Fanconi Anemia Cells Research Articles

Overcoming reprogramming resistance of Fanconi anemia cells

AbstractFanconi anemia (FA) is a recessive syndrome characterized by progressive fatal BM failure and chromosomal instability. FA cells have inactivating mutations in a signaling pathway that is critical for maintaining genomic integrity and protecting cells from the DNA damage caused by cross-linking agents. Transgenic expression of the implicated genes corrects the phenotype of hematopoietic cells, but previous attempts at gene therapy have failed largely because of inadequate numbers of hematopoietic stem cells available for gene correction. Induced pluripotent stem cells (iPSCs) constitute an alternate source of autologous cells that are amenable to ex vivo expansion, genetic correction, and molecular characterization. In the present study, we demonstrate that reprogramming leads to activation of the FA pathway, increased DNA double-strand breaks, and senescence. We also demonstrate that defects in the FA DNA-repair pathway decrease the reprogramming efficiency of murine and human primary cells. FA pathway complementation reduces senescence and restores the reprogramming efficiency of somatic FA cells to normal levels. Disease-specific iPSCs derived in this fashion maintain a normal karyotype and are capable of hematopoietic differentiation. These data define the role of the FA pathway in reprogramming and provide a strategy for future translational applications of patient-specific FA iPSCs.

Abstract 4695: TGF-β enforces genomic instability in liver cancers

Abstract Background: Over 90% of liver cancers, from viral hepatitis to alcohol injury, occur against a background of chronic liver disease and cirrhosis. Genomic stability, G1/S cell cycle arrest through c-Myc and CDK suppression, have been some of the mechanisms proposed in tumor suppression by TGF-β. The Smad3/4 adaptor protein, β2SP, is emerging as potent regulator of tumor genesis by its ability to affect TGF-β tumor suppressor function. Deletion of β2SP results in a dramatic and spontaneous formation of liver cancer and gastrointestinal cancers. Moreover, β2SP+/− and β2SP+/− /Smad3+/− mutant mice phenocopy human Beckwith-Wiedemann syndrome (BWS), a hereditary human cancer stem cell syndrome imprinting disorder with loss of p57, increase in IGF2, and an association with 800 fold increase of cancers that include those of the liver. Delayed liver regeneration and extensive DNA damage was observed in β2SP heterozygote mice. In addition, spectrins have been observed to associate with FANC G and D, DNA interstrand cross links, and Smad3/4 to transcriptionally regulate FANC genes. Hypothesis: We therefore hypothesised that TGF-β is a crucial enforcer of genomic stability, and that β2SP and/or Smad3/4 are key mediators in suppressing liver injury and cancer through modulation of the DNA repair pathway. Materials & Methods: To determine the role of β2SP and Smad3 in the DNA damage response, and whether β2SP and β2SP/Smad3 loss plays a causal role in the downregulation of the Fanc/Brca pathway, we used our mouse models as well as the mouse embryonic fibroblasts. Oxidative DNA damage was induced by H2O2 treatment and assayed tail length analysis. Irradiation was induced by 8Gy IR and DNA was studied by immunofluorescence of DNA damage sensors H2AX, 532BP1, and pChk2. DNA repair was assayed by Mdc1, NBS1, and Rad51 foci formation at different time points after irradiation. Results: β2SP+/− and β2SP+/−/Smad3+/− mice exhibited an increased prevalence of HCC and GI cancer and many phenotypic characteristics observed in BWS patients. Loss of β2SP results in premature replicative senescence with spontaneous DNA damage. β2SP deficiency results in increased sensitivity to exogenous DNA damage and insufficient DNA repair. Depletion of TGF-β adaptor protein β2SP impairs the loading of repair proteins and repair of DNA double-strand breaks. Loss of FancD2 expression in HCC and GI cancer cell lines correlates with loss of β2SP. Moreover, β2SP−/− and β2SP+/−/Smad3+/− cell lines accumulate DNA damage that is further exacerbated by DNA cross-linking agents such as mitomycin C similar to P57 null cells and Fanconi anemia cell lines. Conclusions: TGF-β confers genomic stability through regulation of DNA impairment of the Fanc/Brca DNA damage response and interstrand link repair and inactivation of TGF- ≤ pathway results in alcohol toxicity, cirrhosis and liver cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4695. doi:1538-7445.AM2012-4695

