RESUMO
Anti-CD38 monoclonal antibodies (mAbs) represent a breakthrough in the treatment of multiple myeloma (MM), yet some patients fail to respond or progress quickly with this therapy, highlighting the need for novel approaches. In this study we compared the preclinical efficacy of SAR442085, a next-generation anti-CD38 mAb with enhanced affinity for activating Fcγ receptors (FcγR), with first-generation anti-CD38 mAb daratumumab and isatuximab. In surface plasmon resonance and cellular binding assays, we found that SAR442085 had higher binding affinity than daratumumab and isatuximab for FcγRIIa (CD32a) and FcγRIIIa (CD16a). SAR442085 also exhibited better in vitro antibody-dependent cellular cytotoxicity (ADCC) against a panel of MM cells expressing variable CD38 receptor densities including MM patients' primary plasma cells. The enhanced ADCC of SAR442085 was confirmed using NK-92 cells bearing low and high affinity FcγRIIIa (CD16a)-158F/V variants. Using MM patients' primary bone marrow cells, we confirmed that SAR442085 had an increased ability to engage FcγRIIIa, resulting in higher natural killer (NK) cell activation and degranulation against primary plasma cells than preexisting Fc wild-type anti-CD38 mAbs. Finally, using huFcgR transgenic mice that express human Fcγ receptors under the control of their human regulatory elements, we demonstrated that SAR442085 had higher NK cell-dependent in vivo antitumor efficacy and better survival than daratumumab and isatuximab against EL4 thymoma or VK*MYC myeloma cells overexpressing human CD38. These results highlight the preclinical efficacy of SAR442085 and support the current evaluation of this next-generation anti-CD38 antibody in phase I clinical development in patients with relapsed/refractory MM.
Assuntos
ADP-Ribosil Ciclase 1/antagonistas & inibidores , Antineoplásicos Imunológicos/farmacologia , Células da Medula Óssea , Glicoproteínas de Membrana/antagonistas & inibidores , Mieloma Múltiplo , Proteínas de Neoplasias/antagonistas & inibidores , ADP-Ribosil Ciclase 1/metabolismo , Animais , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Linhagem Celular Tumoral , Células HEK293 , Humanos , Glicoproteínas de Membrana/metabolismo , Camundongos Transgênicos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Proteínas de Neoplasias/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Immunotherapy is revolutionizing cancer treatment; however, complete responses are achieved in only a small fraction of patients and tumor types. Thus, there is an urgent need for predictive preclinical models to drive rational immunotherapeutic drug development, treatment combinations, and to minimize failures in clinical trials. Humanized mouse models (HIS) have been developed to study and modulate the interactions between immune components and tumors of human origin. In this review, we discuss recent advances in the 'humanization' of mouse models to improve the quality of human immune cell reconstitution. We also highlight new insights into the basic mechanisms, and provide a preclinical evaluation of onco-immunotherapies, as well as the limitations thereof, which constitute drivers for the improvement of the models to increase their translational power.
