RESUMO
Promoting residential cells, particularly endogenous neural stem and progenitor cells (NSPCs), for tissue regeneration represents a potential strategy for the treatment of spinal cord injury (SCI). However, adult NSPCs differentiate mainly into glial cells and contribute to glial scar formation at the site of injury. Gsx1 is known to regulate the generation of excitatory and inhibitory interneurons during embryonic development of the spinal cord. In this study, we show that lentivirus-mediated expression of Gsx1 increases the number of NSPCs in a mouse model of lateral hemisection SCI during the acute stage. Subsequently, Gsx1 expression increases the generation of glutamatergic and cholinergic interneurons and decreases the generation of GABAergic interneurons in the chronic stage of SCI. Importantly, Gsx1 reduces reactive astrogliosis and glial scar formation, promotes serotonin (5-HT) neuronal activity, and improves the locomotor function of the injured mice. Moreover, RNA sequencing (RNA-seq) analysis reveals that Gsx1-induced transcriptome regulation correlates with NSPC signaling, NSPC activation, neuronal differentiation, and inhibition of astrogliosis and scar formation. Collectively, our study provides molecular insights for Gsx1-mediated functional recovery and identifies the potential of Gsx1 gene therapy for injuries in the spinal cord and possibly other parts of the central nervous system.
Assuntos
Perfilação da Expressão Gênica/métodos , Vetores Genéticos/administração & dosagem , Proteínas de Homeodomínio/genética , Traumatismos da Medula Espinal/terapia , Animais , Diferenciação Celular , Linhagem Celular , Modelos Animais de Doenças , Redes Reguladoras de Genes , Terapia Genética , Lentivirus/genética , Camundongos , Camundongos Transgênicos , Células-Tronco Neurais , Recuperação de Função Fisiológica , Análise de Sequência de RNA , Traumatismos da Medula Espinal/genética , Traumatismos da Medula Espinal/fisiopatologiaRESUMO
Topoisomerase II beta (Top2b) is an enzyme that alters the topologic states of DNA during transcription. Top2b deletion in early retinal progenitor cells causes severe defects in neural differentiation and affects cell survival in all retinal cell types. However, it is unclear whether the observed severe phenotypes are the result of cell-autonomous/primary defects or non-cell-autonomous/secondary defects caused by alterations of other retinal cells. Using photoreceptor cells as a model, we first characterized the phenotypes in Top2b conditional knockout. Top2b deletion leads to malformation of photoreceptor outer segments (OSs) and synapses accompanied by dramatic cell loss at late-stage photoreceptor differentiation. Then, we performed mosaic analysis with shRNA-mediated Top2b knockdown in neonatal retina using in vivo electroportation to target rod photoreceptors in neonatal retina. Top2b knockdown causes defective OS without causing a dramatic cell loss, suggesting a Top2b cell-autonomous function. Furthermore, RNA-seq analysis reveals that Top2b controls the expression of key genes in the photoreceptor gene-regulatory network (e.g., Crx, Nr2e3, Opn1sw, Vsx2) and retinopathy-related genes (e.g., Abca4, Bbs7, Pde6b). Together, our data establish a combinatorial cell-autonomous and non-cell-autonomous role for Top2b in the late stage of photoreceptor differentiation and maturation. © 2017 The Authors Journal of Neuroscience Research Published by Wiley Periodicals, Inc.
Assuntos
DNA Topoisomerases Tipo II/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Redes Reguladoras de Genes/genética , Células Fotorreceptoras/citologia , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Retina/embriologia , Animais , Diferenciação Celular/genética , Feminino , Masculino , Camundongos , Camundongos Knockout , Retina/crescimento & desenvolvimento , Sinapses/genética , Sinapses/metabolismo , Transcrição GênicaRESUMO
The clinical development of FtsZ-targeting benzamide compounds like PC190723 has been limited by poor drug-like and pharmacokinetic properties. Development of prodrugs of PC190723 (e.g., TXY541) resulted in enhanced pharmaceutical properties, which, in turn, led to improved intravenous efficacy as well as the first demonstration of oral efficacy in vivo against both methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA). Despite being efficacious in vivo, TXY541 still suffered from suboptimal pharmacokinetics and the requirement of high efficacious doses. We describe here the design of a new prodrug (TXA709) in which the Cl group on the pyridyl ring has been replaced with a CF3 functionality that is resistant to metabolic attack. As a result of this enhanced metabolic stability, the product of the TXA709 prodrug (TXA707) is associated with improved pharmacokinetic properties (a 6.5-fold-longer half-life and a 3-fold-greater oral bioavailability) and superior in vivo antistaphylococcal efficacy relative to PC190723. We validate FtsZ as the antibacterial target of TXA707 and demonstrate that the compound retains potent bactericidal activity against S. aureus strains resistant to the current standard-of-care drugs vancomycin, daptomycin, and linezolid. These collective properties, coupled with minimal observed toxicity to mammalian cells, establish the prodrug TXA709 as an antistaphylococcal agent worthy of clinical development.
