Fanconi Anemia Group Research Articles

Citrinin Provoke DNA Damage and Cell-Cycle Arrest Related to Chk2 and FANCD2 Checkpoint Proteins in Hepatocellular and Adenocarcinoma Cell Lines.

Citrinin (CIT), a polyketide mycotoxin produced by Penicillium, Aspergillus, and Monascus species, is a contaminant that has been found in various food commodities and was also detected in house dust. Several studies showed that CIT can impair the kidney, liver, heart, immune, and reproductive systems in animals by mechanisms so far not completely elucidated. In this study, we investigated the CIT mode of action on two human tumor cell lines, HepG2 (hepatocellular carcinoma) and A549 (lung adenocarcinoma). Cytotoxic concentrations were determined using an MTT proliferation assay. The genotoxic effect of sub-IC50 concentrations was investigated using the alkaline comet assay and the impact on the cell cycle using flow cytometry. Additionally, the CIT effect on the total amount and phosphorylation of two cell-cycle-checkpoint proteins, the serine/threonine kinase Chk2 and Fanconi anemia (FA) group D2 (FANCD2), was determined by the cell-based ELISA. The data were analyzed using GraphPad Prism statistical software. The CIT IC50 for HepG2 was 107.3 µM, and for A549, it was >250 µM. The results showed that sensitivity to CIT is cell-type dependent and that CIT in sub-IC50 and near IC50 induces significant DNA damage and cell-cycle arrest in the G2/M phase, which is related to the increase in total and phosphorylated Chk2 and FANCD2 checkpoint proteins in HepG2 and A549 cells.

FANCD2 deficiency sensitizes SHH medulloblastoma to radiotherapy via ferroptosis.

Radiotherapy is one of the standard therapeutic regimens for medulloblastoma (MB). Tumor cells utilize DNA damage repair (DDR) mechanisms to survive and develop resistance during radiotherapy. It has been found that targeting DDR sensitizes tumor cells to radiotherapy in several types of cancer, but whether and how DDR pathways are involved in the MB radiotherapy response remain to be determined. Single-cell RNA sequencing was carried out on 38 MB tissues, followed by expression enrichment assays. Fanconi anemia group D2 gene (FANCD2) expression was evaluated in MB samples and public MB databases. The function of FANCD2 in MB cells was examined using cell counting assays (CCK-8), clone formation, lactate dehydrogenase activity, and in mouse orthotopic models. The FANCD2-related signaling pathway was investigated using assays of peroxidation, a malondialdehyde assay, a reduced glutathione assay, and using FerroOrange to assess intracellular iron ions (Fe2+ ). Here, we report that FANCD2 was highly expressed in the malignant sonic hedgehog (SHH) MB subtype (SHH-MB). FANCD2 played an oncogenic role and predicted worse prognosis in SHH-MB patients. Moreover, FANCD2 knockdown markedly suppressed viability, mobility, and growth of SHH-MB cells and sensitized SHH-MB cells to irradiation. Mechanistically, FANCD2 deficiency led to an accumulation of Fe2+ due to increased divalent metal transporter 1 expression and impaired glutathione peroxidase 4 activity, which further activated ferroptosis and reduced proliferation of SHH-MB cells. Using an orthotopic mouse model, we observed that radiotherapy combined with silencing FANCD2 significantly inhibited the growth of SHH-MB cell-derived tumors in vivo. Our study revealed FANCD2 as a potential therapeutic target in SHH-MB and silencing FANCD2 could sensitize SHH-MB cells to radiotherapy via inducing ferroptosis. © 2024 The Pathological Society of Great Britain and Ireland.

