RESUMO
α-Amanitin is one of the primary toxins produced by the poisonous mushroom genus, Amanita. Because it is odorless and tasteless, it is an important cause of death from the consumption of misidentified mushrooms. To study the thermal stability of α-amanitin, novel cell-based assays were developed to measure the toxin's activity, based on the inhibition of RNA polymerase II by α-amanitin. First, an MTT-formazan cell viability assay was used to measure the biological activity of α-amanitin through the inhibition of cellular activity. This method can detect 10 µg/mL of α-amanitin in a time-dependent manner. Second, a more sensitive quantitative PCR approach was developed to examine its inhibition of viral replication. The new RT-qPCR assay enabled the detection of 100 ng/mL. At this level, α-amanitin still significantly reduced adenovirus transcription. Third, a simpler GFP expression-based assay was developed with an equal sensitivity to the RT-qPCR assay. With this assay, aqueous α-amanitin heated at 90 °C for 16 h or treated in the microwave for 3 min retained its biological activity when tested in HEK293 cells, but a slight reduction was observed when tested in Vero cells. Beyond detecting the activity of α-amanitin, the new method has a potential application for detecting the activity of other toxins that are RNA polymerase inhibitors.
Assuntos
Alfa-Amanitina , RNA Polimerase II , Animais , Chlorocebus aethiops , Humanos , Alfa-Amanitina/farmacologia , Células Vero , Células HEK293 , AmanitaRESUMO
Despite the great success of small molecule inhibitors in the treatment of patients with BRAFV600E mutated melanoma, the response to these drugs remains transient and patients eventually relapse within a few months, highlighting the need to develop novel combination therapies based on the understanding of the molecular changes induced by BRAFV600E inhibitors. The acute inhibition of oncogenic signaling can rewire entire cellular signaling pathways and thereby create novel cancer cell vulnerabilities. Here, we demonstrate that inhibition of BRAFV600E oncogenic signaling in melanoma cell lines leads to destabilization of the large subunit of RNA polymerase II POLR2A (polymerase RNA II DNA-directed polypeptide A), thereby preventing its binding to the unconventional prefoldin RPB5 interactor (URI1) chaperone complex and the successful assembly of RNA polymerase II holoenzymes. Furthermore, in melanoma cell lines treated with mitogen-activated protein kinase (MAPK) inhibitors, α-amanitin, a specific and irreversible inhibitor of RNA polymerase II, induced massive apoptosis. Pre-treatment of melanoma cell lines with MAPK inhibitors significantly reduced IC50 values to α-amanitin, creating a state of collateral vulnerability similar to POLR2A hemizygous deletions. Thus, the development of melanoma specific α-amanitin antibody-drug conjugates could represent an interesting therapeutic approach for combination therapies with BRAFV600E inhibitors.
Assuntos
Alfa-Amanitina/farmacologia , Morte Celular/efeitos dos fármacos , Melanoma/metabolismo , Inibidores da Síntese de Ácido Nucleico/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , RNA Polimerase II/metabolismo , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Transdução de Sinais/efeitos dos fármacosRESUMO
Safeguarding cell function and identity following a genotoxic stress challenge entails a tight coordination of DNA damage signaling and repair with chromatin maintenance. How this coordination is achieved and with what impact on chromatin integrity remains elusive. Here, we address these questions by investigating the mechanisms governing the distribution in mammalian chromatin of the histone variant H2A.X, a central player in damage signaling. We reveal that H2A.X is deposited de novo at sites of DNA damage in a repair-coupled manner, whereas the H2A.Z variant is evicted, thus reshaping the chromatin landscape at repair sites. Our mechanistic studies further identify the histone chaperone FACT (facilitates chromatin transcription) as responsible for the deposition of newly synthesized H2A.X. Functionally, we demonstrate that FACT potentiates H2A.X-dependent signaling of DNA damage. We propose that new H2A.X deposition in chromatin reflects DNA damage experience and may help tailor DNA damage signaling to repair progression.
Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/genética , DNA/genética , Proteínas de Grupo de Alta Mobilidade/genética , Histonas/genética , Fatores de Elongação da Transcrição/genética , Alfa-Amanitina/farmacologia , Animais , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular Tumoral , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , DNA/metabolismo , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Proteínas de Grupo de Alta Mobilidade/metabolismo , Histonas/metabolismo , Humanos , Camundongos , Morfolinas/farmacologia , Células NIH 3T3 , Nucleossomos/química , Nucleossomos/efeitos dos fármacos , Nucleossomos/metabolismo , Venenos/farmacologia , Pirimidinas/farmacologia , Pironas/farmacologia , Transdução de Sinais , Fatores de Elongação da Transcrição/metabolismoRESUMO
Colorectal cancer (CRC) is often diagnosed at an advanced stage when tumor cell dissemination has taken place. Chemo- and targeted therapies provide only a limited increase of overall survival for these patients. The major reason for clinical outcome finds its origin in therapy resistance. Escape mechanisms to both chemo- and targeted therapy remain the main culprits. Here, we evaluate major resistant mechanisms and elaborate on potential new therapies. Amongst promising therapies is α-amanitin antibody-drug conjugate targeting hemizygous p53 loss. It becomes clear that a dynamic interaction with the tumor microenvironment exists and that this dictates therapeutic outcome. In addition, CRC displays a limited response to checkpoint inhibitors, as only a minority of patients with microsatellite instable high tumors is susceptible. In this review, we highlight new developments with clinical potentials to augment responses to checkpoint inhibitors.
Assuntos
Antineoplásicos Imunológicos/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Imunoconjugados/farmacologia , Evasão Tumoral/efeitos dos fármacos , Alfa-Amanitina/farmacologia , Alfa-Amanitina/uso terapêutico , Antineoplásicos Imunológicos/uso terapêutico , Neoplasias Colorretais/genética , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/mortalidade , Receptores Coestimuladores e Inibidores de Linfócitos T/antagonistas & inibidores , Receptores Coestimuladores e Inibidores de Linfócitos T/imunologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Resistencia a Medicamentos Antineoplásicos/imunologia , Humanos , Imunoconjugados/uso terapêutico , Imunoterapia/métodos , Instabilidade de Microssatélites/efeitos dos fármacos , Inibidores da Síntese de Ácido Nucleico/farmacologia , Inibidores da Síntese de Ácido Nucleico/uso terapêutico , RNA Polimerase II/antagonistas & inibidores , Resultado do Tratamento , Evasão Tumoral/genética , Evasão Tumoral/imunologia , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologia , Proteína Supressora de Tumor p53/genéticaRESUMO
The episodic pattern of gonadotropin-releasing hormone (GnRH) secretion from the hypothalamus is driven by an integrated network of cells termed the GnRH pulse generator. Cultured and immortalized GnRH neurons also produce a pulsatile pattern of GnRH secretions when grown in the absence of other cell types, suggesting the presence of an intrinsic oscillator mediating GnRH secretion. The mechanisms underlying such pulsatility comprise one of the most tantalizing problems in contemporary neuroendocrinology. In order to study the mechanism by which GnRH is produced in a pulsatile fashion, the autocrine effect of GnRH on GnRH-producing neurons must be eliminated. This may be performed by downregulating the expression of the GnRH receptor. Treatment with three 21-mer exogenous phosphorothioates and transient transfections with an inducible plasmid containing an antisense construct to the GnRH receptor gene decreased GnRH receptor expression further. This resulted in less cytotoxicity compared to inhibition of RNA or protein synthesis with actinomycin D, α-amanitin, puromycin, and cycloheximide. This study shows methods and optimized conditions established for the generation of a stable GT1-7 cell line containing an inducible construct allowing the downregulation of GnRH receptor expression. ABBREVIATIONS: ANOVA: analysis of the variance; DMEM: Dulbecco's modified Eagle's medium; GnRH: gonadotropin-releasing hormone; RXR: retinoid X receptor.
Assuntos
Sobrevivência Celular/efeitos dos fármacos , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Receptores LHRH/metabolismo , Alfa-Amanitina/farmacologia , Animais , Linhagem Celular Transformada , Meios de Cultura , Ciclofosfamida/farmacologia , Dactinomicina/farmacologia , Regulação para Baixo , Técnicas de Silenciamento de Genes , Hormônio Liberador de Gonadotropina/biossíntese , Hormônio Liberador de Gonadotropina/metabolismo , Camundongos , Plasmídeos , Inibidores da Síntese de Proteínas/farmacologia , Puromicina/farmacologia , Receptores LHRH/antagonistas & inibidores , Receptores LHRH/genética , TransfecçãoRESUMO
In the rapidly developing field of targeted cancer therapy there is growing interest towards therapeutics combining two or more compounds to achieve synergistic action and minimize the chance of cancer resistance to treatment. We developed a fibroblast growth factor 2 (FGF2)-conjugate bearing two cytotoxic drugs with independent mode of action: α-amanitin and monomethyl auristatin E. Drugs are covalently attached to the targeting protein in a site-specific manner via maleimide-thiol conjugation and Cu(I)-catalyzed alkyne-azide cycloaddition. The dual warhead conjugate binds to FGF receptor 1 (FGFR1) and utilizes receptor-mediated endocytosis for selective internalization into cancer cells with FGFR1. The developed conjugate displays high cytotoxicity towards all tested FGFR1-positive cell lines. Most importantly, the improved cytotoxic effect of both drugs is observed for lung cancer cell line NCI-H446. The single drug-FGF2 conjugates have no impact on the viability of NCI-H446 cells, whereas the dual warhead-FGF2 conjugate selectively and efficiently kills these FGFR1 positive cancer cells. Due to the diversified mode of action the dual warhead-FGF2 conjugate may overcome the potential acquired resistance of FGFR1-overproducing cancer cells towards single cytotoxic drugs.
