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1.
Nat Commun ; 12(1): 401, 2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33452241

RESUMO

Mechanisms regulating DNA repair processes remain incompletely defined. Here, the circadian factor CRY1, an evolutionally conserved transcriptional coregulator, is identified as a tumor specific regulator of DNA repair. Key findings demonstrate that CRY1 expression is androgen-responsive and associates with poor outcome in prostate cancer. Functional studies and first-in-field mapping of the CRY1 cistrome and transcriptome reveal that CRY1 regulates DNA repair and the G2/M transition. DNA damage stabilizes CRY1 in cancer (in vitro, in vivo, and human tumors ex vivo), which proves critical for efficient DNA repair. Further mechanistic investigation shows that stabilized CRY1 temporally regulates expression of genes required for homologous recombination. Collectively, these findings reveal that CRY1 is hormone-induced in tumors, is further stabilized by genomic insult, and promotes DNA repair and cell survival through temporal transcriptional regulation. These studies identify the circadian factor CRY1 as pro-tumorigenic and nominate CRY1 as a new therapeutic target.


Assuntos
Carcinogênese/genética , Criptocromos/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias de Próstata Resistentes à Castração/genética , Reparo de DNA por Recombinação/genética , Idoso , Antagonistas de Receptores de Andrógenos/farmacologia , Antagonistas de Receptores de Andrógenos/uso terapêutico , Androgênios/metabolismo , Carcinogênese/efeitos dos fármacos , Linhagem Celular Tumoral , Sequenciamento de Cromatina por Imunoprecipitação , Criptocromos/genética , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Conjuntos de Dados como Assunto , Progressão da Doença , Seguimentos , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Humanos , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Regiões Promotoras Genéticas/genética , Estudos Prospectivos , Próstata/patologia , Próstata/cirurgia , Prostatectomia , Neoplasias de Próstata Resistentes à Castração/mortalidade , Neoplasias de Próstata Resistentes à Castração/patologia , Neoplasias de Próstata Resistentes à Castração/terapia , RNA-Seq , Receptores Androgênicos/metabolismo , Reparo de DNA por Recombinação/efeitos dos fármacos , Estudos Retrospectivos
2.
Int J Mol Sci ; 22(2)2021 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-33466757

RESUMO

Chromosomal rearrangements comprise unbalanced structural variations resulting in gain or loss of DNA copy numbers, as well as balanced events including translocation and inversion that are copy number neutral, both of which contribute to phenotypic evolution in organisms. The exquisite genetic assay and gene editing tools available for the model organism Saccharomyces cerevisiae facilitate deep exploration of the mechanisms underlying chromosomal rearrangements. We discuss here the pathways and influential factors of chromosomal rearrangements in S. cerevisiae. Several methods have been developed to generate on-demand chromosomal rearrangements and map the breakpoints of rearrangement events. Finally, we highlight the contributions of chromosomal rearrangements to drive phenotypic evolution in various S. cerevisiae strains. Given the evolutionary conservation of DNA replication and recombination in organisms, the knowledge gathered in the small genome of yeast can be extended to the genomes of higher eukaryotes.


Assuntos
Inversão Cromossômica/genética , Cromossomos Fúngicos/genética , Rearranjo Gênico/genética , Saccharomyces cerevisiae/genética , Translocação Genética/genética , Antibióticos Antineoplásicos , Bleomicina/farmacologia , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Rearranjo Gênico/efeitos dos fármacos , Rearranjo Gênico/efeitos da radiação , Modelos Genéticos , Radiação Ionizante
3.
Environ Mol Mutagen ; 62(2): 143-154, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33496997

RESUMO

Pioglitazone (PIO), an oral hypoglycemic agent, is used in the treatment of type 2 diabetes. Some studies have suggested that an increased risk of bladder cancer with PIO exposure, while the others reported there is no such relationship. Therefore, it is doubtful whether PIO can increase the risk of bladder cancer. The effects of PIO on DNA damage and/or transformation of human bladder cells are not fully known. We investigated the effects of PIO on cytotoxicity, DNA single and double strand breaks and repair and neoplastic transformation in human bladder cells (hTU1) treated with 10, 20, and 40 µM PIO for 24, 48 and 72 hr. PIO decreased cell viability in a concentration-dependent manner. Increased levels of comet parameters showed that PIO and its metabolites can significantly induce DNA double strand breaks at all concentrations tested. PIO also significantly induced the formation of phosphorylated H2AX and p53 binding protein 1 foci. DNA damage was not repaired in a 24 hr recovery period. PIO can also induce malignant transformation of human bladder cells exhibiting loss of contact inhibition and anchorage independent growth. This is the first study to indicate that PIO can induce DNA damage and malignant transformation, reduce or alter the DNA repair capacity in human bladder cells. From these results, we suggest that patients with diabetes treated with PIO may have an increased risk of bladder cancer.


Assuntos
Transformação Celular Neoplásica/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , DNA/efeitos dos fármacos , Pioglitazona/efeitos adversos , Neoplasias da Bexiga Urinária/induzido quimicamente , Bexiga Urinária/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/genética , Células Epiteliais/efeitos dos fármacos , Humanos , Pioglitazona/farmacologia , Neoplasias da Bexiga Urinária/genética
4.
ACS Appl Mater Interfaces ; 12(51): 56874-56885, 2020 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-33326207

RESUMO

Radiotherapy is the main treatment for cancer patients. A major concern in radiotherapy is the radiation resistance of some tumors, such as human nonsmall cell lung cancer. However, the radiation dose delivered to the tumors is often limited by the possibility of collateral damage to surrounding healthy tissues. A new and efficient gadolinium-based nanoparticle, AGuIX, has recently been developed for magnetic resonance imaging-guided radiotherapy and has been proven to act as an efficient radiosensitizer. The amplified radiation effects of AGuIX nanoparticles appear to be due to the emission of low-energy photoelectrons and Auger electron interactions. We demonstrated that AGuIX nanoparticles exacerbated radiation-induced DNA double-strand break damage and reduced DNA repair in the H1299 nonsmall cell lung cancer cell line. Furthermore, we observed a significant improvement in tumor cell damage and growth suppression, under radiation therapy, with the AGuIX nanoparticles in a H1299 mouse xenograft model. This study paves the way for research into the radiosensitization mechanism of AGuIX nanoparticles and provides a scientific basis for the use of AGuIX nanoparticles as radiosensitizing drugs.


Assuntos
Antineoplásicos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Nanopartículas Metálicas/uso terapêutico , Radiossensibilizantes/uso terapêutico , Animais , Antineoplásicos/química , Antineoplásicos/efeitos da radiação , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Gadolínio/química , Gadolínio/efeitos da radiação , Humanos , Pulmão/patologia , Neoplasias Pulmonares/patologia , Masculino , Nanopartículas Metálicas/química , Nanopartículas Metálicas/efeitos da radiação , Camundongos Nus , Radiação Ionizante , Radiossensibilizantes/química , Radiossensibilizantes/efeitos da radiação , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Int J Mol Sci ; 21(24)2020 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-33322336

RESUMO

Survivin, as an antiapoptotic protein often overexpressed in cancer cells, is a logical target for potential cancer treatment. By overexpressing survivin, cancer cells can avoid apoptotic cell death and often become resistant to treatments, representing a significant obstacle in modern oncology. A survivin suppressor, an imidazolium-based compound known as YM-155, is nowadays studied as an attractive anticancer agent. Although survivin suppression by YM-155 is evident, researchers started to report that YM-155 is also an inducer of DNA damage introducing yet another anticancer mechanism of this drug. Moreover, the concentrations of YM-155 for DNA damage induction seems to be far lower than those needed for survivin inhibition. Understanding the molecular mechanism of action of YM-155 is of vital importance for modern personalized medicine involving the selection of responsive patients and possible treatment combinations. This review focuses mainly on the documented effects of YM-155 on DNA damage signaling pathways. It summarizes up to date literature, and it outlines the molecular mechanism of YM-155 action in the context of the DNA damage field.


Assuntos
Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Imidazóis/farmacologia , Naftoquinonas/farmacologia , Animais , Dano ao DNA/efeitos dos fármacos , Humanos , Survivina/metabolismo
6.
Nucleic Acids Res ; 48(16): 9161-9180, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32797166

RESUMO

FANCJ, a DNA helicase and interacting partner of the tumor suppressor BRCA1, is crucial for the repair of DNA interstrand crosslinks (ICL), a highly toxic lesion that leads to chromosomal instability and perturbs normal transcription. In diploid cells, FANCJ is believed to operate in homologous recombination (HR) repair of DNA double-strand breaks (DSB); however, its precise role and molecular mechanism is poorly understood. Moreover, compensatory mechanisms of ICL resistance when FANCJ is deficient have not been explored. In this work, we conducted a siRNA screen to identify genes of the DNA damage response/DNA repair regime that when acutely depleted sensitize FANCJ CRISPR knockout cells to a low concentration of the DNA cross-linking agent mitomycin C (MMC). One of the top hits from the screen was RAP80, a protein that recruits repair machinery to broken DNA ends and regulates DNA end-processing. Concomitant loss of FANCJ and RAP80 not only accentuates DNA damage levels in human cells but also adversely affects the cell cycle checkpoint, resulting in profound chromosomal instability. Genetic complementation experiments demonstrated that both FANCJ's catalytic activity and interaction with BRCA1 are important for ICL resistance when RAP80 is deficient. The elevated RPA and RAD51 foci in cells co-deficient of FANCJ and RAP80 exposed to MMC are attributed to single-stranded DNA created by Mre11 and CtIP nucleases. Altogether, our cell-based findings together with biochemical studies suggest a critical function of FANCJ to suppress incompletely processed and toxic joint DNA molecules during repair of ICL-induced DNA damage.


Assuntos
Proteína BRCA1/genética , Proteínas de Ligação a DNA/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Instabilidade Genômica/genética , Chaperonas de Histonas/genética , RNA Helicases/genética , Rad51 Recombinase/genética , Instabilidade Cromossômica/genética , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Dano ao DNA/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/deficiência , Técnicas de Inativação de Genes , Células HeLa , Chaperonas de Histonas/deficiência , Humanos , Mitomicina/farmacologia , Reparo de DNA por Recombinação/genética
7.
Nucleic Acids Res ; 48(15): 8490-8508, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32687193

RESUMO

Several functions have been proposed for the Escherichia coli DNA polymerase IV (pol IV). Although much research has focused on a potential role for pol IV in assisting pol III replisomes in the bypass of lesions, pol IV is rarely found at the replication fork in vivo. Pol IV is expressed at increased levels in E. coli cells exposed to exogenous DNA damaging agents, including many commonly used antibiotics. Here we present live-cell single-molecule microscopy measurements indicating that double-strand breaks induced by antibiotics strongly stimulate pol IV activity. Exposure to the antibiotics ciprofloxacin and trimethoprim leads to the formation of double strand breaks in E. coli cells. RecA and pol IV foci increase after treatment and exhibit strong colocalization. The induction of the SOS response, the appearance of RecA foci, the appearance of pol IV foci and RecA-pol IV colocalization are all dependent on RecB function. The positioning of pol IV foci likely reflects a physical interaction with the RecA* nucleoprotein filaments that has been detected previously in vitro. Our observations provide an in vivo substantiation of a direct role for pol IV in double strand break repair in cells treated with double strand break-inducing antibiotics.


Assuntos
Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , DNA Polimerase beta/ultraestrutura , Proteínas de Ligação a DNA/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/ultraestrutura , Exodesoxirribonuclease V/ultraestrutura , Recombinases Rec A/genética , Ciprofloxacino/farmacologia , Dano ao DNA/efeitos dos fármacos , DNA Polimerase beta/genética , Reparo do DNA/genética , Replicação do DNA/genética , Escherichia coli/genética , Escherichia coli/ultraestrutura , Exodesoxirribonuclease V/genética , Imagem Individual de Molécula
8.
Nucleic Acids Res ; 48(16): 9109-9123, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32729622

