RESUMO
CD4+ T cells induced from human iPSCs (iCD4+ T cells) offer a therapeutic opportunity for overcoming immune pathologies arising from hematopoietic stem cell transplantation. However, most iCD4+ T cells are conventional helper T cells, which secrete inflammatory cytokines. We induced high-level expression of FOXP3, a master transcription factor of regulatory T cells, in iCD4+ T cells. Human iPSC-derived, FOXP3-induced CD4+ T (iCD4+ Treg-like) cells did not secrete inflammatory cytokines upon activation. Moreover, they showed demethylation of the Treg-specific demethylation region, suggesting successful conversion to immunosuppressive iCD4+ Treg-like cells. We further assessed these iCD4+ Treg-like cells for CAR-mediated immunosuppressive ability. HLA-A2 CAR-transduced iCD4+ Treg-like cells inhibited CD8+ cytotoxic T cell (CTL) division in a mixed lymphocyte reaction assay with A2+ allogeneic CTLs and suppressed xenogeneic graft-versus-host disease (GVHD) in NSG mice treated with A2+ human PBMCs. In most cases, these cells suppressed the xenogeneic GvHD progression as much as natural CD25+CD127- Tregs did.
Assuntos
Doença Enxerto-Hospedeiro , Células-Tronco Pluripotentes Induzidas , Receptores de Antígenos Quiméricos , Linfócitos T Reguladores , Humanos , Doença Enxerto-Hospedeiro/imunologia , Animais , Linfócitos T Reguladores/imunologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Receptores de Antígenos Quiméricos/metabolismo , Receptores de Antígenos Quiméricos/imunologia , Camundongos , Fatores de Transcrição Forkhead/metabolismo , Xenoenxertos , Camundongos Endogâmicos NOD , Modelos Animais de Doenças , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismoRESUMO
The frequent emergence of pathogenic viruses with pandemic potential has posed a significant threat to human health and economy, despite enormous advances in our understanding of infection mechanisms and devising countermeasures through developing various prophylactic and therapeutic strategies. The recent coronavirus disease (COVID-19) pandemic has re-emphasised the importance of rigorous research on virus infection mechanisms and highlighted the need for our preparedness for potential pandemics. Although viruses cannot self-replicate, they tap into host cell factors and processes for their entry, propagation and dissemination. Upon entering the host cells, viruses ingeniously utilise the innate biological functions of the host cell to replicate themselves and maintain their existence in the hosts. Influenza A virus (IAV), which has a negative-sense, single-stranded RNA as its genome, is no exception. IAVs are enveloped viruses with a lipid bilayer derived from the host cell membrane and have a surface covered with the spike glycoprotein haemagglutinin (HA) and neuraminidase (NA). Viral genome is surrounded by an M1 shell, forming a "capsid" in the virus particle. IAV particles use HA to recognise sialic acids on the cell surface of lung epithelial cells for their attachment. After attachment to the cell surface, IAV particles are endocytosed and sorted into the early endosomes. Subsequently, as the early endosomes mature into late endosomes, the endosomal lumen becomes acidified, and the low pH of the late endosomes induces conformational reaggangements in the HA to initiate fusion between the endosomal and viral membranes. Upon fusion, the viral capsid disintegrates and the viral ribonucleoprotein (vRNP) complexes containing the viral genome are released into the cytosol. The process of viral capsid disintegration is called "uncoating". After successful uncoating, the vRNPs are imported into the nucleus by importin α/ß (IMP α/ß), where viral replication and transcription take place and the new vRNPs are assembled. Recently, we have biochemically elucidated the molecular mechanisms of the processes of viral capsid uncoating subsequent viral genome dissociation. In this chapter, we present the molecular details of the viral uncoating process.
