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1.
Mol Cell Proteomics ; 22(2): 100496, 2023 02.
Article in English | MEDLINE | ID: mdl-36640924

ABSTRACT

Transcriptional enhanced associate domain family members 1 to 4 (TEADs) are a family of four transcription factors and the major transcriptional effectors of the Hippo pathway. In order to activate transcription, TEADs rely on interactions with other proteins, such as the transcriptional effectors Yes-associated protein and transcriptional co-activator with PDZ-binding motif. Nuclear protein interactions involving TEADs influence the transcriptional regulation of genes involved in cell growth, tissue homeostasis, and tumorigenesis. Clearly, protein interactions for TEADs are functionally important, but the full repertoire of TEAD interaction partners remains unknown. Here, we employed an affinity purification mass spectrometry approach to identify nuclear interacting partners of TEADs. We performed affinity purification mass spectrometry experiment in parallel in two different cell types and compared a wildtype TEAD bait protein to a nuclear localization sequence mutant that does not localize to the nucleus. We quantified the results using SAINT analysis and found a significant enrichment of proteins linked to DNA damage including X-ray repair cross-complementing protein 5 (XRCC5), X-ray repair cross-complementing protein 6 (XRCC6), poly(ADP-ribose) polymerase 1 (PARP1), and Rap1-interacting factor 1 (RIF1). In cellular assays, we found that TEADs co-localize with DNA damage-induced nuclear foci marked by histone H2AX phosphorylated on S139 (γH2AX) and Rap1-interacting factor 1. We also found that depletion of TEAD proteins makes cells more susceptible to DNA damage by various agents and that depletion of TEADs promotes genomic instability. Additionally, depleting TEADs dampens the efficiency of DNA double-stranded break repair in reporter assays. Our results connect TEADs to DNA damage response processes, positioning DNA damage as an important avenue for further research of TEAD proteins.


Subject(s)
DNA Damage , DNA Repair , TEA Domain Transcription Factors , Humans , Carcinogenesis/metabolism , DNA Repair/physiology , DNA-Binding Proteins/metabolism , Transcription Factors/metabolism , TEA Domain Transcription Factors/metabolism
2.
Nat Cell Biol ; 25(1): 30-41, 2023 Jan.
Article in English | MEDLINE | ID: mdl-36650381

ABSTRACT

Haematopoietic ageing is marked by a loss of regenerative capacity and skewed differentiation from haematopoietic stem cells (HSCs), leading to impaired blood production. Signals from the bone marrow niche tailor blood production, but the contribution of the old niche to haematopoietic ageing remains unclear. Here we characterize the inflammatory milieu that drives both niche and haematopoietic remodelling. We find decreased numbers and functionality of osteoprogenitors at the endosteum and expansion of central marrow LepR+ mesenchymal stromal cells associated with deterioration of the sinusoidal vasculature. Together, they create a degraded and inflamed old bone marrow niche. Niche inflammation in turn drives the chronic activation of emergency myelopoiesis pathways in old HSCs and multipotent progenitors, which promotes myeloid differentiation and hinders haematopoietic regeneration. Moreover, we show how production of interleukin-1ß (IL-1ß) by the damaged endosteum acts in trans to drive the proinflammatory nature of the central marrow, with damaging consequences for the old blood system. Notably, niche deterioration, HSC dysfunction and defective regeneration can all be ameliorated by blocking IL-1 signalling. Our results demonstrate that targeting IL-1 as a key mediator of niche inflammation is a tractable strategy to improve blood production during ageing.