RESUMO

Nuclear factor erythroid 2-related factor 2 (NRF2) is a well-characterized transcription factor that protects cells against oxidative and electrophilic stresses. Emerging evidence has suggested that NRF2 protects cells against DNA damage by mechanisms other than antioxidation, yet the mechanism remains poorly understood. Here, we demonstrate that knockout of NRF2 in cells results in hypersensitivity to ionizing radiation (IR) in the presence or absence of reactive oxygen species (ROS). Under ROS scavenging conditions, induction of DNA double-strand breaks (DSBs) increases the NRF2 protein level and recruits NRF2 to DNA damage sites where it interacts with ATR, resulting in activation of the ATR-CHK1-CDC2 signaling pathway. In turn, this leads to G2 cell cycle arrest and the promotion of homologous recombination repair of DSBs, thereby preserving genome stability. The inhibition of NRF2 by brusatol increased the radiosensitivity of tumor cells in xenografts by perturbing ATR and CHK1 activation. Collectively, our results reveal a novel function of NRF2 as an ATR activator in the regulation of the cellular response to DSBs. This shift in perspective should help furnish a more complete understanding of the function of NRF2 and the DNA damage response.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Fator 2 Relacionado a NF-E2/genética , Reparo de DNA por Recombinação/genética , Células A549 , Animais , Proteína Quinase CDC2/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/radioterapia , Quinase 1 do Ponto de Checagem/genética , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/efeitos da radiação , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Técnicas de Inativação de Genes , Xenoenxertos , Humanos , Camundongos , Quassinas/farmacologia , Tolerância a Radiação/efeitos dos fármacos , Radiação Ionizante , Reparo de DNA por Recombinação/efeitos dos fármacos , Reparo de DNA por Recombinação/efeitos da radiação , Transdução de Sinais/efeitos dos fármacos
9.
Oncogene ; 39(27): 5068-5081, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32528131

RESUMO

Topoisomerase II poisons are one of the most common class of chemotherapeutics used in cancer. We and others had shown that a subset of glioblastomas, the most malignant of all primary brain tumors in adults, is responsive to TOP2 poisons. To identify genes that confer susceptibility to this drug in gliomas, we performed a genome-scale CRISPR knockout screen with etoposide. Genes involved in protein synthesis and DNA damage were implicated in etoposide susceptibility. To define potential biomarkers for TOP2 poisons, CRISPR hits were overlapped with genes whose expression correlates with susceptibility to this drug across glioma cell lines, revealing ribosomal protein subunit RPS11, 16, and 18 as putative biomarkers for response to TOP2 poisons. Loss of RPS11 led to resistance to etoposide and doxorubicin and impaired the induction of proapoptotic gene APAF1 following treatment. The expression of these ribosomal subunits was also associated with susceptibility to TOP2 poisons across cell lines from gliomas and multiple other cancers.


Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Etoposídeo/farmacologia , Glioblastoma/tratamento farmacológico , Proteínas Ribossômicas/metabolismo , Inibidores da Topoisomerase II/farmacologia , Fator Apoptótico 1 Ativador de Proteases/metabolismo , Neoplasias Encefálicas/genética , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , DNA Topoisomerases Tipo II/metabolismo , Doxorrubicina/farmacologia , Técnicas de Inativação de Genes , Glioblastoma/genética , Humanos
10.
Leukemia ; 34(11): 2992-3006, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32572188

RESUMO

Inactivating mutations in TET2 serve as an initiating genetic lesion in the transformation of hematopoietic stem and progenitor cells (HSPCs). Thus, effective therapy for this subset of patients would ideally include drugs that are selectively lethal in TET2-mutant HSPCs, at dosages that spare normal HSPCs. In this study, we tested 129 FDA-approved anticancer drugs in a tet2-deficient zebrafish model and showed that topoisomerase 1 (TOP1)-targeted drugs and PARP1 inhibitors selectively kill tet2-mutant HSPCs. We found that Tet2-deficient murine bone marrow progenitors and CRISPR-Cas9-induced TET2-mutant human AML cells were more sensitive to both classes of drugs compared with matched control cells. The mechanism underlying the selective killing of TET2-mutant blood cells by these drugs was due to aberrantly low levels of tyrosyl-DNA phosphodiesterase 1 (TDP1), an enzyme that is important for removing TOP1 cleavage complexes (TOP1cc). Low TDP1 levels yield sensitivity to TOP1-targeted drugs or PARP1 inhibitors and an inability to remove TOP1 cleavage complexes, leading to DNA double-strand breaks and cell death. The finding that TET2 mutations render HSPCs uniquely vulnerable to disruption of TOP1 and PARP1 activity may therefore represent a unique opportunity to use relatively low dosages of these drugs for the "precision therapy" of TET2-mutant myeloid malignancies.


