RESUMEN

Fanconi anemia (FA) is a human genetic disorder characterized by hypersensitivity to DNA crosslinking agents. Its cellular phenotypes include increased chromosome breakage and a marked cell-cycle delay with 4N DNA content after introduction of interstrand DNA crosslinks (ICL). To further understand the nature of this delay previously described as a G2/M arrest, we introduced ICL specifically during G2 and monitored the cells for passage into mitosis. Our results showed that, even at the highest doses, postreplication ICL produced neither G2/M arrest nor chromosome breakage in FA-A or FA-C cells. This suggests that, similar to wild-type cells, DNA replication is required to trigger both responses. Therefore, the 4N cell DNA content observed in FA cells after ICL treatment also represents incomplete DNA replication and arrest in late S phase. FA fibroblasts from complementation groups A and C were able to recover from the ICL-induced cell-cycle arrest, but took approximately 3 times longer than controls. These results indicate that the FA pathway is required for the efficient resolution of ICL-induced S-phase arrest.

Asunto(s)

Anemia de Fanconi/fisiopatología , Fase S , Trioxsaleno/análogos & derivados , Línea Celular , Rotura Cromosómica , Reactivos de Enlaces Cruzados/farmacología , ADN , Reparación del ADN , Anemia de Fanconi/genética , Fibroblastos , G-Cuádruplex , Fase G2/efectos de los fármacos , Humanos , Mitosis/efectos de los fármacos , Fase S/efectos de los fármacos , Trioxsaleno/farmacología , Rayos Ultravioleta

RESUMEN

The Fanconi anemia (FA) group C gene product (FANCC) functions to protect cells from cytotoxic and genotoxic effects of cross-linking agents. FANCC is also required for optimal activation of STAT1 in response to cytokine and growth factors and for suppressing cytokine-induced apoptosis by modulating the activity of double-stranded RNA-dependent protein kinase. Because not all FANCC mutations affect STAT1 activation, the hypothesis was considered that cross-linker resistance function of FANCC depends on structural elements that differ from those required for the cytokine signaling functions of FANCC. Structure-function studies were designed to test this notion. Six separate alanine-substituted mutations were generated in 3 highly conserved motifs of FANCC. All mutants complemented mitomycin C (MMC) hypersensitive phenotype of FA-C cells and corrected aberrant posttranslational activation of FANCD2 in FA-C mutant cells. However, 2 of the mutants, S249A and E251A, failed to correct defective STAT1 activation. FA-C lymphoblasts carrying these 2 mutants demonstrated a defect in recruitment of STAT1 to the interferon gamma (IFN-gamma) receptor and GST-fusion proteins bearing S249A and E251A mutations were less efficient binding partners for STAT1 in stimulated lymphoblasts. These same mutations failed to complement the characteristic hypersensitive apoptotic responses of FA-C cells to tumor necrosis factor-alpha (TNF-alpha) and IFN-gamma. Cells bearing a naturally occurring FANCC mutation (322delG) that preserves this conserved region showed normal STAT1 activation but remained hypersensitive to MMC. The conclusion is that a central highly conserved domain of FANCC is required for functional interaction with STAT1 and that structural elements required for STAT1-related functions differ from those required for genotoxic responses to cross-linking agents. Preservation of signaling capacity of cells bearing the del322G mutation may account for the reduced severity and later onset of bone marrow failure associated with this mutation.

Asunto(s)

Proteínas de Ciclo Celular , Anemia de Fanconi/genética , Proteínas Nucleares , Proteínas/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Apoptosis/efectos de los fármacos , Línea Celular Transformada/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Proteína del Grupo de Complementación C de la Anemia de Fanconi , Proteínas del Grupo de Complementación de la Anemia de Fanconi , Prueba de Complementación Genética , Humanos , Interferón gamma/farmacología , Mitomicina/farmacología , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fosforilación , Procesamiento Proteico-Postraduccional , Proteínas/genética , Proteínas/fisiología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/fisiología , Factor de Transcripción STAT1 , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transducción de Señal , Relación Estructura-Actividad , Transactivadores/metabolismo , Factor de Necrosis Tumoral alfa/farmacología

RESUMEN

Fanconi anemia (FA) is a genetic disease with birth defects, bone marrow failure, and cancer susceptibility. To date, genes for five of the seven known complementation groups have been cloned. Complementation group D is heterogeneous, consisting of two distinct genes, FANCD1 and FANCD2. Here we report the positional cloning of FANCD2. The gene consists of 44 exons, encodes a novel 1451 amino acid nuclear protein, and has two protein isoforms. Similar to other FA proteins, the FANCD2 protein has no known functional domains, but unlike other known FA genes, FANCD2 is highly conserved in A. thaliana, C. elegans, and Drosophila. Retroviral transduction of the cloned FANCD2 cDNA into FA-D2 cells resulted in functional complementation of MMC sensitivity.