Subject(s)
Bone Marrow , Hematopoietic Stem Cells , Bone Marrow/metabolism , Cell Differentiation , Hematopoiesis , Hematopoietic Stem Cells/metabolism , Stem Cell Niche , Interleukin-1/metabolism
3.
J Exp Med ; 218(7)2021 07 05.
Article in English | MEDLINE | ID: mdl-34032859

ABSTRACT

While young blood can restore many aged tissues, its effects on the aged blood system itself and old hematopoietic stem cells (HSCs) have not been determined. Here, we used transplantation, parabiosis, plasma transfer, exercise, calorie restriction, and aging mutant mice to understand the effects of age-regulated systemic factors on HSCs and their bone marrow (BM) niche. We found that neither exposure to young blood, nor long-term residence in young niches after parabiont separation, nor direct heterochronic transplantation had any observable rejuvenating effects on old HSCs. Likewise, exercise and calorie restriction did not improve old HSC function, nor old BM niches. Conversely, young HSCs were not affected by systemic pro-aging conditions, and HSC function was not impacted by mutations influencing organismal aging in established long-lived or progeroid genetic models. Therefore, the blood system that carries factors with either rejuvenating or pro-aging properties for many other tissues is itself refractory to those factors.


Subject(s)
Aging/physiology , Hematopoietic Stem Cells/cytology , Rejuvenation/physiology , Animals , Bone Marrow/physiology , Mice , Mice, Inbred C57BL , Mice, Inbred NOD , Mice, SCID , Mutation/genetics
4.
J Med Chem ; 62(11): 5330-5357, 2019 06 13.
Article in English | MEDLINE | ID: mdl-31042381

ABSTRACT

Poly(adenosine 5'-diphosphate-ribose) polymerase (PARP) inhibitors are a class of anticancer drugs that block the catalytic activity of PARP proteins. Optimization of our lead compound 1 (( Z)-2-benzylidene-3-oxo-2,3-dihydrobenzofuran-7-carboxamide; PARP-1 IC50 = 434 nM) led to a tetrazolyl analogue (51, IC50 = 35 nM) with improved inhibition. Isosteric replacement of the tetrazole ring with a carboxyl group (60, IC50 = 68 nM) gave a promising new lead, which was subsequently optimized to obtain analogues with potent PARP-1 IC50 values (4-197 nM). PARP enzyme profiling revealed that the majority of compounds are selective toward PARP-2 with IC50 values comparable to clinical inhibitors. X-ray crystal structures of the key inhibitors bound to PARP-1 illustrated the mode of interaction with analogue appendages extending toward the PARP-1 adenosine-binding pocket. Compound 81, an isoform-selective PARP-1/-2 (IC50 = 30 nM/2 nM) inhibitor, demonstrated selective cytotoxic effect toward breast cancer gene 1 ( BRCA1)-deficient cells compared to isogenic BRCA1-proficient cells.


Subject(s)
Adenosine/metabolism , Benzofurans/chemical synthesis , Benzofurans/pharmacology , Drug Design , Poly(ADP-ribose) Polymerase Inhibitors/chemical synthesis , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Poly(ADP-ribose) Polymerases/metabolism , Amino Acid Motifs , Benzofurans/chemistry , Benzofurans/metabolism , Biocatalysis , Cell Line, Tumor , Chemistry Techniques, Synthetic , Humans , Inhibitory Concentration 50 , Models, Molecular , Poly(ADP-ribose) Polymerase Inhibitors/chemistry , Poly(ADP-ribose) Polymerase Inhibitors/metabolism , Poly(ADP-ribose) Polymerases/chemistry , Structure-Activity Relationship
5.
Turk J Anaesthesiol Reanim ; 47(2): 107-111, 2019 Apr.
Article in English | MEDLINE | ID: mdl-31080951

