RESUMO
BACKGROUND: The receptor tyrosine kinase c-KIT plays a key role in normal mast cell development. Point mutations in c-KIT have been associated with sporadic or familial mastocytosis. OBJECTIVES: Two unrelated pairs of apparently identical twins affected by cutaneous mastocytosis attending the Mastocytosis Clinic at the Royal Children's Hospital, Melbourne, provided an opportunity to assess the possible contribution of c-KIT germline mutations or polymorphisms in this disease. METHODS: Tissue biopsy, blood and/or buccal swab specimens were collected from 10 children with mastocytosis. To detect germline mutations/polymorphisms in c-KIT, we studied all coding exons by denaturing high pressure liquid chromatography. Exons showing mismatches were examined by direct sequencing. The influence of the substitution identified was further examined by expressing the variant form of c-KIT in factor-dependent FDC-P1 cells. RESULTS: In both pairs of twins, a heterozygous ATG to CTG transition in codon 541 was observed, resulting in the substitution of a methionine residue in the transmembrane domain by leucine (M541L). In each case, one parent was also heterozygous for this allele. Expression of M541L KIT in FDC-P1 cells enabled them to grow in human KIT ligand (stem cell factor, SCF) but did not confer factor independence. Compared with cells expressing wild-type KIT at a similar level, M541L KIT-expressing cells displayed enhanced growth at low levels of SCF, and heightened sensitivity to the KIT inhibitor, imatinib mesylate. CONCLUSIONS: The data suggest that the single nucleotide polymorphism resulting in the substitution M541L may predispose to paediatric mastocytosis.
Assuntos
Substituição de Aminoácidos , Doenças em Gêmeos/genética , Mastocitose/genética , Polimorfismo Genético , Proteínas Proto-Oncogênicas c-kit/genética , Adolescente , Benzamidas , Proliferação de Células , Criança , Pré-Escolar , Éxons/genética , Feminino , Humanos , Mesilato de Imatinib , Lactente , Masculino , Mastocitose/metabolismo , Piperazinas/farmacologia , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas c-kit/metabolismo , Pirimidinas/farmacologia , Gêmeos MonozigóticosRESUMO
Small molecule protein kinase inhibitors show great promise as anti-cancer agents, however, de novo and acquired resistance present problems. These are reviewed and illustrated using the receptor tyrosine kinase, KIT, as an example. Emerging solutions are presented, such as targeting active kinase conformations.
Assuntos
Antineoplásicos/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Quinases/efeitos dos fármacos , Antineoplásicos/farmacologia , Desenho de Fármacos , Humanos , Estrutura Molecular , Neoplasias/irrigação sanguínea , Neovascularização Patológica/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/química , Relação Estrutura-AtividadeRESUMO
CD151, a member of the tetraspanin family of cell membrane proteins, is widely expressed in epithelial, endothelial and muscle cells as well as platelets and megakaryocytes. Several monoclonal antibodies recognising CD151 in transfected cells and immunoprecipitating typical bands of 28 and 32 kDa from cell lysates have been produced. Surprisingly, these antibodies show different patterns of staining on tissue sections and on haemopoietic cells. Here we show that these differences are at least in part due to masking of certain epitopes in integrin/CD151 complexes. These data have important implications for the use of monoclonal antibodies in studies of the distribution and function of CD151. Of six monoclonal antibodies from four laboratories, 11B1 was found to be the most reliable for detection of CD151 in different cell and tissue contexts.
Assuntos
Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Antígenos CD/imunologia , Antígenos CD/metabolismo , Epitopos/metabolismo , Animais , Antígenos CD/genética , Western Blotting , Células da Medula Óssea/metabolismo , Linhagem Celular , Células Cultivadas , Epitélio/metabolismo , Epitopos/imunologia , Imunofluorescência , Humanos , Técnicas Imunoenzimáticas , Integrina alfa3 , Integrina alfa6 , Integrinas/metabolismo , Substâncias Macromoleculares , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Músculos/metabolismo , Testes de Precipitina , Tetraspanina 24 , TransfecçãoRESUMO
Activating mutations in c-Kit, the receptor for Stem Cell Factor (SCF), have been identified in dysplasias and leukaemias of the mast cell lineage and have been shown to contribute to transformation in model systems. Early myeloid cells also normally express c-Kit and their survival, proliferation and differentiation is promoted by SCF. It might therefore be expected that c-Kit mutations could also be involved in some acute and/or chronic myeloid leukaemias. We have found that mutant c-Kit (and normal c-Kit in the presence of SCF) provides a strong differentiation stimulus in normal and immortalised murine early myeloid cells. Since maturation of haemopoietic cells, with the exception of mast cells, results in down-regulation of c-Kit expression, the transforming effects of mutant receptor may be self-limiting in most lineages. This is consistent with the observation that multipotential progenitor cells from some patients with systemic mastocytosis express mutant c-Kit. However, c-Kit mutations have been observed in a few cases of myelodysplastic syndromes or AML without mast cell features. Oncogenesis involves multiple genetic changes and the phenotype of malignant haemopoietic cells expressing mutant c-Kit may be influenced by co-oncogenic events. For example mutations blocking the differentiative effect of mutant c-Kit might result in AML rather than mastocytosis. Thus the extent to which c-Kit mutations contribute to malignancies of early myeloid phenotype remains unknown, and resolution of this issue is complicated by the heterogeneity of this family of diseases.