Asunto(s)

Anemia de Fanconi/genética , Proteínas Nucleares/genética , Alelos , Secuencia de Aminoácidos , Secuencia de Bases , Western Blotting , Línea Celular , Rotura Cromosómica/genética , Clonación Molecular , Análisis Mutacional de ADN , Proteínas de Drosophila , Compuestos Epoxi/farmacología , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi , Femenino , Perfilación de la Expresión Génica , Prueba de Complementación Genética , Humanos , Masculino , Mitomicina/farmacología , Datos de Secuencia Molecular , Mutación/genética , Proteínas Nucleares/química , Linaje , Fenotipo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , ARN Mensajero/análisis , ARN Mensajero/genética , Alineación de Secuencia , Transducción Genética

RESUMEN

Following introduction of DNA interstrand cross-links (ICLs), mammalian cells display chromosome breakage or cell cycle delay with a 4N DNA content. To further understand the nature of the delay, previously described as a G(2)/M arrest, we developed a protocol to generate ICLs during specific intervals of the cell cycle. Synchronous populations of G(1), S, and G(2) cells were treated with photoactivated 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) and scored for normal passage into mitosis. In contrast to what was found for ionizing radiation, ICLs introduced during G(2) did not result in a G(2)/M arrest, mitotic arrest, or chromosome breakage. Rather, subsequent passage through S phase was required to trigger both chromosome breakage and arrest in the next cell cycle. Similarly, ICLs introduced during G(1) did not cause a G(1)/S arrest. We conclude that DNA replication is required to elicit the cellular responses of cell cycle arrest and genomic instability after psoralen-induced ICLs. In primary human fibroblasts, the 4N DNA content cell cycle arrest triggered by ICLs was long lasting but reversible. Kinetic analysis suggested that these cells could remove up to approximately 2,500 ICLs/genome at an average rate of 11 ICLs/genome/h.

Asunto(s)

Ciclo Celular , Reactivos de Enlaces Cruzados/farmacología , Replicación del ADN , ADN/metabolismo , Trioxsaleno/análogos & derivados , Trioxsaleno/farmacología , Bromodesoxiuridina/metabolismo , Ciclo Celular/efectos de los fármacos , Separación Celular , Células Cultivadas , Medio de Cultivo Libre de Suero/metabolismo , Replicación del ADN/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Fibroblastos/efectos de los fármacos , Citometría de Flujo , Humanos , Cinética , Masculino , Mitosis/efectos de los fármacos , Modelos Biológicos , Factores de Tiempo , Rayos Ultravioleta

RESUMEN

Fanconi anemia (FA) is a rare autosomal recessive disease manifested by bone-marrow failure and an elevated incidence of cancer. Cells taken from patients exhibit spontaneous chromosomal breaks and rearrangements. These breaks and rearrangements are greatly elevated by treatment of FA cells with the use of DNA cross-linking agents. The FA complementation group D gene (FANCD) has previously been localized to chromosome 3p22-26, by use of microcell-mediated chromosome transfer. Here we describe the use of noncomplemented microcell hybrids to identify small overlapping deletions that narrow the FANCD critical region. A 1.2-Mb bacterial-artificial-chromosome (BAC)/P1 contig was constructed, bounded by the marker D3S3691 distally and by the gene ATP2B2 proximally. The contig contains at least 36 genes, including the oxytocin receptor (OXTR), hOGG1, the von Hippel-Lindau tumor-suppressor gene (VHL), and IRAK-2. Both hOGG1 and IRAK-2 were excluded as candidates for FANCD. BACs were then used as probes for FISH analyses, to map the extent of the deletions in four of the noncomplemented microcell hybrid cell lines. A narrow region of common overlapping deletions limits the FANCD critical region to approximately 200 kb. The three candidate genes in this region are TIGR-A004X28, SGC34603, and AA609512.