Fanconi anemia (FA) binding protein FAAP20 stabilizes FA complementation group A (FANCA) and participates in interstrand cross-link repair

The Fanconi anemia (FA) pathway participates in interstrand cross-link (ICL) repair and the maintenance of genomic stability. The FA core complex consists of eight FA proteins and two Fanconi anemia-associated proteins (FAAP24 and FAAP100). The FA core complex has ubiquitin ligase activity responsible for monoubiquitination of the FANCI-FANCD2 (ID) complex, which in turn initiates a cascade of biochemical events that allow processing and removal of cross-linked DNA and thereby promotes cell survival following DNA damage. Here, we report the identification of a unique component of the FA core complex, namely, FAAP20, which contains a RAD18-like ubiquitin-binding zinc-finger domain. Our data suggest that FAAP20 promotes the functional integrity of the FA core complex via its direct interaction with the FA gene product, FANCA. Indeed, somatic knockout cells devoid of FAAP20 displayed the hallmarks of FA cells, including hypersensitivity to DNA cross-linking agents, chromosome aberrations, and reduced FANCD2 monoubiquitination. Taking these data together, our study indicates that FAAP20 is an important player involved in the FA pathway.

Fanconi Anemia and Vaginal Squamous Cell Carcinoma

Fanconi Anemia (FA) is an autosomal recessive disease characterized by chromosome instability, cellular hypersensitivity to DNA cross-linking agents, and increased predisposition to malignancies. We describe here a 28 year-old female with FA and vaginal squamous cell carcinoma treated by radiation therapy alone. The patient developed arm phlebitis, pulmonary fungal infection, and severe rectal bleeding, followed by hypocalcaemia, hypokalemia, vaginal bacterial and fungal infection, with subsequent leg and arm phlebitis, perineal abscess, and sepsis. The patient died 12 weeks later.

P38 MAPK inhibition suppresses the TLR-hypersensitive phenotype in FANCC- and FANCA-deficient mononuclear phagocytes

AbstractFanconi anemia, complementation group C (FANCC)–deficient hematopoietic stem and progenitor cells are hypersensitive to a variety of inhibitory cytokines, one of which, TNFα, can induce BM failure and clonal evolution in Fancc-deficient mice. FANCC-deficient macrophages are also hypersensitive to TLR activation and produce TNFα in an unrestrained fashion. Reasoning that suppression of inhibitory cytokine production might enhance hematopoiesis, we screened small molecules using TLR agonist–stimulated FANCC- and Fanconi anemia, complementation group A (FANCA)–deficient macrophages containing an NF-κB/AP-1–responsive reporter gene (SEAP). Of the 75 small molecules screened, the p38 MAPK inhibitor BIRB 796 and dasatinib potently suppressed TLR8-dependent expression of the reporter gene. Fanconi anemia (FA) macrophages were hypersensitive to the TLR7/8 activator R848, overproducing SEAP and TNFα in response to all doses of the agonist. Low doses (50nM) of both agents inhibited p38 MAPK–dependent activation of MAPKAPK2 (MK2) and suppressed MK2-dependent TNFα production without substantially influencing TNFα gene transcription. Overproduction of TNFα by primary FA cells was likewise suppressed by these agents and involved inhibition of MK2 activation. Because MK2 is also known to influence production and/or sensitivity to 2 other suppressive factors (MIP-1α and IFNγ) to which FA hematopoietic progenitor cells are uniquely vulnerable, targeting of p38 MAPK in FA hematopoietic cells is a rational objective for preclinical evaluation.

