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1.
Cancer Lett ; 555: 216030, 2023 02 28.
Article in English | MEDLINE | ID: mdl-36496104

ABSTRACT

Cytidine deaminase (CDA) catalyzes the deamination of cytidine (C) and deoxycytidine (dC) to uridine and deoxyuridine, respectively. We recently showed that CDA deficiency leads to genomic instability, a hallmark of cancers. We therefore investigated whether constitutive CDA inactivation conferred a predisposition to cancer development. We developed a novel mouse model of Cda deficiency by generating Cda-knockout mice. Cda+/+ and Cda-/- mice did not differ in lifetime phenotypic or behavioral characteristics, or in the frequency or type of spontaneous cancers. However, the frequency of chemically induced tumors in the colon was significantly lower in Cda-/- mice. An analysis of primary kidney cells from Cda-/- mice revealed an excess of C and dC associated with significantly higher frequencies of sister chromatid exchange and ultrafine anaphase bridges and lower Parp-1 activity than in Cda+/+ cells. Our results suggest that, despite inducing genetic instability, an absence of Cda limits the number of chemically induced tumors. These results raise questions about whether a decrease in basal Parp-1 activity can protect against inflammation-driven tumorigenesis; we discuss our findings in light of published data for the Parp-1-deficient mouse model.


Subject(s)
Colonic Neoplasms , Cytidine Deaminase , Animals , Mice , Cytidine Deaminase/genetics , Poly(ADP-ribose) Polymerase Inhibitors , Genomic Instability , Colonic Neoplasms/chemically induced , Colonic Neoplasms/genetics
3.
Sci Rep ; 12(1): 14062, 2022 08 18.
Article in English | MEDLINE | ID: mdl-35982128

ABSTRACT

Cytidine deaminase (CDA), an enzyme of the pyrimidine salvage pathway, deaminates cytidine, deoxycytidine and analogs, such as gemcitabine. Constitutive low levels of CDA activity have been reported in the blood of patients with hematological malignancies or suffering from gemcitabine toxicity. We previously reported that cellular CDA deficiency leads to genetic instability. We therefore hypothesized that constitutive CDA deficiency might confer a predisposition to cancer. We analyzed CDA activity and expression in blood samples from breast cancer (BC) patients with a suspected predisposition to the disease, and in healthy controls. Contrary to our hypothesis, we found that both CDA activity and mRNA levels were higher in blood samples from BC patients than in those from controls, and that this difference was not due to excess neutrophils. CDA activity levels were significantly higher in the serum samples of BC patients treated by radiotherapy (RT) than in those of untreated healthy controls, and hormone therapy in RT-treated BC patients was associated with significantly lower levels of CDA activity. A preliminary analysis of CDA activity in the serum of the very few BC patients who had undergone no treatment other than surgery suggested that the increase in CDA activity might be due to the breast cancer itself. Our findings raise important questions, which should lead to studies to elucidate the origin and significance of the increase in CDA activity in the serum of BC patients, and the impact of hormone therapy.


Subject(s)
Breast Neoplasms , Cytidine Deaminase/metabolism , Disease Susceptibility , Female , Genotype , Hormones , Humans
4.
Cell Mol Life Sci ; 79(8): 465, 2022 Aug 04.
Article in English | MEDLINE | ID: mdl-35925417

ABSTRACT

Identifying new molecular targets for novel anticancer treatments is a major challenge in clinical cancer research. We have shown that cytidine deaminase (CDA) expression is downregulated in about 60% of cancer cells and tissues. In this study, we aimed to develop a new anticancer treatment specifically inhibiting the growth of CDA-deficient tumor cells. High-throughput screening of a chemical library led to the identification of a naphthol derivative, X55, targeting CDA-deficient tumor cells preferentially, without affecting the growth of non-tumoral cells regardless of CDA expression status. Metabolomic profiling revealed that CDA-deficient HeLa cells differed markedly from control HeLa cells. X55 treatment had a moderate effect on control cells, but greatly disturbed the metabolome of CDA-deficient HeLa cells, worsening the deregulation of many metabolites. In particular, the levels of the three oncometabolites, fumarate, succinate and 2-hydroxyglutarate, were significantly lower in CDA-depleted cells, and this decrease in levels was exacerbated by X55 treatment, revealing an unexpected link between CDA deficiency, mitochondrial function and X55 response. Finally, we identified strong downregulation of MAPT (encoding Tau, a microtubule associated protein) expression as a reliable predictive marker for tumor cell X55 sensitivity.


