RESUMO
Tardigrades are microscopic animals renowned for their ability to withstand extreme conditions, including high doses of ionizing radiation (IR). To better understand their radio-resistance, we first characterized induction and repair of DNA double- and single-strand breaks after exposure to IR in the model species Hypsibius exemplaris. Importantly, we found that the rate of single-strand breaks induced was roughly equivalent to that in human cells, suggesting that DNA repair plays a predominant role in tardigrades' radio-resistance. To identify novel tardigrade-specific genes involved, we next conducted a comparative transcriptomics analysis across three different species. In all three species, many DNA repair genes were among the most strongly overexpressed genes alongside a novel tardigrade-specific gene, which we named Tardigrade DNA damage Response 1 (TDR1). We found that TDR1 protein interacts with DNA and forms aggregates at high concentration suggesting it may condensate DNA and preserve chromosome organization until DNA repair is accomplished. Remarkably, when expressed in human cells, TDR1 improved resistance to Bleomycin, a radiomimetic drug. Based on these findings, we propose that TDR1 is a novel tardigrade-specific gene conferring resistance to IR. Our study sheds light on mechanisms of DNA repair helping cope with high levels of DNA damage inflicted by IR.
Assuntos
Reparo do DNA , Proteínas de Ligação a DNA , Radiação Ionizante , Tardígrados , Transcriptoma , Tardígrados/genética , Tardígrados/metabolismo , Animais , Humanos , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Perfilação da Expressão Gênica , Dano ao DNA , Tolerância a Radiação/genéticaRESUMO
Gene-directed enzyme pro-drug therapy (GDEPT) consists of expressing, in tumor cells, a suicide gene which converts a pro-drug into cytotoxic metabolites, in situ. In a previous work, we demonstrated that the combination of the suicide gene CYP2B6TM-RED (a fusion of a triple mutant of CYP2B6 with NADPH cytochrome P450 reductase) and cyclophosphamide (CPA) constituted a powerful treatment for solid tumors. In this work, we investigated the use of mesenchymal stem cells (MSCs) as cellular vehicles for the delivery of our suicide gene. MSCs were genetically engineered ex-vivo to stably express CYP2B6TM-RED. Ex vivo and in vivo investigations showed that MSCs expressing CYP2B6TM-RED were able 1) to bioactivate CPA and produce local cytotoxic metabolites in tumor sites and 2) to destroy neighboring tumor cells through a bystander effect. Intratumoral injections of CYP2B6TM-RED-MSCs and CPA completely eradicated tumors in 33% of mice without recurrence after 6months. Rechallenge experiments demonstrated an efficient immune response. These data suggest that MSCs expressing CYP2B6TM-RED with CPA could represent a promising treatment for solid tumors to test in future clinical trials.
Assuntos
Genes Transgênicos Suicidas/genética , Engenharia Genética/métodos , Terapia Genética/métodos , Vetores Genéticos/genética , Células-Tronco Mesenquimais/fisiologia , Neoplasias/genética , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Modelos Animais de Doenças , Feminino , Vetores Genéticos/administração & dosagem , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/terapiaRESUMO
DNA methyltransferases (DNMT) are promising drug targets in cancer provided that new, more specific, and chemically stable inhibitors are discovered. Among the non-nucleoside DNMT inhibitors, N-phthaloyl-l-tryptophan 1 (RG108) was first identified as inhibitor of DNMT1. Here, 1 analogues were synthesized to understand its interaction with DNMT. The indole, carboxylate, and phthalimide moieties were modified. Homologated and conformationally constrained analogues were prepared. The latter were synthesized from prolinohomotryptophan derivatives through a methodology based amino-zinc-ene-enolate cyclization. All compounds were tested for their ability to inhibit DNMT1 in vitro. Among them, constrained compounds 16-18 and NPys derivatives 10-11 were found to be at least 10-fold more potent than the reference compound. The cytotoxicity on the tumor DU145 cell line of the most potent inhibitors was correlated to their inhibitory potency. Finally, docking studies were conducted in order to understand their binding mode. This study provides insights for the design of the next-generation of DNMT inhibitors.
