RESUMO
The neural crest transcription factor BRN3A is essential for the proliferation and survival of melanoma cells. It is frequently expressed in melanoma but not in normal melanocytes or benign nevi. The mechanisms underlying the aberrant expression of BRN3A are unknown. Here, we investigated the epigenetic regulation of BRN3A in melanocytes and melanoma cell lines treated with DNA methyltransferase (DNMT), histone acetyltransferase (HAT), and histone deacetylase (HDAC) inhibitors. DNMT and HAT inhibition did not significantly alter BRN3A expression levels, whereas panHDAC inhibition by trichostatin A led to increased expression. Treatment with the isoform-specific HDAC inhibitor mocetinostat, but not with PCI-34051, also increased BRN3A expression levels, suggesting that class I HDACs HDAC1, HDAC2, and HDAC3, and class IV HDAC11, were involved in the regulation of BRN3A expression. Transient silencing of HDACs 1, 2, 3, and 11 by siRNAs revealed that, specifically, HDAC2 inhibition was able to increase BRN3A expression. ChIP-Seq analysis uncovered that HDAC2 inhibition specifically increased H3K27ac levels at a distal enhancer region of the BRN3A gene. Altogether, our data suggest that HDAC2 is a key epigenetic regulator of BRN3A in melanocytes and melanoma cells. These results highlight the importance of epigenetic mechanisms in regulating melanoma oncogenes.
Assuntos
Regulação da Expressão Gênica , Histona Desacetilase 2/metabolismo , Melanócitos/metabolismo , Melanoma/etiologia , Melanoma/metabolismo , Fator de Transcrição Brn-3A/genética , Linhagem Celular , Metilação de DNA , Epigênese Genética , Regulação da Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Histona Desacetilase 2/genética , Inibidores de Histona Desacetilases/farmacologia , Humanos , Melanócitos/patologia , Melanoma/patologia , Fator de Transcrição Brn-3A/metabolismoRESUMO
Cellular heterogeneity, for example, the intratumoral coexistence of cancer cells with and without stem cell characteristics, represents a potential root of therapeutic resistance and a significant challenge for modern drug development in glioblastoma (GBM). We propose here that activation of the innate immune system by stimulation of innate immune receptors involved in antiviral and antitumor responses can similarly target different malignant populations of glioma cells. We used short-term expanded patient-specific primary human GBM cells to study the stimulation of the cytosolic nucleic acid receptors melanoma differentiation-associated gene 5 (MDA5) and retinoic acid-inducible gene I (RIG-I). Specifically, we analyzed cells from the tumor core versus "residual GBM cells" derived from the tumor resection margin as well as stem cell-enriched primary cultures versus specimens without stem cell properties. A portfolio of human, nontumor neural cells was used as a control for these studies. The expression of RIG-I and MDA5 could be induced in all of these cells. Receptor stimulation with their respective ligands, p(I:C) and 3pRNA, led to in vitro evidence for an effective activation of the innate immune system. Most intriguingly, all investigated cancer cell populations additionally responded with a pronounced induction of apoptotic signaling cascades revealing a second, direct mechanism of antitumor activity. By contrast, p(I:C) and 3pRNA induced only little toxicity in human nonmalignant neural cells. Granted that the challenge of effective central nervous system (CNS) delivery can be overcome, targeting of RIG-I and MDA5 could thus become a quintessential strategy to encounter heterogeneous cancers in the sophisticated environments of the brain.
Assuntos
Antineoplásicos/farmacologia , Citosol/imunologia , RNA Helicases DEAD-box/imunologia , Glioblastoma/tratamento farmacológico , Glioblastoma/imunologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Apoptose/imunologia , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Citosol/efeitos dos fármacos , Citosol/metabolismo , Proteína DEAD-box 58 , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Imunidade Inata/efeitos dos fármacos , Imunidade Inata/genética , Imunidade Inata/imunologia , Helicase IFIH1 Induzida por Interferon , Ligantes , Receptores Imunológicos , Transdução de Sinais/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Células-Tronco/imunologia , Células-Tronco/metabolismoRESUMO
A key host response to limit microbial spread is the induction of cell death when foreign nucleic acids are sensed within infected cells. In mouse macrophages, transfected DNA or infection with modified vaccinia virus Ankara (MVA) can trigger cell death via the absent in melanoma 2 (AIM2) inflammasome. In this article, we show that nonmyeloid human cell types lacking a functional AIM2 inflammasome still die in response to cytosolic delivery of different DNAs or infection with MVA. This cell death induced by foreign DNA is independent of caspase-8 and carries features of mitochondrial apoptosis: dependence on BAX, APAF-1, and caspase-9. Although it does not require the IFN pathway known to be triggered by infection with MVA or transfected DNA via polymerase III and retinoid acid-induced gene I-like helicases, it shows a strong dependence on components of the DNA damage signaling pathway: cytosolic delivery of DNA or infection with MVA leads to phosphorylation of p53 (serines 15 and 46) and autophosphorylation of ataxia telangiectasia mutated (ATM); depleting p53 or ATM with small interfering RNA or inhibiting the ATM/ATM-related kinase family by caffeine strongly reduces apoptosis. Taken together, our findings suggest that a pathway activating DNA damage signaling plays an important independent role in detecting intracellular foreign DNA, thereby complementing the induction of IFN and activation of the AIM2 inflammasome.
