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1.
Chembiochem ; 2020 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-32424847

RESUMO

Intracellular transport of molecules, macromolecules or materials is a key step in probing cellular structure, function and regulating plethora of physical and chemical events for treating disease. This communication reveals direct cellular uptake of pyridyl-disulfide (Py-Ds) conjugated nonionic and biocompatible macromolecules with the aid of rapid exchange of the highly reactive Py-Ds groups with exofacial cell surface thiols. Confocal microscopy and flow cytometry analysis confirmed highly efficient cellular uptake of Py-Ds appended polymers (> 50% in 15 min) by avoiding lysosome as a consequence of thiol-disulfide exchange in the cell surface. In contrast a control polymer lacking the Py-Ds group followed caveolae mediated endocytosis. Other control polymers containing either the pyridine group (not disulfide) or the disulfide group (not pyridine) revealed significantly low cellular uptake and thus essential role of the highly reactive Py-Ds group was established beyond doubt.

2.
PLoS Pathog ; 16(2): e1008105, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32092124

RESUMO

Epstein-Barr virus (EBV) nuclear oncoprotein EBNA3C is essential for B-cell transformation and development of several B-cell lymphomas particularly those are generated in an immuno-compromised background. EBNA3C recruits ubiquitin-proteasome machinery for deregulating multiple cellular oncoproteins and tumor suppressor proteins. Although EBNA3C is found to be ubiquitinated at its N-terminal region and interacts with 20S proteasome, the viral protein is surprisingly stable in growing B-lymphocytes. EBNA3C can also circumvent autophagy-lysosomal mediated protein degradation and subsequent antigen presentation for T-cell recognition. Recently, we have shown that EBNA3C enhances autophagy, which serve as a prerequisite for B-cell survival particularly under growth deprivation conditions. We now demonstrate that proteasomal inhibition by MG132 induces EBNA3C degradation both in EBV transformed B-lymphocytes and ectopic-expression systems. Interestingly, MG132 treatment promotes degradation of two EBNA3 family oncoproteins-EBNA3A and EBNA3C, but not the viral tumor suppressor protein EBNA3B. EBNA3C degradation induced by proteasomal inhibition is partially blocked when autophagy-lysosomal pathway is inhibited. In response to proteasomal inhibition, EBNA3C is predominantly K63-linked polyubiquitinated, colocalized with the autophagy-lysosomal fraction in the cytoplasm and participated within p62-LC3B complex, which facilitates autophagy-mediated degradation. We further show that the degradation signal is present at the first 50 residues of the N-terminal region of EBNA3C. Proteasomal inhibition reduces the colony formation ability of this important viral oncoprotein, induces apoptotic cell death and increases transcriptional activation of both latent and lytic gene expression which further promotes viral reactivation from EBV transformed B-lymphocytes. Altogether, this study offers rationale to use proteasome inhibitors as potential therapeutic strategy against multiple EBV associated B-cell lymphomas, where EBNA3C is expressed.


Assuntos
Morte Celular Autofágica/efeitos dos fármacos , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Herpesvirus Humano 4/metabolismo , Leupeptinas/farmacologia , Lisossomos/metabolismo , Proteínas Oncogênicas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Proteólise/efeitos dos fármacos , Animais , Antígenos Nucleares do Vírus Epstein-Barr/genética , Células HEK293 , Herpesvirus Humano 4/genética , Humanos , Lisossomos/genética , Camundongos , Proteínas Oncogênicas/genética , Complexo de Endopeptidases do Proteassoma/genética
3.
Sci Adv ; 5(11): eaax9778, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31723605

RESUMO

A homozygous mutation of human tyrosyl-DNA phosphodiesterase 1 (TDP1) causes the neurodegenerative syndrome, spinocerebellar ataxia with axonal neuropathy (SCAN1). TDP1 hydrolyzes the phosphodiester bond between DNA 3'-end and a tyrosyl moiety within trapped topoisomerase I (Top1)-DNA covalent complexes (Top1cc). TDP1 is critical for mitochondrial DNA (mtDNA) repair; however, the role of mitochondria remains largely unknown for the etiology of SCAN1. We demonstrate that mitochondria in cells expressing SCAN1-TDP1 (TDP1H493R) are selectively trapped on mtDNA in the regulatory non-coding region and promoter sequences. Trapped TDP1H493R-mtDNA complexes were markedly increased in the presence of the Top1 poison (mito-SN38) when targeted selectively into mitochondria in nanoparticles. TDP1H493R-trapping accumulates mtDNA damage and triggers Drp1-mediated mitochondrial fission, which blocks mitobiogenesis. TDP1H493R prompts PTEN-induced kinase 1-dependent mitophagy to eliminate dysfunctional mitochondria. SCAN1-TDP1 in mitochondria creates a pathological state that allows neurons to turn on mitophagy to rescue fit mitochondria as a mechanism of survival.