Assuntos
Transformação Celular Neoplásica/genética , Neoplasias Hematológicas/genética , Hematopoese/genética , Proteínas Proto-Oncogênicas c-kit/genética , Animais , Humanos , Leucemia de Mastócitos/genética , Leucemia Mieloide/genética , Camundongos , Mutação , Transtornos Mieloproliferativos/genética , Proteínas Proto-Oncogênicas c-kit/fisiologiaRESUMO
The receptor tyrosine kinase c-Kit and its ligand Stem Cell Factor (SCF) are essential for haemopoiesis, melanogenesis and fertility. SCF acts at multiple levels of the haemopoietic hierarchy to promote cell survival, proliferation, differentiation, adhesion and functional activation. It is of particular importance in the mast cell and erythroid lineages, but also acts on multipotential stem and progenitor cells, megakaryocytes, and a subset of lymphoid progenitors. SCF exists in soluble or transmembrane forms which appear to differ in function. Multiple isoforms of c-Kit also exist as a result of alternate mRNA splicing, proteolytic cleavage and the use of cryptic internal promoters in certain cell types. This review focuses on what is known about the regulation of c-Kit expression, the functions of SCF and c-Kit isoforms, and the nature of the biological responses elicited by this receptor-ligand pair with emphasis on the haemopoietic system.
Assuntos
Proteínas Proto-Oncogênicas c-kit/fisiologia , Fator de Células-Tronco/fisiologia , Animais , Adesão Celular , Diferenciação Celular , Divisão Celular , Movimento Celular , Sobrevivência Celular , Transformação Celular Neoplásica , Regulação da Expressão Gênica , Humanos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiologia , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Fator de Células-Tronco/genética , Fator de Células-Tronco/metabolismoRESUMO
Alternate splicing of mRNA encoding c-KIT results in isoforms which differ in the presence or absence of four amino acids (GNNK) in the juxtamembrane region of the extracellular domain of the receptor. In this study we show that these isoforms of human c-KIT, expressed at similar levels in NIH3T3 cells, display differential effects on various attributes of transformation. The GNNK- isoform strongly promoted anchorage independent growth (colony formation in semi-solid medium), loss of contact inhibition (focus formation), and led to tumorigenicity in nude mice. In contrast, the GNNK+ isoform elicited colony formation but relatively poor focus formation and no tumorigenicity. Saturation binding analysis indicated that the isoforms do not differ significantly in their affinity for the KIT ligand, Steel Factor (SLF). Negligible ligand-independent receptor phosphorylation was observed in either case but, after ligand stimulation, the GNNK- isoform displayed more rapid and extensive tyrosine autophosphorylation and faster internalization. Both isoforms recruited the p85 subunit of phosphatidylinositol 3-kinase and led to similar phosphorylation of its downstream effector c-Akt, but the GNNK- isoform gave rise to more MAP kinase phosphorylation. Thus the c-KIT isoforms display different signalling characteristics and have different transforming activity in NIH3T3 cells.
Assuntos
Transformação Celular Neoplásica/metabolismo , Isoformas de Proteínas/fisiologia , Proteínas Proto-Oncogênicas c-kit/fisiologia , Splicing de RNA , Transdução de Sinais/fisiologia , Células 3T3/patologia , Células 3T3/transplante , Sequência de Aminoácidos , Animais , Adesão Celular , DNA Complementar/genética , Humanos , Camundongos , Camundongos Nus , Fosfatidilinositol 3-Quinases/fisiologia , Isoformas de Proteínas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/fisiologia , Fator de Células-Tronco/fisiologia , Transfecção , Ensaio Tumoral de Célula-TroncoRESUMO
Activating mutations in c-Kit, the receptor for Stem Cell Factor (SCF), have been identified in dysplasias and leukaemias of the mast cell lineage and have been shown to contribute to transformation in model systems. Early myeloid cells also normally express c-Kit and their survival, proliferation and differentiation is promoted by SCE It might therefore be expected that c-Kit mutations could also be involved in some acute and/or chronic myeloid leukaemias. We have found that mutant c-Kit (and normal c-Kit in the presence of SCF) provides a strong differentiation stimulus in normal and immortalised murine early myeloid cells. Since maturation of haemopoietic cells, with the exception of mast cells, results in down-regulation of c-Kit expression, the transforming effects of mutant receptor may be self-limiting in most lineages. This is consistent with the observation that multipotential progenitor cells from some patients with systemic mastocytosis express mutant c-Kit. However, c-Kit mutations have been observed in a few cases of myelodysplastic syndromes or AML without mast cell features. Oncogenesis involves multiple genetic changes and the phenotype of malignant haemopoietic cells expressing mutant c-Kit may be influenced by co-oncogenic events. For example mutations blocking the differentiative effect of mutant c-Kit might result in AML rather than mastocytosis. Thus the extent to which c-Kit mutations contribute to malignancies of early myeloid phenotype remains unknown, and resolution of this issue is complicated by the heterogeneity of this family of diseases.
Assuntos
Neoplasias Hematológicas/genética , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/fisiologia , Doença Aguda , Animais , Transformação Celular Neoplásica/genética , Hematopoese/genética , Células-Tronco Hematopoéticas/citologia , Humanos , Leucemia de Mastócitos/genética , Leucemia Mieloide/genética , Leucemia Mieloide/patologia , Camundongos , Mutação , Transtornos Mieloproliferativos/genéticaRESUMO
The Transmembrane 4 Superfamily member, PETA-3/CD151, is ubiquitously expressed by endothelial cells in vivo. In cultured human umbilical vein endothelial cells PETA-3 is present on the plasma membrane and predominantly localises to regions of cell-cell contact. Additionally, this protein is abundant within an intracellular compartment which accounts for up to 66% of the total PETA-3 expressed. Intracellular PETA-3 showed colocalisation with transferrin receptor and CD63 suggesting an endosomal/lysosomal localisation which was supported by immuno-electronmicroscopy studies. Co-immunoprecipitation experiments investigating possible interactions of PETA-3 with other molecules demonstrated associations with several integrin chains including beta1, beta3, beta4, (alpha)2, (alpha)3, (alpha)5, (alpha)6 and provide the first report of Transmembrane 4 Superfamily association with the (alpha)6beta4 integrin. Using 2-colour confocal microscopy, we demonstrated similar localisation of PETA-3 and integrin chains within cytoplasmic vesicles and endothelial cell junctions. In order to assess the functional implications of PETA-3/integrin associations, the effect of anti-PETA-3 antibodies on endothelial function was examined. Anti-PETA-3 mAb inhibited endothelial cell migration and modulated in vitro angiogenesis, but had no detectable effect on neutrophil transendothelial migration. The broad range of integrin associations and the presence of PETA-3 with integrins both on the plasma membrane and within intracellular vesicles, suggests a primary role for PETA-3 in regulating integrin trafficking and/or function.
Assuntos
Antígenos CD/análise , Membrana Celular/ultraestrutura , Endotélio Vascular/citologia , Antígenos CD/fisiologia , Membrana Celular/fisiologia , Movimento Celular , Células Cultivadas , Endocitose , Endossomos/ultraestrutura , Endotélio Vascular/fisiologia , Endotélio Vascular/ultraestrutura , Humanos , Integrinas/análise , Junções Intercelulares/fisiologia , Junções Intercelulares/ultraestrutura , Microscopia Confocal , Microscopia Imunoeletrônica , Neovascularização Fisiológica , Glicoproteínas da Membrana de Plaquetas/análise , Receptores da Transferrina/análise , Tetraspanina 24 , Tetraspanina 30 , Veias UmbilicaisRESUMO
CD151 (PETA-3/SFA-1) is a member of the transmembrane 4 superfamily (TM4SF) of cell-surface proteins and is expressed abundantly both on the cell surface and in intracellular membranes by the haemopoietic cell lines M07e, HEL and K562. In the presence of mild detergent (CHAPS), CD151 co-immunoprecipitated with integrin alpha 4 beta 1, alpha 5 beta 1, alpha 6 beta 1 and alpha IIb beta 3. The association of CD151 with alpha 4 beta 1 and alpha 5 beta 1 seemed to be constitutive, as it was not modified by treatment of M07e cells with cytokines that regulate integrin function by 'inside-out' signalling. CD151 also associated with other tetraspans in an apparently cell-type-specific fashion, as defined by its co-precipitation with CD9, CD63 and CD81 from M07e cells, but not from K562 cells, which express similar levels of these proteins. F(ab')2 fragments of monoclonal antibodies (mAbs) against CD151 caused homotypic adhesion of HEL and K562 cells that was dependent on energy and cytoskeletal integrity and was augmented in the presence of RGDS peptides. The adhesion was not blocked by function-inhibiting mAbs against beta 1 or beta 3 integrins, suggesting that cell-cell adhesion was not mediated by the binding of integrin to a cell-associated ligand. Furthermore, mAb CD151 did not affect adhesion of the cells to fibronectin, laminin, collagen or fibrinogen, which are ligands for alpha 4 beta 1, alpha 5 beta 1, alpha 6 beta 1 and alpha IIb beta 3 integrins. Taken together, these results indicate that the ligation of CD151 does not induce the up-regulation of integrin avidity, but might act as a component of integrin signalling complexes.
Assuntos
Antígenos CD/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Integrina beta1/metabolismo , Proteínas de Membrana/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Anticorpos Monoclonais/farmacologia , Antígenos CD/biossíntese , Antígenos CD/imunologia , Antígenos CD/fisiologia , Sítios de Ligação , Adesão Celular/imunologia , Membrana Celular/metabolismo , Precipitação Química , Citoplasma/metabolismo , Detergentes , Matriz Extracelular/metabolismo , Humanos , Integrina beta1/biossíntese , Células K562 , Substâncias Macromoleculares , Proteínas de Membrana/biossíntese , Proteínas de Membrana/imunologia , Proteínas de Membrana/fisiologia , Tetraspanina 24 , Células Tumorais CultivadasRESUMO
We show a dramatic downregulation of the stem cell factor (SCF) receptor in different hematopoietic cell lines by murine stroma. Growth of the human erythroid/macrophage progenitor cell line TF-1 is dependent on granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3). However, TF-1 cells clone and proliferate equally well on stroma. Independent stroma-dependent TF-1 clones (TF-1S) were generated on MS-5 stroma. Growth of TF-1S and TF-1 cells on stroma still requires interaction between c-kit (SCF receptor) and its ligand SCF, because antibodies against c-kit inhibit growth to less than 2%. Surprisingly, c-kit receptor expression (RNA and protein) was downregulated by 2 to 3 orders of magnitude in TF-1S and TF-1 cells grown on stroma. This stroma-dependent regulation of the kit receptor in TF-1 was also observed on exposure to kit ligand-negative stroma, thus indicating the need for heterologous receptor ligand interaction. Removal of stroma induced upregulation by 2 to 4 orders of magnitude. Downregulation and upregulation of c-kit expression could also be shown for the megakaryocytic progenitor cell line M-07e and was comparable to that of TF-1, indicating that stroma-dependent regulation of c-kit is a general mechanism. Downregulation may be an economic way to compensate for the increased sensitivity of the c-kit/ligand interaction on stroma. The stroma-dependent c-kit regulation most likely occurs at the transcriptional level, because mechanisms, such as splicing, attenuation, differential promoter usage, or mRNA stability, could be excluded.
