Indiana University-Purdue University Indianapolis (IUPUI) / Continual and recalcitrant inflammation is an extremely common condition in the human prostate and has been found to be associated with a number of prostatic diseases including prostate cancer and benign prostatic hyperplasia (BPH). While much has been described regarding prostate disease resulting from oxygen and nitrogen radicals during inflammation, proliferative mechanisms associated with repair and regeneration are less understood. The Inhibitor of Apoptosis (IAP) family member survivin has been found to be overexpressed during inflammation and associated with prostate cancer progression. Apurinic/apyrimidinic endonuclease 1/redox effector factor 1 (APE1/Ref-1) is a multifunctional protein that is essential in activating inflammatory transcription factors. Because APE1/Ref-1 is expressed and elevated in prostate cancer, we sought to characterize APE1/Ref-1 expression and activity in human prostate cancer cell lines and determine the effect of selective reduction-oxidation (redox) function inhibition on prostate cancer cells in vitro and in vivo. Due to the role of inflammatory transcriptional activators NFĸB and STAT3 in survivin protein expression, and APE1/Ref-1 redox activity regulating their transcriptional activity, we assessed selective inhibition of APE1/Ref-1’s redox function as a novel method to halt prostate cancer cell growth and survival. Our study demonstrates that survivin and APE1/Ref-1 are significantly higher in human prostate cancer specimens compared to noncancerous controls and that APE1/Ref-1 redox specific inhibition with small molecule inhibitors APX3330 and APX2009 decreases prostate cancer cell proliferation and induces cell cycle arrest. Inhibition of APE1/Ref-1 redox function significantly reduced NFĸB transcriptional activity, survivin mRNA and survivin protein levels. These data indicate that APE1/Ref-1 is a key regulator of survivin and a potentially viable target in prostate cancer.

Made available in DSpace on 2014-12-17T14:05:22Z (GMT). No. of bitstreams: 1 LeonamGC_TESE.pdf: 2406262 bytes, checksum: 604659dd8ff62335f952679dda971b6e (MD5) Previous issue date: 2013-03-19 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / Despite advances in antibiotic therapy, bacterial meningitis (BM) remains with high mortality and morbidity rates in worldwide. One important mechanism associated to sequels during disease is the intense inflammatory response which promotes an oxidative burst and release of reactive oxygen species, consequently leading to cell death. Activation of DNA repair enzymes during oxidative stress has been demonstrated in several neurological disorders. APE1/Ref-1 is a multifunctional protein involved in DNA repair and plays a redox function on transcription factors such as NFkB and AP-1.The aim of this study was assess the role of APE1/Ref-1 on inflammatory response and the possibility of its modulation to reduce the sequels of the disease. Firstly it was performed an assay to measure cytokine in cerebrospinal fluid of patients with BM due to Streptococcus pneumoniae and Neisseriae meningitides. Further, a cellular model of inflammation was used to observe the effect of the inhibition of the endonuclease and redox activity of APE1/Ref-1 on cytokine levels. Additionally, APE1/Ref-1 expression in cortex and hippocampus of rat with MB after vitamin B6 treatment was evaluated. Altogether, results showed a similar profile of cytokines in the cerebrospinal fluid of patients from both pathogens, although IFNy showed higher expression in patients with BM caused by S. pneumoniae. On the other hand, inhibitors of APE1/Ref-1 reduced cytokine levels, mainly TNF-?. Reduction of oxidative stress markers was also observed after introduction of inhibitors in the LPS-stimulated cell. In the animal model, BM increased the expression of the protein APE1/Ref-1, while vitamin B6 promoted reduction. Thereby, this data rise important factors to be considered in pathogenesis of BM, e.g., IFNy can be used as prognostic factor during corticosteroid therapy, APE1/Ref-1 can be an important target to modulate the level of inflammation and VIII oxidative stress, and vitamin B6 seems modulates several proteins related to cell death. So, this study highlights a new understanding on the role of APE1/Ref-1 on the inflammation and the oxidative stress during inflammation condition / A meningite bacteriana (MB) ? uma doen?a infecciosa que permanece com altas taxas de mortalidade e morbidade em todo o mundo, principalmente em pa?ses subdesenvolvidos, apesar dos avan?os na antibioticoterapia. Um dos principais mecanismos associados ?s sequelas durante a MB ? a elevada resposta inflamat?ria, que promove uma exacerbada quantidade de esp?cies reativas de oxig?nio (ERO) levando ?s c?lulas a apoptose ou necrose. A ativa??o de enzimas de reparo de DNA durante o estresse oxidativo tem sido demonstrada nas mais diversas desordens. Uma importante enzima envolvida neste processo ? a endonuclease apur?nica/apirimidinica1/fator redox-1 (APE1/Ref-1). Ela ? uma prote?na multifuncional envolvida no reparo de DNA e na redu??o de fatores envolvidos com a resposta inflamat?ria, tais como o fator nuclear kappa B (NFkB) e prote?na ativadora 1 (AP-1). Este estudo teve como objetivo identificar o envolvimento de APE1/Ref-1 na resposta inflamat?ria visando a possibilidade de sua utiliza??o como alvo terap?utico na redu??o de sequelas durante a MB. Para isto, inicialmente foi realizado uma an?lise no perfil de express?o de citocinas em l?quor de pacientes com meningite causada por Streptococcus pneumoniae e Neisseriae meningitidis visando selecionar moduladores inflamat?rios de interesse para ensaios em cultura de c?lula subsequentes. Em seguida, utilizando um modelo celular de indu??o com LPS foi avaliado o efeito da inibi??o da atividade de reparo e redox de APE1 sobre a express?o de citocinas inflamat?rias. Por fim, foi observada a express?o de APE1 no c?rtex (CX) e hipocampo (HC) de ratos com MB frente a uma terapia adjuvante com vitamina B6. Nossos resultados mostraram um perfil de moduladores inflamat?rios muito semelhante no l?quor dos pacientes