Insulin-Like Growth Factor 2 mRNA Binding Protein 3 Suppresses Ferroptosis in Non-Small Cell Lung Cancer via Stabilizing m6A Modification of Fanconi Anemia Group D2 Protein

This study investigated the role of insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) in non-small cell lung cancer (NSCLC) and its association with N6-methyladenosine (m6A) modification. The study analyzes the expression levels and stability of IGF2BP3, as well as its impact on NSCLC cell functions. The findings indicate that IGF2BP3 is upregulated in NSCLC patients and cell lines. Knocking down IGF2BP3 reduces cell proliferation and promotes ferroptosis in A549 and H1299 cells. Additionally, the study reveals that IGF2BP3 regulates the m6A modification of the fanconi anemia group D2 protein (FANCD2) and influences its mRNA stability. Overexpressing FANCD2 counteracts the effects of IGF2BP3 silencing and increases the aggressiveness of NSCLC. Furthermore, treatment with celastrol induces ferroptosis in NSCLC cells and inhibits tumor growth in vivo. In conclusion, these findings suggest that IGF2BP3 acts as an oncogene in NSCLC. Its interaction with FANCD2 through m6A modification suppresses ferroptosis in NSCLC cells. Thus, the IGF2BP3/FANCD2 signaling pathway may serve as a potential therapeutic target for NSCLC.

TXNL4B regulates radioresistance by controlling the PRP3-mediated alternative splicing of FANCI.

Ionizing radiation (IR) has been extensively used for cancer therapy, but the radioresistance hinders and undermines the radiotherapy efficacy in clinics greatly. Here, we reported that the spliceosomal protein thioredoxin-like 4B (TXNL4B) is highly expressed in lung tissues from lung cancer patients with radiotherapy. Lung cancer cells with TXNL4B knockdown illustrate increased sensitivity to IR. Mechanistically, TXNL4B interacts with RNA processing factor 3 (PRP3) and co-localizes in the nucleus post-IR. Nuclear localization of PRP3 promotes the alternative splicing of the Fanconi anemia group I protein (FANCI) transcript variants, FANCI-12 and FANCI-13. PRP3 regulates alternative splicing of FANCI toward the two variants, FANCI-12 and FANCI-13. Radioresistance was greatly enhanced through the combination of PRP31 and PRP8, the critical components of core spliceosome promoted by PRP3. Notably, the inhibition of PRP3 to suppress the production of FANCI-12 would deprive PRP31 and PRP8 of such interaction. As a result, cell cycle G2/M arrest was induced, DNA damage repair was delayed, and radiosensitivity was improved. Collectively, our study highlights potential novel underlying mechanisms of the involvement of TXNL4B and alternative splicing in radioresistance. The results would benefit potential cancer radiotherapy.

Compartmentalization of the SUMO/RNF4 pathway by SLX4 drives DNA repair

SLX4, disabled in the Fanconi anemia group P, is a scaffolding protein that coordinates the action of structure-specific endonucleases and other proteins involved in the replication-coupled repair of DNA interstrand cross-links. Here, we show that SLX4 dimerization and SUMO-SIM interactions drive the assembly of SLX4 membraneless compartments in the nucleus called condensates. Super-resolution microscopy reveals that SLX4 forms chromatin-bound clusters of nanocondensates. We report that SLX4 compartmentalizes the SUMO-RNF4 signaling pathway. SENP6 and RNF4 regulate the assembly and disassembly of SLX4 condensates, respectively. SLX4 condensation per se triggers the selective modification of proteins by SUMO and ubiquitin. Specifically, SLX4 condensation induces ubiquitylation and chromatin extraction of topoisomerase 1 DNA-protein cross-links. SLX4 condensation also induces the nucleolytic degradation of newly replicated DNA. We propose that the compartmentalization of proteins by SLX4 through site-specific interactions ensures the spatiotemporal control of protein modifications and nucleolytic reactions during DNA repair.

SNV/indel hypermutator phenotype in biallelic RAD51C variant: Fanconi anemia.