Assuntos
Apoptose/imunologia , Dano ao DNA/imunologia , DNA Viral/imunologia , Macrófagos/imunologia , Proteínas Nucleares/imunologia , RNA Polimerase III/imunologia , Transdução de Sinais/imunologia , Vaccinia virus/imunologia , Vacínia/imunologia , Animais , Apoptose/genética , Fator Apoptótico 1 Ativador de Proteases/genética , Fator Apoptótico 1 Ativador de Proteases/imunologia , Fator Apoptótico 1 Ativador de Proteases/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia , Caspase 8/genética , Caspase 8/imunologia , Caspase 8/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/imunologia , Proteínas de Ciclo Celular/metabolismo , Citosol , DNA Viral/genética , DNA Viral/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Proteínas de Ligação a DNA/metabolismo , Células HEK293 , Humanos , Inflamassomos/genética , Inflamassomos/imunologia , Inflamassomos/metabolismo , Interferons/genética , Interferons/imunologia , Interferons/metabolismo , Macrófagos/metabolismo , Macrófagos/virologia , Camundongos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilação/genética , Fosforilação/imunologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/imunologia , Proteínas Serina-Treonina Quinases/metabolismo , RNA Polimerase III/genética , RNA Polimerase III/metabolismo , Transdução de Sinais/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/imunologia , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/imunologia , Proteínas Supressoras de Tumor/metabolismo , Vacínia/genética , Vacínia/metabolismo , Vaccinia virus/genética , Vaccinia virus/metabolismo , Proteína X Associada a bcl-2/genéticaRESUMO
The role of immune suppression by regulatory T (Treg) cells in the maintenance of immune homeostasis is well established. However, little is known about how Treg cell function is inhibited on viral infection to allow the development of a protective immune response. As viral RNA is a crucial mediator for activation of antiviral immunity, we examined the effects of immunostimulatory RNA and infection with RNA viruses on Treg cell function. We show that synthetic RNA oligonucleotides potently inhibit Treg cell-induced suppression in a sequence-dependent manner. This effect is entirely dependent on TLR7 activation of APCs and subsequent IL-6 production. In addition, stimulation with the RNA viruses encephalomyocarditis virus and Sendai virus that specifically activate the RNA-sensing helicases melanoma differentiation-associated gene 5 (MDA-5) and retinoic acid-inducible gene I (RIG-I) also blocks Treg cell function. Interestingly, this effect is seen even in the absence of APCs. Consistent with this, both Treg and T effector cells express RIG-I and MDA-5. Using MDA-5-deficient mice, we demonstrate that the loss of Treg cell function on infection with encephalomyocarditis virus is strictly dependent on MDA-5 expression by Treg cells. Thus, we show in this study for the first time that activation of a RIG-I-like helicase on Treg cells blocks their suppressive function.
Assuntos
RNA Helicases DEAD-box/metabolismo , Oligonucleotídeos/imunologia , RNA/imunologia , Linfócitos T Reguladores/imunologia , Animais , Células Apresentadoras de Antígenos , Sequência de Bases , Proteína DEAD-box 58 , Helicase IFIH1 Induzida por Interferon , Interleucina-6/biossíntese , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Vírus de RNA/imunologia , RNA Viral/imunologia , Linfócitos T Reguladores/metabolismo , Linfócitos T Reguladores/virologia , Receptor 7 Toll-Like/metabolismoRESUMO
The ATPase retinoid acid-inducible gene (RIG)-I senses viral RNA in the cytoplasm of infected cells and subsequently activates cellular antiviral defense mechanisms. RIG-I recognizes molecular structures that discriminate viral from host RNA. Here, we show that RIG-I ligands require base-paired structures in conjunction with a free 5'-triphosphate to trigger antiviral signaling. Hitherto unavailable chemically synthesized 5'-triphosphate RNA ligands do not trigger RIG-I-dependent IFN production in cells, and they are unable to trigger the ATPase activity of RIG-I without a base-paired stretch. Consistently, immunostimulatory RNA from cells infected with a virus recognized by RIG-I is sensitive to double-strand, but not single-strand, specific RNases. In vitro, base-paired stretches and the 5'-triphosphate bind to distinct sites of RIG-I and synergize to trigger the induction of signaling competent RIG-I multimers. Strengthening our model of a bipartite molecular pattern for RIG-I activation, we show that the activity of supposedly "single-stranded" 5'-triphosphate RNAs generated by in vitro transcription depends on extended and base-paired by-products inadvertently, but commonly, produced by this method. Together, our findings accurately define a minimal molecular pattern sufficient to activate RIG-I that can be found in viral genomes or transcripts.
Assuntos
Pareamento de Bases , RNA/química , RNA/imunologia , Receptores do Ácido Retinoico/metabolismo , Transdução de Sinais/imunologia , Vírus/imunologia , Adenosina Trifosfatases/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , RNA Polimerases Dirigidas por DNA/metabolismo , Humanos , Ligantes , Camundongos , Ligação Proteica , Multimerização Proteica , Receptores de Reconhecimento de Padrão/metabolismo , Transcrição Gênica , Proteínas Virais/metabolismoRESUMO
Several germline mutations and sequence variants in cancer predisposition genes have been described. Among these, the CDKN2A p.A148T variant appears to be frequent in patients with melanoma, at least in certain ethnic groups. In this case-control study, we evaluated 127 patients with cutaneous melanoma and 128 controls from Southern Brazil, the region with the highest melanoma incidence rates in the country. Using PCR-RFLP, we demonstrate that CDKN2A p.A148T variant was significantly more frequent in patients with melanoma than in controls (12.6% vs 3.9%; P=0.009). There was no association between presence of the polymorphism and tumor thickness, site of the primary tumor, melanoma subtype, age at diagnosis, quantitative and qualitative number of nevi. Patients with a positive family of history for other cancers were particularly prone to carry the CDKN2A p.A148T allele. All patients with p.A148T-positive melanoma reported European ancestry, especially German, and this was confirmed using a panel of ancestry-informative INDELs. Our data suggest that CDKN2A p.A148T is a melanoma susceptibility allele in Southern Brazil and is particularly common in patients with melanoma of predominantly European ancestry.
Assuntos
Genes p16 , Melanoma/genética , Polimorfismo de Nucleotídeo Único , Neoplasias Cutâneas/genética , Substituição de Aminoácidos , Sequência de Bases , Brasil/epidemiologia , Estudos de Casos e Controles , Primers do DNA/genética , Etnicidade/genética , Europa (Continente)/etnologia , Feminino , Frequência do Gene , Predisposição Genética para Doença , Humanos , Mutação INDEL , Masculino , Melanoma/epidemiologia , Melanoma/patologia , Pessoa de Meia-Idade , Epidemiologia Molecular , Estudos Prospectivos , Neoplasias Cutâneas/epidemiologia , Neoplasias Cutâneas/patologiaRESUMO
BACKGROUND: Human renal cell carcinoma (RCC) is very resistant to chemotherapy. ABT-737 is a novel inhibitor of anti-apoptotic proteins of the Bcl-2 family that has shown promise in various preclinical tumour models. RESULTS: We here report a strong over-additive pro-apoptotic effect of ABT-737 and etoposide, vinblastine or paclitaxel but not 5-fluorouracil in cell lines from human RCC. ABT-737 showed very little activity as a single agent but killed RCC cells potently when anti-apoptotic Mcl-1 or, unexpectedly, A1 was targeted by RNAi. This potent augmentation required endogenous Noxa protein since RNAi directed against Noxa but not against Bim or Puma reduced apoptosis induction by the combination of ABT-737 and etoposide or vinblastine. At the level of mitochondria, etoposide-treatment had a similar sensitizing activity and allowed for ABT-737-induced release of cytochrome c. CONCLUSIONS: Chemotherapeutic drugs can overcome protection afforded by Mcl-1 and A1 through endogenous Noxa protein in RCC cells, and the combination of such drugs with ABT-737 may be a promising strategy in RCC. Strikingly, A1 emerged in RCC cell lines as a protein of similar importance as the well-established Mcl-1 in protection against apoptosis in these cells.
