCA-FANCC complex was essential for NPMc stability and biological function. Thus, these findings reveal the potential molecular mechanism involved in the cytoplasmic retention of the leukemic NPMc. Disclosures No relevant conflicts of interest to declare.
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