A Dutch Fanconi AnemiaFANCCFounder Mutation in Canadian Manitoba Mennonites

Fanconi anemia (FA) is a recessive DNA instability disorder associated with developmental abnormalities, bone marrow failure, and a predisposition to cancer. Based on their sensitivity to DNA cross-linking agents, FA cells have been assigned to 15 complementation groups, and the associated genes have been identified. Founder mutations have been found in different FA genes in several populations. The majority of Dutch FA patients belongs to complementation group FA-C. Here, we report 15 patients of Dutch ancestry and a large Canadian Manitoba Mennonite kindred carrying the FANCC c.67delG mutation. Genealogical investigation into the ancestors of the Dutch patients shows that these ancestors lived in four distinct areas in The Netherlands. We also show that the Dutch and Manitoba Mennonite FANCC c.67delG patients share the same haplotype surrounding this mutation, indicating a common founder.

Selective Damage to Antioxidant Gene Promoters in FA Cells

Abstract 2413Oxidative stress has been implicated in the pathogenesis of many human diseases including Fanconi anemia (FA), a genetic disorder associated with bone marrow failure and progression to leukemia and other cancers. Here we show that several major anti-oxidant defense genes, including Glutathione peroxidase 1, Peroxiredoxin 3, Thioredoxin reductase 1, Superoxide dismutases 1, NAD(P)H:quinone oxireductase and Catalase, are down-regulated in bone marrow cells of FA patients. This gene down-regulation is selectively associated with increased oxidative DNA damage in the promoters of these anti-oxidant defense genes. Further, we show that both increased initial damage and reduced repair rate contribute to augmented oxidative DNA damage in FA cells. Using cell-based assays to assess promoter activity and damage repair kinetics, we demonstrate that FA proteins function to protect the promoter DNA from oxidative damage. Mechanistically, FA proteins appeared to act in concert with Brg1, a chromatin-remodeling ATPase subunit of the BAF complex. Specifically, Brg1 binds to the promoters of the anti-oxidant defense genes in steady state. Upon challenge with oxidative stress, FANCA and FANCD2 proteins are recruited to the promoter DNA, which correlates with significant increase in the binding of Brg1 within the promoter regions. Intriguingly, the formation of the FA-Brg1-promoter complex results in a marked decrease in nuclease hypersensitivity and oxidative damage in the promoter DNA in normal cells compared to FA cells. Finally, disassociation of the FA proteins from the Brg1-promoter complex parallels Pol II loading, suggesting a regulatory role for the FA proteins in transcription. Taken together, the study identifies a role of FA proteins in protecting anti-oxidant genes from oxidative damage. Disclosures:No relevant conflicts of interest to declare.

Fancf‐deficient mice are prone to develop ovarian tumours

Fanconi anaemia (FA) is a rare recessive disorder marked by developmental abnormalities, bone marrow failure, and a high risk for the development of leukaemia and solid tumours. The inactivation of FA genes, in particular FANCF, has also been documented in sporadic tumours in non-FA patients. To study whether there is a causal relationship between FA pathway defects and tumour development, we have generated a mouse model with a targeted disruption of the FA core complex gene Fancf. Fancf-deficient mouse embryonic fibroblasts displayed a phenotype typical for FA cells: they showed an aberrant response to DNA cross-linking agents as manifested by G(2) arrest, chromosomal aberrations, reduced survival, and an inability to monoubiquitinate FANCD2. Fancf homozygous mice were viable, born following a normal Mendelian distribution, and showed no growth retardation or developmental abnormalities. The gonads of Fancf mutant mice functioned abnormally, showing compromised follicle development and spermatogenesis as has been observed in other FA mouse models and in FA patients. In a cohort of Fancf-deficient mice, we observed decreased overall survival and increased tumour incidence. Notably, in seven female mice, six ovarian tumours developed: five granulosa cell tumours and one luteoma. One mouse had developed tumours in both ovaries. High-resolution array comparative genomic hybridization (aCGH) on these tumours suggests that the increased incidence of ovarian tumours correlates with the infertility in Fancf-deficient mice and the genomic instability characteristic of FA pathway deficiency.

Abstract A102: Synthetic lethality: ABT-888 and carboplatin drug combinations are synergistic for BRCA1- and BRCA2-deficient mouse and human cells in vitro.