Assuntos
Proteínas de Bactérias/metabolismo , Benzamidas/farmacologia , Benzamidas/farmacocinética , Proteínas do Citoesqueleto/metabolismo , Resistência a Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Pró-Fármacos/farmacologia , Pró-Fármacos/farmacocinética , Animais , Antibacterianos/farmacocinética , Antibacterianos/farmacologia , Células Cultivadas , Daptomicina/farmacologia , Cães , Meia-Vida , Humanos , Linezolida/farmacologia , Meticilina/farmacologia , Staphylococcus aureus Resistente à Meticilina/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana/métodos , Piridinas/farmacologia , Ratos , Infecções Estafilocócicas/tratamento farmacológico , Tiazóis/farmacologia , Vancomicina/farmacologiaRESUMO
Camptothecin (CPT), a topoisomerase (Top) I-targeting drug that stabilizes Top1-DNA covalent adducts, can induce S-phase-specific cytotoxicity due to the arrest of progressing replication forks. However, CPT-induced non-S-phase cytotoxicity is less well characterized. In this study, we have identified topoisomerase IIß (Top2ß) as a specific determinant for CPT sensitivity, but not for many other cytotoxic agents, in non-S-phase cells. First, quiescent mouse embryonic fibroblasts (MEFs) lacking Top2ß were shown to be hypersensitive to CPT with prominent induction of apoptosis. Second, ICRF-187, a Top2 catalytic inhibitor known to deplete Top2ß, specifically sensitized MEFs to CPT. To explore the molecular basis for CPT hypersensitivity in Top2ß-deficient cells, we found that upon CPT exposure, the RNA polymerase II large subunit (RNAP LS) became progressively depleted, followed by recovery to nearly the original level in wild-type MEFs, whereas RNAP LS remained depleted without recovery in Top2ß-deficient cells. Concomitant with the reduction of the RNAP LS level, the p53 protein level was greatly induced. Interestingly, RNAP LS depletion has been well documented to lead to p53-dependent apoptosis. Altogether, our findings support a model in which Top2ß deficiency promotes CPT-induced apoptosis in quiescent non-S-phase cells, possibly due to RNAP LS depletion and p53 accumulation.
Assuntos
Apoptose/efeitos dos fármacos , Camptotecina/farmacologia , DNA Topoisomerases Tipo II/deficiência , Proteínas de Ligação a DNA/deficiência , Fibroblastos/efeitos dos fármacos , Animais , Antineoplásicos/farmacologia , Western Blotting , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , DNA Topoisomerases Tipo II/genética , Proteínas de Ligação a DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Relação Dose-Resposta a Droga , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Fibroblastos/metabolismo , Camundongos , Camundongos Knockout , Subunidades Proteicas/metabolismo , Razoxano/farmacologia , Inibidores da Topoisomerase I/farmacologia , Transcrição Gênica/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismoRESUMO
Studies in animal models have indicated that dietary isothiocyanates (ITCs) exhibit cancer preventive activities through carcinogen detoxification-dependent and -independent mechanisms. The carcinogen detoxification-independent mechanism of cancer prevention by ITCs has been attributed at least in part to their ability to induce apoptosis of transformed (initiated) cells (e.g. through suppression of IκB kinase and nuclear factor κB as well as other proposed mechanisms). In the current studies we show that ITC-induced apoptosis of oncogene-transformed cells involves thiol modification of DNA topoisomerase II (Top2) based on the following observations. 1) siRNA-mediated knockdown of Top2α in both SV40-transformed MEFs and Ras-transformed human mammary epithelial MCF-10A cells resulted in reduced ITC sensitivity. 2) ITCs, like some anticancer drugs and cancer-preventive dietary components, were shown to induce reversible Top2α cleavage complexes in vitro. 3) ITC-induced Top2α cleavage complexes were abolished by co-incubation with excess glutathione. In addition, proteomic analysis revealed that several cysteine residues on human Top2α were covalently modified by benzyl-ITC, suggesting that ITC-induced Top2α cleavage complexes may involve cysteine modification. Interestingly, consistent with the thiol modification mechanism for Top2α cleavage complex induction, the thiol-reactive selenocysteine, but not the non-thiol-reactive selenomethionine, was shown to induce Top2α cleavage complexes. In the aggregate, our results suggest that thiol modification of Top2α may contribute to apoptosis induction in transformed cells by ITCs.