com MB causada pelos pat?genos estudados, embora interferon gama (IFNy) tenha sido VI significativamente mais expresso em pacientes com S. pneumoniae do que N. meningitidis. Quanto ao uso dos inibidores das fun??es, redox e de reparo, de APE1/Ref-1 no modelo in vitro, houve redu??o significativa na express?o de algumas citocinas, principalmente o fator de necrose tumoral-alfa (TNF-?). Al?m disso, os inibidores demonstraram uma redu??o nos n?veis de ERO nas c?lulas estimuladas com LPS. No modelo animal, a express?o prot?ica de APE1/Ref-1, no CX e HC dos ratos, foi modulada ap?s introdu??o da vitamina B6. Portanto, esses dados fornecem um novo olhar para a fisiopatologia da MB, em que citocinas como IFNy podem ser usadas em um diagn?stico diferencial entre meningites causadas por S. pneumoniae e N. meningitidis. A prote?na de reparo de DNA, APE1/Ref-1, parece ser um alvo potencial na modula??o da resposta inflamat?ria e do estresse oxidativo, bem como a terapia adjuvante com vitamina B6 mostra ter um papel sobre a express?o de APE1/Ref-1. Consequentemente, o conhecimento obtido neste estudo pode ser importante na melhoria do progn?stico da MB, al?m de contribuir para entender a associa??o entre o reparo de DNA e inflama??o / 2020-01-01 / 2020-01-01

Made available in DSpace on 2014-12-17T14:10:20Z (GMT). No. of bitstreams: 1 Julliane Tamara Araujo de Melo.pdf: 3651115 bytes, checksum: d9a05a183487ffcfb8bd75edd16acd9d (MD5) Previous issue date: 2010-06-28 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / Riboflavin is a vitamin very important in aerobic organisms, as a precursor of many coenzymes involved in the electron transporter chain. However, after photosensitization of riboflavin with UV or visible light, it generates reactive oxygen species (ROS), which can oxidize the DNA. The repair of oxidative lesions on DNA occurs through the base excision repair pathway (BER), where APE1 endonuclease plays a central role. On the other hand, the nucleotide excision repair pathway (NER) repairs helix-distorting lesions. Recently, it was described the participation of NERproteins in the repair of oxidative damage and in stimulation of repair function fromAPE1. The aim of this research was to evaluate the cytotoxic effects of photosensitized riboflavin (RF*) in cells proficient and deficient in NER, correlating with APE1 expression. For this propose, the cells were treated with RF* and it was performed the cell viability assay, extraction of whole proteins, cells fractionation, immunoblotting, indirect immunofluorescence and analysis of polymorphisms of BER gens. The results evidenced that cells deficient in XPA and CSB proteins were more sensitive to RF*. However, XPC-deficient cells presented similar resistance to MRC5- SV cells, which is proficient in NER. These results indicate that XPA and CSB proteins have an important role on repair of oxidative lesions induced by RF*. Additionally, it was evidenced that single nucleotide polymorphisms (SNPs) in BER enzymes may influence in sensitivity of NER-deficient cell lines. Concerning the APE1 expression, the results showed that expression of this protein after treatment with RF* only changed in XPC-deficient cells. Though, it was observed that APE1 is recruited and is bound to chromatin in MRC5-SV and XPA cells after treatment with RF*. The results also showed the induction of DNA damage after treatment with RF*, through the analysis of-H2AX, since the treatment promoted an increase of endogenous levels of this phosphorylated protein, which acts signaling double strand-break on DNA. On the other hand, in XPC-deficient cells, regardless of resistance of RF*, the endogenous levels of APE1 are extremely reduced when compared with other cell lines and APE1 is not bound to chromatin after treatment with RF*. These results conclude that RF* was able to induce cell death in NERdeficient cells, where XPA and CSB cells were more sensitive when compared with MRC5-SV and XPC-deficient cells. This last result is potentially very interesting, since XPC-deficient cell line presents low levels of APE1. Additionally, the results evidenced that APE1 protein can be involved in the repair of oxidative damage induced by RF*, because APE1 is recruited and bound strongly to chromatin after treatment. / A riboflavina ? uma vitamina de fundamental import?ncia em organismos aer?bios, sendo precursora de importantes coenzimas que participam da cadeia transportadora de el?trons. Contudo, ap?s a sensibiliza??o da riboflavina com luz UV ou luz vis?vel, observou-se a forma??o de esp?cies reativas de oxig?nio (EROs), as quais podem oxidar o DNA. O reparo de les?es oxidativas no DNA ocorre principalmente atrav?s da via de reparo por excis?o de bases (BER), na qual a endonuclease APE1 exerce um papel central. Por sua vez, a via de reparo por excis?o de nucleot?deos (NER) atua reparando les?es no DNA que causam distor??es na dupla h?lice. Recentemente tem sido descrito a participa??o da via NER na remo??o de danos oxidativos e na estimula??o da fun??o de reparo de APE1. Desta forma, o objetivo desta pesquisa foi analisar os efeitos citot?xicos da riboflavina fotossensibilizada (RF*) em c?lulas proficientes e deficientes na via NER, correlacionando ? express?o de APE1. Para tanto, as linhagens proficientes e deficientes no NER foram submetidas ao tratamento com RF* e em seguida foram realizados os ensaios de viabilidade celular, extra??o de prote?nas totais, fracionamento celular, immunoblotting, imunofluoresc?ncia indireta e a an?lise de polimorfismos em genes da via BER. Os resultados evidenciaram perfis de sensibilidade distintos ao estresse oxidativo induzido pela RF*, onde as linhagens XPA e CSB foram mais sens?veis, enquanto a linhagem XPC mostrou resist?ncia similar ? linhagem MRC5-SV, a qual ? proficiente na via NER. Esses resultados indicam que as prote?nas XPA e CSB possuem um importante papel no reparo das les?es oxidativas induzidas pela RF*. Al?m disso, foi demonstrado que polimorfismos em um ?nico nucleot?deo (SNPs) em enzimas do BER podem influenciar na sensibilidade dessas linhagens. Em rela??o ?s an?lises dos n?veis de express?o de APE1, os resultados mostraram que houve altera??o na express?o dessa prote?na ap?s o tratamento com RF* somente na linhagem deficiente em XPC. Por?m, observou-se que APE1 ? recrutada e se torna ligada ? cromatina ap?s o tratamento nas linhagens MRC5-SV e XPA. Os resultados tamb?m comprovaram a indu??o de danos ap?s o tratamento com RF* atrav?s do estudo da prote?na-H2AX, pois o tratamento provocou um aumento nos n?veis end?genos desta prote?na fosforilada, a qual atua na sinaliza??o de quebras de fita dupla no DNA. Por?m, na linhagem XPC, al?m de ter sido observado uma curva de sobreviv?ncia semelhante ? linhagem MRC5-SV, os n?veis end?genos de APE1 s?o significativamente reduzidos quando comparados com as outras linhagens e APE1 n?o se encontra ligada ? cromatina ap?s tratamento com RF*. Conclui-se que a RF* foi capaz de induzir a morte celular em linhagens deficientes no sistema de reparo por excis?o de nucleot?deos, onde as linhagens XPA e CSB foram mais sens?veis quando comparadas ? linhagem normal MRC5-SV e ? linhagem XPC. Este ?ltimo resultado ? potencialmente interessante, considerando que a linhagem XPC apresenta baixos n?veis prot?icos de APE1. Adicionalmente, os resultados comprovaram que a prote?na APE1 pode estar envolvida no reparo de danos oxidativos causados pela RF*, j? que APE1 ? recrutada na cromatina e se liga fortemente a esta ap?s o tratamento.

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2016-03-03T19:55:15Z No. of bitstreams: 1 RayssaKarlaDeMedeirosOliveira_DISSERT.pdf: 5161047 bytes, checksum: b9cdb0f408571e0784106130b004445a (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2016-03-07T22:14:58Z (GMT) No. of bitstreams: 1 RayssaKarlaDeMedeirosOliveira_DISSERT.pdf: 5161047 bytes, checksum: b9cdb0f408571e0784106130b004445a (MD5) / Made available in DSpace on 2016-03-07T22:14:58Z (GMT). No. of bitstreams: 1 RayssaKarlaDeMedeirosOliveira_DISSERT.pdf: 5161047 bytes, checksum: b9cdb0f408571e0784106130b004445a (MD5) Previous issue date: 2014-08-21 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico - CNPq / Prote?nas do reparo por excis?o de bases (BER) t?m sido associadas a fun??es al?m do reparo e DNA. A apur?nica/apirimidinica endonuclease 1 (APE1) ? uma prote?na multifuncional envolvida em diversas atividades celulares como ativa??o redox de fatores de transcri??o, processamento de RNA e reparo de DNA. Alguns trabalhos t?m descrito a a??o da prote?na 8-oxoguanina (OGG1) na corre??o de les?es oxidadas no promotor como passo para a transcri??o de citocinas pro-inflamat?rias. Apesar de ser notadamente importante na ativa??o redox de fatores de transcri??o, como o fator nuclear ?B (NF- ?B) e AP-1, a atividade de reparo de APE1 ainda n?o foi associada ? resposta inflamat?ria. Neste trabalho, foram utilizadas an?lises bioinform?ticas e abordagens experimentais para investigar a rela??o entre a inibi??o do reparo de s?tios ab?sicos no DNA pela MX, mol?cula sint?tica inibidora indireta da atividade de reparo de APE1, e a modula??o de resposta inflamat?ria. Os resultados demonstraram que o tratamento de mon?citos com lipopolissacar?deo (LPS) e MX reduziu a express?o de citocinas, quimiocinas e receptores toll-like, e regulou negativamente processos biol?gicos da imunidade, como ativa??o de macr?fagos, e as vias ativadas pelo (NF-?B), fator de necrose tumoral (TNF-?) e interferon, sem induzir morte celular. A an?lise transcript?mica sugere que o tratamento LPS/MX induz disfun??es mitocondriais, estresse de ret?culo endoplasm?tico e ativa??o de vias de autofagia, provavelmente ativadas pelo comprometimento da energ?tica celular e/ou pelo ac?mulo de danos ao DNA, nuclear e mitocondrial. Adicionalmente, prop?e-se que a atividade de reparo de APE1 ? requerida para a transcri??o de genes inflamat?rios pela intera??o com s?tios ab?sicos no promotores espec?ficos e recrutamento de complexos transcricionais durante a sinaliza??o inflamat?ria. Este trabalho apresenta uma nova perspectiva acerca das intera??es entre a atividade do BER e a modula??o de resposta inflamat?ria, e sugere uma nova atividade para a prote?na APE1 como modular da resposta imune de maneira redox-independente. / Base excision repair (BER) proteins has been associated with functions beyond DNA repair. Apurynic/apyrimidinic endonuclease 1 (APE1) is a multifunctional protein involved in a plethora of cellular activities, such as redox activation of transcription factors, RNA processing and DNA repair. Some studies have described the action of the protein 8-oxoguanine (OGG1) in correcting oxidized lesions in promoters as a step in the transcription of pro-inflammatory cytokines. Despite being especially important in redox activation of transcription factors such as nuclear factor ?B (NF-?B) and AP- 1, the repair activity of APE1 has not yet been associated with the inflammatory response. In this study, experimental and bioinformatic analysis approaches have been used to investigate the relationship between inhibition of the repair of abasic sites in DNA by MX, a synthetic molecule designed to inhibt the repair activity of APE1, and the modulation of the inflammatory response. The results showed that treatment of monocytes with lipopolysaccharide (LPS) and MX reduced the expression of cytokines, chemokines and toll-like receptors, and negatively regulated biological immune processes, as macrophages activation, and NF-?B and tumor necrosis factor (TNF-?) and interferon pathways, without inducing cell death. The transcriptomic analysis suggests that LPS/MX treatment induces mitochondrial dysfunction, endoplasmic reticulum stress and activation of autophagy pathways, probably activated by impairment of cellular energy and/or the accumulation of nuclear and mitochondria DNA damage. Additionally, it is proposed that the repair activity of APE1 is required for transcription of inflammatory genes by interaction with abasic sites at specific promoters and recruitment of transcriptional complexes during inflammatory signaling. This work presents a new perspective on the interactions between the BER activity and the modulation of inflammatory response, and suggests a new activity for APE1 protein as modulator of the immune response in a redox-independent manner.