Assuntos
Alfa-Amanitina/farmacologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Oligopeptídeos/farmacologia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Alfa-Amanitina/química , Animais , Linhagem Celular Tumoral , Endocitose , Fator 2 de Crescimento de Fibroblastos/química , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Camundongos , Modelos Biológicos , Células NIH 3T3 , Oligopeptídeos/química , Estrutura Secundária de Proteína , Transdução de SinaisRESUMO
To study the relationship between chromatin condensation, gene transcription and developmental competence during oocyte maturation and to explore the mechanisms by which meiotic arrest maintenance (MAM) and sexual maturity improve oocyte competence, we examined effects of MAM with roscovitine or db-cAMP on chromatin condensation, gene transcription and developmental potential of NSN or SN oocytes from prepubertal or adult mice. MAM with roscovitine improved the developmental competence and global gene transcription of prepubertal NSN (prep-NSN) and adult-SN oocytes while having no effect on those of prep-SN oocytes. MAM with db-cAMP facilitated neither development nor transcription in any type of oocytes. MAM with either roscovitine or db-cAMP promoted chromatin condensation of prep-NSN oocytes. MAM with roscovitine promoted gene transcription and chromatin condensation simultaneously through inhibiting cyclin-dependent kinase (CDK) 5 and 2, respectively. The results suggested that MAM with roscovitine improved oocyte competence by promoting gene transcription via inhibiting CDK5. Oocyte cytoplasmic maturation is correlated with gene transcription but not with chromatin condensation. The difference in developmental competence between prepubertal NSN and SN oocytes and between prepubertal and adult SN oocytes was because while the former had not, the latter had completed or acquired the ability for transcription of important genes.
Assuntos
Pontos de Checagem do Ciclo Celular , Meiose , Oócitos/citologia , Maturidade Sexual , Alfa-Amanitina/farmacologia , Animais , Bucladesina/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Nucléolo Celular/efeitos dos fármacos , Nucléolo Celular/metabolismo , Cromatina/metabolismo , Células do Cúmulo/citologia , Células do Cúmulo/efeitos dos fármacos , Células do Cúmulo/metabolismo , Quinase 5 Dependente de Ciclina/antagonistas & inibidores , Quinase 5 Dependente de Ciclina/metabolismo , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Desenvolvimento Embrionário/efeitos dos fármacos , Feminino , Isoquinolinas/farmacologia , Camundongos , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , RNA/genética , RNA/metabolismo , RNA Polimerase II/metabolismo , Roscovitina/farmacologia , Maturidade Sexual/efeitos dos fármacos , Sulfonamidas/farmacologia , Transcrição Gênica/efeitos dos fármacosRESUMO
H2A.Z histone variant is an important regulator of gene transcription, which is enriched at regulatory regions but is also found within gene bodies. Recent evidence suggests that active recruitment of H2A.Z within gene bodies is required to induce gene repression. In contrast to this view, we show that global inhibition of transcription results in H2A.Z accumulation at gene transcription start sites, as well as within gene bodies. Our results indicate that accumulation of H2A.Z within repressed genes can also be a consequence of the repression of gene transcription rather than an active mechanism required to establish the repression.
Assuntos
Regulação Neoplásica da Expressão Gênica/genética , Histonas/genética , Sítio de Iniciação de Transcrição , Iniciação da Transcrição Genética , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Alfa-Amanitina/farmacologia , Flavonoides/farmacologia , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HCT116 , Histonas/metabolismo , Humanos , Modelos Genéticos , Nucleossomos/genética , Nucleossomos/metabolismo , Piperidinas/farmacologiaRESUMO
Cancer relapse occurs with substantial frequency even after treatment with curative intent. Here we studied drug-tolerant colonies (DTCs), which are subpopulations of cancer cells that survive in the presence of drugs. Proteomic characterization of DTCs identified stemness- and epithelial-dominant subpopulations, but functional screening suggested that DTC formation was regulated at the transcriptional level independent from protein expression patterns. We consistently found that α-amanitin, an RNA polymerase II (RNAPII) inhibitor, effectively inhibited DTCs by suppressing TAF15 expression, which binds to RNA to modulate transcription and RNA processing. Sequential administration of α-amanitin and cisplatin extended overall survival in a cancer-relapse mouse model, namely peritonitis carcinomatosa. Therefore, post-treatment cancer relapse may occur through non-distinct subpopulations and may be effectively prevented by α-amanitin to disrupt transcriptional machinery, including TAF15.
