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1.
Nucleic Acids Res ; 51(6): 2516-2528, 2023 04 11.
Article in English | MEDLINE | ID: mdl-36652992

ABSTRACT

At meiosis, programmed meiotic DNA double-strand breaks are repaired via homologous recombination, resulting in crossovers (COs). From a large excess of DNA double-strand breaks that are formed, only a small proportion gets converted into COs because of active mechanisms that restrict CO formation. The Fanconi anemia (FA) complex proteins AtFANCM, MHF1 and MHF2 were previously identified in a genetic screen as anti-CO factors that function during meiosis in Arabidopsis thaliana. Here, pursuing the same screen, we identify FANCC as a new anti-CO gene. FANCC was previously only identified in mammals because of low primary sequence conservation. We show that FANCC, and its physical interaction with FANCE-FANCF, is conserved from vertebrates to plants. Further, we show that FANCC, together with its subcomplex partners FANCE and FANCF, regulates meiotic recombination. Mutations of any of these three genes partially rescues CO-defective mutants, which is particularly marked in female meiosis. Functional loss of FANCC, FANCE, or FANCF results in synthetic meiotic catastrophe with the pro-CO factor MUS81. This work reveals that FANCC is conserved outside mammals and has an anti-CO role during meiosis together with FANCE and FANCF.


The Fanconi Anemia (FA) pathway is the subject of intense interest owing to the role of FA as a tumor suppressor. Three FA complex proteins, FANCM, MHF1 and MHF2, were identified as factors that suppress crossover during meiosis in the model plant Arabidopsis thaliana. Here, the authors extended these findings and identified a novel anti-crossover factor and showed that it encodes the plant FANCC homolog, which was previously thought to be vertebrate-specific. They further showed that FANCC regulates meiotic crossover together with two other FA proteins, FANCE and FANCF. This suggests that the FANCC­E­F subcomplex was already regulating DNA repair in the common ancestor of all living eukaryotes.


Subject(s)
Fanconi Anemia Complementation Group C Protein , Fanconi Anemia Complementation Group F Protein , Fanconi Anemia Complementation Group Proteins , Meiosis , Humans , Arabidopsis/genetics , Arabidopsis/metabolism , DNA/metabolism , Fanconi Anemia Complementation Group C Protein/genetics , Fanconi Anemia Complementation Group C Protein/metabolism , Fanconi Anemia Complementation Group F Protein/genetics , Fanconi Anemia Complementation Group F Protein/metabolism , Fanconi Anemia Complementation Group Proteins/genetics , Fanconi Anemia Complementation Group Proteins/metabolism , Homologous Recombination
2.
Nat Struct Mol Biol ; 28(3): 300-309, 2021 03.
Article in English | MEDLINE | ID: mdl-33686268

ABSTRACT

The Fanconi anemia (FA) pathway is essential for the repair of DNA interstrand crosslinks. Central to the pathway is the FA core complex, a ubiquitin ligase of nine subunits that monoubiquitinates the FANCI-FANCD2 (ID) DNA clamp. The 3.1 Å structure of the 1.1-MDa human FA core complex, described here, reveals an asymmetric assembly with two copies of all but the FANCC, FANCE and FANCF subunits. The asymmetry is crucial, as it prevents the binding of a second FANCC-FANCE-FANCF subcomplex that inhibits the recruitment of the UBE2T ubiquitin conjugating enzyme, and instead creates an ID binding site. A single active site then ubiquitinates FANCD2 and FANCI sequentially. We also present the 4.2-Å structures of the human core-UBE2T-ID-DNA complex in three conformations captured during monoubiquitination. They reveal the core-UBE2T complex remodeling the ID-DNA complex, closing the clamp on the DNA before ubiquitination. Monoubiquitination then prevents clamp opening after release from the core.