ABSTRACT

OBJECTIVE: Local infiltration analgesia (LIA) with ropivacaine is increasingly used to provide postoperative analgesia after total knee arthroplasty (TKA). TKA may be performed with or without the use of a tourniquet. The absence of local blood flow when infiltrating local anaesthesia below an inflated tourniquet may affect the rate of systemic absorption, and this may have an effect on the duration and intensity of analgesia as compared with LIA without the use of a tourniquet. The aim of the present study was to investigate the influence of tourniquet use during surgery on the time to first request (TTFR) of opioids and opioid consumption. METHODS: Two historical time-based cohorts (one with and one without tourniquet during surgery) of 300 patients underwent primary TKA under spinal anaesthesia and received LIA to provide postoperative analgesia. The cohorts were compared for TTFR of opioids and opioid consumption. RESULTS: TTFR did not significantly differ between the tourniquet and non-tourniquet groups with a median (25th-75th percentile) of 240 (102-651) and 282 (100-720) min, respectively. The median (25th-75th percentile) oxycodone use was higher in the tourniquet group with 50 (20-90) versus 40 (10-77.5) mg (p=0.01). CONCLUSION: There was no difference in the time to first opioid consumption, suggesting that the presence of an inflated tourniquet during local anaesthetic injection does not alter systemic absorption sufficiently to affect the duration of analgesia. However, the use of a tourniquet was associated with a higher opioid consumption, which is most likely caused by pain resulting from the tourniquet itself.

6.
Reg Anesth Pain Med ; 43(7): 699-704, 2018 Oct.
Article in English | MEDLINE | ID: mdl-29905628

ABSTRACT

BACKGROUND AND OBJECTIVES: Local infiltration analgesia (LIA) with ropivacaine for total knee arthroplasty (TKA) is increasingly used. Despite the high doses of ropivacaine, LIA is considered safe, and this perception is sustained by pharmacokinetic data demonstrating that maximum concentrations of ropivacaine stay well below the toxic threshold in plasma. These pharmacokinetic studies all involve TKA procedures with the use of a tourniquet. Recently, performing TKA without the use of a tourniquet is gaining popularity, but no pharmacokinetic data exist when LIA is administered for TKA without the use of a tourniquet. The purpose of this study was to describe the pharmacokinetic profile of a single-shot ropivacaine (200 mL 0.2%) and 0.75 mg epinephrine (1000 µg/mL) when used for LIA in patients for TKA without a tourniquet. METHODS: In this prospective cohort study, 20 patients treated with LIA for TKA without a tourniquet were studied. Plasma samples were taken at 20, 40, 60, 90, 120, 240, 360, 480, 600, 720, and 1440 minutes after local anesthetic infiltration, in which total and unbound ropivacaine concentrations were determined. RESULTS: Results are given as median (interquartile range [IQR]). Median peak ropivacaine concentration was 1.16 µg/mL (IQR, 0.46); median peak unbound ropivacaine concentration was 0.05 µg/mL (IQR, 0.02). The corresponding times to reach the maximum concentration for total and unbound ropivacaine were 360 (IQR, 240) and 360 (IQR, 360) minutes, respectively. CONCLUSIONS: Although great interindividual variability in ropivacaine concentration was found, both total and unbound maximum serum concentrations remained below the assumed systemic toxic thresholds in all samples. CLINICAL TRIAL REGISTRATION: This study was registered at Netherlands Trial Registry (http://www.trialregister.nl), trial ID NTR6306.


Subject(s)
Anesthesia, Local/trends , Anesthetics, Local/pharmacokinetics , Arthroplasty, Replacement, Knee/trends , Perioperative Care , Ropivacaine/pharmacokinetics , Tourniquets , Aged , Aged, 80 and over , Anesthetics, Local/administration & dosage , Anesthetics, Local/blood , Arthroplasty, Replacement, Knee/adverse effects , Cohort Studies , Female , Humans , Male , Middle Aged , Perioperative Care/methods , Prospective Studies , Ropivacaine/administration & dosage , Ropivacaine/blood
7.
Anesth Analg ; 126(2): 644-647, 2018 02.
Article in English | MEDLINE | ID: mdl-28598917

ABSTRACT

In our hospital, we introduced a system to measure the collective and individual efficacy of brachial plexus and popliteal nerve blocks with the objective to create transparency as an instrument for monitoring and improvement. Initially, individual results were anonymous, but after 1 year anonymity was lifted within the team of anesthesiologists and results are now discussed quarterly. Collective performance of interscalene, supraclavicular, and popliteal blocks improved significantly over time. Sharing and discussing collective and individual performance has resulted in critical self-appraisal and increased willingness to learn from each other and strengthened the team's ambition for further improvement.


