RESUMEN
Dissemination of T cell hybridomas in mice, a model for in vivo migration of memory T cells and for T lymphoma metastasis, depends on the chemokine stromal cell-derived factor-1 (SDF-1) and the integrin LFA-1 and correlates well with invasion into fibroblast cultures. In addition to the known role of the pertussis toxin-sensitive heterotrimeric GTPase G(i), we show that also the pertussis toxin-insensitive GTPase G(q/11) is required for dissemination and invasion. Furthermore, we show that the small GTPases, Cdc42 and RhoA, are involved, and that invasion is blocked by inhibitors of actinomyosin contraction. G(q/11), RhoA, and contraction are specifically required for LFA-1 activation, since 1) they are essential for LFA-1-dependent migration toward low SDF-1 concentrations through ICAM-1-coated filters, but not for migration toward high SDF-1 levels, which is LFA-1 independent; 2) G protein (AlF(4)(-))-induced adhesion to ICAM-1 requires RhoA and contraction; 3) constitutively active G(q) induces aggregation, mediated by LFA-1. We previously reported that binding of this activated LFA-1 to ICAM-1 triggers a signal, transduced by the zeta-associated protein 70 tyrosine kinase, that activates additional LFA-1 molecules. This amplification of LFA-1 activation is essential for invasion. We show here that zeta-associated protein 70-induced LFA-1 activation requires neither Cdc42 and RhoA nor contraction and is thus quite different from that induced by SDF-1. We conclude that two modes of LFA-1 activation, with distinct underlying mechanisms, are required for the in vivo migration of T cell hybridomas.
Asunto(s)
Movimiento Celular/inmunología , Quimiocinas CXC/fisiología , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/fisiología , Proteínas de Unión al GTP Heterotriméricas/fisiología , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Miosinas/fisiología , Linfocitos T/metabolismo , Proteína de Unión al GTP cdc42/fisiología , Proteína de Unión al GTP rhoA/fisiología , Animales , Inhibición de Migración Celular , Movimiento Celular/genética , Quimiocina CXCL12 , Quimiocinas CXC/antagonistas & inhibidores , Dactinomicina/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11 , Proteínas Activadoras de GTPasa/fisiología , Vectores Genéticos/síntesis química , Vectores Genéticos/inmunología , Proteínas de Unión al GTP Heterotriméricas/genética , Hibridomas/citología , Hibridomas/efectos de los fármacos , Hibridomas/metabolismo , Ratones , Células del Estroma/fisiología , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacología , Transfección , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP rhoA/genéticaRESUMEN
The migration of leukocytes into tissues is regulated by chemokines and other chemotactic factors that act on receptors that signal through Gi proteins. It seems likely that the colonization of tissues during dissemination of hematopoietic tumor cells is similarly regulated. In fact, dissemination of a T-cell hybridoma, a model for T lymphoma, was blocked when Gi proteins were inactivated by the S1 catalytic subunit of pertussis toxin that had been transfected into those cells. Pertussis toxin S1 blocked dissemination of MDAY-D2 murine myeloid leukemia cells to the liver and spleen, as in T-cell hybridoma cells, but it did not prevent bone marrow colonization. In contrast, overexpression of a function-defective mutant of the Gq/11 protein blocked dissemination to the bone marrow and also prevented Gq/11 dissemination to the liver and spleen. This indicates that the influx of these myeloid cells into all tissues requires the Gq/11 protein in addition to the Gi protein in the liver and spleen. (Blood. 2000;96:691-698)
Asunto(s)