Assuntos
Antineoplásicos/farmacologia , Compostos de Bifenilo/farmacologia , Carcinoma de Células Renais/patologia , Neoplasias Renais/patologia , Nitrofenóis/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Sulfonamidas/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma de Células Renais/metabolismo , Linhagem Celular Tumoral , Sinergismo Farmacológico , Humanos , Neoplasias Renais/metabolismo , Mitocôndrias/efeitos dos fármacos , Proteína de Sequência 1 de Leucemia de Células Mieloides , Piperazinas/farmacologiaRESUMO
Nestin is an intermediate filament expressed in proliferating neural progenitor cells and has been considered as a stem cell marker. Nestin is also found in melanoma and we recently demonstrated that its expression in melanoma cell lines is regulated by the transcription factors SOX9 and SOX10, but not BRN2. In this study, the expression levels of nestin, BRN2, SOX9 and SOX10 were analysed in tissues of melanoma (n = 78) and melanocytic nevi (n = 26) by immunohistochemistry. All proteins were highly expressed in primary and metastatic melanomas and, apart from BRN2, showed much lower levels in melanocytic nevi. Significant coexpression of nestin with SOX9 and SOX10 was found in primary melanoma confirming our in vitro data. Correlation analysis with clinicopathological data revealed that nestin was significantly associated with presence of ulceration in primary tumors and SOX9 with more advanced stage of disease. Our data reveal that SOX9 and SOX10 are highly expressed in melanoma and seem to have a regulatory role in nestin expression. The association with ulceration and advanced-stage tumors, respectively, suggests that nestin and SOX9 may be negative prognostic markers in melanoma.
Assuntos
Proteínas de Filamentos Intermediários/metabolismo , Melanoma/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fatores de Transcrição SOX9/metabolismo , Fatores de Transcrição SOXE/metabolismo , Neoplasias Cutâneas/metabolismo , Biomarcadores Tumorais/metabolismo , Biópsia , Proteínas de Homeodomínio/metabolismo , Humanos , Melanoma/patologia , Metástase Neoplásica , Nestina , Fatores do Domínio POU/metabolismo , Neoplasias Cutâneas/patologiaRESUMO
Cathelicidin is strongly expressed in lesional skin in psoriasis and may play an important role as both an antimicrobial peptide and as an autoinflammatory mediator in this chronic skin disease. The mechanism of increased cathelicidin in psoriatic keratinocytes is not known, but recent observations have found that psoriasis has abundant Th17 cells that produce IL-17A and IL-22. We found that human keratinocytes stimulated with supernatants from T cells isolated from lesional psoriatic skin increased expression of cathelicidin when stimulated in the presence of 1,25-dihydroxyvitamin D(3) (1,25D(3)). This increase was signaled through the IL-17RA. In vitro, IL-17A, but not IL-22, enhanced cathelicidin mRNA and peptide expression in keratinocytes dependent on the presence of 1,25D(3). At the same time, coincubation with 1,25D(3) blocked induction of human beta-defensin 2 (HBD2), IL-6, and IL-8, which are other target genes of IL-17A. Act1, an adaptor associated with IL-17RA and essential for IL-17A signaling, mediated cathelicidin induction, as its suppression by small interfering RNA inhibited HBD2 and cathelicidin. Both, 1,25D(3) and IL-17A signaled cathelicidin induction through MEK-ERK. These results suggest that increased IL-17A in psoriatic skin increases cathelicidin through a vitamin D(3)-, Act1-, and MEK-ERK-dependent mechanism. Therapy targeting this cathelicidin-regulating system might be beneficial in patients suffering from psoriasis.