Assuntos
DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , Ftalimidas/síntese química , Triptofano/análogos & derivados , Domínio Catalítico , Linhagem Celular Tumoral , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/química , Humanos , Simulação de Acoplamento Molecular , Ácidos Ftálicos/síntese química , Ácidos Ftálicos/química , Ácidos Ftálicos/farmacologia , Ftalimidas/química , Ftalimidas/farmacologia , Estereoisomerismo , Relação Estrutura-Atividade , Triptofano/síntese química , Triptofano/química , Triptofano/farmacologiaRESUMO
In order to discover new inhibitors of the DNA methyltransferase 3A/3L complex, we used a medium-throughput nonradioactive screen on a random collection of 1120 small organic compounds. After a primary hit detection against DNA methylation activity of the murine Dnmt3A/3L catalytic complex, we further evaluated the EC50 of the 12 most potent hits as well as their cytotoxicity on DU145 prostate cancer cultured cells. Interestingly, most of the inhibitors showed low micromolar activities and little cytotoxicity. Dichlone, a small halogenated naphthoquinone, classically used as pesticide and fungicide, showed the lowest EC50 at 460 nM. We briefly assessed the selectivity of a subset of our new inhibitors against hDNMT1 and bacterial Dnmts, including M. SssI and EcoDam, and the protein lysine methyltransferase PKMT G9a and the mode of inhibition. Globally, the tested molecules showed a clear preference for the DNA methyltransferases, but poor selectivity among them. Two molecules including Dichlone efficiently reactivated YFP gene expression in a stable HEK293 cell line by promoter demethylation. Their efficacy was comparable to the DNMT inhibitor of reference 5-azacytidine.
Assuntos
DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , Metilação de DNA/efeitos dos fármacos , DNA/metabolismo , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/análise , Inibidores Enzimáticos/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Morte Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA Metiltransferase 3A , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Células HEK293 , Humanos , Camundongos , Estrutura Molecular , Bibliotecas de Moléculas Pequenas/análise , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Retinoic acid receptor ß 2 (RARß2) is a tumor suppressor gene whose loss of expression is recurrent in prostate cancers. Here we studied the epigenetic mechanisms leading to its stable silencing. First, we characterized all RARß isoforms in 6 human tumor cell lines (prostate DU145, LNCaP, PC3, lung A549, breast Hs578T, and colon HCT116) by RT-PCR and Western blot. We excluded loss of heterozygosity (2D-FISH) and loss of RARa expression, an upstream regulator, as origin of RARß2 silencing. All data concluded to an epigenetic silencing. In agreement, a DNA methylation inhibitor restored its expression. Second RARß2 loss of expression was found associated with different epigenetic profiles in LNCaP and DU145 cells. According to bisulfite sequencing and ChIP analysis, we observed heavy methylation (97%) of the RARß2 promoter with repressive histone mark H3K9me3 in LNCaP. While DNA methylation and polycomb repression are described to be mutually exclusive at CpG-rich promoters, we observed that in DU145, moderate DNA methylation (36%) and H3K9me3 mark were present concomitantly with H3K27me3, a signature of polycomb repression. In summary, we provide new insights on how the RARß2 promoter is silenced, reveal the existence of two distinct repressive chromatin profiles at the same locus, and support a polycomb-mediated epigenetic repression process in prostate cancer.
Assuntos
Metilação de DNA , Receptores do Ácido Retinoico/metabolismo , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Linhagem Celular Tumoral , Cromatina/efeitos dos fármacos , Ilhas de CpG/efeitos dos fármacos , Ilhas de CpG/genética , Metilação de DNA/efeitos dos fármacos , Decitabina , Inibidores Enzimáticos/farmacologia , Epigênese Genética/fisiologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Inativação Gênica/efeitos dos fármacos , Histonas/genética , Histonas/metabolismo , Humanos , Neoplasias/genética , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Receptores do Ácido Retinoico/genéticaRESUMO
DNA methyltransferases (DNMTs) are responsible for DNA methylation, an epigenetic modification involved in gene regulation. Families of conjugates of procainamide, an inhibitor of DNMT1, were conceived and produced by rapid synthetic pathways. Six compounds resulted in potent inhibitors of the murine catalytic Dnmt3A/3L complex and of human DNMT1, at least 50 times greater than that of the parent compounds. The inhibitors showed selectivity for C5 DNA methyltransferases. The cytotoxicity of the inhibitors was validated on two tumour cell lines (DU145 and HCT116) and correlated with the DNMT inhibitory potency. The inhibition potency of procainamide conjugated to phthalimide through alkyl linkers depended on the length of the linker; the dodecane linker was the best.
Assuntos
Antineoplásicos/farmacologia , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Procainamida/análogos & derivados , Procainamida/química , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , DNA (Citosina-5-)-Metiltransferases/metabolismo , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/química , Humanos , Camundongos , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
DNA methylation is involved in the regulation of gene expression and plays an important role in normal developmental processes and diseases, such as cancer. DNA methyltransferases are the enzymes responsible for DNA methylation on the position 5 of cytidine in a CpG context. In order to identify and characterize novel inhibitors of these enzymes, we developed a fluorescence-based throughput screening by using a short DNA duplex immobilized on 96-well plates. We have screened 114 flavones and flavanones for the inhibition of the murine catalytic Dnmt3a/3L complex and found 36 hits with IC(50) values in the lower micromolar and high nanomolar ranges. The assay, together with inhibition tests on two other methyltransferases, structure-activity relationships and docking studies, gave insights on the mechanism of inhibition. Finally, two derivatives effected zebrafish embryo development, and induced a global demethylation of the genome, at doses lower than the control drug, 5-azacytidine.
Assuntos
DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , Desenvolvimento Embrionário/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Peixe-Zebra/embriologia , Animais , Sequência de Bases , Cristalografia por Raios X , DNA (Citosina-5-)-Metiltransferases/química , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Bibliotecas de Moléculas Pequenas/químicaRESUMO
In mammals DNA methylation occurs at position 5 of cytosine in a CpG context and regulates gene expression. It plays an important role in diseases and inhibitors of DNA methyltransferases (DNMTs)--the enzymes responsible for DNA methylation--are used in clinics for cancer therapy. The most potent inhibitors are 5-azacytidine and 5-azadeoxycytidine. Zebularine (1-(beta-D-ribofuranosyl)-2(1H)- pyrimidinone) is another cytidine analog described as a potent inhibitor that acts by forming a covalent complex with DNMT when incorporated into DNA. Here we bring additional experiments to explain its mechanism of action. First, we observe an increase in the DNA binding when zebularine is incorporated into the DNA, compared to deoxycytidine and 5-fluorodeoxycytidine, together with a strong decrease in the dissociation rate. Second, we show by denaturing gel analysis that the intermediate covalent complex between the enzyme and the DNA is reversible, differing thus from 5-fluorodeoxycytidine. Third, no methylation reaction occurs when zebularine is present in the DNA. We confirm that zebularine exerts its demethylation activity by stabilizing the binding of DNMTs to DNA, hindering the methylation and decreasing the dissociation, thereby trapping the enzyme and preventing turnover even at other sites.
Assuntos
Citidina/análogos & derivados , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , DNA (Citosina-5-)-Metiltransferases/metabolismo , Inibidores Enzimáticos/farmacologia , Animais , Azacitidina/análogos & derivados , Azacitidina/química , Azacitidina/farmacologia , Sequência de Bases , Citidina/química , Citidina/farmacologia , DNA/genética , DNA/metabolismo , DNA (Citosina-5-)-Metiltransferases/química , Decitabina , Humanos , Metilação/efeitos dos fármacos , CamundongosRESUMO
We and others have clearly demonstrated that a topoisomerase I (Top1) inhibitor, such as camptothecin (CPT), coupled to a triplex-forming oligonucleotide (TFO) through a suitable linker can be used to cause site-specific cleavage of the targeted DNA sequence in in vitro models. Here we evaluated whether these molecular tools induce sequence-specific DNA damage in a genome context. We targeted the insulin-like growth factor (IGF)-I axis and in particular promoter 1 of IGF-I and intron 2 of type 1 insulin-like growth factor receptor (IGF-IR) in cancer cells. The IGF axis molecules represent important targets for anticancer strategies, because of their central role in oncogenic maintenance and metastasis processes. We chemically attached 2 CPT derivatives to 2 TFOs. Both conjugates efficiently blocked gene expression in cells, reducing the quantity of mRNA transcribed by 70-80%, as measured by quantitative RT-PCR. We confirmed that the inhibitory mechanism of these TFO conjugates was mediated by Top1-induced cleavage through the use of RNA interference experiments and a camptothecin-resistant cell line. In addition, induction of phospho-H2AX foci supports the DNA-damaging activity of TFO-CPT conjugates at specific sites. The evaluated conjugates induce a specific DNA damage at the target gene mediated by Top1.