Assuntos
Alfa-Amanitina/administração & dosagem , Resistência a Medicamentos/efeitos dos fármacos , Inibidores Enzimáticos/administração & dosagem , Neoplasias Peritoneais/tratamento farmacológico , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Alfa-Amanitina/farmacologia , Animais , Linhagem Celular Tumoral , Cisplatino/administração & dosagem , Cisplatino/uso terapêutico , Regulação para Baixo , Inibidores Enzimáticos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HCT116 , Células HT29 , Células HeLa , Humanos , Células MCF-7 , Camundongos , Neoplasias Peritoneais/genética , Neoplasias Peritoneais/metabolismo , Proteômica/métodos , Prevenção Secundária , Transcrição Gênica/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Long non-coding RNAs (lncRNAs) can regulate the transcript levels of genes in the same genomic region. These locally acting lncRNAs have been found deregulated in human disease and some have been shown to harbour quantitative trait loci (eQTLs) in autoimmune diseases. However, lncRNAs linked to the transcription of candidate risk genes in loci associated to rheumatoid arthritis (RA) have not yet been identified. The TRAF1 and C5 risk locus shows evidence of multiple eQTLs and transcription of intergenic non-coding sequences. Here, we identified a non-coding transcript (C5T1lncRNA) starting in the 3' untranslated region (UTR) of C5. RA-relevant cell types express C5T1lncRNA and RNA levels are further enhanced by specific immune stimuli. C5T1lncRNA is expressed predominantly in the nucleus and its expression correlates positively with C5 mRNA in various tissues (P=0.001) and in peripheral blood mononuclear cells (P=0.02) indicating transcriptional co-regulation. Knockdown results in a concurrent decrease in C5 mRNA levels but not of other neighbouring genes. Overall, our data show the identification of a novel lncRNA C5T1lncRNA that is fully located in the associated region and influences transcript levels of C5, a gene previously linked to RA pathogenesis.
Assuntos
Artrite Reumatoide/genética , DNA Intergênico/genética , Fibroblastos/metabolismo , Predisposição Genética para Doença , RNA Longo não Codificante/genética , RNA Mensageiro/genética , Alfa-Amanitina/farmacologia , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Linhagem Celular Tumoral , DNA Intergênico/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Loci Gênicos , Genótipo , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Inibidores da Síntese de Ácido Nucleico/farmacologia , Polimorfismo de Nucleotídeo Único , Cultura Primária de Células , RNA Longo não Codificante/antagonistas & inibidores , RNA Longo não Codificante/metabolismo , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Membrana Sinovial/citologia , Membrana Sinovial/efeitos dos fármacos , Membrana Sinovial/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
Long noncoding RNAs (lncRNAs) that map to intragenic regions of the human genome with the same (intronic lncRNAs) or opposite orientation (antisense lncRNAs) relative to protein-coding mRNAs have been largely dismissed from biochemical and functional characterization due to the belief that they are mRNA precursors, byproducts of RNA splicing or simply transcriptional noise. In this work, we used a custom microarray to investigate aspects of the biogenesis, processing, stability, evolutionary conservation, and cellular localization of â¼ 6,000 intronic lncRNAs and â¼ 10,000 antisense lncRNAs. Most intronic (2,903 of 3,427, 85%) and antisense lncRNAs (4,945 of 5,214, 95%) expressed in HeLa cells showed evidence of 5' cap modification, compatible with their transcription by RNAP II. Antisense lncRNAs (median t1/2 = 3.9 h) were significantly (p < 0.0001) more stable than mRNAs (median t1/2 = 3.2 h), whereas intronic lncRNAs (median t1/2 = 2.1 h) comprised a more heterogeneous class that included both stable (t1/2 > 3 h) and unstable (t1/2 < 1 h) transcripts. Intragenic lncRNAs display evidence of evolutionary conservation, have little/no coding potential and were ubiquitously detected in the cytoplasm. Notably, a fraction of the intronic and antisense lncRNAs (13 and 15%, respectively) were expressed from loci at which the corresponding host mRNA was not detected. The abundances of a subset of intronic/antisense lncRNAs were correlated (r ≥ |0.8|) with those of genes encoding proteins involved in cell division and DNA replication. Taken together, the findings of this study contribute novel biochemical and genomic information regarding intronic and antisense lncRNAs, supporting the notion that these classes include independently transcribed RNAs with potentials for exerting regulatory functions in the cell.
Assuntos
Perfilação da Expressão Gênica/métodos , Genoma Humano/genética , Íntrons/genética , RNA Antissenso/genética , RNA Longo não Codificante/genética , Alfa-Amanitina/farmacologia , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Linhagem Celular Tumoral , Metilases de Modificação do DNA/antagonistas & inibidores , Metilases de Modificação do DNA/metabolismo , Dactinomicina/farmacologia , Decitabina , Células HeLa , Humanos , Células MCF-7 , Inibidores da Síntese de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos/métodos , RNA Polimerase II/antagonistas & inibidores , RNA Polimerase II/metabolismo , Estabilidade de RNA , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
TP53, a well-known tumour suppressor gene that encodes p53, is frequently inactivated by mutation or deletion in most human tumours. A tremendous effort has been made to restore p53 activity in cancer therapies. However, no effective p53-based therapy has been successfully translated into clinical cancer treatment owing to the complexity of p53 signalling. Here we demonstrate that genomic deletion of TP53 frequently encompasses essential neighbouring genes, rendering cancer cells with hemizygous TP53 deletion vulnerable to further suppression of such genes. POLR2A is identified as such a gene that is almost always co-deleted with TP53 in human cancers. It encodes the largest and catalytic subunit of the RNA polymerase II complex, which is specifically inhibited by α-amanitin. Our analysis of The Cancer Genome Atlas (TCGA) and Cancer Cell Line Encyclopedia (CCLE) databases reveals that POLR2A expression levels are tightly correlated with its gene copy numbers in human colorectal cancer. Suppression of POLR2A with α-amanitin or small interfering RNAs selectively inhibits the proliferation, survival and tumorigenic potential of colorectal cancer cells with hemizygous TP53 loss in a p53-independent manner. Previous clinical applications of α-amanitin have been limited owing to its liver toxicity. However, we found that α-amanitin-based antibody-drug conjugates are highly effective therapeutic agents with reduced toxicity. Here we show that low doses of α-amanitin-conjugated anti-epithelial cell adhesion molecule (EpCAM) antibody lead to complete tumour regression in mouse models of human colorectal cancer with hemizygous deletion of POLR2A. We anticipate that inhibiting POLR2A will be a new therapeutic approach for human cancers containing such common genomic alterations.
Assuntos
Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Genes p53/genética , Proteína Supressora de Tumor p53/deficiência , Alfa-Amanitina/efeitos adversos , Alfa-Amanitina/química , Alfa-Amanitina/farmacologia , Alfa-Amanitina/uso terapêutico , Animais , Anticorpos/química , Anticorpos/imunologia , Antígenos de Neoplasias/imunologia , Domínio Catalítico , Moléculas de Adesão Celular/imunologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/patologia , Bases de Dados Genéticas , Modelos Animais de Doenças , Molécula de Adesão da Célula Epitelial , Feminino , Deleção de Genes , Dosagem de Genes/genética , Humanos , Imunoconjugados/efeitos adversos , Imunoconjugados/química , Imunoconjugados/imunologia , Imunoconjugados/uso terapêutico , Camundongos , Subunidades Proteicas/química , Subunidades Proteicas/deficiência , Subunidades Proteicas/genética , RNA Polimerase II/antagonistas & inibidores , RNA Polimerase II/química , RNA Polimerase II/deficiência , RNA Polimerase II/genética , Proteína Supressora de Tumor p53/biossíntese , Proteína Supressora de Tumor p53/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The cellular genomes are continuously damaged by reactive oxygen species (ROS) from aerobic processes. The impact of DNA damage depends on the specific site as well as the cellular state. The steady-state level of DNA damage is the net result of continuous formation and subsequent repair, but it is unknown to what extent heterogeneous damage distribution is caused by variations in formation or repair of DNA damage. Here, we used a restriction enzyme/qPCR based method to analyze DNA damage in promoter and coding regions of four nuclear genes: the two house-keeping genes Gadph and Tbp, and the Ndufa9 and Ndufs2 genes encoding mitochondrial complex I subunits, as well as mt-Rnr1 encoded by mitochondrial DNA (mtDNA). The distribution of steady-state levels of damage varied in a site-specific manner. Oxidative stress induced damage in nDNA to a similar extent in promoter and coding regions, and more so in mtDNA. The subsequent removal of damage from nDNA was efficient and comparable with recovery times depending on the initial damage load, while repair of mtDNA was delayed with subsequently slower repair rate. The repair was furthermore found to be independent of transcription or the transcription-coupled repair factor CSB, but dependent on cellular ATP. Our results demonstrate that the capacity to repair DNA is sufficient to remove exogenously induced damage. Thus, we conclude that the heterogeneous steady-state level of DNA damage in promoters and coding regions is caused by site-specific DNA damage/modifications that take place under normal metabolism.
Assuntos
Trifosfato de Adenosina/metabolismo , Dano ao DNA , Enzimas Reparadoras do DNA/metabolismo , Reparo do DNA , DNA/análise , Alfa-Amanitina/farmacologia , Animais , Linhagem Celular , Reparo do DNA/efeitos dos fármacos , Genes Essenciais , Genes Mitocondriais , Camundongos , Camundongos Endogâmicos C57BL , Fases de Leitura Aberta , Oxirredução , Estresse Oxidativo , Proteínas de Ligação a Poli-ADP-Ribose , Regiões Promotoras Genéticas , Transcrição GênicaRESUMO
Toxins could be effective anticancer drugs, if their selective delivery into cancer cells could be achieved. We have shown that the energy of membrane-associated folding of water-soluble membrane peptides of the pHLIP (pH low insertion peptide) family could be used to move cell-impermeable cargo across the lipid bilayer into the cytoplasm of cancer cells. Here we present the results of a study of pHLIP-mediated cellular delivery of a polar cell-impermeable toxin, α-amanitin, an inhibitor of RNA polymerase II. We show that pHLIP can deliver α-amanitin into cells in a pH-dependent fashion and induce cell death within 48 h. Translocation capability could be tuned by conjugating amanitin to the C-terminus of pHLIP via linkers of different hydrophobicities that could be cleaved in the cytoplasm. pHLIP-SPDP-amanitin, which exhibits 4-5 times higher antiproliferative ability at pH 6 than at pH 7.4, was selected as the best construct. The major mechanism of amanitin delivery is direct translocation (flip) across a membrane by pHLIP and cleavage of the S-S bond in the cytoplasm. The antiproliferative effect was monitored on four different human cancer cell lines. pHLIP-mediated cytoplasmic delivery of amanitin could create great opportunities to use the toxin as a potent pH-selective anticancer agent, which predominantly targets highly proliferative cancer cells at low extracellular pH values.
Assuntos
Alfa-Amanitina/farmacologia , Antineoplásicos/farmacologia , Portadores de Fármacos/farmacologia , Proteínas de Membrana/química , Alfa-Amanitina/química , Sequência de Aminoácidos , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Membrana Celular/química , Proliferação de Células/efeitos dos fármacos , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos/métodos , Ensaios de Seleção de Medicamentos Antitumorais , Células HeLa/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Cinética , Proteínas de Membrana/farmacologia , Dados de Sequência Molecular , Transporte ProteicoRESUMO
We have previously shown the differential expression of versican in the mouse uterus under ovarian hormone influence. We also demonstrated there is not a direct correlation between mRNA levels and protein expression, suggesting posttranscriptional events, such as alteration in mRNA stability. This posttranscriptional effect may result in the elongation and stabilization of transcripts poly(A) tail. Thus, the aim of this study was to analyze whether estradiol (E2) regulates versican mRNA stability and expression in a dose-related and time-dependent manner. For this purpose female mice were ovariectomized and treated with a single injection of 0.1 or 10 µg E2. To block transcription a group of females received a single injection of alpha-amanitin before hormone administration. Uterine tissues were collected 30 min, 1, 3, 6, 12 and 24 h after treatments and processed for quantitative real time PCR (qPCR), RACE-PAT Assay and immunohistochemistry. qPCR showed that versican mRNA levels are higher than control from 3 to 24 h after E2 administration, whereas after transcription inhibition versican mRNA unexpectedly increases within 3 h, which can be explained when transcriptional blockers alter the degradation rate of the transcript, resulting in the superinduction of this mRNA. Accordingly, analysis of versican transcript poly(A) tail evidenced a longer product 3 h after treatment, but not after 12 h. Versican immunoreaction becomes conspicuous in the superficial stroma only 3 h after E2 injection, whereas the whole stroma is immunoreactive from 6 h onward. These results demonstrate that E2 modulates versican at the transcriptional and posttranscriptional levels in a time-dependent manner.
Assuntos
Estradiol/farmacologia , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Útero/efeitos dos fármacos , Útero/metabolismo , Versicanas/genética , Alfa-Amanitina/farmacologia , Animais , Relação Dose-Resposta a Droga , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Poli A , Poliadenilação/efeitos dos fármacos , Fatores de Tempo , Versicanas/metabolismoRESUMO
Old and young donors cells show different ability to homologous recombination (shown on the first stage--the chromosome transference) in vitro, that we suppose could be the reasons of the genome instability in aging. Homologous recombination, induced by X-radiation, is limited in cells taken from donors older than 70 years. Alpha-amanitin, the RNA-polymerize II repressor, in toxic doze, could induce the chromosome transference in the cells from all studied groups: from old and young donors and donors with repair process defect (with BRCA 1, 2 mutations). Summarized effect of X-radiation and alpha-amanitin does not increase the induction of the chromosome transference.
Assuntos
Alfa-Amanitina/farmacologia , Aberrações Cromossômicas , Reparo de DNA por Recombinação , Doadores de Tecidos , Raios X/efeitos adversos , Fatores Etários , Aberrações Cromossômicas/efeitos dos fármacos , Aberrações Cromossômicas/efeitos da radiação , Dano ao DNA , Instabilidade Genômica/efeitos dos fármacos , Instabilidade Genômica/efeitos da radiação , Humanos , Modelos Genéticos , Mutação , Inibidores da Síntese de Ácido Nucleico/farmacologia , RNA Polimerase II/fisiologia , Reparo de DNA por Recombinação/efeitos dos fármacos , Reparo de DNA por Recombinação/efeitos da radiaçãoRESUMO
Double-strand DNA breaks (DSBs) are continuously induced in cells by endogenously generated free radicals and exogenous genotoxic agents such as ionizing radiation. DSBs activate the kinase activity in sensor proteins such as ATM and DNA-PK, initiating a complex DNA damage response that coordinates various DNA repair pathways to restore genomic integrity. In this study, we report the unexpected finding that homologous chromosomes contact each other at the sites of DSBs induced by either radiation or the endonuclease I-PpoI in human somatic cells. Contact involves short segments of homologous chromosomes and is centered on a DSB in active genes but does not occur at I-PpoI sites in intergenic DNA. I-PpoI-induced contact between homologous genes is abrogated by the transcriptional inhibitors actinomycin D and α-amanitin and requires the kinase activity of ATM but not DNA-PK. Our findings provide documentation of a common transcription-related and ATM kinase-dependent mechanism that induces contact between allelic regions of homologous chromosomes at sites of DSBs in human somatic cells.
Assuntos
Cromossomos Humanos , Dano ao DNA , Fase G1 , Fase de Repouso do Ciclo Celular , Alfa-Amanitina/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/fisiologia , Células Cultivadas , Proteína Quinase Ativada por DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Dactinomicina/farmacologia , Humanos , Hibridização in Situ Fluorescente , Proteínas Serina-Treonina Quinases/fisiologia , Radiação Ionizante , Transcrição Gênica , Proteínas Supressoras de Tumor/fisiologiaRESUMO
BACKGROUND: Human epithelial cell adhesion molecule (EpCAM) is overexpressed in many cancers. Anti-EpCAM antibodies have shown promise in preclinical studies, but showed no tumor regression in a recent phase II clinical trial. Therefore, we generated a novel anti-EpCAM antibody-drug conjugate and assessed whether it showed enhanced antitumor effects. METHODS: Chemical cross-linking was conducted to covalently conjugate α-amanitin, a toxin known to inhibit DNA transcription, with chiHEA125, a chimerized anti-EpCAM monoclonal antibody, to generate the antibody-drug conjugate α-amanitin-glutarate-chiHEA125 (chiHEA125-Ama). Antiproliferative activity of chiHEA125-Ama was tested in human pancreatic (BxPc-3 and Capan-1), colorectal (Colo205), breast (MCF-7), and bile duct (OZ) cancer cell lines in vitro using [(3)H]-thymidine incorporation assay. Antitumor activity of chiHEA125-Ama was assessed in vivo in immunocompromised mice bearing subcutaneous human BxPc-3 pancreatic carcinoma xenograft tumors (n = 66 mice). Cell proliferation and apoptosis were evaluated in xenograft tumors by immunohistochemistry. All statistical tests were two-sided. RESULTS: In all cell lines, chiHEA125-Ama reduced cell proliferation (mean half maximal inhibitory concentration [IC(50)] = 2.5 × 10(-10) to 5.4 × 10(-12) M). A single dose of chiHEA125-Ama inhibited BxPc-3 xenograft tumor growth (chiHEA125 [control, n = 4 mice] vs. chiHEA125-Ama [n = 6 mice], dose of 15 mg/kg with respect to IgG and 50 µg/kg with respect to α-amanitin, mean relative increase in tumor volume on day 16 = 884% vs. -79%, difference = 963%, 95% CI = 582% to 1344%, P = .019). Two higher doses of chiHEA125-Ama (100 µg/kg with respect to α-amanitin), administered 1 week apart (n = 10 mice per group), led to complete tumor regression in nine of 10 (90%) mice compared with chiHEA125, during the observation period of 16 days; increased apoptosis and reduced cell proliferation were observed in mice treated with chiHEA125-Ama. CONCLUSION: This preclinical study suggests that anti-EpCAM antibody conjugates with α-amanitin have the potential to be highly effective therapeutic agents for pancreatic carcinomas and various EpCAM-expressing malignancies.
