RESUMO
Autotaxin [ATX (NPP-2)], originally isolated as a tumor motility-stimulating protein, has recently been shown to augment tumor aggressiveness. Specifically, atx-transfected, ras-transformed NIH3T3 cell lines have been shown to be more invasive, tumorigenic, and metastatic than mock-transfected ras-transformed control cells. In addition, the atx-transfected ras-transformed cell lines appeared to produce tumors that were much more hyperemic than those formed by appropriate control cells. This observation led to the present study, in which we demonstrate that ATX modulates angiogenesis both directly and indirectly. We have used a murine in vivo angiogenesis model in which treated Matrigel plugs are injected s.c. into athymic nude BALB/c mice. Using the same transfected cell lines as before, we found that mixing atx-transfected ras-transformed NIH3T3 cells into the Matrigel resulted in greater new blood vessel formation than control cells. Similarly, mixing purified ATX into the Matrigel resulted in new blood vessel formation within the plug, similar to that produced by vascular endothelial growth factor. Mechanistically, ATX is not a strong chemoattractant for human endothelial cells (HUVECs); however, it strongly stimulates motility in human coronary artery smooth muscle cells. In addition, ATX stimulates HUVECs grown on Matrigel to form tubules, much like vascular endothelial growth factor. Both of these normal cell types are shown to express and secrete ATX. In HUVECs, ATX expression is up-regulated by basic fibroblast growth factor in a time-dependent manner. This up-regulation also extends to secretion of enzymatically active protein, as demonstrated by Western blot analysis and quantification of type-1 phosphodiesterase activity. These results establish the presence of ATX in HUVECs and coronary artery smooth muscle cells and specify ATX as a novel angiogenic factor, suggesting that ATX could contribute to the metastatic cascade through multiple mechanisms, perhaps by supporting an invasive microenvironment for both normal and tumor cells.
Assuntos
Indutores da Angiogênese/fisiologia , Glucose-6-Fosfato Isomerase/fisiologia , Glicoproteínas/fisiologia , Complexos Multienzimáticos , Neovascularização Patológica/fisiopatologia , Células 3T3/efeitos dos fármacos , Células 3T3/fisiologia , Indutores da Angiogênese/genética , Indutores da Angiogênese/farmacologia , Animais , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Linhagem Celular Transformada , DNA Complementar/genética , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiologia , Feminino , Glucose-6-Fosfato Isomerase/genética , Glucose-6-Fosfato Isomerase/farmacologia , Glicoproteínas/genética , Glicoproteínas/farmacologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neovascularização Fisiológica/efeitos dos fármacos , Neovascularização Fisiológica/fisiologia , Fosfodiesterase I , Diester Fosfórico Hidrolases , Pirofosfatases , Proteínas Recombinantes/farmacologia , TransfecçãoRESUMO
Autotaxin (ATX) is a recently described member of the nucleotide pyrophosphatase/phosphodiesterase (NPP) family of proteins with potent tumor cell motility-stimulating activity. Like other NPPs, ATX is a glycoprotein with peptide sequences homologous to the catalytic site of bovine intestinal alkaline phosphodiesterase (PDE) and the loop region of an EF-hand motif. The PDE active site of ATX has been associated with the motility-stimulating activity of ATX. In this study, we examined the roles of the EF-hand loop region and of divalent cations on the enzymatic activities of ATX. Ca(2+) or Mg(2+) was each demonstrated to increase the PDE activity of ATX in a concentration-dependent manner, whereas incubation of ATX with chelating agents abolished this activity, indicating a requirement for divalent cations. Non-linear regression analysis of enzyme kinetic data indicated that addition of these divalent cations increases reaction velocity predominantly through an effect on V(max.) Three mutant proteins, Ala(740)-, Ala(742)-, and Ala(751)-ATX, in the EF-hand loop region of ATX had enzymatic activity comparable to that of the wild-type protein. A deletion mutation of the entire loop region resulted in slightly reduced PDE activity but normal motility-stimulating activity. However, the PDE activity of this same deletion mutant remained sensitive to augmentation by cations, strongly implying that cations exert their effect by interactions outside of the EF-hand loop region.