Assuntos
Camptotecina/farmacologia , Dano ao DNA/efeitos dos fármacos , Fator de Crescimento Insulin-Like I/antagonistas & inibidores , Receptor IGF Tipo 1/antagonistas & inibidores , Inibidores da Topoisomerase I , Animais , Antineoplásicos Fitogênicos , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Inibidores Enzimáticos , Humanos , Fator de Crescimento Insulin-Like I/genética , Regiões Promotoras Genéticas , Ratos , Receptor IGF Tipo 1/genéticaRESUMO
Reversal of the multidrug-resistant (MDR) phenotype is very important for chemotherapy success. In fact, the expression of the MDR1 gene-encoded P-glycoprotein (P-gp) actively expels antitumor agents such as daunomycin (DNM) out of the cells, resulting in drug resistance. We show that upon conjugation to triplex-forming oligonucleotides, it is possible to address DNM in resistant cells (MCF7-R and NIH-MDR-G185). The oligonucleotide moiety of the conjugate changes the cellular penetration properties of the antitumor agent that is no more the target of P-gp in resistant cells. We observe an accumulation of conjugated DNM in cells up to 72 h. For more efficient delivery in the cells' nuclei, transfectant agents must be used. In addition, the conjugate recognizes a sequence located in exon 3 of MDR1, and it inhibits its gene expression as measured both by Western blot and by reverse transcription-polymerase chain reaction.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , DNA/farmacologia , Daunorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Oligonucleotídeos/síntese química , Oligonucleotídeos/farmacologia , Animais , Sequência de Bases , Linhagem Celular Tumoral , DNA/síntese química , Daunorrubicina/química , Regulação para Baixo/efeitos dos fármacos , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Cinética , Camundongos , Dados de Sequência Molecular , Células NIH 3T3 , Espectrometria de Fluorescência , TransfecçãoRESUMO
DNA and RNA oligomers that contain stretches of guanines can associate to form stable secondary structures including G-quadruplexes. Our study shows that the (UUAAAAGAAAAGGGGGGAU) RNA sequence, from the human immunodeficiency virus type 1 (HIV-1 polypurine tract or PPT sequence) forms in vitro a stable folded structure involving the G-run. We have investigated the ability of pyrimidine peptide nucleic acid (PNA) oligomers targeted to the PPT sequence to invade the folded RNA and exhibit biological activity at the translation level in vitro and in cells. We find that PNAs can form stable complexes even with the structured PPT RNA target at neutral pH. We show that T-rich PNAs, namely the tridecamer-I PNA (C4T4CT4) forms triplex structures whereas the C-rich tridecamer-II PNA (TC6T4CT) likely forms a duplex with the target RNA. Interestingly, we find that both C-rich and T-rich PNAs arrested in vitro translation elongation specifically at the PPT target site. Finally, we show that T-rich and C-rich tridecamer PNAs that have been identified as efficient and specific blockers of translation elongation in vitro, specifically inhibit translation in streptolysin-O permeabilized cells where the PPT target sequence has been introduced upstream the reporter luciferase gene.
Assuntos
HIV-1/genética , Oligonucleotídeos Antissenso/farmacologia , Elongação Traducional da Cadeia Peptídica/efeitos dos fármacos , Ácidos Nucleicos Peptídicos/farmacologia , RNA Viral/efeitos dos fármacos , Proteínas de Bactérias , Sequência de Bases , Ensaio de Desvio de Mobilidade Eletroforética , Genes Reporter , Células HeLa , Humanos , Conformação de Ácido Nucleico , Desnaturação de Ácido Nucleico , Oligonucleotídeos Antissenso/química , Ácidos Nucleicos Peptídicos/química , Purinas/análise , RNA Viral/química , Estreptolisinas , Temperatura , Proteínas Virais/biossínteseRESUMO
The serial analysis of gene expression (SAGE) is a powerful method to compare gene expression of mRNA populations. To provide quantitative expression levels on a genome-wide scale, the Cancer Genome Anatomy Project (CGAP) uses SAGE. Over 7 million SAGE tags, from 171 human cell types have been assembled. The growing number of laboratories involved in SAGE research necessitates the use of software that provides statistical analysis of raw data, allowing the rapid visualization and interpretation of results. We have created the first simple tool that performs statistical analysis on SAGE data, identifies the tags differentially expressed and shows the results in a scatter plot. It is freely available and accessible at http://bioserv.rpbs.jussieu.fr/websage/index.php.
