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At least 19 recordsLinked to original sources
PubMed2000

The Fanconi anemia protein FANCF forms a nuclear complex with FANCA, FANCC and FANCG.

Fanconi anemia (FA) is a chromosomal instability syndrome associated with a strong predisposition to cancer, particularly acute myeloid leukemia and squamous cell carcinoma. At the cellular level, FA is characterized by spontaneous chromosomal breakage and a unique hypersensitivity to DNA cross-linking agents. Complementation analysis has indicated that at least seven distinct genes are involved in the pathogenesis of FA. Despite the identification of four of these genes (FANCA, FANCC, FANCF and FANCG), the nature of the 'FA pathway' has remained enigmatic, as the FA proteins lack sequence homologies or motifs that could point to a molecular function. To further define this pathway, we studied the subcellular localizations and mutual interactions of the FA proteins, including the recently identified FANCF protein, in human lymphoblasts. FANCF was found predominantly in the nucleus, where it complexes with FANCA, FANCC and FANCG. These interactions were detected in wild-type and FA-D lymphoblasts, but not in lymphoblasts of other FA complementation groups. This implies that each of the FA proteins, except FANCD, is required for these complexes to form. Similarly, we show that the interaction between FANCA and FANCC is restricted to wild-type and FA-D cells. Furthermore, we document the subcellular localization of FANCA and the FANCA/FANCG complex in all FA complementation groups. Our results, along with published data, culminate in a model in which a multi-protein FA complex serves a nuclear function to maintain genomic integrity.

Antibody Specificity↗
PubMed2000

Aberrant Fanconi anaemia protein profiles in acute myeloid leukaemia cells.

Fanconi anaemia (FA) is an autosomal recessive disease strongly predisposing to bone marrow failure and acute myeloid leukaemia (AML). Four FA genes, corresponding to complementation groups A, C, F and G, have been cloned, but the molecular functions of the corresponding proteins are unknown. The high risk of AML in FA patients suggests that the 'FA pathway' helps to prevent AML in non-FA individuals. We examined 10 AML cell lines, as well as primary cells from 15 AML patients representing the French-American-British subclasses M1-M5a, for possible deficiencies in the 'FA pathway'. Cellular lysates were analysed for the presence of the FA proteins FANCA, FANCC, FANCF and FANCG, as well as the complexes reported to be formed between these proteins, using immunoprecipitation and Western blot analysis. Aberrant protein profiles were observed in five of the 10 cell lines and in 11 of the 15 primary AML samples. Aberrations, that included absence or reduced presence of FA proteins and/or their complexes, were noted in the subclasses M1-M4, but not in M5a (n = 3). Our results suggest that a significant proportion of general AML is characterized by a disturbance of the 'FA pathway' that may represent an early event in the development of this type of leukaemia.

Acute Disease↗
PubMed2002

Heterogeneous activation of the Fanconi anemia pathway by patient-derived FANCA mutants.

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.

Amino Acid Substitution↗
PubMed2000

Association of complementation group and mutation type with clinical outcome in fanconi anemia. European Fanconi Anemia Research Group.

Fanconi anemia (FA) is a clinically and genetically heterogeneous disorder. Clinical care is complicated by variable age at onset and severity of hematologic symptoms. Recent advances in the molecular biology of FA have allowed us to investigate the relationship between FA genotype and the nature and severity of the clinical phenotype. Two hundred forty-five patients from all 7 known complementation groups (FA-A to FA-G) were studied. Mutations were detected in one of the cloned FANC genes in 169 patients; in the remainder the complementation group was assigned by cell fusion or Western blotting. A range of qualitative and quantitative clinical parameters was compared for each complementation group and for different classes of mutation. Significant phenotypic differences were found. FA-G patients had more severe cytopenia and a higher incidence of leukemia. Somatic abnormalities were less prevalent in FA-C, but more common in the rare groups FA-D, FA-E, and FA-F. In FA-A, patients homozygous for null mutations had an earlier onset of anemia and a higher incidence of leukemia than those with mutations producing an altered protein. In FA-C, there was a later age of onset of aplastic anemia and fewer somatic abnormalities in patients with the 322delG mutation, but there were more somatic abnormalities in patients with IVS4 + 4A --> T. This study indicates that FA patients with mutations in the FANCG gene and patients homozygous for null mutations in FANCA are high-risk groups with a poor hematologic outcome and should be considered as candidates both for frequent monitoring and early therapeutic intervention. (Blood. 2000;96:4064-4070)

