RESUMEN
STAD cells are the adherent parental apoptotic line from which two sublines were cloned that differed in their response to suspended culturing conditions, one clone STAD.APO is apoptotic and the other STAD.ARR goes into cell cycle arrest. Using this system we have found that the addition of soluble collagen can rescue STAD and STAD.APO cells from anoikis, and it can also affect STAD.ARR cells by overcoming the suspension induced cell cycle arrest. In contrast, when cells were cultured with a soluble anti-beta1 integrin mAb 33B6, the apoptotic clones again were rescued from anoikis, but the cell cycle arresting clone remained quiescent. This result was somewhat surprising as it is generally accepted that cytoskeletal rearrangements that accompany integrin mediated adhesion and cell shape changes are required for the abrogation of anoikis, and it was unexpected that differences in the mechanism used for integrin triggering would yield variable results on growth regulation. This observation led us to further examine whether the addition of a monovalent anti-beta1 integrin agent could produce similar results as intact mAb. Therefore we employed Fab fragments of 33B6 in our culturing assay and found that indeed monovalent binding was capable of saving STAD and STAD.APO cells from anoikis but did not have an effect on STAD.ARR cells. Therefore in this study we have observed that integrin mediated dependent survival can occur by mere ligation of the beta1 integrin subunit, but that cell cycle arrest due to suspended conditions can not. Thus integrins can play differential roles in cell fate decisions and mediate these effects by different mechanisms.
Asunto(s)
Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Anoicis , Ciclo Celular , Integrina beta1/metabolismo , Integrinas/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología , Actinas/metabolismo , Anoicis/efectos de los fármacos , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Adhesión Celular , Ciclo Celular/efectos de los fármacos , División Celular , Tamaño de la Célula , Colágeno/metabolismo , Colágeno/farmacología , Matriz Extracelular/metabolismo , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Expresión Génica , Humanos , Fragmentos Fab de Inmunoglobulinas/inmunología , Fragmentos Fab de Inmunoglobulinas/farmacología , Integrina beta1/inmunología , Integrinas/inmunología , Cinética , Microscopía Fluorescente , Fenotipo , Proteínas Tirosina Quinasas/metabolismo , Células Tumorales CultivadasRESUMEN
The cyclic hexapeptide CWLDVC (TBC 772) is an antagonist of alpha4 integrins and a potent inhibitor of lymphocyte interactions with fibronectin, vascular cell adhesion molecule-1, and muscosal vascular addressin cell adhesion molecule-1 (MAdCAM-1). As such, peptide TBC 772 effectively inhibits the activation of freshly isolated human T lymphocytes stimulated with purified vascular cell adhesion molecule-1 coimmobilized with anti-CD3 mAb. The influence of peptide binding on distinct sites of the alpha4beta1 complex was determined by flow cytometry and cellular adhesion assays employing a panel of mAbs. Binding of the alpha4-specific mAb L25 and the beta1-specific mAb 33B6 was not altered by the peptide; however, binding of mAb 19H8, which is specific for a combinatorial epitope of alpha4beta1, was dramatically inhibited. Treatment of lymphocytes with the peptide caused an increase in a ligand-induced epitope on beta1 integrin defined by mAb 15/7. In T cell activation studies using coimmobilized anti-CD3 mAb and the anti-integrin mAbs, the peptide had broader inhibitory activity, suppressing costimulation induced by all the integrin mAbs. The peptide was not generally toxic and was integrin selective in its suppressive activity, as coactivation by ligation of CD3 in conjunction with CD28 or CD26 was not affected. These results suggest that the antagonist peptide CWLDVC can effectively neutralize integrin coactivation systems by a mechanism independent of competitive binding.