Abstract Background: Individuals with an inherited BRCA1 or BRCA2 (BRCA) mutation have an elevated risk of developing breast and ovarian cancer, and this BRCA defect is also present in a subset of sporadic tumors. Tumors do not have homologous recombination repair (HRR) to repair double-stranded breaks (DSBs), but they still can use other DNA repair pathways to survive. Therefore, we hypothesized that crosstalk between DNA repair pathways complements HRR in BRCA-deficient cells, leading to tumor survival. Treatment with either a poly(ADP)ribose polymerase (PARP) inhibitor (PARPi) or a platinum drug induced BRCA-deficient cell lethality by different mechanisms. In addition, combining both drugs has been reported to be synergistic in BRCA mouse models. However, the mechanism of this synergism and whether this synergism occurs in human cells are still unclear. Moreover, not all the patients respond to a PARPi chemotherapy alone. Hence, our objectives were: 1) to study the mechanism by which a PARPi combined with a platinum drug is synthetic lethal in human BRCA1 or BRCA-deficient cells; 2) to assess PARP activity as a biomarker for chemosensitivity. Methods: We compared the cytotoxic effect of ABT-888 (a PARPi) with cisplatin or carboplatin (platinum drugs) alone versus combinations by examining Brca-proficient and -deficient mouse embryonic stem (ES) cell survival using clonogenic assays. The effect of ABT-888 and carboplatin combination treatment on human cell growth was tested in HCC1937 (a triple negative BRCA1-deficient breast cancer line) and EUFA423F (a BRCA2-deficient Fanconi anemia cell line), along with their BRCA complemented lines. Synergism, antagonistic or additive drug interactions were assessed. Drug(s)-induced DSBs, HRR and apoptosis were examined and endogenous PARP activity was determined. Results: Each monotherapy killed or inhibited more Brca/BRCA-deficient cells than the corresponding Brca/BRCA-proficient cells. In Brca-deficient cells, the ABT-888 and carboplatin combination was more synergistic than ABT-888 and cisplatin. All paired BRCA-deficient or complemented cells had the same level of DNA damage following treatment with ABT-888 and/or Carboplatin. BRCA-deficient cells lack HRR, while BRCA-proficient cells use HRR. Failure to repair DSBs in BRCA-deficient cells triggered apoptosis, which was synergistic in the BRCA1-deficient cells. Moreover, higher endogenous PARP activity in Brca/BRCA-deficient cells than their respective Brca/BRCA proficient cells correlated with increased chemosensitivity. Conclusion: Our data suggest that combining ABT-888 and carboplatin will likely be more successful than monotherapy in treating most BRCA-associated cancers. In addition, high endogenous PARP activity could be used as a marker to identify PARPi susceptible tumors. Lack of HRR combined with a strong dependence on PARP activity for repair of platinum lesions led to synthetic lethality in BRCA-deficient cells. A randomized phase II trial has recently been initiated to test this drug combination in patients with BRCA-associated breast cancer and we are currently investigating γH2AX as a pharmacodynamic marker in hair follicles. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A102.

GS-Nitroxide (JP4 - 039) Mediated Radioprotection of Human Fanconi Anemia Cell Lines

Fanconi anemia (FA) is an inherited disorder characterized by defective DNA repair and cellular sensitivity to DNA cross-linking agents. Clinically, FA is associated with high risk for marrow failure, leukemia, and head and neck squamous cell carcinoma (HNSCC). Radiosensitivity in FA patients compromises the utilization of total body irradiation for hematopoietic stem cell transplantation and radiation therapy for HNSCC, during which a radioprotector for the surrounding tissue would be valuable.

GS-Nitroxide (JP4-039)-Mediated Radioprotection of Human Fanconi Anemia Cell Lines