Assuntos
Vírus da Influenza A , Humanos , Vírus da Influenza A/genética , Replicação Viral , Capsídeo , Proteínas do Capsídeo , VírionRESUMO
The deacetylase HDAC6 has tandem catalytic domains and a zinc finger domain (ZnF) binding ubiquitin (Ub). While the catalytic domain has an antiviral effect, the ZnF facilitates influenza A virus (IAV) infection and cellular stress responses. By recruiting Ub via the ZnF, HDAC6 promotes the formation of aggresomes and stress granules (SGs), dynamic structures associated with pathologies such as neurodegeneration. IAV subverts the aggresome/HDAC6 pathway to facilitate capsid uncoating during early infection. To target this pathway, we generate designed ankyrin repeat proteins (DARPins) binding the ZnF; one of these prevents interaction with Ub in vitro and in cells. Crystallographic analysis shows that it blocks the ZnF pocket where Ub engages. Conditional expression of this DARPin reversibly impairs infection by IAV and Zika virus; moreover, SGs and aggresomes are downregulated. These results validate the HDAC6 ZnF as an attractive target for drug discovery.
Assuntos
Vírus da Influenza A , Influenza Humana , Infecção por Zika virus , Zika virus , Desacetilase 6 de Histona/metabolismo , Humanos , Vírus da Influenza A/metabolismo , Ubiquitina/metabolismo , Zika virus/metabolismoRESUMO
Epstein-Barr virus-associated lymphoproliferative disease (EBV-LPD) is frequently fatal. Innate immunity plays a key role in protecting against pathogens and cancers. The stimulator of interferon genes (STING) is regarded as a key adaptor protein allowing DNA sensors recognizing exogenous cytosolic DNA to activate the type I interferon signaling cascade. In terms of EBV tumorigenicity, the role of STING remains elusive. Here we showed that treatment with the STING inhibitor, C-176, suppressed EBV-induced transformation in peripheral blood mononuclear cells. In an EBV-LPD mouse model, C-176 treatment also inhibited tumor formation and prolonged survival. Treatment with B cells alone did not affect EBV transformation, but suppression of EBV-induced transformation was observed in the presence of T cells. Even without direct B cell-T cell contact in a transwell system, the inhibitor reduced the transformation activity, indicating that intercellular communication by humoral factors was critical to prevent EBV-induced transformation. These findings suggest that inhibition of STING signaling pathway with C-176 could be a new therapeutic target of EBV-LPD.
Assuntos
Antineoplásicos/administração & dosagem , Transformação Celular Viral/efeitos dos fármacos , Infecções por Vírus Epstein-Barr/tratamento farmacológico , Linfoma de Células B/prevenção & controle , Proteínas de Membrana/antagonistas & inibidores , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Infecções por Vírus Epstein-Barr/imunologia , Células HEK293 , Herpesvirus Humano 4 , Humanos , Células Jurkat , Linfoma de Células B/imunologia , Linfoma de Células B/virologia , Camundongos , Análise de Sobrevida , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Because of their close biological similarity to humans, non-human primate (NHP) models are very useful for the development of induced pluripotent stem cell (iPSC)-based cell and regenerative organ transplantation therapies. However, knowledge on the establishment, differentiation, and genetic modification of NHP-iPSCs, especially rhesus macaque iPSCs, is limited. We succeeded in establishing iPSCs from the peripheral blood of rhesus macaques (Rh-iPSCs) by combining the Yamanaka reprograming factors and two inhibitors (GSK-3 inhibitor [CHIR 99021] and MEK1/2 inhibitor [PD0325901]) and differentiated the cells into functional macrophages through hematopoietic progenitor cells. To confirm feasibility of the Rh-iPSC-derived macrophages as a platform for bioassays to model diseases, we knocked out TRIM5 gene in Rh-iPSCs by CRISPR-Cas9, which is a species-specific HIV resistance factor. TRIM5 knockout (KO) iPSCs had the same differentiation potential to macrophages as did Rh-iPSCs, but the differentiated macrophages showed a gain of sensitivity to HIV infection in vitro. Our reprogramming, gene editing, and differentiation protocols used to obtain Rh-iPSC-derived macrophages can be applied to other gene mutations, expanding the number of NHP gene therapy models.