We previously reported a fetus with Fanconi anemia (FA), complementation group O due to compound heterozygous variants involving RAD51C. Interestingly, the trio exome sequencing analysis also detected eight apparent de novo mosaic variants with variant allele fraction (VAF) ranging between 11.5 and 37%. Here, using whole genome sequencing and a 'home-brew' variant filtering pipeline and DeepMosaic module, we investigated the number and signature of de novo heterozygous and mosaic variants and the hypothesis of a rare phenomenon of hypermutation. Eight-hundred-thirty apparent de novo SNVs and 21 de novo indels had VAFs below 37.41% and were considered postzygotic somatic mosaic variants. The VAFs showed a bimodal distribution, with one component having an average VAF of 25% (range: 18.7-37.41%) (n = 446), representing potential postzygotic first mitotic events, and the other component with an average VAF of 12.5% (range 9.55-18.69%) (n = 384), describing potential second mitotic events. No increased rate of CNV formation was observed. The mutational pattern analysis for somatic single base substitution showed SBS40, SBS5, and SBS3 as the top recognized signatures. SBS3 is a known signature associated with homologous recombination-based DNA damage repair error. Our data demonstrate that biallelic RAD51C variants show evidence for defective genomic DNA damage repair and thereby result in a hypermutator phenotype with the accumulation of postzygotic de novo mutations, at least in the prenatal period. This 'genome hypermutator phenomenon' might contribute to the observed hematological manifestations and the predisposition to tumors in patients with FA. We propose that other FA groups should be investigated for genome-wide de novo variants.

High molecular diagnostic yields and novel phenotypic expansions involving syndromic anorectal malformations.

Evidence suggests that genetic factors contribute to the development of anorectal malformations (ARMs). However, the etiology of the majority of ARMs cases remains unclear. Exome sequencing (ES) may be underutilized in the diagnostic workup of ARMs due to uncertainty regarding its diagnostic yield. In a clinical database of ~17,000 individuals referred for ES, we identified 130 individuals with syndromic ARMs. A definitive or probable diagnosis was made in 45 of these individuals for a diagnostic yield of 34.6% (45/130). The molecular diagnostic yield of individuals who initially met criteria for VACTERL association was lower than those who did not (26.8% vs 44.1%; p = 0.0437), suggesting that non-genetic factors may play an important role in this subset of syndromic ARM cases. Within this cohort, we identified two individuals who carried de novo pathogenic frameshift variants in ADNP, two individuals who were homozygous for pathogenic variants in BBS1, and single individuals who carried pathogenic or likely pathogenic variants in CREBBP, EP300, FANCC, KDM6A, SETD2, and SMARCA4. The association of these genes with ARMs was supported by previously published cases, and their similarity to known ARM genes as demonstrated using a machine learning algorithm. These data suggest that ES should be considered for all individuals with syndromic ARMs in whom a molecular diagnosis has not been made, and that ARMs represent a low penetrance phenotype associated with Helsmoortel-van der Aa syndrome, Bardet-Biedl syndrome 1, Rubinstein-Taybi syndromes 1 and 2, Fanconi anemia group C, Kabuki syndrome 2, SETD2-related disorders, and Coffin-Siris syndrome 4.

Expression of SASP, DNA Damage Response, and Cell Proliferation Factors in Early Gastric Neoplastic Lesions: Correlations and Clinical Significance.

The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING)-mediated senescence-associated secretory phenotype (SASP) pathway has recently been identified in the suppression and promotion of cancers. However, its practical role in carcinogenesis remains to be comprehensively elucidated. Here, we describe an investigation analysing SASP activity and its correlations with DNA damage response (DDR), genomic mutations, and cell proliferation in gastric carcinogenesis among 30 cases with available endoscopic submucosal dissection (ESD) specimens of early neoplastic lesions (including low-grade dysplasia [LGD], high-grade dysplasia [HGD], and intramucosal carcinoma). The positive cells of senescence-associated β-galactosidase staining and cGAS, STING, interferon-regulatory factor 3 (IRF3), and signal transducer and activator of transcription 6 (STAT6) expression levels using immunostaining were elevated in HGD and in cancers. Similarly, increased expression of the Fanconi anemia group D2 (FANCD2) protein, tumour suppressor p53 binding protein 1 (TP53BP1), and replication protein A (RPA2) (i.e., primary DDR factors) was detected in HGD and in cancers; these increased expression levels were closely correlated with high expression of Ki67 and minichromosome maintenance complex component 7 (MCM7) proteins. Moreover, genomic mutations in TP53 gene were detected in 56.67% of the evaluated cases (17/30) using next-generation sequencing, and positive staining was verified in HGD and in cancers. Statistical analysis revealed that cell proliferation closely correlated with the expression of DDR factors, of which TP53BP1 was positively associated with SASP factors and IRF3 was positively correlated with cell proliferation. In addition, an analysis evaluating clinical features demonstrated that STAT6-positive cases showed a longer progression-free survival time than STAT6-negative cases. Our evaluation, conducted using a limited number of specimens, suggests SASP may be prevalent in early gastric neoplastic lesions and could be activated by accelerated cell proliferation-induced DDR. The clinical significance of SASP still needs to be determined.