Asunto(s)

Deleción Cromosómica , Cromosomas Humanos Par 3/genética , Anemia de Fanconi/genética , Southern Blotting , Línea Celular , Rotura Cromosómica/genética , Mapeo Contig , ADN Complementario/genética , ADN-Formamidopirimidina Glicosilasa , Etiquetas de Secuencia Expresada , Anemia de Fanconi/patología , Prueba de Complementación Genética , Ligamiento Genético/genética , Marcadores Genéticos/genética , Humanos , Células Híbridas , Hibridación Fluorescente in Situ , Quinasas Asociadas a Receptores de Interleucina-1 , N-Glicosil Hidrolasas/genética , N-Glicosil Hidrolasas/fisiología , Proteínas Quinasas/genética , Proteínas Quinasas/fisiología , Lugares Marcados de Secuencia

RESUMEN

The gene for maleylacetoacetate isomerase (MAAI) (EC 5.2.1.2) was the last gene in the mammalian phenylalanine/tyrosine catabolic pathway to be cloned. We have isolated the human and murine genes and determined their genomic structure. The human gene spans a genomic region of approximately 10 kb, has 9 exons ranging from 50 to 528 bp in size, and was mapped to 14q24.3-14q31.1 using fluorescence in situ hybridization. The complete catabolic pathway of phenylalanine/tyrosine is normally restricted to liver and kidney, but the maleylacetoacetate isomerase gene is expressed ubiquitously. This suggests a possible second role for the MAAI protein different from phenylalanine/tyrosine catabolism. We have searched for mutations in the maleylacetoacetate isomerase gene in four cases of unexplained severe liver failure in infancy with clinical similarities to hereditary tyrosinemia type I (pseudotyrosinemia). Several amino acid changes were identified, but all were found to retain MAAI activity and thus represent protein polymorphisms. We conclude that MAAI deficiency is not a common cause of the pseudotyrosinemic phenotype.

Asunto(s)

Genes/genética , cis-trans-Isomerasas/genética , Alelos , Errores Innatos del Metabolismo de los Aminoácidos/enzimología , Errores Innatos del Metabolismo de los Aminoácidos/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Bandeo Cromosómico , Mapeo Cromosómico , Cromosomas Humanos Par 14/genética , ADN/química , ADN/genética , Femenino , Expresión Génica , Variación Genética , Humanos , Hibridación Fluorescente in Situ , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , ARN/genética , ARN/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Distribución Tisular , Tirosina/sangre

RESUMEN

Bacterial Artificial Chromosomes (BACs) have been used to complement a metabolic defect and to transfer a drug resistance marker into mammalian cells by electroporation. The selectable markers are stable and the recipient cells have BAC DNA integrated into the chromosomes as shown by fluorescent in situ hybridization, PCR and Southern hybridization.

Asunto(s)

Cromosomas Bacterianos/genética , Electroporación , Prueba de Complementación Genética , Secuencia de Bases , Línea Celular , Cartilla de ADN/genética , Técnicas de Transferencia de Gen , Marcadores Genéticos , Técnicas Genéticas , Humanos , Hibridación Fluorescente in Situ , Reacción en Cadena de la Polimerasa

RESUMEN

Cells from individuals with Fanconi anemia (FA) arrest excessively in the G2/M cell cycle compartment after exposure to low doses of DNA cross-linking agents. The relationship of this abnormality to the fundamental genetic defect in such cells is unknown, but many investigators have speculated that the various FA genes directly regulate cell cycle checkpoints. We tested the hypothesis that the protein encoded by the FA group C complementing gene (FAC) functions to control a cell cycle checkpoint and that cells from group C patients (FA[C]) have abnormalities of cell cycle regulation directly related to the genetic mutation. We found that retroviral transduction of FA(C) lymphoblasts with wild-type FAC cDNA resulted in normalization of the cell cycle response to low-dose mitomycin C (MMC). However, when DNA damage was quantified in terms of cytogenetic damage or cellular cytotoxicity, we found similar degrees of G2/M arrest in response to equitoxic amounts of MMC in FA(C) cells as well as in normal lymphoblasts. Similar results were obtained using isogenic pairs of uncorrected, FAC- or mock-corrected (neo only) FA(C) cell lines. To test the function of other checkpoints we examined the effects of hydroxyurea (HU) and ionizing radiation on cell cycle kinetics of FA(C) and normal lymphoblasts as well as with isogenic pairs of uncorrected, FAC-corrected, or mock-corrected FA(C) cell lines. In all cases the cell cycle response of FA(C) and normal lymphoblasts to these two agents were identical. Based on these studies we conclude that the aberrant G2/M arrest that typifies the response of FA(C) cells to low doses of cross-linking agents does not represent an abnormal cell cycle response but instead represents a normal cellular response to the excessive DNA damage that results in FA(C) cells following exposure to low doses of cross-linking agents.