Subject(s)
Anesthetics, Local/standards , Autonomic Nerve Block/standards , Clinical Competence/standards , Monitoring, Intraoperative/standards , Surveys and Questionnaires/standards , Anesthetics, Local/administration & dosage , Autonomic Nerve Block/trends , Humans , Monitoring, Intraoperative/trends
8.
Nature ; 512(7513): 198-202, 2014 Aug 14.
Article in English | MEDLINE | ID: mdl-25079315

ABSTRACT

Haematopoietic stem cells (HSCs) self-renew for life, thereby making them one of the few blood cells that truly age. Paradoxically, although HSCs numerically expand with age, their functional activity declines over time, resulting in degraded blood production and impaired engraftment following transplantation. While many drivers of HSC ageing have been proposed, the reason why HSC function degrades with age remains unknown. Here we show that cycling old HSCs in mice have heightened levels of replication stress associated with cell cycle defects and chromosome gaps or breaks, which are due to decreased expression of mini-chromosome maintenance (MCM) helicase components and altered dynamics of DNA replication forks. Nonetheless, old HSCs survive replication unless confronted with a strong replication challenge, such as transplantation. Moreover, once old HSCs re-establish quiescence, residual replication stress on ribosomal DNA (rDNA) genes leads to the formation of nucleolar-associated γH2AX signals, which persist owing to ineffective H2AX dephosphorylation by mislocalized PP4c phosphatase rather than ongoing DNA damage. Persistent nucleolar γH2AX also acts as a histone modification marking the transcriptional silencing of rDNA genes and decreased ribosome biogenesis in quiescent old HSCs. Our results identify replication stress as a potent driver of functional decline in old HSCs, and highlight the MCM DNA helicase as a potential molecular target for rejuvenation therapies.


Subject(s)
Cellular Senescence/physiology , DNA Replication/physiology , Hematopoietic Stem Cells/pathology , Stress, Physiological , Animals , Cell Proliferation , Cellular Senescence/genetics , DNA Damage/genetics , DNA, Ribosomal/genetics , Female , Gene Expression Regulation , Hematopoietic Stem Cells/cytology , Histones/genetics , Histones/metabolism , Male , Mice , Mice, Inbred C57BL , Minichromosome Maintenance Proteins/genetics
9.
Exp Hematol ; 41(11): 915-23, 2013 Nov.
Article in English | MEDLINE | ID: mdl-24067363

ABSTRACT

Blood homeostasis is maintained by a rare population of quiescent hematopoietic stem cells (HSCs) that self-renew and differentiate to give rise to all lineages of mature blood cells. In contrast to most other blood cells, HSCs are preserved throughout life, and the maintenance of their genomic integrity is therefore paramount to ensure normal blood production and to prevent leukemic transformation. HSCs are also one of the few blood cells that truly age and exhibit severe functional decline in old organisms, resulting in impaired blood homeostasis and increased risk for hematologic malignancies. In this review, we present the strategies used by HSCs to cope with the many genotoxic insults that they commonly encounter. We briefly describe the DNA-damaging insults that can affect HSC function and the mechanisms that are used by HSCs to prevent, survive, and repair DNA lesions. We also discuss an apparent paradox in HSC biology, in which the genome maintenance strategies used by HSCs to protect their function in fact render them vulnerable to the acquisition of damaging genetic aberrations.


Subject(s)
Cell Proliferation , DNA Damage , Hematopoietic Stem Cells/metabolism , Hematopoietic Stem Cells/physiology , Oxidative Stress/physiology , Cell Differentiation/genetics , Cell Differentiation/physiology , Cell Survival/genetics , Cell Survival/physiology , Cellular Microenvironment/genetics , Cellular Microenvironment/physiology , DNA Repair , Hematopoietic Stem Cells/cytology , Humans , Models, Biological
10.
Leuk Res ; 37(8): 862-7, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23507195

