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Alan D Dandrea - One of the best experts on this subject based on the ideXlab platform.
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heterogeneous activation of the fanconi anemia pathway by patient derived fanca mutants
Human Molecular Genetics, 2002Co-Authors: Daiki Adachi, Toshiyasu Taniguchi, Keiko Nakasato, Alan D Dandrea, Hiroshi Yagasaki, Shigetaka Asano, Takayuki YamashitaAbstract:Fanconi anemia (FA) is an autosomal recessive disorder of hematopoiesis characterized by hypersensitivity to DNA crosslinkers such as mitomycin C (MMC). There is growing evidence for a model of the FA pathway, wherein a nuclear multiprotein complex of five FA proteins (FANCA, C, E, F and G) regulates activation of FANCD2 into a monoubiquitinated form, which, collaborating with the BRCA1 machinery, affects cellular response to DNA damage. However, the role of the FA pathway in defective DNA damage response caused by various mutant forms of FA proteins has not been fully assessed. In the present study, 21 patient-derived FANCA mutants with a missense or a small in-frame deletion were expressed in FANCA-deficient fibroblasts and examined for complementation of MMC sensitivity and for reconstitution of the FA pathway: FANCA phosphorylation, interaction with FANCC, FANCF and FANCG and nuclear localization and FANCD2 monoubiquitination. The altered FANCA proteins complemented MMC sensitivity at different grades: five proteins (group I) behaved like wild-type FANCA, whereas the other proteins were either mildly (group II, n=4) or severely (group III, n = 12) impaired. Group I proteins showed an apparently normal reconstitution of the FA pathway, thus they may be pathogenic by reducing endogenous expression or possibly benign polymorphisms. Reconstitution of the FA pathway by group II and III mutants closely correlated with cellular sensitivity to MMC. The different activation of the FA pathway may partly account for the phenotypic variation seen in FA patients.
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the chinese hamster fancg xrcc9 mutant nm3 fails to express the monoubiquitinated form of the fancd2 protein is hypersensitive to a range of dna damaging agents and exhibits a normal level of spontaneous sister chromatid exchange
Carcinogenesis, 2001Co-Authors: J. B. Wilson, Alan D Dandrea, Kelly L Trueman, Raymond E Meyn, A. Stuckert, Mark A Johnson, Peter E. Bryant, Nigel J JonesAbstract:Fanconi anemia (FA) is a human autosomal disorder characterized by cancer susceptibility and cellular sensitivity to DNA crosslinking agents such as mitomycin C and diepoxybutane. Six FA genes have been cloned including a gene designated XRCC9 (for X-ray Repair Cross Complementing), isolated using a mitomycin C-hypersensitive Chinese hamster cell mutant termed UV40, and subsequently found to be identical to FANCG. A nuclear complex containing the FANCA, FANCC, FANCE, FANCF and FANCG proteins is needed for the activation of a sixth FA protein FANCD2. When monoubiquitinated, the FANCD2 protein co-localizes with the breast cancer susceptibility protein BRCA1 in DNA damage induced foci. In this study, we have assigned NM3, a nitrogen mustardhypersensitive Chinese hamster mutant to the same genetic complementation group as UV40. NM3, like human FA cell lines (but unlike UV40) exhibits a normal spontaneous level of sister chromatid exchange. We show that both NM3 and UV40 are also hypersensitive to other DNA crosslinking agents (including diepoxybutane and chlorambucil) and to non-crosslinking DNA damaging agents (including bleomycin, streptonigrin and EMS), and that all these sensitivities are all corrected upon transfection of the human FANCG/XRCC9 cDNA. Using immunoblotting, NM3 and UV40 were found not to express the active monoubiquitinated isoform of the FANCD2 protein, although expression of the FANCD-L isoform was restored in the FANCG cDNA transformants, correlating with the correction of mutagen-sensitivity. These data indicate that cellular resistance to these DNA damaging agents requires FANCG and that the FA gene pathway, via its activation of FANCD2 and that protein’s subsequent interaction with
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interaction of the fanconi anemia proteins and brca1 in a common pathway
Molecular Cell, 2001Co-Authors: Irene Garciahiguera, Toshiyasu Taniguchi, Markus Grompe, Shridar Ganesan, Stephen M Meyn, Cynthia Timmers, James Hejna, Alan D DandreaAbstract:Abstract Fanconi anemia (FA) is a human autosomal recessive cancer susceptibility disorder characterized by cellular sensitivity to mitomycin C and ionizing radiation. Although six FA genes (for subtypes A, C, D2, E, F, and G) have been cloned, their relationship to DNA repair remains unknown. In the current study, we show that a nuclear complex containing the FANCA, FANCC, FANCF, and FANCG proteins is required for the activation of the FANCD2 protein to a monoubiquitinated isoform. In normal (non-FA) cells, FANCD2 is monoubiq-uitinated in response to DNA damage and is targeted to nuclear foci (dots). Activated FANCD2 protein colocalizes with the breast cancer susceptibility protein, BRCA1, in ionizing radiation–induced foci and in synaptonemal complexes of meiotic chromosomes. The FANCD2 protein, therefore, provides the missing link between the FA protein complex and the cellular BRCA1 repair machinery. Disruption of this pathway results in the cellular and clinical phenotype common to all FA subtypes.