Asunto(s)

Cafeína/farmacología , Reactivos de Enlaces Cruzados/farmacología , Daño del ADN/efectos de los fármacos , ADN/efectos de los fármacos , Anemia de Fanconi/patología , Fase G2/efectos de los fármacos , Hidroxiurea/farmacología , Linfocitos/efectos de los fármacos , Metafase/efectos de los fármacos , Mitomicina/farmacología , Línea Celular Transformada , ADN/efectos de la radiación , Daño del ADN/efectos de la radiación , ADN Complementario/genética , Anemia de Fanconi/genética , Fase G2/efectos de la radiación , Humanos , Linfocitos/patología , Linfocitos/efectos de la radiación , Metafase/efectos de la radiación , Transfección

RESUMEN

Fanconi anemia is a rare autosomal recessive disease characterized by developmental defects of the thumb and radius, childhood onset of pancytopenic anemia and increased risk of leukemia. At least five complementation groups (A-E) have been defined but only the FAC gene has been cloned. Cells can be assigned to complementation group C by direct mutation analysis. To facilitate the search for additional FA genes and to measure the frequency of complementation groups, we have established new genetically marked immortalized FA-A and FA-D fibroblast cell lines and show their usefulness as universal fusion donors. These reference FA cell lines facilitated somatic cell fusion analysis and enabled us to assign the complementation group in 16 unrelated FA patients from North America. The majority of patients, belong to FA complementation group A (69%), followed by FA-C (18%), FA-D (4%) and FA-B or FA-E (9%).

Asunto(s)

Proteínas de Ciclo Celular , Proteínas de Unión al ADN , Anemia de Fanconi/genética , Proteínas Nucleares , Fusión Celular , Supervivencia Celular , Análisis Mutacional de ADN , Compuestos Epoxi/toxicidad , Proteína del Grupo de Complementación C de la Anemia de Fanconi , Proteínas del Grupo de Complementación de la Anemia de Fanconi , Genes , Prueba de Complementación Genética , Ligamiento Genético , Humanos , Mitomicina/toxicidad , América del Norte , Proteínas/genética , Transfección

RESUMEN

Cells from patients with Fanconi anemia (FA) show decreased viability and decreased chromosome stability after treatment with DNA cross-linking agents, compared to normal cells. FA cells also show a relative accumulation at the G2/M transition after such treatment. This has suggested a possible checkpoint abnormality. In the studies presented here, treatment with hydroxyurea, caffeine or inhibitors of cell cycle kinases did not reveal abnormalities in survival or chromosome stability in FA-A or FA-D cells. Chromosomal breaks introduced by hydrogen peroxide or methyl methanesulfonate accumulated to the same extent in FA-A or FA-D cells as in normal cells. We conclude that FA-A and FA-D cells respond normally to agents known to alter the cell cycle or introduce DNA strand breaks. FA cells process strand breaks and a variety of DNA monoadducts normally. Our results are compatible with repair of DNA crosslinks being slower in FA than in normal cells and FA cells having normal cell cycle checkpoints.

Asunto(s)

Cafeína/farmacología , Ciclo Celular/efectos de los fármacos , Estimulantes del Sistema Nervioso Central/farmacología , Reactivos de Enlaces Cruzados/farmacología , Anemia de Fanconi/patología , Antibióticos Antineoplásicos/farmacología , Antineoplásicos Alquilantes/farmacología , Células Cultivadas/efectos de los fármacos , Rotura Cromosómica , Aductos de ADN , Inhibidores Enzimáticos/farmacología , Anemia de Fanconi/genética , Humanos , Peróxido de Hidrógeno/farmacología , Hidroxiurea/farmacología , Metilmetanosulfonato/farmacología , Mitomicina/farmacología , Oxidantes/farmacología

RESUMEN

Fanconi anemia (FA) is an autosomal recessive chromosome instability syndrome characterized by progressive bone marrow (BM) failure, skeletal defects, and increased susceptibility to malignancy. FA cells are hypersensitive to DNA cross-linking agents, oxygen and have cell cycle abnormalities. To develop an animal model of the disease we generated mice homozygous for a targeted deletion of exon 9 of the murine FA complementation group C gene (fac). Mutant mice had normal neonatal viability and gross morphology, but their cells had the expected chromosome breakage and DNA cross-linker sensitivity. Surprisingly, male and female mutant mice had reduced numbers of germ cells and females had markedly impaired fertility. No anemia was detectable in the peripheral blood during the first year of life, but the colony forming capacity of marrow progenitor cells was abnormal in vitro in mutant mice. Progenitor cells from fac knock-out mice were hypersensitive to interferon gamma. This previously unrecognized phenotype may form the basis for BM failure in human FA.