Assuntos
Glucose-6-Fosfato Isomerase/metabolismo , Glicoproteínas/metabolismo , Complexos Multienzimáticos , Sequência de Aminoácidos , Animais , Biotransformação/efeitos dos fármacos , Células COS , Cátions Bivalentes/farmacologia , Quelantes/farmacologia , Dados de Sequência Molecular , Fosfodiesterase I , Diester Fosfórico Hidrolases/metabolismo , Estrutura Terciária de Proteína , Pirofosfatases/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Secreted motility-stimulating factors are often expressed and secreted at low concentrations that are difficult to detect by Northern or Western blot analysis. Autotaxin (ATX) is a tumor-secreted autocrine motility-stimulating factor that has been associated with tumor invasion and metastatic potential. Although ATX has a number of enzymatic activities, it is most sensitively detected by its induced chemotactic response. After transfecting ATX cDNA into NIH3T3 fibroblasts, we developed a motility-based method to screen the resulting cloned cells for secretion of active protein. We placed the cloned and transfected cells into the bottom wells of a modified Boyden chamber and placed responding cells (A2058 human melanoma cells) into the upper wells. After overnight incubation, the membrane that separated the two chambers was removed and stained. Simple densitometry measurements were sufficiently accurate to determine which clones secreted active protein. Utilizing this method, 4 positive cell lines were chosen out of 36 tested clones. Further tests on the expanded cell lines determined that all 4 were secreting ATX. Thus, this modified Boyden chamber assay appears to provide a rapid and highly adaptable means to identify cells that secrete motility-stimulating factors.
Assuntos
Bioensaio/métodos , Quimiotaxia , Glucose-6-Fosfato Isomerase/metabolismo , Glicoproteínas/metabolismo , Complexos Multienzimáticos , Células 3T3 , Animais , Movimento Celular , Meios de Cultivo Condicionados , Glucose-6-Fosfato Isomerase/genética , Glicoproteínas/genética , Humanos , Melanoma/enzimologia , Melanoma/metabolismo , Camundongos , Fosfodiesterase I , Diester Fosfórico Hidrolases , Pirofosfatases , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade , Transfecção , Células Tumorais CultivadasRESUMO
Autotaxin (ATX), an exo-nucleotide pyrophosphatase and phosphodiesterase, was originally isolated as a potent stimulator of tumor cell motility. In order to study whether ATX expression affects motility-dependent processes such as invasion and metastasis, we stably transfected full-length ATX cDNA into two non-expressing cell lines, parental and ras-transformed NIH3T3 (clone7) cells. The effect of ATX secretion on in vitro cell motility was variable. The ras-transformed, ATX-secreting subclones had enhanced motility to ATX as chemoattractant, but there was little difference in the motility responses of NIH3T3 cells transfected with atx, an inactive mutant gene, or empty vector. In MatrigelTM invasion assays, all subclones, which secreted enzymatically active ATX, demonstrated greater spontaneous and ATX-stimulated invasion than appropriate controls. This difference in invasiveness was not caused by differences in gelatinase production, which was constant within each group of transfectants. In vivo studies with athymic nude mice demonstrated that injection of atx-transfected NIH3T3 cells resulted in a weak tumorigenic capacity with few experimental metastases. Combination of ATX expression with ras transformation produced cells with greatly amplified tumorigenesis and metastatic potential compared to ras-transformed controls. Thus, ATX appears to augment cellular characteristics necessary for tumor aggressiveness.
Assuntos
Movimento Celular , Transformação Celular Neoplásica/patologia , Glucose-6-Fosfato Isomerase/metabolismo , Glicoproteínas/metabolismo , Complexos Multienzimáticos , Neoplasias Experimentais/patologia , Neoplasias Experimentais/secundário , Proteína Oncogênica p21(ras)/fisiologia , Células 3T3 , Animais , Adesão Celular , Divisão Celular , Linhagem Celular Transformada , Transformação Celular Neoplásica/metabolismo , Feminino , Glucose-6-Fosfato Isomerase/biossíntese , Glicoproteínas/biossíntese , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica , Neoplasias Experimentais/metabolismo , Fosfodiesterase I , Diester Fosfórico Hidrolases , Pirofosfatases , TransfecçãoRESUMO
Cyclocreatine (1-carboxymethyl-2-iminoimidazolidine), an analog of creatine and a substrate for creatine kinase (EC 2.7.3.2), inhibits the stimulated motility of tumor cells which possess creatine kinase. A2058-055 human melanoma cells, transfected with a creatine kinase gene, showed an 80-90% reduction in chemotactic response to type IV collagen when incubated overnight in the presence of 10 mM cyclocreatine (p < 0.0001 for n = 8 experiments). This inhibitory effect of cyclocreatine can be partially reversed by addition of creatine to the overnight cell treatment. Non-transfected cells, with very low levels of creatine kinase, were not significantly inhibited. Further experiments utilizing type IV collagen as attractant demonstrated that cyclocreatine inhibited the chemokinetic (91%) and the haptotactic (73%) responses and the in vitro invasion of A2058-055 cells through Matrigel-coated membranes (88%). In addition, motility stimulation of A2058-055 cells by either autotaxin or fibronectin was markedly inhibited by cyclocreatine. DU-145 prostatic tumor cells, which express endogenous creatine kinase, also have a reduced motility response to either autotaxin or epidermal growth factor induced motility in the presence of cyclocreatine.
Assuntos
Antineoplásicos/farmacologia , Movimento Celular/efeitos dos fármacos , Cisplatino/farmacologia , Creatina Quinase/metabolismo , Creatinina/análogos & derivados , Antineoplásicos/antagonistas & inibidores , Quimiotaxia , Creatina/farmacologia , Creatinina/antagonistas & inibidores , Creatinina/farmacologia , Humanos , Masculino , Melanoma/enzimologia , Melanoma/patologia , Invasividade Neoplásica , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/patologia , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/enzimologiaRESUMO
Cell motility is an essential component of tumor progression and metastasis. A number of factors, both autocrine and paracrine, have been found to influence cell motility. In the present study, adenosine and adenine nucleotides directly stimulated chemotaxis of A2058 melanoma cells in the absence of exogenous factors. Three adenosine receptor agonists stimulated motility in the melanoma cells and two adenosine receptor antagonists strongly inhibited the chemotactic response to both adenosine and AMP. The chemotactic stimulation by adenosine and AMP was pertussis toxin sensitive. Otherwise unresponsive Chinese hamster ovary cells which were transfected with the adenosine A1 receptor cDNA acquired the direct, pertussis toxin sensitive, chemotactic response to adenosine, and this response was inhibited by adenosine receptor antagonists. These findings demonstrate that adenosine and adenine nucleotides are capable of stimulating chemotaxis of tumor cells mediated through an adenosine receptor, probably of the A1 subtype. The possibility of antimetastatic therapies based on inhibition of adenosine receptor activity is raised.
Assuntos
Monofosfato de Adenosina/farmacologia , Adenosina/farmacologia , Quimiotaxia , Melanoma Experimental/metabolismo , Receptores Purinérgicos P1/metabolismo , Adenosina/análogos & derivados , Animais , Células CHO , Cricetinae , Relação Dose-Resposta a Droga , Humanos , Toxina Pertussis , Agonistas do Receptor Purinérgico P1 , Antagonistas de Receptores Purinérgicos P1 , Transfecção , Fatores de Virulência de Bordetella/farmacologia , Xantinas/farmacologiaRESUMO
Autotaxin (ATX) is a 125 kDa glycoprotein motility factor and exoenzyme which can catalyze the hydrolysis of either the alpha-beta or at the beta-gamma phosphodiester bond in ATP. Its motility stimulating activity requires an intact 5'-nucleotide phosphodiesterase (PDE) active site. Photolysis-dependent labeling of ATX with alpha-[32P]-8-N3-ATP, lysC digestion, and peptide HPLC resolved two radioactive fractions containing single peptides whose amino-terminal sequences were determined. Peptide A (T210FPNLYTLATG. . .) was derived from the PDE active site and peptide B (Y318GPFGPEMTNP. . .) was not previously known to be involved in any of the activities of ATX. The differential effect of NaCl concentration on the labeling of these two peptides, as well as on the two reaction types catalyzed by ATX, allows a classification of activities which predicts both the position of preferential peptide labeling by bound ATP and also the position of phosphodiester bond hydrolysis.
Assuntos
Trifosfato de Adenosina/metabolismo , Glucose-6-Fosfato Isomerase/metabolismo , Glicoproteínas/metabolismo , Complexos Multienzimáticos , Marcadores de Afinidade , Sequência de Aminoácidos , Sítios de Ligação , Glucose-6-Fosfato Isomerase/química , Glucose-6-Fosfato Isomerase/classificação , Glicoproteínas/química , Glicoproteínas/classificação , Metaloendopeptidases/metabolismo , Dados de Sequência Molecular , Mapeamento de Peptídeos , Fosfodiesterase I , Inibidores de Fosfodiesterase/química , Diester Fosfórico Hidrolases , Pirofosfatases , Proteínas Recombinantes , Cloreto de Sódio/farmacologiaRESUMO
Autotaxin (ATX) is a newly found autocrine tumor cell motility-stimulating factor. ATX is a member of the ecto-phosphodiesterase I (PD-I)/ nucleotide pyrophosphatase family. PD-Ialpha was found as a brain-type ecto-phosphodiesterase I/nucleotide pyrophosphatase. ATX and PD-Ialpha are alternative splicing products from one gene. ATX stimulates motility of A2058 melanoma cells in vitro; however, it has not been known if PD-Ialpha/ATX is expressed in naturally occurred human tumors. In this study, we examined the expression of the human PD-Ialpha/ATX gene in human neuroblastoma tumor tissues and the motility stimulating activity of recombinant ATX on neuroblastoma cells and investigated its transcriptional regulatory mechanism in a human neuroblastoma cell line. The PD-Ialpha/ATX gene was expressed in the primary tumor tissues from neuroblastoma patients to varying degrees. This gene is also expressed in the SMS-KAN neuroblastoma cell line. We identified both isoforms, PD-Ialpha and ATX, in these tumor tissues and SMS-KAN cells. The recombinant ATX stimulated the motility of SMS-KAN cells at low nanomolar concentration. We situated the promoter region, which is essential for its transcription in SMS-KAN cells, at -287 to -254 nucleotides by the promoter activity assay. The gel-shift assay revealed that there exists a nuclear protein in SMS-KAN cells that binds this region. These new insights about autocrine tumor cell motility-stimulating protein will help us to understand the metastatic mechanism of human neuroblastoma.
