RESUMEN
BACKGROUND: Gastrointestinal stromal tumour (GIST) is mainly initialised by receptor tyrosine kinase gene mutations. Although the tyrosine kinase inhibitor imatinib mesylate considerably improved the outcome of patients, imatinib resistance still remains a major therapeutic challenge in GIST therapy. Herein we evaluated the clinical impact of microRNAs in imatinib-treated GISTs. METHODS: The expression levels of microRNAs were quantified using microarray and RT-qPCR in GIST specimens from patients treated with neoadjuvant imatinib. The functional roles of miR-125a-5p and PTPN18 were evaluated in GIST cells. PTPN18 expression was quantified by western blotting in GIST samples. RESULTS: We showed that overexpression levels of miR-125a-5p and miR-107 were associated with imatinib resistance in GIST specimens. Functionally, miR-125a-5p expression modulated imatinib sensitivity in GIST882 cells with a homozygous KIT mutation but not in GIST48 cells with double KIT mutations. Overexpression of miR-125a-5p suppressed PTPN18 expression, and silencing of PTPN18 expression increased cell viability in GIST882 cells upon imatinib treatment. PTPN18 protein levels were significantly lower in the imatinib-resistant GISTs and inversely correlated with miR-125a-5p. Furthermore, several microRNAs were significantly associated with metastasis, KIT mutational status and survival. CONCLUSIONS: Our findings highlight a novel functional role of miR-125a-5p on imatinib response through PTPN18 regulation in GIST.
Asunto(s)
Antineoplásicos/uso terapéutico , Benzamidas/uso terapéutico , Neoplasias Gastrointestinales/tratamiento farmacológico , Tumores del Estroma Gastrointestinal/tratamiento farmacológico , MicroARNs/fisiología , Piperazinas/uso terapéutico , Pirimidinas/uso terapéutico , Línea Celular Tumoral , Resistencia a Antineoplásicos , Neoplasias Gastrointestinales/genética , Neoplasias Gastrointestinales/mortalidad , Tumores del Estroma Gastrointestinal/genética , Tumores del Estroma Gastrointestinal/mortalidad , Humanos , Mesilato de Imatinib , Mutación , Proteínas Tirosina Fosfatasas no Receptoras/genética , Proteínas Tirosina Fosfatasas no Receptoras/fisiología , Proteínas Proto-Oncogénicas c-kit/genética , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genéticaRESUMEN
There is emerging evidence that cancer stem cells (CSCs), like normal tissue stem cells, are regulated by a niche formed of mesenchymal cells. In this review we summarize the current knowledge of the role of cancer associated fibroblasts (CAFs) in a tentative CSC niche. We also discuss findings from our own studies showing that CAF derived factors have a strong stimulatory effect on the stem cell properties of breast cancer cells. Based on recent literature we conclude that CAFs are strong modulators of the stem cell properties of cancer cells. This effect is likely to be particularly relevant under circumstances of early stages of tumor cell dissemination and metastasis.
Asunto(s)
Fibroblastos/fisiología , Neoplasias/patología , Células Madre Neoplásicas/fisiología , Animales , Humanos , Metástasis de la Neoplasia , FenotipoRESUMEN
The term 'platelet-derived growth factor' (PDGF) refers to a family of disulphide-bonded dimeric isoforms that are important for growth, survival and function in several types of connective tissue cell. So far, three different PDGF chains have been identified - the classical PDGF-A and PDGF-B and the recently identified PDGF-C. PDGF isoforms (PDGF-AA, AB, BB and CC) exert their cellular effects by differential binding to two receptor tyrosine kinases. The PDGF alpha-receptor (PDGFR-alpha) binds to all three PDGF chains, whereas the beta-receptor (PDGFR-beta) binds only to PDGF-B. Gene-targeting studies using mice have shown that the genes for PDGF-A and PDGF-B, as well as the two PDGFR genes, are essential for normal development. Furthermore, overexpression of PDGFs is linked to different pathological conditions, including malignancies, atherosclerosis and fibroproliferative diseases. Here we have identify and characterize a fourth member of the PDGF family, PDGF-D. PDGF-D has a two-domain structure similar to PDGF-C and is secreted as a disulphide-linked homodimer, PDGF-DD. Upon limited proteolysis, PDGF-DD is activated and becomes a specific agonistic ligand for PDGFR-beta. PDGF-DD is the first known PDGFR-beta-specific ligand, and its unique receptor specificity indicates that it may be important for development and pathophysiology in several organs.