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the fanconi anemia proteins fanca and fancg stabilize each other and promote the nuclear accumulation of the fanconi anemia complex
Blood, 2000Co-Authors: Irene Garciahiguera, Yanan Kuang, Jessica Denham, Alan D DandreaAbstract:Fanconi anemia (FA) is an autosomal recessive cancer susceptibility syndrome with 8 complementation groups. Four of the FA genes have been cloned, and at least 3 of the encoded proteins, FANCA, FANCC, and FANCG/XRCC9, interact in a multisubunit protein complex. The FANCG protein binds directly to the amino terminal nuclear localization sequence (NLS) of FANCA, suggesting that FANCG plays a role in regulating FANCA nuclear accumulation. In the current study the functional consequences of FANCG/FANCA binding were examined. Correction of an FA-G cell line with the FANCG complementary DNA (cDNA) resulted in FANCA/FANCG binding, prolongation of the cellular half-life of FANCA, and an increase in the nuclear accumulation of the FA protein complex. Similar results were obtained upon correction of an FA-A cell line, with a reciprocal increase in the half-life of FANCG. Patient-derived mutant forms of FANCA, containing an intact NLS sequence but point mutations in the carboxy-terminal leucine zipper region, bound FANCG in the cytoplasm. The mutant forms failed to translocate to the nucleus of transduced cells, thereby suggesting a model of coordinated binding and nuclear translocation. These results demonstrate that the FANCA/FANCG interaction is required to maintain the cellular levels of both proteins. Moreover, at least one function of FANCG and FANCA is to regulate the nuclear accumulation of the FA protein complex. Failure to accumulate the nuclear FA protein complex results in the characteristic spectrum of clinical and cellular abnormalities observed in FA.
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carboxy terminal region of the fanconi anemia protein fancg xrcc9 is required for functional activity
Blood, 2000Co-Authors: Yanan Kuang, Irene Garciahiguera, Anna Moran, Michelle A Mondoux, Martin Digweed, Alan D DandreaAbstract:Fanconi anemia (FA) is an autosomal recessive cancer susceptibility syndrome with eight complementation groups. Four of the FA genes have been cloned, and at least three of the encoded proteins, FANCA, FANCC, and FANCG/XRCC9, interact in a nuclear complex, required for the maintenance of normal chromosome stability. In the current study, mutant forms of the FANCA and FANCG proteins have been generated and analyzed with respect to protein complex formation, nuclear translocation, and functional activity. The results demonstrate that the amino terminal two-thirds of FANCG (FANCG amino acids 1-428) binds to the amino terminal nuclear localization signal (NLS) of the FANCA protein. On the basis of 2-hybrid analysis, the FANCA/FANCG binding is a direct protein-protein interaction. Interestingly, a truncated mutant form of the FANCG protein, lacking the carboxy terminus, binds in a complex with FANCA and translocates to the nucleus; however, this mutant protein fails to bind to FANCC and fails to correct the mitomycin C sensitivity of an FA-G cell line. Taken together, these results demonstrate that binding of FANCG to the amino terminal FANCA NLS sequence is necessary but not sufficient for the functional activity of FANCG. Additional amino acid sequences at the carboxy terminus of FANCG are required for the binding of FANCC in the complex.
Alan D. D'andrea - One of the best experts on this subject based on the ideXlab platform.
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FANCC The Fanconi anemia protein, FANCE, promotes the nuclear accumulation of
2013Co-Authors: Toshiyasu Taniguchi, Alan D. D'andreaAbstract:ABSTRACT Fanconi Anemia is an autosomal recessive disorder characterized by aplastic anemia, cancer susceptibility, and cellular sensitivity to mitomycin C. The six known Fanconi Anemia gene products (FANCA, FANCC, FANCD2, FANCE, FANCF and FANCGproteins) interact in a common pathway. The monoubiquitination and nuclear foci formation of FANCD2 are essential for the function of this pathway. FANCA, FANCC, FANCG and FANCF proteins form a multisubunit nuclear complex (FA complex)required for FANCD2 monoubiquitination. Since FANCE and FANCC interact in vitroand FANCE is required for the FANCD2 monoubiquitination, we reasoned that FANCE is a component of the FA complex in vivo. Here we demonstrate that retroviral transduction of FA-E cells with the FANCE cDNA restores the nuclear accumulation of FANCC protein, FANCA-FANCC complex formation, the monoubiquitination and nuclear foci formation of FANCD2, and mitomycin C resistance. HA-tagged FANCE protein localizes diffusely in the nucleus. In normal cells, HA-tagged FANCE protein co-immunoprecipitates with FANCA, FANCC, and FANCG but not with FANCD2. Our data indicate that FANCE is a component of the nuclear FA complex in vivo and is required for the monoubiquitination of FANCD2 and the downstream events in the FA pathway.From bloodjournal.hematologylibrary.org by guest on June 4, 2013. For personal use only.