Subject(s)
Cytidine Deaminase , Naphthols , Cytidine Deaminase/genetics , Cytidine Deaminase/metabolism , HeLa Cells , Humans
5.
Sci Rep ; 10(1): 13907, 2020 08 17.
Article in English | MEDLINE | ID: mdl-32807821

ABSTRACT

Cytidine deaminase (CDA) deficiency causes pyrimidine pool disequilibrium. We previously reported that the excess cellular dC and dCTP resulting from CDA deficiency jeopardizes genome stability, decreasing basal poly(ADP-ribose) polymerase 1 (PARP-1) activity and increasing ultrafine anaphase bridge (UFB) formation. Here, we investigated the mechanism underlying the decrease in PARP-1 activity in CDA-deficient cells. PARP-1 activity is dependent on intracellular NAD+ concentration. We therefore hypothesized that defects of the NAD+ salvage pathway might result in decreases in PARP-1 activity. We found that the inhibition or depletion of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the NAD+ salvage biosynthesis pathway, mimicked CDA deficiency, resulting in a decrease in basal PARP-1 activity, regardless of NAD+ levels. Furthermore, the expression of exogenous wild-type NAMPT fully restored basal PARP-1 activity and prevented the increase in UFB frequency in CDA-deficient cells. No such effect was observed with the catalytic mutant. Our findings demonstrate that (1) the inhibition of NAMPT activity in CDA-proficient cells lowers basal PARP-1 activity, and (2) the expression of exogenous wild-type NAMPT, but not of the catalytic mutant, fully restores basal PARP-1 activity in CDA-deficient cells; these results strongly suggest that basal PARP-1 activity in CDA-deficient cells decreases due to a reduction of NAMPT activity.


Subject(s)
Cytidine Deaminase/deficiency , NAD/metabolism , Poly (ADP-Ribose) Polymerase-1/metabolism , Cytidine Deaminase/metabolism , Cytokines/antagonists & inhibitors , Cytokines/genetics , Cytokines/metabolism , HeLa Cells , Humans , Mutation/genetics , Niacinamide/metabolism , Nicotinamide Phosphoribosyltransferase/antagonists & inhibitors , Nicotinamide Phosphoribosyltransferase/genetics , Nicotinamide Phosphoribosyltransferase/metabolism
6.
Open Biol ; 10(5): 190259, 2020 05.
Article in English | MEDLINE | ID: mdl-32400307

ABSTRACT

Topoisomerase IIα (Topo IIα), a well-conserved double-stranded DNA (dsDNA)-specific decatenase, processes dsDNA catenanes resulting from DNA replication during mitosis. Topo IIα defects lead to an accumulation of ultrafine anaphase bridges (UFBs), a type of chromosome non-disjunction. Topo IIα has been reported to resolve DNA anaphase threads, possibly accounting for the increase in UFB frequency upon Topo IIα inhibition. We hypothesized that the excess UFBs might also result, at least in part, from an impairment of the prevention of UFB formation by Topo IIα. We found that Topo IIα inhibition promotes UFB formation without affecting the global disappearance of UFBs during mitosis, but leads to an aberrant UFB resolution generating DNA damage within the next G1. Moreover, we demonstrated that Topo IIα inhibition promotes the formation of two types of UFBs depending on cell cycle phase. Topo IIα inhibition during S-phase compromises complete DNA replication, leading to the formation of UFB-containing unreplicated DNA, whereas Topo IIα inhibition during mitosis impedes DNA decatenation at metaphase-anaphase transition, leading to the formation of UFB-containing DNA catenanes. Thus, Topo IIα activity is essential to prevent UFB formation in a cell-cycle-dependent manner and to promote DNA damage-free resolution of UFBs.


Subject(s)
DNA Topoisomerases, Type II/metabolism , Poly-ADP-Ribose Binding Proteins/metabolism , Razoxane/pharmacology , Anaphase , Chromosome Segregation , DNA Damage , DNA Replication/drug effects , HeLa Cells , Humans , Nondisjunction, Genetic , Poly-ADP-Ribose Binding Proteins/antagonists & inhibitors
7.
Nat Commun ; 8(1): 693, 2017 09 25.
Article in English | MEDLINE | ID: mdl-28947735

ABSTRACT

Cells from Bloom's syndrome patients display genome instability due to a defective BLM and the downregulation of cytidine deaminase. Here, we use a genome-wide RNAi-synthetic lethal screen and transcriptomic profiling to identify genes enabling BLM-deficient and/or cytidine deaminase-deficient cells to tolerate constitutive DNA damage and replication stress. We found a synthetic lethal interaction between cytidine deaminase and microtubule-associated protein Tau deficiencies. Tau is overexpressed in cytidine deaminase-deficient cells, and its depletion worsens genome instability, compromising cell survival. Tau is recruited, along with upstream-binding factor, to ribosomal DNA loci. Tau downregulation decreases upstream binding factor recruitment, ribosomal RNA synthesis, ribonucleotide levels, and affects ribosomal DNA stability, leading to the formation of a new subclass of human ribosomal ultrafine anaphase bridges. We describe here Tau functions in maintaining survival of cytidine deaminase-deficient cells, and ribosomal DNA transcription and stability. Moreover, our findings for cancer tissues presenting concomitant cytidine deaminase underexpression and Tau upregulation open up new possibilities for anti-cancer treatment.Cytidine deaminase (CDA) deficiency leads to genome instability. Here the authors find a synthetic lethal interaction between CDA and the microtubule-associated protein Tau deficiencies, and report that Tau depletion affects rRNA synthesis, ribonucleotide pool balance, and rDNA stability.


Subject(s)
Bloom Syndrome/genetics , DNA, Ribosomal/metabolism , tau Proteins/physiology , Bloom Syndrome/pathology , Cell Survival , Cytidine Deaminase/deficiency , Down-Regulation , Genomic Instability , HeLa Cells , Humans , RecQ Helicases/genetics , Up-Regulation , tau Proteins/genetics , tau Proteins/metabolism
8.
Cell Cycle ; 16(11): 1128-1135, 2017 Jun 03.
Article in English | MEDLINE | ID: mdl-28463527

ABSTRACT

Bloom Syndrome (BS) is a rare genetic disease characterized by high levels of chromosomal instability and an increase in cancer risk. Cytidine deaminase (CDA) expression is downregulated in BS cells, leading to an excess of cellular dC and dCTP that reduces basal PARP-1 activity, compromising optimal Chk1 activation and reducing the efficiency of downstream checkpoints. This process leads to the accumulation of unreplicated DNA during mitosis and, ultimately, ultrafine anaphase bridge (UFB) formation. BS cells also display incomplete sister chromatid disjunction when depleted of cohesin. Using a combination of fluorescence in situ hybridization and chromosome spreads, we investigated the possible role of CDA deficiency in the incomplete sister chromatid disjunction in cohesin-depleted BS cells. The decrease in basal PARP-1 activity in CDA-deficient cells compromised sister chromatid disjunction in cohesin-depleted cells, regardless of BLM expression status. The observed incomplete sister chromatid disjunction may be due to the accumulation of unreplicated DNA during mitosis in CDA-deficient cells, as reflected in the changes in centromeric DNA structure associated with the decrease in basal PARP-1 activity. Our findings reveal a new function of PARP-1 in sister chromatid disjunction during mitosis.


Subject(s)
Chromatids/metabolism , Cytidine Deaminase/deficiency , Nondisjunction, Genetic , Poly(ADP-ribose) Polymerases/metabolism , Sister Chromatid Exchange , Cell Cycle Proteins , Centromere/metabolism , Cytidine Deaminase/metabolism , DNA/chemistry , DNA-Binding Proteins , HeLa Cells , Humans , Metaphase , Models, Biological , Nuclear Proteins/metabolism , Phosphoproteins/metabolism
9.
Clin Cancer Res ; 23(8): 2116-2126, 2017 04 15.
Article in English | MEDLINE | ID: mdl-27601591

