Genetic disruption of both Fancc and Fancg in mice recapitulates the hematopoietic manifestations of Fanconi anemia.

Fanconi anemia (FA) is an inherited chromosomal instability syndrome characterized by bone marrow failure, myelodysplasia (MDS), and acute myeloid leukemia (AML). Eight FA proteins associate in a nuclear core complex to monoubiquitinate FANCD2/FANCI in response to DNA damage. Additional functions have been described for some of the core complex proteins; however, in vivo genetic proof has been lacking. Here we show that double-mutant Fancc(-/-);Fancg(-/-) mice develop spontaneous hematologic sequelae including bone marrow failure, AML, MDS and complex random chromosomal abnormalities that the single-mutant mice do not. This genetic model provides evidence for unique core complex protein function independent of their ability to monoubiquitinate FANCD2/FANCI. Importantly, this model closely recapitulates the phenotypes found in FA patients and may be useful as a preclinical platform to evaluate the molecular pathogenesis of spontaneous bone marrow failure, MDS and AML in FA.

AMD3100 synergizes with G-CSF to mobilize repopulating stem cells in Fanconi anemia knockout mice.

Fanconi anemia (FA) is a heterogeneous inherited disorder characterized by a progressive bone marrow (BM) failure and susceptibility to myeloid leukemia. Genetic correction using gene-transfer technology is one potential therapy. A major hurdle in applying this technology in FA patients is the inability of granulocyte colony-stimulating factor (G-CSF) to mobilize sufficient numbers of hematopoietic stem (HSC)/progenitor cells (HPC) from the BM to the peripheral blood. Whether the low number of CD34(+) cells is a result of BM hypoplasia or an inability of G-CSF to adequately mobilize FA HSC/HPC remains incompletely understood. Here we use competitive repopulation of lethally irradiated primary and secondary recipients to show that in two murine models of FA, AMD3100 synergizes with G-CSF resulting in a mobilization of HSC, whereas G-CSF alone fails to mobilize stem cells even in the absence of hypoplasia.

An in vivo analysis of MMC-induced DNA damage and its repair.

Mitomycin C (MMC) induces various types of DNA damages that cause significant cytotoxicity to cells. Accordingly, repair of MMC-induced damages involves multiple repair pathways such as nucleotide excision repair, homologous recombination repair and translesion bypass repair pathways. Nonetheless, repair of the MMC-induced DNA damages in mammals have not been fully delineated. In this study, we investigated potential roles for Xeroderma pigmentosum (XP) proteins in the repair of MMC-induced DNA damages using an assay that detects the ssDNA patches generated following treatment with MMC or 8'-methoxy-psoralen (8-MOP) + UVA (ultraviolet light A). Human wild-type cells formed distinctive ssDNA foci following treatment with MMC or 8-MOP + UVA, but not with those inducing alkylation damage, oxidative damage or strand-break damage, suggesting that the foci represent ssDNA patches formed during the crosslink repair. In contrast to wild-type cells, mutant defective in XPE orXPG did not form the ssDNA foci following MMC treatment, while XPF mutant cells showed a significantly delayed response in forming the foci. A positive role for XPG in the repair of MMC-induced DNA damages was further supported by observations that cells treated with MMC induced a tight association of XPG with chromatin, and a targeted inhibition of XPG abolished MMC-induced ssDNA foci formation, rendering cells hypersensitive to MMC. Together, our results suggest that XPG along with XPE and XPF play unique role(s) in the repair of MMC-induced DNA damages.

A role for the Fanconi anemia C protein in maintaining the DNA damage-induced G2 checkpoint.

Fanconi anemia (FA) is a complex, heterogeneous genetic disorder composed of at least 11 complementation groups. The FA proteins have recently been found to functionally interact with the cell cycle regulatory proteins ATM and BRCA1; however, the function of the FA proteins in cell cycle control remains incompletely understood. Here we show that the Fanconi anemia complementation group C protein (Fancc) is necessary for proper function of the DNA damage-induced G2/M checkpoint in vitro and in vivo. Despite apparently normal induction of the G2/M checkpoint after ionizing radiation, murine and human cells lacking functional FANCC did not maintain the G2 checkpoint as compared with wild-type cells. The increased rate of mitotic entry seen in Fancc-/-mouse embryo fibroblasts correlated with decreased inhibitory phosphorylation of cdc2 kinase on tyrosine 15. An increased inability to maintain the DNA damage-induced G2 checkpoint was observed in Fancc -/-; Trp53 -/-cells compared with Fancc -/-cells, indicating that Fancc and p53 cooperated to maintain the G2 checkpoint. In contrast, genetic disruption of both Fancc and Atm did not cooperate in the G2 checkpoint. These data indicate that Fancc and p53 in separate pathways converge to regulate the G2 checkpoint. Finally, fibroblasts lacking FANCD2 were found to have a G2 checkpoint phenotype similar to FANCC-deficient cells, indicating that FANCD2, which is activated by the FA complex, was also required to maintain the G2 checkpoint. Because a proper checkpoint function is critical for the maintenance of genomic stability and is intricately related to the function and integrity of the DNA repair process, these data have implications in understanding both the function of FA proteins and the mechanism of genomic instability in FA.

Oxidative stress/damage induces multimerization and interaction of Fanconi anemia proteins.