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Screening of small molecule inhibitors of the Fanconi Anemia-BRCA pathway
Cancer Research, 2005Co-Authors: Toshiyasu Taniguchi, Maria Vasserman, Deborah Chirnomas, Alan D. D'andreaAbstract:4995 Fanconi anemia (FA) is a cancer-susceptibility syndrome characterized by hypersensitivity to DNA crosslinking agents, such as cisplatin and mitomycin C. All 9 known FA proteins cooperate with breast/ovarian cancer susceptibility gene products (BRCA1 and BRCA2) in a common DNA damage-activated signaling pathway called the Fanconi anemia(FA)-BRCA pathway. Seven FA proteins (FANCA, FANCB, FANCC, FANCE, FANCF, FANCG and FANCL) are components of a multi-subunit ubiquitin ligase complex (FA complex) required for monoubiquitination of FANCD2. After DNA damage, FANCD2 is monoubiquitinated and targeted to BRCA1/BRCA2-containing nuclear foci in an ATR kinase-dependent fashion. Importantly, the pathway is inactivated in a wide variety of human cancers by methylation of the FANCF gene. This inactivation causes cisplatin sensitivity in some ovarian cancer cell lines, suggesting an important role of the pathway in cisplatin sensitivity of human tumors. We hypothesized that inhibitors of the FA-BRCA pathway will sensitize cancer cells to cisplatin and, therefore, may be useful for the treatment of cisplatin-resistant cancer. We developed a high-throughput small molecule screen using cells harboring a Green Fluorescent Protein tagged FANCD2 (GFP-FANCD2) and assaying for GFP-FANCD2 nuclear foci formation as a readout, because FANCD2 foci formation is a surrogate marker of the integrity of the pathway. We plated cells with GFP-FANCD2 onto 384-well plates, added small molecules, gamma-irradiated the cells and scored the GFP-FANCD2 nuclear foci. Wells lacking GFP-FANCD2 nuclear foci were scored as positive. We have screened more than 5000 chemicals so far, and found 30 positive chemicals, including kinase inhibitors (wortmannin, H-9, alsterpaullone), geldanamycin, and curcumin. We also found that wortmannin, H-9, and alsterpaullone inhibit ATR-dependent phosphorylation of Chk1. These results together with our previous finding that ATR is required for the activation of the pathway suggest that these drugs inhibit the pathway through inhibition of ATR. Furthermore, we confirmed that alsterpaullone sensitized an ovarian cancer cell line in an FA-BRCA pathway-dependent manner. These findings support the rationale for further screening for FA-BRCA pathway inhibitors.
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Monoubiquitinated FANCD2 Is Both Necessary and Sufficient for Mitomycin C Resistance in the Absence of a Functional Fanconi Anemia Core Complex.
Blood, 2004Co-Authors: Akiko Shimamura, Rebecca J. Leary, Maria Stotsky, Lisa A. Moreau, Alan D. D'andreaAbstract:Monoubiquitination of the Fanconi anemia (FA) protein, FANCD2, is necessary for resistance to MMC/DEB-induced chromosomal breakage, which is the diagnostic feature of Fanconi anemia. The upstream Fanconi proteins FANCA, FANCB, FANCC, FANCE, FANCF, FANCG, and FANCL are necessary for FANCD2 monoubiquitination and Fanconi pathway function. We posed the question whether FANCD2 monoubiquitination is sufficient to restore the Fanconi pathway function in the absence of the upstream FA proteins. We constructed a chimeric monoubiquitinated FANCD2 protein by fusing the FANCD2 cDNA sequence contiguously (in-frame) with a ubiquitin coding sequence. This FANCD2-ubiquition fusion protein (D2-ubi) was stably expressed in FANCD2 − / −, FANCA − / −, FANCC − / −, and FANCG − / − human patient-derived cell lines. D2-ubi expression corrected the MMC hypersensitivity of these FA cell lines in the absence of a functional upstream FA core complex. By western blot analysis, the D2-ubi protein migrated at a slower rate than the endogenous monoubiquitinated FANCD2 protein, consistent with its higher molecular mass. An even slower migrating D2-ubi form (D2-ubi-L) was detectable in cells harboring an intact Fanconi core protein complex (i.e., FA-D2 cells) but was absent in cells lacking functional Fanconi proteins FANCA, FANCC, or FANCG. These results indicate the D2-ubi can be further monoubiquitinated on lysine 561, but only in the presence of an intact FA complex. Furthermore, D2-ubi-L was upregulated in response to DNA damage in transfected FA-D2 and wild type cells. Like endogenous FANCD2, D2-ubi formed nuclear foci in the presence of the upstream Fanconi core complex. In the absence of the FA core complex, D2-ubi was localized diffusely throughout the nucleus, but unlike endogenous FANCD2, D2-ubi was retained within the nucleus following detergent permeabilization of FA-A, FA-C, and FA-G cells. These results further support the epistatic relationship of the FA genes in a linear pathway. In summary, these data indicate that a constitutively monoubiquitinated FANCD2 polypeptide is able to bypass defects in the upstream FA core complex to restore Fanconi pathway function. These studies identify FANCD2 ubiquitination as a potential therapeutic target for patients lacking the upstream FA core complex.
