RESUMO
Large-scale chromosomal deletions are a prevalent and defining feature of cancer. A high degree of tumor-type and subtype specific recurrencies suggest a selective oncogenic advantage. However, due to their large size it has been difficult to pinpoint the oncogenic drivers that confer this advantage. Suitable functional genomics approaches to study the oncogenic driving capacity of large-scale deletions are limited. Here, we present an effective technique to engineer large-scale deletions by CRISPR-Cas9 and create isogenic cell line models. We simultaneously induce double-strand breaks (DSBs) at two ends of a chromosomal arm and select the cells that have lost the intermittent region. Using this technique, we induced large-scale deletions on chromosome 11q (65 Mb) and chromosome 6q (53 Mb) in neuroblastoma cell lines. A high frequency of successful deletions (up to 30% of selected clones) and increased colony forming capacity in the 11q deleted lines suggest an oncogenic advantage of these deletions. Such isogenic models enable further research on the role of large-scale deletions in tumor development and growth, and their possible therapeutic potential.
Assuntos
Sistemas CRISPR-Cas , DNA/metabolismo , Neuroblastoma/genética , Linhagem Celular Tumoral , Aberrações Cromossômicas , Deleção Cromossômica , HumanosRESUMO
Invariant natural killer T cells (iNKT cells) express a semi-invariant T cell receptor that recognizes certain glycolipids (including α-galactosylceramide, αGC) bound to CD1d, and can induce potent antitumor responses. Here, we assessed whether αGC could enhance the efficacy of a GM-CSF-producing tumor cell vaccine in the transgenic SV40 T antigen-driven TRAMP prostate cancer model. In healthy mice, we initially found that optimal T cell responses were obtained with αGC-pulsed TRAMP-C2 cells secreting GM-CSF and milk fat globule epidermal growth factor protein-8 (MFG-E8) with an RGD to RGE mutation (GM-CSF/RGE TRAMP-C2), combined with systemic low dose IL-12. In a therapeutic model, transgenic TRAMP mice were then castrated at ~ 20 weeks, followed by treatment with the combination vaccine. Untreated mice succumbed to tumor by ~ 40 weeks, but survival was markedly prolonged by vaccine treatment, with most mice surviving past 80 weeks. Prostates in the treated mice were heavily infiltrated with T cells and iNKT cells, which both secreted IFNγ in response to tumor cells. The vaccine was not effective if the αGC, IL-12, or GM-CSF secretion was eliminated. Finally, immunized mice were fully resistant to challenge with TRAMP-C2 cells. Together these findings support further development of therapeutic vaccines that exploit iNKT cell activation.
Assuntos
Vacinas Anticâncer , Células T Matadoras Naturais , Neoplasias da Próstata , Masculino , Camundongos , Animais , Humanos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Ativação Linfocitária , Galactosilceramidas , Interleucina-12/farmacologia , Neoplasias da Próstata/terapia , Neoplasias da Próstata/metabolismo , Vacinas Combinadas/farmacologia , Antígenos Virais de Tumores , Família de Proteínas EGF/metabolismo , Família de Proteínas EGF/farmacologia , Oligopeptídeos/farmacologia , Camundongos Endogâmicos C57BLRESUMO
