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Bogliolo M, Lyakhovich A, Callen E et al.Histone H2AX and Fanconi anemia FANCD2 function in the same pathway to maintain chromosome stability. EMBO J 26:1340-1351

Group of Mutagenesis, Department of Genetics and Microbiology, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, Spain.
The EMBO Journal (Impact Factor: 10.43). 04/2007; 26(5):1340-51. DOI: 10.1038/sj.emboj.7601574
Source: PubMed

ABSTRACT

Fanconi anemia (FA) is a chromosome fragility syndrome characterized by bone marrow failure and cancer susceptibility. The central FA protein FANCD2 is known to relocate to chromatin upon DNA damage in a poorly understood process. Here, we have induced subnuclear accumulation of DNA damage to prove that histone H2AX is a novel component of the FA/BRCA pathway in response to stalled replication forks. Analyses of cells from H2AX knockout mice or expressing a nonphosphorylable H2AX (H2AX(S136A/S139A)) indicate that phosphorylated H2AX (gammaH2AX) is required for recruiting FANCD2 to chromatin at stalled replication forks. FANCD2 binding to gammaH2AX is BRCA1-dependent and cells deficient or depleted of H2AX show an FA-like phenotype, including an excess of chromatid-type chromosomal aberrations and hypersensitivity to MMC. This MMC hypersensitivity of H2AX-deficient cells is not further increased by depleting FANCD2, indicating that H2AX and FANCD2 function in the same pathway in response to DNA damage-induced replication blockage. Consequently, histone H2AX is functionally connected to the FA/BRCA pathway to resolve stalled replication forks and prevent chromosome instability.