FANCC deficiency mediates microglial pyroptosis and secondary neuronal apoptosis in spinal cord contusion

BackgroundTraumatic spinal cord injury (SCI)-induced neuroinflammation results in secondary neurological destruction and functional disorder. Previous findings showed that microglial pyroptosis plays a crucial role in neuroinflammation. Thus, it is necessary to conduct a comprehensive investigation of the mechanisms associated with post-SCI microglial pyroptosis. The Fanconi Anemia Group C complementation group gene (FANCC) has been previously reported to have an anti-inflammation effect; however, whether it can regulate microglial pyroptosis remains unknown. Therefore, we probed the mechanism associated with FANCC-mediated microglial pyroptosis and neuroinflammation in vitro and in vivo in SCI mice.MethodsMicroglial pyroptosis was assessed by western blotting (WB) and immunofluorescence (IF), whereas microglial-induced neuroinflammation was evaluated by WB, Enzyme-linked immunosorbent assays and IF. Besides, flow cytometry, TdT-mediated dUTP Nick-End Labeling staining and WB were employed to examine the level of neuronal apoptosis. Morphological changes in neurons were assessed by hematoxylin–eosin and Luxol Fast Blue staining. Finally, locomotor function rehabilitation was analyzed using the Basso Mouse Scale and Louisville Swim Scale.ResultsOverexpression of FANCC suppressed microglial pyroptosis via inhibiting p38/NLRP3 expression, which in turn reduced neuronal apoptosis. By contrast, knockdown of FANCC increased the degree of neuronal apoptosis by aggravating microglial pyroptosis. Besides, increased glial scar formation, severe myelin sheath destruction and poor axon outgrowth were observed in the mice transfected with short hairpin RNA of FANCC post SCI, which caused reduced locomotor function recovery.ConclusionsTaken together, a previously unknown role of FANCC was identified in SCI, where its deficiency led to microglia pyroptosis, neuronal apoptosis and neurological damage. Mechanistically, FANCC mediated microglia pyroptosis and the inflammatory response via regulating the p38/NLRP3 pathway.

Upregulation of Ferroptosis-Related Fanconi Anemia Group D2 is a Poor Prognostic Factor and an Indicator of Tumor Immune Cell Infiltration in Lung Adenocarcinoma.

Fanconi anemia (FA) group D2 (FANCD2) is a ferroptosis-related gene crucial for DNA damage repair and negative ferroptosis regulation. Our study aimed to evaluate its prognostic value as well as its association with ferroptosis and immune infiltration in lung adenocarcinoma (LUAD). Transcriptome sequencing data, clinical information, and immunohistochemistry data were collected from the TCGA, GEO, and HPA databases, respectively, for three independent cohorts. Univariate and multivariate analyses were used to assess the correlations between FANCD2 expression and overall survival or clinicopathological parameters. cBioPortal was utilized to investigate the FANCD2 alteration status. Gene and protein networks based on FANCD2 interactions were generated using GeneMANIA and STRING, respectively. Based on the CancerSEA database, the function of FANCD2 was explored at the single-cell level. The relationships between FANCD2 expression levels and tumor-infiltrating immune cells and their equivalent gene signatures were analyzed using TIMER, GEPIA, TISIDB, and ssGSEA databases. CIBERSORT was used to analyze the relevance of the infiltration of 24 types of immune cells. The results revealed that FANCD2 expression was significantly upregulated in LUAD and lung squamous cell carcinoma (LUSC) tissues than that in normal tissues. Further, the overexpression of FANCD2 was closely associated with poor survival for Patients with LUAD but not for patients with LUSC. FANCD2 expression levels were related to tumor-infiltrating immune cells and their matching gene signatures, including CD8+ T cells, natural killer (NK) cells, dendritic cells (DC), and Th2 cells in cases of LUAD. Therefore, FANCD2 was identified as a crucial molecule underlying the synergistic effects of ferroptosis and immunotherapy for Patients with LUAD.