The majority of the proteins involved in processing of DNA double-strand breaks (DSBs) accumulate at the damage sites. Real-time imaging and analysis of these processes, triggered by the so-called microirradiation using UV lasers or heavy particle beams, yielded valuable insights into the underlying DSB repair mechanisms. To study the temporal organization of DSB repair responses triggered by a more clinically-relevant DNA damaging agent, we developed a system coined X-ray multi-microbeam microscope (XM3), capable of simultaneous high dose-rate (micro)irradiation of large numbers of cells with ultra-soft X-rays and imaging of the ensuing cellular responses. Using this setup, we analyzed the changes in real-time kinetics of MRE11, MDC1, RNF8, RNF168 and 53BP1-proteins involved in the signaling axis of mammalian DSB repair-in response to X-ray and UV laser-induced DNA damage, in non-cancerous and cancer cells and in the presence or absence of a photosensitizer. Our results reveal, for the first time, the kinetics of DSB signaling triggered by X-ray microirradiation and establish XM3 as a powerful platform for real-time analysis of cellular DSB repair responses.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Imagem com Lapso de Tempo/métodos , Raios X , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Humanos , Proteína Homóloga a MRE11 , Microscopia Eletrônica de Varredura , Osteossarcoma/metabolismo , Epitélio Pigmentado Ocular/metabolismo , Transdução de Sinais , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Raios UltravioletaRESUMO
Most proteins involved in the DNA double-strand break response (DSBR) accumulate at the damage sites, where they perform functions related to damage signaling, chromatin remodeling and repair. Over the last two decades, studying the accumulation of many DSBR proteins provided information about their functionality and underlying mechanisms of action. However, comparison and systemic interpretation of these data is challenging due to their scattered nature and differing experimental approaches. Here, we extracted, analyzed and compared the available results describing accumulation of 79 DSBR proteins at sites of DNA damage, which can be further explored using Cumulus (http://www.dna-repair.live/cumulus/)-the accompanying interactive online application. Despite large inter-study variability, our analysis revealed that the accumulation of most proteins starts immediately after damage induction, occurs in parallel and peaks within 15-20 min. Various DSBR pathways are characterized by distinct accumulation kinetics with major non-homologous end joining proteins being generally faster than those involved in homologous recombination, and signaling and chromatin remodeling factors accumulating with varying speeds. Our meta-analysis provides, for the first time, comprehensive overview of the temporal organization of the DSBR in mammalian cells and could serve as a reference for future mechanistic studies of this complex process.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , DNA/genética , Recombinação Homóloga , Animais , DNA/metabolismo , Humanos , Cinética , Transdução de SinaisRESUMO
DNA double-strand breaks (DSBs) are known to be powerful inducers of homologous recombination (HR), but single-strand breaks (nicks) have also been shown to trigger HR. Both DSB- and nick-induced HR ((nick)HR) are exploited in advanced genome-engineering approaches based on the bacterial RNA-guided nuclease Cas9. However, the mechanisms of (nick)HR are largely unexplored. Here, we applied Cas9 nickases to study (nick)HR in mammalian cells. We find that (nick)HR is unaffected by inhibition of major damage signaling kinases and that it is not suppressed by nonhomologous end-joining (NHEJ) components, arguing that nick processing does not require a DSB intermediate to trigger HR. Relative to a single nick, nicking both strands enhances HR, consistent with a DSB intermediate, even when nicks are induced up to â¼1kb apart. Accordingly, HR and NHEJ compete for repair of these paired nicks, but, surprisingly, only when 5' overhangs or blunt ends can be generated. Our study advances the understanding of molecular mechanisms driving nick and paired-nick repair in mammalian cells and clarify phenomena associated with Cas9-mediated genome editing.