Abnormalities, Multiple↗
PubMed2001

Current knowledge on the pathophysiology of Fanconi anemia: from genes to phenotypes.

Fanconi anemia (FA) is an autosomal recessive disease characterized by congenital anomalies, bone marrow failure, and leukemia susceptibility. FA cells show chromosome instability and hypersensitivity to DNA cross-linking agents such as mitomycin C. Recent studies indicate that there are at least 8 genetically distinct FA groups (A, B, C, D1, D2, E, F, G). To date, 6 genes (for A, C, D2, E, F, and G) have been cloned. In this review, we describe the structures and functions of FA proteins. Increasing evidence indicates that the multiple FA proteins cooperate in a biochemical pathway and/or a multimer complex. FANCD2, a downstream component of the FA pathway, has recently been shown to be ubiquitinated in response to DNA damage and to translocate to nuclear foci containing BRCA1, a breast cancer susceptibility gene product, suggesting a role for this protein in DNA repair functions. We also describe 2 emerging issues: genotype-phenotype relationships and mosaicism. The FA pathway is likely to play a critical role as a caretaker of genomic integrity in hematopoietic stem cells. Clarifying the molecular basis of this disease may provide new insights into the pathogenesis of bone marrow failure syndromes and myeloid malignancies.

Adolescent↗
PubMed2002

FANCE: the link between Fanconi anaemia complex assembly and activity.

The Fanconi anaemia (FA) nuclear complex (composed of the FA proteins A, C, G and F) is essential for protection against chromosome breakage. It activates the downstream protein FANCD2 by monoubiquitylation; this then forges an association with the BRCA1 protein at sites of DNA damage. Here we show that the recently identified FANCE protein is part of this nuclear complex, binding both FANCC and FANCD2. Indeed, FANCE is required for the nuclear accumulation of FANCC and provides a critical bridge between the FA complex and FANCD2. Disease-associated FANCC mutants do not bind to FANCE, cannot accumulate in the nucleus and are unable to prevent chromosome breakage.

Active Transport, Cell Nucleus↗
PubMed2007

The Fanconi anemia (FA) pathway confers glioma resistance to DNA alkylating agents.

DNA alkylating agents including temozolomide (TMZ) and 1,3-bis[2-chloroethyl]-1-nitroso-urea (BCNU) are the most common form of chemotherapy in the treatment of gliomas. Despite their frequent use, the therapeutic efficacy of these agents is limited by the development of resistance. Previous studies suggest that the mechanism of this resistance is complex and involves multiple DNA repair pathways. To better define the pathways contributing to the mechanisms underlying glioma resistance, we tested the contribution of the Fanconi anemia (FA) DNA repair pathway. TMZ and BCNU treatment of FA-proficient cell lines led to a dose- and time-dependent increase in FANCD2 mono-ubiquitination and FANCD2 nuclear foci formation, both hallmarks of FA pathway activation. The FA-deficient cells were more sensitive to TMZ/BCNU relative to their corrected, isogenic counterparts. To test whether these observations were pertinent to glioma biology, we screened a panel of glioma cell lines and identified one (HT16) that was deficient in the FA repair pathway. This cell line exhibited increased sensitivity to TMZ and BCNU relative to the FA-proficient glioma cell lines. Moreover, inhibition of FA pathway activation by a small molecule inhibitor (curcumin) or by small interference RNA suppression caused increased sensitivity to TMZ/BCNU in the U87 glioma cell line. The BCNU sensitizing effect of FA inhibition appeared additive to that of methyl-guanine methyl transferase inhibition. The results presented in this paper underscore the complexity of cellular resistance to DNA alkylating agents and implicate the FA repair pathway as a determinant of this resistance.