Assuntos
Peptídeos Catiônicos Antimicrobianos/biossíntese , Colecalciferol/farmacologia , Interleucina-17/fisiologia , Queratinócitos/imunologia , Queratinócitos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Antibacterianos/biossíntese , Peptídeos Catiônicos Antimicrobianos/genética , Catelicidinas , Linhagem Celular , Células Cultivadas , Humanos , Mediadores da Inflamação/fisiologia , Queratinócitos/enzimologia , Sistema de Sinalização das MAP Quinases/imunologia , Psoríase/enzimologia , Psoríase/imunologia , Psoríase/patologia , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/metabolismo , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/fisiologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/imunologiaRESUMO
Ebola virus (EBOV) causes a deadly hemorrhagic fever in humans and non-human primates. There is currently no FDA-approved vaccine or medication to counter this disease. Here, we report on the design, synthesis and anti-viral activities of two classes of compounds which show high potency against EBOV in both in vitro cell culture assays and in vivo mouse models Ebola viral disease. These compounds incorporate the structural features of cationic amphiphilic drugs (CAD), i.e they possess both a hydrophobic domain and a hydrophilic domain consisting of an ionizable amine functional group. These structural features enable easily diffusion into cells but once inside an acidic compartment their amine groups became protonated, ionized and remain trapped inside the acidic compartments such as late endosomes and lysosomes. These compounds, by virtue of their lysomotrophic functions, blocked EBOV entry. However, unlike other drugs containing a CAD moiety including chloroquine and amodiaquine, compounds reported in this study display faster kinetics of accumulation in the lysosomes, robust expansion of late endosome/lysosomes, relatively more potent suppression of lysosome fusion with other vesicular compartments and inhibition of cathepsins activities, all of which play a vital role in anti-EBOV activity. Furthermore, the diazachrysene 2 (ZSML08) that showed most potent activity against EBOV in in vitro cell culture assays also showed significant survival benefit with 100% protection in mouse models of Ebola virus disease, at a low dose of 10â¯mg/kg/day. Lastly, toxicity studies in vivo using zebrafish models suggest no developmental defects or toxicity associated with these compounds. Overall, these studies describe two new pharmacophores that by virtue of being potent lysosomotrophs, display potent anti-EBOV activities both in vitro and in vivo animal models of EBOV disease.
Assuntos
Antivirais/química , Crisenos/química , Ebolavirus/efeitos dos fármacos , Doença pelo Vírus Ebola/tratamento farmacológico , Animais , Antivirais/farmacologia , Antivirais/toxicidade , Crisenos/farmacologia , Crisenos/toxicidade , Lisossomos/metabolismo , Camundongos , Tensoativos , Internalização do Vírus/efeitos dos fármacos , Peixe-ZebraRESUMO
Melanoma cells share many biological properties with neural crest stem cells. Here we show that the homeodomain transcription factor MSX1, which is significantly correlated with melanoma disease progression, reprograms melanocytes and melanoma cells toward a neural crest precursor-like state. MSX1-reprogrammed normal human melanocytes express the neural crest marker p75 and become multipotent. MSX1 induces a phenotypic switch in melanoma, which is characterized by an oncogenic transition from an E-cadherin-high nonmigratory state toward a ZEB1-high invasive state. ZEB1 up-regulation is responsible for the MSX1-induced migratory phenotype in melanoma cells. Depletion of MSX1 significantly inhibits melanoma metastasis in vivo. These results show that neural crest-like reprogramming achieved by a single factor is a critical process for melanoma progression.
Assuntos
Transformação Celular Neoplásica/patologia , Reprogramação Celular/fisiologia , Fator de Transcrição MSX1/fisiologia , Melanócitos/patologia , Melanoma/patologia , Neoplasias Cutâneas/patologia , Animais , Antígenos CD/metabolismo , Caderinas/metabolismo , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular , Derme/citologia , Derme/patologia , Progressão da Doença , Células HEK293 , Células-Tronco Embrionárias Humanas , Humanos , Estimativa de Kaplan-Meier , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/secundário , Fator de Transcrição MSX1/genética , Melanoma/mortalidade , Melanoma/secundário , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas do Tecido Nervoso/metabolismo , Crista Neural/fisiologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Neoplasias Cutâneas/mortalidade , Ensaios Antitumorais Modelo de Xenoenxerto , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismoRESUMO
Significant advancements of mutation-based targeted therapy and immune checkpoint blockade have been achieved in melanoma. Nevertheless, acquired resistance and nonresponders to therapy require different strategies. An innovative approach is presented here that is based on the combination of innate immune system activation and simultaneous targeting of the oncogene urokinase-type plasminogen activator receptor (uPAR). We generated two triphosphate-conjugated siRNAs targeting uPAR (ppp-uPAR) by in vitro transcription. Specific uPAR knockdown and simultaneous activation of the retinoic acid-inducible gene 1 (RIG-I) was shown in different human melanoma cells, fibroblasts, and melanocytes. The compounds induced massive apoptosis in melanoma cells, whereas fibroblasts and melanocytes were less sensitive. The effects were less pronounced when the IFN receptor was blocked. Treatment with ppp-uPAR led to accumulation of p53 and induction of RIG-I-dependent proapoptotic signaling. The apoptotic effects induced by ppp-uPAR were maintained in melanoma cell lines that had acquired double resistance to B-RAF and MEK/extracellular signal-regulated kinase inhibition. Systemic intraperitoneal application of ppp-uPAR in nude mice significantly reduced growth of human melanoma xenografts and elicited a systemic innate immune response with increased serum cytokine levels. Our data suggest that ppp-uPAR represents a therapeutically attractive compound that may help overcome the strong therapy resistance of melanoma.
