RESUMO
Many targeted cancer therapies rely on biomarkers assessed by scoring of immunohistochemically (IHC)-stained tissue, which is subjective, semiquantitative, and does not account for expression heterogeneity. We describe an image analysis-based method for quantitative continuous scoring (QCS) of digital whole-slide images acquired from baseline human epidermal growth factor receptor 2 (HER2) IHC-stained breast cancer tissue. Candidate signatures for patient stratification using QCS of HER2 expression on subcellular compartments were identified, addressing the spatial distribution of tumor cells and tumor-infiltrating lymphocytes. Using data from trastuzumab deruxtecan-treated patients with HER2-positive and HER2-negative breast cancer from a phase 1 study (NCT02564900; DS8201-A-J101; N = 151), QCS-based patient stratification showed longer progression-free survival (14.8 vs 8.6 months) with higher prevalence of patient selection (76.4 vs 56.9%) and a better cross-validated log-rank p value (0.026 vs 0.26) than manual scoring based on the American Society of Clinical Oncology / College of American Pathologists guidelines. QCS-based features enriched the HER2-negative subgroup by correctly predicting 20 of 26 responders.
Assuntos
Neoplasias da Mama , Seleção de Pacientes , Receptor ErbB-2 , Trastuzumab , Humanos , Feminino , Receptor ErbB-2/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Trastuzumab/uso terapêutico , Pessoa de Meia-Idade , Biomarcadores Tumorais/metabolismo , Adulto , Imunoconjugados/uso terapêutico , Antineoplásicos Imunológicos/uso terapêutico , Idoso , Imuno-Histoquímica , Camptotecina/análogos & derivadosRESUMO
Endothelium differentiates in response to tissue-specific signals; brain endothelium expresses tight junctions and transporters which are absent from other endothelia. The promoter of the tight junction protein occludin exhibited strong activity in a brain endothelial cell line, hCMEC/D3 but was inactive in lung endothelial cells. Expression of occludin in brain endothelium corresponded with binding of Sp3 to a minimal promoter segment close to the transcription-start site. However, in lung endothelium Sp-transcription factors did not bind to this site although they are present in the cell nucleus. In contrast, repression of occludin in lung endothelium was associated with the binding of YY1 to a remote site in the promoter region, which was functionally inactive in brain endothelium. The work identified a group of transcription factors including Sp3 and YY1, which differentially interact with the occludin promoter to induce expression of occludin in brain endothelium and repression in other endothelia. The mechanism controlling occludin expression is similar to that which controls tissue-specific expression of the transferrin receptor in brain endothelium, leading to a scheme for endothelial differentiation, in which activation or repression of tissue-specific proteins is maintained by a set of transcription factors which include Sp3 and YY1.
Assuntos
Endotélio Vascular/metabolismo , Regulação da Expressão Gênica , Proteínas de Membrana/genética , Fator de Transcrição Sp3/metabolismo , Fator de Transcrição YY1/metabolismo , Encéfalo/irrigação sanguínea , Linhagem Celular , Células Endoteliais/citologia , Endotélio Vascular/citologia , Humanos , Ocludina , Fator de Transcrição Sp3/genética , Fator de Transcrição YY1/genéticaRESUMO
Brain endothelium has a distinctive phenotype, including high expression of transferrin receptor, p-glycoprotein, claudin-5 and occludin. Dermal endothelium expresses lower levels of the transferrin receptor and it is absent from lung endothelium. All three endothelia were screened for transcription factors that bind the transferrin receptor promoter and show different patterns of binding between the endothelia. The transcription factor YY1 has distinct DNA-binding activities in brain endothelium and non-brain endothelium. The target-sites on the transferrin receptor promotor for YY1 lie in close proximity to those of the transcription initiation complex containing TFIID, so the two transcription factors potentially compete or interfere. Notably, the DNA-binding activity of TFIID was the converse of YY1, in different endothelia. YY1 knockdown reduced transferrin receptor expression in brain endothelium, but not in dermal endothelium, implying that YY1 is involved in tissue-specific regulation of the transferrin receptor. Moreover a distinct YY1 variant is present in brain endothelium and it associates with Sp3. A model is presented, in which expression from the transferrin receptor gene in endothelium requires the activity of both TFIID and Sp3, but whether the gene is transcribed in different endothelia, is related to the balance between activating and suppressive forms of YY1.