Assuntos
Regulação Neoplásica da Expressão Gênica , Glucose-6-Fosfato Isomerase/genética , Glicoproteínas/genética , Complexos Multienzimáticos , Neuroblastoma/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Movimento Celular/efeitos dos fármacos , Pré-Escolar , Relação Dose-Resposta a Droga , Glucose-6-Fosfato Isomerase/química , Glucose-6-Fosfato Isomerase/metabolismo , Glucose-6-Fosfato Isomerase/farmacologia , Glicoproteínas/química , Glicoproteínas/metabolismo , Glicoproteínas/farmacologia , Humanos , Lactente , Recém-Nascido , Fosfodiesterase I , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/genética , Regiões Promotoras Genéticas , Pirofosfatases , Proteínas Recombinantes/farmacologia , Células Tumorais CultivadasRESUMO
Autotaxin (ATX) is an extracellular enzyme and an autocrine motility factor that stimulates pertussis toxin-sensitive chemotaxis in human melanoma cells at picomolar to nanomolar concentrations. This 125-kDa glycoprotein contains a peptide sequence identified as the catalytic site in type I alkaline phosphodiesterases (PDEs), and it possesses 5'-nucleotide PDE (EC 3.1.4.1) activity (Stracke, M. L., Krutzsch, H. C., Unsworth, E. J., Arestad, A., Cioce, V., Schiffmann, E., and Liotta, L. (1992) J. Biol. Chem. 267, 2524-2529; Murata, J., Lee, H. Y., Clair, T., Krutsch, H. C., Arestad, A. A., Sobel, M. E., Liotta, L. A., and Stracke, M. L. (1994) J. Biol. Chem. 269, 30479-30484). ATX binds ATP and is phosphorylated only on threonine. Thr210 at the PDE active site of ATX is required for phosphorylation, 5'-nucleotide PDE, and motility-stimulating activities (Lee, H. Y., Clair, T., Mulvaney, P. T., Woodhouse, E. C., Aznavoorian, S., Liotta, L. A., and Stracke, M. L. (1996) J. Biol. Chem. 271, 24408-24412). In this article we report that the phosphorylation of ATX is a transient event, being stable at 0 degrees C but unstable at 37 degrees C, and that ATX has adenosine-5'-triphosphatase (ATPase; EC 3.6.1.3) and ATP pyrophosphatase (EC 3.6.1.8) activities. Thus ATX catalyzes the hydrolysis of the phosphodiester bond on either side of the beta-phosphate of ATP. ATX also catalyzes the hydrolysis of GTP to GDP and GMP, of either AMP or PPi to Pi, and the hydrolysis of NAD to AMP, and each of these substrates can serve as a phosphate donor in the phosphorylation of ATX. ATX possesses no detectable protein kinase activity toward histone, myelin basic protein, or casein. These results lead to the proposal that ATX is capable of at least two alternative reaction mechanisms, threonine (T-type) ATPase and 5'-nucleotide PDE/ATP pyrophosphatase, with a common site (Thr210) for the formation of covalently bound reaction intermediates threonine phosphate and threonine adenylate, respectively.
