Fanconi Anemia Cells Research Articles

Cytokines, inducers and inhibitors modulate MMP-2 and MMP‑9 secretion by human Fanconi anemia immortalized fibroblasts.

Acute myeloid leukemia and head and neck squamous cell carcinomas are the major causes of mortality and morbidity in Fanconi anemia (FA) patients. Matrix metalloproteinases (MMPs), particularly MMP-2 and MMP-9, have been implicated in tumor invasion and metastasis. Various cytokines, mitogens, growth factors, inducers and inhibitors control MMP activities. We investigated the roles of these in the regulation of MMP-2 and MMP-9 in human immortalized fibroblasts from FA. Human FA immortalized fibroblast cell lines FA-A:PD220 and FA-D2:PD20 were grown in minimum essential medium (MEM) supplemented with 15% fetal bovine serum (FBS) and antibiotics in 24-well tissue culture plates. At near confluence, the cells were washed with phosphate‑buffered saline (PBS) and incubated in serum-free media with the following: phorbol 12-myristate 13-acetate (PMA) at 10-100ng/ml; tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) at 0.1-25ng/ml; lipopolysaccharide (LPS) at 10-100µg/ml; epigallocatechin gallate (EGCG) and doxycycline (Dox) at 10-100µM without and with PMA; a nutrient mixture (NM) without and with PMA at 10-1,000µg/ml; actinomycin-D and cyclohexamide at 2and4µM; retinoic acid and dexamethasone at 50µM. After 24h, media were removed and analyzed for MMP-2 and MMP-9 by zymography. Both FA cell lines expressed only MMP-2 and responded similarly to cytokines, mitogens, inducers and inhibitors. PMA potently stimulated MMP-9 and had a moderate effect on MMP-2. TNF-α showed variable effects on MMP-2 and significantly enhanced MMP-9. IL-1β enhanced MMP-2 slightly and MMP-9 significantly. LPS had a moderate stimulatory effect on MMP-2 and no effect on MMP-9. EGCG, Dox and NM, without and with PMA, downregulated MMP-2 and MMP-9 expression. Actinomycin-D, retinoic acid and dexamethasone also had inhibitory effects on MMP-2. Our results showed that cytokines, mitogens and inhibitors modulated FA fibroblast MMP-2 and MMP-9 expression, suggesting the clinical use of MMP inhibitors, particularly such potent and non-toxic ones as the NM and its component EGCG in the management of FA cancers.

Phenotypic correction of Fanconi anemia cells in the murine bone marrow after carrier cell mediated delivery of lentiviral vector.

Fanconi anemia (FA) is an autosomal-recessive disorder associated with hematopoietic failure and it is a candidate for hematopoietic stem cell (HSC)-directed gene therapy. However, the characteristically reduced HSC numbers found in FA patients, their ineffective mobilization from the marrow, and re-oxygenation damage during ex vivo manipulation have precluded clinical success using conventional in vitro approaches. We previously demonstrated that lentiviral vector (LV) particles reversibly attach to the cell surface where they gain protection from serum complement neutralization. We reasoned that cellular delivery of LV to the bone marrow niche could avoid detrimental losses during FA HSC mobilization and in vitro modification. Here, we demonstrate that a VSV-G pseudotyped lentivector, carrying the FANCC transgene, can be transmitted from carrier to bystander cells. In cell culture and transplantation models of FA, we further demonstrate that LV carrier cells migrate along SDF-1α gradients and transfer vector particles that stably integrate and phenotypically correct the characteristic DNA alkylator sensitivity in murine and human FA-deficient target bystander cells. Altogether, we demonstrate that cellular homing mechanisms can be harnessed for the functional phenotype correction in murine FA hematopoietic cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-016-0431-z) contains supplementary material, which is available to authorized users.

The PTEN phosphatase functions cooperatively with the Fanconi anemia proteins in DNA crosslink repair.

Fanconi anemia (FA) is a genetic disease characterized by bone marrow failure and increased cancer risk. The FA proteins function primarily in DNA interstrand crosslink (ICL) repair. Here, we have examined the role of the PTEN phosphatase in this process. We have established that PTEN-deficient cells, like FA cells, exhibit increased cytotoxicity, chromosome structural aberrations, and error-prone mutagenic DNA repair following exposure to ICL-inducing agents. The increased ICL sensitivity of PTEN-deficient cells is caused, in part, by elevated PLK1 kinase-mediated phosphorylation of FANCM, constitutive FANCM polyubiquitination and degradation, and the consequent inefficient assembly of the FA core complex, FANCD2, and FANCI into DNA repair foci. We also establish that PTEN function in ICL repair is dependent on its protein phosphatase activity and ability to be SUMOylated, yet is independent of its lipid phosphatase activity. Finally, via epistasis analysis, we demonstrate that PTEN and FANCD2 function cooperatively in ICL repair.