Assuntos
Regulação para Baixo , Regulação da Expressão Gênica , Células-Tronco Hematopoéticas/metabolismo , Proteínas Proto-Oncogênicas c-kit/genética , Células Estromais/fisiologia , Animais , Sequência de Bases , Northern Blotting , Divisão Celular , Linhagem Celular , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Interleucina-3/farmacologia , Camundongos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas c-kit/metabolismo , RNA Mensageiro/metabolismo , Proteínas Recombinantes , Fator de Células-Tronco/metabolismo , Transcrição GênicaRESUMO
Cell-to-cell junction structures play a key role in cell growth rate control and cell polarization. In endothelial cells (EC), these structures are also involved in regulation of vascular permeability and leukocyte extravasation. To identify novel components in EC intercellular junctions, mAbs against these cells were produced and selected using a morphological screening by immunofluorescence microscopy. Two novel mAbs, LIA1/1 and VJ1/16, specifically recognized a 25-kD protein that was selectively localized at cell-cell junctions of EC, both in the primary formation of cell monolayers and when EC reorganized in the process of wound healing. This antigen corresponded to the recently cloned platelet-endothelial tetraspan antigen CD151/PETA-3 (platelet-endothelial tetraspan antigen-3), and was consistently detected at EC cell-cell contact sites. In addition to CD151/PETA-3, two other members of the tetraspan superfamily, CD9 and CD81/ TAPA-1 (target of antiproliferative antibody-1), localized at endothelial cell-to-cell junctions. Biochemical analysis demonstrated molecular associations among tetraspan molecules themselves and those of CD151/ PETA-3 and CD9 with alpha3 beta1 integrin. Interestingly, mAbs directed to both CD151/PETA-3 and CD81/ TAPA-1 as well as mAb specific for alpha3 integrin, were able to inhibit the migration of ECs in the process of wound healing. The engagement of CD151/PETA-3 and CD81/TAPA-1 inhibited the movement of individual ECs, as determined by quantitative time-lapse video microscopy studies. Furthermore, mAbs against the CD151/PETA-3 molecule diminished the rate of EC invasion into collagen gels. In addition, these mAbs were able to increase the adhesion of EC to extracellular matrix proteins. Together these results indicate that CD81/TAPA-1 and CD151/PETA-3 tetraspan molecules are components of the endothelial lateral junctions implicated in the regulation of cell motility, either directly or by modulation of the function of the associated integrin heterodimers.
Assuntos
Antígenos CD/fisiologia , Movimento Celular/fisiologia , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Integrinas/fisiologia , Junções Intercelulares/fisiologia , Glicoproteínas de Membrana , Proteínas de Membrana , Animais , Anticorpos Monoclonais/metabolismo , Antígenos CD/metabolismo , Linhagem Celular , Células Cultivadas , Colágeno , Matriz Extracelular , Géis , Humanos , Integrina alfa3beta1 , Camundongos , Camundongos Endogâmicos BALB C , Tetraspanina 24 , Tetraspanina 28 , Tetraspanina 29RESUMO
By using reverse transcription-PCR, in situ hybridization, enzyme-linked immunosorbent assay and immunocytochemistry, we have studied the production of granulocyte-macrophage colony-stimulating factor (GM-CSF) in human lung mast cells induced by cross-linkage of high-affinity Fc epsilon receptors (Fc epsilonRI). We have also confirmed the bioactivity of GM-CSF released from lung mast cells by investigating the effect of the supernatant from lung mast cells activated with anti-IgE on the release of eosinophil cationic protein (ECP) from eosinophils. Mast cells were purified using affinity magnetic selection with monoclonal antibody (mAb) YB5.B8 (93-99% pure). Purified mast cells were precultured with IgE for 16 h before challenge with 1 microg/ml anti-IgE with or without stem cell factor (SCF). Eosinophils were purified by immunomagnetic negative selection (> 98% pure). The activation of mast cells via Fc epsilonRI enhanced the intensity of the GM-CSF signal within 2 h and the cells produced GM-CSF protein 4 h after the activation. In the absence of recombinant human (rh) SCF, anti-IgE induced a median GM-CSF response of 202 (< 15 to approximately 681) pg/10(6) mast cells/24 h, whereas in the presence of rhSCF the median IgE-dependent GM-CSF release was 356 (152 to approximately 1216) pg/10(6) mast cells/24 h. This difference was statistically significant (p = 0.0029, n = 12). In contrast, mast cells produced only a small amount of GM-CSF in the absence of anti-IgE. The mast cell supernatant induced ECP release from eosinophils and the release was significantly inhibited by blocking mAb against GM-CSF. These findings indicate that human mast cells are an important cellular source of GM-CSF and as such may contribute to chronic eosinophil-mediated inflammation.
Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos/biossíntese , Imunoglobulina E/fisiologia , Pulmão/imunologia , Pulmão/metabolismo , Mastócitos/metabolismo , Ribonucleases , Proteínas Sanguíneas/metabolismo , Sistema Livre de Células/fisiologia , Proteínas Granulares de Eosinófilos , Eosinófilos/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Imunoglobulina E/metabolismo , Cinética , Pulmão/citologia , Mastócitos/imunologia , RNA Mensageiro/biossíntese , Receptores de IgE/fisiologiaRESUMO
Ectopic expression of the normal murine receptor tyrosine kinase, c-Kit, in NIH3T3 cells induced many phenotypic changes characteristic of transformation including anchorage-independent growth, focus formation and tumorigenicity in nude mice. Although transformation was largely dependent on the presence of recombinant murine Steel Factor (SLF), the ligand to the c-Kit receptor, anchorage independent growth did occur at a low frequency in the absence of added factor, and this could not be inhibited by neutralising antibodies or by SLF anti-sense mRNA. Clones from factor-independent colonies in semi-solid agar displayed a narrow range of c-Kit surface protein levels (4.3-6.4 x 10(4) receptors/cell) which was relatively high compared with the pool from which they were derived. Analysis of a larger series of random clones derived from adherent cultures expressing different levels of c-Kit demonstrated a positive correlation between SLF-dependent, anchorage-independent growth and c-Kit protein and mRNA expression levels (respectively, Rs = 0.58, P < 0.01; and Rs = 0.53, P < 0.01) with consistent colony formation observed with clones having > 2.5 x 10(4) receptors/cell. Interestingly, two of the three clones expressing the highest levels of c-Kit protein and mRNA produced few or no colonies in the presence or absence of SLF. Sequential overexpression of human c-KIT in NIH3T3 cells using a dihydrofolate reductase (DHFR)-encoding vector and gene co-amplification through methotrexate selection, which resulted in pools expressing up to 1.5 x 10(5) receptors/cell, confirmed that high receptor densities resulted in a decrease in colony numbers. Thus, analysis of clonal and selected populations has indicated that an optimal level of c-Kit is required for transformation of NIH3T3 cells in the presence of SLF, and that some ligand-independent transformation occurs.
Assuntos
Transformação Celular Neoplásica , Proteínas Proto-Oncogênicas c-kit/fisiologia , Células 3T3 , Animais , Divisão Celular , Ligantes , Metotrexato/farmacologia , Camundongos , Proteínas Proto-Oncogênicas c-kit/análiseRESUMO
The cDNAs encoding wild type (WT) human receptor tyrosine kinase c-Kit and a constitutively activated mutant, V816Kit, were introduced into granulocyte-macrophage colony-stimulating factor (GM-CSF )-dependent early murine hemopoietic cells, which had been transformed with activated Myb. WTKit cells were able to grow in the presence of the human ligand for Kit, stem cell factor (SCF ), but displayed reduced growth and clonogenic potential in either SCF or GM-CSF compared with the parental cells in GM-CSF. In contrast, V816Kit cells grew without factor at a higher rate than the parental cells in GM-CSF and displayed increased clonogenicity. Dissection of the growth characteristics in liquid culture showed that in the presence of appropriate factors, the different populations had similar proliferation rates, but that V816Kit profoundly increased cell survival compared with WTKit or parental cells. This suggests that the signals transduced by WTKit activated with SCF, and by V816Kit, were not identical. Also, WTKit and V816Kit-expressing cells both varied from the early myeloid progenitor phenotype of the parental cells and gave rise to a small number of large to giant adherent cells that expressed macrophage (alpha-naphthyl acetate) esterase and neutrophil (naphtol-AS-D-chloroacetate) esterase, were highly phagocytic and phenotypically resembled histiocytes. Thus, WTKit activated by SCF and V816Kit were able to induce differentiation in a proportion of Myb-transformed myeloid cells. The factor independent V816Kit cells, unlike the parental and WTKit expressing cells, were shown to produce tumors of highly mitotic, invasive cells at various stages of differentiation in syngeneic mice. These results imply that constitutively activated Kit can promote the development of differentiated myeloid tumors and that its oncogenic effects are not restricted to lineages (mast cell and B-cell acute lymphoblastic leukemia), which have been reported previously. Furthermore, the mixed populations of cells in culture and in the tumors phenotypically resembled the leukemic cells from patients with monocytic leukemia with histiocytic differentiation (acute myeloid leukemia-M5c), a newly proposed subtype of myeloid leukemia.