4.
Mol Biol Cell ; 30(12): 1463-1476, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30995168

RESUMO

Though many cancers are known to show up-regulation of nonmuscle myosin (NM) IIA and IIB, the mechanism by which NMIIs aid in cancer development remains unexplored. Here we demonstrate that tumor-generating, fibroblast-like cells isolated from 3-methylcholanthrene (3MC)-induced murine tumor exhibit distinct phospho-dependent localization of NMIIA and NMIIB at the perinuclear area and tip of the filopodia and affect cell migration differentially. While NMIIA-KD affects protrusion dynamics and increases cell directionality, NMIIB-KD lowers migration speed and increases filopodial branching. Strategically located NMIIs at the perinuclear area colocalize with the linker of nucleoskeleton and cytoskeleton (LINC) protein Nesprin2 and maintain the integrity of the nuclear-actin cap. Interestingly, knockdown of NMIIs results in altered expression of genes involved in epithelial-to-mesenchymal transition, angiogenesis, and cellular senescence. NMIIB-KD cells display down-regulation of Gsc and Serpinb2, which is strikingly similar to Nesprin2-KD cells as assessed by quantitative PCR analysis. Further gene network analysis predicts that NMIIA and NMIIB may act on similar pathways but through different regulators. Concomitantly, knockdown of NMIIA or NMIIB lowers the growth rate and tumor volume of 3MC-induced tumor in vivo. Altogether, these results open a new window to further investigate the effect of LINC-associated perinuclear actomyosin complex on mechanoresponsive gene expression in the growing tumor.


Assuntos
Carcinogênese/genética , Movimento Celular/genética , Regulação Neoplásica da Expressão Gênica , Miosina não Muscular Tipo IIA/metabolismo , Miosina não Muscular Tipo IIB/metabolismo , Actinas/metabolismo , Animais , Proliferação de Células , Metilcolantreno , Camundongos , Quinase de Cadeia Leve de Miosina/metabolismo
5.
J Med Chem ; 62(7): 3428-3446, 2019 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-30897325

RESUMO

To overcome chemical limitations of camptothecin (CPT), we report design, synthesis, and validation of a quinoline-based novel class of topoisomerase 1 (Top1) inhibitors and establish that compound 28 ( N-(3-(1 H-imidazol-1-yl)propyl)-6-(4-methoxyphenyl)-3-(1,3,4-oxadiazol-2-yl)quinolin-4-amine) exhibits the highest potency in inhibiting human Top1 activity with an IC50 value of 29 ± 0.04 nM. Compound 28 traps Top1-DNA cleavage complexes (Top1ccs) both in the in vitro cleavage assays and in live cells. Point mutation of Top1-N722S fails to trap compound 28-induced Top1cc because of its inability to form a hydrogen bond with compound 28. Unlike CPT, compound 28 shows excellent plasma serum stability and is not a substrate of P-glycoprotein 1 (permeability glycoprotein) advancing its potential anticancer activity. Finally, we provide evidence that compound 28 overcomes the chemical instability of CPT in human breast adenocarcinoma cells through generation of persistent and less reversible Top1cc-induced DNA double-strand breaks as detected by γH2AX foci immunostaining after 5 h of drug removal.

6.
Nucleic Acids Res ; 46(11): 5601-5617, 2018 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-29718323

RESUMO

Human tyrosyl-DNA phosphodiesterases (TDP) hydrolyze the phosphodiester bond between DNA and the catalytic tyrosine of Top1 to excise topoisomerase I cleavage complexes (Top1cc) that are trapped by camptothecin (CPT) and by genotoxic DNA alterations. Here we show that the protein arginine methyltransferase PRMT5 enhances the repair of Top1cc by direct binding to TDP1 and arginine dimethylation of TDP1 at residues R361 and R586. Top1-induced replication-mediated DNA damage induces TDP1 arginine methylation, enhancing its 3'- phosphodiesterase activity. TDP1 arginine methylation also increases XRCC1 association with TDP1 in response to CPT, and the recruitment of XRCC1 to Top1cc DNA damage foci. PRMT5 knockdown cells exhibit defective TDP1 activity with marked elevation in replication-coupled CPT-induced DNA damage and lethality. Finally, methylation of R361 and R586 stimulate TDP1 repair function and promote cell survival in response to CPT. Together, our findings provide evidence for the importance of PRMT5 for the post-translational regulation of TDP1 and repair of Top1cc.


Assuntos
Reparo do DNA , DNA Topoisomerases Tipo I/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Animais , Arginina/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Dano ao DNA , Replicação do DNA , Células HEK293 , Humanos , Metilação , Camundongos , Diester Fosfórico Hidrolases/química , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/metabolismo
7.
Dalton Trans ; 47(18): 6557-6569, 2018 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-29697103

RESUMO

The present study aims to formulate a common synthetic strategy for preparing quantum dots (QDs) in a greener way by using combination of popular methods, viz. a colloidal method with suitable capping agent and low molecular weight gel based synthesis. Pyridine dicarboxylic acid (PDC) in presence of AlCl3 forms a stable metallogel, which serves as an excellent medium for selective ZnS QD synthesis. The aromatic pyridine moiety, well known for being a capping agent, indeed plays its part in the run up to QD synthesis. To the best of our knowledge, this is the first example of a metallogel based doped ZnS QD synthesis. Altering the doping material and its composition changes the properties of the QDs, but herein we also tried to establish how these changes affect the gel morphology and stability of both gel and QDs. We further demonstrate, by using live cell confocal microscopy, the delivery of QDs Cu ZnS and MnZnS nanomaterials in the nucleus and the cytoplasm of human breast cancer cells (MCF7), implicating the use of metallogel based QDs for bio-imaging and bio-labeling.


Assuntos
Imagem Óptica , Compostos Organometálicos/química , Pontos Quânticos/química , Cloreto de Alumínio , Compostos de Alumínio/química , Sobrevivência Celular/efeitos dos fármacos , Cloretos/química , Cobre/química , Géis/síntese química , Géis/química , Géis/farmacologia , Humanos , Células MCF-7 , Manganês/química , Microscopia Confocal , Compostos Organometálicos/síntese química , Compostos Organometálicos/farmacologia , Tamanho da Partícula , Ácidos Picolínicos/química , Espectroscopia de Infravermelho com Transformada de Fourier , Sulfetos/química , Propriedades de Superfície , Células Tumorais Cultivadas , Zinco/química , Compostos de Zinco/química
8.
J Med Chem ; 61(3): 804-817, 2018 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-29290109

RESUMO

Camptothecin (CPT) selectively traps topoisomerase 1-DNA cleavable complexes (Top1cc) to promote anticancer activity. Here, we report the design and synthesis of a new class of neutral porphyrin derivative 5,10-bis(4-carboxyphenyl)-15, 20-bis(4-dimethylaminophenyl)porphyrin (compound 8) as a potent catalytic inhibitor of human Top1. In contrast to CPT, compound 8 reversibly binds with the free enzyme and inhibits the formation of Top1cc and promotes reversal of the preformed Top1cc with CPT. Compound 8 induced inhibition of Top1cc formation in live cells was substantiated by fluorescence recovery after photobleaching (FRAP) assays. We established that MCF7 cells treated with compound 8 trigger proteasome-mediated Top1 degradation, accumulate higher levels of reactive oxygen species (ROS), PARP1 cleavage, oxidative DNA fragmentation, and stimulate apoptotic cell death without stabilizing apoptotic Top1-DNA cleavage complexes. Finally, compound 8 shows anticancer activity by targeting cellular Top1 and preventing the enzyme from directly participating in the apoptotic process.


Assuntos
Apoptose/efeitos dos fármacos , Clivagem do DNA/efeitos dos fármacos , DNA Topoisomerases Tipo I/metabolismo , Porfirinas/química , Porfirinas/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Biocatálise/efeitos dos fármacos , Quebras de DNA/efeitos dos fármacos , Humanos , Células MCF-7 , Espécies Reativas de Oxigênio/metabolismo , Inibidores da Topoisomerase I/química , Inibidores da Topoisomerase I/farmacologia
9.
Small ; 13(15)2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28134490

RESUMO

Aggregation-induced emission (AIE) is commonly observed in irregular bulk form. Herein, unique aggregation properties of an AIE-active complex into branched supramolecular wires are reported for the first time. Mono-cyclometalated Ir(III) complex shows in-plane J-aggregation at the air-water interface owing to the restriction of intramolecular vibration of bidentate phenylpyridinato and intramolecular rotations of monodentate triphenylphosphine ligands at air-water interface. As a consequence, a large enhancement of luminescence comparable to the solid state is obtained from the monolayers of supramolecular wires. This unique feature is utilized for the fabrication of light-emitting diodes with low threshold voltage using supramolecular wires as active layer. This study opens up the need of ordered assembly of AIE complexes to achieve optimal luminescence characteristics.

10.
Nucleic Acids Res ; 44(17): 8363-75, 2016 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-27466387

RESUMO

Topoisomerase 1 (Top1) is essential for removing the DNA supercoiling generated during replication and transcription. Anticancer drugs like camptothecin (CPT) and its clinical derivatives exert their cytotoxicity by reversibly trapping Top1 in covalent complexes on the DNA (Top1cc). Poly(ADP-ribose) polymerase (PARP) catalyses the addition of ADP-ribose polymers (PAR) onto itself and Top1. PARP inhibitors enhance the cytotoxicity of CPT in the clinical trials. However, the molecular mechanism by which PARylation regulates Top1 nuclear dynamics is not fully understood. Using live-cell imaging of enhanced green fluorescence tagged-human Top1, we show that PARP inhibitors (Veliparib, ABT-888) delocalize Top1 from the nucleolus to the nucleoplasm, which is independent of Top1-PARP1 interaction. Using fluorescence recovery after photobleaching and subsequent fitting of the data employing kinetic modelling we demonstrate that ABT-888 markedly increase CPT-induced bound/immobile fraction of Top1 (Top1cc) across the nuclear genome, suggesting Top1-PARylation counteracts CPT-induced stabilization of Top1cc. We further show Trp205 and Asn722 of Top1 are critical for subnuclear dynamics. Top1 mutant (N722S) was restricted to the nucleolus in the presence of CPT due to its deficiency in the accumulation of CPT-induced Top1-PARylation and Top1cc formation. This work identifies ADP-ribose polymers as key determinant for regulating Top1 subnuclear dynamics.


Assuntos
Camptotecina/farmacologia , Núcleo Celular/metabolismo , DNA Topoisomerases Tipo I/metabolismo , Poli Adenosina Difosfato Ribose/metabolismo , Benzimidazóis/farmacologia , Nucléolo Celular/efeitos dos fármacos , Nucléolo Celular/metabolismo , Núcleo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , DNA/metabolismo , Difusão , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Recuperação de Fluorescência Após Fotodegradação , Proteínas de Fluorescência Verde/metabolismo , Células HCT116 , Humanos , Cinética , Proteínas Mutantes/metabolismo , Plasmídeos/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia
11.
Eur J Med Chem ; 102: 540-51, 2015 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-26312433

RESUMO

DNA topoisomerase I is a potential chemotherapeutic target. Here, we designed and synthesized a library comprising of hydantoin and thiohydantoin derivatives and tested them against human and Leishmania Top1. One of the thiohydantoin compounds with substituted thiophenyl as the central moiety (compound 15) exhibited potent inhibition of human Top1 (HTop1) through stabilization of Top1-DNA cleavage complexes and showed selective anticancer activity against human cervical carcinoma (HeLa) and breast carcinoma (MCF-7) cell lines. Molecular modeling studies with HTop1 rationalized the inhibitory mechanism of compound 15.


Assuntos
Antineoplásicos/farmacologia , DNA Topoisomerases Tipo I/metabolismo , Desenho de Fármacos , Tioidantoínas/farmacologia , Inibidores da Topoisomerase I/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Células HEK293 , Células HeLa , Humanos , Células MCF-7 , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade , Tioidantoínas/síntese química , Tioidantoínas/química , Inibidores da Topoisomerase I/síntese química , Inibidores da Topoisomerase I/química
12.
DNA Repair (Amst) ; 19: 114-29, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24856239

RESUMO

TDP1 and TDP2 were discovered and named based on the fact they process 3'- and 5'-DNA ends by excising irreversible protein tyrosyl-DNA complexes involving topoisomerases I and II, respectively. Yet, both enzymes have an extended spectrum of activities. TDP1 not only excises trapped topoisomerases I (Top1 in the nucleus and Top1mt in mitochondria), but also repairs oxidative damage-induced 3'-phosphoglycolates and alkylation damage-induced DNA breaks, and excises chain terminating anticancer and antiviral nucleosides in the nucleus and mitochondria. The repair function of TDP2 is devoted to the excision of topoisomerase II- and potentially topoisomerases III-DNA adducts. TDP2 is also essential for the life cycle of picornaviruses (important human and bovine pathogens) as it unlinks VPg proteins from the 5'-end of the viral RNA genome. Moreover, TDP2 has been involved in signal transduction (under the former names of TTRAP or EAPII). The DNA repair partners of TDP1 include PARP1, XRCC1, ligase III and PNKP from the base excision repair (BER) pathway. By contrast, TDP2 repair functions are coordinated with Ku and ligase IV in the non-homologous end joining pathway (NHEJ). This article summarizes and compares the biochemistry, functions, and post-translational regulation of TDP1 and TDP2, as well as the relevance of TDP1 and TDP2 as determinants of response to anticancer agents. We discuss the rationale for developing TDP inhibitors for combinations with topoisomerase inhibitors (topotecan, irinotecan, doxorubicin, etoposide, mitoxantrone) and DNA damaging agents (temozolomide, bleomycin, cytarabine, and ionizing radiation), and as novel antiviral agents.


Assuntos
DNA Topoisomerases Tipo I/genética , Neoplasias/genética , Proteínas Nucleares/genética , Diester Fosfórico Hidrolases/genética , Transdução de Sinais/genética , Fatores de Transcrição/genética , Animais , Antineoplásicos/farmacologia , Antivirais/farmacologia , Bovinos , Reparo do DNA por Junção de Extremidades/genética , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Proteínas Nucleares/efeitos dos fármacos , Proteínas Nucleares/metabolismo , Diester Fosfórico Hidrolases/efeitos dos fármacos , Diester Fosfórico Hidrolases/metabolismo , Picornaviridae/enzimologia , Picornaviridae/genética , Picornaviridae/patogenicidade , RNA Viral/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/efeitos dos fármacos , Fatores de Transcrição/metabolismo
13.
Nucleic Acids Res ; 42(7): 4435-49, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24493735

RESUMO

Poly(ADP-ribose) polymerases (PARP) attach poly(ADP-ribose) (PAR) chains to various proteins including themselves and chromatin. Topoisomerase I (Top1) regulates DNA supercoiling and is the target of camptothecin and indenoisoquinoline anticancer drugs, as it forms Top1 cleavage complexes (Top1cc) that are trapped by the drugs. Endogenous and carcinogenic DNA lesions can also trap Top1cc. Tyrosyl-DNA phosphodiesterase 1 (TDP1), a key repair enzyme for trapped Top1cc, hydrolyzes the phosphodiester bond between the DNA 3'-end and the Top1 tyrosyl moiety. Alternative repair pathways for Top1cc involve endonuclease cleavage. However, it is unknown what determines the choice between TDP1 and the endonuclease repair pathways. Here we show that PARP1 plays a critical role in this process. By generating TDP1 and PARP1 double-knockout lymphoma chicken DT40 cells, we demonstrate that TDP1 and PARP1 are epistatic for the repair of Top1cc. The N-terminal domain of TDP1 directly binds the C-terminal domain of PARP1, and TDP1 is PARylated by PARP1. PARylation stabilizes TDP1 together with SUMOylation of TDP1. TDP1 PARylation enhances its recruitment to DNA damage sites without interfering with TDP1 catalytic activity. TDP1-PARP1 complexes, in turn recruit X-ray repair cross-complementing protein 1 (XRCC1). This work identifies PARP1 as a key component driving the repair of trapped Top1cc by TDP1.