Asunto(s)
Linfocinas , Factor de Crecimiento Derivado de Plaquetas/química , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/química , Secuencia de Aminoácidos , Animales , Baculoviridae/metabolismo , Northern Blotting , Clonación Molecular , Cisteína/química , ADN Complementario/metabolismo , Dimerización , Relación Dosis-Respuesta a Droga , Humanos , Inmunohistoquímica , Insectos , Ligandos , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Filogenia , Unión Proteica , Estructura Terciaria de Proteína , ARN Mensajero/metabolismo , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Distribución TisularRESUMEN
Platelet-derived growth factors (PDGFs) are important in many types of mesenchymal cell. Here we identify a new PDGF, PDGF-C, which binds to and activates the PDGF alpha-receptor. PDGF-C is activated by proteolysis and induces proliferation of fibroblasts when overexpressed in transgenic mice. In situ hybridization analysis in the murine embryonic kidney shows preferential expression of PDGF-C messenger RNA in the metanephric mesenchyme during epithelial conversion. Analysis of kidneys lacking the PDGF alpha-receptor shows selective loss of mesenchymal cells adjacent to sites of expression of PDGF-C mRNA; this is not found in kidneys from animals lacking PDGF-A or both PDGF-A and PDGF-B, indicating that PDGF-C may have a unique function.
Asunto(s)
Endopeptidasas/metabolismo , Factor de Crecimiento Derivado de Plaquetas/genética , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Animales , Células COS , Células Epiteliales/química , Células Epiteliales/enzimología , Expresión Génica/fisiología , Regulación del Desarrollo de la Expresión Génica , Humanos , Hibridación in Situ , Insectos , Riñón/química , Riñón/embriología , Riñón/enzimología , Ligandos , Linfocinas , Mesodermo/química , Mesodermo/enzimología , Ratones , Datos de Secuencia Molecular , Miocardio/química , Miocardio/enzimología , Factor de Crecimiento Derivado de Plaquetas/química , Factor de Crecimiento Derivado de Plaquetas/farmacología , Estructura Terciaria de Proteína , ARN Mensajero/análisis , Conejos , Homología de Secuencia de Aminoácido , Transgenes/fisiologíaRESUMEN
Signaling through receptor tyrosine kinases (RTKs) is a major mechanism for intercellular communication during development and in the adult organism, as well as in disease-associated processes. The phosphorylation status and signaling activity of RTKs is determined not only by the kinase activity of the RTK but also by the activities of protein tyrosine phosphatases (PTPs). This review discusses recently identified PTPs that negatively regulate various RTKs and the role of PTP inhibition in ligand-induced RTK activation. The contributions of PTPs to ligand-independent RTK activation and to RTK inactivation by other classes of receptors are also surveyed. Continued investigation into the involvement of PTPs in RTK regulation is likely to unravel previously unrecognized layers of RTK control and to suggest novel strategies for interference with disease-associated RTK signaling.
Asunto(s)
Proteínas Tirosina Fosfatasas/fisiología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal/fisiología , Comunicación Celular/fisiología , Dimerización , Humanos , Modelos Animales , Fosforilación , Proteínas Tirosina Quinasas Receptoras/química , Especificidad por SustratoRESUMEN
Platelet-derived growth factor is a potent mitogen for cells of mesenchymal origin. It is made up of two polypeptide chains (A and B) combined in three disulfide-linked dimeric forms (AA, AB, and BB). Here, the biosynthesis and proteolytic processing of the two homodimeric forms of PDGF (AA and BB) were studied in CHO cells stably transfected with A-chain (short splice version) or B-chain cDNA. PDGF-AA was processed to a 30-kD molecule which was secreted from the cells. In contrast, PDGF-BB formed two structurally distinct end products; a minor secreted 30-kD form and a major cell-associated 24-kD form. Immunocytochemical studies at light- and electron-microscopical levels revealed presence of PDGF in the Golgi complex, in lysosomes, and to a smaller extent in the ER. From analysis of cells treated with brefeldin A, an inhibitor of ER to Golgi transport, it was concluded that dimerization occurs in the ER, whereas the proteolytic processing of PDGF-AA and PDGF-BB precursors normally occurs in a compartment distal to the ER. Exposure of the cultures to the lysosomal inhibitor chloroquine led to an increased cellular accumulation of PDGF-BB, as determined both by metabolic labeling experiments and immunocytochemical methods, indicating that the retained form of PDGF-BB is normally degraded in lysosomes. Structural analysis of the two end products of PDGF-BB revealed that the secreted 30-kD form is a dimer of peptides processed as the B-chain of PDGF purified from human platelets, and that the retained 24-kD form is made up of subunits additionally processed in the NH2-terminus. Also, the 24-kD form was shown to be composed of proteolytic fragments held together by disulfide bridges. Taken together these findings suggest that the newly synthesized PDGF A- and B-chains are dimerized in the ER and thereafter transferred to the Golgi complex for proteolytic processing. From there, PDGF-AA is carried in vesicles to the cell surface for release extracellularly by exocytosis. A smaller part of PDGF-BB (the 30-kD form) is handled in a similar way, whereas the major part (the 24-kD form) is generated by additional proteolysis in the Golgi complex, from which it is slowly carried over to lysosomes for degradation.
Asunto(s)
Aparato de Golgi/metabolismo , Lisosomas/metabolismo , Factor de Crecimiento Derivado de Plaquetas/biosíntesis , Procesamiento Proteico-Postraduccional , Animales , Brefeldino A , Células CHO , Cloroquina/farmacología , Cricetinae , Ciclopentanos/farmacología , Electroforesis en Gel de Poliacrilamida , Técnica del Anticuerpo Fluorescente , Microscopía Inmunoelectrónica , Factor de Crecimiento Derivado de Plaquetas/química , Factor de Crecimiento Derivado de Plaquetas/genética , Procesamiento Proteico-Postraduccional/efectos de los fármacos , TransfecciónRESUMEN
The transmembrane protein-tyrosine phosphatase (PTP) DEP-1 (density-enhanced phosphatase) is a candidate tumor suppressor in the colon epithelium. We have explored the function of DEP-1 in colon epithelial cells by inducible re-expression in a DEP-1-deficient human colon cancer cell line. Density-enhanced phosphatase-1 re-expression led to profound inhibition of cell proliferation and cell migration, and was associated with cytoskeletal rearrangements. These effects were dependent on the PTP activity of DEP-1 as they were not observed with cells expressing the catalytically inactive DEP-1 C1239S variant. shRNA-mediated suppression of DEP-1 in a colon epithelial cell line with high endogenous DEP-1 levels enhanced proliferation, further supporting the antiproliferative function of DEP-1. Nutrients, which are considered to be chemoprotective with respect to colon cancer development, including butyrate, green tea and apple polyphenols, had the capacity to elevate transcription of endogenous DEP-1 mRNA and expression of DEP-1 protein. Upregulation of DEP-1 expression, and in turn inhibition of cell growth and migration may present a previously unrecognized mechanism of chemoprevention by nutrients.
Asunto(s)
Adenocarcinoma/patología , Adenoma/patología , Anticarcinógenos/farmacología , Colon/citología , Neoplasias del Colon/patología , Células Epiteliales/efectos de los fármacos , Proteínas de Neoplasias/fisiología , Proteínas Tirosina Fosfatasas/fisiología , Adenocarcinoma/enzimología , Adenoma/enzimología , Butiratos/farmacología , División Celular/efectos de los fármacos , Línea Celular Tumoral/citología , Línea Celular Tumoral/efectos de los fármacos , Línea Celular Tumoral/enzimología , Movimiento Celular/efectos de los fármacos , Células Cultivadas/citología , Células Cultivadas/efectos de los fármacos , Células Cultivadas/enzimología , Quimiocina CXCL12 , Quimiocinas CXC/farmacología , Colon/enzimología , Neoplasias del Colon/enzimología , Regulación hacia Abajo , Inducción Enzimática/efectos de los fármacos , Células Epiteliales/citología , Células Epiteliales/enzimología , Flavonoides/farmacología , Humanos , Lisofosfolípidos/farmacología , Malus/química , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Fenoles/farmacología , Extractos Vegetales/farmacología , Polifenoles , Proteína Fosfatasa 1 , Proteínas Tirosina Fosfatasas/biosíntesis , Proteínas Tirosina Fosfatasas/genética , Interferencia de ARN , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Neoplásico/biosíntesis , ARN Neoplásico/genética , ARN Interferente Pequeño/farmacología , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Té/química , Transcripción Genética/efectos de los fármacos , Transfección , Regulación hacia Arriba/efectos de los fármacosRESUMEN
High-grade gliomas, including glioblastomas, are malignant brain tumors for which improved treatment is urgently needed. Genetic studies have demonstrated the existence of biologically distinct subsets. Preliminary studies have indicated that platelet-derived growth factor (PDGF) receptor signaling contributes to the growth of some of these tumors. In this study, human high-grade glioma primary cultures were analysed for sensitivity to treatment with the PDGF receptor inhibitor imatinib/Glivec/Gleevec/STI571. Six out of 15 cultures displayed more than 40% growth inhibition after imatinib treatment, whereas seven cultures showed less than 20% growth inhibition. In the sensitive cultures, apoptosis contributed to growth inhibition. Platelet-derived growth factor receptor status correlated with imatinib sensitivity. Supervised analyses of gene expression profiles and real-time PCR analyses identified expression of the chemokine CXCL12/SDF-1 (stromal cell-derived factor 1) as a predictor of imatinib sensitivity. Exogenous addition of CXCL12 to imatinib-insensitive cultures conferred some imatinib sensitivity. Finally, coregulation of CXCL12 and PDGF alpha-receptor was observed in glioblastoma biopsies. We have thus defined the characteristics of a novel imatinib-sensitive subset of glioma cultures, and provided evidence for a functional relationship between imatinib sensitivity and chemokine signaling. These findings will assist in the design and evaluation of clinical trials exploring therapeutic effects of imatinib on malignant brain tumors.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias Encefálicas/tratamiento farmacológico , Glioma/tratamiento farmacológico , Piperazinas/farmacología , Pirimidinas/farmacología , Benzamidas , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Quimiocina CXCL12 , Quimiocinas CXC/biosíntesis , Quimiocinas CXC/genética , Femenino , Glioma/metabolismo , Glioma/patología , Humanos , Mesilato de Imatinib , Masculino , Persona de Mediana Edad , Receptores del Factor de Crecimiento Derivado de Plaquetas/antagonistas & inhibidores , Células Tumorales CultivadasRESUMEN
A non-receptor-binding mutant of the platelet-derived growth factor (PDGF) A chain, PDGF-0, was generated by exchanging 7 amino acids in the sequence. The mutant chains formed dimers that were similar to wild-type PDGF-AA with regard to stability and rate of processing to the mature 30-kDa secreted forms. Moreover, the mutant chains formed disulfide-bonded heterodimers with the PDGF B chain in NIH 3T3 cells heterodimer underwent the same processing and secretion as PDGF-AB. Transfection of c-sis-expressing 3T3 cells with PDGF-0 significantly inhibited the transformed phenotype of these cells, as determined by the following criteria. (i) Compared with PDGF-0-negative clones, PDGF-0-producing clones showed a reverted morphology. (ii) Clones producing PDGF-0 grew more slowly than PDGF-0-negative clones, with a fivefold difference in cell number after 14 days in culture. (iii) The expression of PDGF-0 completely inhibited the ability of the c-sis-expressing 3T3 cells to form colonies in soft agar; this inhibition was overcome by the addition of recombinant PDGF-BB to the culture medium, showing that the lack of colony formation of these cells was not due to a general unresponsiveness to PDGF. The specific expression of a PDGF-0/PDGF wild-type heterodimer in COS cells revealed that the affinity of the mutant heterodimer for the PDGF alpha receptor was decreased by approximately 50-fold compared with that of PDGF-AA. Thus, we show that a non-receptor-binding PDGF A-chain mutant neutralizes in a trans-dominant manner the autocrine transforming potential of the c-sis/PDGF B chain by forming low-affinity heterodimers with wild-type PDGF chains. This method of specifically antagonizing the effect of PDGF may be useful in investigations of the role of PDGF in normal and pathological conditions.
