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1.
Proc Natl Acad Sci U S A ; 117(37): 22953-22961, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32868446

RESUMO

The DNA-dependent protein kinase (DNA-PK), which is composed of the KU heterodimer and the large catalytic subunit (DNA-PKcs), is a classical nonhomologous end-joining (cNHEJ) factor. Naïve B cells undergo class switch recombination (CSR) to generate antibodies with different isotypes by joining two DNA double-strand breaks at different switching regions via the cNHEJ pathway. DNA-PK and the cNHEJ pathway play important roles in the DNA repair phase of CSR. To initiate cNHEJ, KU binds to DNA ends and recruits and activates DNA-PK. Activated DNA-PK phosphorylates DNA-PKcs at the S2056 and T2609 clusters. Loss of T2609 cluster phosphorylation increases radiation sensitivity but whether T2609 phosphorylation has a role in physiological DNA repair remains elusive. Using the DNA-PKcs 5A mouse model carrying alanine substitutions at the T2609 cluster, here we show that loss of T2609 phosphorylation of DNA-PKcs does not affect the CSR efficiency. Yet, the CSR junctions recovered from DNA-PKcs 5A/5A B cells reveal increased chromosomal translocations, extensive use of distal switch regions (consistent with end resection), and preferential usage of microhomology-all signs of the alternative end-joining pathway. Thus, these results uncover a role of DNA-PKcs T2609 phosphorylation in promoting cNHEJ repair pathway choice during CSR.


Assuntos
Proteína Quinase Ativada por DNA/genética , Proteína Quinase Ativada por DNA/metabolismo , Switching de Imunoglobulina/genética , Animais , Linfócitos B/imunologia , Reparo do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Feminino , Rearranjo Gênico , Humanos , Switching de Imunoglobulina/fisiologia , Região de Troca de Imunoglobulinas/genética , Imunoglobulinas/genética , Autoantígeno Ku/metabolismo , Masculino , Camundongos , Camundongos da Linhagem 129 , Fosforilação , Recombinação Genética/genética , Translocação Genética
2.
Nucleic Acids Res ; 48(17): 9710-9723, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32890395

RESUMO

Two DNA repair pathways operate at DNA double strand breaks (DSBs): non-homologous end-joining (NHEJ), that requires two adjacent DNA ends for ligation, and homologous recombination (HR), that resects one DNA strand for invasion of a homologous duplex. Faithful repair of replicative single-ended DSBs (seDSBs) is mediated by HR, due to the lack of a second DNA end for end-joining. ATM stimulates resection at such breaks through multiple mechanisms including CtIP phosphorylation, which also promotes removal of the DNA-ends sensor and NHEJ protein Ku. Here, using a new method for imaging the recruitment of the Ku partner DNA-PKcs at DSBs, we uncover an unanticipated role of ATM in removing DNA-PKcs from seDSBs in human cells. Phosphorylation of DNA-PKcs on the ABCDE cluster is necessary not only for DNA-PKcs clearance but also for the subsequent MRE11/CtIP-dependent release of Ku from these breaks. We propose that at seDSBs, ATM activity is necessary for the release of both Ku and DNA-PKcs components of the NHEJ apparatus, and thereby prevents subsequent aberrant interactions between seDSBs accompanied by DNA-PKcs autophosphorylation and detrimental commitment to Lig4-dependent end-joining.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades/fisiologia , Proteína Quinase Ativada por DNA/metabolismo , Autoantígeno Ku/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/genética , Camptotecina/farmacologia , Linhagem Celular , Reparo do DNA por Junção de Extremidades/efeitos dos fármacos , DNA Ligase Dependente de ATP/genética , DNA Ligase Dependente de ATP/metabolismo , DNA de Cadeia Simples , Proteína Quinase Ativada por DNA/genética , Humanos , Autoantígeno Ku/genética , Proteína Homóloga a MRE11/genética , Proteína Homóloga a MRE11/metabolismo , Fosforilação , Inibidores da Topoisomerase I/farmacologia
3.
PLoS One ; 15(8): e0228002, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32764831

RESUMO

Irinotecan specifically targets topoisomerase I (topoI), and is used to treat various solid tumors, but only 13-32% of patients respond to the therapy. Now, it is understood that the rapid rate of topoI degradation in response to irinotecan causes irinotecan resistance. We have published that the deregulated DNA-PKcs kinase cascade ensures rapid degradation of topoI and is at the core of the drug resistance mechanism of topoI inhibitors, including irinotecan. We also identified CTD small phosphatase 1 (CTDSP1) (a nuclear phosphatase) as a primary upstream regulator of DNA-PKcs in response to topoI inhibitors. Previous reports showed that rabeprazole, a proton pump inhibitor (PPI) inhibits CTDSP1 activity. The purpose of this study was to confirm the effects of rabeprazole on CTDSP1 activity and its impact on irinotecan-based therapy in colon cancer. Using differentially expressing CTDSP1 cells, we demonstrated that CTDSP1 contributes to the irinotecan sensitivity by preventing topoI degradation. Retrospective analysis of patients receiving irinotecan with or without rabeprazole has shown the effects of CTDSP1 on irinotecan response. These results indicate that CTDSP1 promotes sensitivity to irinotecan and rabeprazole prevents this effect, resulting in drug resistance. To ensure the best chance at effective treatment, rabeprazole may not be a suitable PPI for cancer patients treated with irinotecan.


Assuntos
Neoplasias Colorretais/metabolismo , DNA Topoisomerases Tipo I/metabolismo , Rabeprazol/metabolismo , Linhagem Celular Tumoral , Neoplasias do Colo/metabolismo , Neoplasias Colorretais/fisiopatologia , DNA , DNA Topoisomerases Tipo I/fisiologia , Proteína Quinase Ativada por DNA/metabolismo , Resistência a Medicamentos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/fisiologia , Humanos , Irinotecano/metabolismo , Irinotecano/farmacologia , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/metabolismo , Inibidores da Bomba de Prótons/farmacologia , Rabeprazol/farmacologia , Estudos Retrospectivos , Inibidores da Topoisomerase I/farmacologia
4.
Oncogene ; 39(34): 5633-5648, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32661323

RESUMO

Cervical cancer (CC) remains highest in the mortality of female reproductive system cancers, while cisplatin (CDDP) resistance is the one of main reasons for the lethality. Preceding evidence has supported that karyopherins are associated with chemoresistance. In this study, we simultaneously compared CDDP-incomplete responders with CDDP-complete responders of CC patients and CDDP-insensitive CC cell lines with CDDP-sensitive group. We finally identified that DNA-PKcs (PRKDC) was related to CDDP sensitivity after overlapping in CC sample tissues and CC cell lines. Further functional assay revealed that targeting PRKDC by shRNA and NU7026 (specific PRKDC inhibitor) could enhance CDDP sensitivity in vitro and in vivo, which was mediated by impairing DNA damage repair pathway in CC. Mechanistically, we found that PRKDC was transcriptionally upregulated by CCAAT/enhancer-binding protein delta (CEBPD), while intriguingly, CDDP treatment strengthened the transcriptional activity of CEBPD to PRKDC. We further disclosed that Importin 4 (IPO4) augmented the nuclear translocation of CEBPD through nuclear localization signals (NLS) to activate PRKDC-mediated DNA damage repair in response to CDDP. Moreover, we demonstrated that IPO4 and CEBPD knockdown improved CDDP-induced cytotoxicity in vitro and in vivo. Together, we shed the novel insight into the role of IPO4 in chemosensitivity and provide a clinical translational potential to enhance CC chemosensitivity since the IPO4-CEBPD-PRKDC axis is actionable via NU7026 (PRKDC inhibitor) or targeting IPO4 in combination with CDDP.


Assuntos
Proteína delta de Ligação ao Facilitador CCAAT/genética , Cisplatino/farmacologia , Reparo do DNA/efeitos dos fármacos , Proteína Quinase Ativada por DNA/genética , Proteínas de Membrana Transportadoras/genética , Neoplasias do Colo do Útero/genética , Transporte Ativo do Núcleo Celular/genética , Animais , Antineoplásicos/farmacologia , Proteína delta de Ligação ao Facilitador CCAAT/metabolismo , Linhagem Celular Tumoral , Cromonas/farmacologia , Dano ao DNA , Reparo do DNA/genética , Proteína Quinase Ativada por DNA/antagonistas & inibidores , Proteína Quinase Ativada por DNA/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Células HeLa , Humanos , Proteínas de Membrana Transportadoras/metabolismo , Camundongos Nus , Morfolinas/farmacologia , Interferência de RNA , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/terapia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
5.
Mol Cell Biol ; 40(13)2020 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-32284347

