Generation of mutant cells with constitutively active beta 2-integrin LFA-1.
To elucidate the molecular mechanisms behind regulation of integrin adhesiveness, we have developed a system for the generation of mutant cell lines where the adhesive functions of specific molecules were modulated. The human lymphatic leukemia cell line HPB-ALL was chemically mutagenized and rare cells expressing constitutively active beta 2-integrin LFA-1 (alpha L beta 2, CD11a/CD18) were selected from the pool of mutagenized cells, by repeated cycles of binding to immobilized ICAM-1. The selected population showed 20-fold more binding to ICAM-1 compared to the starting population. A sub-clone of the selected population, termed HAP4, was further analyzed. In contrast to the original cell line, HAP4 had constitutively active LFA-1 and adherence to ICAM-1 was insensitive to inhibition by okadaic acid and cytochalasin B or to inhibition of ATP synthesis. The activation of LFA-1 appeared specific since the cells did not show a general increase in adhesiveness. No major aberration in cell surface carbohydrate expression was found and the expression of cell surface molecules including CD43, CD4, CD8, and MHC class I and II was normal. However, apparent cell surface expression of integrin beta 1 chain was lost and alpha 4 chain was greatly diminished. Our study demonstrates the feasibility of generating cells with mutated adhesive functions and the characterized cells will serve as tools to further dissect the mechanisms underlying integrin regulation.
Hedman
H
H
Department of Cell and Molecular Biology, University of Umeå, Sweden.
Lundgren
E
E
D018821:CD18 Antigens / Q000096:biosynthesis / Q000378:metabolism*; D002469:Cell Separation; D002999:Clone Cells; D005786:Gene Expression Regulation / Q000276:immunology; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism; D016169:Lymphocyte Function-Associated Antigen-1 / Q000096:biosynthesis / Q000378:metabolism*; D016296:Mutagenesis / Q000276:immunology*; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000235:genetics / Q000378:metabolism; D013234:Stem Cells; D014162:Transfection / Q000276:immunology; D014407:Tumor Cells, Cultured
D018821:CD18 Antigens; D016169:Lymphocyte Function-Associated Antigen-1; D018799:Intercellular Adhesion Molecule-1