Loss of p53 suppresses replication stress-induced DNA damage in ATRX-deficient neuroblastoma

Genetic aberrations are present in the ATRX gene in older high-risk neuroblastoma (NB) patients with very poor clinical outcomes. Its loss-of-function (LoF) facilitates the alternative lengthening of telomeres (ALT) pathway in tumor cells and is strongly linked to replication stress (RS) and DNA damage through G-quadruplex (G4) DNA secondary structures. However, limited information is available on ATRX alteration-related NB tumorigenesis. We herein knocked out (KO) ATRX in MYCN-amplified (NGP) and MYCN single copy (SK-N-AS) NB cells with wild-type (wt) and truncated TP53 at the C terminus, respectively, using CRISPR/Cas9 technologies. The loss of ATRX increased DNA damage and G4 formation related to RS in TP53 wt isogenic ATRX KO NGP cells, but not in SK-N-AS clones. A gene set enrichment analysis (GSEA) showed that the gene sets related to DNA double-strand break repair, negative cell cycle regulation, the G2M checkpoint, and p53 pathway activation were enriched in NGP clones. The accumulation of DNA damage activated the ATM/CHK2/p53 pathway, leading to cell cycle arrest in NGP clones. Interestingly, ATRX loss did not induce RS related to DNA damage response (DDR) in TP53-truncated SK-N-AS cells. p53 inactivation abrogated cell cycle arrest and reduced G4 accumulation in NGP clones. The loss of p53 also induced G4 DNA helicases or Fanconi anemia group D2 protein (FANCD2) with ATRX deficiency, suggesting that ATRX maintained genome integrity and p53 deficiency attenuated RS-induced DNA damage in NB cells featuring inactivated ATRX by regulating DNA repair mechanisms and replication fork stability.

Acetylation modulates the Fanconi anemia pathway by protecting FAAP20 from ubiquitin-mediated proteasomal degradation

Fanconi anemia (FA) is a chromosome instability syndrome of children caused by inherited mutations in one of FA genes, which together constitute a DNA interstrand cross-link (ICL) repair, or the FA pathway. Monoubiquitination of Fanconi anemia group D2 protein (FANCD2) by the multisubunit ubiquitin E3 ligase, the FA core complex, is an obligate step in activation of the FA pathway, and its activity needs to be tightly regulated. FAAP20 is a key structural component of the FA core complex, and regulated proteolysis of FAAP20 mediated by prolyl cis-trans isomerization and phosphorylation at a consensus phosphodegron motif is essential for preserving the integrity of the FA core complex, and thus FANCD2 monoubiquitination. However, how ubiquitin-dependent FAAP20 degradation is modulated to fine-tune FA pathway activation remains largely un-known. Here, we present evidence that FAAP20 is acetylated by the acetyltransferase p300/CBP on lysine 152, the key residue that when polyubiquitinated results in the degradation of FAAP20. Acetylation or mutation of the lysine residue stabilizes FAAP20 by preventing its ubiquitination, thereby protecting it from proteasome-dependent FAAP20 degradation. Consequently, disruption of the FAAP20 acetylation pathway impairs FANCD2 activation. Together, our study reveals a competition mechanism between ubiquitination and acetylation of a common lysine residue that controls FAAP20 stability and highlights a complex balancing between different posttranslational modifications as a way to refine the FA pathway signaling required for DNA ICL repair and genome stability.

