Discovery of novel hematopoietic cell adhesion molecules from human bone marrow stromal cell membrane protein extracts by a new cell-blotting technique.
In an attempt to define the role of cell adhesion molecules (CAMs) within the bone marrow (BM) microenvironment in normal hematopoiesis and in leukemia development, a novel cell-blotting technique that involved cell adhesion to protein bands after separation by lithium dodecyl sulfate-polyacrylamide gel electrophoresis (LDS-PAGE) and blotting onto polyvinylidene difluoride (PVDF) membrane has been developed. Human BM stromal cell membrane fractions have been prepared from Dexter-type cultures after cell lysis by sonification and differential centrifugations of the sonification contents. The 20,000 g pellets representing membrane fractions have been solubilized by 2% Triton X-100, 0.575% LDS, and 8 mol/L urea in sequential order. The protein extracts are fractionated by LDS-PAGE and screened for CAMs by the new cell-blotting technique. This led to identification of nine protein bands in lanes containing LDS extracts showing adhesion of KG1a (CD34+ progenitor myeloid) cells. Evidence that the BM proteins exhibiting KG1a cell adhesion are novel CAMs is based on the observations that these proteins, in comparison with known CAMs, specifically VCAM-1, CD54, and CD44, show (1) contrasting detergent-solubility properties, (2) different temperature requirement for mediating cell adhesion function, and (3) markedly distinct electrophoretic mobilities. The various cell types tested, notably KG1a, NALM-6, WIL-2, Ramos, HS-Sultan, K562, JY B lymphoblastoid cells, and T lymphoblasts, showed distinctive patterns of binding to different subsets of BM CAMs. These results demonstrate a new approach to studies of molecular mechanisms that may determine specificity of hematopoietic cellular localization within BM microenvironment and may play an important role in controlling hematopoiesis.
Seshi
B
B
Department of Pathology and Laboratory Medicine, University of Rochester School of Medicine and Dentistry, NY 14642.
D001853:Bone Marrow / Q000737:chemistry*; D001854:Bone Marrow Cells; D002352:Carrier Proteins / Q000378:metabolism; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules / Q000032:analysis* / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry*; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D006410:Hematopoiesis; D006801:Humans; D018960:Hyaluronan Receptors; D018799:Intercellular Adhesion Molecule-1; D008565:Membrane Proteins / Q000032:analysis* / Q000378:metabolism; D008854:Microscopy, Electron; D011145:Polyvinyls; D011956:Receptors, Cell Surface / Q000378:metabolism; D016201:Receptors, Lymphocyte Homing / Q000378:metabolism; D019010:Vascular Cell Adhesion Molecule-1; D005465:Fluorocarbon Polymers
D002352:Carrier Proteins; D015815:Cell Adhesion Molecules; D018960:Hyaluronan Receptors; D018799:Intercellular Adhesion Molecule-1; D008565:Membrane Proteins; D011145:Polyvinyls; D011956:Receptors, Cell Surface; D016201:Receptors, Lymphocyte Homing; D019010:Vascular Cell Adhesion Molecule-1; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers