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1.
Nat Commun ; 10(1): 5349, 2019 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-31836706

RESUMO

Increased levels and non-telomeric roles have been reported for shelterin proteins, including RAP1 in cancers. Herein using Rap1 null mice, we provide the genetic evidence that mammalian Rap1 plays a major role in hematopoietic stem cell survival, oncogenesis and response to chemotherapy. Strikingly, this function of RAP1 is independent of its association with the telomere or with its known partner TRF2. We show that RAP1 interacts with many members of the DNA damage response (DDR) pathway. RAP1 depleted cells show reduced interaction between XRCC4/DNA Ligase IV and DNA-PK, and are impaired in DNA Ligase IV recruitment to damaged chromatin for efficient repair. Consistent with its role in DNA damage repair, RAP1 loss decreases double-strand break repair via NHEJ in vivo, and consequently reduces B cell class switch recombination. Finally, we discover that RAP1 levels are predictive of the success of chemotherapy in breast and colon cancer.


Assuntos
Antineoplásicos/farmacologia , Carcinogênese/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Proteínas rap1 de Ligação ao GTP/metabolismo , Animais , Carcinogênese/efeitos dos fármacos , Carcinogênese/patologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Dano ao DNA , DNA Ligase Dependente de ATP/metabolismo , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/efeitos da radiação , Proteína Quinase Ativada por DNA/metabolismo , Fluoruracila/farmacologia , Raios gama , Instabilidade Genômica/efeitos dos fármacos , Instabilidade Genômica/efeitos da radiação , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos da radiação , Humanos , Camundongos Knockout , Mutagênicos/toxicidade , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/efeitos da radiação , Proteínas Proto-Oncogênicas c-myc/metabolismo , Análise de Sobrevida
2.
Nat Commun ; 10(1): 5718, 2019 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-31844045

RESUMO

Maintaining stability of replication forks is important for genomic integrity. However, it is not clear how replisome proteins contribute to fork stability under replication stress. Here, we report that ATAD5, a PCNA unloader, plays multiple functions at stalled forks including promoting its restart. ATAD5 depletion increases genomic instability upon hydroxyurea treatment in cultured cells and mice. ATAD5 recruits RAD51 to stalled forks in an ATR kinase-dependent manner by hydroxyurea-enhanced protein-protein interactions and timely removes PCNA from stalled forks for RAD51 recruitment. Consistent with the role of RAD51 in fork regression, ATAD5 depletion inhibits slowdown of fork progression and native 5-bromo-2'-deoxyuridine signal induced by hydroxyurea. Single-molecule FRET showed that PCNA itself acts as a mechanical barrier to fork regression. Consequently, DNA breaks required for fork restart are reduced by ATAD5 depletion. Collectively, our results suggest an important role of ATAD5 in maintaining genome integrity during replication stress.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Replicação do DNA/genética , Proteínas de Ligação a DNA/metabolismo , Instabilidade Genômica/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Rad51 Recombinase/metabolismo , ATPases Associadas a Diversas Atividades Celulares/genética , Bromodesoxiuridina/metabolismo , Linhagem Celular Tumoral , Quebras de DNA/efeitos dos fármacos , Reparo do DNA , Replicação do DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Citometria de Fluxo , Transferência Ressonante de Energia de Fluorescência , Técnicas de Silenciamento de Genes , Instabilidade Genômica/efeitos dos fármacos , Células HEK293 , Humanos , Hidroxiureia/farmacologia , Ligação Proteica/efeitos dos fármacos , RNA Interferente Pequeno/metabolismo , Imagem Individual de Molécula
3.
BMC Genomics ; 20(1): 579, 2019 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-31299901

RESUMO

BACKGROUND: Replication stress (RS) gives rise to DNA damage that threatens genome stability. RS can originate from different sources that stall replication by diverse mechanisms. However, the mechanism underlying how different types of RS contribute to genome instability is unclear, in part due to the poor understanding of the distribution and characteristics of damage sites induced by different RS mechanisms. RESULTS: We use ChIP-seq to map γH2AX binding sites genome-wide caused by aphidicolin (APH), hydroxyurea (HU), and methyl methanesulfonate (MMS) treatments in human lymphocyte cells. Mapping of γH2AX ChIP-seq reveals that APH, HU, and MMS treatments induce non-random γH2AX chromatin binding at discrete regions, suggesting that there are γH2AX binding hotspots in the genome. Characterization of the distribution and sequence/epigenetic features of γH2AX binding sites reveals that the three treatments induce γH2AX binding at largely non-overlapping regions, suggesting that RS may cause damage at specific genomic loci in a manner dependent on the fork stalling mechanism. Nonetheless, γH2AX binding sites induced by the three treatments share common features including compact chromatin, coinciding with larger-than-average genes, and depletion of CpG islands and transcription start sites. Moreover, we observe significant enrichment of SINEs in γH2AX sites in all treatments, indicating that SINEs may be a common barrier for replication polymerases. CONCLUSIONS: Our results identify the location and common features of genome instability hotspots induced by different types of RS, and help in deciphering the mechanisms underlying RS-induced genetic diseases and carcinogenesis.


Assuntos
Mapeamento Cromossômico , Replicação do DNA/genética , Histonas/metabolismo , Estresse Fisiológico/genética , Afidicolina/farmacologia , Sítios de Ligação , Linhagem Celular , Genoma Humano/genética , Instabilidade Genômica/efeitos dos fármacos , Humanos , Hidroxiureia/farmacologia , Estresse Fisiológico/efeitos dos fármacos , Ácidos Sulfínicos/farmacologia
4.
Appl Microbiol Biotechnol ; 103(16): 6495-6504, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31218376

RESUMO

The present study aimed to evaluate the influence of induction conditions (IPTG concentration, temperature, and induction time) on the plasmid pQE-30 stability and 503 antigen expression of Leishmania i. chagasi in Escherichia coli M15. Batch cultures were performed at 37 °C and induced by the addition of different IPTG concentrations (0.01 to 1.5 mM). Subsequently, experiments were carried out at different temperatures (27 to 42 °C), evaluating the influence of induction time (0.5 to 6 h after the start of the culture). The results showed that IPTG toxicity caused a metabolic stress in the cells and, consequently, the microorganism growth reduced. The induction with IPTG may also be associated with the plasmid pQE-30 instability, due to metabolic burden imposed by the recombinant protein expression. The optimal conditions for 503 antigen expression of Leishmania i. chagasi in Escherichia coli M15 were an IPTG concentration of 1.0 mM, temperature of 37 °C, and induction time of 2 h. The maximum antigen concentration obtained was 0.119 ± 0.009 g/L, about seven times higher than the lowest concentration. Therefore, the results showed that 503 antigen can be produced in laboratory; however, it requires more studies to minimize the plasmid instability and improve to industrial scale.


Assuntos
Antígenos de Protozoários/biossíntese , Escherichia coli/metabolismo , Expressão Gênica , Leishmania/genética , Proteínas Recombinantes/biossíntese , Ativação Transcricional , Antígenos de Protozoários/genética , Escherichia coli/genética , Instabilidade Genômica/efeitos dos fármacos , Isopropiltiogalactosídeo/metabolismo , Plasmídeos , Proteínas Recombinantes/genética , Temperatura
5.
Int J Mol Sci ; 20(7)2019 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-30970566

RESUMO

The mammalian mediator complex subunit 28 (MED28) is overexpressed in a variety of cancers and it regulates cell migration/invasion and epithelial-mesenchymal transition. However, transcription factors that increase MED28 expression have not yet been identified. In this study, we performed a luciferase reporter assay to identify and characterize the prospective transcription factors, namely E2F transcription factor 1, nuclear respiratory factor 1, E-26 transforming sequence 1, and CCAAT/enhancer-binding protein ß, which increased MED28 expression. In addition, the release from the arrest at the G1-S or G2-M phase transition after cell cycle synchronization using thymidine or nocodazole, respectively, showed enhanced MED28 expression at the G1-S transition and mitosis. Furthermore, the overexpression of MED28 significantly decreased the duration of interphase and mitosis. Conversely, a knockdown of MED28 using si-RNA increased the duration of interphase and mitosis. Of note, the overexpression of MED28 significantly increased micronucleus and nuclear budding in HeLa cells. In addition, flow cytometry and fluorescence microscopy analyses showed that the overexpression of MED28 significantly increased aneuploid cells. Taken together, these results suggest that MED28 expression is increased by oncogenic transcription factors and its overexpression disturbs the cell cycle, which results in genomic instability and aneuploidy.


Assuntos
Instabilidade Genômica , Complexo Mediador/genética , Complexo Mediador/metabolismo , Fatores de Transcrição/metabolismo , Aneuploidia , Ciclo Celular/efeitos dos fármacos , Instabilidade Genômica/efeitos dos fármacos , Células HEK293 , Células HeLa , Humanos , Células MCF-7 , Nocodazol/farmacologia , Regiões Promotoras Genéticas , Timidina/farmacologia , Regulação para Cima
6.
Curr Genet ; 65(4): 913-917, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30963245

RESUMO

Oxidative stress has been implicated in a variety of human diseases. One plausible mechanism is that reactive active species can induce DNA damages and jeopardize genome integrity. To explore how oxidative stress results in global genomic instability in cells, our current study examined the genomic alterations caused by H2O2 exposure at the whole genome level in yeast. Using SNP microarrays and genome sequencing, we mapped H2O2-induced genomic alterations in the yeast genome ranging from point mutations and mitotic recombination to chromosomal aneuploidy. Our results suggested most H2O2-induced mitotic recombination events were the result of DNA double-stand breaks generated by hydroxyl radicals. Moreover, the mutagenic effect of H2O2 was shown to be largely dependent on DNA polymerase ζ. Lastly, we showed that H2O2 exposure allows rapid phenotypic evolution in yeast strains. Our findings indicate DNA lesions resulting from H2O2 may be general factors that drive genome instability and phenotypic evolution in organisms.


Assuntos
Dano ao DNA/efeitos dos fármacos , Instabilidade Genômica/genética , Peróxido de Hidrogênio/farmacologia , Estresse Oxidativo/genética , Aneuploidia , Replicação do DNA/efeitos dos fármacos , Replicação do DNA/genética , Instabilidade Genômica/efeitos dos fármacos , Humanos , Mitose/efeitos dos fármacos , Mitose/genética , Mutagênese/genética , Análise de Sequência com Séries de Oligonucleotídeos , Estresse Oxidativo/efeitos dos fármacos , Polimorfismo de Nucleotídeo Único/genética , Saccharomyces cerevisiae/genética , Sequenciamento Completo do Genoma
7.
Nucleic Acids Res ; 47(13): e73, 2019 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-30949695

RESUMO

Regions of genomic instability are not random and often co-localize with DNA sequences that can adopt alternative DNA structures (i.e. non-B DNA, such as H-DNA). Non-B DNA-forming sequences are highly enriched at translocation breakpoints in human cancer genomes, representing an endogenous source of genetic instability. However, a further understanding of the mechanisms involved in non-B DNA-induced genetic instability is needed. Small molecules that can modulate the formation/stability of non-B DNA structures, and therefore the subsequent mutagenic outcome, represent valuable tools to study DNA structure-induced genetic instability. To this end, we have developed a tunable Förster resonance energy transfer (FRET)-based assay to detect triplex/H-DNA-destabilizing and -stabilizing ligands. The assay was designed by incorporating a fluorophore-quencher pair in a naturally-occurring H-DNA-forming sequence from a chromosomal breakpoint hotspot in the human c-MYC oncogene. By tuning triplex stability via buffer composition, the assay functions as a dual-reporter that can identify stabilizers and destabilizers, simultaneously. The assay principle was demonstrated using known triplex stabilizers, BePI and coralyne, and a complementary oligonucleotide to mimic a destabilizer, MCRa2. The potential of the assay was validated in a 384-well plate with 320 custom-assembled compounds. The discovery of novel triplex stabilizers/destabilizers may allow the regulation of genetic instability in human genomes.


Assuntos
Alcaloides de Berberina/farmacologia , Pontos de Quebra do Cromossomo , DNA/efeitos dos fármacos , Transferência Ressonante de Energia de Fluorescência/métodos , Genes myc , Instabilidade Genômica/efeitos dos fármacos , Indóis/farmacologia , Conformação de Ácido Nucleico/efeitos dos fármacos , Piridinas/farmacologia , Alcanossulfonatos/análise , Compostos Azo/análise , Tampões (Química) , Dicroísmo Circular , Relação Dose-Resposta a Droga , Doxorrubicina/farmacologia , Corantes Fluorescentes/análise , Genes myc/efeitos dos fármacos , Genoma Humano , Humanos , Ligantes , Oligodesoxirribonucleotídeos/química
8.
J Ethnopharmacol ; 237: 171-181, 2019 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-30890359

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Cynarin is an artichoke phytochemical that possesses a variety of pharmacological features including free-radical scavenging and antioxidant activity. The origin of artichoke species appears to be Mediterranean region. Two of these species, globe artichoke (Cynara cardunculus var. scolymus L.) and cardoon (Cynara cardunculus var. altilis DC), are widely cultivated and consumed. This vegetable, as the basis of the mediterranean diet, has been used as herbal medicine for its therapeutic effects since ancient times. Therefore, this study was performed to determine genotoxic and antigenotoxic effects of cynarin against MMC (mitomycin C) and H2O2 (hydrogen peroxide) induced genomic instability using chromosome aberrations (CAs), sister chromatid exchanges (SCEs), micronucleus (MN), and comet assays in human lymphocytes. MATERIALS AND METHODS: Lymphocytes obtained from two healthy volunteers (1 male and 1 female) were exposed to different concentrations of cynarin (12-194 µM) alone and the combination of cynarin and MMC (0.60 µM) or cynarin and H2O2 (100 µM, only for comet assay). RESULTS: Cynarin alone did not induce significant genotoxic effect in the CA, SCE (except 194 µM), MN, and comet assays. The combination of some concentrations of cynarin and MMC decreased the frequency of CAs, SCEs and MN induced by MMC. Furthermore, the combination of cynarin and H2O2 reduced all comet parameters at all the concentrations compared to H2O2 alone. While the highest concentrations of cynarin significantly decreased mitotic index (MI), the combination of cynarin and MMC increased the reduction of MI induced by MMC alone. CONCLUSION: All the results obtained in this study demonstrated that cynarin exhibited antigenotoxic effects rather than genotoxic effects. It is believed that cynarin can act as a potential chemo-preventive against genotoxic agents.


Assuntos
Anticarcinógenos/farmacologia , Cinamatos/farmacologia , Adulto , Células Cultivadas , Aberrações Cromossômicas/induzido quimicamente , Ensaio Cometa , Dano ao DNA , Feminino , Instabilidade Genômica/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio , Linfócitos/efeitos dos fármacos , Masculino , Testes para Micronúcleos , Mitomicina , Mutagênicos , Adulto Jovem
9.
Environ Mol Mutagen ; 60(6): 559-562, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30848522

RESUMO

Cell line-based in vitro testing has been widely used as an important component of the genotoxicity testing battery; however, the use of cell lines is constrained by several limitations, including the genetic drift and variability. A study recently reported in the literature comprehensively examined genomic changes in a large number of cell lines and reported extensive genetic variations within the same cell lines across passage numbers and laboratories, even for single-cell derived subclones. The primary objective of this communication is to raise awareness and stimulate discussion within the genotoxicity testing community of the extent of genetic variability of cell lines in general and how these variables could potentially influence the results and reproducibility of genotoxicity testing. Meanwhile, some recommendations for good cell culture practices are highlighted to mitigate, at least to some extent, the concern about genetic variation. Environ. Mol. Mutagen. 60:559-562, 2019. © 2019 Wiley Periodicals, Inc.


Assuntos
Instabilidade Genômica/efeitos dos fármacos , Instabilidade Genômica/genética , Mutagênicos/toxicidade , Animais , Linhagem Celular , Variação Genética/efeitos dos fármacos , Variação Genética/genética , Humanos , Testes de Mutagenicidade/métodos , Reprodutibilidade dos Testes
10.
Adv Protein Chem Struct Biol ; 115: 157-201, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30798931

RESUMO

Genomically instable cancers are characterized by progressive loss and gain of chromosomal fragments, and the acquisition of complex genomic rearrangements. Such cancers, including triple-negative breast cancers and high-grade serous ovarian cancers, typically show aggressive behavior and lack actionable driver oncogenes. Increasingly, oncogene-induced replication stress or defective replication fork maintenance is considered an important driver of genomic instability. Paradoxically, while replication stress causes chromosomal instability and thereby promotes cancer development, it intrinsically poses a threat to cellular viability. Apparently, tumor cells harboring high levels of replication stress have evolved ways to cope with replication stress. As a consequence, therapeutic targeting of such compensatory mechanisms is likely to preferentially target cancers with high levels of replication stress and may prove useful in potentiating chemotherapeutic approaches that exert their effects by interfering with DNA replication. Here, we discuss how replication stress drives chromosomal instability, and the cell cycle-regulated mechanisms that cancer cells employ to deal with replication stress. Importantly, we discuss how mechanisms involving DNA structure-specific resolvases, cell cycle checkpoint kinases and mitotic processing of replication intermediates offer possibilities in developing treatments for difficult-to-treat genomically instable cancers.


Assuntos
Antineoplásicos/farmacologia , Replicação do DNA/efeitos dos fármacos , Instabilidade Genômica/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Estresse Fisiológico/efeitos dos fármacos , Replicação do DNA/genética , Instabilidade Genômica/genética , Humanos , Neoplasias/genética , Estresse Fisiológico/genética
11.
Nucleic Acids Res ; 47(7): 3521-3535, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-30668788

RESUMO

Oxidative DNA damage is a threat to genome stability. Using a genetic system in yeast that allows detection of mitotic recombination, we found that the frequency of crossovers is greatly elevated when cells are treated with hydrogen peroxide (H2O2). Using a combination of microarray analysis and genomic sequencing, we mapped the breakpoints of mitotic recombination events and other chromosome rearrangements at a resolution of about 1 kb. Gene conversions and crossovers were the two most common types of events, but we also observed deletions, duplications, and chromosome aneuploidy. In addition, H2O2-treated cells had elevated rates of point mutations (particularly A to T/T to A and C to G/G to C transversions) and small insertions/deletions (in/dels). In cells that underwent multiple rounds of H2O2 treatments, we identified a genetic alteration that resulted in improved H2O2 tolerance by amplification of the CTT1 gene that encodes cytosolic catalase T. Lastly, we showed that cells grown in the absence of oxygen have reduced levels of recombination. This study provided multiple novel insights into how oxidative stress affects genomic instability and phenotypic evolution in aerobic cells.


Assuntos
Catalase/genética , Dano ao DNA/efeitos dos fármacos , Conversão Gênica/genética , Estresse Oxidativo/efeitos dos fármacos , Cromossomos Fúngicos/genética , Citosol/enzimologia , Reparo do DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Genoma Fúngico/efeitos dos fármacos , Instabilidade Genômica/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Mitose/genética , Mutação Puntual/genética , Saccharomyces cerevisiae/genética
12.
Mutat Res ; 814: 15-22, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30682723

RESUMO

Patients with type 2 diabetes mellitus (T2DM) are associated with an elevated, but poorly understood baseline of genomic instability (GIN). Expert panels are still debating on whether hyperglycemia is the key element in conferring this high GIN. Since high blood glucose and low blood folate are prevalent in T2DM, we hypothesized that high glucose may work with low folate to induce GIN. Using NCM460, CCD841 and L02 cell lines as in vitro cell models, we investigated the genotoxic effects of high sugars (HS; 1-2% glucose, fructose, galactose or sucrose) alone or in combination with folate deficiency (23 nM, FD) over a course of 7 days by the cytokinesis block micronucleus assay. We found that HS is nongenotoxic to NCM460, CCD841 and L02 cells. However, the combination of HS and FD induced significantly higher levels of micronuclei, nucleoplasmic bridges and nuclear buds. Our in vitro work demonstrates that HS is non-genotoxic under folate repletive condition, but is genotoxic under FD condition. These results provide preclinal proof of concept that concomitant hyperglycemia and low folate may explain, at least in part, the high baseline of GIN in T2DM patients, suggesting that folate levels should be kept under control in order to limit the risk of GIN and carcinogenesis in T2DM.


Assuntos
Metabolismo dos Carboidratos/efeitos dos fármacos , Dano ao DNA , Deficiência de Ácido Fólico/patologia , Ácido Fólico/farmacologia , Açúcares/farmacologia , Metabolismo dos Carboidratos/genética , Células Cultivadas , Relação Dose-Resposta a Droga , Deficiência de Ácido Fólico/metabolismo , Frutose/farmacologia , Galactose/farmacologia , Instabilidade Genômica/efeitos dos fármacos , Humanos , Testes para Micronúcleos , Testes de Mutagenicidade , Sacarose/farmacologia
13.
Environ Toxicol Pharmacol ; 66: 62-68, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30605873

RESUMO

With the known facts of deltamethrin toxicity in aquaculture, efforts have been made to ameliorate its toxicity with supplementation of Silybum marianum plant extract in Cyprinus carpio. For validating its efficacy, standard techniques of histopathology, anti-oxidant profile and RAPD-PCR were used. By performing acute toxicity bioassay, 96 h LC50 of the pesticide to C. carpio was determined and was found to be 2 µg/L. Histopathological alterations were comprised of nuclear alterations, vacuolisation and oedema in the hepatocytes. Chronic exposure to the toxicant induced significant changes in antioxidant defense system (CAT, SOD, GSH and GST levels), lipid peroxidation being prominent. Diet supplementation with silymarin appeared to modulate the oxidative stress, histopathological alterations and genotoxic damage caused by the pesticide in the fish. RAPD-PCR studies revealed deltamethrin induced toxicity and its effective amelioration in form of restoration of bands which were lost in toxicant exposed DNA profile.


Assuntos
Carpas/fisiologia , Inseticidas/toxicidade , Fígado/efeitos dos fármacos , Cardo-Mariano , Nitrilos/toxicidade , Extratos Vegetais/farmacologia , Substâncias Protetoras/farmacologia , Piretrinas/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Antioxidantes , Instabilidade Genômica/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/patologia , Peroxidação de Lipídeos/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Estresse Oxidativo/efeitos dos fármacos
14.
Genes Chromosomes Cancer ; 58(6): 365-372, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30578714

RESUMO

Rare cases of hematological precursor neoplasms fulfill the diagnostic criteria of mixed phenotype acute leukemia (MPAL), characterized by expression patterns of at least two hematopoietic lineages, for which a highly aggressive behavior was reported. We present a series of 11 pediatric non-leukemic MPAL identified among 146 precursor lymphoblastic lymphomas included in the prospective trial Euro-LBL 02. Paraffin-embedded biopsies of 10 cases were suitable for molecular analyses using OncoScan assay (n = 7), fluorescence in situ hybridization (FISH; n = 7) or both (n = 5). Except for one case with biallelic KMT2A (MLL) breaks, all cases analyzed by FISH lacked the most common translocations defining molecular subsets of lymphoblastic leukemia/lymphomas. Two non-leukemic B-myeloid MPALs showed the typical genomic profile of hyperdiploid precursor B-cell lymphoblastic leukemia with gains of chromosomes 4, 6, 10, 14, 18, and 21. One B-T MPAL showed typical aberrations of T-cell lymphoblastic lymphoma, such as copy number neutral loss of heterozygosity (CNN-LOH) at 9p targeting a 9p21.3 deletion of CDKN2A and 11q12.2-qter affecting the ATM gene. ATM was also mutated in a T-myeloid MPAL case with additional loss at 7q21.2-q36.3 and mutation of NRAS, two alterations common in myeloid disorders. No recurrent regions of CNN-LOH were observed. The outcome under treatment was good with all patients being alive in first complete remission after treatment according to a protocol for precursor lymphoblastic lymphoma (follow-up 3-10 years, median: 4.9 years). In summary, the present series of non-leukemic MPALs widely lacked recurrently reported translocations in lymphoid/myeloid neoplasias and showed heterogeneous spectrum of chromosomal imbalances.


Assuntos
Fenótipo , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Translocação Genética , Adolescente , Antineoplásicos/efeitos adversos , Antineoplásicos/uso terapêutico , Proteínas Mutadas de Ataxia Telangiectasia/genética , Criança , Pré-Escolar , Inibidor p16 de Quinase Dependente de Ciclina/genética , Feminino , GTP Fosfo-Hidrolases/genética , Instabilidade Genômica/efeitos dos fármacos , Histona-Lisina N-Metiltransferase/genética , Humanos , Masculino , Proteínas de Membrana/genética , Proteína de Leucina Linfoide-Mieloide/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia
15.
Chem Res Toxicol ; 31(12): 1290-1292, 2018 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-30521319

RESUMO

Evaluating carcinogenic mechanisms is a challenging part of hazard identification, as mechanistic data are both voluminous and diverse. An evaluation approach based on 10 key characteristics of human carcinogens provides a holistic and unbiased way to tackle this challenge.


Assuntos
Carcinógenos/química , Biomarcadores/metabolismo , Testes de Carcinogenicidade , Carcinógenos/toxicidade , Dano ao DNA/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Instabilidade Genômica/efeitos dos fármacos , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , Estresse Oxidativo/efeitos dos fármacos
16.
Nature ; 563(7732): 522-526, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30464262

RESUMO

Limited DNA end resection is the key to impaired homologous recombination in BRCA1-mutant cancer cells. Here, using a loss-of-function CRISPR screen, we identify DYNLL1 as an inhibitor of DNA end resection. The loss of DYNLL1 enables DNA end resection and restores homologous recombination in BRCA1-mutant cells, thereby inducing resistance to platinum drugs and inhibitors of poly(ADP-ribose) polymerase. Low BRCA1 expression correlates with increased chromosomal aberrations in primary ovarian carcinomas, and the junction sequences of somatic structural variants indicate diminished homologous recombination. Concurrent decreases in DYNLL1 expression in carcinomas with low BRCA1 expression reduced genomic alterations and increased homology at lesions. In cells, DYNLL1 limits nucleolytic degradation of DNA ends by associating with the DNA end-resection machinery (MRN complex, BLM helicase and DNA2 endonuclease). In vitro, DYNLL1 binds directly to MRE11 to limit its end-resection activity. Therefore, we infer that DYNLL1 is an important anti-resection factor that influences genomic stability and responses to DNA-damaging chemotherapy.


Assuntos
Proteína BRCA1/deficiência , Dineínas do Citoplasma/metabolismo , DNA/metabolismo , Genes BRCA1 , Proteína Homóloga a MRE11/metabolismo , Reparo de DNA por Recombinação , Proteína BRCA1/genética , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Aberrações Cromossômicas , Dano ao DNA/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Edição de Genes , Instabilidade Genômica/efeitos dos fármacos , Recombinação Homóloga/efeitos dos fármacos , Humanos , Mutação , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Platina/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Ligação Proteica , Reparo de DNA por Recombinação/efeitos dos fármacos , Fatores de Transcrição/metabolismo
17.
J Mol Cell Cardiol ; 125: 174-184, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30385152

RESUMO

Diet-induced metabolic acidosis is associated with the impairment of bone metabolism and an increased risk of a number of chronic noncommunicable diseases, such as type 2 diabetes mellitus and hypertension. Low serum bicarbonate is associated with high mortality in healthy older individuals. Recently, we demonstrated that both coupling factor 6 (CF6)-overexpressing transgenic (TG) and high salt-fed mice which had sustained intracellular acidosis, due to enhanced proton import through ecto-F1Fo complex and/or reduced proton export through Na+-K+ ATPase inhibition, displayed shortened lifespan and early senescence-associated phenotypes such as signs of hair greying and alopecia, weight loss, and/or reduced organ mass. In this study, we searched causative genes of proton-induced aging in CF6-overexpressing TG and high salt-fed mice. We discovered NM_026333 as a novel anti-aging gene which was downregulated in the heart and kidney in both types of mice. NM_026333 protein consists of 269 amino acids with transmembrane region (90-193aa). Induction of NM_026333 or recombinant protein rescued TG cells and CF6-treated human cells from aging hallmarks of impaired autophagy, genomic instability, and epigenetic alteration. NM_026333 protein directly bound plasma membrane Na+-Ca2+ exchanger 1 (NCX1) to suppress its reverse mode, and cancelled proton-induced epigenetic regression of Atg7 that was caused by H3K4 and H4K20 tri-methylation via suppression of demethylase and H4K5 acetylation via suppression of nuclear HDAC3-HDAC4-emerin system. NM_026333 also attenuated proton-induced impaired formation of autolysosome, an increase in nuclear acetylated LC3 II, and acetylation of Atg7. These effects reappeared by NCX1 inhibitor. Furthermore, NCX1 inhibitor extended lifespan compared with vehicle-treatment in TG mice. This study will shed light on novel aging mechanism and provide implications in a target for anti-aging therapy.


Assuntos
ATPases Mitocondriais Próton-Translocadoras/metabolismo , Fatores Acopladores da Fosforilação Oxidativa/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Envelhecimento/efeitos dos fármacos , Animais , Autofagia/genética , Autofagia/fisiologia , Membrana Celular/metabolismo , Células Cultivadas , Imunoprecipitação da Cromatina , Epigenômica , Instabilidade Genômica/efeitos dos fármacos , Instabilidade Genômica/genética , Células HEK293 , Humanos , Camundongos , ATPases Mitocondriais Próton-Translocadoras/genética , Fatores Acopladores da Fosforilação Oxidativa/genética , Prótons , Transdução de Sinais/efeitos dos fármacos
18.
Annu Rev Microbiol ; 72: 209-230, 2018 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-30200850

RESUMO

By targeting essential cellular processes, antibiotics provoke metabolic perturbations and induce stress responses and genetic variation in bacteria. Here we review current knowledge of the mechanisms by which these molecules generate genetic instability. They include production of reactive oxygen species, as well as induction of the stress response regulons, which lead to enhancement of mutation and recombination rates and modulation of horizontal gene transfer. All these phenomena influence the evolution and spread of antibiotic resistance. The use of strategies to stop or decrease the generation of resistant variants is also discussed.