RESUMO
Clinical successes demonstrated by chimeric antigen receptor T-cell immunotherapy have facilitated further development of T-cell immunotherapy against wide variety of diseases. One approach is the development of "off-the-shelf" T-cell sources. Technologies to generate T-cells from pluripotent stem cells (PSCs) may offer platforms to produce "off-the-shelf" and synthetic allogeneic T-cells. However, low differentiation efficiency and poor scalability of current methods may compromise their utilities. Here we show improved differentiation efficiency of T-cells from induced PSCs (iPSCs) derived from an antigen-specific cytotoxic T-cell clone, or from T-cell receptor (TCR)-transduced iPSCs, as starting materials. We additionally describe feeder-free differentiation culture systems that span from iPSC maintenance to T-cell proliferation phases, enabling large-scale regenerated T-cell production. Moreover, simultaneous addition of SDF1α and a p38 inhibitor during T-cell differentiation enhances T-cell commitment. The regenerated T-cells show TCR-dependent functions in vitro and are capable of in vivo anti-tumor activity. This system provides a platform to generate a large number of regenerated T-cells for clinical application and investigate human T-cell differentiation and biology.
Assuntos
Técnicas de Cultura de Células/métodos , Imunoterapia Adotiva/métodos , Células-Tronco Pluripotentes Induzidas/fisiologia , Neoplasias/terapia , Linfócitos T Citotóxicos/transplante , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Quimiocina CXCL12/metabolismo , Meios de Cultura/metabolismo , Meios de Cultura/farmacologia , Feminino , Humanos , Imidazóis/farmacologia , Camundongos , Neoplasias/imunologia , Piridinas/farmacologia , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T Citotóxicos/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Non-invasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) and invasive encapsulated follicular variant of papillary thyroid carcinoma (EFV-PTC) are indistinguishable preoperatively. CD26 expression in follicular tumor-uncertain malignant potential (FT-UMP) is reported to be clearly higher than in that without capsular invasion. To verify the diagnostic significance of CD26 immunostaining in EFV-PTC, we examined the expression pattern of CD26 in non-invasive EFV-PTC (NIFTP) and invasive EFV-PTC. We performed immunohistochemical analysis using CD26 antibody for 37 NIFTPs and 54 EFV-PTCs (34 minimally invasive EFV-PTCs and 20 widely invasive EFV-PTCs). Most NIFTP samples showed an apical membranous pattern or a cytoplasmic diffuse pattern of expression. Invasive EFV-PTCs more frequently showed a cytoplasmic dot-like pattern, and the labeling indices of tumor cells with cytoplasmic dot-like patterns were significantly higher than those in NIFTPs. The sizes of dots seen in NIFTPs (mean: 1.12 µm) were significantly smaller than in invasive EFV-PTCs (1.33 µm), minimally invasive EFV-PTC (1.27 µm), and widely invasive EFV-PTC (1.38 µm). We, therefore, conclude that cytoplasmic diffuse and/or cytoplasmic dot-like CD26 expression, particularly the larger CD26-positive dots, could be useful markers for capsular invasion in EFV-PTC. CD26 immunostaining, using cell blocks or cytological specimens, may preoperatively distinguish between NIFTP and invasive EFV-PTC.
Assuntos
Dipeptidil Peptidase 4/metabolismo , Câncer Papilífero da Tireoide/diagnóstico , Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/diagnóstico , Adulto , Idoso , Biomarcadores Tumorais/metabolismo , Núcleo Celular/metabolismo , Núcleo Celular/patologia , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Câncer Papilífero da Tireoide/metabolismo , Câncer Papilífero da Tireoide/patologia , Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/patologiaRESUMO
Influenza A virus is a pathogen of great medical impact. To develop novel antiviral strategies, it is essential to understand the molecular aspects of virus-host cell interactions in detail. During entry, the viral ribonucleoproteins (vRNPs) that carry the RNA genome must be released from the incoming particle before they can enter the nucleus for replication. The uncoating process is facilitated by histone deacetylase 6 (ref.1). However, the precise mechanism of shell opening and vRNP debundling is unknown. Here, we show that transportin 1, a member of the importin-ß family proteins, binds to a PY-NLS2 sequence motif close to the amino terminus of matrix protein (M1) exposed during acid priming of the viral core. It promotes the removal of M1 and induces disassembly of vRNP bundles. Next, the vRNPs interact with importin-α/ß and enter the nucleus. Thus, influenza A virus uses dual importin-ßs for distinct steps in host cell entry.