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Disruption of the Fanconi anemia-BRCA pathway in cisplatin-sensitive ovarian tumors.
Nature medicine, 2003Co-Authors: Toshiyasu Taniguchi, Marc Tischkowitz, Shirley V. Hodgson, Hans Joenje, Najim Ameziane, Christopher Mathew, Samuel C. Mok, Alan D. D'andreaAbstract:Ovarian tumor cells are often genomically unstable and hypersensitive to cisplatin. To understand the molecular basis for this phenotype, we examined the integrity of the Fanconi anemia-BRCA (FANC-BRCA) pathway in those cells. This pathway regulates cisplatin sensitivity and is governed by the coordinate activity of six genes associated with Fanconi anemia (FANCA, FANCC, FANCD2, FANCE, FANCF and FANCG) as well as BRCA1 and BRCA2 (FANCD1). Here we show that the FANC-BRCA pathway is disrupted in a subset of ovarian tumor lines. Mono-ubiquitination of FANCD2, a measure of the function of this pathway, and cisplatin resistance were restored by functional complementation with FANCF, a gene that is upstream in this pathway. FANCF inactivation in ovarian tumors resulted from methylation of its CpG island, and acquired cisplatin resistance correlated with demethylation of FANCF. We propose a model for ovarian tumor progression in which the initial methylation of FANCF is followed by FANCF demethylation and ultimately results in cisplatin resistance.
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The Fanconi anemia protein, FANCE, promotes the nuclear accumulation of FANCC.
Blood, 2002Co-Authors: Toshiyasu Taniguchi, Alan D. D'andreaAbstract:Fanconi anemia is an autosomal recessive disorder characterized by aplastic anemia, cancer susceptibility, and cellular sensitivity to mitomycin C. The 6 known Fanconi anemia gene products (FANCA, FANCC, FANCD2, FANCE, FANCF, and FANCG proteins) interact in a common pathway. The monoubiquitination and nuclear foci formation of FANCD2 are essential for the function of this pathway. FANCA, FANCC, FANCG, and FANCF proteins form a multisubunit nuclear complex (FA complex) required for FANCD2 monoubiquitination. Because FANCE and FANCC interact in vitro and FANCE is required for FANCD2 monoubiquitination, we reasoned that FANCE is a component of the FA complex in vivo. Here we demonstrate that retroviral transduction of Fanconi anemia subtype E (FA-E) cells with the FANCE cDNA restores the nuclear accumulation of FANCC protein, FANCA–FANCC complex formation, monoubiquitination and nuclear foci formation of FANCD2, and mitomycin C resistance. Hemagglutinin (HA)-tagged FANCE protein localizes diffusely in the nucleus. In normal cells, HA-tagged FANCE protein coimmunoprecipitates with FANCA, FANCC, and FANCG but not with FANCD2. Our data indicate that FANCE is a component of the nuclear FA complex in vivo and is required for the monoubiquitination of FANCD2 and the downstream events in the FA pathway.
Nigel J Jones - One of the best experts on this subject based on the ideXlab platform.
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Fanconi anemia complementation group FANCD2 protein serine 331 phosphorylation is important for fanconi anemia pathway function and BRCA2 interaction.
Cancer Research, 2009Co-Authors: J. B. Wilson, Nigel J Jones, Yuxuan Xiao, Diane S. Krause, Xiaoyong Chen, Gary M KupferAbstract:Fanconi anemia is a cancer-prone inherited bone marrow failure and cancer susceptibility syndrome with at least 13 complementation groups (FANCA, FANCB, FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ, FANCL, FANCM, and FANCN). Our laboratory has previously described several regulatory phosphorylation events for core complex member proteins FANCG and FANCA by phosphorylation. In this study, we report a novel phosphorylation site serine 331 (S331) of FANCD2, the pivotal downstream player of the Fanconi anemia pathway. Phosphorylation of S331 is important for its DNA damage–inducible monoubiquitylation, resistance to DNA cross-linkers, and in vivo interaction with FANCD1/BRCA2. A phosphomimetic mutation at S331 restores all of these phenotypes to wild-type. In vitro and in vivo experiments show that phosphorylation of S331 is mediated by CHK1, the S-phase checkpoint kinase implicated in the Fanconi anemia DNA repair pathway. [Cancer Res 2009;69(22):8775–83]
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the chinese hamster fancg xrcc9 mutant nm3 fails to express the monoubiquitinated form of the fancd2 protein is hypersensitive to a range of dna damaging agents and exhibits a normal level of spontaneous sister chromatid exchange
Carcinogenesis, 2001Co-Authors: J. B. Wilson, Alan D Dandrea, Kelly L Trueman, Raymond E Meyn, A. Stuckert, Mark A Johnson, Peter E. Bryant, Nigel J JonesAbstract:Fanconi anemia (FA) is a human autosomal disorder characterized by cancer susceptibility and cellular sensitivity to DNA crosslinking agents such as mitomycin C and diepoxybutane. Six FA genes have been cloned including a gene designated XRCC9 (for X-ray Repair Cross Complementing), isolated using a mitomycin C-hypersensitive Chinese hamster cell mutant termed UV40, and subsequently found to be identical to FANCG. A nuclear complex containing the FANCA, FANCC, FANCE, FANCF and FANCG proteins is needed for the activation of a sixth FA protein FANCD2. When monoubiquitinated, the FANCD2 protein co-localizes with the breast cancer susceptibility protein BRCA1 in DNA damage induced foci. In this study, we have assigned NM3, a nitrogen mustardhypersensitive Chinese hamster mutant to the same genetic complementation group as UV40. NM3, like human FA cell lines (but unlike UV40) exhibits a normal spontaneous level of sister chromatid exchange. We show that both NM3 and UV40 are also hypersensitive to other DNA crosslinking agents (including diepoxybutane and chlorambucil) and to non-crosslinking DNA damaging agents (including bleomycin, streptonigrin and EMS), and that all these sensitivities are all corrected upon transfection of the human FANCG/XRCC9 cDNA. Using immunoblotting, NM3 and UV40 were found not to express the active monoubiquitinated isoform of the FANCD2 protein, although expression of the FANCD-L isoform was restored in the FANCG cDNA transformants, correlating with the correction of mutagen-sensitivity. These data indicate that cellular resistance to these DNA damaging agents requires FANCG and that the FA gene pathway, via its activation of FANCD2 and that protein’s subsequent interaction with
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The Chinese hamster FANCG/XRCC9 mutant NM3 fails to express the monoubiquitinated form of the FANCD2 protein, is hypersensitive to a range of DNA damaging agents and exhibits a normal level of spontaneous sister chromatid exchange.