Asunto(s)
Transformación Celular Neoplásica/genética , Factor de Crecimiento Derivado de Plaquetas/genética , Células 3T3 , Animales , Línea Celular , Células Cultivadas , Cromatografía Líquida de Alta Presión , Clonación Molecular , Humanos , Cinética , Ratones , Mutación , Fenotipo , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Pruebas de Precipitina , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Transfección , Células Tumorales Cultivadas , Regulación hacia ArribaRESUMEN
The structure of the human receptor for platelet-derived growth factor (PDGF) has been deduced through cDNA cloning. A 5.45-kilobase-pair cDNA clone predicts a 1,106-amino-acid polypeptide, including the cleavable signal sequence. The overall amino acid sequence similarity with the murine PDGF receptor is 85%. After transcription of the cDNA and translation in vitro, a PDGF receptor antiserum was used to immunoprecipitate a product of predicted size, which also could be phosphorylated in vitro. Stable introduction of the cDNA into Chinese hamster ovary (CHO) cells led to the expression of a 190-kilodalton component, which was immunoprecipitated by the PDGF receptor antiserum; this most probably represents the mature PDGF receptor. Binding assays with different 125I-labeled dimeric forms of PDGF A and B chains showed that the PDGF receptor expressed in CHO cells bound PDGF-BB and, to a lesser extent, PDGF-AB, but not PDGF-AA.
Asunto(s)
Clonación Molecular , ADN/genética , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Receptores de Superficie Celular/genética , Transcripción Genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Genes , Humanos , Sustancias Macromoleculares , Ratones , Datos de Secuencia Molecular , Biosíntesis de Proteínas , Receptores de Superficie Celular/metabolismo , Receptores del Factor de Crecimiento Derivado de Plaquetas , Homología de Secuencia de Ácido Nucleico , Especificidad de la Especie , TransfecciónRESUMEN
Growth, differentiation, apoptosis and movement of cells are regulated, in part, by polypeptide growth factors, or cytokines, which exert their effects by binding to cell surface receptors on the target cells. Recent observations have indicated that a common mechanism of activation of several classes of such receptors is ligand-induced dimerization or oligomerization of the receptors.
Asunto(s)
Receptores de Factores de Crecimiento/química , Dimerización , Diseño de Fármacos , Humanos , Ligandos , Receptores de Factores de Crecimiento/antagonistas & inhibidoresRESUMEN
Most solid malignancies display interstitial hypertension and a poor uptake of anticancer drugs. Platelet-derived growth factor (PDGF) and the cognate tyrosine kinase receptors are expressed in many tumors. Signaling through PDGFbeta receptors was shown recently to increase interstitial fluid pressure (IFP) in dermis after anaphylaxis-induced lowering of IFP. In this study, we show that treatment with the selective PDGF receptor kinase inhibitor, STI571, formerly known as CGP57148B, decreased the interstitial hypertension and increased capillary-to-interstitium transport of 51Cr-EDTA in s.c. growing rat PROb colonic carcinomas. Furthermore, treatment with an antagonistic PDGF-B oligonucleotide aptamer decreased interstitial hypertension in these tumors. PDGFbeta receptors were expressed in blood vessels and stromal cells but not in the tumor cells of PROb colonic carcinomas. Our study indicates a previously unrecognized role of PDGF receptors in tumor biology, although similar effects of PDGF on IFP have been demonstrated previously in the dermis. The data suggest interference with PDGF receptors, or their ligands, as a novel strategy to increase drug uptake and therapeutic effectiveness of cancer chemotherapy.