Assuntos
Antibacterianos/efeitos adversos , Bactérias/efeitos dos fármacos , Variação Genética/efeitos dos fármacos , Adaptação Biológica , Bactérias/genética , Instabilidade Genômica/efeitos dos fármacos , Mutação , Espécies Reativas de Oxigênio/metabolismo , Recombinação Genética , Seleção Genética/efeitos dos fármacos , Estresse Fisiológico
19.
Free Radic Biol Med ; 129: 97-106, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30223018

RESUMO

A genetic analysis of synthetic lethal interactions in yeast revealed that the mutation of SOD1, encoding an antioxidant enzyme that scavenges superoxide anion radical, impaired the growth of a set of mutants defective in homologous recombination (HR) pathway. Hence, SOD1 inhibition has been proposed as a promising approach for the selective killing of HR-deficient cancer cells. However, we show that the deletion of RAD51 and SOD1 is not synthetic lethal but displays considerably slow growth and synergistic sensitivity to both reactive oxygen species (ROS)- and DNA double-strand break (DSB)-generating drugs in the budding yeast Saccharomyces cerevisiae. The function of Sod1 in regard to Rad51 is dependent on Ccs1, a copper chaperone for Sod1. Sod1 deficiency aggravates genomic instability in conjunction with the absence of Rad51 by inducing DSBs and an elevated mutation frequency. Inversely, lack of Rad51 causes a Sod1 deficiency-derived increase of intracellular ROS levels. Taken together, our results indicate that there is a significant and specific crosstalk between two major cellular damage response pathways, ROS signaling and DSB repair, for cell survival.


Assuntos
DNA Fúngico/genética , Regulação Fúngica da Expressão Gênica , Genoma Fúngico , Instabilidade Genômica/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Superóxido Dismutase-1/genética , 4-Nitroquinolina-1-Óxido/farmacologia , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , DNA Fúngico/metabolismo , Recombinação Homóloga , Peróxido de Hidrogênio/farmacologia , Hidroxiureia/farmacologia , Metanossulfonato de Metila/farmacologia , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutação , Paraquat/farmacologia , Fleomicinas/farmacologia , Quinolonas/farmacologia , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Espécies Reativas de Oxigênio/agonistas , Espécies Reativas de Oxigênio/metabolismo , Reparo de DNA por Recombinação/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Superóxido Dismutase-1/deficiência
20.
PLoS One ; 13(9): e0204858, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30261076

RESUMO

L-arginine supplementation has been related to increased maximum strength and improvement of hemodynamic parameters in several diseases. The aim of our study was to evaluate the effect of L-arginine supplementation and resistance training on muscle mass, hemodynamic function and DNA damage in healthy rats subjected to a low-arginine concentration diet. Twenty three Wistar rats (290-320g) were divided into 4 groups: Sedentary (SED-Arg, n = 6), Sedentary+Arg (SED+Arg, n = 6), Resistance Training (RT-Arg, n = 5), Resistance Training+Arg (RT+Arg, n = 6). Trained animals performed resistance training protocol in a squat apparatus adapted for rats (4 sets of 10-12 repetitions, 90s of interval, 4x/week, 65-75% of One Maximum Repetition, for 8 weeks). Comet assay was performed to measure DNA damage in leukocytes. The resistance training induced higher muscle mass in trained groups. The L-arginine supplementation increased both gastrocnemius and left ventricle to body mass ratio and increased left ventricle contractility without changing hemodynamic variables. The SED+Arg group showed higher concentration of extracellular heat shock protein 72 (eHSP72) and total testosterone, as well as lower uric acid concentration in blood versus SED-Arg group. The administration of isolated L-arginine supplementation and its association with resistance training promoted less damage in leukocytes DNA. In conclusion, the L-arginine supplementation showed synergistic effect with resistance training regarding leukocyte genomic stability in a low-L-arginine diet scenario.


Assuntos
Arginina/farmacologia , Suplementos Nutricionais , Instabilidade Genômica/efeitos dos fármacos , Músculo Esquelético/metabolismo , Contração Miocárdica/efeitos dos fármacos , Condicionamento Físico Animal , Animais , Dano ao DNA , Leucócitos/metabolismo , Masculino , Músculo Esquelético/crescimento & desenvolvimento , Músculo Esquelético/patologia , Tamanho do Órgão/efeitos dos fármacos , Ratos , Ratos Wistar
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