Carcinogenesis, 2001Co-Authors: J. B. Wilson, Alan D. D'andrea, Kelly L Trueman, Raymond E Meyn, A. Stuckert, Mark A Johnson, Peter E. Bryant, Nigel J JonesAbstract:Fanconi anemia (FA) is a human autosomal disorder characterized by cancer susceptibility and cellular sensitivity to DNA crosslinking agents such as mitomycin C and diepoxybutane. Six FA genes have been cloned including a gene designated XRCC9 (for X-ray Repair Cross Complementing), isolated using a mitomycin C-hypersensitive Chinese hamster cell mutant termed UV40, and subsequently found to be identical to FANCG. A nuclear complex containing the FANCA, FANCC, FANCE, FANCF and FANCG proteins is needed for the activation of a sixth FA protein FANCD2. When monoubiquitinated, the FANCD2 protein co-localizes with the breast cancer susceptibility protein BRCA1 in DNA damage induced foci. In this study, we have assigned NM3, a nitrogen mustardhypersensitive Chinese hamster mutant to the same genetic complementation group as UV40. NM3, like human FA cell lines (but unlike UV40) exhibits a normal spontaneous level of sister chromatid exchange. We show that both NM3 and UV40 are also hypersensitive to other DNA crosslinking agents (including diepoxybutane and chlorambucil) and to non-crosslinking DNA damaging agents (including bleomycin, streptonigrin and EMS), and that all these sensitivities are all corrected upon transfection of the human FANCG/XRCC9 cDNA. Using immunoblotting, NM3 and UV40 were found not to express the active monoubiquitinated isoform of the FANCD2 protein, although expression of the FANCD-L isoform was restored in the FANCG cDNA transformants, correlating with the correction of mutagen-sensitivity. These data indicate that cellular resistance to these DNA damaging agents requires FANCG and that the FA gene pathway, via its activation of FANCD2 and that protein’s subsequent interaction with
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The Chinese hamster FANCG/XRCC9 mutant NM3 fails to express the monoubiquitinated form of the FANCD2 protein, is hypersensitive to a range of DNA damaging agents and exhibits a normal level of spontaneous sister chromatid exchange.
Carcinogenesis, 2001Co-Authors: James B. Wilson, Alan D. D'andrea, Kelly L Trueman, Raymond E Meyn, A. Stuckert, Mark A Johnson, Peter E. Bryant, Simon May, Nigel J JonesAbstract:Fanconi anemia (FA) is a human autosomal disorder characterized by cancer susceptibility and cellular sensitivity to DNA crosslinking agents such as mitomycin C and diepoxybutane. Six FA genes have been cloned including a gene designated XRCC9 (for X-ray Repair Cross Complementing), isolated using a mitomycin C-hypersensitive Chinese hamster cell mutant termed UV40, and subsequently found to be identical to FANCG. A nuclear complex containing the FANCA, FANCC, FANCE, FANCF and FANCG proteins is needed for the activation of a sixth FA protein FANCD2. When monoubiquitinated, the FANCD2 protein co-localizes with the breast cancer susceptibility protein BRCA1 in DNA damage induced foci. In this study, we have assigned NM3, a nitrogen mustard-hypersensitive Chinese hamster mutant to the same genetic complementation group as UV40. NM3, like human FA cell lines (but unlike UV40) exhibits a normal spontaneous level of sister chromatid exchange. We show that both NM3 and UV40 are also hypersensitive to other DNA crosslinking agents (including diepoxybutane and chlorambucil) and to non-crosslinking DNA damaging agents (including bleomycin, streptonigrin and EMS), and that all these sensitivities are all corrected upon transfection of the human FANCG/XRCC9 cDNA. Using immunoblotting, NM3 and UV40 were found not to express the active monoubiquitinated isoform of the FANCD2 protein, although expression of the FANCD-L isoform was restored in the FANCG cDNA transformants, correlating with the correction of mutagen-sensitivity. These data indicate that cellular resistance to these DNA damaging agents requires FANCG and that the FA gene pathway, via its activation of FANCD2 and that protein's subsequent interaction with BRCA1, is involved in maintaining genomic stability in response not only to DNA interstrand crosslinks but also a range of other DNA damages including DNA strand breaks. NM3 and other "FA-like" Chinese hamster mutants should provide an important resource for the study of these processes in mammalian cells.