Asunto(s)
Neoplasias del Colon/metabolismo , Receptores del Factor de Crecimiento Derivado de Plaquetas/antagonistas & inhibidores , Animales , Antineoplásicos/farmacología , Benzamidas , Permeabilidad Capilar/efectos de los fármacos , Permeabilidad Capilar/fisiología , Neoplasias del Colon/irrigación sanguínea , Ácido Edético/farmacocinética , Inhibidores Enzimáticos/farmacología , Proteínas de la Matriz Extracelular/metabolismo , Mesilato de Imatinib , Microdiálisis , Oligonucleótidos/farmacología , Fosforilación/efectos de los fármacos , Piperazinas/farmacología , Proteínas Proto-Oncogénicas/metabolismo , Pirimidinas/farmacología , Ratas , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Especificidad por Sustrato , Receptor 1 de Factores de Crecimiento Endotelial VascularRESUMEN
Antithrombin is a plasma protein of the serpin superfamily that may occur as several conformational variants. The native form of antithrombin is a major regulator of blood clotting. In the present study, we have identified the mechanism underlying the antiangiogenic action of a heat-denatured form, denoted latent antithrombin. Fibroblast growth factor (FGF)-induced angiogenesis in the chick embryo and angiogenesis in mouse fibrosarcoma tumors were inhibited by treatment with latent antithrombin at 1 mg/kg/day. Thermolysin-cleaved and native antithrombin were less efficient in these respects. Treatment with latent antithrombin induced apoptosis of cultured endothelial cells and inhibited cell migration toward FGF-2. Under these conditions, FGF-2-stimulated FGF receptor kinase activity was unaffected. However, actin reorganization, activation of focal adhesion kinase, and focal adhesion formation were disturbed by latent antithrombin treatment of FGF-2-stimulated endothelial cells. These data indicate that latent antithrombin induces apoptosis of endothelial cells by disrupting cell-matrix interactions through uncoupling of focal adhesion kinase.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Antitrombinas/farmacología , Apoptosis/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Fibrosarcoma/irrigación sanguínea , Neovascularización Patológica/tratamiento farmacológico , Alantoides/irrigación sanguínea , Alantoides/efectos de los fármacos , Animales , Adhesión Celular/efectos de los fármacos , Adhesión Celular/fisiología , Comunicación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Inhibición de Migración Celular , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Células Cultivadas , Embrión de Pollo , Corion/irrigación sanguínea , Corion/efectos de los fármacos , Factores de Crecimiento Endotelial/antagonistas & inhibidores , Endotelio Vascular/citología , Matriz Extracelular/efectos de los fármacos , Femenino , Factor 2 de Crecimiento de Fibroblastos/antagonistas & inhibidores , Fibrosarcoma/tratamiento farmacológico , Fibrosarcoma/patología , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Linfocinas/antagonistas & inhibidores , Ratones , Ratones Endogámicos C57BL , Neovascularización Patológica/patología , Proteínas Tirosina Quinasas , Proteínas Tirosina Quinasas Receptoras , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos , Receptores de Factores de Crecimiento de Fibroblastos , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
In the present investigation, we have transfected a human malignant glioma cell line, U-1242 MG, and derived clones that produce transforming growth factor alpha (TGF-alpha) in an inducible manner using the tetracycline suppressible vector system. TGF-alpha expression was confirmed by Northern analysis, by ELISA, and by immunoprecipitation of metabolically labeled cells. The functional activity of the induced protein was proven by the finding of epidermal growth factor receptor (EGFR) tyrosine phosphorylation on induction of TGF-alpha. A clear effect on cell motility, i.e., cell scattering and an increased phagokinetic track area of individual glioma cells, was demonstrated. The fact that the EGFR tyrosine kinase activation was independent of cell density suggests that autocrine activation of the EGFR kinase occurred at the single-cell level. These findings are of interest, because increased cell motility is most likely a requirement for glioma cell invasion in vivo. The results imply that as a result of coexpression of EGFR and its ligand, individual glioma cells are capable of acting as independent autocrine locomotory units.