RESUMO

The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) has well-established roles in DNA double-strand break repair, and recently, nonrepair functions have also been reported. To better understand its cellular functions, we deleted DNA-PKcs from HeLa and A549 cells using CRISPR/Cas9. The resulting cells were radiation sensitive, had reduced expression of ataxia-telangiectasia mutated (ATM), and exhibited multiple mitotic defects. Mechanistically, nocodazole-induced upregulation of cyclin B1, anillin, and securin was decreased in DNA-PKcs-deficient cells, as were phosphorylation of Aurora A on threonine 288, phosphorylation of Polo-like kinase 1 (PLK1) on threonine 210, and phosphorylation of targeting protein for Xenopus Klp2 (TPX2) on serine 121. Moreover, reduced nocodazole-induced expression of anillin, securin, and cyclin B1 and phosphorylation of PLK1, Aurora A, and TPX2 were rescued by inhibition of the anaphase-promoting complex/cyclosome (APC/C) by proTAME, which prevents binding of the APC/C-activating proteins Cdc20 and Cdh1 to the APC/C. Altogether, our studies suggest that loss of DNA-PKcs prevents inactivation of the APC/C in nocodazole-treated cells.


Assuntos
Ciclossomo-Complexo Promotor de Anáfase/antagonistas & inibidores , Antineoplásicos/farmacologia , Proteínas Contráteis/genética , Proteína Quinase Ativada por DNA/genética , Inibidores Enzimáticos/farmacologia , Nocodazol/farmacologia , Células A549 , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Animais , Aurora Quinase A/metabolismo , Sistemas CRISPR-Cas , Carbamatos/farmacologia , Proteínas de Ciclo Celular/metabolismo , Proteínas Contráteis/metabolismo , Proteína Quinase Ativada por DNA/metabolismo , Diaminas/farmacologia , Regulação para Baixo/efeitos dos fármacos , Células HeLa , Humanos , Fosforilação/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Regulação para Cima/efeitos dos fármacos , Xenopus
6.
Nat Commun ; 11(1): 1270, 2020 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-32152280

RESUMO

Prolonged cell survival occurs through the expression of specific protein isoforms generated by alternate splicing of mRNA precursors in cancer cells. How alternate splicing regulates tumor development and resistance to targeted therapies in cancer remain poorly understood. Here we show that RNF113A, whose loss-of-function causes the X-linked trichothiodystrophy, is overexpressed in lung cancer and protects from Cisplatin-dependent cell death. RNF113A is a RNA-binding protein which regulates the splicing of multiple candidates involved in cell survival. RNF113A deficiency triggers cell death upon DNA damage through multiple mechanisms, including apoptosis via the destabilization of the prosurvival protein MCL-1, ferroptosis due to enhanced SAT1 expression, and increased production of ROS due to altered Noxa1 expression. RNF113A deficiency circumvents the resistance to Cisplatin and to BCL-2 inhibitors through the destabilization of MCL-1, which thus defines spliceosome inhibitors as a therapeutic approach to treat tumors showing acquired resistance to specific drugs due to MCL-1 stabilization.


Assuntos
Proteínas de Ligação a DNA/genética , Genes Ligados ao Cromossomo X , Spliceossomos/metabolismo , Síndromes de Tricotiodistrofia/genética , Células A549 , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/patologia , Processamento Alternativo/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Sobrevivência Celular/genética , Cisplatino/farmacologia , Citoproteção/efeitos dos fármacos , Dano ao DNA/genética , Proteína Quinase Ativada por DNA/metabolismo , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Íntrons/genética , Camundongos Endogâmicos NOD , Camundongos SCID , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteínas de Neoplasias/metabolismo , Fosforilação/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Subunidades Proteicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo
7.
Cell Mol Life Sci ; 77(20): 4133-4142, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31932854

RESUMO

The transcription factors of the nuclear factor κB (NF-κB) family play a pivotal role in the cellular response to DNA damage. Genotoxic stress-induced activation of NF-κB differs from the classical canonical pathway by shuttling of the NF-κB Essential Modifier (IKKγ/NEMO) subunit through the nucleus. Here, we show that DNA-dependent protein kinase (DNA-PK), an enzyme involved in DNA double-strand break (DSB) repair, triggers the phosphorylation of NEMO by genotoxic stress, thereby enabling shuttling of NEMO through the nucleus with subsequent NF-κB activation. We identified serine 43 of NEMO as a DNA-PK phosphorylation site and point mutation of this serine to alanine led to a complete block of NF-κB activation by ionizing radiation (IR). Blockade of DNA-PK by a specific shRNA or by DNA-PKcs-deficient cells abrogated NEMO entry into the nucleus, as well. Accordingly, SUMOylation of NEMO, a prerequisite of nuclear NEMO, was abolished. Based on these observations, we propose a model in which NEMO phosphorylation by DNA-PK provides the first step in the nucleocytoplasmic trafficking of NEMO.