Assuntos
Encéfalo/citologia , Endotélio/metabolismo , Receptores da Transferrina/metabolismo , Fatores de Transcrição/metabolismo , Pareamento de Bases , Western Blotting , Extratos Celulares , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Derme/citologia , Células Endoteliais/citologia , Imunofluorescência , Humanos , Pulmão/citologia , Fenótipo , Regiões Promotoras Genéticas , Ligação Proteica , Isoformas de Proteínas/metabolismo , Transporte Proteico , Receptores da Transferrina/genética , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição Sp3/metabolismo , Fator de Transcrição TFIID/metabolismo , Fator de Transcrição YY1/metabolismoRESUMO
Depletion of immunosuppressive tumor-associated macrophages (TAMs) or reprogramming toward a proinflammatory activation state represent different strategies to therapeutically target this abundant myeloid population. In this study, we report that inhibition of colony-stimulating factor-1 receptor (CSF-1R) signaling sensitizes TAMs to profound and rapid reprogramming in the presence of a CD40 agonist before their depletion. Despite the short-lived nature of macrophage hyperactivation, combined CSF-1R+CD40 stimulation of macrophages is sufficient to create a proinflammatory tumor milieu that reinvigorates an effective T cell response in transplanted tumors that are either responsive or insensitive to immune checkpoint blockade. The central role of macrophages in regulating preexisting immunity is substantiated by depletion experiments, transcriptome analysis of ex vivo sorted TAMs, and gene expression profiling of whole tumor lysates at an early treatment time point. This approach enabled the identification of specific combination-induced changes among the pleiotropic activation spectrum of the CD40 agonist. In patients, CD40 expression on human TAMs was detected in mesothelioma and colorectal adenocarcinoma.
Assuntos
Imunidade , Macrófagos/imunologia , Neoplasias/imunologia , Neoplasias/patologia , Animais , Antígenos CD40/agonistas , Antígenos CD40/metabolismo , Linfócitos T CD8-Positivos/imunologia , Feminino , Humanos , Inflamação/patologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Modelos Biológicos , Fenótipo , Receptor de Fator Estimulador de Colônias de Macrófagos/antagonistas & inibidores , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismoRESUMO
Immunohistochemical (IHC) α-synuclein (Asyn) pathology in peripheral biopsies may be a biomarker of Parkinson disease (PD). The multi-center Systemic Synuclein Sampling Study (S4) is evaluating IHC Asyn pathology within skin, colon and submandibular gland biopsies from 60 PD and 20 control subjects. Asyn pathology is being evaluated by a blinded panel of specially trained neuropathologists. Preliminary work assessed 2 candidate immunoperoxidase methods using a set of PD and control autopsy-derived sections from formalin-fixed, paraffin-embedded blocks of the 3 tissues. Both methods had 100% specificity; one, utilizing the 5C12 monoclonal antibody, was more sensitive in skin (67% vs 33%), and was chosen for further use in S4. Four trainee neuropathologists were trained to perform S4 histopathology readings; in subsequent testing, their scoring was compared to that of the trainer neuropathologist on both glass slides and digital images. Specificity and sensitivity were both close to 100% with all readers in all tissue types on both glass slides and digital images except for skin, where sensitivity averaged 75% with digital images and 83.5% with glass slides. Semiquantitative (0-3) density score agreement between trainees and trainer averaged 67% for glass slides and 62% for digital images.
Assuntos
Histocitoquímica/métodos , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Sinucleínas/metabolismo , Idoso , Idoso de 80 Anos ou mais , Biópsia , Colo/metabolismo , Feminino , Humanos , Masculino , Fibras Nervosas/metabolismo , Fibras Nervosas/patologia , Guias de Prática Clínica como Assunto , Estudos de Amostragem , Sensibilidade e Especificidade , Pele/metabolismo , Pele/patologia , Glândula Submandibular/metabolismo , Glândula Submandibular/patologiaRESUMO
The progeny of T lymphocytes responding to immunization mostly die rapidly, leaving a few long-lived survivors functioning as immune memory. Thus, control of this choice of death versus survival is critical for immune memory. There are indications that reactive radicals may be involved in this death pathway. We now show that, in mice lacking inducible nitric oxide synthase (iNOS), higher frequencies of both CD4 and CD8 memory T cells persist in response to immunization, even when iNOS(+/+) APCs are used for immunization. Postactivation T cell death by neglect is reduced in iNOS(-/-) T cells, and levels of the antiapoptotic proteins Bcl-2 and Bcl-xL are increased. Inhibitors of the iNOS-peroxynitrite pathway also enhance memory responses and block postactivation death by neglect in both mouse and human T cells. However, early primary immune responses are not enhanced, which suggests that altered survival, rather than enhanced activation, is responsible for the persistent immunity observed. Thus, in primary immune responses, iNOS in activated T cells autocrinely controls their susceptibility to death by neglect to determine the level of persisting CD4 and CD8 T cell memory, and modulation of this pathway can enhance the persistence of immune memory in response to vaccination.
