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1.
FEBS Lett ; 595(5): 595-603, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33423298

RESUMO

We have previously demonstrated that Fanconi anemia (FA) proteins work in concert with other FA and non-FA proteins to mediate stalled replication fork restart. Previous studies suggest a connection between the FA protein FANCD2 and the non-FA protein mechanistic target of rapamycin (mTOR). A recent study showed that mTOR is involved in actin-dependent DNA replication fork restart, suggesting possible roles in the FA DNA repair pathway. In this study, we demonstrate that during replication stress mTOR interacts and cooperates with FANCD2 to provide cellular stability, mediate stalled replication fork restart, and prevent nucleolytic degradation of the nascent DNA strands. Taken together, this study unravels a novel functional cross-talk between two important mechanisms: mTOR and FA DNA repair pathways that ensure genomic stability.


Assuntos
Reparo do DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Anemia de Fanconi/genética , Fibroblastos/metabolismo , Serina-Treonina Quinases TOR/genética , Afidicolina/farmacologia , Sobrevivência Celular/efeitos dos fármacos , DNA/genética , DNA/metabolismo , Anemia de Fanconi/metabolismo , Anemia de Fanconi/patologia , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/deficiência , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Genoma Humano , Instabilidade Genômica , Humanos , Hidroxiureia/farmacologia , Mitomicina/farmacologia , Cultura Primária de Células , Ligação Proteica/efeitos dos fármacos , Transdução de Sinais , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/metabolismo
2.
Neoplasma ; 68(1): 40-52, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32940045

RESUMO

Fanconi anemia complementation group D2 (FANCD2) has been associated with the sensitivity of tumor cells to DNA crosslinking damaging agents in certain solid tumors. However, its role in nasopharyngeal carcinoma (NPC) is still unclear. In the present study, the role of FANCD2 in the response of NPC CNE-2 cells to radiation was investigated. A CNE-2 cell model with stable FANCD2 silencing was constructed by lentiviral transfection. Fluorescence quantitative PCR and western blotting were used to evaluate FANCD2 expression in CNE-2 cells. The biological impact of FANCD2 silencing on the response of CNE-2 cells to radiation was investigated in vitro and in vivo. The microarray technology, western blotting, and immunohistochemistry were used to analyze the proteins involved in related pathways after irradiation to investigate the underlying mechanism. Lentivirus-mediated shRNA interference stably silenced the FANCD2 gene in CNE-2 cells. In vitro, in the FANCD2-silenced group, cell proliferation was significantly inhibited, apoptosis was increased, and the cell cycle was arrested at the G2/M phase after irradiation. In vivo, FANCD2 silencing slowed tumor growth, as the volume and weight of the xenograft tumors were significantly decreased. Both in vitro and in vivo, the differentially expressed genes NUPR1, FLI1, and FGF21 were downregulated in the FANCD2-silenced group. Our results show that FANCD2 silencing affected the sensitivity of CNE-2 cells to ionizing radiation by regulating cell proliferation, apoptosis, and cell cycle distribution. The mechanism might be associated with changes in NUPR1, FLI1, and FGF21 protein expression due to the FANCD2 silencing. This study provides a promising target for NPC radiotherapy.


Assuntos
Proteína do Grupo de Complementação D2 da Anemia de Fanconi , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas , Animais , Apoptose/efeitos da radiação , Linhagem Celular Tumoral , Proliferação de Células/efeitos da radiação , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/deficiência , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Humanos , Carcinoma Nasofaríngeo/genética , Carcinoma Nasofaríngeo/radioterapia , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/radioterapia , RNA Interferente Pequeno/genética , Tolerância a Radiação , Radiação Ionizante
3.
Stem Cell Res ; 40: 101550, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31472450

RESUMO

Members of the Fanconi anemia (FA) protein family are involved in multiple cellular processes including response to DNA damage and oxidative stress. Here we show that a major FA protein, Fancd2, plays a role in mitochondrial biosynthesis through regulation of mitochondrial translation. Fancd2 interacts with Atad3 and Tufm, which are among the most frequently identified components of the mitochondrial nucleoid complex essential for mitochondrion biosynthesis. Deletion of Fancd2 in mouse hematopoietic stem and progenitor cells (HSPCs) leads to increase in mitochondrial number, and enzyme activity of mitochondrion-encoded respiratory complexes. Fancd2 deficiency increases mitochondrial protein synthesis and induces mitonuclear protein imbalance. Furthermore, Fancd2-deficient HSPCs show increased mitochondrial respiration and mitochondrial reactive oxygen species. By using a cell-free assay with mitochondria isolated from WT and Fancd2-KO HSPCs, we demonstrate that the increased mitochondrial protein synthesis observed in Fancd2-KO HSPCs was directly linked to augmented mitochondrial translation. Finally, Fancd2-deficient HSPCs are selectively sensitive to mitochondrial translation inhibition and depend on augmented mitochondrial translation for survival and proliferation. Collectively, these results suggest that Fancd2 restricts mitochondrial activity through regulation of mitochondrial translation, and that augmented mitochondrial translation and mitochondrial respiration may contribute to HSC defect and bone marrow failure in FA.