Assuntos
Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Animais , Linhagem Celular Tumoral , DNA Topoisomerases Tipo I/metabolismo , Epistasia Genética , Humanos , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/genética , Poli(ADP-Ribose) Polimerases/química , Poli(ADP-Ribose) Polimerases/genética , Domínios e Motivos de Interação entre Proteínas , Sumoilação , Proteína 1 Complementadora Cruzada de Reparo de Raio-X
14.
DNA Repair (Amst) ; 13: 1-9, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24355542

RESUMO

Tyrosyl-DNA-phosphodiesterase 1 (TDP1) repairs 3'-blocking DNA lesions by catalytically hydrolyzing the tyrosyl-DNA-phosphodiester bond of trapped topoisomerase I (Top1) cleavage complexes (Top1cc). It also removes 3'-blocking residues derived from oxidative damage or incorporation of chain terminating anticancer and antiviral nucleosides. Thus, TDP1 is regarded as a determinant of resistance to Top1 inhibitors and chain terminating nucleosides, and possibly of genomic stability. In the 60 cell lines of the NCI Developmental Therapeutic Anticancer Screen (the NCI-60), whose whole genome transcriptome and mutations have recently been characterized, we discovered two human lung cancer cell lines deficient for TDP1 (NCI_H522 and HOP_62). HOP_62 shows undetectable TDP1 mRNA and NCI_H522 bears a homozygous deleterious mutation of TDP1 at a highly conserved amino acid residue (K292E). Absence of TDP1 protein and lack of TDP1 catalytic activity were demonstrated in cell lysates from both cell lines. Lack of TDP1 expression in HOP_62 was shown to be due to TDP1 promoter hypermethylation. Our study provides insights into the possible inactivation of TDP1 in cancers and its relationship to cellular response to Top1-targeted drugs. It also reveals two TDP1 knockout lung cancer cell lines for further TDP1 functional analyses.


Assuntos
DNA Topoisomerases Tipo I/metabolismo , Variação Genética , Neoplasias Pulmonares/genética , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Sequência de Aminoácidos , Linhagem Celular Tumoral , Sequência Conservada , Metilação de DNA , DNA Topoisomerases Tipo I/genética , Epigênese Genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação , National Cancer Institute (U.S.) , Regiões Promotoras Genéticas , Estados Unidos
15.
Cancer Res ; 72(21): 5588-99, 2012 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-23118055

RESUMO

Small-molecule inhibitors of PARP are thought to mediate their antitumor effects as catalytic inhibitors that block repair of DNA single-strand breaks (SSB). However, the mechanism of action of PARP inhibitors with regard to their effects in cancer cells is not fully understood. In this study, we show that PARP inhibitors trap the PARP1 and PARP2 enzymes at damaged DNA. Trapped PARP-DNA complexes were more cytotoxic than unrepaired SSBs caused by PARP inactivation, arguing that PARP inhibitors act in part as poisons that trap PARP enzyme on DNA. Moreover, the potency in trapping PARP differed markedly among inhibitors with niraparib (MK-4827) > olaparib (AZD-2281) >> veliparib (ABT-888), a pattern not correlated with the catalytic inhibitory properties for each drug. We also analyzed repair pathways for PARP-DNA complexes using 30 genetically altered avian DT40 cell lines with preestablished deletions in specific DNA repair genes. This analysis revealed that, in addition to homologous recombination, postreplication repair, the Fanconi anemia pathway, polymerase ß, and FEN1 are critical for repairing trapped PARP-DNA complexes. In summary, our study provides a new mechanistic foundation for the rational application of PARP inhibitors in cancer therapy.


Assuntos
Reparo do DNA/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases , Animais , Antineoplásicos/farmacologia , Aves , Linhagem Celular , Técnicas de Inativação de Genes , Humanos , Immunoblotting , Ftalazinas/farmacologia , Piperazinas/farmacologia , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/efeitos dos fármacos , Poli(ADP-Ribose) Polimerases/metabolismo , RNA Interferente Pequeno , Transfecção
16.
J Biol Chem ; 287(16): 12848-57, 2012 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-22375014