Assuntos
Adenosina Trifosfatases/metabolismo , Glucose-6-Fosfato Isomerase/química , Glicoproteínas/química , Complexos Multienzimáticos , Diester Fosfórico Hidrolases/metabolismo , Pirofosfatases/metabolismo , Ligação Competitiva , Guanosina Trifosfato/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Fosfodiesterase I , Fosforilação , Células Tumorais CultivadasRESUMO
While nucleotides have a well-established role in intracellular metabolism, ATP and other nucleotides also have important extracellular roles in receptor-mediated signal transduction (34, 35). Extracellular or cell surface proteins capable of binding ATP and hydrolyzing phosphoester bonds of nucleotides are known to exist but their function has remained obscure. Our recent data point to a structure-function correlation between PDE activity and motility stimulation by ATX, indicating a biologically important functional role for the ecto/exophosdiesterases in the stimulation of cellular motility. Data from studies with PC-1 and gp130RB13-6 have suggested that cell surface PDE's may also play roles in cellular differentiation. Extracellular PDE activities, in combination with other nucleotidases, may result in ecto-nucleotidase cascades (36-38). These data suggest that ecto-/exo-enzymes may catalyze extracellular biochemical reactions that are important in the regulation of cell behavior.
Assuntos
Movimento Celular , Glucose-6-Fosfato Isomerase/química , Glucose-6-Fosfato Isomerase/metabolismo , Glicoproteínas/química , Glicoproteínas/metabolismo , Complexos Multienzimáticos , Neoplasias/patologia , Diester Fosfórico Hidrolases/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Fatores Quimiotáticos/metabolismo , Glucose-6-Fosfato Isomerase/farmacologia , Glicoproteínas/farmacologia , Humanos , Melanoma , Dados de Sequência Molecular , Metástase Neoplásica , Neoplasias/metabolismo , Fosfodiesterase I , Diester Fosfórico Hidrolases/química , Fosforilação , Mutação Puntual , Pirofosfatases , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Homologia de Sequência de Aminoácidos , Células Tumorais CultivadasRESUMO
We report the first correlation of Nm23 sequence and its tumor metastasis-suppressive capacity using site-directed mutagenesis and an in vitro tumor cell motility assay. MDA-MB-435 human breast carcinoma cells were transfected with a control expression vector (pCMVBamneo), the vector containing the wild type nm23-H1, or the nm23-H1 vector encoding mutations at the following amino acids: serine 44, a phosphorylation site; proline 96, the k-pn mutation in the Drosophila nm23 homolog that causes developmental defects; histidine 118, involved in Nm23's nucleoside diphosphate kinase activity; and serine 120, a site of mutation in human neuroblastomas and phosphorylation. The wild type nm23-H1 transfectants were 44-98% less motile to serum and 86-99% less motile to autotaxin than control vector transfectants. The proline 96 k-pn, serine 120 to glycine, and to a lesser extent serine 120 to alanine mutant nm23-H1-transfected cell lines exhibited motility levels at or above the control transfectants, indicating that these mutations can abrogate the motility-suppressive phenotype of nm23-H1. No effect was observed on cellular proliferation, nor were the serine 44 to alanine nm23-H1 mutant transfectants motile, demonstrating the specificity of the data. The data identify the first structural motifs of nm23-H1 that influence its metastasis suppressive effect and suggest complex biochemical associations or activities in the Nm23 suppressive pathway.
Assuntos
Proteínas Monoméricas de Ligação ao GTP , Núcleosídeo-Difosfato Quinase/metabolismo , Prolina , Serina , Fatores de Transcrição/fisiologia , Alanina , Sequência de Aminoácidos , Animais , Neoplasias da Mama , Divisão Celular , Quimiotaxia , Drosophila , Feminino , Glicina , Histidina , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Nucleosídeo NM23 Difosfato Quinases , Neuroblastoma/genética , Núcleosídeo-Difosfato Quinase/biossíntese , Fosforilação , Reação em Cadeia da Polimerase , Proteínas Recombinantes/metabolismo , Fatores de Transcrição/biossíntese , Transfecção , Células Tumorais CultivadasRESUMO
A family of extracellular type I phosphodiesterases has recently been isolated by cDNA cloning, but a physiological function linked to the phosphodiesterase active site has remained unknown. We now present evidence that the phosphodiesterase catalytic site, 201YMRPVYPTKTFPN213, is essential for the motility stimulating activity of autotaxin (ATX), one member of the exophosphodiesterase family. Native ATX possesses phosphodiesterase activity at neutral and alkaline pH, binds ATP noncovalently, and undergoes threonine phosphorylation. Homogeneously purified recombinant ATX, based on the teratocarcinoma sequence, retains these same activities. A single amino acid in the phosphodiesterase catalytic site, Thr210, is found to be necessary for motility stimulation, phosphodiesterase activity, and phosphorylation. Two mutant recombinant proteins, Ala210- and Asp210-ATX, lack motility stimulation and lack both enzymatic activities; Ser210-ATX possesses intermediate activities. Another mutation, with the adjacent lysine (Lys209) changed to Leu209-ATX, possesses normal motility stimulation with sustained phosphodiesterase activity but exhibits no detectable phosphorylation. This mutation eliminates the phosphorylation reaction and indicates that the dephosphorylated state is an active motility-stimulating form of the ATX molecule. By demonstrating that the phosphodiesterase enzymatic site is linked to motility stimulation, these data reveal a novel role for this family of exo/ecto-enzymes and open up the possibility of extracellular enzymatic cascades as a regulatory mechanism for cellular motility.