Fanconi anemia (FA) is an inherited disorder characterized by defective DNA repair and cellular sensitivity to DNA crosslinking agents. Clinically, FA is associated with high risk for marrow failure, leukemia and head and neck squamous cell carcinoma (HNSCC). Radiosensitivity in FA patients compromises the use of total-body irradiation for hematopoietic stem cell transplantation and radiation therapy for HNSCC. A radioprotector for the surrounding tissue would therefore be very valuable during radiotherapy for HNSCC. Clonogenic radiation survival curves were determined for pre- or postirradiation treatment with the parent nitroxide Tempol or JP4-039 in cells of four FA patient-derived cell lines and two transgene-corrected subclonal lines. FancG(-/-) (PD326) and FancD2(-/-) (PD20F) patient lines were more sensitive to the DNA crosslinking agent mitomycin C (MMC) than their transgene-restored subclonal cell lines (both P < 0.0001). FancD2(-/-) cells were more radiosensitive than the transgene restored subclonal cell line (ñ = 2.0 ± 0.7 and 4.7 ± 2.2, respectively, P = 0.03). In contrast, FancG(-/-) cells were radioresistant relative to the transgene-restored subclonal cell line (ñ = 9.4 ± 1.5 and 2.2 ± 05, respectively, P = 0.001). DNA strand breaks measured by the comet assay correlated with radiosensitivity. Cell lines from a Fanc-C and Fanc-A patients showed radiosensitivity similar to that of Fanc-D2(-/-) cells. A fluorophore-tagged JP4-039 (BODIPY-FL) analog targeted the mitochondria of the cell lines. Preirradiation or postirradiation treatment with JP4-039 at a lower concentration than Tempol significantly increased the radioresistance and stabilized the antioxidant stores of all cell lines. Tempol increased the toxicity of MMC in FancD2(-/-) cells. These data provide support for the potential clinical use of JP4-039 for normal tissue radioprotection during chemoradiotherapy in FA patients.

The role of stat1b in zebrafish hematopoiesis

STAT1 mediates response to interferons and regulates immunity, cell proliferation, apoptosis, and sensitivity of Fanconi Anemia cells to apoptosis after interferon signaling; the roles of STAT1 in embryos, however, are not understood. To explore embryonic functions of STAT1, we investigated stat1b, an unstudied zebrafish co-ortholog of human STAT1. Zebrafish stat1a encodes all five domains of the human STAT1-alpha splice form but, like the human STAT1-beta splice variant, stat1b lacks a complete transactivation domain; thus, two unlinked zebrafish paralogs encode protein forms translated from two splice variants of a single human gene, as expected by sub-functionalization after genome duplication. Phylogenetic and conserved synteny studies showed that stat1b and stat1a arose as duplicates in the teleost genome duplication (TGD) and clarified the evolutionary origin of STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B and STAT6 by tandem and genome duplication. RT-PCR revealed maternal expression of stat1a and stat1b. In situ hybridization detected stat1b but not stat1a expression in embryonic hematopoietic tissues. Morpholino knockdown of stat1b, but not stat1a, decreased expression of the myeloid and granulocyte markers spi and mpo and increased expression of the hematopoietic progenitor marker scl, the erythrocyte marker gata1, and hemoglobin. These results suggest that zebrafish Stat1b promotes myeloid development at the expense of erythroid development.

Direct Inhibition of TNF-α Promoter Activity by Fanconi Anemia Protein FANCD2

Fanconi anemia (FA), an inherited disease, is associated with progressive bone marrow failure, predisposition to cancer, and genomic instability. Genes corresponding to 15 identified FA complementation groups have been cloned, and each gene product functions in the response to DNA damage induced by cross-linking agents and/or in protection against genome instability. Interestingly, overproduction of inflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and aberrant activation of NF-κB-dependent transcriptional activity have been observed in FA cells. Here we demonstrated that FANCD2 protein inhibits NF-κB activity in its monoubiquitination-dependent manner. Furthermore, we detected a specific association between FANCD2 and an NF-κB consensus element in the TNF-α promoter by electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP) assay. Therefore, we propose FANCD2 deficiency promotes transcriptional activity of the TNF-α promoter and induces overproduction of TNF-which then sustains prolonged inflammatory responses. These results also suggest that artificial modulation of TNFα production could be a promising therapeutic approach to FA.

Functional and physical interaction between the mismatch repair and FA-BRCA pathways.