Fanconi anemia (FANC) is a heterogeneous genetic disorder characterized by a hypersensitivity to DNA-damaging agents, chromosomal instability, and defective DNA repair. Eight FANC genes have been identified so far, and five of them (FANCA, -C, -E, -F, and -G) assemble in a multinuclear complex and function at least in part in a complex to activate FANCD2 by monoubiquitination. Here we show that FANCA and FANCG are redox-sensitive proteins that are multimerized and/or form a nuclear complex in response to oxidative stress/damage. Both FANCA and FANCG proteins exist as monomers under non-oxidizing conditions, whereas they become multimers following H2O2 treatment. Treatment of cells with oxidizing agent not only triggers the multimeric complex of FANCA and FANCG in vivo but also induces the interaction between FANCA and FANCG. N-Ethylmaleimide treatment abolishes multimerization and interaction of FANCA and FANCG in vitro. Taken together, our results lead us to conclude that FANCA and FANCG uniquely respond to oxidative damage by forming complex(es) via intermolecular disulfide linkage(s), which may be crucial in forming such complexes and in determining their function.

A positive role for the Ku complex in DNA replication following strand break damage in mammals.

Ku70-Ku80 complex is the regulatory subunit of DNA-dependent protein kinase (DNA-PK) and plays an essential role in double-strand break repair following ionizing radiation (IR). It preferentially interacts with chromosomal breaks and protects DNA ends from nuclease attack. Here we show evidence that cells defective in Ku80 exhibit a significantly slow S phase progression following DNA damage. IR-induced retardation in S phase progression in Ku80-/- cells was not due to the lack of DNA-PK kinase activity because both wild-type cells and DNA-PKcs-deficient cells showed no such symptom. Instead, proliferating cell nuclear antigen (PCNA) dissociated from chromosomes following IR in Ku80-deficient cells but not in wild-type or DNA-PKcs-deficient cells. Treatment of HeLa cells with IR induced colocalization of the Ku complex with PCNA on chromosomes. Together, these results suggest that binding of the Ku complex at chromosomal breaks may be necessary to maintain the sliding clamps (PCNA) on chromatin, which would allow cells to resume DNA replication without a major delay following IR.

Fanconi anemia type C and p53 cooperate in apoptosis and tumorigenesis.

Fanconi anemia (FA) is a recessive genomic instability syndrome characterized by developmental defects, progressive bone marrow failure, and cancer. FA is genetically heterogeneous, however; the proteins encoded by different FA loci interact functionally with each other and with the BRCA1, BRCA2, and ATM gene products. Although patients with FA are highly predisposed to the development of myeloid leukemia and solid tumors, the alterations in biochemical pathways responsible for the progression of tumorigenesis in these patients remain unknown. FA cells are hypersensitive to a range of genotoxic and cellular stresses that activate signaling pathways mediating apoptosis. Here we show that ionizing radiation (IR) induces modestly elevated levels of p53 in cells from FA type C (Fancc) mutant mice and that inactivation of Trp53 rescues tumor necrosis factor alpha-induced apoptosis in myeloid cells from Fancc-/- mice. Further, whereas Fancc-/- mice failed to form hematopoietic or solid malignancies, mice mutant at both Fancc and Trp53 developed tumors more rapidly than mice mutant at Trp53 alone. This shortened latency was associated with the appearance of tumor types that are found in patients with FA but not in mice mutant at Trp53 only. Collectively, these data demonstrate that p53 and Fancc interact functionally to regulate apoptosis and tumorigenesis in Fancc-deficient cells.

Retroviral-mediated expression of recombinant Fancc enhances the repopulating ability of Fancc-/- hematopoietic stem cells and decreases the risk of clonal evolution.

Fanconi anemia (FA) is a chromosomal instability disorder characterized by a progressive bone marrow (BM) failure and an increased incidence of myeloid leukemias. Children with FA are currently being enrolled in clinical trials to evaluate the safety of retroviral-mediated gene transfer. Previously, we used Fancc(-/-) mice to show that Fancc(-/-) hematopoietic stem cells (HSCs) have a profound defect in repopulating ability. Here, we examined whether retroviral-mediated gene transfer of recombinant Fancc (rFancc) would restore the repopulating ability of Fancc(-/-) HSC to wild-type levels. Fancc(-/-) HSCs transduced with a retrovirus encoding rFancc exhibited a repopulating ability that approached wild-type levels. Interestingly, approximately 30% of primary recipients (7 of 22) transplanted with uncorrected Fancc(-/-) cells developed a range of hematopoietic abnormalities including pancytopenia and BM hypoplasia similar to individuals with FA. Hematopoietic abnormalities were detected in only 1 of 22 mice transplanted with Fancc(-/-) cells transduced with a retrovirus encoding rFancc. Moreover, several mice with hematopoietic defects had progenitors that displayed a marked resistance to IFN-gamma, TNF-alpha, and MIP-1alpha compared to both Fancc(-/-) progenitors, which are uniquely hypersensitive to these cytokines, and wild-type progenitors. These data are analogous to studies using progenitors from patients with myelodysplasia and provide functional support for clonal evolution in these mice. Collectively, these data show that gene transfer can enhance HSC repopulating ability and suppresses the tendency for clonal evolution. These studies also reveal potential detrimental effects of ex vivo manipulation for untransduced Fancc(-/-) HSCs.