Assuntos
Quebras de DNA de Cadeia Dupla , Endonucleases/metabolismo , Recombinação Homóloga , Reparo de DNA por Recombinação , Animais , Linhagem Celular , Dano ao DNA , Reparo do DNA por Junção de Extremidades , Replicação do DNA , Técnicas de Inativação de Genes , Humanos , Camundongos , Motivos de Nucleotídeos , Troca de Cromátide IrmãRESUMO
Colorectal cancer (CRC) development is in most cases marked by the accumulation of genomic alterations including gain of the entire q-arm of chromosome 13. This aberration occurs in 40%-60% of all CRC and is associated with progression from adenoma to carcinoma. To date, little is known about the effect of the 13q amplicon on the expression of the therein located genes and their functional relevance. We therefore aimed to identify candidate genes at the 13q amplicon that contribute to colorectal adenoma to carcinoma progression in a gene dosage-dependent manner. Integrative analysis of whole genome expression and DNA copy number signatures resulted in the identification of 36 genes on 13q of which significant overexpression in carcinomas compared with adenomas was linked to a copy number gain. Five genes showing high levels of overexpression in carcinomas versus adenomas were further tested by quantitative reverse transcription-PCR in two independent sample sets of colorectal tumors (n = 40 and n = 47). DIS3 and LRCH1 revealed significant overexpression in carcinomas compared with adenomas in a 13q gain dependent manner. Silencing of DIS3 affected important tumorigenic characteristics such as viability, migration, and invasion. In conclusion, significant overexpression of DIS3 and LRCH1 associated with adenoma to carcinoma progression is linked to the CRC specific gain of 13q. The functional relevance of this copy number aberration was corroborated for DIS3, thereby identifying this gene as novel candidate oncogene contributing to the 13q-driven adenoma to carcinoma progression.
Assuntos
Adenoma/genética , Carcinoma/genética , Cromossomos Humanos Par 13/genética , Neoplasias Colorretais/genética , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Dosagem de Genes , Adenoma/metabolismo , Adenoma/patologia , Carcinoma/metabolismo , Carcinoma/patologia , Linhagem Celular Tumoral , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Progressão da Doença , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Estudos ProspectivosRESUMO
One of the hallmarks of cancer is genomic instability controlled by cell cycle checkpoints. The G1 and G2 checkpoints allow DNA damage responses, whereas the mitotic checkpoint enables correct seggregation of the sister chromosomes to prevent aneuploidy. Cancer cells often lack a functional G1 arrest and rely on G2 arrest for DNA damage responses. WEE1 kinase is an important regulator of the G2 checkpoint and is overexpressed in various cancer types. Inhibition of WEE1 is a promising strategy in cancer therapy in combination with DNA-damaging agents, especially when cancer cells harbor p53 mutations, as it causes mitotic catastrophy when DNA is not repaired during G2 arrest. Cancer cell response to WEE1 inhibition monotherapy has also been demonstrated in various types of cancer, including p53 wild-type cancers. We postulate that chromosomal instability can explain tumor response to WEE1 monotherapy. Therefore, chromosomal instability may need to be taken into account when determining the most effective strategy for the use of WEE1 inhibitors in cancer therapy.
Assuntos
Antineoplásicos/uso terapêutico , Proteínas de Ciclo Celular/antagonistas & inibidores , Instabilidade Genômica/efeitos dos fármacos , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Proteínas Nucleares/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Animais , HumanosRESUMO
UNLABELLED: Hepatitis C virus (HCV)-specific immune effector responses can cause liver damage in chronic infection. Hepatic stellate cells (HSC) are the main effectors of liver fibrosis. TGFß, produced by HCV-specific CD8(+) T cells, is a key regulatory cytokine modulating HCV-specific effector T cells. Here we studied TGFß as well as other factors produced by HCV-specific intrahepatic lymphocytes (IHL) and peripheral blood cells in hepatic inflammation and fibrogenesis. This was a cross-sectional study of two well-defined groups of HCV-infected subjects with slow (≤ 0.1 Metavir units/year, n = 13) or rapid (n = 6) liver fibrosis progression. HCV-specific T-cell responses were studied using interferon-gamma (IFNγ)-ELISpot ±monoclonal antibodies (mAbs) blocking regulatory cytokines, along with multiplex, enzyme-linked immunosorbent assay (ELISA) and multiparameter fluorescence-activated cell sorting (FACS). The effects of IHL stimulated with HCV-core peptides on HSC expression of profibrotic and fibrolytic genes were determined. Blocking regulatory cytokines significantly raised detection of HCV-specific effector (IFNγ) responses only in slow fibrosis progressors, both in the periphery (P = 0.003) and liver (P = 0.01). Regulatory cytokine blockade revealed HCV-specific IFNγ responses strongly correlated with HCV-specific TGFß, measured before blockade (R = 0.84, P = 0.0003), with only a trend to correlation with HCV-specific IL-10. HCV-specific TGFß was produced by CD8 and CD4 T cells. HCV-specific TGFß, not interleukin (IL)-10, inversely correlated with liver inflammation (R = -0.63, P = 0.008) and, unexpectedly, fibrosis (R = -0.46, P = 0.05). In addition, supernatants from HCV-stimulated IHL of slow progressors specifically increased fibrolytic gene expression in HSC and treatment with anti-TGFß mAb abrogated such expression. CONCLUSION: Although TGFß is considered a major profibrogenic cytokine, local production of TGFß by HCV-specific T cells appeared to have a protective role in HCV-infected liver, together with other T-cell-derived factors, ameliorating HCV liver disease progression.