Fanconi anemia (FA) is a rare genetic disorder characterized by bone marrow failure and an increased risk for leukemia and cancer. Fifteen proteins thought to function in the repair of DNA interstrand crosslinks (ICLs) comprise what is known as the FA-BRCA pathway. Activation of this pathway leads to the monoubiquitylation and chromatin localization of FANCD2 and FANCI. It has previously been shown that FANCJ interacts with the mismatch repair (MMR) complex MutLα. Here we show that FANCD2 interacts with the MMR proteins MSH2 and MLH1. FANCD2 monoubiquitylation, foci formation and chromatin loading are greatly diminished in MSH2-deficient cells. Human or mouse cells lacking MSH2 or MLH1 display increased sensitivity and radial formation in response to treatment with DNA crosslinking agents. Studies in human cell lines and Drosophila mutants suggest an epistatic relationship between FANCD2, MSH2 and MLH1 with regard to ICL repair. Surprisingly, the interaction between MSH2 and MLH1 is compromised in multiple FA cell lines, and FA cell lines exhibit deficient MMR. These results suggest a significant role for MMR proteins in the activation of the FA pathway and repair of ICLs. In addition, we provide the first evidence for a defect in MMR in FA cell lines.

Anemia de Fanconi: diagnóstico citogenético de 40 casos.

Fanconi Anemia (FA) is a rare recessive disorder clinically characterized by progressive bone marrow failure, diverse congenital malformations and increased predisposition to cancer. Given the late onset of anemia, relatively to other cytopenias, and the high variability in the phenotype, a correct clinical diagnosis is difficult, and may be delayed or even missed. This fact may be prejudicial to patients, due to the need of avoiding exposure to toxic agents, programming the transplantation of hematopoietic progenitor cells and screening of neoplasia associated with the disease. Given the high genetic variability (thirteen complementation groups have been identified, each with genes presenting several different mutations), a rapid molecular diagnosis is not possible. However, there is an urgent need for a timely and correct diagnosis, due to the early evolution of the disease towards malignancy and to the early need of finding compatible donors for future hematopoietic stem cell transplantation. Fortunately, the hypersensitivity of FA cells to the clastogenic (chromosome breaking) effect of DNA cross-linking agents, in particular to diepoxybutane (DEB), provides a unique marker for the diagnosis. At present, cytogenetic analysis for detection of DEB-induced chromosome instability is the gold-standard test for the diagnosis of FA. In the present work we present the results from the DEB induced chromosome instability studies performed in the Laboratory of Cytogenetics of ICBAS between 1992 and 2009. Blood samples from 222 patients were obtained from different hospitals mainly from the north and centre of Portugal. This population includes not only patients with clinical suspicion of FA, but also patients presented with thrombocytopenia, pancitopenia or aplastic anemia, for confirmation/exclusion of FA. Two samples of amniotic fluid were also obtained for pre-natal diagnosis. A total of 34 FA patients were diagnosed. Cytogenetic studies were also performed in blood samples from AF relatives, which allowed the diagnosis of 6 new cases, 5 of them corresponding to asymptomatic individuals. In the total population of FA patients studied, 25% belong to the gypsy ethnic group. Periodic cytogenetic studies were also performed in blood samples from AF patients post transplantation, which confirmed the elimination of the original hematopoietic DEB sensitive cells.

Protective effect of acetyl-l-carnitine and α-lipoic acid against the acute toxicity of diepoxybutane to human lymphocytes

The biotransformation and oxidative stress may contribute to 1,2:3,4-diepoxybutane (DEB)-induced toxicity to human lymphocytes of Fanconi Anemia (FA) patients. Thus, the identification of putative inhibitors of bioactivation, as well as the determination of the protective role of oxidant defenses, on DEB-induced toxicity, can help to understand what is failing in FA cells. In the present work we studied the contribution of several biochemical pathways for DEB-induced acute toxicity in human lymphocyte suspensions, by using inhibitors of epoxide hydrolases, inhibitors of protective enzymes as glutathione S-transferase and catalase, the depletion of glutathione (GSH), and the inhibition of protein synthesis; and a variety of putative protective compounds, including antioxidants, and mitochondrial protective agents. The present study reports two novel findings: (i) it was clearly evidenced, for the first time, that the acute exposure of freshly isolated human lymphocytes to DEB results in severe GSH depletion and loss of ATP, followed by cell death; (ii) acetyl-l-carnitine elicits a significant protective effect on DEB induced toxicity, which was potentiated by α-lipoic acid. Collectively, these findings contribute to increase our knowledge of DEB-induce toxicity and will be very useful when applied in studies with lymphocytes from FA patients, in order to find out a protective agent against spontaneous and DEB-induced chromosome instability.