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    • "In turn, FANCD2 is also phosphorylated at S222 by ATM in response to IR and oxidative stress and this step is required for a proper S phase checkpoint activation [3] [4]. Both FANCD2 and FANCI are then monoubiquitinated by the upstream FA core complex (formed by FANCA, B, C, D1, E, F, G, M and the E3 ubiquitin ligase FANCL) and targeted to chromatin in nuclear foci at the sites of stalled replication forks in a BRCA1 dependent manner [5]. FANCD2 is involved in resolving stalled replication forks by coordinating a complex mechanism that involves three classic DNA repair pathways: translesion synthesis (TLS), nucleotide excision repair (NER) and homologous recombination [22] [24]. "
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    ABSTRACT: Fanconi anemia (FA) is a rare, clinically heterogeneous autosomal recessive or X-linked genetic disease characterized by chromosome fragility, congenital malformations and cancer susceptibility. FA patients are usually radiosensitive when exposed to radiotherapy but the role of the FA in response to ionizing radiation (IR) is controversial. Here we have investigated IR-induced activation of the FA pathway by systematically analyzing monoubiquitination of the central protein FANCD2 and subsequent recruitment to stalled replication forks in primary fibroblasts. We developed an immunolabelling method to simultaneously visualize IR-induced FANCD2 and γH2AX foci in S-phase. We observed FANCD2 foci formation in a subset of IR-induced γH2AX foci in S-phase cells. This was observed at doses of IR ranging from 0.1 to 5.0 Gy in a dose dependent non-threshold fashion. Our results indicate that minimum doses of IR can produce replication fork stalling and FA pathway activation during S-phase in primary cells.
    Full-text · Article · Jun 2015 · Mutation Research/Genetic Toxicology and Environmental Mutagenesis
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    • "Fanconi anemia (FA) is a rare chromosome instability syndrome that predisposes to bone marrow failure, developmental abnormalities, and a high risk for the development of cancer, such as hematological malignancies, solid tumors of the head and neck region, and gynecological tumors (2-5). The FA protein is a multifunctional protein composed of 15 of the FA complementation groups (FANC A-C, D1, D2, E, F, G, I, J, L, M, N, O, and P) (6-8), and is involved in cell cycle, DNA damage and repair, apoptosis, gene transcription, and gene stability through common FA/breast cancer susceptibility gene (BRCA) cellular pathways (9). As an adaptor protein, FANCF interacts with the FANCC/FANCE subunit through its N-terminal, and with the FANCA/FANCG subunit through its C-terminal. "
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    ABSTRACT: Fanconi anemia complementation group F protein (FANCF) is a key factor, which maintains the function of FA/BRCA, a DNA damage response pathway. However, the functional role of FANCF in breast cancer has not been elucidated. We performed a specific FANCF-shRNA knockdown of endogenous FANCF in vitro. Cell viability was measured with a CCK-8 assay. DNA damage was assessed with an alkaline comet assay. Apoptosis, cell cycle, and drug accumulation were measured by flow cytometry. The expression levels of protein were determined by Western blot using specific antibodies. Based on these results, we used cell migration and invasion assays to demonstrate a crucial role for FANCF in those processes. FANCF shRNA effectively inhibited expression of FANCF. We found that proliferation of FANCF knockdown breast cancer cells (MCF-7 and MDA-MB-435S) was significantly inhibited, with cell cycle arrest in the S phase, induction of apoptosis, and DNA fragmentation. Inhibition of FANCF also resulted in decreased cell migration and invasion. In addition, FANCF knockdown enhanced sensitivity to doxorubicin in breast cancer cells. These results suggest that FANCF may be a potential target for molecular, therapeutic intervention in breast cancer.
    Full-text · Article · Dec 2013 · Brazilian journal of medical and biological research = Revista brasileira de pesquisas medicas e biologicas / Sociedade Brasileira de Biofisica ... [et al.]
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    • "Cells from patients with FA are particularly sensitive to DNA interstrand cross-link-inducing agents such as MMC and diepoxybutane [7]. The FA protein is a multifunctional protein composed of 15 FA complementation groups (FANC A-C, D1, D2, E, F, G, I, J, L, M, N, O, and P) [8], and is involved in the cell cycle, DNA damage and repair, apoptosis, gene transcription, and gene stability through common FA/breast cancer susceptibility gene (BRCA) cellular pathways [9]. In particular, the FA complex plays a critical role in the cell's response to chemotherapy-induced DNA damage. "
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    ABSTRACT: Fanconi anemia complementation group F (FANCF) is a key factor to maintaining the function of Fanconi anaemia/BRCA (FA/BRCA) pathway, a DNA-damage response pathway. However, the functional role of FANCF in breast cancer has not been elucidated. In the present study, we evaluated the chemosensitization effect of FANCF in breast cancer cells. We performed specific knockdown of the endogenous FANCF in breast cancer cells by transfecting the cells with an FANCF short hairpin RNA (shRNA) vector. Cell viability was measured with a Cell Counting Kit-8, and DNA damage was assessed with the alkaline comet assay. The apoptosis, cell cycle, and drug accumulation were measured by flow cytometric analysis. Protein expression levels were determined by Western blot analysis, using specific antibodies. The analyses of two breast cancer cell lines (MCF-7 and MDA-MB-435S) demonstrated that the FANCF shRNA could effectively block the FA/BRCA pathway through the inhibition of Fanconi anemia complementation group D2 ubiquitination. Moreover, FANCF silencing potentiated the sensitivity of cells to mitomycin C (MMC), where combined FANCF shRNA/MMC treatment inhibited cell proliferation, induced S-phase arrest, apoptosis, and DNA fragmentation, and reduced the mitochondrial membrane potential, compared with MMC treatment alone. Taken together, this study demonstrates that the inhibition of FANCF by its shRNA leads to a synergistic enhancement of MMC cytotoxicity in breast cancer cells. These results suggest that the inhibition of the FA/BRCA pathway is a useful adjunct to cytotoxic chemotherapy for the treatment of breast cancer.
    Full-text · Article · Sep 2013 · Journal of Breast Cancer
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