Subject(s)
DNA/metabolism , Fanconi Anemia Complementation Group Proteins/chemistry , Fanconi Anemia Complementation Group Proteins/metabolism , Multienzyme Complexes/chemistry , Multienzyme Complexes/metabolism , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/metabolism , Binding Sites , Cryoelectron Microscopy , DNA/chemistry , DNA/ultrastructure , Fanconi Anemia Complementation Group C Protein/metabolism , Fanconi Anemia Complementation Group D2 Protein/metabolism , Fanconi Anemia Complementation Group E Protein/metabolism , Fanconi Anemia Complementation Group F Protein/metabolism , Fanconi Anemia Complementation Group Proteins/ultrastructure , HEK293 Cells , Humans , Models, Molecular , Multienzyme Complexes/ultrastructure , Reproducibility of Results , Ubiquitin-Conjugating Enzymes/metabolism , Ubiquitin-Protein Ligases/ultrastructure , Ubiquitination , Ubiquitins/metabolism
3.
Cell Death Dis ; 10(9): 666, 2019 09 11.
Article in English | MEDLINE | ID: mdl-31511498

ABSTRACT

Adriamycin(ADR) is still considered to be one of the most effective agents in the treatment of breast cancer (BrCa), its efficacy is compromised by intrinsic resistance or acquire characteristics of multidrug resistance. At present, there are few genetic alterations that can be exploited as biomarkers to guide targeted use of ADR in clinical. Therefore, exploring the determinants of ADR sensitivity is pertinent for their optimal clinical application. TP53 is the most frequently mutated gene in human BrCa, p53 mutation has been reported to be closely related to ADR resistance, whereas the underlying mechanisms that cause endogenous ADR resistance in p53-mutant BrCa cells are not completely understood. The aim of the present study was to investigate the potential roles of miRNA in the response to ADR in p53-mutated breast cancer. Here, we report that BrCa cells expressing mutp53 are more resistant to ADR than cells with wild-type p53 (wtp53). The DNA repair protein- Fanconi anemia complementation group F protein (FANCF) and the translesion synthesis DNA polymerase REV1 protein is frequently abundant in the context of mutant p53 of BrCa. By targeting two key factors, miR-30c increases the sensitivity of BrCa cells to ADR. Furthermore, p53 directly activates the transcription of miR-30c by binding to its promoter. Subsequent analyses revealed that p53 regulates REV1 and FANCF by modulating miR-30c expression. Mutation of the p53 abolished this response. Consistently, reduced miR-30c expression is highly correlated with human BrCa with p53 mutational status and is associated with poor survival. We propose that one of the pathways affected by mutant p53 to increase intrinsic resistance to ADR involves miR-30c downregulation and the consequent upregulation of FANCF and REV1. The novel miRNA-mediated pathway that regulates chemoresistance in breast cancer will facilitate the development of novel therapeutic strategies.


Subject(s)
Antibiotics, Antineoplastic/pharmacology , Breast Neoplasms/genetics , Doxorubicin/pharmacology , Fanconi Anemia Complementation Group F Protein/metabolism , MicroRNAs/metabolism , Nucleotidyltransferases/metabolism , Tumor Suppressor Protein p53/genetics , 3' Untranslated Regions , Animals , Antibiotics, Antineoplastic/therapeutic use , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Proliferation/genetics , Cell Survival/drug effects , Cell Survival/genetics , DNA Damage/drug effects , DNA Damage/genetics , Doxorubicin/therapeutic use , Drug Resistance, Neoplasm/drug effects , Drug Resistance, Neoplasm/genetics , Fanconi Anemia Complementation Group F Protein/genetics , Female , Humans , Mice , Mice, Inbred BALB C , Mice, Nude , MicroRNAs/genetics , Mutation , Nucleotidyltransferases/genetics , Transplantation, Heterologous , Tumor Suppressor Protein p53/chemistry , Tumor Suppressor Protein p53/metabolism
4.
BMC Med Genet ; 20(1): 122, 2019 07 09.
Article in English | MEDLINE | ID: mdl-31288759

ABSTRACT

BACKGROUND: Fanconi anemia (FA) is a heterogeneous genetic disorder characterized by congenital anomalies, early-onset bone marrow failure, and a high predisposition to cancers. Up to know, different genes involved in the DNA repair pathway, mainly FANCA genes, have been identified to be affected in patients with FA. CASE PRESENTATION: Here, we report clinical, laboratory and genetic findings in a 3.5-year-old Iranian female patient, a product of a consanguineous marriage, who was suspicious of FA, observed with short stature, microcephaly, skin hyperpigmentation, anemia, thrombocytopenia and hypo cellular bone marrow. Therefore, Next Generation Sequencing was performed to identify the genetic cause of the disease in this patient. Results revealed a novel, private, homozygous frameshift mutation in the FANCF gene (NM_022725: c. 534delG, p. G178 fs) which was confirmed by Sanger sequencing in the proband. CONCLUSION: Such studies may help uncover the exact pathomechanisms of this disorder and establish the genotype-phenotype correlations by identification of more mutations in this gene. It is the first report of a mutation in the FANCF gene in Iranian patients with Fanconi anemia. This new mutation correlates with a hematological problem (pancytopenia), short stature, and microcephaly and skin hyperpigmentation. Until now, no evidence of malignancy was detected.