Assuntos
Antígenos de Neoplasias/metabolismo , Apoptose/efeitos dos fármacos , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/metabolismo , Dieta , Isotiocianatos/farmacologia , Compostos de Sulfidrila/metabolismo , Animais , Linhagem Celular Transformada , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cisteína/metabolismo , Dano ao DNA , Fragmentação do DNA/efeitos dos fármacos , DNA Topoisomerases Tipo II/deficiência , Proteínas de Ligação a DNA/deficiência , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Técnicas de Silenciamento de Genes , Inativação Gênica/efeitos dos fármacos , Histonas/metabolismo , Humanos , Camundongos , Nucleossomos/efeitos dos fármacos , Nucleossomos/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas ras/metabolismoRESUMO
Nkx6.1 plays an essential role during the embryonic development of the spinal cord. However, its role in the adult and injured spinal cord is not well understood. Here we show that lentivirus-mediated Nkx6.1 expression in the adult injured mouse spinal cord promotes cell proliferation and activation of endogenous neural stem/progenitor cells (NSPCs) at the acute phase of injury. In the chronic phase, Nkx6.1 increases the number of interneurons, reduces the number of reactive astrocytes, minimizes glial scar formation, and represses neuroinflammation. Transcriptomic analysis reveals that Nkx6.1 upregulates the sequential expression of genes involved in cell proliferation, neural differentiation, and Notch signaling pathway, downregulates genes and pathways involved in neuroinflammation, reactive astrocyte activation, and glial scar formation. Together, our findings support the potential role of Nkx6.1 in neural regeneration in the adult injured spinal cord.
Assuntos
Gliose/metabolismo , Proteínas de Homeodomínio/biossíntese , Células-Tronco Neurais/metabolismo , Doenças Neuroinflamatórias/metabolismo , Traumatismos da Medula Espinal/metabolismo , Fatores Etários , Animais , Feminino , Gliose/patologia , Gliose/prevenção & controle , Células HEK293 , Humanos , Locomoção/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Doenças Neuroinflamatórias/prevenção & controle , Traumatismos da Medula Espinal/patologiaRESUMO
Reversible topoisomerase I (Top1)-DNA cleavage complexes are the key DNA lesion induced by anticancer camptothecins (CPTs) (e.g. topotecan and irinotecan) as well as structurally perturbed DNAs (e.g. oxidatively damaged, UV-irradiated, or alkylated DNA). It has been proposed that Top1 cleavage complexes arrest advancing replication forks, triggering the formation of DNA double strand breaks (DSBs) because of replication fork runoff at the Top1 cleavage complex sites on the leading strand. In this study, we show that the formation of replication-dependent DSBs requires the ubiquitin-proteasome pathway in CPT-treated cells. First, the proteasome inhibitor MG-132 specifically inhibited CPT-induced but not ionizing radiation- or hydroxyurea-induced DSBs as revealed by both the neutral comet assay and measurements of the specific DNA damage signals (e.g. gamma-H2AX, phosphorylated ataxia telangiectasia mutated (Ser-1981), and phosphorylated Chk2 (Ser-33/35)) that are characteristic for DSBs. Knocking down the 20 S proteasome maturation protein also supported the requirement of the proteasome activity for CPT-induced DSBs. Second, CPT-induced DSB signals were shown to require ubiquitin, ubiquitin-activating enzyme (E1), a CUL-3-based ubiquitin ligase (E3), and the formation of Lys-48-linked polyubiquitin chains on Top1. Third, immunocytochemical studies revealed that the CPT-induced formation of gamma-H2AX foci occurred at the replication forks and was attenuated by co-treatment with the proteasome inhibitor MG-132. In the aggregate, these results support a replication fork collision model in which Top1 cleavage complexes at the arrested replication forks are degraded by proteasome prior to replication fork runoff on the leading strand to generate DSBs.