Toshiyasu Taniguchi - One of the best experts on this subject based on the ideXlab platform.
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Abstract IA13: The Fanconi anemia-BRCA pathway and chemosensitivity of cancer cells
Therapeutic Opportunities from DNA Repair Deficiency, 2013Co-Authors: Toshiyasu TaniguchiAbstract:The Fanconi anemia-BRCA pathway has emerged as an important pathway in cancer biology. Fanconi anemia (FA) is a rare genetic disease characterized by chromosomal instability, cancer-susceptibility and cellular sensitivity to interstrand DNA crosslink (ICL)-inducing agents. Sixteen FA genes have been identified (FANCA, FANCB, FANCC, FANCD1/BRCA2, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ/BRIP1, FANCL, FANCM, FANCN/PALB2, FANCO/RAD51, FANCP/SLX4 and FANCQ/XPF). The FA proteins and breast/ovarian cancer susceptibility proteins, BRCA1 and BRCA2, cooperate in a common pathway required for cellular resistance to ICL-inducing agents. This pathway is called the FA-BRCA pathway or FA pathway. The main function of the pathway is to coordinate multiple DNA repair mechanisms (including homologous recombination) during ICL repair. Inactivation of this pathway in cancer cells can lead to sensitivity to anti-cancer ICL-inducing agents, such as cisplatin, while reactivation of this pathway is implicated in acquired resistance to ICL-inducing agents. Since a part of the FA-BRCA pathway regulates homologous recombination, integrity of the pathway is also important for cellular resistance to PARP inhibitor. Therefore, inhibition of the FA-BRCA pathway is an attractive therapeutic strategy to overcome drug resistance of tumor cells. In this talk, I will introduce what the FA-BRCA pathway is and how this pathway is inactivated in human cancer. I will also mention our finding that secondary BRCA1/2 mutations, which restore normal function of BRCA1/2 proteins, are involved in acquired drug resistance of BRCA1/2-mutated cancer. Citation Format: Toshiyasu Taniguchi. The Fanconi anemia-BRCA pathway and chemosensitivity of cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr IA13.
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FANCC The Fanconi anemia protein, FANCE, promotes the nuclear accumulation of
2013Co-Authors: Toshiyasu Taniguchi, Alan D. D'andreaAbstract:ABSTRACT Fanconi Anemia is an autosomal recessive disorder characterized by aplastic anemia, cancer susceptibility, and cellular sensitivity to mitomycin C. The six known Fanconi Anemia gene products (FANCA, FANCC, FANCD2, FANCE, FANCF and FANCGproteins) interact in a common pathway. The monoubiquitination and nuclear foci formation of FANCD2 are essential for the function of this pathway. FANCA, FANCC, FANCG and FANCF proteins form a multisubunit nuclear complex (FA complex)required for FANCD2 monoubiquitination. Since FANCE and FANCC interact in vitroand FANCE is required for the FANCD2 monoubiquitination, we reasoned that FANCE is a component of the FA complex in vivo. Here we demonstrate that retroviral transduction of FA-E cells with the FANCE cDNA restores the nuclear accumulation of FANCC protein, FANCA-FANCC complex formation, the monoubiquitination and nuclear foci formation of FANCD2, and mitomycin C resistance. HA-tagged FANCE protein localizes diffusely in the nucleus. In normal cells, HA-tagged FANCE protein co-immunoprecipitates with FANCA, FANCC, and FANCG but not with FANCD2. Our data indicate that FANCE is a component of the nuclear FA complex in vivo and is required for the monoubiquitination of FANCD2 and the downstream events in the FA pathway.From bloodjournal.hematologylibrary.org by guest on June 4, 2013. For personal use only.
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The Fanconi anemia-BRCA Pathway and Cancer
The DNA Damage Response: Implications on Cancer Formation and Treatment, 2009Co-Authors: Toshiyasu TaniguchiAbstract:The Fanconi anemia (FA)-BRCA pathway has emerged as an important pathway in cancer biology. Fanconi anemia (FA) is a rare genetic disease characterized by aplastic anemia, developmental defects, cancer susceptibility and cellular hypersensitivity to interstrand DNA crosslinking agents. Thirteen FA genes have been identified (FANCA, FANCB, FANCC, FANCD1/BRCA2, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ/BRIP1, FANCL, FANCM, and FANCN/PALB2). The FA proteins and breast/ovarian cancer susceptibility proteins (BRCA1 and BRCA2) cooperate in a common DNA damage-activated signaling pathway (the FA-BRCA pathway) which regulates DNA repair and is required for cellular resistance to DNA crosslinking agents. Inactivation of this pathway has been found in a wide variety of human cancers and is implicated in the sensitivity of cancer cells to anti-cancer DNA crosslinking agents, such as cisplatin and mitomycin C. Reactivation of this pathway is implicated in acquired resistance to DNA crosslinking agents. Therefore, inhibition of the FA-BRCA pathway is an attractive therapeutic strategy to overcome DNA-crosslinker resistance of tumor cells.