Assuntos
Linfócitos T CD4-Positivos/enzimologia , Linfócitos T CD8-Positivos/enzimologia , Morte Celular/fisiologia , Memória Imunológica/fisiologia , Óxido Nítrico Sintase/metabolismo , Clorometilcetonas de Aminoácidos/metabolismo , Animais , Antígenos CD/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Humanos , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-2/metabolismo , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo II , Ácido Peroxinitroso/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Baço/citologia , Subpopulações de Linfócitos T , Proteína bcl-XRESUMO
Blockade of colony-stimulating factor-1 receptor (CSF-1R) enables the therapeutic targeting of tumor-associated macrophages (TAM) in cancer patients. Various CSF-1R inhibitors, mAbs, and tyrosine kinase inhibitors are currently evaluated in early clinical trials. Presence of an alternative survival signal, such as GM-CSF, rescues human monocyte-derived macrophages from CSF-1R inhibitor-induced apoptosis. In this study, we sought to identify additional factors that mediate resistance to CSF-1R-blocking antibody RG7155 (emactuzumab). We investigated the impact of hypoxia, macrophage-polarizing cytokines IL4 and IL10, and genetic alterations within the CSF1R locus and mitochondrial DNA. Among all investigated factors, only IL4 completely rescued viability of RG7155-treated macrophages in vitro This RG7155-resistant population was characterized by a substantially increased mannose receptor-1 (CD206) expression. Analysis of CD206 and the hemoglobin scavenger receptor CD163 expression on normal tissue allowed for discrimination of distinct macrophage populations according to localization and frequency. In emactuzumab-treated cancer patients, we found a significant reduction of CSF-1R, CD204, and CD163 mRNA levels in contrast to a less pronounced decrease of CD206 expression by transcriptome analysis of tumor biopsies. However, we detected in normal skin tissue, which shows lower IL4 mRNA expression compared with melanoma tissue, significant reduction of CD206+ dermal macrophages in RG7155-treated skin biopsies. These results suggest that in cancers where the cytokines IL4 and GM-CSF are sufficiently expressed to induce very high CD206 expression on macrophages, CSF-1R inhibition may not deplete CD206hi TAM. This observation can help to identify those patients most likely to benefit from CSF-1R-targeting agents. Mol Cancer Ther; 15(12); 3077-86. ©2016 AACR.
Assuntos
Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Anticorpos Monoclonais Humanizados , Biomarcadores , Biópsia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Citocinas/metabolismo , Resistência a Medicamentos , Humanos , Imunofenotipagem , Monócitos/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Fenótipo , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Pele/citologiaRESUMO
Dysregulated cellular apoptosis and resistance to cell death are hallmarks of neoplastic initiation and disease progression. Therefore, the development of agents that overcome apoptosis dysregulation in tumor cells is an attractive therapeutic approach. Activation of the extrinsic apoptotic pathway is strongly dependent on death receptor (DR) hyperclustering on the cell surface. However, strategies to activate DR5 or DR4 through agonistic antibodies have had only limited clinical success. To pursue an alternative approach for tumor-targeted induction of apoptosis, we engineered a bispecific antibody (BsAb), which simultaneously targets fibroblast-activation protein (FAP) on cancer-associated fibroblasts in tumor stroma and DR5 on tumor cells. We hypothesized that bivalent binding to both FAP and DR5 leads to avidity-driven hyperclustering of DR5 and subsequently strong induction of apoptosis in tumor cells but not in normal cells. Here, we show that RG7386, an optimized FAP-DR5 BsAb, triggers potent tumor cell apoptosis in vitro and in vivo in preclinical tumor models with FAP-positive stroma. RG7386 antitumor efficacy was strictly FAP dependent, was independent of FcR cross-linking, and was superior to conventional DR5 antibodies. In combination with irinotecan or doxorubicin, FAP-DR5 treatment resulted in substantial tumor regression in patient-derived xenograft models. FAP-DR5 also demonstrated single-agent activity against FAP-expressing malignant cells, due to cross-binding of FAP and DR5 across tumor cells. Taken together, these data demonstrate that RG7386, a novel and potent antitumor agent in both mono- and combination therapies, overcomes limitations of previous DR5 antibodies and represents a promising approach to conquer tumor-associated resistance to apoptosis. Mol Cancer Ther; 15(5); 946-57. ©2016 AACR.