Assuntos
Etiquetas de Sequências Expressas , Perfilação da Expressão Gênica/métodos , RNA Mensageiro/biossíntese , Software , Linhagem Celular Tumoral , Gráficos por Computador , Interpretação Estatística de Dados , Feminino , Humanos , Internet , Pessoa de Meia-IdadeRESUMO
Recently, we showed that antisense peptide nucleic acids (PNA) containing a short pyrimidine stretch (C(4)TC(3)) invade Ha-ras mRNA hairpin structures to form highly stable duplex and triplex complexes that contribute to the arrest of translation elongation. The antisense PNA targeted to codon 74 of Ha-ras was designed to bind in antiparallel configuration (the N-terminal of the PNA faces the 3'-end of target mRNA), as PNA/RNA duplexes are most stable in this configuration. In order to show that different sequences in the coding region could be targeted successfully with antisense PNAs, we extended our study to three other purine-rich targets. We show that the tridecamer PNA (targeted to codon 149) containing a CTC(3)T pyrimidine stretch forms with the complementary oligoribonucleotide (ORN) a stable (PNA)(2)/ORN triplex at neutral pH (T(m) = 50 degrees C) and arrests Ha-ras mRNA translation elongation. Interestingly, the thermal stability of triplexes formed with PNAs designed to bind to the complementary ORN in a parallel orientation (the N-terminal of the PNA faces the 5'-end of target) was higher than that formed with antiparallel oriented PNAs (T(m) = 58 degrees C). Because parallel and antiparallel PNAs form stable triplexes with target sequence, they act as translation elongation blockers. These duplex-forming and partly triplex-forming PNAs targeted to Ha-ras mRNA also arrested translation elongation at specific polypurine sites contained in the mRNA coding for HIV-integrase protein. Furthermore, the tridecamer PNA containing the C(3)TC(4) motif was more active than a bis-PNA in which the Hoogsteen recognizing strand was linked to the Watson-Crick recognizing strand by a flexible linker. Pyrimidine-rich, short PNAs that form very stable duplexes with target Ha-ras mRNA inhibit translation by a mechanism that does not involve ribosome elongation arrest, whereas PNAs forming duplex and triplex structures arrest ribosome elongation. The remarkable efficacy of the tridecamer PNAs in arresting translation elongation of HIV-1 integrase mRNA is explained by their ability to form stable triplexes at neutral pH with short purine sequences.
Assuntos
Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/farmacologia , Elongação Traducional da Cadeia Peptídica/efeitos dos fármacos , Ácidos Nucleicos Peptídicos/farmacologia , Inibidores da Síntese de Proteínas/química , Inibidores da Síntese de Proteínas/farmacologia , Animais , Sequência de Bases , Ensaio de Desvio de Mobilidade Eletroforética , Integrase de HIV/biossíntese , Integrase de HIV/efeitos dos fármacos , Integrase de HIV/genética , Humanos , Dados de Sequência Molecular , Oligonucleotídeos Antissenso/genética , Elongação Traducional da Cadeia Peptídica/genética , Ácidos Nucleicos Peptídicos/química , Ácidos Nucleicos Peptídicos/genética , Inibidores da Síntese de Proteínas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/biossíntese , Proteínas Proto-Oncogênicas p21(ras)/efeitos dos fármacos , Proteínas Proto-Oncogênicas p21(ras)/genética , Purinas/química , Pirimidinas/química , Ratos , Análise EspectralRESUMO
Recently, we have shown that peptide nucleic acid (PNA) tridecamers targeted to the codon 74, 128 and 149 regions of Ha-ras mRNA arrested translation elongation in vitro. Our data demonstrated for the first time that PNAs with mixed base sequence targeted to the coding region of a messenger RNA could arrest the translation machinery and polypeptide chain elongation. The peculiarity of the complexes formed with PNA tridecamers and Ha-ras mRNA rests upon the stability of PNA-mRNA hybrids, which are not dissociated by cellular proteins or multiple denaturing conditions. In the present study, we show that shorter PNAs such as a dodecamer or an undecamer targeted to the codon 74 region arrest translation elongation in vitro. The 13, 12, and 11-mer PNAs contain eight and the 10-mer PNA seven contiguous pyrimidine residues. Upon binding with parallel Hoogsteen base-pairing to the PNA-RNA duplex, six of the cytosine bases and one thymine base of a second PNA can form C.G*C(+) and T.A*T triplets. Melting experiments show two well-resolved transitions corresponding to the dissociation of the third strand from the core duplex and to melting of duplex at higher temperature. The enzymatic structure mapping of a target 27-mer RNA revealed a hairpin structure that is disrupted upon binding of tri-, dodeca-, undeca- and decamer PNAs. We show that the non-bonded nucleobase overhangs on the RNA stabilize the PNA-RNA hybrids and probably assist the PNA in overcoming the stable secondary structure of the RNA target. The great stability of PNA-RNA duplex and triplex structures allowed us to identify both 1:1 and 2:1 PNA-RNA complexes using matrix-assisted laser desorption/ionization time-of -flight mass spectrometry. Therefore, it is possible to successfully target mixed sequences in structured regions of messenger RNA with short PNA oligonucleotides that form duplex and triplex structures that can arrest elongating ribosomes.