Rad18 E3 ubiquitin ligase activity mediates Fanconi anemia pathway activation and cell survival following DNA Topoisomerase 1 inhibition

Camptothecin (CPT) and related chemotherapeutic drugs induce formation of DNA Topoisomerase I (Top1) covalent or cleavage complexes (Top1ccs) that block leading-strand DNA synthesis and elicit DNA Double Stranded Breaks (DSB) during S phase. The Fanconi Anemia (FA) pathway is implicated in tolerance of CPT-induced DNA damage yet the mechanism of FA pathway activation by Top1 poisons has not been studied. We show here that the FA core complex protein FANCA and monoubiquitinated FANCD2 (an effector of the FA pathway) are rapidly mobilized to chromatin in response to CPT treatment in several human cancer cell lines and untransformed primary human dermal fibroblasts. FANCD2 depletion using siRNA leads to impaired recovery from CPT-induced inhibition or DNA synthesis, persistence of γH2AX (a DSB marker) and reduced cell survival following CPT treatment. The E3 ubiquitin ligase Rad18 is necessary for CPT-induced recruitment of FANCA and FANCD2 to chromatin. Moreover, Rad18-depletion recapitulates the DNA synthesis and survival defects of FANCD2-deficiency in CPT-treated cells. It is well-established that Rad18 promotes FA pathway activation and DNA damage tolerance in response to bulky DNA lesions via a mechanism involving PCNA monoubiquitination. In contrast, PCNA monoubiquitination is not involved in Rad18-mediated FA pathway activation or cell survival following acquisition of CPT-induced DSB. Moreover, while Rad18 is implicated in recombinational repair of DSB via an E3 ligase-independent mechanism, we demonstrate that Rad18 E3 ligase activity is essential for appropriate FA pathway activation and DNA damage tolerance after CPT treatment. Taken together, our results define a novel pathway of Rad18-dependent DSB repair that is dissociable from known Rad18-mediated DNA repair mechanisms based on its independence from PCNA ubiquitination and requirement for E3 ligase activity.

Abstract 3924: Failure of localization of αII spectrin to telomeres after DNA interstrand cross-link damage in Fanconi anemia cells may be an important factor in their chromosome instability

Abstract A hallmark of the cancer prone genetic disorder, Fanconi anemia (FA), is genomic instability characterized by both chromosome instability and defects in DNA interstrand crosslink (ICL) repair. The chromosome instability, in particular after exposure to ICLs, suggests telomere dysfunction. We have demonstrated that the structural protein, nonerythroid α spectrin (αIISp), is present in the nucleus of normal human cells where it is critical in repair of DNA ICLs and maintenance of chromosome stability and that in FA cells levels of αIISp are reduced due to its increased cleavage by μ-calpain, which correlate with reduced ICL repair and increased chromosomal instability. The goal of this study was to determine whether αIISp is important for telomere function after DNA ICL damage and whether telomere dysfunction in FA cells after damage can be corrected when levels of αIISp are restored to normal. Normal and FA complementation group A (FA-A) lymphoblastoid cells were treated with a DNA ICL agent, 8-methoxypsoralen plus UVA light or mitomycin C and studied using immunofluorescence and in situ hybridization (IF-FISH). Cells were probed with anti-α-spectrin and either a telomeric probe (PNA) or antibodies against two telomeric proteins, TRF1 or TRF2. The results showed that 47-50% of αIISp foci colocalized with the PNA probe, TRF1 or TRF2 in telomeres in ICL damaged but not undamaged normal cells and that this colocalization was specific for the S phase. This indicated that αIISp may play an important role in telomere function after ICLs and this view was strengthened by data showing that αIISp has markedly enhanced binding for TRF1 and TFR2 after ICL damage. IF-FISH showed that the ICL repair protein, XPF, also had enhanced association with telomeres after ICL formation. In FA-A cells, no localization of XPF to telomeres was observed; however, after αIISp levels were returned to normal by siRNA knockdown of μ-calpain, αIISp as well as XPF associated with telomeres after ICLs. The chromosomal aberrations (i.e., breaks and end fusions) observed in FA-A cells after ICL damage were also corrected. The present results suggest that, after ICL damage, αIISp associates with telomeres in S phase and this association may be important in repair of ICLs in replicating telomeres. In FA-A cells, failure of localization of αIISp and XPF to telomeres after ICL damage could lead to unrepaired ICLs, telomere dysfunction and production of the chromosomal aberrations observed in these cells. These studies show that αIISp may play an important role in telomere function after DNA damage and during telomere replication. Correction of the deficiency in αIISp in FA cells by knocking down μ-calpain may be significant in reversing telomere dysfunction and chromosomal instability after ICL damage and could have important therapeutic relevance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3924. doi:10.1158/1538-7445.AM2011-3924