Assuntos
Imunização/métodos , Melanoma/genética , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , Neoplasias Cutâneas/genética , Animais , Apoptose/genética , Sobrevivência Celular/genética , Modelos Animais de Doenças , Feminino , Humanos , Melanócitos/citologia , Melanócitos/patologia , Melanoma/patologia , Melanoma/terapia , Camundongos , Camundongos Knockout , Camundongos Nus , RNA Interferente Pequeno/genética , Distribuição Aleatória , Receptores de Superfície Celular , Valores de Referência , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/terapia , Células Tumorais CultivadasRESUMO
DNA-binding molecules that recognize specific sequences offer a high potential for the understanding of chromatin structure and associated biological processes in addition to their therapeutic potential, e.g. as positioning agents for validated anticancer drugs. A prerequisite for the development of DNA-binding molecules is the availability of appropriate methods to assess their binding properties quantitatively at the desired target sequence in the human genome. We have further developed a capture assay to assess triplex-forming oligonucleotide (TFO) binding efficiency quantitatively. This assay is based on bifunctional, psoralen and biotin-conjugated, TFOs and real-time PCR analysis. We have applied this novel quantification method to address two issues that are relevant for DNA-binding molecules. First, we have compared directly the extent of TFO-binding in three experimental settings with increasing similarity to the situation in vivo, i.e. naked genomic DNA, isolated cell nuclei, or whole cells. This comparison allows us to characterize factors that influence genomic triplex formation, e.g. chromosomal DNA organization or intracellular milieu. In isolated nuclei, the binding was threefold lower compared to naked DNA, consistent with a decreased target accessibility int he nucleosomal environment. Binding was detected in whole cells, indicating that the TFO enters the nucleus and binds to its target in intact cells in vivo, but the efficiency was decreased (tenfold) compared to nuclei. Secondly, we applied the method to characterize the binding properties of two different TFOs targeting the same sequence. We found that an antiparallel-binding GT-containing TFO bound more efficiently, but with less target sequence selectivity compared to a parallel-binding CU-containing TFO. Collectively, a sensitive method to characterize genomic triplex formation was described. This may be useful for the determination of factors driving TFO binding efficiency and, thus, may improve the usefulness of triplex-mediated gene targeting for studies of chromatin structure as well as for therapeutic antigene strategies.
Assuntos
DNA/química , Conformação de Ácido Nucleico , Sequência de Bases , Linhagem Celular Tumoral , Primers do DNA , Furocumarinas/química , Humanos , Reação em Cadeia da PolimeraseRESUMO
Psoriasis vulgaris is a common T cell-mediated inflammatory skin disease with a suspected autoimmune pathogenesis. The human leukocyte antigen (HLA) class I allele, HLA-C*06:02, is the main psoriasis risk gene. Epidermal CD8(+) T cells are essential for psoriasis development. Functional implications of HLA-C*06:02 and mechanisms of lesional T cell activation in psoriasis, however, remained elusive. Here we identify melanocytes as skin-specific target cells of an HLA-C*06:02-restricted psoriatic T cell response. We found that a Vα3S1/Vß13S1 T cell receptor (TCR), which we had reconstituted from an epidermal CD8(+) T cell clone of an HLA-C*06:02-positive psoriasis patient specifically recognizes HLA-C*06:02-positive melanocytes. Through peptide library screening, we identified ADAMTS-like protein 5 (ADAMTSL5) as an HLA-C*06:02-presented melanocytic autoantigen of the Vα3S1/Vß13S1 TCR. Consistent with the Vα3S1/Vß13S1-TCR reactivity, we observed numerous CD8(+) T cells in psoriasis lesions attacking melanocytes, the only epidermal cells expressing ADAMTSL5. Furthermore, ADAMTSL5 stimulation induced the psoriasis signature cytokine, IL-17A, in CD8(+) T cells from psoriasis patients only, supporting a role as psoriatic autoantigen. This unbiased analysis of a TCR obtained directly from tissue-infiltrating CD8(+) T cells reveals that in psoriasis HLA-C*06:02 directs an autoimmune response against melanocytes through autoantigen presentation. We propose that HLA-C*06:02 may predispose to psoriasis via this newly identified autoimmune pathway.