Assuntos
Proteína do Grupo de Complementação D2 da Anemia de Fanconi/deficiência , Anemia de Fanconi/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Mitocôndrias/metabolismo , Biossíntese de Proteínas , ATPases Associadas a Diversas Atividades Celulares/genética , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Animais , Proliferação de Células , Sobrevivência Celular , Anemia de Fanconi/genética , Anemia de Fanconi/fisiopatologia , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Técnicas de Inativação de Genes , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Fator Tu de Elongação de Peptídeos/genética , Fator Tu de Elongação de Peptídeos/metabolismo , Ligação Proteica
4.
J Biol Chem ; 294(43): 15623-15637, 2019 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-31434739

RESUMO

Defects in the Fanconi anemia (FA) DNA damage-response pathway result in genomic instability, developmental defects, hematopoietic failure, cancer predisposition, and metabolic disorders. The endogenous sources of damage contributing to FA phenotypes and the links between FA and metabolic disease remain poorly understood. Here, using mice lacking the Fancd2 gene, encoding a central FA pathway component, we investigated whether the FA pathway protects against metabolic challenges. Fancd2-/- and wildtype (WT) mice were fed a standard diet (SD), a diet enriched in fat, cholesterol, and cholic acid (Paigen diet), or a diet enriched in lipid alone (high-fat diet (HFD)). Fancd2-/- mice developed hepatobiliary disease and exhibited decreased survival when fed a Paigen diet but not a HFD. Male Paigen diet-fed mice lacking Fancd2 had significant biliary hyperplasia, increased serum bile acid concentration, and increased hepatic pathology. In contrast, female mice were similarly impacted by Paigen diet feeding regardless of Fancd2 status. Upon Paigen diet challenge, male Fancd2-/- mice had altered expression of genes encoding hepatic bile acid transporters and cholesterol and fatty acid metabolism proteins, including Scp2/x, Abcg5/8, Abca1, Ldlr, Srebf1, and Scd-1 Untargeted lipidomic profiling in liver tissue revealed 132 lipid species, including sphingolipids, glycerophospholipids, and glycerolipids, that differed significantly in abundance depending on Fancd2 status in male mice. We conclude that the FA pathway has sex-specific impacts on hepatic lipid and bile acid metabolism, findings that expand the known functions of the FA pathway and may provide mechanistic insight into the metabolic disease predisposition in individuals with FA.


Assuntos
Bile/metabolismo , Dieta , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/deficiência , Metabolismo dos Lipídeos , Fígado/metabolismo , Caracteres Sexuais , Animais , Colesterol/metabolismo , Dano ao DNA , Doenças do Sistema Digestório/metabolismo , Suscetibilidade a Doenças , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Comportamento Alimentar , Feminino , Regulação da Expressão Gênica , Cinética , Metabolismo dos Lipídeos/genética , Masculino , Camundongos
5.
Biochem Biophys Res Commun ; 514(3): 713-719, 2019 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-31078270

RESUMO

Fanconi anemia (FA) is a genetic disorder characterized by congenital malfunction, bone marrow failure and hypersensitivity to DNA damage. FANCD2 protein play the central role in FA pathway. To study the in vivo role of FANCD2, we generated and characterized a new Fancd2 knockout mouse strain with 7bp deletion in Fancd2 gene 5' terminus using Crispr-Cas9 in congenic C57BL/6J background. This Fancd2-/- mice displayed similar but overall more severe manifestation than the previous ES cell targeted Fancd2 model. These features include increased embryonic and postnatal lethality rate, higher incidence of microphthalmia, and more severe hypogonadism. The anemia we observed in this Fancd2-/- mice has not been described in other FA models. Further study indicated that the hematopoiesis deficiency was associated with increased apoptotic cell death, G2/M phase arrest and hypersensitivity to MMC and IR damage of Fancd2-/- bone marrow progenitor cells. Collectively, the resulting Fancd2-/- mice with higher resemblance of FA patient symptoms, will be useful in understand the parthenogenesis of pancytopenia and bone marrow failure in FA.