Abstract 2503: Distinct radiosensitivities of human FancG-/- and FancD2-/- cell lines

Abstract Background: Fanconi Anemia is an autosomal recessive disorder marked by a dysfunction of DNA repair, an increased sensitivity to DNA crosslinking agents, and some patients poorly tolerating ionizing irradiation. We determined the radiosensitivity of two Fanconi patient cell lines and we determined if the nitroxide radioprotector Tempo or a novel mitochondrial targeted nitroxide JP4-039 could alter their radiosensitivity. Materials and Methods: Fanconi patient derived fibroblastic cell lines, PD326 and PD20F, and their respective transgene restored cell lines FancG and FancD2 were irradiated to doses ranging from 0 to 8 Gy in the presence or absence of Tempo or JP4-039 (10 µm), plated, and colonies greater than 50 cells were counted eight days later. Results: The PD20F cell line was more radiosensitive compared to the restored FancD2 cell line (n = 1.6 + 0.2 and 10.7 + 3.1, respectively, p = 0.0423). In contrast the PD326 cell line was more radioresistant than its restored FancG cell line as seen by an increased shoulder on the survival curve (n = 8.1 + 1.4 and 2.2 + 0.3, respectively, p = 0.0174). JP4-039 but not Tempo increased radioresistance in the PD326 (n = 15.7 + 2.5, p = 0.0373), and FancG cell lines (10.2 + 2.1, p = 0.0151). Conclusions: The human Fanconi Anemia cell line PD326 is radioresistant when compared to its transgene restored FancG cell line, and this radioresistance is increased by treatment with the mitochondrial targeted nitroxide JP4-039. In contrast, PD20F is more radiosensitive when compared to its transgene restored FancD2 cell line. Testing the efficacy of JP4-039 and Tempo on mitigating the effects of mitomycin C treatment is being evaluated. Supported by 2U19A1068021-06 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2503. doi:10.1158/1538-7445.AM2011-2503

Coordinated action of the Fanconi anemia and ataxia telangiectasia pathways in response to oxidative damage

Fanconi anemia (FA) and ataxia telangiectasia (AT) share common traits such chromosomal instability and proneness to hematological cancers. Both AT and FA cell lines, and patients, are characterized by abnormally high levels of oxidative stress markers. The key FA protein FANCD2 is phosphorylated on Ser 222 by ATM after ionizing radiation (IR), thus allowing normal activation of the S-phase checkpoint, and ATM cells are known to be hypersensitive to oxidative damage. In this work we show that FANCD2 deficient cells have a defective S-phase checkpoint after Hydrogen Peroxide (H 2O 2) induced oxidative damage. ATM dependent phosphorylation of FANCD2 at the S222 residue is necessary for normal S-phase checkpoint activation after oxidative stress, while FANCD2 monoubiquitination at K561 is dispensable. We also show that FANCD2 is not required for base excision repair of 8-oxoG and other DNA lesions (abasic sites, uracils), while treatments that exclusively induce 8-oxoG, but not DNA double strand breaks, fail to activate FANCD2 monoubiquitination, thus indicating that the known accumulation of 8-oxoG in FA cells reflects an overproduction of ROS rather than defective processing of oxidized bases. We conclude that the handling of DNA damage after H 2O 2-induced oxidative stress requires the coordinated action of FANCD2 and ATM.