Subject(s)
Fanconi Anemia Complementation Group F Protein/genetics , Fanconi Anemia/genetics , Genetic Association Studies , Genetic Predisposition to Disease/genetics , Sequence Deletion , Base Sequence , Child, Preschool , Consanguinity , Fanconi Anemia/physiopathology , Fanconi Anemia Complementation Group F Protein/metabolism , Female , Genotype , High-Throughput Nucleotide Sequencing , Homozygote , Humans , Iran , Pancytopenia/genetics , Pedigree , Sequence Analysis, Protein
5.
J Biomed Sci ; 22: 77, 2015 Sep 18.
Article in English | MEDLINE | ID: mdl-26385482

ABSTRACT

BACKGROUND: Cisplatin is one of the most commonly used chemotherapy agent for lung cancer. The therapeutic efficacy of cisplatin is limited by the development of resistance. In this study, we test the effect of RNA interference (RNAi) targeting Fanconi anemia (FA)/BRCA pathway upstream genes on the sensitivity of cisplatin-sensitive (A549 and SK-MES-1) and -resistant (A549/DDP) lung cancer cells to cisplatin. RESULT: Using small interfering RNA (siRNA), knockdown of FANCF, FANCL, or FANCD2 inhibited function of the FA/BRCA pathway in A549, A549/DDP and SK-MES-1 cells, and potentiated sensitivity of the three cells to cisplatin. The extent of proliferation inhibition induced by cisplatin after knockdown of FANCF and/or FANCL in A549/DDP cells was significantly greater than in A549 and SK-MES-1 cells, suggesting that depletion of FANCF and/or FANCL can reverse resistance of cisplatin-resistant lung cancer cells to cisplatin. Furthermore, knockdown of FANCL resulted in higher cisplatin sensitivity and dramatically elevated apoptosis rates compared with knockdown of FANCF in A549/DDP cells, indicating that FANCL play an important role in the repair of cisplatin-induced DNA damage. CONCLUSION: Knockdown of FANCF, FANCL, or FANCD2 by RNAi could synergize the effect of cisplatin on suppressing cell proliferation in cisplatin-resistant lung cancer cells through inhibition of FA/BRCA pathway.


Subject(s)
BRCA1 Protein , Cisplatin/pharmacology , Drug Resistance, Neoplasm , Fanconi Anemia Complementation Group D2 Protein , Fanconi Anemia Complementation Group F Protein , Fanconi Anemia Complementation Group L Protein , Lung Neoplasms , RNA Interference , Signal Transduction , BRCA1 Protein/antagonists & inhibitors , BRCA1 Protein/genetics , BRCA1 Protein/metabolism , Cell Line, Tumor , Drug Resistance, Neoplasm/drug effects , Drug Resistance, Neoplasm/genetics , Fanconi Anemia Complementation Group D2 Protein/antagonists & inhibitors , Fanconi Anemia Complementation Group D2 Protein/genetics , Fanconi Anemia Complementation Group D2 Protein/metabolism , Fanconi Anemia Complementation Group F Protein/antagonists & inhibitors , Fanconi Anemia Complementation Group F Protein/genetics , Fanconi Anemia Complementation Group F Protein/metabolism , Fanconi Anemia Complementation Group L Protein/antagonists & inhibitors , Fanconi Anemia Complementation Group L Protein/genetics , Fanconi Anemia Complementation Group L Protein/metabolism , Humans , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Signal Transduction/drug effects , Signal Transduction/genetics
6.
Mol Med Rep ; 11(6): 4605-10, 2015 Jun.
Article in English | MEDLINE | ID: mdl-25647473

ABSTRACT

The Fanconi anemia (FA)­associated proteins FANCF and FANCD2 are important components of the FA pathway of DNA crosslink repair. FANCF and FANCD2 have been found to be involved in drug­resistant multiple myeloma, ovarian cancer, non­small­cell lung cancer, and head and neck cancer. However, it is unclear whether these two genes participate in adriamycin (ADR)­resistant leukemia. Therefore, the aim of the current study was to investigate FANCF and FANCD2 expression in drug­resistant and drug­sensitive leukemia cells. Western blot analysis revealed enhanced FANCF expression and monoubiquitination of FANCD2 in ADR­resistant cells. Additionally, it was observed that drug­resistant cells had reduced DNA damage compared with drug­sensitive cells. The results of this study indicate that the FA pathway may confer leukemia resistance to ADR via enhanced DNA interstrand crosslink repair.