Antineoplastic Agents, Alkylating↗
PubMed2001

Direct interactions of the five known Fanconi anaemia proteins suggest a common functional pathway.

Fanconi anaemia (FA) is an autosomal recessive inherited disorder associated with a progressive aplastic anaemia, diverse congenital abnormalities and cancer. The condition is genetically heterogeneous, with at least seven complementation groups (A-G) described. Cells from individuals who are homozygous for mutations in FA genes are characterized by chromosomal instability and hypersensitivity to DNA interstrand crosslinking agents. These features suggest a possible role for the encoded proteins in the recognition or repair of these lesions, but neither their function nor whether they operate in a concerted or discrete functional pathways is known. The recent cloning of the FANCF and FANCE genes has allowed us to investigate the interaction of the proteins encoded by five of the seven complementation groups of FA. We used the yeast two-hybrid system and co-immunoprecipitation analysis to test the 10 possible pairs of proteins for direct interaction. In addition to the previously described binding of FANCA to FANCG, we now demonstrate direct interaction of FANCF with FANCG, of FANCC with FANCE and a weaker interaction of FANCE with both FANCA and FANCG. These findings show that the newly identified FANCE protein is an integral part of the FA pathway, and support the concept of a functional link between all known proteins encoded by the genes that are mutated in this disorder. These proteins may act either as a multimeric complex or by sequential recruitment of subsets of the proteins in a common pathway that protects the genomic integrity of mammalian cells.

Animals↗
PubMed2003

Fanconi anemia protein complex: mapping protein interactions in the yeast 2- and 3-hybrid systems.

Fanconi anemia (FA) is an autosomal recessive syndrome characterized by progressive bone marrow failure and cancer predisposition. Eight FA complementation groups have been identified. The FANCA, FANCC, FANCE, FANCF, and FANCG proteins form a nuclear complex required for the monoubiquination of the FANCD2 protein. To investigate the architecture of the FA protein complex, the yeast 2-hybrid system was used to map contact points of the FANCA/FANCG, FANCC/FANCE, and FANCF/FANCG interactions. FANCG was shown to interact with both the amino-terminus of FANCA and the carboxyl-terminal region of FANCF. A FANCG mutant truncated at the carboxyl-terminus retained the ability to interact with FANCA. The interaction between FANCG and FANCF was ablated by a Leu71Pro mutant of FANCG. A central region of FANCE was sufficient for FANCC binding. A Leu554Pro mutant of FANCC failed to interact with FANCE. To further examine complex assembly, the yeast 3-hybrid system was used to investigate the ability of FANCG to act as a molecular bridge in mediating interaction between other FA proteins. FANCG was able to mediate interaction between FANCA and FANCF, as well as between monomers of FANCA. Direct interaction between FANCE and FANCD2 was also demonstrated in the yeast 2-hybrid system. This interaction involving an amino-terminal region of FANCD2 may provide a link between the FA protein complex and its downstream targets.

Binding Sites↗
PubMed2003

Gene therapy for fanconi anemia.

Fanconi anemia is a hereditary syndrome of bone marrow failure, congenital anomalies, and a predisposition to malignancy. Most patients die from bone marrow failure. Cells from patients display a heightened sensitivity to DNA cross-linking agents with increased chromosomal breakage and increased cytotoxicity. Bone marrow from patients with Fanconi anemia have decreased numbers of hematopoietic progenitors when grown in culture. Transfer of the normal Fanconi anemia cDNA into cells from patients corrects the laboratory abnormalities, suggesting that gene transfer may prevent or reverse the bone marrow failure. Advances in gene transfer into human hematopoietic cells make this approach seem feasible. However, decreased numbers of stem cell targets may represent a significant obstacle. In addition, new insights on potential toxicities related to gene transfer have heightened a cautious approach. Fanconi anemia represents a prototype disorder for gene therapy and highlights the difficulties in adapting this technology to human disease.