Assuntos
Movimento Celular , Glucose-6-Fosfato Isomerase/metabolismo , Glicoproteínas/metabolismo , Melanoma/patologia , Complexos Multienzimáticos , Diester Fosfórico Hidrolases/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Catálise , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fosfodiesterase I , Diester Fosfórico Hidrolases/genética , Pirofosfatases , Proteínas Recombinantes/metabolismo , Células Tumorais CultivadasRESUMO
Distinctions between chemotaxis and haptotaxis of cells to extracellular matrix proteins have not been defined in terms of mechanisms or signaling pathways. Migration of A2058 human melanoma cells to soluble (chemotaxis) and substratum-bound (haptotaxis) vitronectin, mediated by alphav beta3, provided a system with which to address these questions. Both chemotaxis and haptotaxis were completely inhibited by treatment with RGD-containing peptides. Chemotaxis was abolished by a blocking antibody to alphavbeta3 (LM609), whereas haptotaxis was inhibited only by approximately 50%, suggesting involvement of multiple receptors and/or signaling pathways. However, blocking antibodies to alphavbeta5, also present on A2058 cells, did not inhibit. Pertussis toxin treatment of cells inhibited chemotaxis by >80%, but did not inhibit haptotaxis. Adhesion and spreading over vitronectin induced the phosphorylation of paxillin on tyrosine. In cells migrating over substratum-bound vitronectin, tyrosine phosphorylation of paxillin increased 5-fold between 45 min and 5 h. Dilutions of anti- alphavbeta3 that inhibited haptotaxis also inhibited phosphorylation of paxillin (by approximately 50%) and modestly reduced cell spreading. In contrast, soluble vitronectin (50-100 microg/ml) did not induce tyrosine phosphorylation of paxillin. The data suggest that soluble vitronectin stimulates chemotaxis predominantly through a G protein-mediated pathway that is functionally linked to alphavbeta3. Haptotaxis is analogous to directional cell spreading and requires alphavbeta3-mediated tyrosine phosphorylation of paxillin.
Assuntos
Movimento Celular/fisiologia , Quimiotaxia/fisiologia , Receptores de Vitronectina/fisiologia , Transdução de Sinais , Sequência de Aminoácidos , Anticorpos/farmacologia , Adesão Celular , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Quimiotaxia/efeitos dos fármacos , Proteínas do Citoesqueleto/metabolismo , Proteínas da Matriz Extracelular , Proteínas de Ligação ao GTP/metabolismo , Humanos , Cinética , Melanoma , Oligopeptídeos/farmacologia , Paxilina , Toxina Pertussis , Fosfoproteínas/metabolismo , Fosforilação , Fosfotirosina/análise , Receptores de Vitronectina/antagonistas & inibidores , Receptores de Vitronectina/imunologia , Células Tumorais Cultivadas , Fatores de Virulência de Bordetella/farmacologia , Vitronectina/farmacologiaRESUMO
Autotaxin, a potent human tumor cell motility-stimulating exophosphodiesterase, was isolated and cloned from the human teratocarcinoma cell line NTera2D1. The deduced amino acid sequence for the teratocarcinoma autotaxin has 94% identity to the melanoma-derived protein, 90% identity to rat brain phosphodiesterase I/nucleotide pyrophosphatase (PD-I alpha), and 44% identity to the plasma cell membrane marker PC-I. Utilizing polymerase chain reaction screening of the CEPH YAC library, we localized the autotaxin gene to human chromosome 8q23-24. Northern blot analysis of relative mRNA from multiple human tissues revealed that autotaxin mRNA steady state expression is most abundant in brain, placenta, ovary, and small intestine.