ABSTRACT

Myelodysplastic syndromes (MDS) often transform into acute leukemia (AL-MDS), although its prognostic details have not been examined thoroughly. We retrospectively analyzed the prognosis of 189 AL-MDS patients. Ninety-four patients received best supportive care (BSC), and 94 patients received disease-modifying therapies (DMT) that included chemotherapy (CHT) for 65 patients, allogeneic stem-cell transplantation (allo-SCT) for 21 patients, and other therapies for 8 patients. The median survival time was 142 days. In patients treated with BSC, platelet count alone was an independent prognostic factor. In younger patients treated with DMT (<60 years, N=25), allo-SCT was an independent prognostic factor associated with longer survival. In older patients treated with DMT (≥60 years, N=69), the therapy type did not affect survival, and performance status and MDS-specific comorbidity index were independent prognostic factors.


Subject(s)
Cell Transformation, Neoplastic , Leukemia, Myeloid/therapy , Myelodysplastic Syndromes/therapy , Acute Disease , Adult , Age Factors , Aged , Aged, 80 and over , Drug Therapy , Female , Hematopoietic Stem Cell Transplantation/methods , Humans , Leukemia, Myeloid/etiology , Male , Middle Aged , Multivariate Analysis , Myelodysplastic Syndromes/complications , Palliative Care , Prognosis , Retrospective Studies , Risk Factors , Survival Analysis , Time Factors , Transplantation, Homologous
11.
Dis Model Mech ; 6(1): 40-7, 2013 Jan.
Article in English | MEDLINE | ID: mdl-23268537

ABSTRACT

Fanconi anaemia (FA) is a rare autosomal recessive or X-linked inherited disease characterised by an increased incidence of bone marrow failure (BMF), haematological malignancies and solid tumours. Cells from individuals with FA show a pronounced sensitivity to DNA interstrand crosslink (ICL)-inducing agents, which manifests as G2-M arrest, chromosomal aberrations and reduced cellular survival. To date, mutations in at least 15 different genes have been identified that cause FA; the products of all of these genes are thought to function together in the FA pathway, which is essential for ICL repair. Rapidly following the discovery of FA genes, mutant mice were generated to study the disease and the affected pathway. These mutant mice all show the characteristic cellular ICL-inducing agent sensitivity, but only partially recapitulate the developmental abnormalities, anaemia and cancer predisposition seen in individuals with FA. Therefore, the usefulness of modelling FA in mice has been questioned. In this Review, we argue that such scepticism is unjustified. We outline that haematopoietic defects and cancer predisposition are manifestations of FA gene defects in mice, albeit only in certain genetic backgrounds and under certain conditions. Most importantly, recent work has shown that developmental defects in FA mice also arise with concomitant inactivation of acetaldehyde metabolism, giving a strong clue about the nature of the endogenous lesion that must be repaired by the functional FA pathway. This body of work provides an excellent example of a paradox in FA research: that the dissimilarity, rather than the similarity, between mice and humans can provide insight into human disease. We expect that further study of mouse models of FA will help to uncover the mechanistic background of FA, ultimately leading to better treatment options for the disease.


Subject(s)
Fanconi Anemia/genetics , Animals , Congenital Abnormalities/genetics , Disease Models, Animal , Fanconi Anemia/etiology , Fanconi Anemia/metabolism , Fanconi Anemia Complementation Group Proteins/genetics , Fanconi Anemia Complementation Group Proteins/metabolism , Genes, Lethal , Humans , Mice , Mice, Mutant Strains , Mutation , Neoplasms/genetics , Species Specificity , Telomere/genetics
12.
J Pathol ; 226(1): 28-39, 2012 Jan.
Article in English | MEDLINE | ID: mdl-21915857

ABSTRACT

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.