Subject(s)
Fanconi Anemia Complementation Group D2 Protein/genetics , Fanconi Anemia Complementation Group F Protein/genetics , Cell Line, Tumor , DNA Damage/drug effects , Doxorubicin/pharmacology , Drug Resistance, Neoplasm , Fanconi Anemia Complementation Group D2 Protein/metabolism , Fanconi Anemia Complementation Group F Protein/metabolism , Gene Expression Regulation/drug effects , Humans , K562 Cells , Leukemia/metabolism , Leukemia/pathology , S Phase Cell Cycle Checkpoints/drug effects , Signal Transduction/drug effects , Ubiquitination/drug effects
7.
Leuk Lymphoma ; 56(3): 755-62, 2015 Mar.
Article in English | MEDLINE | ID: mdl-24996439

ABSTRACT

The Fanconi anemia/BRCA (FA/BRCA) pathway plays a vital role in DNA damage repair induced by DNA cross-linking agents and is closely related to drug response in cancer treatment. Here we demonstrate that the FA/BRCA pathway contributes to acquired drug resistance in adriamycin (ADR)-resistant leukemia cell lines, and disruption of this pathway partially reverses the drug resistance. We observed that ADR-resistant cells have reduced DNA interstrand cross-links (ICL) compared with ADR-sensitive cells. Western blot studies demonstrated enhanced FA protein expression in ADR-resistant cells. Using siRNA to knock down FANCF in K562/R drug-resistant cells showed increases in sensitivity to ADR and ADR-induced DNA damage, and demonstrated a direct relationship between the FA/BRCA pathway and drug sensitivity. Overexpression of FANCF in K562 drug-sensitive cells partially reproduced the drug-resistant phenotype. These results show that the FA/BRCA pathway is involved in acquired ADR resistance of leukemia cells. The FA/BRCA pathway may be a new target to reverse ADR resistance in leukemia treatment.


Subject(s)
BRCA1 Protein/metabolism , BRCA2 Protein/metabolism , Doxorubicin/pharmacology , Fanconi Anemia Complementation Group F Protein/metabolism , Antibiotics, Antineoplastic/pharmacology , Apoptosis/drug effects , Apoptosis/genetics , BRCA1 Protein/genetics , BRCA2 Protein/genetics , Blotting, Western , Cell Cycle/drug effects , Cell Cycle/genetics , Cell Survival/drug effects , Cell Survival/genetics , DNA Damage , DNA Repair , Dose-Response Relationship, Drug , Drug Resistance, Neoplasm/genetics , Fanconi Anemia Complementation Group F Protein/genetics , Gene Expression , Humans , K562 Cells , Leukemia/genetics , Leukemia/metabolism , Leukemia/pathology , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/drug effects , Signal Transduction/genetics
8.
Int J Oncol ; 45(1): 129-38, 2014 Jul.
Article in English | MEDLINE | ID: mdl-24789349

ABSTRACT

The Fanconi anemia/BRCA (FA/BRCA) DNA damage repair pathway plays a pivotal role in the cellular response to DNA alkylating agents and greatly influences drug response in cancer treatment. However, the molecular mechanisms underlying the FA/BRCA pathway reversed resistance have received limited attention. In the present study, we investigated the effect of Fanconi anemia complementation group F protein (FANCF), a critical factor of the FA/BRCA pathway, on cancer cell apoptosis induced by DNA alkylating agents such as mitomycin c (MMC). We found that FANCF shRNA potentiated MMC-induced cytotoxicity and apoptosis in MCF-7 and MDA-MB-231 breast cancer cells. At a mechanistic level, FANCF shRNA downregulated the anti-apoptotic protein Bcl-2 and upregulated the pro-apoptotic protein Bax, accompanied by release of cyt-c and smac into the cytosol in MMC-treated cells. Furthermore, activation of caspase-3 and -9, other than caspase-8, cleavage of poly(ADP ribose) polymerase (PARP), and a decrease of mitochondrial membrane potential (MMP) indicated that involvement of the mitochondrial apoptotic pathway in FANCF silencing of MMC-treated breast cancer cells. A decrease in IAP family proteins XIAP and survivin were also observed following FANCF silencing in MMC-treated breast cancer cells. Notably, FANCF shRNA was able to increase p53 levels through activation of the JNK pathway in MMC-treated breast cancer cells. Furthermore, p53 inhibition using pifithrin-α abolished the induction of caspase-3 and PARP by FANCF shRNA and MMC, indicating that MMC-induced apoptosis is substantially enhanced by FANCF shRNA via p53-dependent mechanisms. To our knowledge, we provide new evidence for the potential application of FANCF as a chemosensitizer in breast cancer therapy.