Assuntos
Vírus da Influenza A/fisiologia , Influenza Humana/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas Virais/metabolismo , Internalização do Vírus , beta Carioferinas/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Núcleo Celular/virologia , Humanos , Vírus da Influenza A/genética , Influenza Humana/genética , Influenza Humana/virologia , Ribonucleoproteínas/genética , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismo , Proteínas Virais/genética , Replicação ViralRESUMO
Influenza A virus (IAV) is an enveloped virus with a segmented single-stranded negative-strand RNA genome. In general, the role of virally encapsidated host cell proteins in the viral life cycle is unclear. The virion contains abundant ubiquitin molecules some of which have been identified as unanchored polyubiquitin chains. These ubiquitin chains have been postulated to play a role in recruiting histone deacetylase 6 (HDAC6) to the cytosolic surface of late endosomes (LEs), promoting IAV uncoating via aggresome processing-a cellular machinery that disposes of protein waste. HDAC6, a class II HDAC, is unusual because it resides mostly in the cytosol instead of the nucleus. It is a unique protein consisting of two catalytic domains (CDs) and a zinc-finger ubiquitin-binding domain (ZnF-UBP) close to its C-terminus. This ZnF-UBP recognizes the unconjugated ubiquitin C-terminus (di-Gly motif) with very high affinity. Biochemical analyses showed that free di-Gly motifs are present in the form of unanchored ubiquitin inside IAV virions. These motifs are exposed following low pH-triggered viral fusion at the LEs and attract HDAC6 transiently to the cytosolic surface of vesicles. The binding of the two components promotes viral uncoating via HDAC6 interaction with cellular motor proteins dynein and myosin II and the viral M1 capsid. The cellular mechanism involved is related to aggresome processing, a pathway that promotes degradation of misfolded protein aggregates. K63-linked ubiquitin chains are thought to be the trigger for aggresome processing, though it is still not clear whether such types of chains are prevalent within the IAV capsid. Here, we present methods using purified ZnF-UBP domain of HDAC6 to immunoprecipitate viral unanchored ubiquitin chains, which can then be used for further biochemical analyses of ubiquitin chain linkage.
Assuntos
Proteínas de Transporte/isolamento & purificação , Vírus da Influenza A/metabolismo , Poliubiquitina/isolamento & purificação , Vírion/metabolismo , Animais , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Linhagem Celular , Expressão Gênica , Desacetilase 6 de Histona/genética , Desacetilase 6 de Histona/metabolismo , Humanos , Modelos Moleculares , Poliubiquitina/química , Poliubiquitina/metabolismo , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Ubiquitina/química , Ubiquitina/isolamento & purificação , Ubiquitina/metabolismoRESUMO
We report crystal structures of zebrafish histone deacetylase 6 (HDAC6) catalytic domains in tandem or as single domains in complex with the (R) and (S) enantiomers of trichostatin A (TSA) or with the HDAC6-specific inhibitor nexturastat A. The tandem domains formed, together with the inter-domain linker, an ellipsoid-shaped complex with pseudo-twofold symmetry. We identified important active site differences between both catalytic domains and revealed the binding mode of HDAC6 selective inhibitors. HDAC inhibition assays with (R)- and (S)-TSA showed that (R)-TSA was a broad-range inhibitor, whereas (S)-TSA had moderate selectivity for HDAC6. We identified a uniquely positioned α-helix and a flexible tryptophan residue in the loop joining α-helices H20 to H21 as critical for deacetylation of the physiologic substrate tubulin. Using single-molecule measurements and biochemical assays we demonstrated that HDAC6 catalytic domain 2 deacetylated α-tubulin lysine 40 in the lumen of microtubules, but that its preferred substrate was unpolymerized tubulin.