Assuntos
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteína Quinase Ativada por DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Quinase I-kappa B/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , NF-kappa B/metabolismo , Alanina/metabolismo , Animais , Dano ao DNA/fisiologia , Células HEK293 , Humanos , Camundongos , Células NIH 3T3 , Fosforilação/fisiologia , Serina/metabolismo , Transdução de Sinais/fisiologia
8.
Sci Adv ; 6(2): eaay0922, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31934630

RESUMO

The repair of DNA double-strand breaks occurs through nonhomologous end joining or homologous recombination in vertebrate cells-a choice that is thought to be decided by a competition between DNA-dependent protein kinase (DNA-PK) and the Mre11/Rad50/Nbs1 (MRN) complex but is not well understood. Using ensemble biochemistry and single-molecule approaches, here, we show that the MRN complex is dependent on DNA-PK and phosphorylated CtIP to perform efficient processing and resection of DNA ends in physiological conditions, thus eliminating the competition model. Endonucleolytic removal of DNA-PK-bound DNA ends is also observed at double-strand break sites in human cells. The involvement of DNA-PK in MRN-mediated end processing promotes an efficient and sequential transition from nonhomologous end joining to homologous recombination by facilitating DNA-PK removal.


Assuntos
Proteína Quinase Ativada por DNA/metabolismo , DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Complexos Multiproteicos/metabolismo , Linhagem Celular , Humanos , Imagem Individual de Molécula
9.
Cell Mol Biol Lett ; 25: 2, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31988640

RESUMO

This review focuses on DNA-dependent protein kinase (DNA-PK), which is the key regulator of canonical non-homologous end-joining (NHEJ), the predominant mechanism of DNA double-strand break (DSB) repair in mammals. DNA-PK consists of the DNA-binding Ku70/80 heterodimer and the catalytic subunit DNA-PKcs. They assemble at DNA ends, forming the active DNA-PK complex, which initiates NHEJ-mediated DSB repair. Paradoxically, both Ku and DNA-PKcs are associated with telomeres, and they play crucial roles in protecting the telomere against fusions. Herein, we discuss possible mechanisms and contributions of Ku and DNA-PKcs in telomere regulation.


Assuntos
Proteína Quinase Ativada por DNA/metabolismo , Ribonucleoproteína Nuclear Heterogênea A1/metabolismo , Telomerase/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Telômero/metabolismo , Animais , Reparo do DNA por Junção de Extremidades/genética , DNA Topoisomerases Tipo II/metabolismo , Proteína Quinase Ativada por DNA/química , Proteína Quinase Ativada por DNA/genética , Humanos , Autoantígeno Ku/metabolismo , Telômero/genética
10.
Cell Rep ; 30(4): 1208-1222.e9, 2020 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-31995759

RESUMO

Protein arginine methyltransferase 1 (PRMT1) is overexpressed in various human cancers and linked to poor response to chemotherapy. Various PRMT1 inhibitors are currently under development; yet, we do not fully understand the mechanisms underpinning PRMT1 involvement in tumorigenesis and chemoresistance. Using mass spectrometry-based proteomics, we identified PRMT1 as regulator of arginine methylation in ovarian cancer cells treated with cisplatin. We showed that DNA-dependent protein kinase (DNA-PK) binds to and phosphorylates PRMT1 in response to cisplatin, inducing its chromatin recruitment and redirecting its enzymatic activity toward Arg3 of histone H4 (H4R3). On chromatin, the DNA-PK/PRMT1 axis induces senescence-associated secretory phenotype through H4R3me2a deposition at pro-inflammatory gene promoters. Finally, PRMT1 inhibition reduces the clonogenic growth of cancer cells exposed to low doses of cisplatin, sensitizing them to apoptosis. While unravelling the role of PRMT1 in response to genotoxic agents, our findings indicate the possibility of targeting PRMT1 to overcome chemoresistance in cancer.