First molecular-cytogenetic characterization of Fanconi anemia fragile sites in primary lymphocytes of FA-D2 patients in different stages of the disease.

BackgroundFanconi anemia (FA) is a chromosomal instability syndrome characterized by increased frequency of chromosomal breakages, chromosomal radial figures and accelerated telomere shortening. In this work we performed detailed molecular-cytogenetic characterization of breakpoints in primary lymphocytes of FA-D2 patients in different stages of the disease using fluorescent in situ hybridization.ResultsWe found that chromosomal breakpoints co-localize on the molecular level with common fragile sites, whereas their distribution pattern depends on the severity of the disease. Telomere quantitative fluorescent in situ hybridization revealed that telomere fusions and radial figures, especially radials which involve telomere sequences are the consequence of critically shortened telomeres that increase with the disease progression and could be considered as a predictive parameter during the course of the disease. Sex chromosomes in FA cells are also involved in radial formation indicating that specific X chromosome regions share homology with autosomes and also could serve as repair templates in resolving DNA damage.ConclusionsFA-D2 chromosomal breakpoints co-localize with common fragile sites, but their distribution pattern depends on the disease stage. Telomere fusions and radials figures which involve telomere sequences are the consequence of shortened telomeres, increase with disease progression and could be of predictive value.

Nuclear alpha spectrin: Critical roles in DNA interstrand cross-link repair and genomic stability.

Non-erythroid alpha spectrin (αIISp) is a structural protein which we have shown is present in the nucleus of human cells. It interacts with a number of nuclear proteins such as actin, lamin, emerin, chromatin remodeling factors, and DNA repair proteins. αIISp's interaction with DNA repair proteins has been extensively studied. We have demonstrated that nuclear αIISp is critical in DNA interstrand cross-link (ICL) repair in S phase, in both genomic (non-telomeric) and telomeric DNA, and in maintenance of genomic stability following ICL damage to DNA. We have proposed that αIISp acts as a scaffold aiding to recruit repair proteins to sites of damage. This involvement of αIISp in ICL repair and telomere maintenance after ICL damage represents new and critical functions for αIISp. These studies have led to development of a model for the role of αIISp in DNA ICL repair. They have been aided by examination of cells from patients with Fanconi anemia (FA), a repair-deficient genetic disorder in which a deficiency in αIISp leads to defective ICL repair in genomic and telomeric DNA, telomere dysfunction, and chromosome instability following DNA ICL damage. We have shown that loss of αIISp in FA cells is due to increased breakdown by the protease, µ-calpain. Importantly, we have demonstrated that this deficiency can be corrected by knockdown of µ-calpain and restoring αIISp levels to normal. This corrects a number of the phenotypic deficiencies in FA after ICL damage. These studies suggest a new and unexplored direction for therapeutically restoring genomic stability in FA cells and for correcting numerous phenotypic deficiencies occurring after ICL damage. Developing a more in-depth understanding of the importance of the interaction of αIISp with other nuclear proteins could significantly enhance our knowledge of the consequences of loss of αIISp on critical nuclear processes.

Impaired mitophagy in Fanconi anemia is dependent on mitochondrial fission.

Fanconi anemia (FA) is a rare genetic disorder associated with bone-marrow failure, genome instability and cancer predisposition. Recently, we and others have demonstrated dysfunctional mitochondria with morphological alterations in FA cells accompanied by high reactive oxygen species (ROS) levels. Mitochondrial morphology is regulated by continuous fusion and fission events and the misbalance between these two is often accompanied by autophagy. Here, we provide evidence of impaired autophagy in FA. We demonstrate that FA cells have increased number of autophagic (presumably mitophagic) events and accumulate dysfunctional mitochondria due to an impaired ability to degrade them. Moreover, mitochondrial fission accompanied by oxidative stress (OS) is a prerequisite condition for mitophagy in FA and blocking this pathway may release autophagic machinery to clear dysfunctional mitochondria.