Subject(s)
Fanconi Anemia Complementation Group F Protein/genetics , Granulosa Cell Tumor/genetics , Luteoma/genetics , Ovarian Neoplasms/genetics , Animals , Comparative Genomic Hybridization , Disease Models, Animal , Fanconi Anemia Complementation Group F Protein/deficiency , Female , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout
13.
DNA Repair (Amst) ; 10(12): 1252-61, 2011 Dec 10.
Article in English | MEDLINE | ID: mdl-22036606

ABSTRACT

Fanconi anemia (FA) is a heritable disease characterized by bone marrow failure, congenital abnormalities, and cancer predisposition. The 15 identified FA genes operate in a molecular pathway to preserve genomic integrity. Within this pathway the FA core complex operates as an ubiquitin ligase that activates the complex of FANCD2 and FANCI to coordinate DNA repair. The FA core complex is formed by at least 12 proteins. However, only the FANCL subunit displays ubiquitin ligase activity. FANCA and FANCG are members of the FA core complex for which no other functions have been described than to participate in protein interactions. In this study we generated mice with combined null alleles for Fanca and Fancg to identify extended functions for these genes by characterizing the double mutant mice and cells. Double mutant a(-/-)/g(-/-) mice were born at near Mendelian frequencies without apparent developmental abnormalities. Histological analysis of a(-/-)/g(-/-) mice revealed a Leydig cell hyperplasia and frequent vacuolization of Sertoli cells in testes, while ovaries were depleted from developing follicles and displayed an interstitial cell hyperplasia. These gonadal aberrations were associated with a compromised fertility of a(-/-)/g(-/-) males and females. During the first year of life a(-/-)/g(-/-) did not develop malignancies or bone marrow failure. At the cellular level a(-/-)/g(-/-), Fanca(-/-), and Fancg(-/-) cells proved equally compromised in DNA crosslink and homology-directed repair. Overall the phenotype of a(-/-)/g(-/-) double knockout mice and cells appeared highly similar to the phenotype of Fanca or Fancg single knockouts. The lack of an augmented phenotype suggest that null mutations in Fanca or Fancg are fully epistatic, making additional important functions outside of the FA core complex highly unlikely.


Subject(s)
Epistasis, Genetic/genetics , Fanconi Anemia Complementation Group A Protein/genetics , Fanconi Anemia Complementation Group G Protein/genetics , Fanconi Anemia/genetics , Mutation/genetics , Active Transport, Cell Nucleus/drug effects , Animals , Bone Marrow Cells/cytology , Cell Line , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Chromosome Breakage/drug effects , Embryo, Mammalian , Female , Fertility/genetics , Fibroblasts/cytology , Fluorobenzenes/pharmacology , Hematologic Tests , Humans , Male , Mice , Ovary/metabolism , Phthalazines/pharmacology , Poly(ADP-ribose) Polymerase Inhibitors , Stem Cells/drug effects , Stem Cells/metabolism , Testis/metabolism
14.
Hum Mol Genet ; 18(18): 3484-95, 2009 Sep 15.
Article in English | MEDLINE | ID: mdl-19561169

ABSTRACT

The Fanconi anemia (FA) core complex member FANCM remodels synthetic replication forks and recombination intermediates. Thus far, only one FA patient with FANCM mutations has been described, but the relevance of these mutations for the FA phenotype is uncertain. To provide further experimental access to the FA-M complementation group we have generated Fancm-deficient mice by deleting exon 2. FANCM deficiency caused hypogonadism in mice and hypersensitivity to cross-linking agents in mouse embryonic fibroblasts (MEFs), thus phenocopying other FA mouse models. However, Fancm(Delta2/Delta2) mice also showed unique features atypical for FA mice, including underrepresentation of female Fancm(Delta2/Delta2) mice and decreased overall and tumor-free survival. This increased cancer incidence may be correlated to the role of FANCM in the suppression of spontaneous sister chromatid exchanges as observed in MEFs. In addition, FANCM appeared to have a stimulatory rather than essential role in FANCD2 monoubiquitination. The FA-M mouse model presented here suggests that FANCM functions both inside and outside the FA core complex to maintain genome stability and to prevent tumorigenesis.