Asunto(s)
Glioblastoma/patología , Factor de Crecimiento Transformador alfa/fisiología , Anticuerpos Monoclonales/farmacología , Northern Blotting , Movimiento Celular/fisiología , Ensayo de Inmunoadsorción Enzimática , Receptores ErbB/inmunología , Receptores ErbB/metabolismo , Receptores ErbB/fisiología , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Ligandos , Fosforilación , Pruebas de Precipitina , ARN Mensajero/metabolismo , Transfección , Factor de Crecimiento Transformador alfa/biosíntesis , Factor de Crecimiento Transformador alfa/genética , Células Tumorales Cultivadas , Tirosina/metabolismoRESUMEN
Dermatofibrosarcoma protuberans (DFSP) and giant cell fibroblastoma (GCF) are recurrent, infiltrative skin tumors that presently are treated with surgery. DFSP and GCF tumors are genetically characterized by chromosomal rearrangements fusing the collagen type Ialpha1 (COLIA1) gene to the platelet-derived growth factor B-chain (PDGFB) gene. It has been shown that the resulting COL1A1/PDGF-B fusion protein is processed to mature PDGF-BB. Autocrine PDGF receptor stimulation has therefore been predicted to contribute to DFSP and GCF tumor development and growth. Here we demonstrate presence of activated PDGF receptors in primary cultures derived from six different DFSP and GCF tumors. Three of the primary cultures were further characterized; their in vitro growth displayed an increased sensitivity to treatment with the PDGF receptor tyrosine kinase inhibitor STI571, as compared with normal fibroblasts. Transplantable tumors, displaying a DFSP-like histology, were established from one of the DFSP primary cultures. Treatment of tumor-bearing severe combined immunodeficient mice with STI571 reduced tumor growth. The growth-inhibitory effects in vitro and in vivo occurred predominantly through induction of tumor cell apoptosis. Our study demonstrates growth-inhibitory effects of PDGF receptor antagonists on human DFSP- and GCF-derived tumor cells and demonstrates that autocrine PDGF receptor stimulation provides antiapoptotic signals contributing to the growth of these cells. These findings suggest targeting of PDGF receptors as a novel treatment strategy for DFSP and GCF.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Dermatofibrosarcoma/patología , Piperazinas/farmacología , Pirimidinas/farmacología , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/antagonistas & inhibidores , Adulto , Animales , Benzamidas , División Celular/efectos de los fármacos , División Celular/fisiología , Preescolar , Dermatofibrosarcoma/irrigación sanguínea , Dermatofibrosarcoma/tratamiento farmacológico , Femenino , Fibrosarcoma/irrigación sanguínea , Fibrosarcoma/tratamiento farmacológico , Fibrosarcoma/patología , Tumores de Células Gigantes/irrigación sanguínea , Tumores de Células Gigantes/tratamiento farmacológico , Tumores de Células Gigantes/patología , Inhibidores de Crecimiento/farmacología , Humanos , Mesilato de Imatinib , Lactante , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Ratones SCID , Neovascularización Patológica/tratamiento farmacológico , Proteínas de Fusión Oncogénica/biosíntesis , Proteínas de Fusión Oncogénica/genética , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/fisiología , Neoplasias Cutáneas/irrigación sanguínea , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Dermatofibrosarcoma protuberans (DFSP) displays chromosomal rearrangements involving chromosome 17 and 22, which fuse the collagen type Ialpha1 (COLIA1) gene to the platelet-derived growth factor (PDGF) B-chain (PDGFB) gene. To characterize the functional and structural properties of the COLIA1/PDGFB fusion protein, we generated a stable NIH3T3 cell line that contained a tumor-derived chimeric gene resulting from a COIA1 intron 7-PDGFB intron 1 fusion. Expression of the fusion protein led to morphological transformation and increased growth rate of these cells. The PDGF receptor kinase inhibitor CGP57148B reversed the transformed phenotype and reduced the growth rate of COLIA1/PDGFB-expressing cells but had no effects on control cells. The presence of dimeric COLIA1/PDGFB precursors was demonstrated through PDGFB immunoprecipitations of metabolically labeled cells and also by PDGFB immunoprecipitations followed by immunoblotting with COLIA1 antibodies. Pulse-chase studies demonstrated that the COLIA1/PDGFB precursor was processed to an end product that was indistinguishable from wild-type PDGF-BB. Finally, COLIA1/PDGFB-expressing cells generated tumors after s.c. injection into nude mice, and tumor growth was reduced by treatment with CGP57148B. We conclude that the COLIA1/PDGFB fusion associated with DFSP contributes to tumor development through ectopic production of PDGF-BB and the formation of an autocrine loop. Our findings, thus, suggest that PDGF receptors could be a target for pharmacological treatment of DFSP and giant cell fibroblastoma, e.g., through the use of PDGF receptor kinase inhibitors such as CGP57148B.