Assuntos
Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Ácidos Hidroxâmicos/farmacologia , Tubulina (Proteína)/metabolismo , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/metabolismo , Acetilação/efeitos dos fármacos , Animais , Biocatálise , Desacetilase 6 de Histona , Inibidores de Histona Desacetilases/química , Histona Desacetilases/química , Humanos , Ácidos Hidroxâmicos/química , Modelos Moleculares , Relação Estrutura-Atividade , Tubulina (Proteína)/química , Peixe-Zebra , Proteínas de Peixe-Zebra/químicaRESUMO
Lysine deacetylases inhibitors (KDACIs) are used in basic research, and many are being investigated in clinical trials for treatment of cancer and other diseases. However, their specificities in cells are incompletely characterized. Here we used quantitative mass spectrometry (MS) to obtain acetylation signatures for 19 different KDACIs, covering all 18 human lysine deacetylases. Most KDACIs increased acetylation of a small, specific subset of the acetylome, including sites on histones and other chromatin-associated proteins. Inhibitor treatment combined with genetic deletion showed that the effects of the pan-sirtuin inhibitor nicotinamide are primarily mediated by SIRT1 inhibition. Furthermore, we confirmed that the effects of tubacin and bufexamac on cytoplasmic proteins result from inhibition of HDAC6. Bufexamac also triggered an HDAC6-independent, hypoxia-like response by stabilizing HIF1-α, providing a possible mechanistic explanation of its adverse, pro-inflammatory effects. Our results offer a systems view of KDACI specificities, providing a framework for studying function of acetylation and deacetylases.
Assuntos
Inibidores Enzimáticos/química , Inibidores de Histona Desacetilases/química , Histona Desacetilases/química , Espectrometria de Massas/métodos , Mapeamento de Peptídeos/métodos , Mapeamento de Interação de Proteínas/métodos , Acetilação , Sequência de Aminoácidos , Sítios de Ligação , Células Cultivadas , Humanos , Dados de Sequência Molecular , Ligação ProteicaRESUMO
During cell entry, capsids of incoming influenza A viruses (IAVs) must be uncoated before viral ribonucleoproteins (vRNPs) can enter the nucleus for replication. After hemagglutinin-mediated membrane fusion in late endocytic vacuoles, the vRNPs and the matrix proteins dissociate from each other and disperse within the cytosol. Here, we found that for capsid disassembly, IAV takes advantage of the host cell's aggresome formation and disassembly machinery. The capsids mimicked misfolded protein aggregates by carrying unanchored ubiquitin chains that activated a histone deacetylase 6 (HDAC6)-dependent pathway. The ubiquitin-binding domain was essential for recruitment of HDAC6 to viral fusion sites and for efficient uncoating and infection. That other components of the aggresome processing machinery, including dynein, dynactin, and myosin II, were also required suggested that physical forces generated by microtubule- and actin-associated motors are essential for IAV entry.
Assuntos
Capsídeo/metabolismo , Histona Desacetilases/fisiologia , Vírus da Influenza A/fisiologia , Influenza Humana/virologia , Internalização do Vírus , Animais , Linhagem Celular Tumoral , Núcleo Celular/virologia , Complexo Dinactina , Dineínas/metabolismo , Técnicas de Inativação de Genes , Desacetilase 6 de Histona , Histona Desacetilases/genética , Interações Hospedeiro-Patógeno , Humanos , Influenza Humana/genética , Influenza Humana/metabolismo , Fusão de Membrana/genética , Fusão de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Miosina Tipo II/metabolismo , Ligação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Interferência de RNA , Ribonucleoproteínas/metabolismo , Ubiquitina/química , Ubiquitina/metabolismo , Replicação ViralRESUMO
Angioimmunoblastic T cell lymphoma (AITL) is a distinct subtype of peripheral T cell lymphoma characterized by generalized lymphadenopathy and frequent autoimmune-like manifestations. Although frequent mutations in TET2, IDH2 and DNMT3A, which are common to various hematologic malignancies, have been identified in AITL, the molecular pathogenesis specific to this lymphoma subtype is unknown. Here we report somatic RHOA mutations encoding a p.Gly17Val alteration in 68% of AITL samples. Remarkably, all cases with the mutation encoding p.Gly17Val also had TET2 mutations. The RHOA mutation encoding p.Gly17Val was specifically identified in tumor cells, whereas TET2 mutations were found in both tumor cells and non-tumor hematopoietic cells. RHOA encodes a small GTPase that regulates diverse biological processes. We demonstrated that the Gly17Val RHOA mutant did not bind GTP and also inhibited wild-type RHOA function. Our findings suggest that impaired RHOA function in cooperation with preceding loss of TET2 function contributes to AITL-specific pathogenesis.