Animals↗
PubMed2001

Function of the Fanconi anemia pathway in Fanconi anemia complementation group F and D1 cells.

OBJECTIVE: Fanconi anemia (FA) is a human autosomal-recessive cancer susceptibility disorder characterized by multiple congenital abnormalities, progressive bone marrow failure, and cellular sensitivity to mitomycin C (MMC). FA has at least eight complementation groups (A, B, C, D1, D2, E, F, G), and six of the FA genes have been cloned. Several FA proteins, including FANCA, FANCC, FANCF, and FANCG, interact in a nuclear complex, and this complex is required for the activation (monoubiquitination) of the downstream FANCD2 protein. Activation of FANCD2 results in the assembly of FANCD2/BRCA1 foci. The aim of this study was to analyze the FA pathway in several FA patient-derived cell lines. MATERIALS AND METHODS: We generated an antibody to FANCF and analyzed FANCF expression in human lymphoblasts corresponding to all known FA subtypes. We systematically analyzed the FA pathway (FANCD2 monoubiquitination and assembly of FANCD2 nuclear foci) in patient-derived FA-F and FA-D1 cell lines. RESULTS: FANCF protein expression is normal in cells derived from all FA complementation groups except FA-F and does not vary during cell cycle progression. FANCF, but not FANCD2, is a component of the nuclear FA protein complex and appears to stabilize other subunits of the complex. FANCF is required for the monoubiquitination of the FANCD2 protein following ionizing radiation. FANCD2 is monoubiquitinated in FA-D1 cells, even though these cells are highly sensitive to MMC. CONCLUSIONS: The recently cloned FANCF protein is required for FANCD2 activation, and the yet uncloned FANCD1 protein functions further downstream or independently of the FA pathway.

Cell Line↗
PubMed2003

Evaluation of Fanconi Anemia genes in familial breast cancer predisposition.

Fanconi Anemia (FA) is an autosomal recessive syndrome characterized by congenital abnormalities, progressive bone marrow failure, and susceptibility to cancer. FA has eight known complementation groups and is caused by mutations in at least seven genes. Biallelic BRCA2 mutations were shown recently to cause FA-D1. Monoallelic (heterozygous) BRCA2 mutations confer a high risk of breast cancer and are a major cause of familial breast cancer. To investigate whether heterozygous variants in other FA genes are high penetrance breast cancer susceptibility alleles, we screened germ-line DNA from 88 BRCA1/2-negative families, each with at least three cases of breast cancer, for mutations in FANCA, FANCC, FANCD2, FANCE, FANCF, and FANCG. Sixty-nine sequence variants were identified of which 25 were exonic. None of the exonic variants resulted in translational frameshifts or nonsense codons and 14 were polymorphisms documented previously. Of the remaining 11 exonic variants, 2 resulted in synonymous changes, and 7 were present in controls. Only 2 conservative missense variants, 1 in FANCA and 1 in FANCE, were each found in a single family and were not present in 300 controls. The results indicate that FA gene mutations, other than in BRCA2, are unlikely to be a frequent cause of highly penetrant breast cancer predisposition.

Breast Neoplasms↗
PubMed2005

The FA/BRCA pathway is involved in melphalan-induced DNA interstrand cross-link repair and accounts for melphalan resistance in multiple myeloma cells.

Melphalan, a DNA cross-linker, is one of the most widely used and effective drugs in the treatment of multiple myeloma (MM). In this report, we demonstrate that enhanced interstrand cross-link (ICL) repair via the Fanconi anemia (FA)/BRCA pathway contributes to acquired drug resistance in melphalan-resistant myeloma cell lines, and disruption of this pathway reverses drug resistance. Using the alkaline comet assay (single-cell gel electrophoresis), we observed that melphalan-resistant cells have reduced ICL formation and enhanced ICL repair compared with melphalan-sensitive cells. Cell-cycle studies demonstrated that enhanced ICL repair released cells from melphalan-induced cell-cycle delay. Using siRNA to knock down FANCF in 8226/LR5 and U266/LR6 drug-resistant cells demonstrated a direct relationship between ICL repair capacity and drug sensitivity. Overexpression of FANCF in 8226/S and U266/S drug-sensitive cells partially reproduced the drug-resistant phenotype. These data show that enhanced DNA repair via the Fanconi anemia/BRCA pathway is involved in acquired melphalan resistance. Our findings provide for a new target to enhance response to DNA cross-linking agents in cancer treatment.