Potential Doxorubicin-Mediated Dual-Targeting Chemotherapy in FANC/BRCA-Deficient Tumors via Modulation of Cellular Formaldehyde Concentration.

Doxorubicin (DOX) is a widely used classical broad-spectrum anticancer drug. The major mechanism of DOX-mediated anticancer activity at clinically relevant concentrations is believed to be via DNA double-strand breaks due to topoisomerase IIα. However, other mechanisms by which DOX causes cytotoxicity have been proposed, including formaldehyde-dependent virtual interstrand cross-linking (ICL) formation. In this study, a method was established whereby cytotoxicity caused by virtual ICL derived from DOX is turned on and off using a cell culture system. Using this strategy, DOX-mediated cytotoxicity in Fanconi anemia group gene (FANC)/breast cancer susceptibility gene (BRCA)-deficient cells increased up to 70-fold compared to that in cells proficient in DNA repair pathways by increasing intracellular formaldehyde (FA) concentration. This approach also demonstrated that cytotoxicity introduced by DOX-mediated FA-dependent virtual ICL is completely independent of the toxicity induced by topoisomerase II inhibition at the cellular level. The potential of dual-targeting by DOX treatment was verified using an acid-specific FA donor. Overall, anticancer therapy targeting tumors deficient in the FANC/BRCA pathway may be possible by minimizing DOX-induced toxicity in normal cells.

Enhanced transcription activity of histone acetyltransferase HBO1 after genotoxic stress

Chromatin remodeling is induced at sites of DNA damage as part of the damage response to allow DNA repair protein accumulation and stability of function. Histone posttranslational modifications are required to orchestrate DNA damage response, and ubiquitination of histone H2A-K15 at DNA double-strand break sites is involved in the recruitment of DNA repair protein 53BP1 to DNA damage sites. Acetylation of histone H4K16 promotes homologous recombination repair by inducing dissociation of 53BP1, while deacetylation inhibits the homologous recombination repair by promoting accumulation of 53BP1 at DNA damage sites. Chromatin remodeling is indispensable for accumulation of numerous DNA damage repair proteins and their subsequent dissociation, and we investigated the possibility of new DNA damage response pathway. In this study, we used qPCR, western blotting and survival assays to demonstrate that genotoxic stress, both biochemical and radiological, specifically upregulated the expression of histone acetyltransferase binding to ORC (HBO1) protein in the histone acetyltransferase MYST family, which colocalized with the DNA damage response protein, Fanconi anemia group D2 (FANCD2). HBO1 expression was upregulated via phosphoinositide-3-kinase-related kinase protein, which plays a crucial role as a DNA damage response signal. These results suggest that HBO1induced by genotoxic stress has a critical role in DNA damage responses.

Circular RNA circFANCL motivates the glioma progression via the action on the miR-337-3p/HMGB1 signal axis.

Circular RNAs (circRNAs) are significant modulatory molecules in the developing process of glioma. Our study will emphasize on exploring the function of circRNA Fanconi anemia group L protein (circFANCL) and the specific mechanism in glioma. The quantitative real-time polymerase chain reaction (qRT-PCR) was implemented for detecting circFANCL, microRNA-337-3p (miR-337-3p) and high mobility group box-1 (HMGB1). Cell proliferation analysis was assessed using Cell Counting Kit-8 (CCK-8) and colony formation assays. Flow cytometry was applied to determine cell cycle and apoptosis. All protein detection was completed by western blot. Animal experiment was performed to investigate the role of circFANCL in vivo. Dual-luciferase reporter, RNA immunoprecipitation (RIP) and RNA pull-down assays were collectively conducted for analyzing the targeted combination. CircFANCL was signally increased in glioma tissues and cells, and the up-regulated circFANCL could predict poor prognosis in clinical glioma patients. Down-regulated circFANCL induced the proliferation inhibition, cell cycle arrest and apoptosis promotion of glioma cells in vitro, and inhibited tumor growth in vivo. Regarding the mechanism, circFANCL served as a sponge of miR-337-3p that was a tumor suppressor in glioma and circFANCL targeted miR-337-3p to regulate HMGB1 that was a target gene of miR-337-3p. Furthermore, HMGB1 down-regulation was responsible for the repression of glioma progression caused by knockdown of circFANCL. Through the illustration of oncogenic function of circFANCL in glioma by the miR-337-3p/HMGB1 axis, we believed that circFANCL might be a great target in the early diagnosis and late treatment of glioma.