Assuntos
Proteínas de Ligação a DNA/genética , Linfadenopatia Imunoblástica/genética , Linfoma de Células T Periférico/genética , Proteínas Proto-Oncogênicas/genética , Proteína rhoA de Ligação ao GTP/genética , Animais , Sequência de Bases , Bromodesoxiuridina , DNA (Citosina-5-)-Metiltransferases/genética , DNA Metiltransferase 3A , Dioxigenases , Exoma/genética , Humanos , Isocitrato Desidrogenase/genética , Células Jurkat , Linfoma de Células T Periférico/patologia , Camundongos , Dados de Sequência Molecular , Mutação de Sentido Incorreto/genética , Células NIH 3T3 , Análise de Sequência de DNARESUMO
Induced pluripotent stem (iPS) cells are an attractive source for potential cell-replacement therapy. However, transplantation of differentiated products harbors the risk of teratoma formation, presenting a serious health risk. Thus, we characterized Nanog-expressing (undifferentiated) cells remaining after induction of differentiation by cytological examination. To induce differentiation of iPS cells, we generated embryoid bodies (EBs) derived from iPS cells carrying a Nanoggreen fluorescent protein(GFP) reporter and then injected GFP-positive and GFP negative EBs into nude mice. GFP-positive EB transplantation resulted in the formation of immature teratoma grade 3, but no tumors were induced by GFP-negative EB. GFP positive cells revealed significantly lower cytoplasmic area and higher nucleus/cytoplasm ratio than those of GFP negative cells. Our results suggest that morphological analysis might be a useful method for distinguishing between tumorigenic and nontumorigenic iPS cells.
Assuntos
Transformação Celular Neoplásica , Células-Tronco Pluripotentes Induzidas/patologia , Transplante de Células-Tronco/efeitos adversos , Teratoma/etiologia , Teratoma/patologia , Animais , Diferenciação Celular , Células Cultivadas , Corpos Embrioides/citologia , Proteínas de Fluorescência Verde , Células-Tronco Pluripotentes Induzidas/citologia , Camundongos , Camundongos NusRESUMO
Cancer stem cells (CSCs) possess the ability for self-renewal, differentiation, and tumorigenesis and play a role in cancer recurrence and metastasis. CSCs are usually sorted in analysis into side population (SP) cells using ultraviolet (UV) laser (350 nm) excitation; they cannot be stained with Hoechst 33342 because of their efflux ability. However, it is difficult to avoid cell damage using a UV laser. Therefore, we attempted to isolate CSCs using a violet laser (407 nm) excitation to avoid cellular DNA damage. We sorted SP cells and main population (MP) cells from a human endometrial cancer cell line using the FACSAria system equipped with a violet laser and analyzed the biological properties of these cells. SP cells exhibited drug efflux, self-renewal, differentiation abilities, and tumorigenicity. It was found that v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) expression was significantly higher in SP cells than in MP cells. Our results suggest that CSCs exist in the SP fraction sorted using the FACSAria system equipped with a violet laser, which presents a useful tool to isolate small populations of viable putative CSCs from solid tumors and can be used to identify and characterize CSCs.
Assuntos
Carcinoma Endometrioide/genética , Carcinoma Endometrioide/patologia , Separação Celular/métodos , Neoplasias do Endométrio/patologia , Lasers Semicondutores , Células-Tronco Neoplásicas , Células da Side Population , Animais , Linhagem Celular Tumoral , Separação Celular/instrumentação , Transformação Celular Neoplásica , Neoplasias do Endométrio/genética , Feminino , Expressão Gênica , Humanos , Mutação , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/patologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Ratos , Células da Side Population/citologia , Células da Side Population/patologia , Raios UltravioletaRESUMO
Risk of leukemia relapse after T cell-depleted hematopoietic stem cell transplantation is lower in the "HLA-C mismatched" recipient-donor combinations. This might be attributable to increased natural killing by allogeneic NK cells carrying a KIR that does not bind to HLA-C on target cells (HLA-C-uncoupled KIR). Considering a new strategy of allogeneic NK cell transfer with rituximab to treat B-cell lymphomas, however, it is unknown whether the HLA-C matching status also affects rituximab-mediated antibody-dependent cellular cytotoxicity (ADCC). To address this issue, we investigated the levels of ADCC by purified NK cells carrying an HLA-C-uncoupled KIR, where the NK cell donors had either matched or mismatched HLA-C combination with target cells. Purified NK cells carrying an HLA-C-uncoupled KIR consistently showed enhanced ADCC against target cells when NK cell donors had an HLA-C-mismatch. When NK cell donors did not have an HLA-C mismatch, it was inconsistent whether HLA-C-uncoupled KIR caused ADCC enhancement. When the levels of ADCC by whole NK cells were compared, there were substantial differences among the donors regardless of the HLA-C matching status. Subjects with HLA-C mismatch may not have an advantage when cytoimmunotherapy using allogeneic NK cells is considered in combination with rituximab.