RESUMO

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) repairs topoisomerase I cleavage complexes (Top1cc) by hydrolyzing their 3'-phosphotyrosyl DNA bonds and repairs bleomycin-induced DNA damage by hydrolyzing 3'-phosphoglycolates. Yeast Tdp1 has also been implicated in the repair of topoisomerase II-DNA cleavage complexes (Top2cc). To determine whether vertebrate Tdp1 is involved in the repair of various DNA end-blocking lesions, we generated Tdp1 knock-out cells in chicken DT40 cells (Tdp1-/-) and Tdp1-complemented DT40 cells with human TDP1. We found that Tdp1-/- cells were not only hypersensitive to camptothecin and bleomycin but also to etoposide, methyl methanesulfonate (MMS), H(2)O(2), and ionizing radiation. We also show they were deficient in mitochondrial Tdp1 activity. In biochemical assays, recombinant human TDP1 was found to process 5'-phosphotyrosyl DNA ends when they mimic the 5'-overhangs of Top2cc. Tdp1 also processes 3'-deoxyribose phosphates generated from hydrolysis of abasic sites, which is consistent with the hypersensitivity of Tdp1-/- cells to MMS and H(2)O(2). Because recent studies established that CtIP together with BRCA1 also repairs topoisomerase-mediated DNA damage, we generated dual Tdp1-CtIP-deficient DT40 cells. Our results show that Tdp1 and CtIP act in parallel pathways for the repair of Top1cc and MMS-induced lesions but are epistatic for Top2cc. Together, our findings reveal a broad involvement of Tdp1 in DNA repair and clarify the role of human TDP1 in the repair of Top2-induced DNA damage.


Assuntos
Antígenos de Neoplasias/metabolismo , Dano ao DNA/fisiologia , Reparo do DNA/fisiologia , DNA Topoisomerases Tipo II/metabolismo , DNA Topoisomerases Tipo I/metabolismo , Proteínas de Ligação a DNA/metabolismo , Neoplasias/enzimologia , Diester Fosfórico Hidrolases/metabolismo , Alquilação/fisiologia , Sequência de Aminoácidos , Animais , Proteínas de Transporte/metabolismo , Células Cultivadas , Galinhas , DNA de Cadeia Simples/metabolismo , Humanos , Dados de Sequência Molecular , Neoplasias/genética , Proteínas Nucleares/metabolismo , Diester Fosfórico Hidrolases/genética , Proteínas de Ligação a Poli-ADP-Ribose , Especificidade por Substrato/fisiologia , Vertebrados
17.
Proc Natl Acad Sci U S A ; 107(46): 19790-5, 2010 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-21041670

RESUMO

Human tyrosyl-DNA phosphodiesterase (TDP1) hydrolyzes the phosphodiester bond at a DNA 3'-end linked to a tyrosyl moiety and has been implicated in the repair of topoisomerase I (Top1)-DNA covalent complexes. TDP1 can also hydrolyze other 3'-end DNA alterations including 3'-phosphoglycolate and 3'-abasic sites, and exhibits 3'-nucleosidase activity indicating it may function as a general 3'-end-processing DNA repair enzyme. Here, using laser confocal microscopy, subcellular fractionation and biochemical analyses we demonstrate that a fraction of the TDP1 encoded by the nuclear TDP1 gene localizes to mitochondria. We also show that mitochondrial base excision repair depends on TDP1 activity and provide evidence that TDP1 is required for efficient repair of oxidative damage in mitochondrial DNA. Together, our findings provide evidence for TDP1 as a novel mitochondrial enzyme.


Assuntos
Mitocôndrias/enzimologia , Diester Fosfórico Hidrolases/metabolismo , Animais , Sequência de Bases , Extratos Celulares , Linhagem Celular Tumoral , Dano ao DNA , DNA Ligase Dependente de ATP , DNA Ligases/metabolismo , Reparo do DNA , DNA Mitocondrial/metabolismo , Humanos , Camundongos , Dados de Sequência Molecular , Estresse Oxidativo , Proteínas de Ligação a Poli-ADP-Ribose , Transporte Proteico , Proteínas de Xenopus
18.
EMBO J ; 28(23): 3667-80, 2009 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-19851285

RESUMO

Human tyrosyl-DNA phosphodiesterase (TDP1) hydrolyzes the phosphodiester bond at a DNA 3' end linked to a tyrosyl moiety. This type of linkage is found at stalled topoisomerase I (Top1)-DNA covalent complexes, and TDP1 has been implicated in the repair of such complexes. Here we show that Top1-associated DNA double-stranded breaks (DSBs) induce the phosphorylation of TDP1 at S81. This phosphorylation is mediated by the protein kinases: ataxia-telangiectasia-mutated (ATM) and DNA-dependent protein kinase (DNA-PK). Phosphorylated TDP1 forms nuclear foci that co-localize with those of phosphorylated histone H2AX (gammaH2AX). Both Top1-induced replication- and transcription-mediated DNA damages induce TDP1 phosphorylation. Furthermore, we show that S81 phosphorylation stabilizes TDP1, induces the formation of XRCC1 (X-ray cross-complementing group 1)-TDP1 complexes and enhances the mobilization of TDP1 to DNA damage sites. Finally, we provide evidence that TDP1-S81 phosphorylation promotes cell survival and DNA repair in response to CPT-induced DSBs. Together; our findings provide a new mechanism for TDP1 post-translational regulation by ATM and DNA-PK.