Assuntos
DNA Topoisomerases Tipo I/metabolismo , Proteínas de Ligação a DNA/genética , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Mutações Sintéticas Letais , Inibidores da Topoisomerase I/farmacologia , Animais , Animais Geneticamente Modificados , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Genótipo , Humanos , Camundongos , Camundongos Knockout , Fenótipo , Topotecan/farmacologia , Peixe-Zebra
11.
PLoS One ; 15(6): e0234859, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32589683

RESUMO

Proteins are covalently trapped on DNA to form DNA-protein cross-links (DPCs) when cells are exposed to DNA-damaging agents. Aldehyde compounds produce common types of DPCs that contain proteins in an undisrupted DNA strand. Tyrosyl-DNA phosphodiesterase 1 (TDP1) repairs topoisomerase 1 (TOPO1) that is trapped at the 3'-end of DNA. In the present study, we examined the contribution of TDP1 to the repair of formaldehyde-induced DPCs using a reverse genetic strategy with chicken DT40 cells. The results obtained showed that cells deficient in TDP1 were sensitive to formaldehyde. The removal of formaldehyde-induced DPCs was slower in tdp1-deficient cells than in wild type cells. We also found that formaldehyde did not produce trapped TOPO1, indicating that trapped TOPO1 was not a primary cytotoxic DNA lesion that was generated by formaldehyde and repaired by TDP1. The formaldehyde treatment resulted in the accumulation of chromosomal breakages that were more prominent in tdp1-deficient cells than in wild type cells. Therefore, TDP1 plays a critical role in the repair of formaldehyde-induced DPCs that are distinct from trapped TOPO1.


Assuntos
Reparo do DNA , DNA Topoisomerases Tipo I/metabolismo , DNA/metabolismo , Formaldeído/toxicidade , Diester Fosfórico Hidrolases/metabolismo , Animais , Linhagem Celular , Galinhas , Quebra Cromossômica/efeitos dos fármacos , DNA/química , Quebras de DNA/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , DNA Topoisomerases Tipo I/química , Decitabina/toxicidade , Mitomicina/toxicidade , Diester Fosfórico Hidrolases/genética
12.
Nucleic Acids Res ; 48(12): 6654-6671, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32501506

RESUMO

DNA double-stranded breaks (DSBs) trigger human genome instability, therefore identifying what factors contribute to DSB induction is critical for our understanding of human disease etiology. Using an unbiased, genome-wide approach, we found that genomic regions with the ability to form highly stable DNA secondary structures are enriched for endogenous DSBs in human cells. Human genomic regions predicted to form non-B-form DNA induced gross chromosomal rearrangements in yeast and displayed high indel frequency in human genomes. The extent of instability in both analyses is in concordance with the structure forming ability of these regions. We also observed an enrichment of DNA secondary structure-prone sites overlapping transcription start sites (TSSs) and CCCTC-binding factor (CTCF) binding sites, and uncovered an increase in DSBs at highly stable DNA secondary structure regions, in response to etoposide, an inhibitor of topoisomerase II (TOP2) re-ligation activity. Importantly, we found that TOP2 deficiency in both yeast and human leads to a significant reduction in DSBs at structure-prone loci, and that sites of TOP2 cleavage have a greater ability to form highly stable DNA secondary structures. This study reveals a direct role for TOP2 in generating secondary structure-mediated DNA fragility, advancing our understanding of mechanisms underlying human genome instability.


Assuntos
Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , DNA Topoisomerases Tipo II/ultraestrutura , Conformação de Ácido Nucleico/efeitos dos fármacos , Sítios de Ligação/genética , Fator de Ligação a CCCTC/genética , DNA/genética , DNA/ultraestrutura , Reparo do DNA/genética , DNA Topoisomerases Tipo II/genética , Etoposídeo/farmacologia , Genoma Humano/genética , Instabilidade Genômica/genética , Humanos , Sítio de Iniciação de Transcrição/efeitos dos fármacos
13.
Nat Med ; 26(7): 1063-1069, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32483361

RESUMO

The mucosal epithelium is a common target of damage by chronic bacterial infections and the accompanying toxins, and most cancers originate from this tissue. We investigated whether colibactin, a potent genotoxin1 associated with certain strains of Escherichia coli2, creates a specific DNA-damage signature in infected human colorectal cells. Notably, the genomic contexts of colibactin-induced DNA double-strand breaks were enriched for an AT-rich hexameric sequence motif, associated with distinct DNA-shape characteristics. A survey of somatic mutations at colibactin target sites of several thousand cancer genomes revealed notable enrichment of this motif in colorectal cancers. Moreover, the exact double-strand-break loci corresponded with mutational hot spots in cancer genomes, reminiscent of a trinucleotide signature previously identified in healthy colorectal epithelial cells3. The present study provides evidence for the etiological role of colibactin in human cancer.