Assuntos
Proteína do Grupo de Complementação D2 da Anemia de Fanconi/deficiência , Anemia de Fanconi/patologia , Anemia/patologia , Animais , Sequência de Bases , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Células da Medula Óssea/efeitos da radiação , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Feminino , Hematopoese/efeitos dos fármacos , Hematopoese/efeitos da radiação , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitomicina/farmacologia , Fenótipo , Radiação Ionizante , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Células-Tronco/efeitos da radiação
6.
Nat Commun ; 9(1): 3915, 2018 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-30254368

RESUMO

Fanconi anemia (FA) is a bone marrow failure (BMF) syndrome that arises from mutations in a network of FA genes essential for DNA interstrand crosslink (ICL) repair and replication stress tolerance. While allogeneic stem cell transplantation can replace defective HSCs, interventions to mitigate HSC defects in FA do not exist. Remarkably, we reveal here that Lnk (Sh2b3) deficiency restores HSC function in Fancd2-/- mice. Lnk deficiency does not impact ICL repair, but instead stabilizes stalled replication forks in a manner, in part, dependent upon alleviating blocks to cytokine-mediated JAK2 signaling. Lnk deficiency restores proliferation and survival of Fancd2-/- HSCs, while reducing replication stress and genomic instability. Furthermore, deletion of LNK in human FA-like HSCs promotes clonogenic growth. These findings highlight a new role for cytokine/JAK signaling in promoting replication fork stability, illuminate replication stress as a major underlying origin of BMF in FA, and have strong therapeutic implications.


Assuntos
Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Anemia de Fanconi/genética , Instabilidade Genômica/genética , Células-Tronco Hematopoéticas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Adaptadoras de Transdução de Sinal , Animais , Transplante de Medula Óssea , Proliferação de Células/genética , Células Cultivadas , Reparo do DNA/genética , Replicação do DNA/genética , Anemia de Fanconi/metabolismo , Anemia de Fanconi/terapia , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/deficiência , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Masculino , Proteínas de Membrana , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Knockout
7.
Nat Commun ; 9(1): 2280, 2018 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-29891926

RESUMO

Defects in DNA repair can cause various genetic diseases with severe pathological phenotypes. Fanconi anemia (FA) is a rare disease characterized by bone marrow failure, developmental abnormalities, and increased cancer risk that is caused by defective repair of DNA interstrand crosslinks (ICLs). Here, we identify the deubiquitylating enzyme USP48 as synthetic viable for FA-gene deficiencies by performing genome-wide loss-of-function screens across a panel of human haploid isogenic FA-defective cells (FANCA, FANCC, FANCG, FANCI, FANCD2). Thus, as compared to FA-defective cells alone, FA-deficient cells additionally lacking USP48 are less sensitive to genotoxic stress induced by ICL agents and display enhanced, BRCA1-dependent, clearance of DNA damage. Consequently, USP48 inactivation reduces chromosomal instability of FA-defective cells. Our results highlight a role for USP48 in controlling DNA repair and suggest it as a potential target that could be therapeutically exploited for FA.


Assuntos
Reparo do DNA/genética , Reparo do DNA/fisiologia , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Proteases Específicas de Ubiquitina/genética , Proteases Específicas de Ubiquitina/metabolismo , Proteína BRCA1/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular , Instabilidade Cromossômica , Dano ao DNA , Anemia de Fanconi/terapia , Proteína do Grupo de Complementação A da Anemia de Fanconi/deficiência , Proteína do Grupo de Complementação A da Anemia de Fanconi/genética , Proteína do Grupo de Complementação A da Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação C da Anemia de Fanconi/deficiência , Proteína do Grupo de Complementação C da Anemia de Fanconi/genética , Proteína do Grupo de Complementação C da Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/deficiência , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação G da Anemia de Fanconi/deficiência , Proteína do Grupo de Complementação G da Anemia de Fanconi/genética , Proteína do Grupo de Complementação G da Anemia de Fanconi/metabolismo , Proteínas de Grupos de Complementação da Anemia de Fanconi/deficiência , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Técnicas de Inativação de Genes , Terapia Genética , Histonas/metabolismo , Humanos , Mutação , Rad51 Recombinase/metabolismo , Proteases Específicas de Ubiquitina/deficiência , Ubiquitinação
8.
Nature ; 553(7687): 171-177, 2018 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-29323295

RESUMO

Haematopoietic stem cells renew blood. Accumulation of DNA damage in these cells promotes their decline, while misrepair of this damage initiates malignancies. Here we describe the features and mutational landscape of DNA damage caused by acetaldehyde, an endogenous and alcohol-derived metabolite. This damage results in DNA double-stranded breaks that, despite stimulating recombination repair, also cause chromosome rearrangements. We combined transplantation of single haematopoietic stem cells with whole-genome sequencing to show that this damage occurs in stem cells, leading to deletions and rearrangements that are indicative of microhomology-mediated end-joining repair. Moreover, deletion of p53 completely rescues the survival of aldehyde-stressed and mutated haematopoietic stem cells, but does not change the pattern or the intensity of genome instability within individual stem cells. These findings characterize the mutation of the stem-cell genome by an alcohol-derived and endogenous source of DNA damage. Furthermore, we identify how the choice of DNA-repair pathway and a stringent p53 response limit the transmission of aldehyde-induced mutations in stem cells.