Assuntos
Antineoplásicos/farmacologia , Senescência Celular/efeitos dos fármacos , Cromatina/metabolismo , Cisplatino/farmacologia , Proteína Quinase Ativada por DNA/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Repressoras/metabolismo , Arginina/metabolismo , Senescência Celular/genética , Imunoprecipitação da Cromatina , Cromatografia Líquida , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/efeitos da radiação , Proteína Quinase Ativada por DNA/genética , Células HEK293 , Células HeLa , Histonas/metabolismo , Humanos , Espectrometria de Massas , Metilação , NF-kappa B/metabolismo , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Proteína-Arginina N-Metiltransferases/genética , Proteoma/química , Proteoma/metabolismo , RNA-Seq , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Espectrometria de Massas em Tandem
11.
Basic Res Cardiol ; 115(2): 11, 2020 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-31919590

RESUMO

DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is a novel inducer to promote mitochondrial apoptosis and suppress tumor growth in a variety of cells although its role in cardiovascular diseases remains obscure. This study was designed to examine the role of DNA-PKcs in cardiac ischemia reperfusion (IR) injury and mitochondrial damage. Cardiomyocyte-specific DNA-PKcs knockout (DNA-PKcsCKO) mice were subjected to IR prior to assessment of myocardial function and mitochondrial apoptosis. Our data revealed that IR challenge, hypoxia-reoxygenation (HR) or H2O2-activated DNA-PKcs through post-transcriptional phosphorylation in murine hearts or cardiomyocytes. Mice deficient in DNA-PKcs in cardiomyocytes were protected against cardiomyocyte death, infarct area expansion and cardiac dysfunction. DNA-PKcs ablation countered IR- or HR-induced oxidative stress, mPTP opening, mitochondrial fission, mitophagy failure and Bax-mediated mitochondrial apoptosis, possibly through suppression of Bax inhibitor-1 (BI-1) activity. A direct association between DNA-PKcs and BI-1 was noted where DNA-PKcs had little effect on BI-1 transcription but interacted with BI-1 to promote its degradation. Loss of DNA-PKcs stabilized BI-1, thus offering resistance of mitochondria and cardiomyocytes against IR insult. Moreover, DNA-PKcs ablation-induced beneficial cardioprotection against IR injury was mitigated by concurrent knockout of BI-1. Double deletion of DNA-PKcs and BI-1 failed to exert protection against global IR injury and mitochondrial damage, confirming a permissive role of BI-1 in DNA-PKcs deletion-elicited cardioprotection against IR injury. DNA-PKcs serves as a novel causative factor for mitochondrial damage via suppression of BI-1, en route to the onset and development of cardiac IR injury.


Assuntos
Apoptose , Proteína Quinase Ativada por DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias Cardíacas/enzimologia , Infarto do Miocárdio/enzimologia , Traumatismo por Reperfusão Miocárdica/enzimologia , Miócitos Cardíacos/enzimologia , Animais , Células Cultivadas , Proteína Quinase Ativada por DNA/deficiência , Proteína Quinase Ativada por DNA/genética , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Homeostase , Humanos , Masculino , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias Cardíacas/patologia , Infarto do Miocárdio/genética , Infarto do Miocárdio/patologia , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/patologia , Miócitos Cardíacos/patologia , Estabilidade Proteica , Proteólise , Transdução de Sinais
12.
J Clin Invest ; 130(1): 258-271, 2020 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-31581151

RESUMO

Potentiating radiotherapy and chemotherapy by inhibiting DNA damage repair is proposed as a therapeutic strategy to improve outcomes for patients with solid tumors. However, this approach risks enhancing normal tissue toxicity as much as tumor toxicity, thereby limiting its translational impact. Using NU5455, a newly identified highly selective oral inhibitor of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) activity, we found that it was indeed possible to preferentially augment the effect of targeted radiotherapy on human orthotopic lung tumors without influencing acute DNA damage or a late radiation-induced toxicity (fibrosis) to normal mouse lung. Furthermore, while NU5455 administration increased both the efficacy and the toxicity of a parenterally administered topoisomerase inhibitor, it enhanced the activity of doxorubicin released locally in liver tumor xenografts without inducing any adverse effect. This strategy is particularly relevant to hepatocellular cancer, which is treated clinically with localized drug-eluting beads and for which DNA-PKcs activity is reported to confer resistance to treatment. We conclude that transient pharmacological inhibition of DNA-PKcs activity is effective and tolerable when combined with localized DNA-damaging therapies and thus has promising clinical potential.