Assuntos
Adutos de DNA , Quebras de DNA de Cadeia Dupla , DNA Topoisomerases Tipo I/química , DNA Topoisomerases Tipo I/metabolismo , DNA/química , DNA/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Afidicolina/metabolismo , DNA/efeitos dos fármacos , DNA/efeitos da radiação , Adutos de DNA/química , Adutos de DNA/metabolismo , Replicação do DNA , DNA Topoisomerases Tipo I/genética , Inibidores Enzimáticos/metabolismo , Células HeLa , Histonas/genética , Histonas/metabolismo , Humanos , Leupeptinas/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/fisiologia , Ubiquitina/metabolismoRESUMO
G-quadruplex stabilizers such as telomestatin and HXDV bind with exquisite specificity to G-quadruplexes, but not to triplex, duplex, or single-stranded DNAs. Studies have suggested that the antiproliferative and possibly anti-tumor activities of these compounds are linked to their inhibitory effect on telomerase and/or telomere function. In the current studies, we show that HXDV, a synthetic analog of telomestatin, exhibits antiproliferative activity against both telomerase-positive and -negative cells and induces robust apoptosis within 16 h of treatment, suggesting a mode of action independent of telomerase. HXDV was also shown to inhibit cell cycle progression causing M-phase cell cycle arrest, as evidenced by accumulation of cells with 4 n DNA content, increased mitotic index, separated centrosomes, elevated histone H3 phosphorylation at Ser-10 (an M-phase marker), and defective chromosome alignment and spindle fiber assembly (revealed by time-lapse microscopy). The M-phase arrest caused by HXDV paralleled with reduction in the expression level of the major M-phase checkpoint regulator Aurora A. All these cellular effects appear to depend on the G-quadruplex binding activity of HXDV as its non-G-quadruplex binding analog, TXTLeu, is completely devoid of all these effects. In the aggregate, our results suggest that HXDV, which exhibits anti-proliferative and apoptotic activities, is also a novel M-phase blocker, with a mode of action dependent on its G-quadruplex binding activity.
Assuntos
Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Divisão Celular/efeitos dos fármacos , Quadruplex G/efeitos dos fármacos , Antineoplásicos/química , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Compostos Macrocíclicos/química , Compostos Macrocíclicos/farmacologia , Microscopia , Telomerase/genética , Telomerase/fisiologiaRESUMO
Genistein is a bioflavonoid enriched in soy products. However, high levels of maternal soy consumption have been linked to the development of infant leukemia ALL and AML. The majority of infant leukemia is linked to mixed lineage leukemia gene (MLL) translocations. Previous studies have implicated topoisomerase II (Top2) in genistein-induced infant leukemia. In order to understand the roles of the two Top2 isozymes in and the molecular mechanism for genistein-induced infant leukemia, we carried out studies in vitro using purified recombinant human Top2 isozymes, as well as studies in cultured mouse myeloid progenitor cells (32Dc13) and Top2beta knockout mouse embryonic fibroblasts (MEFs). First, we showed that genistein efficiently induced both Top2alpha and Top2beta cleavage complexes in the purified system as well as in cultured mouse cells. Second, genistein induced proteasomal degradation of Top2beta in 32Dc13 cells. Third, the genistein-induced DNA double-strand break (DSB) signal, gamma-H2AX, was dependent on the Top2beta isozyme and proteasome activity. Fourth, the requirement for Top2beta and proteasome activity was mirrored in genistein-induced DNA sequence rearrangements, as monitored by a DNA integration assay. Together, our results suggest a model in which genistein-induced Top2beta cleavage complexes are processed by proteasome, leading to the exposure of otherwise Top2beta-concealed DSBs and subsequent chromosome rearrangements, and implicate a major role of Top2beta and proteasome in genistein-induced infant leukemia.