Assuntos
Alfa-Amanitina/farmacologia , Antígenos de Neoplasias/metabolismo , Antineoplásicos/farmacologia , Biomarcadores Tumorais/metabolismo , Carcinoma/tratamento farmacológico , Moléculas de Adesão Celular/metabolismo , Inibidores Enzimáticos/farmacologia , Imunoconjugados/farmacologia , Neoplasias Pancreáticas/tratamento farmacológico , Alfa-Amanitina/imunologia , Inibidores da Angiogênese/farmacologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Antígenos de Neoplasias/imunologia , Antineoplásicos/imunologia , Biomarcadores Tumorais/imunologia , Carcinoma/imunologia , Carcinoma/patologia , Moléculas de Adesão Celular/imunologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Quimera , Neoplasias do Colo/tratamento farmacológico , Inibidores Enzimáticos/imunologia , Molécula de Adesão da Célula Epitelial , Feminino , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Imunoconjugados/imunologia , Imuno-Histoquímica , Fígado/enzimologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/patologia , Regulação para Cima/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus implicated in lymphomas, such as Burkitt's lymphoma, Hodgkin's lymphoma, and NK/T cell lymphoma. MicroRNAs (miRNAs) are 19-25 nucleotide long single-stranded RNAs involved in post-transcriptional gene regulation. miRNAs are mainly transcribed by RNA polymerase II (pol II) to have stem-loop structures and subsequently processed by Drosha and Dicer. EBV miRNAs are expressed in B cells, nasopharyngeal carcinoma cells, and gastric carcinoma cells infected with EBV. EBV miRNAs can be divided into two groups: BHRF1 miRNAs and BART miRNAs. In this study, we investigated the biogenesis of EBV miRNAs. Treatment of the SNU-719 EBV-positive gastric cancer cell line with α-amanitin at a concentration that selectively inhibits RNA polymerase II activity decreased the expression levels of BART miRNAs. The expression levels of BART miRNAs were also reduced by RNA interference targeting Drosha and Dicer. Two of each C/EBPß and c-Myc binding sites are located upstream of the proposed initiation sites for primary BART miRNA transcripts. Knockdown of C/EBPß but not c-Myc using siRNAs reduced BART miRNA expression by 25-55% compared with the control. These results suggest that BART miRNAs are transcribed by pol II and undergo a similar biogenesis process with cellular miRNAs.
Assuntos
MicroRNAs/biossíntese , RNA Viral/biossíntese , Alfa-Amanitina/farmacologia , Linfócitos B/metabolismo , Sequência de Bases , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Linhagem Celular Tumoral , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Regulação Viral da Expressão Gênica , Técnicas de Silenciamento de Genes , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/fisiologia , Humanos , MicroRNAs/genética , Dados de Sequência Molecular , Regiões Promotoras Genéticas , RNA Polimerase II/antagonistas & inibidores , RNA Polimerase II/metabolismo , RNA Viral/genética , Ribonuclease III/genética , Ribonuclease III/metabolismo , Transcrição Gênica , Latência ViralRESUMO
In mammals, the sperm deliver mRNA of unknown function into the oocytes during fertilization. The role of sperm microRNAs (miRNAs) in preimplantation development is unknown. miRNA profiling identified six miRNAs expressed in the sperm and the zygotes but not in the oocytes or preimplantation embryos. Sperm contained both the precursor and the mature form of one of these miRNAs, miR-34c. The absence of an increased level of miR-34c in zygotes derived from α-amanitin-treated oocytes and in parthenogenetic oocytes supported a sperm origin of zygotic miR-34c. Injection of miR-34c inhibitor into zygotes inhibited DNA synthesis and significantly suppressed first cleavage division. A 3' UTR luciferase assay and Western blotting demonstrated that miR-34c regulates B-cell leukemia/lymphoma 2 (Bcl-2) expression in the zygotes. Coinjection of anti-Bcl-2 antibody in zygotes partially reversed but injection of Bcl-2 protein mimicked the effect of miR-34c inhibition. Oocyte activation is essential for the miR-34c action in zygotes, as demonstrated by a decrease in 3'UTR luciferase reporter activity and Bcl-2 expression after injection of precursor miR-34c into parthenogenetic oocytes. Our findings provide evidence that sperm-borne miR-34c is important for the first cell division via modulation of Bcl-2 expression.