Assuntos
Linfócitos T CD8-Positivos/metabolismo , Hepacivirus/imunologia , Células Estreladas do Fígado/metabolismo , Hepatite C Crônica/metabolismo , Cirrose Hepática/imunologia , Linfócitos T Reguladores/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Adulto , Idoso , Linfócitos T CD8-Positivos/imunologia , Colágeno Tipo I/genética , Cadeia alfa 1 do Colágeno Tipo I , Estudos Transversais , Progressão da Doença , Feminino , Expressão Gênica , Hepatite C Crônica/imunologia , Hepatite C Crônica/patologia , Humanos , Interferon gama/metabolismo , Interleucina-10/metabolismo , Fígado/imunologia , Fígado/metabolismo , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Masculino , Metaloproteinase 1 da Matriz/genética , Pessoa de Meia-Idade , Linfócitos T Reguladores/imunologia , Proteínas do Core Viral/imunologiaRESUMO
Fanconi anaemia (FA) is a rare chromosomal-instability syndrome caused by mutations of any of the 22 known FA DNA-repair genes. FA individuals have an increased risk of head-and-neck squamous-cell-carcinomas (HNSCC), often fatal. Systemic intolerance to standard cisplatin-based protocols due to somatic-cell hypersensitivity underscores the urgent need to develop novel therapies. Here, we performed unbiased siRNA screens to unveil genetic interactions synthetic-lethal with FA-pathway deficiency in FA-patient HNSCC cell lines. We identified based on differential-lethality scores between FA-deficient and FA-proficient cells, next to common-essential genes such as PSMC1, PSMB2, and LAMTOR2, the otherwise non-essential RBBP9 gene. Accordingly, low dose of the FDA-approved RBBP9-targeting drug Emetine kills FA-HNSCC. Importantly both RBBP9-silencing as well as Emetine spared non-tumour FA cells. This study provides a minable genome-wide analyses of vulnerabilities to address treatment challenges in FA-HNSCC. Our investigation divulges a DNA-cross-link-repair independent lead, RBBP9, for targeted treatment of FA-HNSCCs without systemic toxicity.
Assuntos
Anemia de Fanconi , Neoplasias de Cabeça e Pescoço , Carcinoma de Células Escamosas de Cabeça e Pescoço , Humanos , Proteínas de Ciclo Celular/genética , DNA , Emetina/uso terapêutico , Anemia de Fanconi/genética , Anemia de Fanconi/patologia , Estudo de Associação Genômica Ampla , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Neoplasias/genética , RNA Interferente Pequeno/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/genéticaRESUMO
Homologous recombination (HR) is an essential, widely conserved mechanism that utilizes a template for accurate repair of DNA breaks. Some early HR models, developed over five decades ago, anticipated single-strand breaks (nicks) as initiating lesions. Subsequent studies favored a more double-strand break (DSB)-centered view of HR initiation and at present this pathway is primarily considered to be associated with DSB repair. However, mounting evidence suggests that nicks can indeed initiate HR directly, without first being converted to DSBs. Moreover, recent studies reported on novel branches of nick-initiated HR (nickHR) that rely on single-, rather than double-stranded repair templates and that are characterized by mechanistically and genetically unique properties. The physiological significance of nickHR is not well documented, but its high-fidelity nature and low mutagenic potential are relevant in recently developed, precise gene editing approaches. Here, we review the evidence for stimulation of HR by nicks, as well as the data on the interactions of nickHR with other DNA repair pathways and on its mechanistic properties. We conclude that nickHR is a bona-fide pathway for nick repair, sharing the molecular machinery with the canonical HR but nevertheless characterized by unique properties that secure its inclusion in DNA repair models and warrant future investigations.