Assuntos
Acetaldeído/metabolismo , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Etanol/metabolismo , Etanol/farmacologia , Instabilidade Genômica/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/patologia , Mutação , Álcool Desidrogenase/deficiência , Álcool Desidrogenase/genética , Álcool Desidrogenase/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Reparo do DNA por Junção de Extremidades , Etanol/administração & dosagem , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Anemia de Fanconi/patologia , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/deficiência , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Feminino , Deleção de Genes , Genes p53/genética , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Autoantígeno Ku/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Reparo de DNA por Recombinação/efeitos dos fármacos , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Sequenciamento Completo do Genoma
10.
Exp Hematol ; 48: 79-86, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-27915139

RESUMO

Fanconi anemia (FA) results from mutations in the genes necessary for DNA damage repair and often leads to progressive bone marrow failure. Although the exhaustion of the bone marrow leads to cytopenias in FA patients as they age, evidence from human FA and mouse model fetal livers suggests that hematopoietic defects originate in utero, which may lead to deficient seeding of the bone marrow. To address this possibility, we examined the consequences of loss of Fancd2, a central component of the FA pathway. Examination of embryonic day 14.5 (E14.5) Fancd2 knockout (KO) fetal livers showed a decrease in total cellularity and specific declines in long-term and short-term hematopoietic stem cell (LT-HSC and ST-HSC, respectively) numbers. Fancd2 KO fetal liver cells display similar functional defects to Fancd2 adult bone marrow cells, including reduced colony-forming units, increased mitomycin C sensitivity, increased LT-HSC apoptosis, and heavily impaired competitive repopulation, implying that these defects are intrinsic to the fetal liver and are not dependent on the accumulation of DNA damage during aging. Telomere shortening, an aging-related mechanism proposed to contribute to HSC apoptosis and bone marrow failure in FA, was not observed in Fancd2 KO fetal livers. In summary, loss of Fancd2 yields significant defects to fetal liver hematopoiesis, particularly the HSC population, which mimics key phenotypes from adult Fancd2 KO bone marrow independently of aging-accrued DNA damage.


Assuntos
Proteína do Grupo de Complementação D2 da Anemia de Fanconi/deficiência , Feto , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Animais , Apoptose/genética , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Dano ao DNA , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Transplante de Tecido Fetal , Genótipo , Hematopoese , Células-Tronco Hematopoéticas/efeitos dos fármacos , Fígado/embriologia , Fígado/metabolismo , Camundongos , Camundongos Knockout , Mitomicina/farmacologia , Células-Tronco
11.
Blood ; 128(24): 2774-2784, 2016 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-27756748

RESUMO

Fanconi anemia (FA) is an inherited bone marrow failure disorder associated with a high incidence of leukemia and solid tumors. Bone marrow transplantation is currently the only curative therapy for the hematopoietic complications of this disorder. However, long-term morbidity and mortality remain very high, and new therapeutics are badly needed. Here we show that the widely used diabetes drug metformin improves hematopoiesis and delays tumor formation in Fancd2-/- mice. Metformin is the first compound reported to improve both of these FA phenotypes. Importantly, the beneficial effects are specific to FA mice and are not seen in the wild-type controls. In this preclinical model of FA, metformin outperformed the current standard of care, oxymetholone, by improving peripheral blood counts in Fancd2-/- mice significantly faster. Metformin increased the size of the hematopoietic stem cell compartment and enhanced quiescence in hematopoietic stem and progenitor cells. In tumor-prone Fancd2-/-Trp53+/- mice, metformin delayed the onset of tumors and significantly extended the tumor-free survival time. In addition, we found that metformin and the structurally related compound aminoguanidine reduced DNA damage and ameliorated spontaneous chromosome breakage and radials in human FA patient-derived cells. Our results also indicate that aldehyde detoxification might be one of the mechanisms by which metformin reduces DNA damage in FA cells.