Mustard Seed (Brassica nigra) Extract Exhibits Antiproliferative Effect against Human Lung Cancer Cells through Differential Regulation of Apoptosis, Cell Cycle, Migration, and Invasion.

Lung cancer is the primary cause of cancer-related death worldwide, and development of novel lung cancer preventive and therapeutic agents are urgently needed. Brassica nigra (black mustard) seeds are commonly consumed in several Asian and African countries. Mustard seeds previously exhibited significant anticancer activities against several cancer types. In the present study, we have investigated various cellular and molecular mechanisms of anticancer effects of an ethanolic extract of B. nigra seeds against A549 and H1299 human non-small cell lung cancer cell lines. B. nigra extract showed a substantial growth-inhibitory effect as it reduced the viability and clonogenic survival of A549 and H1299 cells in a concentration-dependent manner. B. nigra extract induced cellular apoptosis in a time- and concentration-dependent fashion as evidenced from increased caspase-3 activity. Furthermore, treatment of both A549 and H1299 cells with B. nigra extract alone or in combination with camptothecin induced DNA double-strand breaks as evidenced by upregulation of γH2A histone family member X, Fanconi anemia group D2 protein, Fanconi anemia group J protein, ataxia-telangiectesia mutated and Rad3-related protein. Based on cell cycle analysis, B. nigra extract significantly arrested A549 and H1299 cells at S and G2/M phases. Additionally, B. nigra extract suppressed the migratory and invasive properties of both cell lines, downregulated the expression of matrix metalloproteinase-2 (MMP2), MMP9, and Snail and upregulated the expression of E-cadherin at mRNA and protein levels. Taken together, these findings indicate that B. nigra seed extract may have an important anticancer potential against human lung cancer which could be mediated through simultaneous and differential regulation of proliferation, apoptosis, DNA damage, cell cycle, migration, and invasion.

FANCL gene mutations in premature ovarian insufficiency.

The Fanconi anemia (FA) pathway is mainly involved in DNA interstrand crosslinks (ICLs) repair in the genome. Several FA genes, including FANCD1/BRCA2, FANCM, and FANCU/XRCC2, have been identified as causative genes for premature ovary insufficiency (POI). Fanconi anemia group L protein (FANCL) cooperates with FANCT/UBE2T to ubiquitinate the FANCI-D2 dimer, which is a crucial event in the process of ICLs repair. Fancl-knockout mice phenocopy human POI, but the role of FANCL mutations in POI pathogenesis has not been confirmed. In the present work, potentially pathogenic mutations in the FANCL gene were screened in 200 Chinese patients with idiopathic POI and in 200 matched controls. Two novel heterozygous frameshift mutations, c.1048_1051delGTCT (p.Gln350Valfs*18) and c.739dupA (p.Met247Asnfs*4), were identified in the FANCL gene in POI patients but not in controls. Wild-type FANCL protein was predominantly localized in the nuclei, while both mutant FANCL proteins were retained in the cytoplasm. In addition, the FANCL variants exhibited impaired ubiquitin-ligase activity and compromised DNA repair ability after mitomycin C treatment. Furthermore, the FANCL variants were deleterious and might be associated with haploinsufficiency. Our results show that FANCL mutations are potentially causative for POI by disrupting DNA damage repair processes.