Subject(s)
Alkylating Agents/pharmacology , Breast Neoplasms/metabolism , Fanconi Anemia Complementation Group F Protein/antagonists & inhibitors , Mitochondria/metabolism , Mitomycin/pharmacology , Apoptosis/drug effects , Breast Neoplasms/pathology , Cell Line, Tumor , Fanconi Anemia Complementation Group F Protein/metabolism , Female , Gene Expression Regulation, Neoplastic , Humans , MCF-7 Cells , Mitochondria/drug effects , RNA, Small Interfering/metabolism , Signal Transduction
9.
Oncol Rep ; 29(5): 1721-9, 2013 May.
Article in English | MEDLINE | ID: mdl-23440494

ABSTRACT

In the present study, we downregulated FANCF expression by small interfering RNA (siRNA) in OVCAR ovarian cancer cells to address the effects of decreased FANCF expression on the function of the Fanconi anemia (FA)/breast cancer susceptibility gene (BRCA) pathway. Furthermore, we investigated whether this method increases the sensitivity of OVCAR3 cells to adriamycin (ADM) and the possible mechanism(s). We found that silencing of FANCF inactivated the FA/BRCA pathway by decreasing the monoubiquitination and focus formation of FANCD2 and reduced the function of the FA/BRCA pathway, resulting in the inhibition of cell proliferation, increased cell apoptosis and DNA damage in OVCAR3 cells. Moreover, we observed that silencing of FANCF enhanced the antiproliferative effect of ADM in OVCAR3 cells and increased ADM intracellular accumulation consequently sensitizing OVCAR3 cells to ADM. Furthermore, silencing of FANCF increased cell apoptosis of OVCAR3 cells which was caused by decreased mitochondrial membrane potential (MMP)-induced DNA damage, activated Jun N-terminal kinase (JNK), increased release of cytochrome c, increased expression of cleaved caspase-3 and poly(ADP-ribose) polymerase (PARP) dependent on JNK activation following treatment of ADM. Collectively, we confirm that silencing of FANCF sensitizes OVCAR3 ovarian cancer cells to ADM, suggesting that FANCF may serve as a potential target for therapeutic strategies in the treatment of ovarian cancer.


Subject(s)
Apoptosis/drug effects , Doxorubicin/pharmacology , Fanconi Anemia Complementation Group F Protein/genetics , JNK Mitogen-Activated Protein Kinases/genetics , Ovarian Neoplasms/genetics , Ovarian Neoplasms/therapy , RNA, Small Interfering/genetics , Apoptosis/genetics , BRCA1 Protein/genetics , BRCA1 Protein/metabolism , Caspase 3/genetics , Caspase 3/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Combined Modality Therapy , Cytochromes c/genetics , Cytochromes c/metabolism , DNA Damage/drug effects , DNA Damage/genetics , Down-Regulation/drug effects , Down-Regulation/genetics , Fanconi Anemia/genetics , Fanconi Anemia/metabolism , Fanconi Anemia Complementation Group F Protein/metabolism , Female , Humans , JNK Mitogen-Activated Protein Kinases/metabolism , Membrane Potential, Mitochondrial/drug effects , Membrane Potential, Mitochondrial/genetics , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/metabolism , Poly(ADP-ribose) Polymerases/genetics , Poly(ADP-ribose) Polymerases/metabolism , RNA Interference , Signal Transduction/drug effects , Signal Transduction/genetics
10.
PLoS One ; 7(8): e44254, 2012.
Article in English | MEDLINE | ID: mdl-22952942