Assuntos
Carcinogênese/patologia , Anemia de Fanconi/tratamento farmacológico , Anemia de Fanconi/patologia , Hematopoese/efeitos dos fármacos , Metformina/farmacologia , Aldeídos/metabolismo , Animais , Contagem de Células Sanguíneas , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/patologia , Carcinogênese/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Quebra Cromossômica , Dano ao DNA , Dieta , Anemia de Fanconi/sangue , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/deficiência , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Guanidinas/farmacologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/patologia , Humanos , Inativação Metabólica/efeitos dos fármacos , Metformina/administração & dosagem , Camundongos , Poli I-C/farmacologia
12.
Oncotarget ; 7(42): 68449-68472, 2016 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-27637088

RESUMO

We tested the effect of expression of the Human Papilloma Virus (HPV E7) oncogene on hematopoiesis in long-term bone marrow cultures (LTBMCs) derived from K14E7 (FVB) Fancd2-/- (129/Sv), K14E7 Fancd2+/+, Fancd2-/-, and control (FVB X 129/Sv) Fl mice. K14E7 Fancd2-/- and Fancd2-/- LTBMCs showed decreased duration of production of total nonadherent hematopoietic cells and progenitors forming day 7 and day 14 multilineage CFU-GEMM colonies in secondary cultures (7 wks and 8 wks respectively) compared to cultures from K14E7 Fancd2+/+ (17 wks) or control mice (18 wks) p < 0.0001. Marrow stromal cell lines derived from both K14E7 Fancd2-/- and Fancd2-/- cultures were radiosensitive, as were IL-3 dependent hematopoietic progenitor cell lines derived from K14E7 Fancd2-/- cultures. In contrast, Fancd2-/- mouse hematopoietic progenitor cell lines and fresh marrow were radioresistant. K14E7 Fancd2-/- mouse freshly explanted bone marrow expressed no detectable K14 or E7; however, LTBMCs produced K14 positive factor-independent (FI) clonal malignant plasmacytoma forming cell lines in which E7 was detected in the nucleus with p53 and Rb. Transfection of an E6/E7 plasmid into Fancd2-/-, but not control Fancd2+/+ IL-3 dependent hematopoietic progenitor cell lines, increased cloning efficiency, cell growth, and induced malignant cell lines. Therefore, the altered radiobiology of hematopoietic progenitor cells and malignant transformation in vitro by K14E7 expression in cells of the Fancd2-/- genotype suggests a potential role of HPV in hematopoietic malignancies in FA patients.


Assuntos
Células da Medula Óssea/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Células-Tronco Hematopoéticas/metabolismo , Plasmocitoma/genética , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Sobrevivência Celular/efeitos da radiação , Células Cultivadas , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/deficiência , Hematopoese/genética , Humanos , Queratina-14/genética , Queratina-14/metabolismo , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/metabolismo , Plasmocitoma/metabolismo , Plasmocitoma/patologia , Células Estromais/metabolismo , Células Estromais/efeitos da radiação , Fatores de Tempo
13.
Toxins (Basel) ; 8(7)2016 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-27399778

RESUMO

Epidemiological studies have found a positive association between coffee consumption and a lower risk of cardiovascular disorders, some cancers, diabetes, Parkinson and Alzheimer disease. Coffee consumption, however, has also been linked to an increased risk of developing some types of cancer, including bladder cancer in adults and leukemia in children of mothers who drink coffee during pregnancy. Since cancer is driven by the accumulation of DNA alterations, the ability of the coffee constituent caffeic acid to induce DNA damage in cells may play a role in the carcinogenic potential of this beverage. This carcinogenic potential may be exacerbated in cells with DNA repair defects. People with the genetic disease Fanconi Anemia have DNA repair deficiencies and are predisposed to several cancers, particularly acute myeloid leukemia. Defects in the DNA repair protein Fanconi Anemia D2 (FANCD2) also play an important role in the development of a variety of cancers (e.g., bladder cancer) in people without this genetic disease. This communication shows that cells deficient in FANCD2 are hypersensitive to the cytotoxicity (clonogenic assay) and DNA damage (γ-H2AX and 53BP1 focus assay) induced by caffeic acid and by a commercial lyophilized coffee extract. These data suggest that people with Fanconi Anemia, or healthy people who develop sporadic mutations in FANCD2, may be hypersensitive to the carcinogenic activity of coffee.


Assuntos
Ácidos Cafeicos/toxicidade , Café/toxicidade , Dano ao DNA , Reparo do DNA , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/deficiência , Anemia de Fanconi/patologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Histonas/metabolismo , Humanos , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo
14.
Nucleic Acids Res ; 44(14): 6803-16, 2016 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-27179029