ABSTRACT

Purpose: One of the main challenges in cancer therapy is the identification of molecular mechanisms mediating resistance or sensitivity to treatment. Cytidine deaminase (CDA) was reported to be downregulated in cells derived from patients with Bloom syndrome, a genetic disease associated with a strong predisposition to a wide range of cancers. The purpose of this study was to determine whether CDA deficiency could be associated with tumors from the general population and could constitute a predictive marker of susceptibility to antitumor drugs.Experimental Design: We analyzed CDA expression in silico, in large datasets for cancer cell lines and tumors and in various cancer cell lines and primary tumor tissues using IHC, PDXs, qRT-PCR, and Western blotting. We also studied the mechanism underlying CDA silencing and searched for molecules that might target specifically CDA-deficient tumor cells using in silico analysis coupled to classical cellular experimental approaches.Results: We found that CDA expression is downregulated in about 60% of cancer cells and tissues. We demonstrate that DNA methylation is a prevalent mechanism of CDA silencing in tumors. Finally, we show that CDA-deficient tumor cells can be specifically targeted with epigenetic treatments and with the anticancer drug aminoflavone.Conclusions: CDA expression status identifies new subgroups of cancers, and CDA deficiency appears to be a novel and relevant predictive marker of susceptibility to antitumor drugs, opening up new possibilities for treating cancer. Clin Cancer Res; 23(8); 2116-26. ©2016 AACR.


Subject(s)
Biomarkers, Tumor/analysis , Cytidine Deaminase/deficiency , Neoplasms/enzymology , Animals , Blotting, Western , Cell Line, Tumor , Drug Resistance, Neoplasm/physiology , Humans , Immunohistochemistry , Mice , Real-Time Polymerase Chain Reaction , Xenograft Model Antitumor Assays
10.
J Cell Sci ; 129(16): 3167-77, 2016 08 15.
Article in English | MEDLINE | ID: mdl-27383768

ABSTRACT

Cytidine deaminase (CDA) deficiency induces an excess of cellular dCTP, which reduces basal PARP-1 activity, thereby compromising complete DNA replication, leading to ultrafine anaphase bridge (UFB) formation. CDA dysfunction has pathological implications, notably in cancer and in Bloom syndrome. It remains unknown how reduced levels of PARP-1 activity and pyrimidine pool imbalance lead to the accumulation of unreplicated DNA during mitosis. We report that a decrease in PARP-1 activity in CDA-deficient cells impairs DNA-damage-induced Chk1 activation, and, thus, the downstream checkpoints. Chemical inhibition of the ATR-Chk1 pathway leads to UFB accumulation, and we found that this pathway was compromised in CDA-deficient cells. Our data demonstrate that ATR-Chk1 acts downstream from PARP-1, preventing the accumulation of unreplicated DNA in mitosis, and, thus, UFB formation. Finally, delaying entry into mitosis is sufficient to prevent UFB formation in both CDA-deficient and CDA-proficient cells, suggesting that both physiological and pathological UFBs are derived from unreplicated DNA. Our findings demonstrate an unsuspected requirement for a balanced nucleotide pool for optimal Chk1 activation both in unchallenged cells and in response to genotoxic stress.


Subject(s)
Anaphase , Checkpoint Kinase 1/metabolism , Pyrimidines/metabolism , Ataxia Telangiectasia Mutated Proteins/metabolism , Cell Cycle Checkpoints , Cytidine Deaminase/metabolism , DNA Damage , DNA Replication , Enzyme Activation , HeLa Cells , Humans , Models, Biological , Poly(ADP-ribose) Polymerases/metabolism , S Phase
11.
PLoS Genet ; 11(7): e1005384, 2015 Jul.
Article in English | MEDLINE | ID: mdl-26181065