Multifunctional Fanconi proteins, inflammation and the Fanconi phenotype

Multifunctional Fanconi proteins, inflammation and the Fanconi phenotype

Upregulated LINE-1 Activity in the Fanconi Anemia Cancer Susceptibility Syndrome Leads to Spontaneous Pro-inflammatory Cytokine Production

Fanconi Anemia (FA) is a genetic disorder characterized by elevated cancer susceptibility and pro-inflammatory cytokine production. Using SLX4FANCP deficiency as a working model, we questioned the trigger for chronic inflammation in FA. We found that absence of SLX4 caused cytoplasmic DNA accumulation, including sequences deriving from active Long INterspersed Element-1 (LINE-1), triggering the cGAS-STING pathway to elicit interferon (IFN) expression. In agreement, absence of SLX4 leads to upregulated LINE-1 retrotransposition. Importantly, similar results were obtained with the FANCD2 upstream activator of SLX4. Furthermore, treatment of FA cells with the Tenofovir reverse transcriptase inhibitor (RTi), that prevents endogenous retrotransposition, decreased both accumulation of cytoplasmic DNA and pro-inflammatory signaling. Collectively, our data suggest a contribution of endogenous RT activities to the generation of immunogenic cytoplasmic nucleic acids responsible for inflammation in FA. The additional observation that RTi decreased pro-inflammatory cytokine production induced by DNA replication stress-inducing drugs further demonstrates the contribution of endogenous RTs to sustaining chronic inflammation. Altogether, our data open perspectives in the prevention of adverse effects of chronic inflammation in tumorigenesis.

558. Targeted Gene Therapy in CD34+ Cells from Healthy Donors and Fanconi Anemia Patients

Gene targeting is rapidly progressing thanks to the development of improved nucleases and donor constructs, and represents a new gene therapy-based strategy for the treatment of inherited diseases, such as Fanconi anemia (FA). Because FA can be generated by multiple mutations in up to 19 different genes, we focused our interests in the insertion of therapeutic FA genes in safe harbor loci, since this approach could serve as a platform for treating all FA subtypes and pathogenic mutations. In previous studies we demonstrated the feasibility of correcting the phenotype of hematopoietic progenitors through the genome editing and reprogramming of fibroblasts from FA patients. Because of the limited repopulating properties so far reported for iPSC-derived hematopoietic precursor cells, here we aimed to investigate the possibility of conducting a similar gene targeting approach using human primary CD34+ cells. In a first set of experiments we investigated the efficiency of targeting the AAVS1 locus in cord blood HSPCs. Pre-stimulated CD34+ cells were transduced with integrase-defective lentiviral vectors (IDLV) encoding an EGFP reporter gene under the control of the PGK promoter, flanked by sequences homologous to the AAVS1 target site. The cells were then nucleofected with AAVS1 -specific ZFN mRNAs. The average targeting efficiencies ranged from 10-20% both in in vitro cultured cells and NSG mice-repopulating cells. In subsequent experiments, the EGFP reporter gene was replaced by the hFANCA therapeutic gene. Gene targeting experiments conducted in lymphoblast cell lines and primary CD34+ cells from FA-A patients reverted the characteristic hypersensitivity of FA cells to mitomycin C (MMC), and also restored the formation of FANCD2 foci after DNA damage, evidencing the phenotypic correction of these cells. Our data confirm the efficacy of AAVS1-gene targeting in hHSPCs and demonstrate the feasibility of conducting these approaches in primary CD34+ cells from FA patients.

P53 downregulates the Fanconi anaemia DNA repair pathway.

Germline mutations affecting telomere maintenance or DNA repair may, respectively, cause dyskeratosis congenita or Fanconi anaemia, two clinically related bone marrow failure syndromes. Mice expressing p53Δ31, a mutant p53 lacking the C terminus, model dyskeratosis congenita. Accordingly, the increased p53 activity in p53Δ31/Δ31 fibroblasts correlated with a decreased expression of 4 genes implicated in telomere syndromes. Here we show that these cells exhibit decreased mRNA levels for additional genes contributing to telomere metabolism, but also, surprisingly, for 12 genes mutated in Fanconi anaemia. Furthermore, p53Δ31/Δ31 fibroblasts exhibit a reduced capacity to repair DNA interstrand crosslinks, a typical feature of Fanconi anaemia cells. Importantly, the p53-dependent downregulation of Fanc genes is largely conserved in human cells. Defective DNA repair is known to activate p53, but our results indicate that, conversely, an increased p53 activity may attenuate the Fanconi anaemia DNA repair pathway, defining a positive regulatory feedback loop.

