Robert Christopher Spiro

Subsets of hairy cell leukemia defined by unique membrane proteins

Abstract Leukemic cell membrane proteins of 27 patients with hairy cell leukemia were evaluated by SDS gel electrophoresis in order to identify subsets of these patients and to define molecules for isolation and the development of diagnostic reagents. [35S]methionine metabolic labeling, gel electrophoresis of isolated membranes and fluorography demonstrated about 100 membrane proteins. Variations in the amount of synthesis of two principal proteins, p35 and p15 (35,000 and 15,000 dalton proteins respectively), defined, in relative terms, three subtypes of hairy cell leukemias: (a) p35+, p15− cells, (b) p35−, p15+ cells, and (c) p35−, p15− cells. At this time, it is not known whether these subsets represent various related disease entities or stages in the progression of one disorder. Although p35 migrated in the gels with one component of the HLA-DR (p23,30) protein complex, immunoprecipitation studies with anti-p23, 30 serum indicated that it probably was not derived from HLA-DR antigen, which was, in fact, synthesized in addition to p35 on all hairy cells which were tested. Cultured lymphoblastoid cell lines established from the peripheral blood of hairy cell leukemia patients were substantially different in membrane protein patterns from the fresh leukemic cells. In particular, p35 and p15 were minimally expressed by cultured cells.

Identification of hairy cell leukemia subset defining p35 as the human homologue of Ii

A molecule defining a subset of patients with hairy cell leukemia (HCL) on the basis of being abundantly labeled with [35S]methionine, was demonstrated to be the human homologue of murine Ii, a glycoprotein which lacks alloantigenic variation and is associated non-covalently with Ia antigens. In one-dimensional SDS-polyacrylamide gradient gel electrophoresis, the HCL-subset-defining molecule migrated with HLA-DR molecules which were immunoprecipitated with a specific heteroantiserum. These molecules were further defined in two-dimensional, SDS and non-equilibrium pH gradient electrophoresis of either membrane preparations or immunoprecipitates formed with various antibodies. [35S]methionine-labeling of the HCL-subset-defining molecule was greater in hairy leukemic cells than in lymphoblastoid cell lines. The subset-defining species was associated non-covalently with HLA-DR alpha and beta chains and ran electrophoretically at a position described for murine and human Ii molecules (in terms of pI and weight). Metabolic labeling of HLA-A,-B and -DR was also increased in HCL cells relative to lymphoblastoid cell lines. A separate protein, of 41,000 mol. wt and pI of 7-8, resembled another Ii-associated molecule which has been described in murine and human studies.

Comparison of membrane proteins of burkitt's lymphoma and EBV-transformed B lymphoblast cell lines and of con A-activated T lymphocytes and T lymphoblast cell lines

Abstract The aim of this study was to examine the heterogeneity of membrane proteins of several B and T lymphoblast cell lines and activated normal T lymphocytes using SDS gel electrophoresis of [ 35 S]methionine metabolically labeled cells. In part, we hoped to test the hypothesis that human lymphocytic malignancies are “frozen” representatives of various stages in the differentiation paths of normal lymphoid cells. The membrane protein patterns of Burkitts lymphoma cell lines were very similar to each other and to those of EBV-transformed B lymphoblastoid cell lines from healthy persons. The American Burkitts lymphoma cell line Ramos lacked p29, 34 (HLA-DR) antigen, as was confirmed with immunoprecipitation studies of this complex and of p44, 12 (HLA-A, and -B antigens and β 2 -microglobulin) from each of the cell lines. Heterogeneity in the electrophoretic mobility of HLA-DR antigen was apparent, reflecting size or charge variations. Small degrees of membrane protein heterogeneity among the T lymphoblast cultured cell lines was also evident. Proteins of mol. wt 90,000, 75,000, 18,000 and 16,000 were more heavily labeled on some cell lines than on others. This heterogeneity is consistent with the hypothesis that these tumors might be derived from cells of varying maturational stages or from different subsets of T lymphocytes. In contrast to the T lymphoblast lines, Con A-activated T lymphocytes on each successive day of stimulation expressed dramatically different membrane proteins. Proteins of mol. wt 30,000 and 36,000 were found on day 2 cells; mol. wt 55,000 proteins were dominant on days 1, 2 and 3; a group of proteins of mol. wt about 120,000 was present on cells surviving a lytic process on day 4. Although many proteins were shared between Con A blasts and cultured T cell lines, the absence from the cultured lines of proteins which were dominantly synthesized by mitogen-activated lymphoblasts was consistent with the view that long-term cultured lymphoblasts represent a more primitive, undifferentiated, immature T-cell population, while the changes seen with Con A activation represent metabolically controlled changes consequent to activation of one or more subsets of mature T cells. While many proteins were shared among membrane proteins of T lymphoid tumors and mitogen-activated, normal T lymphocytes, the differences were so striking that simple parallelisms between these lymphoid tumors and activated sets of normal lymphocytes could not be proposed.