Assuntos
Carcinoma Hepatocelular , Proteína Quinase Ativada por DNA/antagonistas & inibidores , Neoplasias Hepáticas Experimentais , Proteínas de Neoplasias/antagonistas & inibidores , Inibidores de Proteínas Quinases , Animais , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/enzimologia , Carcinoma Hepatocelular/patologia , Proteína Quinase Ativada por DNA/metabolismo , Doxorrubicina/farmacologia , Humanos , Neoplasias Hepáticas Experimentais/tratamento farmacológico , Neoplasias Hepáticas Experimentais/enzimologia , Neoplasias Hepáticas Experimentais/patologia , Células MCF-7 , Camundongos , Proteínas de Neoplasias/metabolismo , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Biochem Biophys Res Commun ; 521(3): 668-673, 2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31679687

RESUMO

BACKGROUND: Presence of unperfused regions containing cells under hypoxia and nutrient starvation; contributes to radioresistance in solid human tumors. We have previously reported that cultured cells; under nutrient starvation show resistance to ionizing radiation compare with cells under normal; condition, and that nutrient starvation increases ATM activity, which causes cellular resistance to; ionizing radiation (Murata et al., BBRC2018). For further investigation of molecular mechanisms; underlying radioresistance of cells under nutrient starvation, effects of nutrient starvation on activity; of DNA-PKcs have been investigated because both DNA-PKcs and ATM belong to the PIKK family; and are required for DNA DSBs repair. In addition to DNA-PKcs, effects of nutrient starvation on; activities of FoxO3a and its regulators Akt, MST1 and AMPK have been investigated because FoxO3a; mediates cellular responses to stress and is activated under nutrient starvation. METHODS: A human glioblastoma cell line, T98G was used to examine the effects of nutrient starvation on activities and expression of DNA-PKcs, Akt, MST1, FoxO3a, NDR1, and AMPK. To elucidate; signal transduction pathways for FoxO3a activation under nutrient starvation, we examined effects of; specific inhibitors or siRNA for DNA-PKcs or Akt on activities and expression of MST1, FoxO3, NDR1, andAMPK. RESULTS: Under nutrient starvation, phosphorylations of DNA-PKcs at Ser2056, Akt at Ser473, MST at Thr183, FoxO3a at Ser413, NDR1 at Ser281 and Thr282, and AMPK at Thr172 were increased, which suggests their activation. Nutrient starvation did not affect expression of DNA-PKcs, Akt, MST1, or NDR1, with decreased expression of FoxO3a and increased expression of AMPK. Inhibition; of DNA-PK suppressed phosphorylation of Akt under nutrient starvation. Inhibition of DNA-PK or; Akt suppressed phosphorylations of MST1, FoxO3a, and NDR1 under nutrient starvation, which; suggests DNA-PKcs and Akt activate MST1, FoxO3a, and NDR1. Inhibition of DNA-PK did not; suppress phosphorylation ofAMPK under nutrient starvation. CONCLUSION: Our data suggest that DN-PKcs is activated under nutrient starvation and activates AktMST1, FoxO3a, and NDR1.


Assuntos
Proteína Quinase Ativada por DNA/metabolismo , Ativação Enzimática , Proteína Forkhead Box O3/metabolismo , Glioblastoma/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Linhagem Celular Tumoral , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Nutrientes/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Inanição/metabolismo
14.
Chem Biol Interact ; 315: 108898, 2020 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-31715134

RESUMO

Usnic acid, a dibenzofuran derivative found in many lichen species, is reported to have anticancer activity against human gastric cancer. We investigated the molecular alterations associated with anticancer effects of usnic acid against human gastric adenocarcinoma AGS and gastric carcinoma SNU-1 cells. Usnic acid (10-25 µM) treatment to these cells caused a significant increase in mitochondrial membrane depolarization and apoptotic cells. Apoptosis induction was accompanied by an increase in the ratio of Bax:Bcl-2 expression and cleaved-PARP. Usnic acid increased the comet tail length and tail DNA in alkaline comet assay indicating DNA double-strand breaks which was also evidenced by an increase in γH2A.X (Ser139) phosphorylation. The expression of DNA damage response proteins including DNA-PKcs, pATM (Ser1981), Chk-2 and p53 were increased. Further, N-acetyl cysteine, a known reactive oxygen species (ROS) scavenger, reversed the effects of usnic acid on expression of DNA damage response proteins and γH2A.X (Ser139) phosphorylation. This reversal was also observed in comet assay in a time and dose-dependent manner suggesting that usnic acid-induced DNA damage was caused by ROS. In addition, the non-toxic concentrations (1-10 µM) of usnic acid inhibited colony forming potential of AGS cells indicating its anti-proliferation activity. More importantly, the concentration of usnic acid that caused significant death in gastric cancer cells, did not show any considerable toxicity to normal human embryonic kidney HEK293 cells, human keratinocyte HaCaT cells and mouse primary gastric cells. Collectively, these results for the first time demonstrated the selective apoptotic effect of usnic acid (10-25 µM) through ROS generation and DNA damage on human gastric cancer cells accompanied with upregulation of γH2A.X (Ser139) phosphorylation, DNA-PKcs and p53.