Subject(s)
Fanconi Anemia Complementation Group Proteins/deficiency , Fanconi Anemia Complementation Group Proteins/metabolism , Alleles , Animals , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Cell Transformation, Neoplastic/pathology , Cells, Cultured , Fanconi Anemia/genetics , Fanconi Anemia/metabolism , Fanconi Anemia/pathology , Fanconi Anemia Complementation Group Proteins/genetics , Female , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Ovary/abnormalities , Ovary/metabolism , Phenotype , Sister Chromatid Exchange , Survival Rate , Testis/abnormalities , Testis/metabolism
15.
Blood ; 114(1): 174-80, 2009 Jul 02.
Article in English | MEDLINE | ID: mdl-19423727

ABSTRACT

FANCM is a component of the Fanconi anemia (FA) core complex and one FA patient (EUFA867) with biallelic mutations in FANCM has been described. Strikingly, we found that EUFA867 also carries biallelic mutations in FANCA. After correcting the FANCA defect in EUFA867 lymphoblasts, a "clean" FA-M cell line was generated. These cells were hypersensitive to mitomycin C, but unlike cells defective in other core complex members, FANCM(-/-) cells were proficient in monoubiquitinating FANCD2 and were sensitive to the topoisomerase inhibitor camptothecin, a feature shared only with the FA subtype D1 and N. In addition, FANCM(-/-) cells were sensitive to UV light. FANCM and a C-terminal deletion mutant rescued the cross-linker sensitivity of FANCM(-/-) cells, whereas a FANCM ATPase mutant did not. Because both mutants restored the formation of FANCD2 foci, we conclude that FANCM functions in an FA core complex-dependent and -independent manner.


Subject(s)
DNA Helicases/genetics , DNA Helicases/metabolism , Fanconi Anemia Complementation Group D2 Protein/genetics , Fanconi Anemia Complementation Group D2 Protein/metabolism , Fanconi Anemia/genetics , Fanconi Anemia/metabolism , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Camptothecin/pharmacology , Cell Line, Tumor , Cross-Linking Reagents/pharmacology , DNA Helicases/deficiency , Drug Resistance/genetics , Drug Resistance/physiology , Fanconi Anemia Complementation Group A Protein/genetics , Fanconi Anemia Complementation Group A Protein/metabolism , Gene Expression , Humans , Mutation , Radiation Tolerance/genetics , Radiation Tolerance/physiology , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Transfection , Ubiquitination/genetics , Ultraviolet Rays
16.
Cell Oncol ; 29(3): 211-8, 2007.
Article in English | MEDLINE | ID: mdl-17452773

ABSTRACT

To identify the gene underlying Fanconi anemia (FA) complementation group I we studied informative FA-I families by a genome-wide linkage analysis, which resulted in 4 candidate regions together encompassing 351 genes. Candidates were selected via bioinformatics and data mining on the basis of their resemblance to other FA genes/proteins acting in the FA pathway, such as: degree of evolutionary conservation, presence of nuclear localization signals and pattern of tissue-dependent expression. We found a candidate, KIAA1794 on chromosome 15q25-26, to be mutated in 8 affected individuals previously assigned to complementation group I. Western blots of endogenous FANCI indicated that functionally active KIAA1794 protein is lacking in FA-I individuals. Knock-down of KIAA1794 expression by siRNA in HeLa cells caused excessive chromosomal breakage induced by mitomycin C, a hallmark of FA cells. Furthermore, phenotypic reversion of a patient-derived cell line was associated with a secondary genetic alteration at the KIAA1794 locus. These data add up to two conclusions. First, KIAA1794 is a FA gene. Second, this gene is identical to FANCI, since the patient cell lines found mutated in this study included the reference cell line for group I, EUFA592.


Subject(s)
Fanconi Anemia Complementation Group Proteins/genetics , Adolescent , Adult , Base Sequence , Cell Line , Child , Chromosomal Instability/genetics , Fanconi Anemia Complementation Group D2 Protein/metabolism , Female , Genome, Human/genetics , HeLa Cells , Humans , Male , Molecular Sequence Data , Mutation/genetics , Pedigree , Phenotype , Ubiquitin/metabolism
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