Assuntos
Neoplasias/imunologia , Evasão Tumoral/imunologia , Microambiente Tumoral/imunologia , Animais , Modelos Animais de Doenças , Humanos , Sistema Imunitário/citologia , Sistema Imunitário/imunologia , Sistema Imunitário/metabolismo , Hospedeiro Imunocomprometido/genética , Hospedeiro Imunocomprometido/imunologia , Imunoterapia/métodos , Camundongos , Camundongos Transgênicos , Neoplasias/genética , Neoplasias/patologia , Neoplasias/terapia , Pesquisa , Evasão Tumoral/genética , Microambiente Tumoral/genéticaRESUMO
BACKGROUND: The proteins of the galectin family are implicated in many cellular processes, including cell interactions, polarity, intracellular trafficking, and signal transduction. In human and mouse, galectin-7 is almost exclusively expressed in stratified epithelia, notably in the epidermis. Galectin-7 expression is also altered in several human tumors of epithelial origin. This study aimed at dissecting the consequences of galectin-7 overexpression on epidermis structure and functions in vivo. METHODS: We established transgenic mice specifically overexpressing galectin-7 in the basal epidermal keratinocytes and analyzed the consequences on untreated skin and after UVB irradiation or mechanical injury. RESULTS: The intercellular cohesion of the epidermis is impaired in transgenic animals, with gaps developing between adjacent keratinocytes, associated with loss of adherens junctions. The epidermal architecture is aberrant with perturbations in the multilayered cellular organisation of the tissue, and structural defects in the basement membrane. These transgenic animals displayed a reduced re-epithelialisation potential following superficial wound, due to a defective collective migration of keratinocytes. Finally, a single mild dose of UVB induced an abnormal apoptotic response in the transgenic epidermis. CONCLUSION: These results indicate that an excess of galectin-7 leads to a destabilisation of adherens junctions associated with defects in epidermal repair. As this phenotype shares similarities with that of galectin-7 null mutant mice, we conclude that a critical level of this protein is required for maintaining proper epidermal homeostasis. This study brings new insight into the mode of action of galectins in normal and pathological situations.
Assuntos
Epiderme/metabolismo , Galectinas/genética , Junções Intercelulares/metabolismo , Cicatrização , Animais , Western Blotting , Linhagem Celular , Células Epidérmicas , Epiderme/efeitos da radiação , Camundongos , Camundongos Transgênicos , Raios UltravioletaRESUMO
BACKGROUND: Tobacco smoke predisposes humans and animals to develop lung tumors, but the molecular events responsible for this are poorly understood. We recently showed that signaling mechanisms triggered by smoke in lung cells could lead to the activation of a growth factor signaling pathway, thereby promoting hyperproliferation of lung epithelial cells. Hyperproliferation is considered a premalignant change in the lung, in that increased rates of DNA synthesis are associated with an increased number of DNA copying errors, events that are exacerbated in the presence of tobacco smoke carcinogens. Despite the existence of DNA repair mechanisms, a small percentage of these errors go unrepaired and can lead to tumorigenic mutations. The results of our previous study showed that an early event following smoke exposure was the generation of oxygen radicals through the activation of NADPH oxidase. Although it was clear that these radicals transduced signals through the epidermal growth factor receptor (EGFR), and that this was mediated by TACE-dependent cleavage of amphiregulin, it remained uncertain how oxygen radicals were able to activate TACE. PRINCIPAL FINDINGS: In the present study, we demonstrate for the first time that phosphorylation of TACE at serine/threonine residues by tobacco smoke induces amphiregulin release and EGFR activation. TACE phosphorylation is triggered in smoke-exposed lung cells by the ROS-induced activation of PKC through the action of SRC kinase. Furthermore, we identified PKCε as the PKC isoform involved in smoke-induced TACE activation and hyperproliferation of lung cells. CONCLUSIONS: Our data elucidate new signaling paradigms by which tobacco smoke promotes TACE activation and hyperproliferation of lung cells.
Assuntos
Proteínas ADAM/metabolismo , Pulmão/enzimologia , Pulmão/patologia , Nicotiana/química , Lesões Pré-Cancerosas/patologia , Proteína Quinase C-épsilon/metabolismo , Fumar/efeitos adversos , Proteína ADAM17 , Animais , Brônquios/patologia , Proliferação de Células , Células Cultivadas , Ativação Enzimática , Células Epiteliais/enzimologia , Células Epiteliais/patologia , Receptores ErbB/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Isoenzimas/metabolismo , Camundongos , Modelos Biológicos , Fosforilação , Lesões Pré-Cancerosas/enzimologia , Espécies Reativas de Oxigênio/metabolismo , Quinases da Família src/metabolismoRESUMO
Periostin is considered to be a matricellular protein with expression typically confined to cells of mesenchymal origin. Here, by using in situ hybridization, we show that periostin is specifically up-regulated in bronchial epithelial cells of asthmatic subjects, and in vitro, we show that periostin protein is basally secreted by airway epithelial cells in response to IL-13 to influence epithelial cell function, epithelial-mesenchymal interactions, and extracellular matrix organization. In primary human bronchial epithelial cells stimulated with periostin and epithelial cells overexpressing periostin, we reveal a function for periostin in stimulating the TGF-beta signaling pathway in a mechanism involving matrix metalloproteinases 2 and 9. Furthermore, conditioned medium from the epithelial cells overexpressing periostin caused TGF-beta-dependent secretion of type 1 collagen by airway fibroblasts. In addition, mixing recombinant periostin with type 1 collagen in solution caused a dramatic increase in the elastic modulus of the collagen gel, indicating that periostin alters collagen fibrillogenesis or cross-linking and leads to stiffening of the matrix. Epithelial cell-derived periostin in asthma has roles in TGF-beta activation and collagen gel elasticity in asthma.
Assuntos
Asma/metabolismo , Brônquios/patologia , Moléculas de Adesão Celular/fisiologia , Colágeno Tipo I/biossíntese , Células Epiteliais/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Asma/patologia , Brônquios/citologia , Células Cultivadas , Colágeno Tipo I/química , Colágeno Tipo I/metabolismo , Elasticidade , Géis , Humanos , Metaloproteinases da MatrizRESUMO
BACKGROUND: Previously, we found that mast cell tryptases and carboxypeptidase A3 (CPA3) are differentially expressed in the airway epithelium in asthmatic subjects. We also found that asthmatic subjects can be divided into 2 subgroups ("T(H)2 high" and "T(H)2 low" asthma) based on epithelial cell gene signatures for the activity of T(H)2 cytokines. OBJECTIVES: We sought to characterize intraepithelial mast cells (IEMCs) in asthma. METHODS: We performed gene expression profiling in epithelial brushings and stereology-based quantification of mast cell numbers in endobronchial biopsy specimens from healthy control and asthmatic subjects before and after treatment with inhaled corticosteroids (ICSs). We also performed gene expression and protein quantification studies in cultured airway epithelial cells and mast cells. RESULTS: By means of unsupervised clustering, mast cell gene expression in the airway epithelium related closely to the expression of IL-13 signature genes. The levels of expression of mast cell genes correlate positively with lung function improvements with ICSs. IEMC density was 2-fold higher than normal in subjects with T(H)2-high asthma compared with that seen in subjects with T(H)2-low asthma or healthy control subjects (P = .015 for both comparisons), and these cells were characterized by expression of tryptases and CPA3 but not chymase. IL-13 induced expression of stem cell factor in cultured airway epithelial cells, and mast cells exposed to conditioned media from IL-13-activated epithelial cells showed downregulation of chymase but no change in tryptase or CPA3 expression. CONCLUSION: IEMC numbers are increased in subjects with T(H)2-high asthma, have an unusual protease phenotype (tryptase and CPA3 high and chymase low), and predict responsiveness to ICSs. IL-13-stimulated production of stem cell factor by epithelial cells potentially explains mast cell accumulation in T(H)2-high asthmatic epithelium.
Assuntos
Asma , Células Epiteliais/imunologia , Mastócitos/imunologia , Peptídeo Hidrolases/classificação , Peptídeo Hidrolases/metabolismo , Células Th2/imunologia , Adulto , Asma/imunologia , Asma/fisiopatologia , Carboxipeptidases A/genética , Carboxipeptidases A/metabolismo , Células Cultivadas , Quimases/genética , Quimases/metabolismo , Citocinas/genética , Citocinas/metabolismo , Citocinas/farmacologia , Células Epiteliais/citologia , Células Epiteliais/enzimologia , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Interleucina-13/genética , Interleucina-13/metabolismo , Interleucina-13/farmacologia , Masculino , Mastócitos/citologia , Mastócitos/enzimologia , Pessoa de Meia-Idade , Peptídeo Hidrolases/genética , Fenótipo , Fator de Células-Tronco , Triptases/genética , Triptases/metabolismo , Adulto JovemRESUMO
Galectins, a family of beta-galactoside binding lectins, have recently emerged as novel regulators of tissue homeostasis. Galectin-7 is predominantly expressed in stratified epithelia, especially in epidermis. We report here the generation of galectin-7-deficient mice that are viable and do not display phenotypical abnormalities in skin structure or expression of epidermal markers. However, these mice show unique defects in the maintenance of epidermal homeostasis in response to environmental challenges. First, after UVB irradiation in vivo, the apoptotic response is prematurely triggered and lasts longer in the mutant epidermis. This result contrasts with the proapoptotic role that had been proposed for galectin-7. Second, wound-healing experiments in vivo revealed that galectin-7-deficient mice displayed a reduced reepithelialization potential compared with wild-type littermates. This effect could be attributed to a defect in cell migration. Because galectin-7 is located in the podosomes of keratinocytes migrating out of skin explants in culture, we propose that this glycan-binding protein may directly influence cell/extracellular matrix interactions. Finally, we also detected an unexpected intense hyperproliferative reaction consecutive to both types of stress in galectin-7-deficient mice. Together, these studies provide the first genetic evidence showing that galectin-7 can modulate keratinocyte apoptosis, proliferation, and migration during skin repair.
Assuntos
Epiderme/lesões , Epiderme/metabolismo , Galectinas/metabolismo , Homeostase , Animais , Apoptose/fisiologia , Biomarcadores/metabolismo , Cortactina/metabolismo , Células Epidérmicas , Epiderme/efeitos da radiação , Galectinas/genética , Humanos , Camundongos , Camundongos Knockout , Raios UltravioletaRESUMO
Airway inflammation and epithelial remodeling are two key features of asthma. IL-13 and other cytokines produced during T helper type 2 cell-driven allergic inflammation contribute to airway epithelial goblet cell metaplasia and may alter epithelial-mesenchymal signaling, leading to increased subepithelial fibrosis or hyperplasia of smooth muscle. The beneficial effects of corticosteroids in asthma could relate to their ability to directly or indirectly decrease epithelial cell activation by inflammatory cells and cytokines. To identify markers of epithelial cell dysfunction and the effects of corticosteroids on epithelial cells in asthma, we studied airway epithelial cells collected from asthmatic subjects enrolled in a randomized controlled trial of inhaled corticosteroids, from healthy subjects and from smokers (disease control). By using gene expression microarrays, we found that chloride channel, calcium-activated, family member 1 (CLCA1), periostin, and serine peptidase inhibitor, clade B (ovalbumin), member 2 (serpinB2) were up-regulated in asthma but not in smokers. Corticosteroid treatment down-regulated expression of these three genes and markedly up-regulated expression of FK506-binding protein 51 (FKBP51). Whereas high baseline expression of CLCA1, periostin, and serpinB2 was associated with a good clinical response to corticosteroids, high expression of FKBP51 was associated with a poor response. By using airway epithelial cells in culture, we found that IL-13 increased expression of CLCA1, periostin, and serpinB2, an effect that was suppressed by corticosteroids. Corticosteroids also induced expression of FKBP51. Taken together, our findings show that airway epithelial cells in asthma have a distinct activation profile and identify direct and cell-autonomous effects of corticosteroid treatment on airway epithelial cells that relate to treatment responses and can now be the focus of specific mechanistic studies.
Assuntos
Corticosteroides/uso terapêutico , Asma/tratamento farmacológico , Asma/genética , Células Epiteliais/fisiologia , Perfilação da Expressão Gênica , Genoma Humano , Administração por Inalação , Corticosteroides/administração & dosagem , Asma/patologia , Broncoscopia , Moléculas de Adesão Celular/genética , Canais de Cloreto/genética , Células Epiteliais/patologia , Humanos , Hipersensibilidade , Inflamação/genética , Inflamação/fisiopatologia , Análise de Sequência com Séries de Oligonucleotídeos , Valores de Referência , Serpinas/genética , Fumar/patologiaRESUMO
BACKGROUND: Lung cancer is the leading cause of cancer death in the world, and greater than 90% of lung cancers are cigarette smoke-related. Current treatment options are inadequate, because the molecular basis of cigarette-induced lung cancer is poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show that human primary or immortalized bronchial epithelial cells exposed to cigarette smoke for eight days in culture rapidly proliferate, show anchorage-independent growth, and form tumors in nude mice. Using this model of the early stages of smoke-induced tumorigenesis, we examined the molecular changes leading to lung cancer. We observed that the embryonic signaling pathways mediated by Hedgehog and Wnt are activated by smoke. Pharmacological inhibition of these pathways blocked the transformed phenotype. CONCLUSIONS/SIGNIFICANCE: These experiments provide a model in which the early stages of smoke-induced tumorigenesis can be elicited, and should permit us to identify molecular changes driving this process. Results obtained so far indicate that smoke-induced lung tumors are driven by activation of two embryonic regulatory pathways, Hedgehog (Hh) and Wnt. Based on the current and emerging availability of drugs to inhibit Hh and Wnt signaling, it is possible that an understanding of the role of Hh and Wnt in lung cancer pathogenesis will lead to the development of new therapies.
Assuntos
Proteínas Hedgehog/metabolismo , Neoplasias Pulmonares/etiologia , Neoplasias Pulmonares/metabolismo , Fumar/efeitos adversos , Proteínas Wnt/metabolismo , Animais , Brônquios/metabolismo , Brônquios/patologia , Adesão Celular , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Proteínas Hedgehog/antagonistas & inibidores , Humanos , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Nus , Modelos Biológicos , Fenótipo , Transdução de Sinais/efeitos dos fármacos , Fumaça/efeitos adversos , Fumar/metabolismo , Sulindaco/farmacologia , Transplante Heterólogo , Alcaloides de Veratrum/farmacologia , Proteínas Wnt/antagonistas & inibidoresRESUMO
OBJECTIVE: Magainin II belongs to a family of antimicrobial peptides and has been shown to exhibit antibiotic activity in a wide range of organisms. Recent studies have also reported a significant antitumor effect of magainin II against various cancer cell lines and tumor mice models. In this study, we evaluated the cytotoxic and antiproliferative potency of magainin II in bladder tumor cells and normal fibroblasts. METHODS: The antiproliferative and cytotoxic effect of magainin II was quantified by colorimetric WST-1-, bromodeoxyuridine (BrdU)-, and lactic dehydrogenase (LDH) assays in three bladder cancer cell lines (RT4, 647V, and 486P) and in the murine fibroblast cell line 3T3 as well as in a primary culture from human fibroblasts. The median inhibitory concentration (IC50) values were determined for each assay, representing the concentration at which cell viability was reduced by 50%. Scanning electron microscopy (SEM) was used to visualize the morphologic effects of magainin II on bladder tumor cells and fibroblasts. RESULTS: Magainin II inhibited cell proliferation of bladder cancer cells in a dose-dependent manner. The average IC50 of magainin II against all bladder cancer cell lines was 198.1 microM (range, 52.4-484.03 microM) for the WST-1 assay and 75.2 microM (range, 31.0-135.3 microM) for the BrdU assay. The normal murine and human fibroblast cell lines were not affected by magainin II and their IC50 could not be determined at the concentrations of magainin II tested. LDH release was increased in all bladder tumor cell lines in the presence of magainin II, whereas normal fibroblasts showed no cell lysis. SEM demonstrated lethal membrane perforation by peptide pore formation in bladder cancer cells, but not in fibroblasts. CONCLUSION: Magainin II peptide exerts cytotoxic and antiproliferative efficacy by pore formation in bladder cancer cells but has no effect on normal murine or human fibroblasts. Magainin II may offer a novel therapeutic strategy in the treatment of bladder cancer with potentially low cytotoxic effects on normal cells.