TGF-β Inhibition Rescues Hematopoietic Stem Cell Defects and Bone Marrow Failure in Fanconi Anemia

Fanconi anemia (FA) is an inherited DNA repair disorder characterized by progressive bone marrow failure (BMF) from hematopoietic stem and progenitor cell (HSPC) attrition. A greater understanding of the pathogenesis of BMF could improve the therapeutic options for FA patients. Using a genome-wide shRNA screen in human FA fibroblasts, we identify transforming growth factor-β (TGF-β) pathway-mediated growth suppression as a cause of BMF in FA. Blocking the TGF-β pathway improves the survival of FA cells and rescues the proliferative and functional defects of HSPCs derived from FA mice and FA patients. Inhibition of TGF-β signaling in FA HSPCs results inelevated homologous recombination (HR) repair with a concomitant decrease in non-homologous end-joining (NHEJ), accounting for the improvement in cellular growth. Together, our results suggest that elevated TGF-β signaling contributes to BMF in FA by impairing HSPC function and may be a potential therapeutic target for the treatment of FA.

TGF-β Pathway Inhibition Rescues the Function of Hematopoietic Stem and Progenitor Cells Derived from Patients with Fanconi Anemia

Introduction: Fanconi anemia (FA) is the most common inherited bone marrow failure syndrome. FA patients develop bone marrow failure during the first decade of life, and frequently require an allogeneic or unrelated donor bone marrow transplant. FA patients also develop other hematologic manifestations, including myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) due to clonal evolution. FA is caused by biallelic mutation in one of eighteen FANC genes, the products of which cooperate in the FA/BRCA DNA repair pathway and regulate cellular resistance to DNA cross-linking agents. Bone marrow failure in FA is attributable to an impaired hematopoietic stem and progenitor cell (HSPC) pool. HSPCs in FA patients and FA mice exhibit reduced cell number and compromised stem cell function. Recent studies suggest that bone marrow failure in FA and impaired HSPC function result from the genotoxicity of endogenous cross-linking agents or from physiological stress. A greater understanding of the mechanisms of impairment of HSPC function could improve the therapeutic options for FA patients. Using a whole genome-wide shRNA screen, we have recently identified that the canonical transforming growth factor-β (TGF-β) pathway plays an important growth suppressive role in FA and targeting this pathway can reduce the genotoxic stress-induced growth inhibition of FA cells. Here, we investigated the possible suppressive function of the TGF-β pathway in HSPCs derived from patients with FA.Methods: We performed in vitro colony-forming assays using primary FA patient- derived bone marrow CD34+ cells which were either transduced with shRNA targeting SMAD3 or treated with the anti-human TGF-β neutralizing antibody GC1008. FA-like HSPCs were generated by stably knocking down FANCD2 with lentivirus encoded shRNA in primary human cord blood CD34+ cells. An in vivo engraftment assay was performed by transplanting the FA-like HSPCs into irradiated NSG mice.Results: The primary human FA bone marrow cells displayed elevated mRNA expression of multiple TGF-β pathway components. The TGF-β pathway inhibition, by knockdown of SMAD3 or anti-human TGF-β neutralizing antibody GC1008, rescued the in vitro clonogenic defects of primary CD34+ cells from bone marrow of five different FA patients. Similarly, the TGF-β pathway disruption by depletion of SMAD3 or GC1008 antibody in primary FA-like HSPCs, also rescued their clonogenic defect, and partially restored genotoxic stress-induced growth inhibition. Further, as the very low number of CD34+ cells in FA patients did not allow efficient xenograft assay to analyze in vivo clonogenicity, we performed a surrogate in vivo xenograft assay using FA-like primary CD34+ cells. Importantly, blockade of the TGF-β pathway by GC1008 antibody treatment enhanced the engraftment potential of primary FA-like CD34+ cells in vivo. Collectively, these results demonstrated that increased TGF-β pathway signaling impairs the hematopoietic function of primary human FA HSPCs.Conclusions: The TGF-β pathway signaling is increased in primary FA patient-derived hematopoietic cells and blockade of this pathway can restore the function of human FA-deficient primary HSPCs. The TGF-β signaling pathway-mediated growth suppression may account, at least in part, for bone marrow failure in FA. This work suggests that the TGF-β signaling pathway provides a novel therapeutic target for the treatment of bone marrow failure in FA. DisclosuresNo relevant conflicts of interest to declare.