Assuntos
DNA Topoisomerases Tipo I/metabolismo , Genisteína/efeitos adversos , Isoenzimas/metabolismo , Leucemia Mieloide Aguda/induzido quimicamente , Complexo de Endopeptidases do Proteassoma/metabolismo , Recombinação Genética/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , DNA/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla , Humanos , Lactente , Leucemia Mieloide Aguda/enzimologia , Leucemia Mieloide Aguda/genética , CamundongosRESUMO
Drugs that target DNA topoisomerase II (Top2), including etoposide (VP-16), doxorubicin, and mitoxantrone, are among the most effective anticancer drugs in clinical use. However, Top2-based chemotherapy has been associated with higher incidences of secondary malignancies, notably the development of acute myeloid leukemia in VP-16-treated patients. This association is suggestive of a link between carcinogenesis and Top2-mediated DNA damage. We show here that VP-16-induced carcinogenesis involves mainly the beta rather than the alpha isozyme of Top2. In a mouse skin carcinogenesis model, the incidence of VP-16-induced melanomas in the skin of 7,12-dimethylbenz[a]anthracene-treated mice is found to be significantly higher in TOP2beta(+) than in skin-specific top2beta-knockout mice. Furthermore, VP-16-induced DNA sequence rearrangements and double-strand breaks (DSBs) are found to be Top2beta-dependent and preventable by cotreatment with a proteasome inhibitor, suggesting the importance of proteasomal degradation of the Top2beta-DNA cleavage complexes in VP-16-induced DNA sequence rearrangements. VP-16 cytotoxicity in transformed cells expressing both Top2 isozymes is, however, found to be primarily Top2alpha-dependent. These results point to the importance of developing Top2alpha-specific anticancer drugs for effective chemotherapy without the development of treatment-related secondary malignancies.
Assuntos
Antineoplásicos/efeitos adversos , DNA Topoisomerases Tipo II/fisiologia , Isoenzimas/fisiologia , Segunda Neoplasia Primária/induzido quimicamente , Animais , Dano ao DNA , Modelos Animais de Doenças , Desenho de Fármacos , Etoposídeo/efeitos adversos , Isoenzimas/antagonistas & inibidores , Melanoma Experimental/tratamento farmacológico , Camundongos , Camundongos Knockout , Segunda Neoplasia Primária/tratamento farmacológico , Segunda Neoplasia Primária/etiologia , Inibidores de Proteases/farmacologia , Inibidores da Topoisomerase IIRESUMO
Mice lacking topoisomerase IIbeta (TopIIbeta) are known to exhibit a perinatal death phenotype. In the current study, transcription profiles of the brains of wild-type and top2beta knockout mouse embryos were generated. Surprisingly, only a small number (1 to 4%) of genes were affected in top2beta knockout embryos. However, the expression of nearly 30% of developmentally regulated genes was either up- or down-regulated. By contrast, the expression of genes encoding general cell growth functions and early differentiation markers was not affected, suggesting that TopIIbeta is not required for early differentiation programming but is specifically required for the expression of developmentally regulated genes at later stages of differentiation. Consistent with this notion, immunohistochemical analysis of brain sections showed that TopIIbeta and histone deacetylase 2, a known TopIIbeta-interacting protein, were preferentially expressed in neurons which are in their later stages of differentiation. Chromatin immunoprecipitation analysis of the developing brains revealed TopIIbeta binding to the 5' region of a number of TopIIbeta-sensitive genes. Further studies of a TopIIbeta-sensitive gene, Kcnd2, revealed the presence of TopIIbeta in the transcription unit with major binding near the promoter region. Together, these results support a role of TopIIbeta in activation/repression of developmentally regulated genes at late stages of neuronal differentiation.
Assuntos
Encéfalo/fisiologia , Diferenciação Celular/fisiologia , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Animais , Encéfalo/citologia , DNA Topoisomerases Tipo II/genética , Proteínas de Ligação a DNA/genética , Embrião de Mamíferos/anatomia & histologia , Embrião de Mamíferos/fisiologia , Perfilação da Expressão Gênica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/citologia , Neurônios/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , Transcrição GênicaRESUMO
Doxorubicin is among the most effective and widely used anticancer drugs in the clinic. However, cardiotoxicity is one of the life-threatening side effects of doxorubicin-based therapy. Dexrazoxane (Zinecard, also known as ICRF-187) has been used in the clinic as a cardioprotectant against doxorubicin cardiotoxicity. The molecular basis for doxorubicin cardiotoxicity and the cardioprotective effect of dexrazoxane, however, is not fully understood. In the present study, we showed that dexrazoxane specifically abolished the DNA damage signal gamma-H2AX induced by doxorubicin, but not camptothecin or hydrogen peroxide, in H9C2 cardiomyocytes. Doxorubicin-induced DNA damage was also specifically abolished by the proteasome inhibitors bortezomib and MG132 and much reduced in top2beta(-/-) mouse embryonic fibroblasts (MEF) compared with TOP2beta(+/+) MEFs, suggesting the involvement of proteasome and DNA topoisomerase IIbeta (Top2beta). Furthermore, in addition to antagonizing Top2 cleavage complex formation, dexrazoxane also induced rapid degradation of Top2beta, which paralleled the reduction of doxorubicin-induced DNA damage. Together, our results suggest that dexrazoxane antagonizes doxorubicin-induced DNA damage through its interference with Top2beta, which could implicate Top2beta in doxorubicin cardiotoxicity. The specific involvement of proteasome and Top2beta in doxorubicin-induced DNA damage is consistent with a model in which proteasomal processing of doxorubicin-induced Top2beta-DNA covalent complexes exposes the Top2beta-concealed DNA double-strand breaks.