Antineoplastic Agents, Alkylating↗
PubMed2006

No evidence of significant silencing of Fanconi genes FANCF and FANCB or Nijmegen breakage syndrome gene NBS1 by DNA hyper-methylation in sporadic childhood leukaemia.

Fanconi anaemia (FA) and Nijmegen breakage syndrome (NBS) carry a high risk of haematological cancer. Affected cellular pathways may be modulated in sporadic malignancies and silencing of FANCF through methylation has been shown to cause somatic disruption of the FA pathway. Combined bisulphite restriction analysis for methylation of FANCF, FANCB and NBS1 was used to investigate 81 sporadic acute childhood leukaemias. No methylation was detected at any associated CpG sites analysed. This does not exclude very low levels of FANCF, FANCB or NBS1 methylation, but suggests other factors are responsible for chemo-sensitivity and chromosomal instability in sporadic childhood leukaemia.

Acute Disease↗
PubMed2004

Hallmarks of 'BRCAness' in sporadic cancers.

Germline mutations in the BRCA1, BRCA2 and Fanconi anaemia genes confer cancer susceptibility, and the proteins encoded by these genes have distinct functions in related DNA-repair processes. Emerging evidence indicates that these processes are disrupted by numerous mechanisms in sporadic cancers. Collectively, there are properties that define 'BRCAness' - that is, traits that some sporadic cancers share with those occurring in either BRCA1- or BRCA2-mutation carriers. These common properties might have important implications for the clinical management of these cancers.

BRCA1 Protein↗
PubMed2004

CpG methylation of the FHIT, FANCF, cyclin-D2, BRCA2 and RUNX3 genes in Granulosa cell tumors (GCTs) of ovarian origin.

BACKGROUND: Granulosa cell tumors (GCTs) are relatively rare and are subtypes of the sex-cord stromal neoplasms. Methylation induced silencing in the promoters of genes such as tumor suppressor genes, DNA repair genes and pro-apoptotic genes is recognised as a critical factor in cancer development. METHODS: We examined the role of promoter hypermethylation, an epigenetic alteration that is associated with the silencing tumor suppressor genes in human cancer, by studying 5 gene promoters in 25 GCTs cases by methylation specific PCR and RT-PCR. In addition, the compatible tissues (normal tissues distant from lesion) from three non-astrocytoma patients were also included as the control. RESULTS: Frequencies of methylation in GCTs were 7/25 (28 % for FHIT), 6/25 (24% for FNACF), 3/25 (12% for Cyclin D2), 1/25 (4% for BRCA2) and 14/25 (56%) in RUNX3 genes. Correlation of promoter methylation with clinical characteristics and other genetic changes revealed that overall promoter methylation was higher in more advanced stage of the disease. Promoter methylation was associated with gene silencing in GCT cell lines. Treatment with methylation or histone deacetylation-inhibiting agents resulted in profound reactivation of gene expression. CONCLUSIONS: These results may have implications in better understanding the underlying epigenetic mechanisms in GCT development, provide prognostic indicators, and identify important gene targets for treatment.

Acid Anhydride Hydrolases↗
PubMed2003

FANCF methylation contributes to chemoselectivity in ovarian cancer.

A new model of ovarian cancer tumor progression implicates aberrant FANCF promoter methylation that is associated with gene silencing and disruption of the Fanconi-anemia-BRCA pathway. Disruption of the pathway occurs de novo in ovarian cancers and may contribute to selective sensitivity to platinum salts.

Alleles↗