Assuntos
Quebras de DNA , Reparo de DNA por Recombinação , Animais , DNA/metabolismo , Reparo do DNA por Junção de Extremidades , Eucariotos/genética , Eucariotos/metabolismo , HumanosRESUMO
Hyperthermia - application of supra-physiological temperatures to cells, tissues or organs - is a pleiotropic treatment that affects most aspects of cellular metabolism, but its effects on DNA are of special interest in the context of cancer research and treatment. Hyperthermia inhibits repair of various DNA lesions, including double-strand breaks (DSBs), making it a powerful radio- and chemosensitizer, with proven clinical efficacy in therapy of various types of cancer, including tumors of head and neck, bladder, breast and cervix. Among the challenges for hyperthermia-based therapies are the transient character of its effects, the technical difficulties in maintaining uniformly elevated tumor temperature and the acquisition of thermotolerance. Approaches to reduce or eliminate these challenges could simplify the application of hyperthermia, boost its efficacy and improve treatment outcomes. Here we show that a single, short treatment with a relatively low dose of HSP90 inhibitor Ganetespib potentiates cytotoxic as well as radio- and chemosensitizing effects of hyperthermia and reduces thermotolerance in cervix cancer cell lines. Ganetespib alone, applied at this low dose, has virtually no effect on survival of non-heated cells. Our results thus suggest that HSP90 inhibition can be a safe, simple and efficient approach to improving hyperthermia treatment efficacy and reducing thermotolerance, paving the way for in vivo studies.
RESUMO
Cis-diamminedichloroplatinum(II) (cisplatin, cDDP) is an effective chemotherapeutic agent that induces DNA double strand breaks (DSBs), primarily in replicating cells. Generally, such DSBs can be repaired by the classical or backup non-homologous end joining (c-NHEJ/b-NHEJ) or homologous recombination (HR). Therefore, inhibiting these pathways in cancer cells should enhance the efficiency of cDDP treatments. Indeed, inhibition of HR by hyperthermia (HT) sensitizes cancer cells to cDDP and in the Netherlands this combination is a standard treatment option for recurrent cervical cancer after previous radiotherapy. Additionally, cDDP has been demonstrated to disrupt c-NHEJ, which likely further increases the treatment efficacy. However, if one of these pathways is blocked, DSB repair functions can be sustained by the Poly-(ADP-ribose)-polymerase1 (PARP1)-dependent b-NHEJ. Therefore, disabling b-NHEJ should, in principle, further inhibit the repair of cDDP-induced DNA lesions and enhance the toxicity of thermochemotherapy. To explore this hypothesis, we treated a panel of cancer cell lines with HT, cDDP and a PARP1-i and measured various end-point relevant in cancer treatment. Our results demonstrate that PARP1-i does not considerably increase the efficacy of HT combined with standard, commonly used cDDP concentrations. However, in the presence of a PARP1-i, ten-fold lower concentration of cDDP can be used to induce similar cytotoxic effects. PARP1 inhibition may thus permit a substantial lowering of cDDP concentrations without diminishing treatment efficacy, potentially reducing systemic side effects.