Assuntos
Quebras de DNA de Cadeia Dupla , Doxorrubicina/antagonistas & inibidores , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/enzimologia , Razoxano/farmacologia , Animais , Antibióticos Antineoplásicos/antagonistas & inibidores , Antibióticos Antineoplásicos/toxicidade , DNA Topoisomerases Tipo II/química , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Doxorrubicina/toxicidade , Interações Medicamentosas , Cardiopatias/induzido quimicamente , Cardiopatias/enzimologia , Cardiopatias/prevenção & controle , Histonas/metabolismo , Humanos , Camundongos , Modelos Moleculares , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma , Conformação Proteica , Inibidores da Topoisomerase IIRESUMO
Etoposide-induced treatment-related acute myelogenous leukemia (t-AML) is characterized by rearrangements of the mixed lineage leukemia (MLL) gene with one of its >50 partner genes, most probably as a consequence of etoposide-induced DNA double-strand breaks (DSBs). Recent studies have shown that etoposide-induced DSBs occur predominantly within the breakpoint cluster region (bcr) of the MLL gene. However, bcr-specific DSBs induced by etoposide are not topoisomerase II-linked but the result of apoptotic nuclease-mediated DNA cleavage. Here, we test the involvement of caspase-activated DNase (CAD) and other apoptotic components in etoposide-induced gene rearrangements using two methods. First, we measured the effect of etoposide on the integration frequency of a transfected plasmid. Etoposide strongly stimulated plasmid integration in CAD cDNA-complemented mouse embryonic fibroblasts (MEFs) but not in CAD knockout (KO) MEFs. Consistently, down-regulation of ICAD (inhibitor of CAD, also required for proper folding of CAD) in an HT29-derived cell line, which leads to decreased CAD activity, significantly reduced etoposide-induced plasmid integration. Second, we used long-template inverse PCR to focus on gene rearrangements at the MLL locus. Etoposide stimulated MLL fusion product formation in CAD cDNA-complemented MEFs but not in CAD KO MEFs. Together, these results suggest that CAD and other apoptotic components may play an important role in etoposide-induced t-AML.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Desoxirribonucleases/metabolismo , Etoposídeo/farmacologia , Leucemia Mieloide Aguda/induzido quimicamente , Leucemia Mieloide Aguda/enzimologia , Animais , Apoptose/fisiologia , Desoxirribonucleases/antagonistas & inibidores , Rearranjo Gênico/efeitos dos fármacos , Células HT29 , Humanos , Camundongos , Camundongos Knockout , Proteína de Leucina Linfoide-Mieloide/genéticaRESUMO
Failure to establish neuromuscular junctions is a major phenotype of top2beta knockout mice. However, the precise mechanism for this defect is not known. In the current study, we have investigated the role of TopIIbeta in cultured neurons. We showed that the TopII inhibitor ICRF-193 significantly blocked neurite outgrowth and growth cone formation in cultured cerebellar granule neurons (CGNs), dorsal root ganglions (DRGs) and cortical neurons (CNs). In addition, ICRF-193 also blocked neurite outgrowth and growth cone formation of PC12 cells undergoing NGF-induced differentiation. Isolated cortical neurons from top2beta knockout embryos elaborated shorter neurites than did those from their wild type counterparts, confirming the role of TopIIbeta in neurite outgrowth. Together, these results demonstrate a critical role of TopIIbeta in neurite outgrowth in cultured neurons. Furthermore, we demonstrated that neurons derived from top2beta knockout mice failed to form contacts with muscle cells in co-cultures. These results suggest that the defect in establishing neuromuscular junctions in top2beta knockout mice could be due to the lack of TopIIbeta-mediated neurite outgrowth.