ABSTRACT

Fanconi anemia complementation group-F (FANCF) is a key factor to maintain the function of FA/BRCA, a DNA-damage response pathway. However, the functional role of FANCF in breast cancer has not been elucidated. In this study, we examined the effects and mechanisms of FANCF-RNAi on the sensitivity of breast cancer cells to mitoxantrone (MX). FANCF silencing by FANCF-shRNA blocked functions of FA/BRCA pathway through inhibition of FANCD2 mono-ubiquitination in breast cancer cell lines MCF-7 and T-47D. In addition, FANCF shRNA inhibited cell proliferation, induced apoptosis, and chromosome fragmentation in both breast cancer cells. We also found that FANCF silencing potentiated the sensitivity to MX in breast cancer cells, accompanying with an increase in intracellular MX accumulation and a decrease in BCRP expression. Furthermore, we found that the blockade of FA/BRCA pathway by FANCF-RNAi activated p38 and JNK MAPK signal pathways in response to MX treatment. BCRP expression was restored by p38 inhibitor SB203580, but not by JNK inhibitor SP600125. FANCF silencing increased JNK and p38 mediated activation of p53 in MX-treated breast cancer cells, activated the mitochondrial apoptosis pathway. Our findings indicate that FANCF shRNA potentiates the sensitivity of breast cancer cells to MX, suggesting that FANCF may be a potential target for therapeutic strategies for the treatment of breast tumors.


Subject(s)
Breast Neoplasms/enzymology , Fanconi Anemia Complementation Group F Protein/genetics , Gene Silencing/drug effects , JNK Mitogen-Activated Protein Kinases/metabolism , MAP Kinase Signaling System/drug effects , Mitoxantrone/pharmacology , p38 Mitogen-Activated Protein Kinases/metabolism , ATP Binding Cassette Transporter, Subfamily G, Member 2 , ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/metabolism , Apoptosis/drug effects , BRCA1 Protein/metabolism , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Line, Tumor , Cell Proliferation/drug effects , Down-Regulation/drug effects , Down-Regulation/genetics , Drug Screening Assays, Antitumor , Enzyme Activation/drug effects , Fanconi Anemia/enzymology , Fanconi Anemia/pathology , Fanconi Anemia Complementation Group D2 Protein/metabolism , Fanconi Anemia Complementation Group F Protein/metabolism , Female , Gene Expression Regulation, Neoplastic/drug effects , Gene Knockdown Techniques , Humans , Mitochondria/drug effects , Mitochondria/metabolism , Mitoxantrone/therapeutic use , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , RNA, Small Interfering/metabolism , Tumor Suppressor Protein p53/metabolism , Ubiquitination/drug effects , Up-Regulation/drug effects , Up-Regulation/genetics
11.
Mol Cell Biochem ; 365(1-2): 29-35, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22297615

ABSTRACT

Curcumin and resveratrol were evaluated for their potential to cause reversal of promoter hypermethylation and associated gene expression of FANCF in SiHa cell line. Methylation specific PCR along with bisulphite sequencing revealed the demethylation of 12 CpG sites out of 15 CpG sites spanning ?280 to ?432 region of FANCF promoter after treatment with curcumin and fivefold up regulation of FANCF gene expression as shown by qRT-PCR. In vitro methylation assay also showed that M.SssI an analogue of DNMT1 was effectively inhibited at 50 lM concentration of curcumin. Resveratrol was not found to be effective in causing reversal of promoter hypermethylation of FANCF gene when used at 20 lM for 4 days in SiHa cell line.


Subject(s)
Curcumin/pharmacology , DNA Methylation , Fanconi Anemia Complementation Group F Protein/metabolism , Promoter Regions, Genetic , Transcription, Genetic/drug effects , Azacitidine/analogs & derivatives , Azacitidine/pharmacology , Base Sequence , Cell Line, Tumor , Cell Survival/drug effects , DNA (Cytosine-5-)-Methyltransferase 1 , DNA (Cytosine-5-)-Methyltransferases/antagonists & inhibitors , DNA (Cytosine-5-)-Methyltransferases/chemistry , DNA, Complementary/chemistry , DNA-Cytosine Methylases/chemistry , Decitabine , Epigenesis, Genetic , Fanconi Anemia Complementation Group F Protein/genetics , Humans , Inhibitory Concentration 50 , Resveratrol , Sequence Analysis, DNA , Stilbenes/pharmacology
12.
DNA Repair (Amst) ; 10(2): 149-58, 2011 Feb 07.
Article in English | MEDLINE | ID: mdl-21109493