No description available.

Made available in DSpace on 2014-12-17T14:03:36Z (GMT). No. of bitstreams: 1 JullianeTAM_TESE_Parcial.pdf: 7138229 bytes, checksum: a06700fccf67cc2db086f62bf86db506 (MD5) Previous issue date: 2014-02-26 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / studies using UV as a source of DNA damage. However, even though unrepaired UV-induced DNA damages are related to mutagenesis, cell death and tumorigenesis, they do not explain phenotypes such as neurodegeneration and internal tumors observed in patients with syndromes like Xeroderma Pigmentosum (XP) and Cockayne Syndrome (CS) that are associated with NER deficiency. Recent evidences point to a role of NER in the repair of 8-oxodG, a typical substrate of Base Excision Repair (BER). Since deficiencies in BER result in genomic instability, neurodegenerative diseases and cancer, it was investigated in this research the impact of XPC deficiency on BER functions in human cells. It was analyzed both the expression and the cellular localization of APE1, OGG1 e PARP-1, the mainly BER enzymes, in different NER-deficient human fibroblasts. The endogenous levels of these enzymes are reduced in XPC deficient cells. Surprisingly, XP-C fibroblasts were more resistant to oxidative agents than the other NER deficient fibroblasts, despite presenting the highest of 8-oxodG. Furthermore, subtle changes in the nuclear and mitochondrial localization of APE1 were detected in XP-C fibroblasts. To confirm the impact of XPC deficiency in the regulation of APE1 and OGG1 expression and activity, we constructed a XPC-complemented cell line. Although the XPC complementation was only partial, we found that XPC-complemented cells presented increased levels of OGG1 than XPC-deficient cells. The extracts from XPC-complemented cells also presented an elevated OGG1 enzimatic activity. However, it was not observed changes in APE1 expression and activity in the XPCcomplemented cells. In addition, we found that full-length APE1 (37 kDa) and OGG1- ? are in the mitochondria of XPC-deficient fibroblasts and XPC-complemented fibroblasts before and after induction of oxidative stress. On the other hand, the expression of APE1 and PARP-1 are not altered in brain and liver of XPC knockout mice. However, XPC deficiency changed the APE1 localization in hypoccampus and hypothalamus. We also observed a physical interaction between XPC and APE1 proteins in human cells. In conclusion, the data suggest that XPC protein has a role in the regulation of OGG1 expression and activity in human cells and is involved mainly in the regulation of APE1 localization in mice. Aditionally, the response of NER deficient cells under oxidative stress may not be only associated to the NER deficiency per se, but it may include the new functions of NER enzymes in regulation of expression and cell localization of BER proteins / A maior parte do nosso conhecimento sobre a via de Reparo de Excis?o Nucleot?deos (NER) vem de estudos usando a luz ultravioleta (UV) como fonte de danos no DNA. Contudo, embora os danos no DNA causados pela luz UV sejam relacionados ? ocorr?ncia de mutag?nese, morte celular e tumorig?nese, eles n?o justificam fen?tipos como neurodegenera??o e tumorig?nese observados em pacientes com s?ndromes como Xeroderma Pigmentosum (XP) e S?ndrome de Cockayne (CS), as quais s?o associadas ? defici?ncia na via NER. Adicionalmente, evid?ncias mais recentes indicam o envolvimento da via NER no reparo de 8-oxodG, um substrato t?pico da via de Reparo por Excis?o de Bases (BER). Uma vez que a defici?ncia na via BER resulta em instabilidade gen?mica, doen?as neurodegenerativas e c?ncer, foi investigado neste trabalho o impacto da defici?ncia em XPC nas fun??es da via BER em c?lulas humanas. Foram realizadas an?lises da express?o e da localiza??o celular de APE1, OGG1 e PARP-1, principais enzimas da via BER, em fibroblastos humanos deficientes na via NER. Os resultados demonstraram que os n?veis end?genos de APE1, PARP-1 e OGG1 s?o reduzidos nos fibroblastos deficientes em XPC, os quais foram mais resistentes a diferentes tipos de agentes oxidantes e apresentaram n?veis elevados de 8-oxodG quando comparados aos demais fibroblastos deficientes na via NER. Adicionalmente, altera??es sutis na localiza??o nuclear e mitocondrial de APE1 foram observadas nos fibroblastos deficientes em XPC. Para confirmar o impacto da defici?ncia de XPC na regula??o da express?o e atividade de APE1 e OGG1, foi constru?da uma linhagem complementada com XPC. Embora a complementa??o tenha sido parcial, foi poss?vel observar que os fibroblastos parcialmente complementados com XPC apresentaram n?veis maiores de express?o de OGG1 quando comparados aos fibroblastos deficientes em XPC. Os extratos dos fibroblastos parcialmente complementados com XPC tamb?m apresentaram uma elevada atividade enzim?tica de OGG1. Contudo, n?o foram observadas mudan?as na express?o e atividade de APE1 nos fibroblastos parcialmente complementados com XPC. Adicionalmente, foi poss?vel verificar a presen?a da forma completa de APE1 (37 kDa) e de OGG1-? na mitoc?ndria dos fibroblastos deficientes em XPC e parcialmente complementados com XPC. Por outro lado, observou-se que a express?o de APE1 e PARP-1 n?o ? alterada no c?rebro e f?gado de camundongos knockouts para XPC. Contudo, a defici?ncia em XPC resultou em mudan?as na localiza??o celular de APE1 no hipocampo e hipot?lamo. Ainda, foi observada a ocorr?ncia de uma intera??o f?sica entre as prote?nas XPC e APE1 em c?lulas humanas. Em conclus?o, os dados sugerem que a prote?na XPC possui um papel na regula??o da express?o e da atividade de OGG1 em c?lulas humanas e est? envolvida na regula??o da localiza??o celular de APE1 principalmente em camundongos. Adicionalmente, as respostas celulares dos fibroblastos deficientes na via NER ao estresse oxidativo podem n?o estar associadas ? defici?ncia na via NER per se, mas podem incluir novas fun??es das enzimas da via NER na regula??o da express?o e localiza??o celular das prote?nas da via BER / 2020-01-01