Assuntos
Neoplasias Colorretais/genética , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Peptídeos/farmacologia , Policetídeos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/microbiologia , Neoplasias Colorretais/patologia , Células Epiteliais/efeitos dos fármacos , Escherichia coli/patogenicidade , Humanos , Mutação/efeitos dos fármacos , Motivos de Nucleotídeos/efeitos dos fármacos
14.
Toxicology ; 440: 152441, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32433928

RESUMO

4-Aminobiphenyl (4-ABP), a well-known human carcinogen, has been shown to cause oxidative DNA damage and induce miR-630 expression in HepG2 cells treated with 18.75 µM-300 µM for 24 h. However, the underlying mechanism regarding the epigenetic regulation of miR-630 on DNA damage repair in liver cells is still not understood and needs to be investigated. In present study, our results showed that miR-630 was upregulated, resulting in mediating a decrease of DNA homologous recombination (HR) repair in L-02, HepG2 or Hep3B cells. Results from a luciferase reporting experiment showed that RAD18 and MCM8 were the potential targets of miR-630 during DNA damage induction. The downregulation of RAD18 or MCM8 by miR-630 was accompanied by inhibition of HR repair. Conversely, inhibiting miR-630 enhanced the expression of RAD18 and MCM8, and rescued HR repair. Additionally, we proved that the transcription factor CREB was related to miR-630 biogenesis in liver cells. Moreover, the levels of CREB, miR-630 expression, and double-strand breaks (DSBs) were attenuated by 5 mM N-acetyl-L-cysteine (NAC) pretreatment, indicating that reactive oxygen species (ROS)-dependent CREB-miR-630 was involved in DSB repair. These findings indicated that the ROS/CREB/-miR-630 axis plays a relevant role in the regulation of RAD18 and MCM8 in HR repair, which may facilitate our understanding of molecular mechanisms regarding the role of miR-630 downregulating DNA damage repair in liver cells.


Assuntos
Compostos de Aminobifenil/farmacologia , Proteínas de Ligação a DNA/antagonistas & inibidores , Fígado/metabolismo , MicroRNAs/metabolismo , Proteínas de Manutenção de Minicromossomo/antagonistas & inibidores , Reparo de DNA por Recombinação/efeitos dos fármacos , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Acetilcisteína/farmacologia , Linhagem Celular , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/biossíntese , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Depuradores de Radicais Livres/farmacologia , Recombinação Homóloga , Humanos , Fígado/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
15.
PLoS One ; 15(5): e0232724, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32374749

RESUMO

DNA damage in the A549 human lung cancer cell line treated with cold plasma irradiation was investigated. We confirmed that cold atmospheric plasma generated reactive oxygen and nitrogen species (RONS) in a liquid, and the intracellular RONS level was increased in plasma-irradiated cells. However, a notable decrease in cell viability was not observed 24 hours after plasma irradiation. Because RONS induce oxidative damage in cells, strand breaks and chemical modification of DNA in the cancer cells were investigated. We found that 8-oxoguanine (8-oxoG) formation as well as DNA strand breaks, which have been thoroughly investigated, were induced by plasma irradiation. In addition, up-regulation of 8-oxoG repair enzyme was observed after plasma irradiation.


Assuntos
Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Quebras de DNA de Cadeia Simples/efeitos dos fármacos , DNA Glicosilases/metabolismo , Reparo do DNA , Guanina/análogos & derivados , Gases em Plasma/farmacologia , Células A549 , Sobrevivência Celular/efeitos dos fármacos , DNA/metabolismo , Guanina/biossíntese , Humanos , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Regulação para Cima
16.
Mol Carcinog ; 59(6): 618-628, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32189406