Assuntos
Apoptose/efeitos dos fármacos , Benzofuranos/farmacologia , Dano ao DNA/efeitos dos fármacos , Proteína Quinase Ativada por DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Histonas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Neoplasias Gástricas/tratamento farmacológico , Animais , Linhagem Celular , Linhagem Celular Tumoral , Feminino , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação/efeitos dos fármacos , Neoplasias Gástricas/metabolismo , Regulação para Cima/efeitos dos fármacos
15.
Biochem Biophys Res Commun ; 522(3): 639-646, 2020 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-31785810

RESUMO

Uveal melanoma (UM) is the most common primary intraocular tumor in adults. Despite of important progress in the local therapy, high radioresistance in primary tumor and chemoresistance in metastatic disease are the major obstacles for UM therapy. Therefore, strategies to overcome resistance to radiation or chemotherapy in UM are urgently needed. In this study, we found that phosphorylation of DNA-PKcs, which is the key factor of non-homologous end joining (NHEJ) pathway, was remarkably overexpressed in ionizing radiation (IR)- and Selumetinib resistant UM cells. Increased amount of NHEJ events were also observed in resistant UM cells. Inhibition of DNA-PKcs by NU7441 significantly impaired DNA repair and re-sensitized resistant UM cells to radiation and Selumetinib both in vitro and in vivo. The results demonstrate increased DNA double strand break repair as a mechanism of resistance to ionizing radiation and Selumetinib, and identify DNA-PKcs as a promising target for radio-and chemotherapy in UM patients.


Assuntos
Benzimidazóis/farmacologia , Cromonas/farmacologia , Proteína Quinase Ativada por DNA/antagonistas & inibidores , Melanoma/tratamento farmacológico , Melanoma/radioterapia , Morfolinas/farmacologia , Neoplasias Uveais/tratamento farmacológico , Neoplasias Uveais/radioterapia , Animais , Benzimidazóis/uso terapêutico , Linhagem Celular Tumoral , Cromonas/uso terapêutico , Proteína Quinase Ativada por DNA/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Feminino , Humanos , Melanoma/metabolismo , Camundongos Endogâmicos BALB C , Morfolinas/uso terapêutico , Fosforilação/efeitos dos fármacos , Radiossensibilizantes/farmacologia , Radiossensibilizantes/uso terapêutico , Neoplasias Uveais/metabolismo
16.
Mol Cell ; 77(5): 1080-1091.e8, 2020 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-31862156

RESUMO

Enzymatic processing of DNA underlies all DNA repair, yet inappropriate DNA processing must be avoided. In vertebrates, double-strand breaks are repaired predominantly by non-homologous end joining (NHEJ), which directly ligates DNA ends. NHEJ has the potential to be highly mutagenic because it uses DNA polymerases, nucleases, and other enzymes that modify incompatible DNA ends to allow their ligation. Using frog egg extracts that recapitulate NHEJ, we show that end processing requires the formation of a "short-range synaptic complex" in which DNA ends are closely aligned in a ligation-competent state. Furthermore, single-molecule imaging directly demonstrates that processing occurs within the short-range complex. This confinement of end processing to a ligation-competent complex ensures that DNA ends undergo ligation as soon as they become compatible, thereby minimizing mutagenesis. Our results illustrate how the coordination of enzymatic catalysis with higher-order structural organization of substrate maximizes the fidelity of DNA repair.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Instabilidade Genômica , Animais , DNA Ligases/genética , DNA Ligases/metabolismo , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Proteína Quinase Ativada por DNA/genética , Proteína Quinase Ativada por DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Autoantígeno Ku/genética , Autoantígeno Ku/metabolismo , Modelos Genéticos , Complexos Multiproteicos , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Imagem Individual de Molécula , Fatores de Tempo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis
17.
Toxicol Appl Pharmacol ; 387: 114855, 2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31830491