Assuntos
Anticorpos Biespecíficos/metabolismo , Anticorpos Biespecíficos/farmacologia , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Gelatinases/metabolismo , Proteínas de Membrana/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Serina Endopeptidases/metabolismo , Animais , Anticorpos Biespecíficos/imunologia , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados , Afinidade de Anticorpos/imunologia , Antineoplásicos/imunologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Endopeptidases , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Gelatinases/imunologia , Humanos , Proteínas de Membrana/imunologia , Camundongos , Ligação Proteica/imunologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/imunologia , Serina Endopeptidases/imunologia , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We have adapted an in vitro model of the human blood-brain barrier, the immortalized human cerebral microvascular endothelial cells (hCMEC/D3), to quantitatively measure protein transcytosis. After validating the receptor-mediated transport using transferrin, the system was used to measure transcytosis rates of antibodies directed against potential brain shuttle receptors. While an antibody to the insulin-like growth factor 1 receptor (IGF1R) was exclusively recycled to the apical compartment, the fate of antibodies to the transferrin receptor (TfR) was determined by their relative affinities at extracellular and endosomal pH. An antibody with reduced affinity at pH5.5 showed significant transcytosis, while pH-independent antibodies of comparable affinities at pH 7.4 remained associated with intracellular vesicular compartments and were finally targeted for degradation.
Assuntos
Anticorpos/metabolismo , Barreira Hematoencefálica/metabolismo , Transcitose , Anticorpos/imunologia , Antígenos CD/imunologia , Linhagem Celular , Humanos , Concentração de Íons de Hidrogênio , Receptor IGF Tipo 1/imunologia , Receptores da Transferrina/imunologiaRESUMO
Although biotherapeutics have vast potential for treating brain disorders, their use has been limited due to low exposure across the blood-brain barrier (BBB). We report that by manipulating the binding mode of an antibody fragment to the transferrin receptor (TfR), we have developed a Brain Shuttle module, which can be engineered into a standard therapeutic antibody for successful BBB transcytosis. Brain Shuttle version of an anti-Aß antibody, which uses a monovalent binding mode to the TfR, increases ß-Amyloid target engagement in a mouse model of Alzheimer's disease by 55-fold compared to the parent antibody. We provide in vitro and in vivo evidence that the monovalent binding mode facilitates transcellular transport, whereas a bivalent binding mode leads to lysosome sorting. Enhanced target engagement of the Brain Shuttle module translates into a significant improvement in amyloid reduction. These findings have major implications for the development of biologics-based treatment of brain disorders.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Transporte Proteico/fisiologia , Anticorpos de Cadeia Única/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Doença de Alzheimer/terapia , Peptídeos beta-Amiloides/imunologia , Precursor de Proteína beta-Amiloide/genética , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/imunologia , Linhagem Celular Transformada , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Macrolídeos/farmacologia , Camundongos , Camundongos Transgênicos , Modelos Imunológicos , Presenilina-1/genética , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/imunologia , Transporte Proteico/efeitos dos fármacos , Receptores da Transferrina/imunologia , Receptores da Transferrina/metabolismo , Anticorpos de Cadeia Única/farmacologia , Anticorpos de Cadeia Única/uso terapêutico , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismo , Fatores de Tempo , Transcitose/efeitos dos fármacos , Transcitose/genética , Transcitose/imunologiaRESUMO
BACKGROUND: The aim of this study was to characterize the hCMEC/D3 cell line, an in vitro model of the human Blood Brain Barrier (BBB) for the expression of brain endothelial specific claudins-3 and -12. FINDINGS: hCMEC/D3 cells express claudins-3 and -12. Claudin-3 is distinctly localized to the TJ whereas claudin -12 is observed in the perinuclear region and completely absent from TJs. We show that the expression of both proteins is lost in cell passage numbers where the BBB properties are no longer fully conserved. Expression and localization of claudin-3 is not modulated by simvastatin shown to improve barrier function in vitro and also recommended for routine hCMEC/D3 culture. CONCLUSIONS: These results support conservation of claudin-3 and -12 expression in the hCMEC/D3 cell line and make claudin-3 a potential marker for BBB characteristics in vitro.