Assuntos
Proteínas de Ciclo Celular/química , Reparo do DNA , Proteína Quinase Ativada por DNA/química , Proteínas de Ligação a DNA/química , Diester Fosfórico Hidrolases/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Supressoras de Tumor/química , Ataxia Telangiectasia/enzimologia , Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia , Carnitina O-Palmitoiltransferase/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/fisiologia , Sobrevivência Celular/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Proteína Quinase Ativada por DNA/metabolismo , Proteína Quinase Ativada por DNA/fisiologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Humanos , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/fisiologia , Fosforilação/genética , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Serina/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Supressoras de Tumor/fisiologia , Proteína 1 Complementadora Cruzada de Reparo de Raio-X
19.
Mol Pharmacol ; 74(5): 1292-307, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18703668

RESUMO

Mitochondria are the principal site for the generation of cellular ATP by oxidative phosphorylation. F0F1-ATP synthase, a complex V of the electron transport chain, is an important constituent of mitochondria-dependent signaling pathways involved in apoptosis. In the present study, we have shown for the first time that 3,3'-diindolylmethane (DIM), a DNA topoisomerase I poison, inhibits mitochondrial F0F1-ATP synthase of Leishmania donovani and induces programmed cell death (PCD), which is a novel insight into the mechanism in protozoan parasites. DIM-induced inhibition of F0F1-ATP synthase activity causes depletion of mitochondrial ATP levels and significant stimulation of mitochondrial reactive oxygen species (ROS) production, followed by depolarization of mitochondrial membrane potential (DeltaPsi(m)). Because DeltaPsi(m) is the driving force for mitochondrial ATP synthesis, loss of DeltaPsi(m) results in depletion of cellular ATP level. The loss of DeltaPsi(m) causes the cellular ROS generation and in turn leads to the oxidative DNA lesions followed by DNA fragmentation. In contrast, loss of DeltaPsi(m) leads to release of cytochrome c into the cytosol and subsequently activates the caspase-like proteases, which lead to oligonucleosomal DNA cleavage. We have also shown that mitochondrial DNA-depleted cells are insensitive to DIM to induce PCD. Therefore, mitochondria are necessary for cytotoxicity of DIM in kinetoplastid parasites. Taken together, our study indicates for the first time that DIM-induced mitochondrial dysfunction by inhibition of F0F1-ATP synthase activity leads to PCD in Leishmania spp. parasites, which could be exploited to develop newer potential therapeutic targets.


Assuntos
Anticarcinógenos/farmacologia , Apoptose/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Indóis/farmacologia , Leishmania donovani/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , ATPases Translocadoras de Prótons/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Replicação do DNA , Ativação Enzimática , Glutationa/metabolismo , Leishmania donovani/enzimologia , Peroxidação de Lipídeos/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Estresse Oxidativo/efeitos dos fármacos
20.
Adv Exp Med Biol ; 625: 103-15, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18365662

RESUMO

Protozoan parasites of the genus Leishmania cause severe diseases that threaten human beings, both for the high mortality rates involved and the economic loss resulting from morbidity, primarily in the tropical and subtropical areas. This ancient eukaryote shows variable genetic diversity in their life cycle, wherein DNA topoisomerases play a key role in cellular processes affecting the topology and organization ofintracellular DNA. Kinetoplastid topoisomerases offer most attractive targets for their structural diversity from other eukaryotic counterparts and their indispensable function in cell biology Therefore, understanding the biology of kinetoplastid topoisomerases and the components and steps involved in this intricate process provide opportunities for target based drug designing against protozoan parasitic diseases.


Assuntos
Inibidores Enzimáticos/farmacologia , Leishmania/efeitos dos fármacos , Leishmania/enzimologia , Leishmaniose/tratamento farmacológico , Inibidores da Topoisomerase , Tripanossomicidas/farmacologia , Animais , DNA Topoisomerases/classificação , Sistemas de Liberação de Medicamentos , Humanos , Leishmania/genética , Leishmaniose/enzimologia , Leishmaniose/parasitologia , Proteínas de Protozoários/efeitos dos fármacos , Proteínas de Protozoários/genética
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