RESUMO

Vanillin is a natural compound endowed with antioxidant and anti-mutagenic properties. We previously identified the vanillin derivative VND3207 with strong radio-protective and antioxidant effects and found that VND3207 confers survival benefit and protection against radiation-induced intestinal injury (RIII) in mice. We also observed that VND3207 treatment enhanced the expression level of the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) in human lymphoblastoid cells with or without γ-irradiation. DNA-PKcs is a critical component of DNA double strand break repair pathway and also regulates mitotic progression by stabilizing spindle formation and preventing mitotic catastrophe in response to DNA damage. In the present study, we found that VND3207 protected intestinal epithelial cells in vitro against ionizing radiation by promoting cell proliferation and inhibiting cell apoptosis. In addition, VND3207 promoted DNA-PKcs activity by increasing autophosphorylation at S2056 site. Consistent with this, VND3207 significantly decreased the number of γH2AX foci and mitotic catastrophe after radiation. DNA-PKcs deficiency abolished these VND3207 radio-protective effects, indicating that DNA-PKcs activation is essential for VND3207 activity. In conclusion, VND3207 promoted intestinal repair following radiation injury by regulating the DNA-PKcs pathway.


Assuntos
Benzaldeídos/farmacologia , Proteína Quinase Dependente de GMP Cíclico Tipo I/metabolismo , Proteína Quinase Ativada por DNA/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Lesões Experimentais por Radiação/prevenção & controle , Protetores contra Radiação/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Proteína Quinase Ativada por DNA/genética , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Células Epiteliais/efeitos da radiação , Raios gama/efeitos adversos , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/patologia , Mucosa Intestinal/efeitos da radiação , Mutação com Perda de Função , Masculino , Camundongos , Fosforilação/efeitos dos fármacos , Lesões Experimentais por Radiação/patologia , Protetores contra Radiação/uso terapêutico
18.
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
19.
Viruses ; 11(10)2019 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-31614688

RESUMO

Most DNA viruses exhibit relatively low rates of spontaneous mutation. However, the molecular mechanisms underlying DNA virus genetic stability remain unclear. In principle, mutation rates should not depend solely on polymerase fidelity, but also on factors such as DNA damage and repair efficiency. Most eukaryotic DNA viruses interact with the cellular DNA damage response (DDR), but the role of DDR pathways in preventing mutations in the virus has not been tested empirically. To address this goal, we serially transferred human adenovirus type 5 in cells in which the telangiectasia-mutated PI3K-related protein kinase (ATM), the ATM/Rad3-related (ATR) kinase, and the DNA-dependent protein kinase (DNA-PK) were chemically inactivated, as well as in control cells displaying normal DDR pathway functioning. High-fidelity deep sequencing of these viral populations revealed mutation frequencies in the order of one-millionth, with no detectable effect of the inactivation of DDR mediators ATM, ATR, and DNA-PK on adenovirus sequence variability. This suggests that these DDR pathways do not play a major role in determining adenovirus genetic diversity.


Assuntos
Adenovírus Humanos/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína Quinase Ativada por DNA/metabolismo , Taxa de Mutação , Proteínas Mutadas de Ataxia Telangiectasia/genética , Linhagem Celular , Dano ao DNA/genética , Reparo do DNA/genética , Proteína Quinase Ativada por DNA/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Transdução de Sinais
20.
Prog Biophys Mol Biol ; 147: 92-102, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31570166

RESUMO

X-ray crystallography and electron microscopy maps resolved to 3-8 Šare generally sufficient for tracing the path of the polypeptide chain in space, while often insufficient for unambiguously registering the sequence on the path (i.e., threading). Frequently, however, additional information is available from other biophysical experiments, physical principles, statistical analyses, and other prior models. Here, we formulate an integrative approach for sequence assignment to a partial backbone model as an optimization problem, which requires three main components: the representation of the system, the scoring function, and the optimization method. The method is implemented in the open source Integrative Modeling Platform (IMP) (https://integrativemodeling.org), allowing a number of different terms in the scoring function. We apply this method to localizing the sequence assignment within a 199-residue disordered region of three structured and sequence unassigned helices in the DNA-PKcs crystallographic structure, using chemical crosslinks, hydrogen deuterium exchange, and sequence connectivity. The resulting ensemble of threading models provides two major solutions, one of which suggests that the crucial ABCDE cluster of phosphorylation sites cannot undergo intra-molecular autophosphorylation without a conformational rearrangement. The ensemble of solutions embodies the most accurate and precise sequence threading given the available information.


Assuntos
Proteína Quinase Ativada por DNA/química , Proteína Quinase Ativada por DNA/metabolismo , Medição da Troca de Deutério , Cristalografia por Raios X , Fosforilação , Conformação Proteica em alfa-Hélice
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