ABSTRACT

Fanconi anemia (FA) is an inherited disease characterized by bone marrow failure, increased cancer risk and hypersensitivity to DNA cross-linking agents, implying a role for this pathway in the maintenance of genomic stability. The central player of the FA pathway is the multi-subunit E3 ubiquitin ligase complex activated through a replication- and DNA damage-dependent mechanism. A consequence of the activation of the complex is the monoubiquitylation of FANCD2 and FANCI, late term effectors in the maintenance of genome integrity. The details regarding the coordination of the FA-dependent response and the DNA replication process are still mostly unknown. We found, by yeast two-hybrid assay and co-immunoprecipitation in human cells, that the core complex subunit FANCF physically interacts with PSF2, a member of the GINS complex essential for both the initiation and elongation steps of DNA replication. In HeLa cells depleted for PSF2, we observed a decreased binding to chromatin of the FA core complex, suggesting that the GINS complex may have a role in either loading or stabilizing the FA core complex onto chromatin. Consistently, GINS and core complex bind chromatin contemporarily upon origin firing and PSF2 depletion sensitizes cells to DNA cross-linking agents. However, depletion of PSF2 is not sufficient to reduce monoubiquitylation of FANCD2 or its localization to nuclear foci following DNA damage. Our results suggest a novel crosstalk between DNA replication and the FA pathway.


Subject(s)
Chromosomal Proteins, Non-Histone/metabolism , DNA Replication , Fanconi Anemia Complementation Group D2 Protein/metabolism , Fanconi Anemia Complementation Group F Protein/metabolism , Fanconi Anemia/metabolism , Chromatin/genetics , Chromatin/physiology , Chromosomal Proteins, Non-Histone/genetics , DNA Damage , Fanconi Anemia/genetics , Fanconi Anemia Complementation Group D2 Protein/genetics , Fanconi Anemia Complementation Group F Protein/genetics , Fanconi Anemia Complementation Group Proteins/genetics , Fanconi Anemia Complementation Group Proteins/metabolism , Genomic Instability , HeLa Cells , Humans , Immunoprecipitation , Protein Binding , Two-Hybrid System Techniques , Ubiquitin-Protein Ligases/metabolism , Ubiquitination
13.
J Biol Chem ; 284(48): 33242-54, 2009 Nov 27.
Article in English | MEDLINE | ID: mdl-19801548

ABSTRACT

The interferon consensus sequence binding protein (ICSBP) is an interferon regulatory transcription factor with leukemia-suppressor activity. ICSBP regulates genes that are involved in phagocyte function, proliferation, and apoptosis. In murine models ICSBP deficiency results in a myeloproliferative disorder (MPD) with increased mature neutrophils. Over time this MPD progresses to acute myeloid leukemia (AML), suggesting that ICSBP deficiency is adequate for MPD, but additional genetic lesions are required for AML. The hypothesis of these studies is that dysregulation of key target genes predisposes to disease progression under conditions of decreased ICSBP expression. To investigate this hypothesis, we used chromatin co-immunoprecipitation to identify genes involved the ICSBP-leukemia suppressor effect. In the current studies, we identify the gene encoding Fanconi F (FANCF) as an ICSBP target gene. FancF participates in a repair of cross-linked DNA. We identify a FANCF promoter cis element, which is activated by ICSBP in differentiating myeloid cells. We also determine that DNA cross-link repair is impaired in ICSBP-deficient myeloid cells in a FancF-dependent manner. This effect is observed in differentiating cells, suggesting that ICSBP protects against the genotoxic stress of myelopoiesis. Decreased ICSBP expression is found in human AML and chronic myeloid leukemia during blast crisis (CML-BC). Our studies suggest that ICSBP deficiency may be functionally important for accumulation of chromosomal abnormalities during disease progression in these myeloid malignancies.