Assuntos
Autoantígenos/imunologia , Autoimunidade/imunologia , Melanócitos/metabolismo , Psoríase/imunologia , Proteínas ADAM/metabolismo , Proteínas ADAMTS , Adulto , Sequência de Aminoácidos , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular , Epiderme/metabolismo , Epiderme/patologia , Epitopos/química , Epitopos/imunologia , Feminino , Antígenos HLA-C/imunologia , Humanos , Masculino , Dados de Sequência Molecular , Peptídeos/química , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismoRESUMO
The combination of psoralens with UVA is used as PUVA therapy for psoriasis and other skin diseases. UVA-induced psoralen/DNA photoadducts act via suppression of DNA replication and cell proliferation, but do not sufficiently repress gene transcription. To explore whether PUVA may also be used for gene repression, psoralen was conjugated to a triplex-forming oligonucleotide (TFO) that targets a gene sequence of ICAM-1, a key molecule in cutaneous inflammation. Triplex formation between TFO and target sequence was detected by non-denaturing gel electrophoresis. UVA-irradiation induced psoralen cross-links at the triplex-duplex junction as verified by denaturing gel electrophoresis. When the target sequence was placed within the transcribed portion of the chloramphenicol acetyltransferase (CAT) gene, TFO inhibited CAT expression in A431 cells. Inhibition was sequence-specific, since a scrambled control oligonucleotide or mismatched or scrambled target sequences failed to inhibit CAT expression. Inhibition was not significant without UVA exposure, but was strongly enhanced by PUVA-mediated cross-links at the TFO target site. These results suggest that TFO may add a new quality to PUVA therapy by transcriptionally repressing pathogenically relevant genes, in addition to antiproliferative PUVA effects. TFO designed to repress only after PUVA activation may allow the development of a cutaneous organ specific strategy for gene repression.
Assuntos
Expressão Gênica/efeitos dos fármacos , Terapia PUVA/métodos , Psoríase/tratamento farmacológico , Psoríase/fisiopatologia , Carcinoma de Células Escamosas , Linhagem Celular Tumoral , Reagentes de Ligações Cruzadas/farmacologia , Furocumarinas/farmacologia , Genes Reporter/efeitos dos fármacos , Humanos , Molécula 1 de Adesão Intercelular/genética , Conformação de Ácido Nucleico , Oligonucleotídeos/química , Oligonucleotídeos/genética , Neoplasias CutâneasRESUMO
The down regulation of gene expression is a promising strategy for molecular medicine and experimental biology. Molecules that bind to the DNA double helix may interfere with gene expression and, in addition to potential therapeutic applications, can be helpful for the investigation of DNA processing, chromatin package, or associated biological processes. Triplex-forming oligonucleotides (TFOs) bind to specific sequences in the DNA double helix via hydrogen bonding interactions. TFOs have been shown to down-regulate gene expression, to induce targeted genomic DNA modifications, to stimulate DNA recombination, and to modulate chromatin organization. Additionally, they may be used as carriers to position DNA-modifying agents to selected sequences. TFO-mediated effects have been mostly described in cell culture, but one study reported TFO activity in a mouse model. Critical issues regarding TFO-based technologies are the development of new oligonucleotide analogues with improved binding affinity, better target selectivity, and sufficient stability in the intracellular environment. A prerequisite for the development of such DNA-binding molecules is the availability of appropriate methods to assess their binding properties quantitatively at the desired target sequence in the genome. This review focuses on recent results regarding gene-inhibitory effects of TFOs in cell culture and methods to evaluate TFO-binding to the desired target sequence in the context of the human genome.