Investigation of the core binding regions of human Werner syndrome and Fanconi anemia group J helicases on replication protein A

Werner syndrome protein (WRN) and Fanconi anemia group J protein (FANCJ) are human DNA helicases that contribute to genome maintenance. They interact with replication protein A (RPA), and these interactions dramatically enhance the unwinding activities of both helicases. Even though the interplay between these helicases and RPA is particularly important in the chemoresistance pathway of cancer cells, the precise binding regions, interfaces, and properties have not yet been characterized. Here we present systematic NMR analyses and fluorescence polarization anisotropy assays of both helicase-RPA interactions for defining core binding regions and binding affinities. Our results showed that two acidic repeats of human WRN bind to RPA70N and RPA70A. For FANCJ, the acidic-rich sequence in the C-terminal domain is the binding region for RPA70N. Our results suggest that each helicase interaction has unique features, although they both fit an acidic peptide into a basic cleft for RPA binding. Our findings shed light on the protein interactions involved in overcoming the DNA-damaging agents employed in the treatment of cancer and thus potentially provide insight into enhancing the efficacy of cancer therapy.

Expression and prognostic significance of Fanconi anemia group D2 protein and breast cancer type 1 susceptibility protein in familial and sporadic breast cancer.

Fanconi anemia group D2 protein (FANCD2) and breast cancer type 1 susceptibility protein (BRCA1), within the FA/BRCA pathway, are involved in the regulation of DNA damage repair, which is associated with breast cancer (BC) progression. The present study aimed to investigate BRCA1 and FANCD2 expression in breast cancer, and to highlight the association with patient clinical characteristics and prognoses. The BRCA1 and FANCD2 proteins were detected by immunohistochemistry in 335 tissue samples obtained from patients with BC, including 141 patients with familial BC (FBC), 147 patients with sporadic breast cancer (SBC) and 47 patients with benign breast tumors. Western blotting was used to detect the FANCD2 ubiquitination level in 56 frozen specimens that were randomly selected from the SBC group. Protein expression of BRCA1 in the FBC group was positively associated with tumor size, lymphatic invasion, Tumor-Node-Metastasis (TNM) stage, estrogen receptor (ER) status and FANCD2 expression. Protein expression of FANCD2 in the SBC group was positively associated with tumor size, TNM stage, ER status and Ki-67 index. Survival analyses revealed that BRCA1 expression was associated with the decreased disease-free survival (DFS) rate of patients with FBC (versus no BRCA1 expression) and that FANCD2 was associated with decreased DFS of patients with SBC (versus no FANCD expression). Univariable and multivariable analyses demonstrated that BRCA1 expression may be an independent prognostic factor in the FBC group. In the SBC group, FANCD2 high expression and low ubiquitination levels were considered as independent prognostic factors. In conclusion, the present study suggested that BRCA1 and FANCD2 expression, and FANCD2 ubiquitination levels, may be considered of novel potential prognostic value in patients with BC.

Comet assay and cytogenetic findings in differential diagnosis of Fanconi anemia

Fanconi anemia (FA) is a complex genetic disease with a variety of congenital and hematological symptoms, including the predisposition for cancer development. The main hallmark of FA cells, an increased chromosomal fragility, in the presence of the DNA-interstrand cross-linking chemicals, mitomycin C or diepoxybutane (DEB), makes the diagnosis of FA much easier. Cytogenetic method can detect the FA patients with highly elevated chromosomal breakage, but also some of the patients with borderline sensitivity to DEB no matter if they have FA or not. These particular circumstances lead us to introduce comet assay along with cytogenetic analysis, in order to determine DNA lesions and chromosomal fragility in untreated and DEB-treated lymphocytes of full blood from seven patients with clinical features of FA. Highly elevated DEB induced chromosomal sensitivity confirmed the diagnosis in five patients (FA group: 0.48-4.47 breaks/cell vs control group: 0.00-0.08 breaks/cell). Borderline DEB sensitivity (FA* group: 0.15-0.44 breaks/cell) was found in the remaining two patients. Results of the comet assay showed higher baseline and DEB-induced DNA damage values (Olive tail moment and tail intensity) in all five FA and one FA