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Antineoplásicos/farmacologia , Neoplasias da Bexiga Urinária/patologia , Proteínas de Xenopus/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Magaininas , Microscopia Eletrônica de Varredura , Neoplasias da Bexiga Urinária/ultraestruturaRESUMO
Chronic inflammation in the airways is associated with dramatic architectural changes in the walls of the airways and in the vasculature they contain. In this study, we show that the adaptive immune system is essential for airway remodeling that occurs in mice that are chronically infected with the respiratory pathogen Mycoplasma pulmonis. Angiogenesis, lymphangiogenesis, and epithelial remodeling were greatly reduced in mice that lacked B cells. Substantiating a role for Ab and airway immune complexes, we found that the transfer of immune serum to B cell-deficient mice could reconstitute pathogen-induced angiogenesis. Inflammatory cells recruited to the infected airways were activated by the humoral response, and this activation correlated with the induction of genes for remodeling factors such as vascular endothelial growth factor-D. The results reveal a novel pathway whereby T cell-dependent humoral immunity to a persistent airway infection can induce inflammation-dependent angiogenesis, lymphangiogenesis, and chronic airway pathology.
Assuntos
Complexo Antígeno-Anticorpo/metabolismo , Infecções por Mycoplasma/imunologia , Infecções por Mycoplasma/patologia , Mycoplasma pulmonis , Sistema Respiratório/irrigação sanguínea , Sistema Respiratório/imunologia , Infecções Respiratórias/imunologia , Infecções Respiratórias/patologia , Animais , Anticorpos Antibacterianos/biossíntese , Linfócitos B/imunologia , Doença Crônica , Inflamação/etiologia , Inflamação/imunologia , Inflamação/patologia , Cinética , Linfangiogênese , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mycoplasma pulmonis/imunologia , Mycoplasma pulmonis/patogenicidade , Neovascularização Patológica , Sistema Respiratório/patologiaRESUMO
Transitional cell carcinoma of the urinary bladder remains life threatening due to the high occurrence of metastases. Emerging evidence suggests that chemokines and their receptors play a critical role in tumor metastases. In our study, we performed a systematic analysis of the mRNA and protein expression levels of all 18 chemokine receptors in normal urothelium and bladder cancer. CXCR4 was the only chemokine receptor whose mRNA expression level was upregulated in bladder cancer cell lines as well as in invasive and locally advanced bladder cancer tissue samples (pT2-pT4). In contrast, superficial bladder tumors (pTa and pT1) displayed low CXCR4 expression levels and normal urothelial cells were negative for CXCR4. Immunohistochemistry of a bladder cancer tissue microarray (TMA) confirmed that a subgroup of invasive bladder cancers revealed a high CXCR4 protein expression, while superficial bladder tumors showed low immunoreactivity. To investigate the functional significance of CXCR4 expression, we performed migration and invasion assays. Exposure of CXCR4-positive bladder cancer cells to CXCL12 in a Boyden chamber type assay provoked a significant increase in migration as well as invasion across a Matrigel barrier. Enhanced migration and invasion were inhibited by a CXCR4-specific blocking antibody. In contrast, normal urothelial cells did not respond to CXCL12 and lacked chemotactic migration. In conclusion, bladder cancer cells express CXCR4 progressively with advanced tumorigenesis and this receptor interacts with CXCL12 to mediate tumor chemotaxis and invasion through connective tissue. These properties identify CXCR4 as a potential target for the attenuation of bladder cancer metastases.