Assuntos
Anticorpos Monoclonais Murinos/administração & dosagem , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Antineoplásicos/administração & dosagem , Antígenos HLA-C/imunologia , Histocompatibilidade , Imunoterapia Adotiva , Células Matadoras Naturais/transplante , Linfoma/terapia , Antígenos CD20/imunologia , Linfócitos B/imunologia , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Antígenos HLA-C/genética , Humanos , Isoantígenos/imunologia , Células Matadoras Naturais/imunologia , Depleção Linfocítica , Linfoma/tratamento farmacológico , RNA Interferente Pequeno/genética , Receptor Cross-Talk , Receptores KIR/metabolismo , Rituximab , Transplante HomólogoRESUMO
OBJECTIVE: It was the aim of this study to evaluate the diagnostic utility of Notch-1 immunocytochemistry in distinguishing endometrial glandular and stromal breakdown (EGBD) from endometrial adenocarcinoma in endometrial cytology. STUDY DESIGN: Samples of normal endometrium, EGBD and endometrial adenocarcinoma were subjected to immunocytochemical staining for Notch-1, and we examined the labeling index (LI) of Notch-1 (the ratio of intranuclear Notch-1-positive cells to total cells). We compared (1) the Notch-1 LI in normal endometrium, (2) the Notch-1 LI between normal endometrium and endometrial adenocarcinoma, and (3) the Notch-1 LI in normal endometrium, EGBD and endometrial adenocarcinoma. RESULTS: In analysis item 1, the LI of Notch-1 was 32.9 ± 8.4, 19.4 ± 8.2 and 12.5 ± 7.5% in proliferative endometrium, secretory endometrium and atrophic endometrium, respectively. In analysis item 2, the LI of Notch-1 in endometrial adenocarcinoma was 45.2 ± 7.4%, which was significantly higher than that in normal endometrium. In analysis item 3, the LI of Notch-1 in EGBD was 31.3 ± 8.3%, which was significantly lower than that in endometrial adenocarcinoma. CONCLUSION: In conclusion, Notch-1 immunocytochemistry is a useful method for distinguishing between EGBD and endometrial carcinoma in endometrial cytology.
Assuntos
Adenocarcinoma/diagnóstico , Neoplasias do Endométrio/diagnóstico , Receptor Notch1/análise , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Adulto , Idoso , Biomarcadores Tumorais/análise , Biópsia/métodos , Contagem de Células , Neoplasias do Endométrio/metabolismo , Neoplasias do Endométrio/patologia , Endométrio/citologia , Endométrio/metabolismo , Endométrio/patologia , Feminino , Humanos , Imuno-Histoquímica/métodos , Pessoa de Meia-Idade , Células Estromais/citologia , Células Estromais/metabolismo , Células Estromais/patologiaRESUMO
Tumor cytology has proven to be inadequate for precise diagnosis of thyroid follicular adenoma. This suggests the need for a molecular approach for its diagnosis. Expression of CD26/DPPIV (dipeptidyl peptidas IV), p53, and PTEN was analyzed in smears or sections obtained from 19 patients with histologically proven thyroid follicular adenoma. Papanicolaou staining, CD26/DPPIV activity staining, and HE staining were performed and the specimens were observed morphologically. Immunohistochemical analysis using antibodies against p53 and PTEN was performed. Genetic mutation of PTEN exons was performed using the laser capture microdissection method. The nuclear area of the CD26/DPPIV-positive cells was significantly larger than that of the CD26/DPPIV-negative cells. p53 expression was not observed any specimen. PTEN expression was observed in 18 of 19 cases. DNA sequence analysis did not reveal mutations in exons 5-9 of PTEN in the immunohistochemically PTEN-negative case. In accordance with our previous reports, we found that observation of concomitant CD26-positive and PTEN-negative status in cases of follicular adenoma suggests a state close to follicular carcinoma or progression to cancer, thus warranting careful follow-up.