Assuntos
Cisplatino/farmacologia , Temperatura Alta , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Reparo do DNA por Junção de Extremidades/efeitos dos fármacos , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Células HeLa , Humanos , Microscopia Confocal , Ratos , Reparo de DNA por Recombinação/efeitos dos fármacos , Imagem com Lapso de Tempo/métodosRESUMO
Purpose: Invariant NKT cells (iNKT) are innate-like CD1d-restricted T cells with immunoregulatory activity in diseases including cancer. iNKT from advanced cancer patients can have reversible defects including IFNγ production, and iNKT IFNγ production may stratify for survival. Previous clinical trials using iNKT cell activating ligand α-galactosylceramide have shown clinical responses. Therefore, a phase I clinical trial was performed of autologous in vitro expanded iNKT cells in stage IIIB-IV melanoma.Experimental Design: Residual iNKT cells [<0.05% of patient peripheral blood mononuclear cell (PBMC)] were purified from autologous leukapheresis product using an antibody against the iNKT cell receptor linked to magnetic microbeads. iNKT cells were then expanded with CD3 mAb and IL2 in vitro to obtain up to approximately 109 cells.Results: Expanded iNKT cells produced IFNγ, but limited or undetectable IL4 or IL10. Three iNKT infusions each were completed on 9 patients, and produced only grade 1-2 toxicities. The 4th patient onward received systemic GM-CSF with their second and third infusions. Increased numbers of iNKT cells were seen in PBMCs after some infusions, particularly when GM-CSF was also given. IFNγ responses to α-galactosylceramide were increased in PBMCs from some patients after infusions, and delayed-type hypersensitivity responses to Candida increased in 5 of 8 evaluated patients. Three patients have died, three were progression-free at 53, 60, and 65 months, three received further treatment and were alive at 61, 81, and 85 months. There was no clear correlation between outcome and immune parameters.Conclusions: Autologous in vitro expanded iNKT cells are a feasible and safe therapy, producing Th1-like responses with antitumor potential. Clin Cancer Res; 23(14); 3510-9. ©2017 AACR.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos/métodos , Imunoterapia , Melanoma/terapia , Células T Matadoras Naturais/transplante , Subpopulações de Linfócitos T/transplante , Transferência Adotiva/métodos , Adulto , Idoso , Complexo CD3/imunologia , Feminino , Galactosilceramidas/imunologia , Humanos , Interferon gama/imunologia , Interferon gama/uso terapêutico , Interleucina-10/imunologia , Interleucina-2/imunologia , Interleucina-4/imunologia , Estimativa de Kaplan-Meier , Ativação Linfocitária/imunologia , Masculino , Melanoma/imunologia , Melanoma/patologia , Pessoa de Meia-Idade , Células T Matadoras Naturais/imunologia , Subpopulações de Linfócitos T/imunologiaRESUMO
The currently available arsenal of anticancer modalities includes many DNA damaging agents that can kill malignant cells. However, efficient DNA repair mechanisms protect both healthy and cancer cells against the effects of treatment and contribute to the development of drug resistance. Therefore, anti-cancer treatments based on inflicting DNA damage can benefit from inhibition of DNA repair. Hyperthermia - treatment at elevated temperature - considerably affects DNA repair, among other cellular processes, and can thus sensitize (cancer) cells to DNA damaging agents. This effect has been known and clinically applied for many decades, but how heat inhibits DNA repair and which pathways are targeted has not been fully elucidated. In this review we attempt to summarize the known effects of hyperthermia on DNA repair pathways relevant in clinical treatment of cancer. Furthermore, we outline the relationships between the effects of heat on DNA repair and sensitization of cells to various DNA damaging agents.