Assuntos
Transformação Celular Neoplásica/genética , Células-Tronco Hematopoéticas/patologia , Histiócitos/citologia , Leucemia Monocítica Aguda/patologia , Oncogenes , Proteínas Proto-Oncogênicas c-kit/biossíntese , Células 3T3 , Animais , Adesão Celular , Diferenciação Celular , Células Clonais , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Células-Tronco Hematopoéticas/citologia , Humanos , Leucemia Monocítica Aguda/genética , Camundongos , Proteínas Proto-Oncogênicas c-kit/genéticaRESUMO
P-glycoprotein (Pgp), the major mediator of multidrug resistance (MDR) has often been implicated as a poor prognostic indicator in acute myeloid leukaemia (AML). We have previously reported that high expression of the receptor tyrosine kinase c-Kit in AML is associated with poor prognosis. To determine whether the MDR phenotype is associated with high c-Kit expression, the monoclonal antibodies UIC-2 and YB5.B8, which identify Pgp and c-Kit, respectively, were used for indirect immunofluorescence labelling of 50 de novo AML specimens. Quantitative dye efflux studies using Rhodamine123 were also carried out to assess the functional drug efflux capability of these samples. Pgp expression by the majority of primary AML was comparable to that seen in subsets of cells from normal bone marrow and Spearman rank analysis showed no relationship with c-Kit expression (rs = 0.20, P = 0.16). However, c-Kit expression did show a significant correlation with Rhodamine123 efflux (rs = 0.57, P = 0.0001), suggesting that the MDR phenotype, Pgp mediated or other, may contribute to the prognostic significance of high c-Kit expression. The monoclonal antibody UIC-2 was used specifically to block Pgp activity of a limited number of leukaemic specimens and cell lines, and evidence of non-Pgp-mediated efflux was found. The existence of alternative mechanisms may explain the relatively low correlation of Pgp expression with dye efflux within the leukaemic samples (rs = 0.47, P = 0.0006) and has implications for prognosis in AML. The c-Kit ligand, stem cell factor, did not influence drug efflux activity of the nine c-Kit-positive AML specimens tested. Thus the correlation between c-Kit and the MDR phenotype in AML is likely to be a consequence of co-expression at a similar stage of differentiation, and may account for the previously observed association of high c-Kit expression with poor outcome.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Resistência a Múltiplos Medicamentos , Leucemia Mieloide/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/efeitos dos fármacos , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Doença Aguda , Antimetabólitos Antineoplásicos/metabolismo , Antimetabólitos Antineoplásicos/farmacologia , Ciclosporina/farmacologia , Daunorrubicina/metabolismo , Daunorrubicina/farmacologia , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Imunossupressores/farmacologia , Leucemia Mieloide/genética , Fenótipo , Proteínas Proto-Oncogênicas c-kit/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-kit/genética , Rodamina 123 , Rodaminas/metabolismo , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
Here we show that the supernatant from activated lung mast cells induced the release of eosinophil cationic protein (ECP) from eosinophils. Lung mast cells were purified using affinity magnetic selection with monoclonal antibody (mAb) YB5.B8 to achieve a final mast cell purity of 93-99%. Eosinophils were purified by immunomagnetic negative selection (>98.0% pure). The supernatant was obtained from lung mast cells activated for 24 h with 1 microg/ml anti-IgE and 50 ng/ml stem cell factor (SCF). Human eosinophils were incubated with various concentrations of the supernatants for 4 h and ECP released was measured by RIA. Using 4 different donors' supernatant from mast cells, each donor's supernatant caused a dose-dependent release of ECP from eosinophils. The dilutant of 1:2 (v/v) of the supernatant induced 657.5 +/- 55.6 ng/10(6) eosinophils of ECP which is statistically significant (p = 0.008, n = 4) compared with the culture medium alone. Anti-interleukin (IL-5 neutralizing mAb, 10 microg/ml, and anti-tumor necrosis factor-alpha (TNF alpha) neutralizing mAb, 10 microg/ml, significantly inhibited the supernatant-induced ECP release in 79.3 +/- 9.4 and 68.2 +/- 14.1% (mean +/- SEM, n = 6, p < 0.005), respectively. Anti-granulocyte/macrophage colony-stimulating factor (GM-CSF) neutralizing mAb, 50 microg/ml, caused 68.0 +/- 6.1% of inhibition (p = 0.002). The isotype negative control had no measurable inhibitory or stimulatory effect for the stimuli. We confirmed that mast cells produce IL-5, GM-CSF and TNF alpha in response to IgE-dependent stimulation by using RT-PCR, in situ hybridization, ELISA and immunocytochemistry. A million of lung mast cells generated 41.4 pg (7.0-273.6) (median with range) of TNF alpha, 252.6 pg (158.7-3,652) of GM-CSF and 735 pg (< 10-2,750) of IL-5 24 h after activation with SCF and anti-IgE. These findings indicate that the human mast cells may contribute to the chronicity of tissue inflammation.