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Screening of small molecule inhibitors of the Fanconi Anemia-BRCA pathway
Cancer Research, 2005Co-Authors: Toshiyasu Taniguchi, Maria Vasserman, Deborah Chirnomas, Alan D. D'andreaAbstract:4995 Fanconi anemia (FA) is a cancer-susceptibility syndrome characterized by hypersensitivity to DNA crosslinking agents, such as cisplatin and mitomycin C. All 9 known FA proteins cooperate with breast/ovarian cancer susceptibility gene products (BRCA1 and BRCA2) in a common DNA damage-activated signaling pathway called the Fanconi anemia(FA)-BRCA pathway. Seven FA proteins (FANCA, FANCB, FANCC, FANCE, FANCF, FANCG and FANCL) are components of a multi-subunit ubiquitin ligase complex (FA complex) required for monoubiquitination of FANCD2. After DNA damage, FANCD2 is monoubiquitinated and targeted to BRCA1/BRCA2-containing nuclear foci in an ATR kinase-dependent fashion. Importantly, the pathway is inactivated in a wide variety of human cancers by methylation of the FANCF gene. This inactivation causes cisplatin sensitivity in some ovarian cancer cell lines, suggesting an important role of the pathway in cisplatin sensitivity of human tumors. We hypothesized that inhibitors of the FA-BRCA pathway will sensitize cancer cells to cisplatin and, therefore, may be useful for the treatment of cisplatin-resistant cancer. We developed a high-throughput small molecule screen using cells harboring a Green Fluorescent Protein tagged FANCD2 (GFP-FANCD2) and assaying for GFP-FANCD2 nuclear foci formation as a readout, because FANCD2 foci formation is a surrogate marker of the integrity of the pathway. We plated cells with GFP-FANCD2 onto 384-well plates, added small molecules, gamma-irradiated the cells and scored the GFP-FANCD2 nuclear foci. Wells lacking GFP-FANCD2 nuclear foci were scored as positive. We have screened more than 5000 chemicals so far, and found 30 positive chemicals, including kinase inhibitors (wortmannin, H-9, alsterpaullone), geldanamycin, and curcumin. We also found that wortmannin, H-9, and alsterpaullone inhibit ATR-dependent phosphorylation of Chk1. These results together with our previous finding that ATR is required for the activation of the pathway suggest that these drugs inhibit the pathway through inhibition of ATR. Furthermore, we confirmed that alsterpaullone sensitized an ovarian cancer cell line in an FA-BRCA pathway-dependent manner. These findings support the rationale for further screening for FA-BRCA pathway inhibitors.
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Disruption of the Fanconi anemia-BRCA pathway in cisplatin-sensitive ovarian tumors.
Nature medicine, 2003Co-Authors: Toshiyasu Taniguchi, Marc Tischkowitz, Shirley V. Hodgson, Hans Joenje, Najim Ameziane, Christopher Mathew, Samuel C. Mok, Alan D. D'andreaAbstract:Ovarian tumor cells are often genomically unstable and hypersensitive to cisplatin. To understand the molecular basis for this phenotype, we examined the integrity of the Fanconi anemia-BRCA (FANC-BRCA) pathway in those cells. This pathway regulates cisplatin sensitivity and is governed by the coordinate activity of six genes associated with Fanconi anemia (FANCA, FANCC, FANCD2, FANCE, FANCF and FANCG) as well as BRCA1 and BRCA2 (FANCD1). Here we show that the FANC-BRCA pathway is disrupted in a subset of ovarian tumor lines. Mono-ubiquitination of FANCD2, a measure of the function of this pathway, and cisplatin resistance were restored by functional complementation with FANCF, a gene that is upstream in this pathway. FANCF inactivation in ovarian tumors resulted from methylation of its CpG island, and acquired cisplatin resistance correlated with demethylation of FANCF. We propose a model for ovarian tumor progression in which the initial methylation of FANCF is followed by FANCF demethylation and ultimately results in cisplatin resistance.
Irene Garciahiguera - One of the best experts on this subject based on the ideXlab platform.