Assuntos
DNA Topoisomerases Tipo II/fisiologia , Proteínas de Ligação a DNA/fisiologia , Neuritos/fisiologia , Neurônios/citologia , Animais , Células Cultivadas , Cerebelo/citologia , Córtex Cerebral/citologia , Técnicas de Cocultura/métodos , DNA Topoisomerases Tipo II/deficiência , Proteínas de Ligação a DNA/deficiência , Dicetopiperazinas , Embrião de Mamíferos , Inibidores Enzimáticos/farmacologia , Feminino , Gânglios Espinais/citologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/genética , Masculino , Camundongos , Camundongos Knockout , Mioblastos/fisiologia , Neuritos/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Piperazinas/farmacologia , RatosRESUMO
NF-κB plays an important role in cancer initiation and progression. CD44, a cell surface glycoprotein, is involved in many cellular processes including cell adhesion, migration and proliferation. However, whether and how the two molecules interact in breast cancer is not clear. In recent years, the up-regulation of CD44 has served as a marker for tumor initiating cells in breast cancer and other cancer types. Despite the important role of CD44 in cellular processes and cancer, the mechanism underlying CD44 up-regulation in cancers remains poorly understood. Previously, we have identified a novel cis-element, CR1, located upstream of the CD44 promoter. We demonstrated that NF-κB and AP-1 are key trans-acting factors that interact with CR1. Here, we further analyzed the role of NF-κB in regulating CD44 expression in triple negative breast cancer cells, MDA-MB-231 and SUM159. Inhibition of NF-κB by Bay-11-7082 resulted in a reduction in CD44 expression. CD44 repression via NF-κB inhibition consequently decreased proliferation and invasiveness of breast cancer cells. These findings provide not only new insight into the molecular mechanism underlying CD44 regulation but also potential therapeutic targets that may help eliminate chemo- and radiation-resistant cancer cells.
Assuntos
Neoplasias da Mama/metabolismo , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Receptores de Hialuronatos/biossíntese , NF-kappa B/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Invasividade NeoplásicaRESUMO
Topoisomerase IIbeta (Top2b) is an enzyme that modulates DNA supercoiling by catalyzing the passage of DNA duplexes through one another. It is ubiquitously expressed in postmitotic cells and known to function during the development of neuromuscular junctions in the diaphragm and the proper formation of laminar structure in the cerebral cortex. However, due to the perinatal death phenotype of the traditional constitutive and brain-specific Top2b knockout mice, the precise in vivo function of Top2b, especially during postnatal neural development, remains to be determined. Using both the constitutive and retina-specific knockout mouse models, we showed that Top2b deficiency resulted in delayed neuronal differentiation, degeneration of the plexiform layers and outer segment of photoreceptors, as well as dramatic reduction in cell number in the retina. Genome-wide transcriptome analysis by RNA sequencing revealed that genes involved in neuronal survival and neural system development were preferentially affected in Top2b-deficient retinas. Collectively, our findings have indicated an important function of Top2b in proper development and the maintenance/survival of postmitotic neurons in the retina.
RESUMO
Topoisomerase IIß (Top2ß)-DNA cleavage complexes are known to arrest elongating RNA polymerase II (RNAPII), triggering a proteasomal degradation of the RNAPII large subunit (RNAPII LS) and Top2ß itself as a prelude to DNA repair. Here, we demonstrate that the degradation of Top2ß occurs through a novel ubiquitin-independent mechanism that requires only 19S AAA ATPases and 20S proteasome. Our results suggest that 19S AAA ATPases play a dual role in sensing the Top2ß cleavage complex and coordinating its degradation by 20S proteasome when RNAPII is persistently stalled by the Top2ß protein roadblock. Clarification of this transcription-associated proteasome pathway could shed light on a general role of 19S AAA ATPases in processing tight protein-DNA complexes during transcription elongation.