ABSTRACT

Genome stability is jeopardized by imbalances of the dNTP pool; such imbalances affect the rate of fork progression. For example, cytidine deaminase (CDA) deficiency leads to an excess of dCTP, slowing the replication fork. We describe here a novel mechanism by which pyrimidine pool disequilibrium compromises the completion of replication and chromosome segregation: the intracellular accumulation of dCTP inhibits PARP-1 activity. CDA deficiency results in incomplete DNA replication when cells enter mitosis, leading to the formation of ultrafine anaphase bridges between sister-chromatids at "difficult-to-replicate" sites such as centromeres and fragile sites. Using molecular combing, electron microscopy and a sensitive assay involving cell imaging to quantify steady-state PAR levels, we found that DNA replication was unsuccessful due to the partial inhibition of basal PARP-1 activity, rather than slower fork speed. The stimulation of PARP-1 activity in CDA-deficient cells restores replication and, thus, chromosome segregation. Moreover, increasing intracellular dCTP levels generates under-replication-induced sister-chromatid bridges as efficiently as PARP-1 knockdown. These results have direct implications for Bloom syndrome (BS), a rare genetic disease combining susceptibility to cancer and genomic instability. BS results from mutation of the BLM gene, encoding BLM, a RecQ 3'-5' DNA helicase, a deficiency of which leads to CDA downregulation. BS cells thus have a CDA defect, resulting in a high frequency of ultrafine anaphase bridges due entirely to dCTP-dependent PARP-1 inhibition and independent of BLM status. Our study describes previously unknown pathological consequences of the distortion of dNTP pools and reveals an unexpected role for PARP-1 in preventing DNA under-replication and chromosome segregation defects.


Subject(s)
Bloom Syndrome/genetics , Cytidine Deaminase/genetics , Poly(ADP-ribose) Polymerases/genetics , Pyrimidines/metabolism , Bloom Syndrome/pathology , Cell Line , Centromere/genetics , Chromosome Fragile Sites/genetics , Chromosome Segregation/genetics , Cytidine Deaminase/deficiency , DNA Replication/genetics , Genomic Instability , Humans , Mitosis/genetics , Poly (ADP-Ribose) Polymerase-1 , Poly(ADP-ribose) Polymerases/biosynthesis , RecQ Helicases/genetics , Sister Chromatid Exchange/genetics
12.
PLoS One ; 7(4): e33905, 2012.
Article in English | MEDLINE | ID: mdl-22563370

ABSTRACT

Centromeres are specialized chromosome domains that control chromosome segregation during mitosis, but little is known about the mechanisms underlying the maintenance of their integrity. Centromeric ultrafine anaphase bridges are physiological DNA structures thought to contain unresolved DNA catenations between the centromeres separating during anaphase. BLM and PICH helicases colocalize at these ultrafine anaphase bridges and promote their resolution. As PICH is detectable at centromeres from prometaphase onwards, we hypothesized that BLM might also be located at centromeres and that the two proteins might cooperate to resolve DNA catenations before the onset of anaphase. Using immunofluorescence analyses, we demonstrated the recruitment of BLM to centromeres from G2 phase to mitosis. With a combination of fluorescence in situ hybridization, electron microscopy, RNA interference, chromosome spreads and chromatin immunoprecipitation, we showed that both BLM-deficient and PICH-deficient prometaphase cells displayed changes in centromere structure. These cells also had a higher frequency of centromeric non disjunction in the absence of cohesin, suggesting the persistence of catenations. Both proteins were required for the correct recruitment to the centromere of active topoisomerase IIα, an enzyme specialized in the catenation/decatenation process. These observations reveal the existence of a functional relationship between BLM, PICH and topoisomerase IIα in the centromere decatenation process. They indicate that the higher frequency of centromeric ultrafine anaphase bridges in BLM-deficient cells and in cells treated with topoisomerase IIα inhibitors is probably due not only to unresolved physiological ultrafine anaphase bridges, but also to newly formed ultrafine anaphase bridges. We suggest that BLM and PICH cooperate in rendering centromeric catenates accessible to topoisomerase IIα, thereby facilitating correct centromere disjunction and preventing the formation of supernumerary centromeric ultrafine anaphase bridges.


Subject(s)
Antigens, Neoplasm/metabolism , Centromere/metabolism , DNA Helicases/metabolism , DNA Topoisomerases, Type II/metabolism , DNA-Binding Proteins/metabolism , RecQ Helicases/metabolism , Anaphase , Cell Cycle Proteins/metabolism , Centromere/chemistry , Centromere/enzymology , Chromosomal Proteins, Non-Histone/metabolism , Chromosomes/metabolism , DNA Helicases/antagonists & inhibitors , DNA Helicases/genetics , DNA, Catenated/metabolism , G2 Phase , HeLa Cells , Humans , Mitosis , Mutagenesis, Site-Directed , Prometaphase , RNA Interference , RNA, Small Interfering/metabolism , RecQ Helicases/antagonists & inhibitors , RecQ Helicases/genetics , Cohesins
13.
Nat Commun ; 2: 368, 2011 Jun 28.
Article in English | MEDLINE | ID: mdl-21712816