Les sites apuriniques/apyrimidiniques (AP) sont des sites de l’ADN hautement mutagène. Les dommages au niveau de ces sites peuvent survenir spontanément ou être induits par une variété d’agents. Chez l’humain, les sites AP sont réparés principalement par APE1, une enzyme de réparation de l’ADN qui fait partie de la voie de réparation par excision de base (BER). APE1 est une enzyme multifonctionnelle; c’est une AP endonucléase, 3’-diestérase et un facteur redox impliqué dans l’activation des facteurs de transcription. Récemment, il a été démontré qu’APE1 interagit avec l’enzyme glycolytique GAPDH. Cette interaction induit l’activation d’APE1 par réduction. En outre, la délétion du gène GAPDH sensibilise les cellules aux agents endommageant l’ADN, induit une augmentation de formation spontanée des sites AP et réduit la prolifération cellulaire. A partir de toutes ces données, il était donc intéressant d’étudier l’effet de la délétion de GAPDH sur la progression du cycle cellulaire, sur la distribution cellulaire d’APE1 et d’identifier la cystéine(s) d’APE1 cible(s) de la réduction par GAPDH. Nos travaux de recherche ont montré que la déficience en GAPDH cause un arrêt du cycle cellulaire en phase G1. Cet arrêt est probablement dû à l’accumulation des dommages engendrant un retard au cours duquel la cellule pourra réparer son ADN. De plus, nous avons observé des foci nucléaires dans les cellules déficientes en GAPDH qui peuvent représenter des agrégats d’APE1 sous sa forme oxydée ou bien des focis de la protéine inactive au niveau des lésions d’ADN. Nous avons utilisé la mutagénèse dirigée pour créer des mutants (Cys en Ala) des sept cystéines d’APE1 qui ont été cloné dans un vecteur d’expression dans les cellules de mammifères. Nous émettons l’hypothèse qu’au moins un mutant ou plus va être résistant à l’inactivation par oxydation puisque l’alanine ne peut pas s’engager dans la formation des ponts disulfures. Par conséquent, on anticipe que l’expression de ce mutant dans les cellules déficientes en GAPDH pourrait restaurer une distribution cellulaire normale de APE1, libérerait les cellules de l’arrêt en phase G1 et diminuerait la sensibilité aux agents endommageant l’ADN. En conclusion, il semble que GAPDH, en préservant l’activité d’APE1, joue un nouveau rôle pour maintenir l’intégrité génomique des cellules aussi bien dans les conditions normales qu’en réponse au stress oxydatif. / Apurinic/apyrimidinic (AP) sites are highly mutagenic DNA lesions occurring either spontaneously or by the action of DNA damaging agents. In human cells, AP sites are processed by the major DNA repair enzyme APE1 through the base excision repair (BER) pathway. APE1 is a multifunctional protein that has AP endonuclease/3’-diesterase activities in addition to its role as a redox factor in activating many transcription factor. Recently, it has been shown that APE1 interacts with the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an interaction that results in the activation of APE1 by reduction. Interestingly, depletion of GAPDH sensitized the cells to DNA damaging agents and induced an increase in spontaneous AP sites frequency. Moreover, cells knocked-down for GAPDH showed defects in proliferation. Here we set up to investigate the effects of GAPDH knockdown on cell cycle progression, APE1 subcellular localization and to identify the cysteine residue(s) of APE1, target(s) of GAPDH reduction. Our studies showed that GAPDH deficient cells arrested in G1 phase of the cell cycle. The defect in cell cycle progression is most probably due to accumulation of DNA damage which activates checkpoints leading to a delay in the cell cycle to allow DNA repair. Furthermore, in GAPDH deficient cells, APE1 formed nuclear foci-like structures that could represent aggregates of the oxidized form of APE1 or inactive APE1 foci on DNA lesions. Using site-directed mutagenesis, we created seven APE1 cysteine to alanine mutants which were cloned into a mammalian expression vector. We expect that at least one of these mutants is likely to resist the inactivation by oxidation as it cannot engage in disulfide bridge formation. Therefore, the expression of this mutant(s) in GAPDH knockdown cells is expected to restore a normal APE1 cellular distribution, rescue the cell cycle defects, and render the cells less sensitive to DNA damaging agents. In conclusion, our results show a new role of GAPDH in maintaining genomic stability under oxidative stress by maintaining APE1 in its functional state.