Assuntos
Glucose-6-Fosfato Isomerase/genética , Glicoproteínas/genética , Complexos Multienzimáticos , Sequência de Aminoácidos , Animais , Sequência de Bases , Movimento Celular , Mapeamento Cromossômico , Cromossomos Artificiais de Levedura , Cromossomos Humanos Par 8 , Clonagem Molecular , DNA Complementar/genética , Expressão Gênica , Genes , Humanos , Melanoma/química , Dados de Sequência Molecular , Fosfodiesterase I , Diester Fosfórico Hidrolases , Pirofosfatases , RNA Mensageiro/genética , Ratos , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Teratocarcinoma/químicaRESUMO
Autotaxin is a 125kD autocrine motility factor that stimulates both random and directed motility in producing the human A2058 melanoma cell line. The recently cloned autotaxin has been demonstrated to bind strongly and specifically to concanavalin A (con A). In this study, we show that the oligosaccharide side chains on autotaxin are exclusively asparagine linked, since N-glycosidase F, but not neuraminidase or O-glycosidase, decreases the protein molecular mass to 100-105kD, which is the calculated molecular mass of the deduced autotaxin polypeptide. Furthermore, removal of oligosaccharide side chains by N-glycosidase F can be performed under mild conditions that retain motility-stimulating activity, suggesting that the oligosaccharide side chains are not necessary for autotaxin to activate its receptor. Finally, when melanoma cells are treated with inhibitors of carbohydrate processing, such as N-methyl-1-deoxynojirimycin, 1-deoxymannojirimycin and swainsonine, they still secrete a motility-stimulating autotaxin. Therefore, the carbohydrate side chains on autotaxin are not necessary to stimulate motility; however, they may still play a role in folding, secretion or maintenance of the active conformation of the protein.
Assuntos
Glucose-6-Fosfato Isomerase/metabolismo , Glucose-6-Fosfato Isomerase/farmacologia , Glicoproteínas/metabolismo , Glicoproteínas/farmacologia , Melanoma/metabolismo , Melanoma/patologia , Complexos Multienzimáticos , Amidoidrolases/metabolismo , Amidoidrolases/farmacologia , Western Blotting , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Concanavalina A/metabolismo , Glucose-6-Fosfato Isomerase/fisiologia , Glicoproteínas/fisiologia , Glicosilação , Humanos , Invasividade Neoplásica , Oligossacarídeos/metabolismo , Oligossacarídeos/farmacologia , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase , Fosfodiesterase I , Diester Fosfórico Hidrolases , Dobramento de Proteína , Pirofosfatases , Estimulação Química , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
A novel immunoglobulin-type protein expressed in blood vessels has been identified. The cDNA for AAMP (angio-associated, migratory cell protein) was first isolated from a human melanoma cell line during a search for motility-associated cell surface proteins. Upon analysis of the tissue distribution of AAMP, it was found to be expressed strongly in endothelial cells, cytotrophoblasts, and poorly differentiated colon adenocarcinoma cells found in lymphatics. The sequence of AAMP predicts a protein (M(r) 49,000) with distant identity (25%) to known proteins. It contains immunoglobulin-like domains [one with multiple homologies to deleted in colon carcinoma (DCC) protein], the WD40 repeat motif, and a heparin-binding consensus sequence. A 1.6-kilobase mRNA transcript of AAMP is detected in tissue culture cell lines and tissues. Affinity-purified polyclonal antibodies, anti-recombinant AAMP, and anti-peptide 189 (AAMP derived) recognize a M(r) 52,000 protein in human tissue and cellular extracts. The protein size is in keeping with the mRNA and predicted sequence. The AAMP-derived peptide, P189, contains a heparin-binding domain (dissociation constant, 14 pmol) and mediates heparin-sensitive cell adhesion. The shared expression of AAMP in endothelial cells, trophoblasts, and tumor cells implies a common function in migrating cells.
Assuntos
Proteínas de Transporte/isolamento & purificação , Endotélio Vascular/química , Heparitina Sulfato/química , Proteínas de Neoplasias/isolamento & purificação , Receptores de Antígenos de Linfócitos B/isolamento & purificação , Proteínas Supressoras de Tumor , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Sequência de Bases , Proteínas de Transporte/química , Proteínas de Transporte/genética , Moléculas de Adesão Celular/química , Receptor DCC , DNA Complementar/química , Heparina/metabolismo , Heparitina Sulfato/metabolismo , Humanos , Melanoma/química , Melanoma/metabolismo , Dados de Sequência Molecular , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , RNA Mensageiro/química , Receptores de Antígenos de Linfócitos B/química , Receptores de Antígenos de Linfócitos B/genética , Receptores de Superfície Celular , Homologia de Sequência , Células Tumorais CultivadasRESUMO
In this review, the role of extracellular factors in the stimulation and regulation of tumor cell motility are discussed. Tumor cells respond in a motile fashion to a variety of external ligands including autocrine motility factors, growth factors, and components of the extracellular matrix. Since tumor cell motility is a necessary component of tumor invasion and metastasis, we speculate that these protein factors could play important regulatory roles in tumor motility at different stages of the metastatic cascade.