The Fanconi Anemia Pathway Protects Genome Integrity from R-loops

Co-transcriptional RNA-DNA hybrids (R loops) cause genome instability. To prevent harmful R loop accumulation, cells have evolved specific eukaryotic factors, one being the BRCA2 double-strand break repair protein. As BRCA2 also protects stalled replication forks and is the FANCD1 member of the Fanconi Anemia (FA) pathway, we investigated the FA role in R loop-dependent genome instability. Using human and murine cells defective in FANCD2 or FANCA and primary bone marrow cells from FANCD2 deficient mice, we show that the FA pathway removes R loops, and that many DNA breaks accumulated in FA cells are R loop-dependent. Importantly, FANCD2 foci in untreated and MMC-treated cells are largely R loop dependent, suggesting that the FA functions at R loop-containing sites. We conclude that co-transcriptional R loops and R loop-mediated DNA damage greatly contribute to genome instability and that one major function of the FA pathway is to protect cells from R loops.

Advances in the understanding of Fanconi Anemia Complementation Group D2 Protein (FANCD2) in human cancer.

Fanconi anemia (FA) is a rare human genetic disease, resulting from dysfunction in any of 17 known complementation proteins: FANC-A, B, C, D1, D2, E, F, G, I, J, L, M, N, O, P, Q & S, and other unknowns. Besides the severe bone marrow failure, an extremely high incidence of cancer as well as many other clinic symptoms associated with FA patients, FA cells are known of insufficiency in homologous recombination, DNA mismatch repair, nucleotide excision repair, translesion DNA synthesis, and other molecular defects, leading to genome instability. Those similar molecular and cellular/tissue features show that all FA proteins function in one common signaling pathway, namely, the FA pathway. The monoubiquitination of FANCD2 is the central step of the FA pathway activation upon DNA damage or during DNA replication. The molecular functions of FANCD2 emerge as a very attractive filed of investigation in cancer research. Herein, we review the recent progresses in FANCD2 functions at these rapidly progressed aspects.

Epigenetic Alterations in Fanconi Anaemia: Role in Pathophysiology and Therapeutic Potential.

Fanconi anaemia (FA) is an inherited disorder characterized by chromosomal instability. The phenotype is variable, which raises the possibility that it may be affected by other factors, such as epigenetic modifications. These play an important role in oncogenesis and may be pharmacologically manipulated. Our aim was to explore whether the epigenetic profiles in FA differ from non-FA individuals and whether these could be manipulated to alter the disease phenotype. We compared expression of epigenetic genes and DNA methylation profile of tumour suppressor genes between FA and normal samples. FA samples exhibited decreased expression levels of genes involved in epigenetic regulation and hypomethylation in the promoter regions of tumour suppressor genes. Treatment of FA cells with histone deacetylase inhibitor Vorinostat increased the expression of DNM3Tβ and reduced the levels of CIITA and HDAC9, PAK1, USP16, all involved in different aspects of epigenetic and immune regulation. Given the ability of Vorinostat to modulate epigenetic genes in FA patients, we investigated its functional effects on the FA phenotype. This was assessed by incubating FA cells with Vorinostat and quantifying chromosomal breaks induced by DNA cross-linking agents. Treatment of FA cells with Vorinostat resulted in a significant reduction of aberrant cells (81% on average). Our results suggest that epigenetic mechanisms may play a role in oncogenesis in FA. Epigenetic agents may be helpful in improving the phenotype of FA patients, potentially reducing tumour incidence in this population.

Impaired TIP60-mediated H4K16 acetylation accounts for the aberrant chromatin accumulation of 53BP1 and RAP80 in Fanconi anemia pathway-deficient cells.