RESUMO
Peripheral T cell homeostasis results from a balance between factors promoting survival and those that trigger deletion of Ag-reactive cells. The cytokine IL-2 promotes T cell survival whereas reactive oxygen species (ROS) sensitize T cells to apoptosis. Two pathways of activated T cell apoptosis-one triggered by Fas ligand and the other by cytokine deprivation-depend on ROS, with the latter also regulated by members of the Bcl-2 family. Notch family proteins regulate several cell-fate decisions in metazoans. Ectopic expression of the Notch1 intracellular domain (NICD) in T cells inhibits Fas-induced apoptosis. The underlying mechanism is not known and the role, if any, of Notch in regulating apoptosis triggered by cytokine deprivation or neglect has not been examined. In this study, we use a Notch1/Fc chimera; a blocking Ab to Notch1 and chemical inhibitors of gamma-secretase to investigate the role of Notch signaling in activated T cells of murine origin. We show that perturbing Notch signaling in activated CD4+/CD8+ T cells maintained in IL-2 results in the accumulation of ROS, reduced Akt/protein kinase B activity, and expression of the antiapoptotic protein Bcl-xL, culminating in apoptosis. A broad-spectrum redox scavenger inhibits apoptosis but T cells expressing mutant Fas ligand are sensitive to apoptosis. Activated T cells isolated on the basis of Notch expression (Notch+) are enriched for Bcl-xL expression and demonstrate reduced susceptibility to apoptosis triggered by neglect or oxidative stress. Furthermore, enforced expression of NICD protects activated T cells from apoptosis triggered by cytokine deprivation. Taken together, these data implicate Notch1 signaling in the cytokine-dependent survival of activated T cells.
Assuntos
Apoptose/fisiologia , Interleucina-2/farmacologia , Ativação Linfocitária , Receptor Notch1/fisiologia , Linfócitos T/fisiologia , Animais , Sobrevivência Celular/fisiologia , Citocinas/farmacologia , Homeostase , Fragmentos Fc das Imunoglobulinas/genética , Camundongos , Camundongos Endogâmicos C57BL , Receptor Notch1/genética , Proteínas Recombinantes de Fusão , Transdução de Sinais , Linfócitos T/citologiaRESUMO
We have investigated the mechanism of IL-7-mediated inhibition of dexamethasone-induced apoptosis in T cells. Broad-spectrum caspase inhibitors block dexamethasone-triggered nuclear fragmentation, but not the loss of mitochondrial transmembrane potential or membrane integrity in CD3(+) mature T cells isolated from adult mouse spleens. IL-7 blocked dexamethasone-induced apoptosis and the processing of caspase-3 and caspase-7. IL-7 also blocked dexamethasone-triggered dephosphorylation of the serine-threonine kinase Akt/PKB and its target, the Ser(136) residue in Bad. The loss of anti-apoptotic proteins Bcl-x(L) and inhibitor of apoptosis protein-2 (IAP-2) was also blocked by IL-7. The protective effect was attenuated by pharmacological inhibitors of phosphatidylinositol-3 kinase (PI3K) with one exception: inhibition of PI3K did not abrogate Bcl-x(L) expression in the presence of IL-7. The anti-apoptotic role of Akt suggested by these experiments was tested by overexpression of constitutively active Akt, which blocked dexamethasone-induced apoptosis and elevated IAP-2 but not Bcl-x(L) levels in a mature T cell line. Thus, IL-7 regulates IAP-2 expression and inhibits dexamethasone-induced apoptosis by activating Akt via PI3K-dependent signaling, but regulates Bcl-x(L)expression via a PI3K-independent pathway in mature T cells.