RESUMO

Targeting DNA repair with small-molecule inhibitors is an attractive strategy for cancer therapy. Majority of DNA double-strand breaks in mammalian cells are repaired through nonhomologous end-joining (NHEJ). It has been shown that small-molecule inhibitors of NHEJ can block efficient repair inside cancer cells, leading to cell death. Previously, we have reported that SCR7, an inhibitor of NHEJ can induce tumor regression in mice. Later studies have shown that different forms of SCR7 can inhibit DNA end-joining in Ligase IV-dependent manner. Recently, we have derivatized SCR7 by introducing spiro ring into core structure. Here, we report the identification of a novel inhibitor of NHEJ, named SCR130 with 20-fold higher efficacy in inducing cytotoxicity in cancer cell lines. SCR130 inhibited DNA end-joining catalyzed by rat tissue extract. Specificity analysis revealed that while SCR130 was specific to Ligase IV, it showed minimal or no effect on Ligase III and Ligase I mediated joining. Importantly, SCR130 exhibited the least cytotoxicity in Ligase IV-null cell line as compared with wild type, confirming Ligase IV-specificity. Furthermore, we demonstrate that SCR130 can potentiate the effect of radiation in cancer cells when used in combination with γ-radiation. Various cellular assays in conjunction with Western blot analysis revealed that treatment with SCR130 led to loss of mitochondrial membrane potential leading to cell death by activating both intrinsic and extrinsic pathways of apoptosis. Thus, we describe a novel inhibitor of NHEJ with higher efficacy and may have the potential to be developed as cancer therapeutic.


Assuntos
Antineoplásicos/farmacologia , Morte Celular , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Reparo do DNA por Junção de Extremidades/efeitos dos fármacos , DNA Ligase Dependente de ATP/antagonistas & inibidores , Pirimidinas/farmacologia , Bases de Schiff/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Células HeLa , Humanos , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Ratos
17.
PLoS Biol ; 18(3): e3000666, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32203529

RESUMO

Ataxia-telangiectasia mutated (ATM) is an apical kinase of the DNA damage response following DNA double-strand breaks (DSBs); however, the mechanisms of ATM activation are not completely understood. Long noncoding RNAs (lncRNAs) are a class of regulatory molecules whose significant roles in DNA damage response have started to emerge. However, how lncRNA regulates ATM activity remains unknown. Here, we identify an inhibitor of ATM activation, lncRNA HITT (HIF-1α inhibitor at translation level). Mechanistically, HITT directly interacts with ATM at the HEAT repeat domain, blocking MRE11-RAD50-NBS1 complex-dependent ATM recruitment, leading to restrained homologous recombination repair and enhanced chemosensitization. Following DSBs, HITT is elevated mainly by the activation of Early Growth Response 1 (EGR1), resulting in retarded and restricted ATM activation. A reverse association between HITT and ATM activity was also detected in human colon cancer tissues. Furthermore, HITTs sensitize DNA damaging agent-induced cell death both in vitro and in vivo. These findings connect lncRNA directly to ATM activity regulation and reveal potential roles for HITT in sensitizing cancers to genotoxic treatment.


Assuntos
Antineoplásicos/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Dano ao DNA , RNA Longo não Codificante/metabolismo , Reparo de DNA por Recombinação/genética , Hidrolases Anidrido Ácido/metabolismo , Animais , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/genética , Sítios de Ligação , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Proteína 1 de Resposta de Crescimento Precoce/genética , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Células HCT116 , Células HeLa , Humanos , Proteína Homóloga a MRE11/metabolismo , Camundongos , Camundongos Nus , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilação , Ligação Proteica , RNA Longo não Codificante/genética , Transcrição Genética/efeitos dos fármacos
18.
Mutat Res ; 849: 503144, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-32087851

RESUMO

Bioflavonoids have a similar chemical structure to etoposide, the well-characterized topoisomerase II (Top2) poison, and evidence shows that they also induce DNA double-strand breaks (DSBs) and promote genome rearrangements. The purpose of this study was to determine the kinetics of bioflavonoid-induced DSB appearance and repair, and their dependence on Top2. Cells were exposed to bioflavonoids individually or in combination in the presence or absence of the Top2 catalytic inhibitor dexrazoxane. The kinetics of appearance and repair of γH2AX foci were measured. In addition, the frequency of resultant MLL-AF9 breakpoint cluster region translocations was determined. Bioflavonoids readily induced the appearance of γH2AX foci, but bioflavonoid combinations did not act additively or synergistically to promote DSBs. Myricetin-induced DSBs were mostly reduced by dexrazoxane, while genistein and quercetin-induced DSBs were only partially, but significantly, reduced. By contrast, luteolin and kaempferol-induced DSBs increased with dexrazoxane pre-treatment. Sensitivity to Top2 inhibition correlated with a significant reduction of bioflavonoid-induced MLL-AF9 translocations. These data demonstrate that myricetin, genistein, and quercetin act most similar to etoposide although with varying Top2-dependence. By contrast, luteolin and kaempferol have distinct kinetics that are mostly Top2-independent. These findings have implications for understanding the mechanisms of bioflavonoid activity and the potential of individual bioflavonoids to promote chromosomal translocations. Further, they provide direct evidence that specific Top2 inhibitors or targeted drugs could be developed that possess less leukemic potential or suppress chromosomal translocations associated with therapy-related and infant leukemias.