Assuntos
Proteínas Sanguíneas/metabolismo , Citocinas/metabolismo , Eosinófilos/imunologia , Mastócitos/imunologia , Ribonucleases , Comunicação Celular , Células Cultivadas , Proteínas Granulares de Eosinófilos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Interleucina-5/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
A quantitative assay of hepatitis B virus (HBV) binding to hepatocyte plasma membranes was adapted to show that leucocyte plasma membranes bind serum-derived HBV saturably, and that this binding is inhibited using synthetic peptides representative of the large envelope protein of HBV. Using a panel of ligand-blocking monoclonal antibodies (mAb) to opsonin receptors, it was shown that the three classes of Fc gamma R and CR3 are not major receptors for HBV on leucocytes or hepatocytes. It was also shown that HBV does not utilize the receptor for IgA, Fc alpha R, for attachment to leucocytes, despite reported sequence homology between the large envelope protein of HBV and the Fc portion of human IgA. Evidence is presented that the receptor for HBV on leucocytes may differ from the hepatocyte receptor(s), based on synthetic peptide inhibition assays of HBV binding. Furthermore, it was observed that glycosaminoglycans influence the HBV-liver and leucocyte interactions, providing evidence that HBV attachment may be a multi-stage process.
Assuntos
Antígenos de Superfície da Hepatite B/metabolismo , Vírus da Hepatite B/metabolismo , Leucócitos/virologia , Fígado/virologia , Precursores de Proteínas/metabolismo , Receptores Virais/metabolismo , Proteínas do Envelope Viral/metabolismo , Adulto , Anticorpos Bloqueadores , Anticorpos Monoclonais , Sítios de Ligação , Membrana Celular/virologia , Glicosaminoglicanos/metabolismo , Vírus da Hepatite B/patogenicidade , Humanos , Técnicas In Vitro , Fígado/citologia , Receptores Imunológicos/antagonistas & inibidores , Receptores Imunológicos/metabolismoRESUMO
It has recently been shown that several members of the tetraspan superfamily, including CD9 and CD63, associate with each other and with beta1 integrins. In this study, we examined the distribution of a recently identified tetraspan, PETA-3 (CD151), and of CD9, CD63, alpha5beta1, and the integrin beta1 chain in normal human tissues by the indirect immunoperoxidase and alkaline phosphatase-anti-alkaline phosphatase techniques. PETA-3 showed a broad distribution and was expressed by endothelium, epithelium, Schwann cells, and dendritic cells and by skeletal, smooth, and cardiac muscle. Expression in skin was mostly restricted to the basal cells of the epidermis and was downregulated on differentiation. In the small intestine, PETA-3 was expressed by crypt and villous enterocytes with a mostly basolateral distribution, but was not detectable on the brush border. CD9 was expressed on the plasma membrane of enterocytes in crypts and at the bases of the villi whereas CD63 demonstrated a unique granular appearance concentrated in the apical cytoplasm below the brush border. The findings of this study show co-localization of PETA-3 with CD9, CD63, alpha5beta1, and beta1 in particular tissues, demonstrating that tetraspan/integrin complexes may occur. However, the lack of co-localization of these antigens in other tissues also implies distinct roles for these molecules.
Assuntos
Antígenos CD/metabolismo , Glicoproteínas de Membrana , Proteínas de Membrana/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Receptores de Fibronectina/metabolismo , Adulto , Humanos , Imuno-Histoquímica , Tetraspanina 29 , Tetraspanina 30RESUMO
Three monoclonal antibodies (Mabs), 7H6, 4B10 and Genzyme Mab, and a commercially-available polyclonal antiserum (Genzyme) to human Stem Cell Factor (SCF) were compared for their ability to detect native and recombinant SCF in a variety of assays, and for blocking of SCF function. All antibodies were found to bind to the membrane bound isoform as well as soluble SCF and to bind to both glycosylated (yeast MGF) and unglycosylated (E. coli SCF) recombinant factor. Mabs 7H6 and 4B10, as well as the polyclonal antiserum could immunoprecipitate membrane-associated SCF and all the antibodies could detect recombinant soluble SCF on western blots, although the binding of all except 7H6 was partially sensitive to reduction. Titration of the antibodies on CHO cells expressing membrane-associated human SCF showed similar dose-dependence for all Mabs with 70% of maximum binding seen at 3, 5 and 8 micrograms/ml for 7H6, 4B10 and Genzyme Mab respectively, however the maximum binding seen with 7H6 was approximately 2-fold greater than with 4B10 and 7-fold greater than Genzyme Mab. Competitive binding experiments of the Mabs on cells expressing membrane SCF gave non-reciprocal blocking in all cases with 7H6 completely blocking 4B10 and Genzyme Mab binding. All antibodies except the Genzyme Mab effectively blocked SCF binding to c-Kit-expressing cells, and were strongly inhibitory in an assay of in vitro haemopoiesis which is believed to depend on adhesive interactions, as well as the "classical' cytokine-receptor interaction, mediated by SCF binding to c-Kit.