ABSTRACT

Defects in DNA replication are associated with genetic instability and cancer development, as illustrated in Bloom syndrome. Features of this syndrome include a slowdown in replication speed, defective fork reactivation and high rates of sister chromatid exchange, with a general predisposition to cancer. Bloom syndrome is caused by mutations in the BLM gene encoding a RecQ helicase. Here we report that BLM deficiency is associated with a strong cytidine deaminase defect, leading to pyrimidine pool disequilibrium. In BLM-deficient cells, pyrimidine pool normalization leads to reduction of sister chromatid exchange frequency and is sufficient for full restoration of replication fork velocity but not the fork restart defect, thus identifying the part of the Bloom syndrome phenotype because of pyrimidine pool imbalance. This study provides new insights into the molecular basis of control of replication speed and the genetic instability associated with Bloom syndrome. Nucleotide pool disequilibrium could be a general phenomenon in a large spectrum of precancerous and cancer cells.


Subject(s)
Bloom Syndrome/genetics , DNA Replication/genetics , Genomic Instability/genetics , Pyrimidines/metabolism , RecQ Helicases/deficiency , Blotting, Western , Cell Line , Cytidine Deaminase/metabolism , DNA Primers/genetics , Humans , Microarray Analysis , Mutagenesis, Site-Directed , Reverse Transcriptase Polymerase Chain Reaction , Sister Chromatid Exchange/genetics , Statistics, Nonparametric
14.
J Nucleic Acids ; 20102010 Sep 08.
Article in English | MEDLINE | ID: mdl-20936166

ABSTRACT

Bloom's syndrome (BS) displays one of the strongest known correlations between chromosomal instability and a high risk of cancer at an early age. BS cells combine a reduced average fork velocity with constitutive endogenous replication stress. However, the response of BS cells to replication stress induced by hydroxyurea (HU), which strongly slows the progression of replication forks, remains unclear due to publication of conflicting results. Using two different cellular models of BS, we showed that BLM deficiency is not associated with sensitivity to HU, in terms of clonogenic survival, DSB generation, and SCE induction. We suggest that surviving BLM-deficient cells are selected on the basis of their ability to deal with an endogenous replication stress induced by replication fork slowing, resulting in insensitivity to HU-induced replication stress.

15.
Mol Cancer Res ; 8(3): 385-94, 2010 Mar.
Article in English | MEDLINE | ID: mdl-20215422

ABSTRACT

Little is known about the functional interaction between the Bloom's syndrome protein (BLM) and the recombinase RAD51 within cells. Using RNA interference technology, we provide the first demonstration that RAD51 acts upstream from BLM to prevent anaphase bridge formation. RAD51 downregulation was associated with an increase in the frequency of BLM-positive anaphase bridges, but not of BLM-associated ultrafine bridges. Time-lapse live microscopy analysis of anaphase bridge cells revealed that BLM promoted cell survival in the absence of Rad51. Our results directly implicate BLM in limiting the lethality associated with RAD51 deficiency through the processing of anaphase bridges resulting from the RAD51 defect. These findings provide insight into the molecular basis of some cancers possibly associated with variants of the RAD51 gene family.


Subject(s)
Neoplasms/genetics , Rad51 Recombinase/genetics , RecQ Helicases/genetics , Anaphase/genetics , Cell Death/genetics , Cell Survival/genetics , Down-Regulation/genetics , Gene Expression Regulation, Neoplastic/genetics , HeLa Cells , Humans , Neoplasms/metabolism , RNA Interference/physiology , RecQ Helicases/metabolism , Sister Chromatid Exchange/genetics
16.
Genet Test ; 12(2): 257-61, 2008 Jun.
Article in English | MEDLINE | ID: mdl-18471088

ABSTRACT

Bloom's syndrome (BS) is a rare autosomal recessive disease predisposing patients to all types of cancers affecting the general population. BS cells display a high level of genetic instability, including a 10-fold increase in the rate of sister chromatid exchanges, currently the only objective criterion for BS diagnosis. We have developed a method for screening the BLM gene for mutations based on direct genomic DNA sequencing. A questionnaire based on clinical information, cytogenetic features, and family history was addressed to physicians prescribing BS genetic screening, with the aim of confirming or guiding diagnosis. We report here four BLM gene mutations, three of which have not been described before. Three of the mutations are frameshift mutations, and the fourth is a nonsense mutation. All these mutations introduce a stop codon, and may therefore be considered to have deleterious biological effect. This approach should make it possible to identify new mutations and to correlate them with clinical information.