Médula Ósea/patología , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/fisiología , Proteínas de Unión al GTP/fisiología , Leucemia Mieloide/patología , Infiltración Leucémica , Hígado/patología , Bazo/patología , Animales , Subunidades alfa de la Proteína de Unión al GTP Gq-G11 , Expresión Génica , Ratones , Ratones Endogámicos DBA , Trasplante de Neoplasias , Toxina del Pertussis , Transfección , Células Tumorales Cultivadas , Factores de Virulencia de Bordetella/genética , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
The ZAP-70 tyrosine kinase is essential for T cell activation by the T cell receptor. We show that ZAP-70 is also required for migration of T cells that is dependent on the integrin LFA-1. Invasion of TAM2D2 T cell hybridoma cells into fibroblast monolayers, which is LFA-1-dependent, was blocked by overexpression of dominant-negative ZAP-70 and by piceatannol but not by herbimycin A. The Syk inhibitor piceatannol blocks the Syk homologue ZAP-70, which is expressed by TAM2D2 cells, with the same dose dependence as the inhibition of invasion. Dominant-negative ZAP-70 completely inhibited the extensive metastasis formation of TAM2D2 cells to multiple organs upon i.v. injection into mice. Migration of TAM2D2 cells through filters coated with the LFA-1 ligand ICAM-1, induced by 1 ng/ml of the chemokine SDF-1, was blocked by anti-LFA-1 mAb and also abrogated by dominant-negative ZAP-70 and piceatannol. In contrast, migration induced by 100 ng/ml SDF-1 was independent of both LFA-1 and ZAP-70. LFA-1 cross-linking induced tyrosine phosphorylation, which was blocked by dominant-negative ZAP-70 and piceatannol. We conclude that LFA-1 engagement triggers ZAP-70 activity that is essential for LFA-1-dependent migration.
Asunto(s)
Movimiento Celular/fisiología , Antígeno-1 Asociado a Función de Linfocito/fisiología , Proteínas Tirosina Quinasas/fisiología , Linfocitos T/metabolismo , Animales , Benzoquinonas , Quimiocina CXCL12 , Quimiocinas CXC/farmacología , Fibroblastos , Expresión Génica/genética , Humanos , Hibridomas/metabolismo , Integrinas/fisiología , Molécula 1 de Adhesión Intercelular/metabolismo , Lactamas Macrocíclicas , Ratones , Metástasis de la Neoplasia/fisiopatología , Fosfotirosina/metabolismo , Quinonas/farmacología , Ratas , Rifabutina/análogos & derivados , Estilbenos/farmacología , Factores de Virulencia de Bordetella/farmacología , Proteína Tirosina Quinasa ZAP-70RESUMEN
HT-29 colon carcinoma cells form liver metastases upon intrasplenic injection, and adhesion to fibronectin under the liver microvascular liver endothelium is likely to be important for metastasis formation. We have therefore studied the integrins involved in fibronectin adhesion. This was not affected by blocking antibodies against the beta1, alpha3, and alpha5 integrin subunits, but it was blocked by an RGD-containing peptide, indicating involvement of RGD-dependent non-beta1 alphaV integrins. Both alphaVbeta5 and alphaVbeta6 were detected on HT-29 cells. Blocking mAb against alphaV, but not against alphaVbeta5, abolished adhesion. From a HT-29 cell lysate, only alphaVbeta6 bound to a fibronectin-Sepharose column. Thus, alphaVbeta6 is the main fibronectin receptor on HT-29 cells, despite the very low levels of alphaVbeta6 and the much higher levels of alphaVbeta5. The HT29 cells did not spread on fibronectin in the absence of serum, not even after a three- to fourfold increase in alphaVbeta6 levels, induced by interleukin 4. The cells did spread on vitronectin. Using immunofluorescence we observed that both on vitronectin and on fibronectin alphaVbeta5 was arranged in a striped pattern, aligned with actin fibers, and not in focal adhesions. On fibronectin, but not on vitronectin, alphaVbeta6 was concentrated in a punctate pattern at the periphery of cell islands.
Asunto(s)
Actinas/análisis , Antígenos de Neoplasias , Fibronectinas/metabolismo , Cadenas beta de Integrinas , Integrinas/análisis , Receptores de Vitronectina/análisis , Secuencia de Aminoácidos , Adhesión Celular/efectos de los fármacos , Adhesión Celular/fisiología , Fibronectinas/farmacología , Citometría de Flujo , Expresión Génica/fisiología , Células HT29/química , Células HT29/citología , Células HT29/metabolismo , Humanos , Integrinas/genética , Interleucina-4/farmacología , Datos de Secuencia Molecular , Oligopéptidos/farmacología , Receptores de Fibronectina/metabolismo , Receptores de Vitronectina/genética , Vitronectina/farmacologíaRESUMEN
The OPAR mouse monoclonal antibody (mAb) directed against rat hepatocytes was previously shown to inhibit adhesion of TA3/Ha mammary carcinoma cells to hepatocytes. The antigen is abundantly present at the surface of hepatocytes beneath the endothelium of liver capillaries where we have observed invasion of carcinoma cells to occur. The OPAR mAb reacted with three major bands on a Western blot of liver plasma membrane proteins. The same proteins were also seen upon immunoprecipitation from iodinated liver plasma membrane proteins. We have isolated OPAR antigens by lectin wheat germ agglutinin (WGA) and OPAR affinity chromatography. Amino acid sequence analysis revealed that two of the bands were alpha 1-macroglobulin and C4-binding protein, which are serum components produced by hepatocytes. The presence of the epitope on distinct proteins and our previous observation that it can be detected in the Golgi apparatus but not in the endoplasmic reticulum, suggested that OPAR reacts with a liver-specific glycoconjugate. Loss of OPAR reactivity after neuraminidase and N-glycosidase F treatment showed that the epitope contains sialic acid residues on N-linked sugar moieties. OPAR also reacted with rat fibronectin, and inhibited adhesion of TA3/St cells to fibronectin. This explains the inhibition by the OPAR mAb of TA3/St cell adhesion to hepatocytes, which we have shown to be due mainly to interaction with hepatocyte surface-associated fibronectin. However, adhesion of the related TA3/Ha cells to hepatocytes, which is mediated by the alpha 6 beta 4 integrin, and does not involve binding to fibronectin, is also inhibited. This suggests that alpha 6 beta 4 on liver-metastasizing carcinoma cells binds to an OPAR epitope-carrying glycoprotein produced by hepatocytes.
Asunto(s)
Antígenos de Carbohidratos Asociados a Tumores/inmunología , Hígado/inmunología , Neoplasias Mamarias Experimentales/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Adhesión Celular/inmunología , Membrana Celular/inmunología , Epítopos/análisis , Fibronectinas/inmunología , Glicósido Hidrolasas , Hígado/citología , Ratones , Datos de Secuencia Molecular , Neuraminidasa , Ratas , Células Tumorales CultivadasRESUMEN
TA3/Ha murine mammary carcinoma cells grow in suspension, do not adhere to extracellular matrix molecules, but do adhere to hepatocytes and form liver metastases upon intraportal injection. Recently we showed that the integrin alpha 6 beta 4 on the TA3/Ha cells is involved in adhesion to hepatocytes. However, despite high cell surface levels of alpha 6 beta 4, TA3/Ha cells do not adhere to the alpha 6 beta 4 ligands laminin and kalinin. Here we show that this is due to the mucin epiglycanin that is highly expressed on TA3/Ha cells. Some monoclonal antibodies generated against epiglycanin induced capping of most of the epiglycanin molecules. TA3/Ha cells treated with these mAb did adhere to laminin and kalinin, and an epithelial monolayer was formed on kalinin, with alpha 6 beta 4 localized in HD1-containing hemidesmosome-like structures and E-cadherin at the cell-cell contact sites. Similar results were obtained after treatment of TA3/Ha cells with O-sialoglycoprotein endopeptidase which removes all epiglycanin. In addition, the enzyme induced E-cadherin-mediated cell-cell aggregation. Both treatments also enhanced the adhesion to hepatocytes, but given the potent antiadhesive effect of epiglycanin it is remarkable that nontreated TA3/Ha cells adhere to hepatocytes at all. We found that during this interaction, epiglycanin was redistributed. We conclude that epiglycanin can completely prevent both intercellular and matrix adhesion, but that this effect can be overcome in certain intercellular interactions because of the induced redistribution of the mucin.