RESUMO

Microsatellite DNAs that form non-B structures are implicated in replication fork stalling, DNA double strand breaks (DSBs) and human disease. Fanconi anemia (FA) is an inherited disorder in which mutations in at least nineteen genes are responsible for the phenotypes of genome instability and cancer predisposition. FA pathway proteins are active in the resolution of non-B DNA structures including interstrand crosslinks, G quadruplexes and DNA triplexes. In FANCJ helicase depleted cells, we show that hydroxyurea or aphidicolin treatment leads to loss of microsatellite polymerase chain reaction signals and to chromosome recombination at an ectopic hairpin forming CTG/CAG repeat in the HeLa genome. Moreover, diverse endogenous microsatellite signals were also lost upon replication stress after FANCJ depletion, and in FANCJ null patient cells. The phenotype of microsatellite signal instability is specific for FANCJ apart from the intact FA pathway, and is consistent with DSBs at microsatellites genome-wide in FANCJ depleted cells following replication stress.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Replicação do DNA/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Genoma Humano , Repetições de Microssatélites/genética , Estresse Fisiológico/genética , Afidicolina/farmacologia , Cromossomos Humanos/genética , Replicação do DNA/efeitos dos fármacos , Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/deficiência , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Reação em Cadeia da Polimerase , Recombinação Genética/genética , Estresse Fisiológico/efeitos dos fármacos , Expansão das Repetições de Trinucleotídeos/genética
15.
Oncogene ; 35(1): 22-34, 2016 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-25893307

RESUMO

Fanconi anemia (FA) is a genetic disease of bone marrow failure, cancer susceptibility, and sensitivity to DNA crosslinking agents. FANCD2, the central protein of the FA pathway, is monoubiquitinated upon DNA damage, such as crosslinkers and replication blockers such as hydroxyurea (HU). Even though FA cells demonstrate unequivocal sensitivity to crosslinkers, such as mitomycin C (MMC), we find that they are largely resistant to HU, except for cells absent for expression of FANCD2. FANCD2, RAD51 and RAD18 form a complex, which is enhanced upon HU exposure. Surprisingly, although FANCD2 is required for this enhanced interaction, its monoubiquitination is not. Similarly, non-ubiquitinated FANCD2 can still support proliferation cell nuclear antigen (PCNA) monoubiquitination. RAD51, but not BRCA2, is also required for PCNA monoubiquitination in response to HU, suggesting that this function is independent of homologous recombination (HR). We further show that translesion (TLS) polymerase PolH chromatin localization is decreased in FANCD2 deficient cells, FANCD2 siRNA knockdown cells and RAD51 siRNA knockdown cells, and PolH knockdown results in HU sensitivity only. Our data suggest that FANCD2 and RAD51 have an important role in PCNA monoubiquitination and TLS in a FANCD2 monoubiquitination and HR-independent manner in response to HU. This effect is not observed with MMC treatment, suggesting a non-canonical function for the FA pathway in response to different types of DNA damage.


Assuntos
Dano ao DNA , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Anemia de Fanconi/tratamento farmacológico , Anemia de Fanconi/genética , Hidroxiureia/farmacologia , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/deficiência , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Hidroxiureia/efeitos adversos , Antígeno Nuclear de Célula em Proliferação/metabolismo , Rad51 Recombinase/metabolismo , Ubiquitina-Proteína Ligases , Ubiquitinação
16.
Stem Cells ; 33(11): 3382-96, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26212365

RESUMO

Fanconi anemia (FA) patients develop bone marrow (BM) failure or leukemia. One standard care for these devastating complications is hematopoietic stem cell transplantation. We identified a group of mesenchymal stromal cells (MSCs)-derived metabolites, glycerophospholipids, and their endogenous inhibitor, 5-(tetradecyloxy)-2-furoic acid (TOFA), as regulators of donor hematopoietic stem and progenitor cells. We provided two pieces of evidence that TOFA could improve hematopoiesis-supporting function of FA MSCs: (a) limiting-dilution cobblestone area-forming cell assay revealed that TOFA significantly increased cobblestone colonies in Fanca-/- or Fancd2-/- cocultures compared to untreated cocultures. (b) Competitive repopulating assay using output cells collected from cocultures showed that TOFA greatly alleviated the abnormal expansion of the donor myeloid (CD45.2+Gr1+Mac1+) compartment in both peripheral blood and BM of recipient mice transplanted with cells from Fanca-/- or Fancd2-/- cocultures. Furthermore, mechanistic studies identified Tlr4 signaling as the responsible pathway mediating the effect of glycerophospholipids. Thus, targeting glycerophospholipid biosynthesis in FA MSCs could be a therapeutic strategy to improve hematopoiesis and stem cell transplantation.