Assuntos
Dano ao DNA/fisiologia , Reparo do DNA/fisiologia , Hipertermia Induzida , Animais , Antineoplásicos/farmacologia , Dano ao DNA/efeitos dos fármacos , Humanos , Neoplasias/terapiaRESUMO
Thousands of DNA breaks occur daily in mammalian cells, including potentially tumorigenic double-strand breaks (DSBs) and less dangerous but vastly more abundant single-strand breaks (SSBs). The majority of SSBs are quickly repaired, but some can be converted to DSBs, posing a threat to the integrity of the genome. Although SSBs are usually repaired by dedicated pathways, they can also trigger homologous recombination (HR), an error-free pathway generally associated with DSB repair. While HR-mediated DSB repair has been extensively studied, the mechanisms of HR-mediated SSB repair are less clear. This chapter describes a protocol to investigate SSB-induced HR in mammalian cells employing the DR-GFP reporter, which has been widely used in DSB repair studies, together with an adapted bacterial CRISPR/Cas system.
Assuntos
Proteínas Associadas a CRISPR/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Desoxirribonuclease I/metabolismo , Genes Reporter , Recombinação Homóloga , Animais , Sequência de Bases , Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas , Linhagem Celular , Clonagem Molecular/métodos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Desoxirribonuclease I/genética , Endonucleases/genética , Endonucleases/metabolismo , Citometria de Fluxo/métodos , Humanos , Dados de Sequência Molecular , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , TransfecçãoRESUMO
Dendritic cells (DCs) orchestrate antibody-mediated responses to combat extracellular pathogens including parasites by initiating T helper cell differentiation. Here we demonstrate that carbohydrate-specific signalling by DC-SIGN drives follicular T helper cell (TFH) differentiation via IL-27 expression. Fucose, but not mannose, engagement of DC-SIGN results in activation of IKKε, which collaborates with type I IFNR signalling to induce formation and activation of transcription factor ISGF3. Notably, ISGF3 induces expression of IL-27 subunit p28, and subsequent IL-27 secreted by DC-SIGN-primed DCs is pivotal for the induction of Bcl-6(+)CXCR5(+)PD-1(hi)Foxp1(lo) TFH cells, IL-21 secretion by TFH cells and T-cell-dependent IgG production by B cells. Thus, we have identified an essential role for DC-SIGN-induced ISGF3 by fucose-based PAMPs in driving IL-27 and subsequent TFH polarization, which might be harnessed for vaccination design.
Assuntos
Moléculas de Adesão Celular/metabolismo , Células Dendríticas/citologia , Fucose/química , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/metabolismo , Interleucina-27/metabolismo , Lectinas Tipo C/metabolismo , Receptores de Superfície Celular/metabolismo , Linfócitos T Auxiliares-Indutores/citologia , Motivos de Aminoácidos , Linfócitos B/citologia , Diferenciação Celular , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Dimerização , Citometria de Fluxo , Humanos , Imunoglobulina G/química , Fator Regulador 7 de Interferon/metabolismo , Leucócitos Mononucleares/citologia , Ativação Linfocitária/imunologia , Manose/química , Proteínas Proto-Oncogênicas c-bcl-6 , Interferência de RNA , Transdução de SinaisRESUMO
Langerhans cells (LCs) lining the stratified epithelia and mucosal tissues are the first antigen presenting cells to encounter invading pathogens, such as viruses, bacteria and fungi. Fungal infections form a health threat especially in immuno-compromised individuals. LCs express C-type lectin Langerin that has specificity for mannose, fucose and GlcNAc structures. Little is known about the role of human Langerin in fungal infections. Our data show that Langerin interacts with both mannan and beta-glucan structures, common cell-wall carbohydrate structures of fungi. We have screened a large panel of fungi for recognition by human Langerin and, strikingly, we observed strong binding of Langerin to a variety of Candida and Saccharomyces species and Malassezia furfur, but very weak binding was observed to Cryptococcus gattii and Cryptococcus neoformans. Notably, Langerin is the primary fungal receptor on LCs, since the interaction of LCs with the different fungi was blocked by antibodies against Langerin. Langerin recognizes both mannose and beta-glucans present on fungal cell walls and our data demonstrate that Langerin is the major fungal pathogen receptor on human LCs that recognizes pathogenic and commensal fungi. Together these data may provide more insight in the role of LCs in fungal infections.