To rescue collapsed replication forks cells utilize homologous recombination (HR)-mediated mechanisms to avoid the induction of gross chromosomal abnormalities that would be generated by non-homologous end joining (NHEJ). Using DNA interstrand crosslinks as a replication barrier, we investigated how the Fanconi anemia (FA) pathway promotes HR at stalled replication forks. FA pathway inactivation results in Fanconi anemia, which is associated with a predisposition to cancer. FANCD2 monoubiquitination and assembly in subnuclear foci appear to be involved in TIP60 relocalization to the chromatin to acetylates histone H4K16 and prevents the binding of 53BP1 to its docking site, H4K20Me2. Thus, FA pathway loss-of-function results in accumulation of 53BP1, RIF1 and RAP80 at damaged chromatin, which impair DNA resection at stalled replication fork-associated DNA breaks and impede HR. Consequently, DNA repair in FA cells proceeds through the NHEJ pathway, which is likely responsible for the accumulation of chromosome abnormalities. We demonstrate that the inhibition of NHEJ or deacetylase activity rescue HR in FA cells.

Cytokine overproduction and crosslinker hypersensitivity are unlinked in Fanconi anemia macrophages.

The Fanconi anemia proteins participate in a canonical pathway that repairs cross-linking agent-induced DNA damage. Cells with inactivated Fanconi anemia genes are universally hypersensitive to such agents. Fanconi anemia-deficient hematopoietic stem cells are also hypersensitive to inflammatory cytokines, and, as importantly, Fanconi anemia macrophages overproduce such cytokines in response to TLR4 and TLR7/8 agonists. We questioned whether TLR-induced DNA damage is the primary cause of aberrantly regulated cytokine production in Fanconi anemia macrophages by quantifying TLR agonist-induced TNF-α production, DNA strand breaks, crosslinker-induced chromosomal breakage, and Fanconi anemia core complex function in Fanconi anemia complementation group C-deficient human and murine macrophages. Although both M1 and M2 polarized Fanconi anemia cells were predictably hypersensitive to mitomycin C, only M1 macrophages overproduced TNF-α in response to TLR-activating signals. DNA damaging agents alone did not induce TNF-α production in the absence of TLR agonists in wild-type or Fanconi anemia macrophages, and mitomycin C did not enhance TLR responses in either normal or Fanconi anemia cells. TLR4 and TLR7/8 activation induced cytokine overproduction in Fanconi anemia macrophages. Also, although TLR4 activation was associated with induced double strand breaks, TLR7/8 activation was not. That DNA strand breaks and chromosome breaks are neither necessary nor sufficient to account for the overproduction of inflammatory cytokines by Fanconi anemia cells suggests that noncanonical anti-inflammatory functions of Fanconi anemia complementation group C contribute to the aberrant macrophage phenotype and suggests that suppression of macrophage/TLR hyperreactivity might prevent cytokine-induced stem cell attrition in Fanconi anemia.

Fanconi anemia cells with unrepaired DNA damage activate components of the checkpoint recovery process.

BackgroundThe FA/BRCA pathway repairs DNA interstrand crosslinks. Mutations in this pathway cause Fanconi anemia (FA), a chromosome instability syndrome with bone marrow failure and cancer predisposition. Upon DNA damage, normal and FA cells inhibit the cell cycle progression, until the G2/M checkpoint is turned off by the checkpoint recovery, which becomes activated when the DNA damage has been repaired. Interestingly, highly damaged FA cells seem to override the G2/M checkpoint. In this study we explored with a Boolean network model and key experiments whether checkpoint recovery activation occurs in FA cells with extensive unrepaired DNA damage.MethodsWe performed synchronous/asynchronous simulations of the FA/BRCA pathway Boolean network model. FA-A and normal lymphoblastoid cell lines were used to study checkpoint and checkpoint recovery activation after DNA damage induction. The experimental approach included flow cytometry cell cycle analysis, cell division tracking, chromosome aberration analysis and gene expression analysis through qRT-PCR and western blot.ResultsComputational simulations suggested that in FA mutants checkpoint recovery activity inhibits the checkpoint components despite unrepaired DNA damage, a behavior that we did not observed in wild-type simulations. This result implies that FA cells would eventually reenter the cell cycle after a DNA damage induced G2/M checkpoint arrest, but before the damage has been fixed. We observed that FA-A cells activate the G2/M checkpoint and arrest in G2 phase, but eventually reach mitosis and divide with unrepaired DNA damage, thus resolving the initial checkpoint arrest. Based on our model result we look for ectopic activity of checkpoint recovery components. We found that checkpoint recovery components, such as PLK1, are expressed to a similar extent as normal undamaged cells do, even though FA-A cells harbor highly damaged DNA.ConclusionsOur results show that FA cells, despite extensive DNA damage, do not loss the capacity to express the transcriptional and protein components of checkpoint recovery that might eventually allow their division with unrepaired DNA damage. This might allow cell survival but increases the genomic instability inherent to FA individuals and promotes cancer.