Asunto(s)
Dermatofibrosarcoma/genética , Proteínas de Fusión Oncogénica/metabolismo , Factor de Crecimiento Derivado de Plaquetas/genética , Neoplasias Cutáneas/genética , Células 3T3 , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Becaplermina , Benzamidas , Transformación Celular Neoplásica/genética , Cósmidos , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Mesilato de Imatinib , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/efectos de los fármacos , Proteínas de Neoplasias/fisiología , Proteínas de Fusión Oncogénica/genética , Piperazinas/farmacología , Piperazinas/uso terapéutico , Factor de Crecimiento Derivado de Plaquetas/biosíntesis , Proteínas Proto-Oncogénicas c-sis , Pirimidinas/farmacología , Pirimidinas/uso terapéutico , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/fisiología , Receptor beta de Factor de Crecimiento Derivado de Plaquetas , Receptores del Factor de Crecimiento Derivado de Plaquetas/efectos de los fármacos , Receptores del Factor de Crecimiento Derivado de Plaquetas/fisiologíaRESUMEN
Cellular growth, differentiation and migration is regulated by protein tyrosine phosphorylation. Receptor-like protein tyrosine phosphatases are thus likely to be key regulators of vital cellular processes. The regulation of these enzymes is in general poorly understood. Ligands have been identified only for a small subset of the receptor-like protein tyrosine phosphatases and in no case has upregulation of the specific activity by extracellular ligands been demonstrated. Prompted by earlier findings of ligands for receptor-like protein tyrosine phosphatases in extracellular matrix we investigated if Matrigel, a preparation of extracellular matrix proteins, contained modulators of the specific activity of the receptor-like protein tyrosine phosphatase DEP-1. Matrigel stimulation of cells increased the specific activity of immunoprecipitated DEP-1. Also, incubation of immunoprecipitated DEP-1 with Matrigel led to an increase in DEP-1 activity, which was blocked by soluble DEP-1 extracellular domain. Finally, immunoprecipitated DeltaECD-DEP-1, a mutant form of DEP-1 lacking most of the extracellular domain, failed to respond to Matrigel stimulation. These experiments identify Matrigel as a source of DEP-1 agonist(s) and provide the first evidence for upregulation of the specific activity of receptor-like protein tyrosine phosphatases by extracellular ligands.
Asunto(s)
Colágeno/farmacología , Laminina/farmacología , Proteínas Tirosina Fosfatasas/metabolismo , Proteoglicanos/farmacología , Regulación hacia Arriba , Animales , Línea Celular , Combinación de Medicamentos , Endotelio Vascular/citología , Humanos , Ligandos , Mutación , Fosforilación , Pruebas de Precipitina , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Tirosina Fosfatasas/química , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores , Porcinos , Células Tumorales CultivadasRESUMEN
The t(5;12) translocation, associated with chronic myelomonocytic leukemia, generates a novel gene encoding a protein, TEL-PDGF beta R, composed of the 154 amino-terminal amino acids of the transcription factor TEL and the transmembrane and intracellular part of the PDGF beta-receptor (PDGF beta R). TEL also occurs as a tumor-associated fusion partner for the tyrosine kinases c-ABL, JAK2 and TRK-C. Previous studies have demonstrated growth promoting activity of TEL-PDGF beta R and also indicated that the TEL moiety activates the tyrosine kinase of the PDGF beta R through the formation of TEL-PDGF beta R oligomers. We demonstrate that tyrosine phosphorylation of the fusion protein can be attenuated through overexpression of the TEL part of TEL-PDGF beta R, suggesting a strategy for antagonizing the signaling of TEL-PDGF beta R, and other TEL-fusion proteins containing tyrosine kinase domains. Comparison of BaF/3 cell lines expressing TEL-PDGF beta R and ligand-stimulated PDGF beta R revealed that only TEL-PDGF beta R expression conferred IL-3-independent growth, suggesting differences in signaling capacity of the two proteins. Finally, tyrosine residues 17 and 27 in TEL-PDGF beta R was identified as autophosphorylation sites in TEL-PDGF beta R.