Asunto(s)

Proteínas de Ciclo Celular , Proteínas de Unión al ADN , Anemia de Fanconi/genética , Células Germinativas/patología , Células Madre Hematopoyéticas/efectos de los fármacos , Infertilidad Femenina/genética , Interferón gamma/farmacología , Proteínas Nucleares , Proteínas/genética , Animales , Secuencia de Bases , Ciclo Celular , Células Cultivadas , Quimiocina CCL4 , Aberraciones Cromosómicas , Ensayo de Unidades Formadoras de Colonias , Reactivos de Enlaces Cruzados/farmacología , Daño del ADN , Exones/genética , Anemia de Fanconi/patología , Proteína del Grupo de Complementación C de la Anemia de Fanconi , Proteínas del Grupo de Complementación de la Anemia de Fanconi , Femenino , Factores de Crecimiento de Célula Hematopoyética/farmacología , Células Madre Hematopoyéticas/patología , Proteínas Inflamatorias de Macrófagos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Monocinas/farmacología , Ovario/patología , Proteínas/fisiología , Proteínas Recombinantes/farmacología , Método Simple Ciego , Testículo/patología , Factor de Necrosis Tumoral alfa/farmacología

RESUMEN

Fanconi anemia (FA) is an autosomal recessive disease characterized by birth defects, progressive bone marrow failure and increased risk for leukemia. FA cells display chromosome breakage and increased cell killing in response to DNA crosslinking agents. At least 5 genes have been defined by cell complementation studies, but only one of these, FAC has been cloned to date. Efforts to map and isolate new FA genes by functional complementation have been hampered by the lack of immortalized FA fibroblast cell lines. Here we report the use of a novel immortalization strategy to create 4 new immortalized FA fibroblast lines, including one from the rare complementation group D.

Asunto(s)

Línea Celular Transformada , Anemia de Fanconi , Fibroblastos/citología , Técnicas de Cultivo de Célula , Fusión Celular , Supervivencia Celular/efectos de los fármacos , Aberraciones Cromosómicas , Compuestos Epoxi/farmacología , Metanosulfonato de Etilo/farmacología , Fibroblastos/efectos de los fármacos , Prueba de Complementación Genética , Humanos , Cariotipificación , Mitomicina/farmacología , Mutágenos/farmacología , Fenotipo , Piel/citología

RESUMEN

Fanconi anaemia (FA) is an autosomal recessive disorder characterized by progressive pancytopenia, short stature, radial ray defects, skin hyperpigmentation and a predisposition to cancer. Cells from FA patients are hypersensitive to cell killing and chromosome breakage induced by DNA cross-linking agents such as mitomycin C (MMC) and diepoxybutane (DEB). Consequently, the defect in FA is thought to be in DNA crosslink repair. Additional cellular phenotypes of FA include oxygen sensitivity, poor cell growth and a G2 cell cycle delay. At least 5 complementation groups for Fanconi anaemia exist, termed A through E. One of the five FA genes, FA(C), has been identified by cDNA complementation, but no other FA genes have been mapped or cloned until now. The strategy of cDNA complementation, which was successful for identifying the FA(C) gene has not yet been successful for cloning additional FA genes. The alternative approach of linkage analysis, followed by positional cloning, is hindered in FA by genetic heterogeneity and the lack of a simple assay for determining complementation groups. In contrast to genetic linkage studies, microcell mediated chromosome transfer utilizes functional complementation to identify the disease bearing chromosome. Here we report the successful use of this technique to map the gene for the rare FA complementation group D (FA(D)).

Asunto(s)

Cromosomas Humanos Par 3 , Anemia de Fanconi/genética , Prueba de Complementación Genética , Línea Celular , Mapeo Cromosómico/métodos , Daño del ADN , Anemia de Fanconi/patología , Humanos