Abstract 3018: FANCF, a Fanconi anemia core protein, functions outside of monoubiquitinating FANCD2 in DNA interstrand crosslink repair

Abstract Genomic instability is a hallmark of the genetic disorder, Fanconi anemia (FA), which is characterized by bone marrow failure, an increased incidence of cancer, congenital abnormalities and a defect in ability to repair DNA interstrand cross-links (ICLs). We have previously shown that FA cells have a deficiency in the structural protein, nonerythroid A spectrin (ASpII), which is critical for repair of DNA ICLs and binds to cross-linked DNA. Eight FA proteins, FANC -A, B, C, E, F, G, L, and M, form a core complex which is essential for monoubiquitination of FANCD2, an important step in ICL repair. However, whether any of these core proteins play additional roles in the ICL repair process is not clearly known. The present study was undertaken to address this question and to examine whether one of these proteins, FANCF, is involved in the ICL repair process with ASpII. Immunofluorescence microscopy was used to determine whether these two proteins co-localize in nuclear foci after normal human cells are damaged with an ICL agent, 8-methylpsoralen plus UVA light (8-MOP). Time course measurements showed that, in normal human cells, FANCF nuclear foci formed over a similar time course as did those of ASpII. This time course was also similar to that of FANCA and the ICL repair protein, XPF. FANCF foci, as well as ASpII, FANCA and XPF foci, were visible 10 hours after damage, peaked at 16 hours and by 24 hours were no longer observed. FANCF foci, over this time course, co-localized with ASpII foci, indicating that FANCF is associated with ASpII during ICL repair. This association was corroborated by co-immunoprecipitation studies which demonstrated that FANCF has enhanced binding to ASpII after ICL damage. This indicates that FANCF is involved in the same steps in the repair process as ASpII. In FA-A cells, FANCF is present as in normal cells, but does not form nuclear foci after ICL damage. Transfection of FA-A cells with a cDNA expressing FANCA, however, led to restoration of ASpII levels to normal and to formation of FANCF nuclear foci, which colocalized with ASpII foci. Our studies indicate that this is due to restoration of ASpII levels to normal. These studies support a model we have proposed in which ASpII acts as a scaffold in the recruitment of proteins (i.e., FANCF, FANCA, and XPF) to sites of ICL damage and that FA proteins, such as FANCA, are needed for maintenance of ASpII stability in the cell. In the transfected FA-A cells, expression of FANCA leads to enhanced stability of ASpII, which then participates in the ICL repair process. These studies indicate that the FA core complex protein, FANCF, has an additional function in ICL repair, besides monoubiquitination of FANCD2, and is involved with ASpII in its role in the repair process and in maintaining genomic stability after DNA ICL damage. Citation Format: Muriel W. Lambert, Deepa Sridharan, Pan Zhang. FANCF, a Fanconi anemia core protein, functions outside of monoubiquitinating FANCD2 in DNA interstrand crosslink repair. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3018. doi:10.1158/1538-7445.AM2015-3018

Functional Significance of Nuclear α Spectrin

Nonerythroid alpha spectrin (αIISp) interacts in the nucleus with an array of different proteins indicating its involvement in a number of diverse functions. However, the significance of these interactions and their functional importance has been a relatively unexplored area. The best documented role of nuclear αIISp is in DNA repair where it is critical for repair of DNA interstrand cross-links (ICLs), acting as a scaffold recruiting proteins to sites of damage in genomic and telomeric DNA. A deficiency in αIISp can importantly impact DNA ICL repair as is seen in cells from patients with the genetic disorder, Fanconi anemia (FA), where loss of αIISp leads to not only defects in repair of both genomic and telomeric DNA but also to telomere dysfunction and chromosome instability. This previously unexplored link between αIISp and telomere function is important in developing an understanding of maintenance of genomic stability after ICL damage. In FA cells, these defects in chromosome instability after ICL damage can be corrected when levels of αIISp are returned to normal by knocking down μ-calpain, a protease which cleaves αIISp. These studies suggest a new direction for correcting a number of the phenotypic defects in FA and could serve as a basis for therapeutic intervention. More in depth, examination of the interactions of αIISp with other proteins in the nucleus is of major importance in development of insights into the interacting key elements involved in the diverse processes occurring in the nucleus and the consequences loss of αIISp has on them.