Assuntos
Apoptose , Dexametasona/antagonistas & inibidores , Interleucina-7/farmacologia , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/fisiologia , Linfócitos T/imunologia , Clorometilcetonas de Aminoácidos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Inibidores de Caspase , Linhagem Celular , Sobrevivência Celular , Células Cultivadas , Inibidores de Cisteína Proteinase/farmacologia , Inibidores Enzimáticos/farmacologia , Proteínas Inibidoras de Apoptose , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Fosfatidilinositol 3-Quinases/fisiologia , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-akt , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/enzimologia , Proteína bcl-XRESUMO
The Notch family of transmembrane receptors have been implicated in a variety of cellular decisions in different cell types. Here we investigate the mechanism underlying Notch-1-mediated anti-apoptotic function in T cells using model cell lines as the experimental system. Ectopic expression of the intracellular domain of Notch-1/activated Notch (AcN1) increases expression of anti-apoptotic proteins of the inhibitors of apoptosis (IAP) family, the Bcl-2 family, and the FLICE-like inhibitor protein (FLIP) and inhibits death triggered by multiple stimuli that activate intrinsic or extrinsic pathways of apoptosis in human and murine T cell lines. Numb inhibited the AcN1-dependent induction of anti-apoptotic proteins and anti-apoptotic function. Using pharmacological inhibitors and dominant-negative approaches, we describe a functional role for phosphatidylinositol 3-kinase (PI3K)-dependent activation of the serine-threonine kinase Akt/PKB in the regulation of AcN1-mediated anti-apoptotic function and the expression of FLIP and IAP family proteins. Using a cell line deficient for the T cell-specific, Src family protein, the tyrosine kinase p56(lck) and by reconstitution approaches we demonstrate that p56(lck) is required for the Notch-1-mediated activation of Akt/PKB function. Furthermore, the Src tyrosine kinase inhibitor, PP2, abrogated ectopically expressed AcN1-mediated anti-apoptotic function and phosphorylation of p56(lck). We present evidence that endogenous Notch-1 associates with p56(lck) and PI3K but that Akt/PKB does not co-immunoprecipitate with the Notch1.p56(lck).PI3K complex. Finally, we demonstrate that the Notch1.p56(lck).PI3K complex is present in primary T cells that have been activated in vitro and sustained in culture with the cytokine interleukin-2.
Assuntos
Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Proteínas Serina-Treonina Quinases , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Linfócitos T/metabolismo , Fatores de Transcrição , Apoptose , Humanos , Células Jurkat , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Receptor Notch1RESUMO
The molecular machinery of apoptosis is evolutionarily conserved with some exceptions. One such example is the Drosophila proapoptotic gene Head involution defective (Hid), whose mammalian homologue is not known. Hid is apoptotic to mammalian cells, and we have examined the mechanism by which Hid induces death. We demonstrate for the first time a role for the extracellular signal-related kinase-1/2 (Erk-1/2) in the regulation of Hid function in mammalian cells. Bcl-2 and an inhibitor of caspase-9 blocked apoptosis, indicative of a role for the mitochondrion in this pathway, and we provide evidence for a role for caspase-8 in Hid-induced apoptosis. Thus, apoptosis was blocked by an inhibitor of caspase-8, deletion of caspase-8 rendered cells resistant to Hid-induced apoptosis, and Hid associated with caspase-8 in cell lysates. The Fas-associated death domain (FADD) was dispensable for the apoptotic function of Hid, indicating that Hid does not require extracellular death receptor signaling for the activation of caspase-8. In activated T cells, the cytokine interleukin-2 blocked caspase-8 processing and apoptosis, suggesting that survival cues from trophic factors may target a Hid-like intermediate present in mammalian cells. Thus, this study shows that Hid engages with conserved components of cellular death machinery and suggests that apoptotic paradigms characterized by FADD-independent activation of caspase-8 may involve a Hid-like molecule in mammalian cells.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Apoptose/fisiologia , Proteínas de Transporte/fisiologia , Caspases/fisiologia , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Neuropeptídeos/fisiologia , Animais , Apoptose/efeitos da radiação , Caspase 8 , Caspase 9 , Caspases/metabolismo , Linhagem Celular , Drosophila , Proteínas de Drosophila , Ativação Enzimática , Proteína de Domínio de Morte Associada a Fas , Humanos , Ativação Linfocitária , Camundongos , Proteína Quinase 3 Ativada por Mitógeno , Raios UltravioletaRESUMO