A proteína APE1 (do inglêsApurinic/Apyrimidinicendonuclease 1/ Redox Factor-1 - APE1/REF-1) é uma enzima multifuncional, cuja expressão encontra-se frequentemente aumentada em gliomas. Além de apresentar atividade no reparo por excisão de base (BER), o gene APE1 também atua como fator de redução, mantendo fatores de transcrição (FTs) em um estado reduzido ativo. A via BER de reparo do DNA tem sido apontada como um possível fator de resistência a terapias baseadas no uso de agentes alquilantes, tais como temozolomida (TMZ). No presente trabalho, utilizou-se a estratégia de inibição da transcrição do gene APE1 pelo método de RNA interferente(siRNA) e tratamento com a droga TMZ nas células de glioblastoma (GBM), T98G (resistente à TMZ) e U87MG (sensível à TMZ), a fim de verificar a influência do silenciamento do gene APE1 sobre as respostas celulares à droga avaliadas por vários ensaios, bem como os efeitos sobre a expressão transcricional dos genes alvos dos FTs regulados por APE1. O silenciamento de APE1 e o tratamento das células T98G com a TMZ foram eficazes no sentido de reduzir a proliferação e a capacidade clonogênica, além de intervir na progressão do ciclo celular com bloqueio na fase S. Tais efeitos foram acompanhados pelo aumento da indução de danos no DNA e da expressão de H2AX fosforilada (H2AX), o que justifica a queda na sobrevivência celular. O mesmo efeito não foi observado nas células U87MG silenciadas para APE1 e tratadas com a TMZ, havendo o predomínio dos efeitos causados pela TMZ, exceto por uma leve indução de danos no DNA e de H2AX. Adicionalmente, nas células T98G silenciadas e tratadas, verificou-se uma moderada indução de apoptose, que foi observada ao longo dos tempos avaliados (1 a 10 dias), com uma leve indução de caspase-3 (5 dias); nessas células, observou-se também a indução (3,8 vezes) de morte celular autofágica (5 dias). Entretanto, nas células U87MG,a indução de apoptose foi baixa e não houve indução de morte por autofagia, sugerindo outros mecanismos de morte envolvidos na eliminação dessas células em resposta ao tratamento com a TMZ. O silenciamento de APE1 causou uma redução acentuada na invasão das células T98G, de forma similar à observada nas células somente tratadas com a TMZ, sendo que a combinação (silenciamento de APE1 e tratamento com a droga) resultou em um efeito aditivo, enquanto que nas células U87MG a combinação foi eficaz no sentido de reduzir a proporção de células invasivas, fato não observado nas condições isoladas. Os genes COX2 e VEGF, alvos dos FT NFB e HIF-1 (regulados por APE1) foram reprimidos nas células T98G enquanto que o gene VEGF foi induzido nas células U87MG, entretanto, tais alterações no padrão de expressão transcricional foram observadas somente em resposta ao tratamento com a TMZ, independentemente do silenciamento de APE1, indicando nenhuma mudança na atividade redox de APE1, possivelmente pela existência de proteínas APE1 remanescentes na célula. Além disso, a expressão proteica de NFBp65(ser563) foi aumentada em ambas as linhagens silenciadas e tratadas com a TMZ, provavelmente devido à inibição da proliferação celular. Em geral, os resultados do presente trabalho demonstraram que a estratégia de inibição do gene APE1 (participante da via BER) mostrou-se potencialmente viável, suportando a contribuição do BER na resistência à TMZ, visto que nas condições testadas, observou-se uma sensibilização das células de GBM, com efeito restrito às células de GBM resistentes (linhagem T98G), sendo pouco eficaz no sentido de sensibilizar as células sensíveis (linhagem U87MG) a esse agente. Assim, há que considerar as características genéticas de cada linhagem de GBM, visto que estas são cruciais para as respostas apresentadas pelas células aos tratamentos empregados. / APE1 (Apurinic/Apyrimidinic endonuclease 1/ Redox Factor-1 - APE1/REF-1) protein is a multifunctional enzyme whose expression is often increased in gliomas. Besides presenting activity in base excision repair (BER), APE1 also acts as a reduction factor, maintaining transcription factors (TFs) in an active reduced state. The BER pathway has been implicated as a possible factor of resistance to therapies based on the use of alkylating agents such as temozolomide (TMZ). In the present study, we have been using a strategy of small interference RNA (siRNA) to down-regulate the APE1 gene under conditions of treatment with TMZ in T98G (resistant to TMZ) and U87MG (sensitive to TMZ), glioblastoma (GBM), in order to determine the effects of APE1 gene silencing on cellular responses to this drug, evaluated by several assays, as well as the effects on the transcriptional expression of target genes of TFs regulated by APE1. APE1 silencing and TMZ treatment was effective to reduce the cell proliferation and clonogenic capacity of T98G cells, in addition to interfering in the cell cycle progression (S-phase arrest). These effects were accompanied by induction of DNA damage and phosphorylation of H2AX (H2AX), which may explain the decrease in cell survival. The same effect was not observed in silenced U87MG and TMZ-treated cells, being observed a predominance of the effects caused by TMZ itself, except for a slight induction of DNA damage and H2AX. Additionally, in silenced T98G and TMZ-treated cells, there was a moderate induction of apoptosis, as observed over time (1 to 10 days), with a slight induction of caspase-3 (on day 5); for those cells, we also observed autophagic induction (3.8 fold) at day 5. However, the induction of apoptosis and autophagy in U87MG cells was very low, suggesting that other mechanisms of cell death might be involved in the elimination of GBM cells under TMZ treatment. APE1 silencing caused a marked reduction on the invasiveness of T98G cells, similarly to that observed in TMZ treated cells, while the combination (APE1 silencing and drug treatment) led to an additive effect. For U87MG, the treatment combination was effective in reducing the proportion of invasive cells, in spite of an absence of any effect produced by each isolated condition tested. Regarding to the expression profile of target genes of TFs regulated by the APE1 redox activity, it was observed that COX2 and VEGF genes, targets of FTs NFB and HIF-1, were down-regulated in T98G while VEGF gene showed induced in U87MG cells; however, such alterations in the transcriptional expression pattern were observed only in response to TMZ treatment, independently of APE1 gene silencing, indicating no change in the APE1 redox activity, possibly due to the presence of APE1 remaining proteins inside cells. In addition, NFBp65(ser563) protein expression was increased in both cell lines (silenced and treated with TMZ), probably due to the reduced cell proliferation rates. In general, the present results show that the strategy of APE1 gene knockdown was potentially viable, supporting the BER contribution of the mechanism of TMZ resistance, since under the conditions tested, there was a sensitization of GBM cells. However, this effect was restricted to the resistant cell line (T98G cells). Thus, it should be considered the genetic characteristics of each GBM cell line, since these are crucial to the cellular responses to the conditions tested in the present work.