Assuntos
Reparo do DNA/efeitos dos fármacos , Flavonoides/toxicidade , Genisteína/toxicidade , Quempferóis/toxicidade , Luteolina/toxicidade , Quercetina/toxicidade , Animais , Linhagem Celular , Pontos de Quebra do Cromossomo/efeitos dos fármacos , Cromossomos de Mamíferos/efeitos dos fármacos , DNA/química , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , Dexrazoxano/farmacologia , Etoposídeo/toxicidade , Histonas/genética , Histonas/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Embrionárias Murinas/ultraestrutura , Inibidores da Topoisomerase II/farmacologia , Translocação Genética/efeitos dos fármacos
19.
Mutat Res ; 849: 503141, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-32087855

RESUMO

The inherent capacity of individuals to efficiently repair ionizing radiation induced DNA double strand breaks (DSBs) may be inherited, however, it is influenced by several epigenetic and environmental factors. A pilot study tested whether chronic low dose natural radiation exposure influences the rejoining of initial DNA DSBs induced by a 2 Gy γ-irradiation in 22 individuals from high (>1.5 mGy/year) and normal (≤1.5 mGy/year) level natural radiation areas (H&NLNRA) of Kerala. Rejoining of DSBs (during 1 h at 37 °C, immediately after irradiation) was evaluated at the chromosome level in the presence and absence of wortmannin (a potent inhibitor of DSB repair in normal human cells) using a cell fusion-induced premature chromosome condensation (PCC) assay. The PCC assay quantitates DSBs in the form of excess chromosome fragments in human G0 lymphocytes without the requirement for cell division. A quantitative difference was observed in the early rejoining of DNA DSBs between individuals from HLNRA and NLNRA, with HLNRA individuals showing a higher (P = 0.05) mean initial repair ratio. The results indicate an influence of chronic low dose natural radiation on initial DNA DSB repair in inhabitants of HLNRA of the Kerala coast.


Assuntos
Radiação de Fundo/efeitos adversos , Bioensaio , Reparo do DNA/efeitos dos fármacos , Raios gama/efeitos adversos , Linfócitos/efeitos da radiação , Adulto , Animais , Células CHO , Fusão Celular , Cromossomos Humanos/efeitos dos fármacos , Cromossomos Humanos/efeitos da radiação , Cricetulus , DNA/genética , DNA/metabolismo , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Humanos , Linfócitos/citologia , Linfócitos/efeitos dos fármacos , Masculino , Projetos Piloto , Cultura Primária de Células , Doses de Radiação , Wortmanina/farmacologia
20.
Cancer Cell ; 37(2): 157-167.e6, 2020 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-32004442

RESUMO

In response to DNA double-strand breaks, MAD2L2-containing shieldin complex plays a critical role in the choice between homologous recombination (HR) and non-homologous end-joining (NHEJ)-mediated repair. Here we show that EZH2 inhibition upregulates MAD2L2 and sensitizes HR-proficient epithelial ovarian cancer (EOC) to poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitor in a CARM1-dependent manner. CARM1 promotes MAD2L2 silencing by driving the switch from the SWI/SNF complex to EZH2 through methylating the BAF155 subunit of the SWI/SNF complex on the MAD2L2 promoter. EZH2 inhibition upregulates MAD2L2 to decrease DNA end resection, which increases NHEJ and chromosomal abnormalities, ultimately causing mitotic catastrophe in PARP inhibitor treated HR-proficient cells. Significantly, EZH2 inhibitor sensitizes CARM1-high, but not CARM-low, EOCs to PARP inhibitors in both orthotopic and patient-derived xenografts.


Assuntos
Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Recombinação Homóloga/efeitos dos fármacos , Neoplasias Ovarianas/tratamento farmacológico , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Antineoplásicos/uso terapêutico , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Reparo do DNA por Junção de Extremidades/efeitos dos fármacos , Inibidores Enzimáticos/uso terapêutico , Feminino , Humanos , Neoplasias Ovarianas/genética , Proteína-Arginina N-Metiltransferases/efeitos dos fármacos , Reparo de DNA por Recombinação/efeitos dos fármacos
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