Assuntos
DNA/farmacologia , Marcação de Genes , Animais , Sequência de Bases , DNA/metabolismo , Regulação para Baixo , Regulação da Expressão Gênica , Humanos , Molécula 1 de Adesão Intercelular/genéticaRESUMO
Re-activation of molecules active in embryogenesis can play an important role in cancer, since they can provide tumor cells with malignant properties. Several members of the family of POU transcription factors are essential for the development of the nervous system and several are expressed in the neural crest, which is the same location where melanocytic cells develop from. Here, the POU transcription factor family and its role in melanocytic transformation and melanoma are reviewed.
Assuntos
Melanócitos/metabolismo , Melanoma/genética , Fatores do Domínio POU/metabolismo , Neoplasias Cutâneas/genética , Humanos , Melanoma/metabolismo , Fatores do Domínio POU/genética , Neoplasias Cutâneas/metabolismoRESUMO
Double-stranded RNA molecules carrying a triphosphate moiety represent a molecular structure by which the host recognizes viral infections. Such RNA molecules can be generated synthetically by chemical synthesis or by in vitro transcription (see Chapter 2, Hornung et al.). Similar to viruses, they initiate an antiviral immune response, e.g., by stimulation of the immune system. Short, double-stranded RNA in the cytosol can also trigger the RNA interference mechanism, which also has been considered as an antiviral response. Notably, synthetic RNAs that are designed to be specific for a certain host mRNA inhibit expression of the respective gene, leading to specific gene silencing. Both effects-gene silencing and immunostimulation-are interesting from a therapeutic perspective, e.g., for cancer therapy. Notably, both effects can be activated by a single molecule, an siRNA carrying a triphosphate moiety. This chapter provides information how to design such compounds with respect to the associated signaling pathways and the techniques to evaluate bifunctional RNAs in the context of tumor therapy.
Assuntos
Neoplasias/terapia , RNA Interferente Pequeno/genética , Animais , Inativação Gênica , Humanos , Neoplasias/genética , Interferência de RNA , RNA Interferente Pequeno/fisiologiaRESUMO
Cyprinid herpesvirus-3 (CyHV-3) is an etiological agent of a notifiable disease that causes high mortality rates affecting both the common and koi carp Cyprinus carpio L. There is no current treatment strategy to save CyHV-3 infected fish. RNA mediated interference (RNAi) is an emerging strategy used for understanding gene function and is a promising method in developing novel therapeutics and antiviral medications. For this study, the possibility of activating the RNAi pathway by the use of small interfering (si)RNAs was tested to inhibit in vitro viral replication of CyHV-3 in common carp brain (CCB) cells. The siRNAs were designed to target either thymidine kinase (TK) or DNA polymerase (DP) genes, which both code for transcripts involved in DNA replication. The inhibition of viral replication caused by the siRNAs was measured by a reporter gene, termed ORF81. Treatment with siRNA targeting either TK or DP genes reduced the release of viral particles from infected CCB cells. However, siRNA targeting DP was most effective at reducing viral release as measured by qPCR.
Assuntos
Antivirais/farmacologia , Carpas/virologia , Herpesviridae/efeitos dos fármacos , Herpesviridae/fisiologia , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Replicação Viral/efeitos dos fármacos , Animais , Células Cultivadas , DNA Viral/análise , Herpesviridae/genética , Testes de Sensibilidade Microbiana , Carga ViralRESUMO
The transcription factor SOX10 (SRY (sex determining region Y)-box 10) has a key role in the embryonic development of melanocytes. Recently, it has been suggested that SOX10 is highly relevant for melanoma development and survival. However, the distinct functions and downstream targets of SOX10 in melanoma remain widely unknown. In this study, we inhibited SOX10 via RNA interference in different human melanoma cell lines and found a significantly reduced invasion capacity in vitro and in the chick embryo model. At later time points, SOX10 inhibition reduced proliferation and induced cell death. We identified melanoma inhibitory activity (MIA) as a direct target gene of SOX10, which is an essential protein for melanoma cell migration and invasion. Expression levels of SOX10 and MIA strictly correlated in melanoma cell lines, and SOX10 inhibition reduced MIA expression and promoter activity. Direct binding of SOX10 to the MIA promoter was demonstrated by electrophoretic mobility shift assay and chromatin immunoprecipitation. Ectopic expression of MIA in SOX10-inhibited melanoma cells restored the invasion capacity, supporting the hypothesis that MIA is responsible for SOX10-mediated melanoma cell invasion. Our data provide evidence for a critical role of SOX10 in melanoma cell invasion through the regulation of MIA and highlight its role as a therapeutic target in melanoma.