Les sites apuriniques/apyrimidiniques (AP) sont des sites de l’ADN hautement mutagène. Les dommages au niveau de ces sites peuvent survenir spontanément ou être induits par une variété d’agents. Chez l’humain, les sites AP sont réparés principalement par APE1, une enzyme de réparation de l’ADN qui fait partie de la voie de réparation par excision de base (BER). APE1 est une enzyme multifonctionnelle; c’est une AP endonucléase, 3’-diestérase et un facteur redox impliqué dans l’activation des facteurs de transcription. Récemment, il a été démontré qu’APE1 interagit avec l’enzyme glycolytique GAPDH. Cette interaction induit l’activation d’APE1 par réduction. En outre, la délétion du gène GAPDH sensibilise les cellules aux agents endommageant l’ADN, induit une augmentation de formation spontanée des sites AP et réduit la prolifération cellulaire. A partir de toutes ces données, il était donc intéressant d’étudier l’effet de la délétion de GAPDH sur la progression du cycle cellulaire, sur la distribution cellulaire d’APE1 et d’identifier la cystéine(s) d’APE1 cible(s) de la réduction par GAPDH. Nos travaux de recherche ont montré que la déficience en GAPDH cause un arrêt du cycle cellulaire en phase G1. Cet arrêt est probablement dû à l’accumulation des dommages engendrant un retard au cours duquel la cellule pourra réparer son ADN. De plus, nous avons observé des foci nucléaires dans les cellules déficientes en GAPDH qui peuvent représenter des agrégats d’APE1 sous sa forme oxydée ou bien des focis de la protéine inactive au niveau des lésions d’ADN. Nous avons utilisé la mutagénèse dirigée pour créer des mutants (Cys en Ala) des sept cystéines d’APE1 qui ont été cloné dans un vecteur d’expression dans les cellules de mammifères. Nous émettons l’hypothèse qu’au moins un mutant ou plus va être résistant à l’inactivation par oxydation puisque l’alanine ne peut pas s’engager dans la formation des ponts disulfures. Par conséquent, on anticipe que l’expression de ce mutant dans les cellules déficientes en GAPDH pourrait restaurer une distribution cellulaire normale de APE1, libérerait les cellules de l’arrêt en phase G1 et diminuerait la sensibilité aux agents endommageant l’ADN. En conclusion, il semble que GAPDH, en préservant l’activité d’APE1, joue un nouveau rôle pour maintenir l’intégrité génomique des cellules aussi bien dans les conditions normales qu’en réponse au stress oxydatif. / Apurinic/apyrimidinic (AP) sites are highly mutagenic DNA lesions occurring either spontaneously or by the action of DNA damaging agents. In human cells, AP sites are processed by the major DNA repair enzyme APE1 through the base excision repair (BER) pathway. APE1 is a multifunctional protein that has AP endonuclease/3’-diesterase activities in addition to its role as a redox factor in activating many transcription factor. Recently, it has been shown that APE1 interacts with the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an interaction that results in the activation of APE1 by reduction. Interestingly, depletion of GAPDH sensitized the cells to DNA damaging agents and induced an increase in spontaneous AP sites frequency. Moreover, cells knocked-down for GAPDH showed defects in proliferation. Here we set up to investigate the effects of GAPDH knockdown on cell cycle progression, APE1 subcellular localization and to identify the cysteine residue(s) of APE1, target(s) of GAPDH reduction. Our studies showed that GAPDH deficient cells arrested in G1 phase of the cell cycle. The defect in cell cycle progression is most probably due to accumulation of DNA damage which activates checkpoints leading to a delay in the cell cycle to allow DNA repair. Furthermore, in GAPDH deficient cells, APE1 formed nuclear foci-like structures that could represent aggregates of the oxidized form of APE1 or inactive APE1 foci on DNA lesions. Using site-directed mutagenesis, we created seven APE1 cysteine to alanine mutants which were cloned into a mammalian expression vector. We expect that at least one of these mutants is likely to resist the inactivation by oxidation as it cannot engage in disulfide bridge formation. Therefore, the expression of this mutant(s) in GAPDH knockdown cells is expected to restore a normal APE1 cellular distribution, rescue the cell cycle defects, and render the cells less sensitive to DNA damaging agents. In conclusion, our results show a new role of GAPDH in maintaining genomic stability under oxidative stress by maintaining APE1 in its functional state.