Assuntos
Diferenciação Celular/fisiologia , Proteína do Grupo de Complementação A da Anemia de Fanconi/deficiência , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/deficiência , Glicerofosfolipídeos/biossíntese , Células-Tronco Hematopoéticas/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Células Cultivadas , Técnicas de Cocultura , Anemia de Fanconi/genética , Proteína do Grupo de Complementação A da Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais/fisiologia
17.
Nature ; 518(7538): 258-62, 2015 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-25642963

RESUMO

Large-scale genomic studies have shown that half of epithelial ovarian cancers (EOCs) have alterations in genes regulating homologous recombination (HR) repair. Loss of HR accounts for the genomic instability of EOCs and for their cellular hyper-dependence on alternative poly-ADP ribose polymerase (PARP)-mediated DNA repair mechanisms. Previous studies have implicated the DNA polymerase θ (Polθ also known as POLQ, encoded by POLQ) in a pathway required for the repair of DNA double-strand breaks, referred to as the error-prone microhomology-mediated end-joining (MMEJ) pathway. Whether Polθ interacts with canonical DNA repair pathways to prevent genomic instability remains unknown. Here we report an inverse correlation between HR activity and Polθ expression in EOCs. Knockdown of Polθ in HR-proficient cells upregulates HR activity and RAD51 nucleofilament assembly, while knockdown of Polθ in HR-deficient EOCs enhances cell death. Consistent with these results, genetic inactivation of an HR gene (Fancd2) and Polq in mice results in embryonic lethality. Moreover, Polθ contains RAD51 binding motifs and it blocks RAD51-mediated recombination. Our results reveal a synthetic lethal relationship between the HR pathway and Polθ-mediated repair in EOCs, and identify Polθ as a novel druggable target for cancer therapy.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , DNA Polimerase Dirigida por DNA/metabolismo , Recombinação Homóloga , Neoplasias Epiteliais e Glandulares/genética , Neoplasias Epiteliais e Glandulares/metabolismo , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Motivos de Aminoácidos , Animais , Carcinoma Epitelial do Ovário , Ciclo Celular , Morte Celular , Linhagem Celular Tumoral , Reparo do DNA por Junção de Extremidades/genética , Replicação do DNA , DNA Polimerase Dirigida por DNA/deficiência , Perda do Embrião , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/deficiência , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Feminino , Instabilidade Genômica , Recombinação Homóloga/genética , Humanos , Camundongos , Terapia de Alvo Molecular , Neoplasias Epiteliais e Glandulares/patologia , Neoplasias Ovarianas/patologia , Ligação Proteica , Rad51 Recombinase/antagonistas & inibidores , Rad51 Recombinase/metabolismo , Reparo de DNA por Recombinação/genética , DNA Polimerase teta
18.
Clin Cancer Res ; 21(8): 1962-72, 2015 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-25609062

RESUMO

PURPOSE: Fanconi anemia is an inherited disorder associated with a constitutional defect in the Fanconi anemia DNA repair machinery that is essential for resolution of DNA interstrand crosslinks. Individuals with Fanconi anemia are predisposed to formation of head and neck squamous cell carcinomas (HNSCC) at a young age. Prognosis is poor, partly due to patient intolerance of chemotherapy and radiation requiring dose reduction, which may lead to early recurrence of disease. EXPERIMENTAL DESIGN: Using HNSCC cell lines derived from the tumors of patients with Fanconi anemia, and murine HNSCC cell lines derived from the tumors of wild-type and Fancc(-/-) mice, we sought to define Fanconi anemia-dependent chemosensitivity and DNA repair characteristics. We utilized DNA repair reporter assays to explore the preference of Fanconi anemia HNSCC cells for non-homologous end joining (NHEJ). RESULTS: Surprisingly, interstrand crosslinker (ICL) sensitivity was not necessarily Fanconi anemia-dependent in human or murine cell systems. Our results suggest that the increased Ku-dependent NHEJ that is expected in Fanconi anemia cells did not mediate relative ICL resistance. ICL exposure resulted in increased DNA damage sensing and repair by PARP in Fanconi anemia-deficient cells. Moreover, human and murine Fanconi anemia HNSCC cells were sensitive to PARP inhibition, and sensitivity of human cells was attenuated by Fanconi anemia gene complementation. CONCLUSIONS: The observed reliance upon PARP-mediated mechanisms reveals a means by which Fanconi anemia HNSCCs can acquire relative resistance to the ICL-based chemotherapy that is a foundation of HNSCC treatment, as well as a potential target for overcoming chemoresistance in the chemosensitive individual.


Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Anemia de Fanconi/complicações , Anemia de Fanconi/genética , Neoplasias de Cabeça e Pescoço/etiologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Animais , Linhagem Celular Tumoral , Proliferação de Células , Cisplatino/farmacologia , Dano ao DNA/efeitos dos fármacos , Reparo do DNA por Junção de Extremidades , DNA Helicases/metabolismo , Modelos Animais de Doenças , Ativação Enzimática , Proteína do Grupo de Complementação C da Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/deficiência , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Técnicas de Inativação de Genes , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Xenoenxertos , Humanos , Autoantígeno Ku , Camundongos , Camundongos Knockout , Fenótipo , Poli(ADP-Ribose) Polimerases/metabolismo , Esferoides Celulares , Células Tumorais Cultivadas
19.
FEBS Lett ; 588(21): 3959-63, 2014 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-25240201

RESUMO

The silkworm Fanconi anemia (FA) pathway is required for normal cellular resistance to mitomycin C (MMC) in silkworms, but little is known about the requirement for repair of other types of DNA damage. Here we report that silkworm cells deficient for FA proteins FancD2 and FancM exhibit normal sensitivities to hydroxyurea (HU) and camptothecin (CPT), although FancM-dependent FancD2 monoubiquitination is induced upon these treatments. Similar results were observed in cells depleted for Rmi1 and Mhf1, which interact with the FancM protein. We also found that Rad51-knockdown cells exhibited normal sensitivity to HU despite induction of double-strand breaks by HU treatment.


Assuntos
Bombyx/citologia , Dano ao DNA , DNA Helicases/metabolismo , Reparo do DNA , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Animais , Camptotecina/farmacologia , Células Cultivadas , DNA Helicases/deficiência , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/deficiência , Histonas/metabolismo , Recombinação Homóloga/efeitos dos fármacos , Humanos , Hidroxiureia/farmacologia , Mitomicina/farmacologia , Fosforilação/efeitos dos fármacos , Ubiquitinação/efeitos dos fármacos
20.
Radiat Res ; 182(1): 35-49, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24932534

RESUMO

The altered DNA damage response pathway in patients with Fanconi anemia (FA) may increase the toxicity of clinical radiotherapy. We quantitated oral cavity mucositis in irradiated Fanconi anemia Fancd2(-/-) mice, comparing this to Fancd2(+/-) and Fancd2(+/+) mice, and we measured distant bone marrow suppression and quantitated the effect of the intraoral radioprotector GS-nitroxide, JP4-039 in F15 emulsion. We found that FA mice were more susceptible to radiation injury and that protection from radiation injury by JP4-039/F15 was observed at all radiation doses. Adult 10-12-week-old mice, of FVB/N background Fancd2(-/-), Fancd2(+/-) and Fancd2(+/+) were head and neck irradiated with 24, 26, 28 or 30 Gy (large fraction sizes typical of stereotactic radiosurgery treatments) and subgroups received intraoral JP4-039 (0.4 mg/mouse in 100 µL F15 liposome emulsion) preirradiation. On day 2 or 5 postirradiation, mice were sacrificed, tongue tissue and femur marrow were excised for quantitation of radiation-induced stress response, inflammatory and antioxidant gene transcripts, histopathology and assay for femur marrow colony-forming hematopoietic progenitor cells. Fancd2(-/-) mice had a significantly higher percentage of oral mucosal ulceration at day 5 after 26 Gy irradiation (59.4 ± 8.2%) compared to control Fancd2(+/+) mice (21.7 ± 2.9%, P = 0.0063). After 24 Gy irradiation, Fancd2(-/-) mice had a higher oral cavity percentage of tongue ulceration compared to Fancd2(+/+) mice irradiated with higher doses of 26 Gy (P = 0.0123). Baseline and postirradiation oral cavity gene transcripts were altered in Fancd2(-/-) mice compared to Fancd2(+/+) controls. Fancd2(-/-) mice had decreased baseline femur marrow CFU-GM, BFUe and CFU-GEMM, which further decreased after 24 or 26 Gy head and neck irradiation. These changes were not seen in head- and neck-irradiated Fancd2(+/+) mice. In radiosensitive Fancd2(-/-) mice, biomarkers of both local oral cavity and distant marrow radiation toxicity were ameliorated by intraoral JP4-039/F15. We propose that Fancd2(-/-) mice are a valuable radiosensitive animal model system, which can be used to evaluate potential radioprotective agents.


Assuntos
Medula Óssea/efeitos dos fármacos , Medula Óssea/efeitos da radiação , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/deficiência , Boca/efeitos da radiação , Mucosite/prevenção & controle , Óxidos de Nitrogênio/farmacologia , Lesões Experimentais por Radiação/prevenção & controle , Animais , Medula Óssea/imunologia , Contagem de Células , Linhagem Celular , Feminino , Fêmur/imunologia , Cabeça/efeitos da radiação , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos da radiação , Lipossomos , Masculino , Camundongos , Boca/efeitos dos fármacos , Pescoço/efeitos da radiação , Óxidos de Nitrogênio/administração & dosagem , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Tolerância a Radiação/efeitos dos fármacos , Protetores contra Radiação/administração & dosagem , Protetores contra Radiação/farmacologia
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