The Bcl-2 family proapoptotic protein, Bax, redistributes to the mitochondrion in response to varied stimuli, triggering loss of mitochondrial integrity and apoptosis. Suppression of MAPK kinase (MEK1) by the reagent UO126 in activated T cells maintained in the cytokine IL-2 disrupts cytoplasmic localization of Bax and cell survival. UO126 triggers mitochondrial translocation of ectopically expressed Bax-GFP, and both UO126 and dominant negative MEK-1 (DN-MEK1) trigger increased apoptosis in Bax-GFP-expressing T cell lines. Because inhibition of PI3K or its target Akt also triggers mitochondrial translocation of Bax in T cells and apoptosis in Bax-transfected cell lines, we generated Bax deletion mutants to identify the region(s) that confers sensitivity to regulation by MEK1 and Akt. A deletion mutant (Bax(1-171)) without the C terminus mitochondrial targeting sequence or an Akt target site (Ser(184)) localizes to the cytoplasm and triggers low level apoptosis that is enhanced by DN-Akt or DN-MEK1. A construct that lacks the first 29 aa (Bax-delta29) largely localizes to mitochondria, is highly apoptogenic, and is not inhibited by Akt or MEK1. Furthermore, Bax-delta29 overcomes IL-2-dependent survival in a T cell line, whereas Bax triggers comparatively low levels of apoptosis in these cells. Cytoplasmic localization and regulation by MEK1 and Akt are restored in a mutant deleted of the first 13 aa (Bax-delta13). Taken together, our results identify a region in the Bax N terminus that determines cellular localization regulated by MEK- and Akt-dependent signaling in T cells.
Assuntos
Regulação para Baixo , MAP Quinase Quinase 1/fisiologia , Fragmentos de Peptídeos/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Animais , Apoptose/genética , Apoptose/imunologia , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Citoplasma/enzimologia , Citoplasma/genética , Citoplasma/metabolismo , Regulação para Baixo/imunologia , Inibidores Enzimáticos/farmacologia , Deleção de Genes , Humanos , Células Jurkat , Ativação Linfocitária/genética , MAP Quinase Quinase 1/antagonistas & inibidores , Camundongos , Mitocôndrias/enzimologia , Mitocôndrias/genética , Mitocôndrias/metabolismo , Fragmentos de Peptídeos/genética , Fosfatidilinositol 3-Quinases/fisiologia , Inibidores de Fosfoinositídeo-3 Quinase , Estrutura Terciária de Proteína/genética , Transporte Proteico/genética , Transporte Proteico/imunologia , Proteínas Proto-Oncogênicas c-akt , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/enzimologia , Subpopulações de Linfócitos T/metabolismo , Proteína X Associada a bcl-2RESUMO
In thymocytes, dexamethasone initiates cytochrome c-dependent processing of caspase-9 and the activation of caspase-3 to trigger apoptotic damage. Using murine thymocytes or a thymocyte cell line WEHI 7.1, we show that this pathway is inhibited by dominant-negative caspase-9, the anti-apoptotic protein Bcl-2, or by blocking components of the mitochondrial permeability transition pore complex (PTPC). We use DIDS (dithiocyanatostilbene-2,2-disulfonic acid), a pharmacological modifier of VDAC (voltage-dependent anion channel) function or ectopic expression of hexokinase-II, to examine the role of the VDAC--a mitochondrial outer membrane protein--in this apoptotic pathway. This approach implicated the VDAC in dexamethasone-mediated cytochrome c release, processing of caspase-9 and caspase-3, the loss of mitochondrial transmembrane potential (Deltapsim), nuclear damage and cell lysis. Inhibiting the adenine nucleotide transporter (ANT), a protein on the mitochondrial inner membrane, also blocks dexamethasone-induced apoptosis, but the ANT regulates caspase-3 processing and nuclear damage but not the mitochondrial efflux of cytochrome c. Collectively, the data identify two separable, but connected events in dexamethasone-induced mitochondrial damage in thymocytes. The first event is an increase in permeability of the mitochondrial outer membrane leading to VDAC-regulated efflux of cytochrome c and initial processing of caspase-9 followed by ANT-dependent caspase-3 processing and apoptotic damage to cells.