Assuntos
Movimento Celular , Glucose-6-Fosfato Isomerase/fisiologia , Glicoproteínas/fisiologia , Substâncias de Crescimento/fisiologia , Complexos Multienzimáticos , Invasividade Neoplásica , Metástase Neoplásica , Neoplasias/patologia , Neoplasias/fisiopatologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Fatores Quimiotáticos/fisiologia , Citocinas/fisiologia , Matriz Extracelular/fisiologia , Glucose-6-Fosfato Isomerase/química , Glicoproteínas/química , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Neoplasias Experimentais/patologia , Neoplasias Experimentais/fisiopatologia , Fosfodiesterase I , Diester Fosfórico Hidrolases , Pirofosfatases , Homologia de Sequência de Aminoácidos , Células Tumorais CultivadasRESUMO
A human cDNA clone encoding autotaxin, a tumor cell motility-stimulating protein, reveals that this protein is an ecto/exo-enzyme with significant homology to the plasma cell membrane differentiation antigen PC-1. ATX is a 125-kDa glycoprotein, previously isolated from a human melanoma cell line (A2058), which elicits chemotactic and chemokinetic responses at picomolar to nanomolar concentrations. Affinity-purified antipeptide antibodies to the ATX peptide, ATX-102, were employed to screen an A2058 cDNA expression library made in lambda gt11. The partial cDNA sequence which was obtained was then extended by utilizing reverse transcriptase on total cellular RNA followed by polymerase chain reaction amplification. The isolated cDNA clone contained 3251 base pairs, and the mRNA message size was approximately 3.3 kilobases. The deduced amino acid sequence of autotaxin matched 30 previously sequenced peptides and comprised a protein of 915 amino acids. Data base analysis of the ATX sequence revealed a 45% amino acid identity (including 30 out of 33 cysteines) with PC-1, a pyrophosphatase/type I phosphodiesterase expressed on the surface of activated B cells and plasma cells. ATX, like PC-1, was found to hydrolyze the type I phosphodiesterase substrate p-nitrophenyl thymidine-5'-monophosphate. Autotaxin now defines a novel motility-regulating function for this class of ecto/exo-enzymes.
Assuntos
Glucose-6-Fosfato Isomerase/biossíntese , Glucose-6-Fosfato Isomerase/química , Glicoproteínas/biossíntese , Glicoproteínas/química , Complexos Multienzimáticos , Diester Fosfórico Hidrolases/química , Sequência de Aminoácidos , Anticorpos , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Primers do DNA , DNA Complementar/metabolismo , Glucose-6-Fosfato Isomerase/isolamento & purificação , Glicoproteínas/isolamento & purificação , Humanos , Melanoma , Dados de Sequência Molecular , Peptídeos/síntese química , Peptídeos/imunologia , Fosfodiesterase I , Reação em Cadeia da Polimerase , Pirofosfatases , Mapeamento por Restrição , Homologia de Sequência de Aminoácidos , Células Tumorais CultivadasRESUMO
The extracellular matrix is a macromolecular network that surrounds cells and makes up a significant portion of their microenvironment. During the processes of invasion and metastasis, tumor cells encounter interstitial stroma and basement membranes many times. In fact, tumor cell response to extracellular matrix components appears to be a major determinant of metastatic potential.
Assuntos
Matriz Extracelular/fisiologia , Metástase Neoplásica/patologia , Neoplasias/patologia , Animais , Adesão Celular , Divisão Celular , Movimento Celular , Matriz Extracelular/patologia , Humanos , Invasividade Neoplásica , Neoplasias/fisiopatologiaRESUMO
Cytoskeletal agents have been demonstrated to inhibit stimulated motility and substrate adherence by the human tumor cell line, A2058. cis-tubulozole, taxol, and cytochalasin D were tested for their effects on chemotaxis in response to a tumor cytokine, autocrine motility factor, and on adherence to several substrata: laminin- and gelatin-coated dishes as well as tissue culture plastic. Cytochalasin D, which inhibits microfilament polymerization, abolished stimulated motility. Taxol, which stabilizes microtubules, decreased stimulated motility to a greater degree than cis-tubulozole, which inhibits microtubular polymerization. In contrast, cis-tubulozole had the greatest inhibitory effect on adherence with a gelatin substratum more affected (100% inhibition) than tissue culture plastic (90%) or laminin substratum (52%). Taxol affected adherence in the same order but less than cis-tubulozole. Cytochalasin D had no significant effect on adherence to laminin with moderate inhibition of adherence to tissue culture plastic or gelatin. These data suggest that, in these tumor cells, microfilaments are more crucial for motility than adherence, but the dynamic polymerization and depolymerization of microtubules are required for both types of cellular activities.