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interaction of the fanconi anemia proteins and brca1 in a common pathway
Molecular Cell, 2001Co-Authors: Irene Garciahiguera, Toshiyasu Taniguchi, Markus Grompe, Shridar Ganesan, Stephen M Meyn, Cynthia Timmers, James Hejna, Alan D DandreaAbstract:Abstract Fanconi anemia (FA) is a human autosomal recessive cancer susceptibility disorder characterized by cellular sensitivity to mitomycin C and ionizing radiation. Although six FA genes (for subtypes A, C, D2, E, F, and G) have been cloned, their relationship to DNA repair remains unknown. In the current study, we show that a nuclear complex containing the FANCA, FANCC, FANCF, and FANCG proteins is required for the activation of the FANCD2 protein to a monoubiquitinated isoform. In normal (non-FA) cells, FANCD2 is monoubiq-uitinated in response to DNA damage and is targeted to nuclear foci (dots). Activated FANCD2 protein colocalizes with the breast cancer susceptibility protein, BRCA1, in ionizing radiation–induced foci and in synaptonemal complexes of meiotic chromosomes. The FANCD2 protein, therefore, provides the missing link between the FA protein complex and the cellular BRCA1 repair machinery. Disruption of this pathway results in the cellular and clinical phenotype common to all FA subtypes.
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the fanconi anemia proteins fanca and fancg stabilize each other and promote the nuclear accumulation of the fanconi anemia complex
Blood, 2000Co-Authors: Irene Garciahiguera, Yanan Kuang, Jessica Denham, Alan D DandreaAbstract:Fanconi anemia (FA) is an autosomal recessive cancer susceptibility syndrome with 8 complementation groups. Four of the FA genes have been cloned, and at least 3 of the encoded proteins, FANCA, FANCC, and FANCG/XRCC9, interact in a multisubunit protein complex. The FANCG protein binds directly to the amino terminal nuclear localization sequence (NLS) of FANCA, suggesting that FANCG plays a role in regulating FANCA nuclear accumulation. In the current study the functional consequences of FANCG/FANCA binding were examined. Correction of an FA-G cell line with the FANCG complementary DNA (cDNA) resulted in FANCA/FANCG binding, prolongation of the cellular half-life of FANCA, and an increase in the nuclear accumulation of the FA protein complex. Similar results were obtained upon correction of an FA-A cell line, with a reciprocal increase in the half-life of FANCG. Patient-derived mutant forms of FANCA, containing an intact NLS sequence but point mutations in the carboxy-terminal leucine zipper region, bound FANCG in the cytoplasm. The mutant forms failed to translocate to the nucleus of transduced cells, thereby suggesting a model of coordinated binding and nuclear translocation. These results demonstrate that the FANCA/FANCG interaction is required to maintain the cellular levels of both proteins. Moreover, at least one function of FANCG and FANCA is to regulate the nuclear accumulation of the FA protein complex. Failure to accumulate the nuclear FA protein complex results in the characteristic spectrum of clinical and cellular abnormalities observed in FA.
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carboxy terminal region of the fanconi anemia protein fancg xrcc9 is required for functional activity
Blood, 2000Co-Authors: Yanan Kuang, Irene Garciahiguera, Anna Moran, Michelle A Mondoux, Martin Digweed, Alan D DandreaAbstract:Fanconi anemia (FA) is an autosomal recessive cancer susceptibility syndrome with eight complementation groups. Four of the FA genes have been cloned, and at least three of the encoded proteins, FANCA, FANCC, and FANCG/XRCC9, interact in a nuclear complex, required for the maintenance of normal chromosome stability. In the current study, mutant forms of the FANCA and FANCG proteins have been generated and analyzed with respect to protein complex formation, nuclear translocation, and functional activity. The results demonstrate that the amino terminal two-thirds of FANCG (FANCG amino acids 1-428) binds to the amino terminal nuclear localization signal (NLS) of the FANCA protein. On the basis of 2-hybrid analysis, the FANCA/FANCG binding is a direct protein-protein interaction. Interestingly, a truncated mutant form of the FANCG protein, lacking the carboxy terminus, binds in a complex with FANCA and translocates to the nucleus; however, this mutant protein fails to bind to FANCC and fails to correct the mitomycin C sensitivity of an FA-G cell line. Taken together, these results demonstrate that binding of FANCG to the amino terminal FANCA NLS sequence is necessary but not sufficient for the functional activity of FANCG. Additional amino acid sequences at the carboxy terminus of FANCG are required for the binding of FANCC in the complex.
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fanconi anemia proteins fanca fancc and fancg xrcc9 interact in a functional nuclear complex
Molecular and Cellular Biology, 1999Co-Authors: Irene Garciahiguera, Yanan Kuang, Jennifer Wasik, Alan D DandreaAbstract:Fanconi anemia (FA) is an autosomal recessive cancer susceptibility syndrome with at least eight complementation groups (A to H). Three FA genes, corresponding to complementation groups A, C, and G, have been cloned, but their cellular function remains unknown. We have previously demonstrated that the FANCA and FANCC proteins interact and form a nuclear complex in normal cells, suggesting that the proteins cooperate in a nuclear function. In this report, we demonstrate that the recently cloned FANCG/XRCC9 protein is required for binding of the FANCA and FANCC proteins. Moreover, the FANCG protein is a component of a nuclear protein complex containing FANCA and FANCC. The amino-terminal region of the FANCA protein is required for FANCG binding, FANCC binding, nuclear localization, and functional activity of the complex. Our results demonstrate that the three cloned FA proteins cooperate in a large multisubunit complex. Disruption of this complex results in the specific cellular and clinical phenotype common to most FA complementation groups.