Assuntos
Adenocarcinoma Folicular/diagnóstico , Adenoma/diagnóstico , Dipeptidil Peptidase 4/genética , PTEN Fosfo-Hidrolase/genética , Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/diagnóstico , Adenocarcinoma Folicular/patologia , Adenoma/patologia , Adulto , Idoso , Diagnóstico Diferencial , Éxons , Feminino , Expressão Gênica , Humanos , Imuno-Histoquímica , Microdissecção e Captura a Laser , Masculino , Pessoa de Meia-Idade , Mutação , Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/patologia , Proteína Supressora de Tumor p53/genéticaRESUMO
Endometrial cancer is one of the most common gynecological malignancies in Japan, where the disease shows an increasing morbidity. However, surgical therapy remains the treatment of choice for endometrial cancers that tend to be insensitive to radiation therapy and chemotherapy. Therefore, novel therapeutic strategies are required. The Notch signaling pathway regulates embryogenesis and cellular development, but deregulated Notch signaling may contribute to tumorigenesis in several cancers. Moreover, γ-secretase inhibitors have been shown to be potent inhibitors of the Notch signaling pathway; they suppress cellular proliferation and induce apoptosis in several cancer cells. In the present study, we investigated the effect of N-[N-(3, 5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester (DAPT, γ-secretase inhibitor) on the cell proliferation and apoptosis in Ishikawa endometrial cancer cells. Real-time PCR detected mRNA derived from NOTCH1 and HES1, which are target genes of the Notch signaling pathway, in Ishikawa endometrial cancer cells. After blocking Notch signaling, cellular proliferation decreased, accompanied by increased expression of p21 mRNA and decreased expression of the cyclin A protein. Furthermore, blockade of Notch signaling induced apoptosis. These results suggest that the Notch signaling pathway may be involved in cell proliferation through cell cycle regulation and apoptosis in Ishikawa endometrial cancer cells. Inhibition of the Notch signaling pathway by γ-secretase inhibitors is expected to be a potential target of novel therapeutic strategies for endometrial cancer.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Dipeptídeos/farmacologia , Neoplasias do Endométrio/patologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Linhagem Celular Tumoral , Neoplasias do Endométrio/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/genética , Humanos , RNA Mensageiro/genética , Receptor Notch1/genética , Transdução de Sinais , Fatores de Transcrição HES-1RESUMO
Notch receptor-mediated signaling is involved in the developmental process and functional modulation of lymphocytes, as well as in mast cell differentiation. Here, we investigated whether Notch signaling is required for antipathogen host defense regulated by mast cells. Mast cells were rarely found in the small intestine of wild-type C57BL/6 mice but accumulated abnormally in the lamina propria of the small-intestinal mucosa of the Notch2-conditional knockout mice in naive status. When transplanted into mast cell-deficient W(sh)/W(sh) mice, Notch2-null bone marrow-derived mast cells were rarely found within the epithelial layer but abnormally localized to the lamina propria, whereas control bone marrow-derived mast cells were mainly found within the epithelial layer. After the infection of Notch2 knockout and control mice with L3 larvae of Strongyloides venezuelensis, the abundant number of mast cells was rapidly mobilized to the epithelial layer in the control mice. In contrast, mast cells were massively accumulated in the lamina propria of the small intestinal mucosa in Notch2-conditional knockout mice, accompanied by impaired eradication of Strongyloides venezuelensis. These findings indicate that cell-autonomous Notch2 signaling in mast cells is required for proper localization of intestinal mast cells and further imply a critical role of Notch signaling in the host-pathogen interface in the small intestine.