A proteína APE1 (do inglêsApurinic/Apyrimidinicendonuclease 1/ Redox Factor-1 - APE1/REF-1) é uma enzima multifuncional, cuja expressão encontra-se frequentemente aumentada em gliomas. Além de apresentar atividade no reparo por excisão de base (BER), o gene APE1 também atua como fator de redução, mantendo fatores de transcrição (FTs) em um estado reduzido ativo. A via BER de reparo do DNA tem sido apontada como um possível fator de resistência a terapias baseadas no uso de agentes alquilantes, tais como temozolomida (TMZ). No presente trabalho, utilizou-se a estratégia de inibição da transcrição do gene APE1 pelo método de RNA interferente(siRNA) e tratamento com a droga TMZ nas células de glioblastoma (GBM), T98G (resistente à TMZ) e U87MG (sensível à TMZ), a fim de verificar a influência do silenciamento do gene APE1 sobre as respostas celulares à droga avaliadas por vários ensaios, bem como os efeitos sobre a expressão transcricional dos genes alvos dos FTs regulados por APE1. O silenciamento de APE1 e o tratamento das células T98G com a TMZ foram eficazes no sentido de reduzir a proliferação e a capacidade clonogênica, além de intervir na progressão do ciclo celular com bloqueio na fase S. Tais efeitos foram acompanhados pelo aumento da indução de danos no DNA e da expressão de H2AX fosforilada (H2AX), o que justifica a queda na sobrevivência celular. O mesmo efeito não foi observado nas células U87MG silenciadas para APE1 e tratadas com a TMZ, havendo o predomínio dos efeitos causados pela TMZ, exceto por uma leve indução de danos no DNA e de H2AX. Adicionalmente, nas células T98G silenciadas e tratadas, verificou-se uma moderada indução de apoptose, que foi observada ao longo dos tempos avaliados (1 a 10 dias), com uma leve indução de caspase-3 (5 dias); nessas células, observou-se também a indução (3,8 vezes) de morte celular autofágica (5 dias). Entretanto, nas células U87MG,a indução de apoptose foi baixa e não houve indução de morte por autofagia, sugerindo outros mecanismos de morte envolvidos na eliminação dessas células em resposta ao tratamento com a TMZ. O silenciamento de APE1 causou uma redução acentuada na invasão das células T98G, de forma similar à observada nas células somente tratadas com a TMZ, sendo que a combinação (silenciamento de APE1 e tratamento com a droga) resultou em um efeito aditivo, enquanto que nas células U87MG a combinação foi eficaz no sentido de reduzir a proporção de células invasivas, fato não observado nas condições isoladas. Os genes COX2 e VEGF, alvos dos FT NFB e HIF-1 (regulados por APE1) foram reprimidos nas células T98G enquanto que o gene VEGF foi induzido nas células U87MG, entretanto, tais alterações no padrão de expressão transcricional foram observadas somente em resposta ao tratamento com a TMZ, independentemente do silenciamento de APE1, indicando nenhuma mudança na atividade redox de APE1, possivelmente pela existência de proteínas APE1 remanescentes na célula. Além disso, a expressão proteica de NFBp65(ser563) foi aumentada em ambas as linhagens silenciadas e tratadas com a TMZ, provavelmente devido à inibição da proliferação celular. Em geral, os resultados do presente trabalho demonstraram que a estratégia de inibição do gene APE1 (participante da via BER) mostrou-se potencialmente viável, suportando a contribuição do BER na resistência à TMZ, visto que nas condições testadas, observou-se uma sensibilização das células de GBM, com efeito restrito às células de GBM resistentes (linhagem T98G), sendo pouco eficaz no sentido de sensibilizar as células sensíveis (linhagem U87MG) a esse agente. Assim, há que considerar as características genéticas de cada linhagem de GBM, visto que estas são cruciais para as respostas apresentadas pelas células aos tratamentos empregados. / APE1 (Apurinic/Apyrimidinic endonuclease 1/ Redox Factor-1 - APE1/REF-1) protein is a multifunctional enzyme whose expression is often increased in gliomas. Besides presenting activity in base excision repair (BER), APE1 also acts as a reduction factor, maintaining transcription factors (TFs) in an active reduced state. The BER pathway has been implicated as a possible factor of resistance to therapies based on the use of alkylating agents such as temozolomide (TMZ). In the present study, we have been using a strategy of small interference RNA (siRNA) to down-regulate the APE1 gene under conditions of treatment with TMZ in T98G (resistant to TMZ) and U87MG (sensitive to TMZ), glioblastoma (GBM), in order to determine the effects of APE1 gene silencing on cellular responses to this drug, evaluated by several assays, as well as the effects on the transcriptional expression of target genes of TFs regulated by APE1. APE1 silencing and TMZ treatment was effective to reduce the cell proliferation and clonogenic capacity of T98G cells, in addition to interfering in the cell cycle progression (S-phase arrest). These effects were accompanied by induction of DNA damage and phosphorylation of H2AX (H2AX), which may explain the decrease in cell survival. The same effect was not observed in silenced U87MG and TMZ-treated cells, being observed a predominance of the effects caused by TMZ itself, except for a slight induction of DNA damage and H2AX. Additionally, in silenced T98G and TMZ-treated cells, there was a moderate induction of apoptosis, as observed over time (1 to 10 days), with a slight induction of caspase-3 (on day 5); for those cells, we also observed autophagic induction (3.8 fold) at day 5. However, the induction of apoptosis and autophagy in U87MG cells was very low, suggesting that other mechanisms of cell death might be involved in the elimination of GBM cells under TMZ treatment. APE1 silencing caused a marked reduction on the invasiveness of T98G cells, similarly to that observed in TMZ treated cells, while the combination (APE1 silencing and drug treatment) led to an additive effect. For U87MG, the treatment combination was effective in reducing the proportion of invasive cells, in spite of an absence of any effect produced by each isolated condition tested. Regarding to the expression profile of target genes of TFs regulated by the APE1 redox activity, it was observed that COX2 and VEGF genes, targets of FTs NFB and HIF-1, were down-regulated in T98G while VEGF gene showed induced in U87MG cells; however, such alterations in the transcriptional expression pattern were observed only in response to TMZ treatment, independently of APE1 gene silencing, indicating no change in the APE1 redox activity, possibly due to the presence of APE1 remaining proteins inside cells. In addition, NFBp65(ser563) protein expression was increased in both cell lines (silenced and treated with TMZ), probably due to the reduced cell proliferation rates. In general, the present results show that the strategy of APE1 gene knockdown was potentially viable, supporting the BER contribution of the mechanism of TMZ resistance, since under the conditions tested, there was a sensitization of GBM cells. However, this effect was restricted to the resistant cell line (T98G cells). Thus, it should be considered the genetic characteristics of each GBM cell line, since these are crucial to the cellular responses to the conditions tested in the present work.