Appearance of cytoskeletal components on the surface of leukemia cells and of lymphocytes transformed by mitogens and Epstein-Barr virus.

Abstract

Lactoperoxidase iodination and two-dimensional electrophoresis of the labeled proteins have demonstrated well-characterized cytoskeletal proteins (actin and tubulins) on the surface of human lymphocytes undergoing blastogenic transformation and of certain malignant human cells. Such proteins could not be detected on the surface of normal resting human lymphocytes. The most prominent cytoskeletal rotein identified on the surface membrane of mitogen-transformed T and B lymphocytes was actin. In Epstein-Barr virus genome-positive Burkitt's lymphoma and lymphoblastoid cell lines and in two leukemia cells, the major iodinated membrane protein components were actin and a,-, a-, and P-tubulins.These proteins were firmly connected to the cytoplasmic skeleton and could not be removed by Triton X-100. Concurrent immunofluorescence studies with specific antibodies and FRab')2 fragments confirmed the appearance of cytoskeletal components on the surface of live and fixed lymphocytes, in parallel with the biochemical data, and indicated that such cytoskeletal proteins formed distinctive patterns on the cell surface, ranging from small patches to large projections. Five-hour labeling with [asS]methionine indicates that all such cells released large quantities of labeled actin and tubulins into the culture medium. These materials were not readsorbed to the membrane surfaces of the cells.The cytoskeletal framework, composed principally of microfilaments and microtubules, is normally confined to the interior of the cell and to the inner surface of the cell membrane (1-4). Earlier studies have documented marked increases in relative rates of synthesis of major skeletal proteins (actin and tubulin) in polyclonally activated normal human T and B lymphocytes, as well as in Epstein-Barr virus (EBV) genome-positive lines and in three leukemia cell lines (5). Such cells also release large quantities of labeled actin and tubulin into the culture medium, with no evidence of a loss of cell viability (5). In view of recent reports of extensive shedding of membrane materials and submembrane anchorage components by mouse mastocytoma cells (6), an attempt has been made to ascertain whether a similar mechanism was operative in the transformed human lymphocytes studied in this laboratory. The results indicate that blastogenic stimulation of normal lymphocytes or malignant transformation of such cells (or both) is associated with the appearance of large concentrations of cytoskeletal proteins on the cell membrane surface. This observation is in marked contrast to the absence of such components on the surface of normal human lymphocytes. MATERIALS AND METHODSEnriched populations of T and B lymphocytes from healthy volunteers were prepared from defibrinated blood (7) and were stimulated in vitro with concanavalin A (Con A) (T cells) or pokeweed mitogen (PWM) (B cells) as described (8). EBV genome-positive cells used in the present study were: (i) Burkitt's lymphoma lines Daudi, Maku, and Solubo; (iH) AW-Ramos and EHRA-Ramos, lines that had...

Cell-Surface Actin Binds Plasminogen and Modulates Neurotransmitter Release from Catecholaminergic Cells

“…As an independent approach, we demonstrated the presence of actin on the catecholaminergic cell surface using FACS analyses. Several reports suggest the presence of a cell-surface form of actin on other cell types (Owen et al, 1978;Bachvaroff et al, 1980;Sanders and Craig, 1983;Moroianu et al, 1993;Dudani and Ganz, 1996;Andronicos and Ranson, 2001;Dudani et al, 2005). Actin mediates autoproteolysis of plasminogen to angiostatin on cancer cells (H. Wang et al, , 2006.…”

Proteins in unexpected locations.

“…In endothelial cells, surface-associated actin could be displaced by heparin or angiogenin (Moroianu et al, 1993). Controls indicated that lymphocytes did not bind actin added exogenously, either in purified form or when incubated in cellconditioned medium (Bachvaroff et al, 1980;Sanders and Craig, 1983). Although actin is present in fetal calf serum, the form of actin on endothelial cells is smooth muscle a-actin, not the same type as found in fetal calf serum (Moroianu et al, 1993).…”

“…At least eight research groups have reported the existence of actin at the external face of the cell surface in lymphocytes (Owen et al, 1978;Bachvaroff et al, 1980;Rubin et al, 1982;Sanders and Craig, 1983), monocytes (Por et al, 1991), endothelial cells (Pardridge et al, 1989;Moroianu et al, 1993), and L cells (Rosenblatt et al, 1985). (Evidence and control experiments will be discussed only for this first example, but may be taken as representative of the other proteins and locations mentioned below.)…”

“…Abundant cytoplasmic proteins such as vimentin were not labeled. Labeled actin was identified by comigration with authentic actin on two-dimensional polyacrylamide gel electrophoresis gels (Bachvaroff et al, 1980;Rubin et al, 1982).…”

“…(Evidence and control experiments will be discussed only for this first example, but may be taken as representative of the other proteins and locations mentioned below.) Actin was localized by three methods: 1) Surface labeling of proteins in live cells was carried out with biotin (Moroianu et al, 1993) or iodina-tion using lactoperoxidase or other methods (Bachvaroff et al, 1980;Rubin et al, 1982). Controls confirmed that iodination was not mediated by endogenous peroxidases and that it did not cause cell permeabilization.…”