Assuntos
Adenosina Trifosfatases/genética , Reparo do DNA , DNA Topoisomerases Tipo II/genética , Proteínas de Ligação a DNA/genética , DNA/genética , Complexo de Endopeptidases do Proteassoma/genética , RNA Polimerase II/genética , Elongação da Transcrição Genética , Adenosina Trifosfatases/metabolismo , Animais , DNA/metabolismo , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/metabolismo , Embrião de Mamíferos , Fibroblastos/citologia , Fibroblastos/metabolismo , Células HeLa , Humanos , Camundongos , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , RNA Polimerase II/metabolismo , UbiquitinaRESUMO
Doxorubicin is believed to cause dose-dependent cardiotoxicity through redox cycling and the generation of reactive oxygen species (ROS). Here we show that cardiomyocyte-specific deletion of Top2b (encoding topoisomerase-IIß) protects cardiomyocytes from doxorubicin-induced DNA double-strand breaks and transcriptome changes that are responsible for defective mitochondrial biogenesis and ROS formation. Furthermore, cardiomyocyte-specific deletion of Top2b protects mice from the development of doxorubicin-induced progressive heart failure, suggesting that doxorubicin-induced cardiotoxicity is mediated by topoisomerase-IIß in cardiomyocytes.
Assuntos
Cardiotoxinas , Doxorrubicina/toxicidade , Renovação Mitocondrial/efeitos dos fármacos , Miócitos Cardíacos , Animais , Apoptose/efeitos dos fármacos , DNA Topoisomerases Tipo II/genética , Proteínas de Ligação a DNA/genética , Insuficiência Cardíaca/induzido quimicamente , Humanos , Camundongos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Proteínas de Ligação a Poli-ADP-Ribose , Espécies Reativas de Oxigênio/metabolismo , Deleção de SequênciaRESUMO
Low doses of anticancer drugs have been shown to enhance antitumor immune response and increase the efficacy of immunotherapy. The molecular basis for such effects remains elusive, although selective depletion of T regulatory cells has been demonstrated. In the current studies, we demonstrate that topotecan (TPT), a topoisomerase I-targeting drug with a well-defined mechanism of action, stimulates major histocompatibility complex class I (MHC I) expression in breast cancer cells through elevated expression/secretion of interferon-ß (IFN-ß) and activation of type I IFN signaling. First, we show that TPT treatment elevates the expression of both total and cell-surface MHC I in breast cancer cells. Second, conditioned media from TPT-treated breast cancer ZR-75-1 cells induce elevated expression of cell-surface MHC I in drug-naïve recipient cells, suggesting the involvement of cytokines and/or other secreted molecules. Consistently, TPT-treated cells exhibit elevated expression of multiple cytokines such as IFN-ß, TNF-α, IL-6 and IL-8. Third, either knocking down the type I interferon receptor subunit 1 (IFNAR1) or addition of neutralizing antibody against IFN-ß results in reduced MHC I expression in TPT-treated cells. Together, these results suggest that TPT induces increased IFN-ß autocrine/paracrine signaling through type I IFN receptor, resulting in the elevated MHC I expression in tumor cells. Studies have also demonstrated that other chemotherapeutic agents (e.g. etoposide, cisplatin, paclitaxel and vinblastine) similarly induce increased IFN-ß secretion and elevated MHC I expression. In addition, conditioned media from γ-irradiated donor cells are shown to induce IFN-ß-dependent MHC I expression in unirradiated recipient cells. In the aggregate, our results suggest that many cancer therapeutics induce elevated tumor antigen presentation through MHC I, which could represent a common mechanism for enhanced antitumor immune response through T cell cytotoxicity during metronomic chemotherapy, as well as increased efficacy of combined chemo- (or radio-)/immuno-therapy.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/radioterapia , Regulação Neoplásica da Expressão Gênica , Genes MHC Classe I , Antígenos de Histocompatibilidade Classe I/biossíntese , Interferon beta/metabolismo , Transdução de Sinais , Antineoplásicos/uso terapêutico , Apoptose , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Humanos , Interferon beta/biossíntese , Interleucina-6/biossíntese , Interleucina-8/biossíntese , NF-kappa B/metabolismo , Topotecan/farmacologia , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Increasing evidence has pointed to activated type I interferon signaling in tumors. However, the molecular basis for such activation and its role in tumorigenesis remain unclear. In the current studies, we report that activation of type I interferon (IFN) signaling in tumor cells is primarily due to elevated secretion of the type I interferon, IFN-ß. Studies in oncogene-transformed cells suggest that oncogenes such as Ras and Src can activate IFN-ß signaling. Significantly, elevated IFN-ß signaling in Ras-transformed mammary epithelial MCF-10A cells was shown to contribute to Ras transformation as evidenced by morphological changes, anchorage-independent growth, and migratory properties. Our results demonstrate for the first time that the type I IFN, IFN-ß, contributes to Ras transformation and support the notion that oncogene-induced cytokines play important roles in oncogene transformation.