Asunto(s)
Antígenos de Superficie/metabolismo , Carcinoma/metabolismo , Adhesión Celular , Integrinas/metabolismo , Neoplasias Mamarias Animales/metabolismo , Glicoproteínas de Membrana/metabolismo , Animales , Cadherinas/metabolismo , Carbohidratos/inmunología , Moléculas de Adhesión Celular/metabolismo , Agregación Celular , Tamaño de la Célula , Células Epiteliales , Epítopos/inmunología , Matriz Extracelular/metabolismo , Integrina alfa6beta4 , Laminina/metabolismo , Hígado/citología , Glicoproteínas de Membrana/inmunología , Metaloendopeptidasas/metabolismo , Ratones , Unión Proteica , Células Tumorales Cultivadas , KalininaRESUMEN
The cell lines TA3/Ha and TA3/St are derived from the same tumor, grow both in suspension and form liver metastases upon intraportal injection. We have studied the interaction of these cell lines with hepatocytes, which is likely to be relevant for liver metastasis formation. Recently we have shown that the integrin alpha 6 beta 4 is involved in adhesion of TA3/Ha cells to hepatocytes. However, we show here that the alpha 6 beta 4-specific antibodies, that inhibit adhesion of TA3/Ha cells, did not affect adhesion of TA3/St cells to hepatocytes. The beta 4 subunit, present at high levels on TA3/Ha cells, was found to be expressed at a much lower level by TA3/St cells. In contrast, TA3/St cells express much more of the beta 1-integrin subunit than TA3/Ha cells. We assessed whether these differences in integrin expression are responsible for the different adhesion mechanisms used by these cell lines. We show that alpha 5 beta 1, which is expressed by TA3/St cells, and not by TA3/Ha cells, is involved in TA3/St adhesion to fibronectin that is associated with the hepatocyte surface. Since fibronectin is the main extracellular matrix component present on the hepatocyte surface underneath the sinusoidal endothelium, alpha 5 beta 1 may be particularly important for metastasis formation in the liver, as compared to other organs.
Asunto(s)
Adhesión Celular , Fibronectinas/metabolismo , Integrinas/metabolismo , Hígado/fisiología , Neoplasias Mamarias Experimentales/fisiopatología , Receptores de Fibronectina/metabolismo , Animales , Línea Celular , Membrana Celular/fisiología , Células Cultivadas , Proteínas de la Matriz Extracelular/metabolismo , Ratones , Ratas , Células Tumorales CultivadasRESUMEN
TA3/Ha mammary carcinoma cells form liver metastases upon intraportal injection. We have studied the interaction between these cells and hepatocytes that is likely to be important for liver metastasis formation. We show that the integrin alpha 6 beta 4, which is highly expressed on TA3 cells, is involved in this interaction. Fab fragments, generated from a polyclonal serum against TA3 cells, inhibited TA3-hepatocyte adhesion. By affinity purification on purified alpha 6 beta 4, we isolated alpha 6 beta 4-specific Fab fragments from this anti-TA3 serum. These antibodies were highly specific for alpha 6 beta 4, as demonstrated by Western blot analysis and immunoprecipitation, and inhibited TA3-hepatocyte adhesion. This shows that alpha 6 beta 4, which thus far has only been implicated in cell-matrix adhesion, can also mediate interactions with cell surfaces. Our results suggest that the high levels of alpha 6 beta 4 often expressed by metastatic carcinoma cells contribute to liver metastasis formation.