Subject(s)
Bloom Syndrome/diagnosis , Bloom Syndrome/genetics , DNA Helicases/genetics , DNA Mutational Analysis/methods , Mutation , Adult , Bloom Syndrome/physiopathology , Child , Child, Preschool , Codon, Nonsense , Female , Frameshift Mutation , Genetic Testing , Genome , Humans , Infant , Male , RecQ Helicases , Sequence Analysis, DNA
17.
Leuk Res ; 31(3): 353-8, 2007 Mar.
Article in English | MEDLINE | ID: mdl-16890283

ABSTRACT

The frequency of acute myeloid leukemia (AML) with balanced chromosomal translocations arising after anticancer therapy with DNA-damaging agents such as DNA topoisomerase II inhibitors has increased over the last two decades. However, factors that predispose to these therapy-related disorders are still poorly defined. It has been reported that DNA double-strand break (DSB) repair by the non-homologous end-joining (NHEJ) pathway is impaired in myeloid leukemia cells. This led us to hypothesize that therapy-related AML (t-AML) may result from individual differences in the repair of DSBs generated by the treatment. We show here that DSB repair is accurate, in vivo, in non-tumoral cells derived from patients who developed t-AML with t(9;11) or t(15;17) translocation after treatment for a first cancer with DNA topoisomerase II inhibitors. These results indicate that a major constitutive defect in the NHEJ pathway is unlikely to predispose to t-AML with balanced chromosomal translocations.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/adverse effects , DNA Breaks, Double-Stranded/drug effects , DNA Repair , DNA Topoisomerases, Type II , Leukemia, Myeloid/genetics , Neoplasms, Second Primary/genetics , Translocation, Genetic/genetics , Acute Disease , Adult , Aged , Cell Line, Tumor , Chromosomes, Human, Pair 11/genetics , Chromosomes, Human, Pair 15/genetics , Chromosomes, Human, Pair 17/genetics , Chromosomes, Human, Pair 9/genetics , DNA Repair/drug effects , Enzyme Inhibitors/pharmacology , Female , Humans , Leukemia, Myeloid/drug therapy , Male , Middle Aged , Neoplasms, Second Primary/drug therapy , Structure-Activity Relationship , Topoisomerase II Inhibitors
18.
Cancer Res ; 64(24): 8954-9, 2004 Dec 15.
Article in English | MEDLINE | ID: mdl-15604258

ABSTRACT

Cdc2 kinase is inactivated when DNA damage occurs during the spindle assembly checkpoint. Here, we show that the level of mitotic Bloom syndrome protein phosphorylation reflects the level of cdc2 activity. A complete inactivation of cdc2 by either introduction of DNA double-strand breaks or roscovitine treatment prevents exit from mitosis. Thus, mitotic cdc2 inactivation plays a major role in the establishment of the mitotic DNA damage checkpoint. In response to mitotic cdc2 inactivation, the M/G(1) transition is delayed after releasing the drug block in nonmalignant cells, whereas tumor cells exit mitosis without dividing and rereplicate their DNA, which results in mitotic catastrophe. This opens the way for new chemotherapeutic strategies.


