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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: 10.75). 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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Available from: Massimo Bogliolo, Sep 01, 2015
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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.
    Brazilian journal of medical and biological research = Revista brasileira de pesquisas medicas e biologicas / Sociedade Brasileira de Biofisica ... [et al.] 12/2013; DOI:10.1590/1414-431X20132938 · 1.08 Impact Factor
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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.
    Journal of Breast Cancer 09/2013; 16(3):291-9. DOI:10.4048/jbc.2013.16.3.291 · 1.32 Impact Factor
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    • "The main functions of the FA/BRCA pathway involve the cell cycle, DNA damage and repair, apoptosis, gene transcription and gene stability. Moreover, this pathway is necessary for cells to respond to DNA damage caused by IR, mitoxantrone (MX), CDDP and ADM (26,27). In the present study, we found that FANCF silencing inhibited proliferation, induced cell apoptosis and DNA damage in OVCAR3 cells, indicating that the function of the FA/BRCA pathway was blocked. "
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    ABSTRACT: In the present study, we downregulated FANCF expression by small interfering RNA (siRNA) in OVCAR ovarian cancer cells to address the effects of decreased FANCF expression on the function of the Fanconi anemia (FA)/breast cancer susceptibility gene (BRCA) pathway. Furthermore, we investigated whether this method increases the sensitivity of OVCAR3 cells to adriamycin (ADM) and the possible mechanism(s). We found that silencing of FANCF inactivated the FA/BRCA pathway by decreasing the monoubiquitination and focus formation of FANCD2 and reduced the function of the FA/BRCA pathway, resulting in the inhibition of cell proliferation, increased cell apoptosis and DNA damage in OVCAR3 cells. Moreover, we observed that silencing of FANCF enhanced the antiproliferative effect of ADM in OVCAR3 cells and increased ADM intracellular accumulation consequently sensitizing OVCAR3 cells to ADM. Furthermore, silencing of FANCF increased cell apoptosis of OVCAR3 cells which was caused by decreased mitochondrial membrane potential (MMP)-induced DNA damage, activated Jun N-terminal kinase (JNK), increased release of cytochrome c, increased expression of cleaved caspase-3 and poly(ADP-ribose) polymerase (PARP) dependent on JNK activation following treatment of ADM. Collectively, we confirm that silencing of FANCF sensitizes OVCAR3 ovarian cancer cells to ADM, suggesting that FANCF may serve as a potential target for therapeutic strategies in the treatment of ovarian cancer.
    Oncology Reports 02/2013; 29(5). DOI:10.3892/or.2013.2295 · 2.19 Impact Factor
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