Assuntos
Receptores CXCR4/genética , Neoplasias da Bexiga Urinária/genética , Neoplasias da Mama , Linhagem Celular Tumoral , Movimento Celular , Progressão da Doença , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Análise de Sequência com Séries de Oligonucleotídeos , Osteossarcoma , Reação em Cadeia da Polimerase , Neoplasias da Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/fisiopatologia , Urotélio/citologia , Urotélio/patologiaRESUMO
Galectins form a family of structurally related carbohydrate binding proteins (lectins) that have been identified in a large variety of metazoan phyla. They are involved in many biological processes such as morphogenesis, control of cell death, immunological response, and cancer. To elucidate the evolutionary history of galectins and galectin-like proteins in chordates, we have exploited three independent lines of evidence: (i) location of galectin encoding genes (LGALS) in the human genome; (ii) exon-intron organization of galectin encoding genes; and (iii) sequence comparison of carbohydrate recognition domains (CRDs) of chordate galectins. Our results suggest that a duplication of a mono-CRD galectin gene gave rise to an original bi-CRD galectin gene, before or early in chordate evolution. The N-terminal and C-terminal CRDs of this original galectin subsequently diverged into two different subtypes, defined by exon-intron structure (F4-CRD and F3-CRD). We show that all vertebrate mono-CRD galectins known to date belong to either the F3- or F4- subtype. A sequence of duplication and divergence events of the different galectins in chordates is proposed.
Assuntos
Galectinas/classificação , Galectinas/genética , Filogenia , Vertebrados/genética , Animais , Cromossomos/genética , Evolução Molecular , HumanosRESUMO
EMMPRIN is a transmembrane glycoprotein expressed at high levels by tumor cells. It has been identified as a tumor-derived factor that can stimulate matrix metalloproteinase expression in fibroblasts and hence facilitate tumor invasion and metastasis. Recent studies have shown that full-length EMMPRIN is released by tumor cells, but the mechanism of release remains unclear. Here, we show that EMMPRIN is released from the surface of NCI-H460 cells via microvesicle shedding. However, these vesicles are unstable and rapidly break down to release bioactive EMMPRIN. Although microvesicle shedding has been considered a constitutive process in tumor cells, our data show that it can be amplified upon cell exposure to PMA, elucidating at least one signalling cascade responsible for EMMPRIN release. This pathway is dependent on protein kinase C, calcium mobilization and mitogen-activated protein kinase kinase (MEK 1/2). Thus, the results outline a novel form of tumor-stromal interaction in which extracellular matrix degradation by fibroblasts is controlled through the microvesicular release of EMMPRIN from tumor cells.
Assuntos
Antígenos CD , Antígenos de Neoplasias , Glicoproteínas de Membrana/fisiologia , Células Estromais/citologia , Basigina , Linhagem Celular Tumoral , Meios de Cultivo Condicionados , Humanos , Imuno-Histoquímica , Metaloproteinase 1 da Matriz/genética , Microscopia Eletrônica , Transcrição GênicaRESUMO
The time course for delivery and transport of two major proteins of herpes simplex virus (HSV) has been determined for mature mouse retinal ganglion cell axons in vivo. Twenty-four hours after intravitreal injection of HSV, valacyclovir was introduced into the drinking water of the mice to inhibit subsequent viral replication. Without treatment, viral spread and replication in periaxonal glial cells confound study of axonal transport. At 2 to 5 days after infection, the animals were sacrificed and contiguous segments of the optic pathway were removed. Immunofluorescence microscopy indicated that the number of infected astrocytes was reduced in the proximal optic nerve and eliminated in the optic tract. Western blots of the retina with antibodies for envelope and capsid components, glycoprotein D (gD) and VP5, respectively, revealed that both components were expressed in retinal homogenates by 2 days. Results of reverse transcription-PCR indicated that there was no gD mRNA present in the treated optic tract 5 days after infection. Therefore, we conclude that gD is transcribed from viral mRNA in the retinal ganglion cell bodies. The gD accumulated in the proximal ganglion cell axon by 2 days and reached the most distal segment after 3 days. The VP5 first appeared in the proximal axons at 4 days, about 48 h after the appearance of gD. Thus, gD entered the axon earlier and independent of VP5. These finding confirm the subassembly model of viral transport in neurons and suggest that there is a 4- to 5-day window for initiation of effective antiviral treatment with valacyclovir.