Subject(s)
Cell Differentiation , Fanconi Anemia Complementation Group F Protein/metabolism , Interferon Regulatory Factors/metabolism , Transcriptional Activation , Animals , Blotting, Western , Cells, Cultured , Chromatin Immunoprecipitation , DNA Repair , Fanconi Anemia Complementation Group F Protein/genetics , Humans , Interferon Regulatory Factors/deficiency , Interferon Regulatory Factors/genetics , Leukemia, Myeloid/genetics , Leukemia, Myeloid/metabolism , Leukemia, Myeloid/pathology , Mice , Mice, Inbred C57BL , Mice, Knockout , Myeloid Progenitor Cells/cytology , Myeloid Progenitor Cells/metabolism , Promoter Regions, Genetic/genetics , Protein Binding , RNA Interference , Reverse Transcriptase Polymerase Chain Reaction , Transfection , U937 Cells
14.
Oncogene ; 27(26): 3641-52, 2008 Jun 12.
Article in English | MEDLINE | ID: mdl-18212739

ABSTRACT

Fanconi anemia (FA) is a human disorder characterized by cancer susceptibility and cellular sensitivity to DNA crosslinks and other damages. Thirteen complementation groups and genes are identified, including BRCA2, which is defective in the FA-D1 group. Eight of the FA proteins, including FANCG, participate in a nuclear core complex that is required for the monoubiquitylation of FANCD2 and FANCI. FANCD2, like FANCD1/BRCA2, is not part of the core complex, and we previously showed direct BRCA2-FANCD2 interaction using yeast two-hybrid analysis. We now show in human and hamster cells that expression of FANCG protein, but not the other core complex proteins, is required for co-precipitation of BRCA2 and FANCD2. We also show that phosphorylation of FANCG serine 7 is required for its co-precipitation with BRCA2, XRCC3 and FANCD2, as well as the direct interaction of BRCA2-FANCD2. These results argue that FANCG has a role independent of the FA core complex, and we propose that phosphorylation of serine 7 is the signalling event required for forming a discrete complex comprising FANCD1/BRCA2-FANCD2-FANCG-XRCC3 (D1-D2-G-X3). Cells that fail to express either phospho-Ser7-FANCG, or full length BRCA2 protein, lack the interactions amongst the four component proteins. A role for D1-D2-G-X3 in homologous recombination repair (HRR) is supported by our finding that FANCG and the RAD51-paralog XRCC3 are epistatic for sensitivity to DNA crosslinking compounds in DT40 chicken cells. Our findings further define the intricate interface between FANC and HRR proteins in maintaining chromosome stability.


Subject(s)
BRCA2 Protein/metabolism , DNA-Binding Proteins/metabolism , Fanconi Anemia Complementation Group D2 Protein/metabolism , Fanconi Anemia Complementation Group G Protein/physiology , Animals , Ataxia Telangiectasia Mutated Proteins , CHO Cells , Cell Cycle Proteins/physiology , Chickens , Chromosomal Instability , Cricetinae , Cricetulus , Fanconi Anemia Complementation Group A Protein/metabolism , Fanconi Anemia Complementation Group F Protein/metabolism , Humans , Phosphorylation , Protein Serine-Threonine Kinases/physiology , Recombination, Genetic , Serine/metabolism
15.
J Mol Med (Berl) ; 85(5): 497-509, 2007 May.
Article in English | MEDLINE | ID: mdl-17221219

ABSTRACT

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.


Subject(s)
Antineoplastic Agents, Alkylating/pharmacology , Brain Neoplasms/drug therapy , DNA Repair/drug effects , Drug Resistance, Neoplasm , Fanconi Anemia Complementation Group Proteins/metabolism , Glioma/drug therapy , Protein Processing, Post-Translational/drug effects , Antineoplastic Agents, Alkylating/therapeutic use , Brain Neoplasms/genetics , Brain Neoplasms/metabolism , Brain Neoplasms/pathology , Carmustine/pharmacology , Cell Line, Tumor , Curcumin/pharmacology , DNA Modification Methylases/antagonists & inhibitors , DNA Modification Methylases/metabolism , DNA Repair Enzymes/antagonists & inhibitors , DNA Repair Enzymes/metabolism , Dacarbazine/analogs & derivatives , Dacarbazine/pharmacology , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , Fanconi Anemia Complementation Group D2 Protein/metabolism , Fanconi Anemia Complementation Group F Protein/metabolism , Fanconi Anemia Complementation Group Proteins/genetics , Glioma/genetics , Glioma/metabolism , Glioma/pathology , Guanine/analogs & derivatives , Guanine/pharmacology , Humans , RNA Interference , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Temozolomide , Time Factors , Transfection , Tumor Suppressor Proteins/antagonists & inhibitors , Tumor Suppressor Proteins/metabolism , Ubiquitins/metabolism
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