Subject(s)
CDC2 Protein Kinase/metabolism , DNA Damage/physiology , Mitosis/physiology , Adenosine Triphosphatases/metabolism , CDC2 Protein Kinase/antagonists & inhibitors , Cell Division/physiology , Chloroquine/pharmacology , DNA/drug effects , DNA/metabolism , DNA Helicases/metabolism , Enzyme Activation , G1 Phase/physiology , HeLa Cells , Humans , Hydroxamic Acids/pharmacology , Mitosis/drug effects , Mitosis/genetics , Phosphorylation/drug effects , Purines/pharmacology , RecQ Helicases , Roscovitine , Subcellular Fractions/metabolism
19.
Nucleic Acids Res ; 31(21): 6272-82, 2003 Nov 01.
Article in English | MEDLINE | ID: mdl-14576316

ABSTRACT

Bloom's syndrome (BS) which associates genetic instability and predisposition to cancer is caused by mutations in the BLM gene encoding a RecQ family 3'-5' DNA helicase. It has been proposed that the generation of genetic instability in BS cells could result from an aberrant non-homologous DNA end joining (NHEJ), one of the two main DNA double-strand break (DSB) repair pathways in mammalian cells, the second major pathway being homologous recombination (HR). Using cell extracts, we report first that Ku70/80 and the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), key factors of the end-joining machinery, and BLM are located in close proximity on DNA and that BLM binds to DNA only in the absence of ATP. In the presence of ATP, BLM is phosphorylated and dissociates from DNA in a strictly DNA-PKcs-dependent manner. We also show that BS cells display, in vivo, an accurate joining of DSBs, reflecting thus a functional NHEJ pathway. In sharp contrast, a 5-fold increase of the HR-mediated DNA DSB repair in BS cells was observed. These results support a model in which NHEJ activation mediates BLM dissociation from DNA, whereas, under conditions where HR is favored, e.g. at the replication fork, BLM exhibits an anti-recombinogenic role.


Subject(s)
Adenosine Triphosphatases/metabolism , Bloom Syndrome/enzymology , DNA Damage , DNA Helicases/metabolism , DNA Repair , DNA/metabolism , Models, Biological , Recombination, Genetic , Adenosine Triphosphatases/deficiency , Adenosine Triphosphatases/genetics , Adenosine Triphosphate/metabolism , Antigens, Nuclear/metabolism , Bloom Syndrome/genetics , Bloom Syndrome/pathology , Cell Line, Transformed , DNA/genetics , DNA Helicases/deficiency , DNA Helicases/genetics , DNA-Activated Protein Kinase , DNA-Binding Proteins/metabolism , HeLa Cells , Humans , Ku Autoantigen , Nuclear Proteins , Phosphorylation , Precipitin Tests , Protein Binding , Protein Serine-Threonine Kinases/metabolism , RecQ Helicases
20.
J Biol Chem ; 277(8): 6280-6, 2002 Feb 22.
Article in English | MEDLINE | ID: mdl-11741924

ABSTRACT

Bloom's syndrome is a rare human autosomal recessive disorder that combines a marked genetic instability and an increased risk of developing all types of cancers and which results from mutations in both copies of the BLM gene encoding a RecQ 3'-5' DNA helicase. We recently showed that BLM is phosphorylated and excluded from the nuclear matrix during mitosis. We now show that the phosphorylated mitotic BLM protein is associated with a 3'-5' DNA helicase activity and interacts with topoisomerase III alpha. We demonstrate that in mitosis-arrested cells, ionizing radiation and roscovitine treatment both result in the reversion of BLM phosphorylation, suggesting that BLM could be dephosphorylated through the inhibition of cdc2 kinase. This was supported further by our data showing that cdc2 kinase activity is inhibited in gamma-irradiated mitotic cells. Finally we show that after ionizing radiation, BLM is not involved in the establishment of the mitotic DNA damage checkpoint but is subjected to a subcellular compartment change. These findings lead us to propose that BLM may be phosphorylated during mitosis, probably through the cdc2 pathway, to form a pool of rapidly available active protein. Inhibition of cdc2 kinase after ionizing radiation would lead to BLM dephosphorylation and possibly to BLM recruitment to some specific sites for repair.


Subject(s)
Adenosine Triphosphatases/genetics , Bloom Syndrome/genetics , DNA Helicases/genetics , Subcellular Fractions/enzymology , Adenosine Triphosphatases/metabolism , Adenosine Triphosphatases/radiation effects , B-Lymphocytes , Bloom Syndrome/enzymology , CDC2 Protein Kinase/antagonists & inhibitors , CDC2 Protein Kinase/radiation effects , Cell Cycle , Cell Line , DNA Helicases/metabolism , DNA Helicases/radiation effects , DNA Topoisomerases, Type I/metabolism , Gamma Rays , Humans , Mitosis , RecQ Helicases
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