Human Leukemic Myeloblasts and Myeloblastoid Cells Contain the Enzyme Cytidine 5 -Monophosphate-Jv-Acetylneuraminic Acid:Galãÿl- 3Galnaca(2-3)-Sialyltransferase1
Total Page：16
File Type：pdf, Size：1020Kb
[CANCER RESEARCH 50. 5003-5007. August 15. 1990] Human Leukemic Myeloblasts and Myeloblastoid Cells Contain the Enzyme Cytidine 5 -Monophosphate-jV-acetylneuraminic Acid:Galßl- 3GalNAca(2-3)-sialyltransferase1 Amita Kanani, D. Robert Sutherland, Eitan Fibach, Kushi L. Matta, Alex Hindenburg, Inka Brockhausen, William Kuhns, Robert N. Taub, Dirk H. van den Eijnden, and Michael A. Baker2 Department of Medicine, Toronto General Hospital, University of Toronto, Ontario M5G 2C4, Canada [A. K., D. R. S., M. A. BJ; Department of Haematology, Hadassah Hospital, Hebrew university, Jerusalem, Israel, IL-91120 [E. F.]; Department of Medicine, Columbia University, New York, New York 10032 [R. N. T.J; Department of Biochemistry, Hospital for Sick Children, Toronto, Ontario, Canada MSG 1X8 [I. B., W. K.J; Roswell Park Memorial Institute, Buffalo, New York 14263 [K. L. MJ; Division of Oncology-Hematology, Winthrop University Hospital, Mineóla, Long Island, New York 11501 [A. H.]; and Department of Medical Chemistry, Vrije Universiteit, Amsterdam, The Netherlands, NL-1007 MC [D. H. v. d. E.J ABSTRACT CML compared to normals have demonstrated decreased ad hesiveness (6), decreased chemotaxis (7), and reduced mem We have examined the role of CMP-NeuAc:Gal£I-3GalNAc-R o(2- brane binding of the chemotactic peptide /V-formylmethionyl- 3)-sialyltransferase in fresh leukemia cells and leukemia-derived cell leucylphenylalanine (8). These altered functions are partially lines. Enzyme activity in normal granulocytes using Gal$l-3GalNAca- reversible by removal of membrane sialic acid with neuramini- o-nitrophenyl as substrate was 1.5 ±0.7 nmol/mg/h whereas activity in morphologically mature granulocytes from 6 patients with chronic mye- dase, suggesting a role for aberrant sialylation in the abnormal logenous leukemia (CML) was 4.2 ±1.6 nmol/mg/h (/' < 0.05). Myelo cell behavior (8). Sialic acids are common constituents of both the O- and /V-linked glycan chains of glycoproteins (9), as well blasts from 5 patients with CML in blast crisis showed enzyme activity levels of 6.5 ±2.5 nmol/mg/h. From 2 patients with CML, both blasts as of many glycolipids (10). They are found in a variety of and granulocytes were obtained, with higher enzyme activity in the linkage patterns to galactose, GalNAc, GlcNAc, or other patients' blasts (7.1 nmol/mg/h) than in their granulocytes (4.9 nmol/mg/ NeuAc moieties. Nevertheless, there seem to be many more h) in both cases, suggesting that the increase in enzyme activity is related sialyltransferases than there are sialic acid linkages, thus sup to the differentiation or proliferation status of the CML cells. However, porting the contention (11) that the activity of these enzymes similarly high enzyme levels were also seen in myeloblasts from acute in various tissues is probably largely regulated by the strict myeloblastic leukemia patients (5.6 ±1.4nmol/mg/h) and in some acute substrate specificity of each sialyltransferase. For example, the myeloblastic leukemia-derived cell lines (KGla and 111,60). suggesting a(2-3)-sialyltransferases (EC 2.4.99.4) which have been puri that increased levels of this enzyme are not directly correlated with the fied from porcine submaxillary gland (12, 13) and human presence of the Ph1 chromosome. This a(2-3)-sialyltransferase activity placenta (14), specifically sialylate the galactosyl residue of can also be detected in normal peripheral blood lymphocytes and exhibits Gal01-3GalNAc-R via an «2-3linkage but cannot synthesize increased activity in chronic lymphocytic leukemia cells and acute lyin- the NeuAca2-3Gal/31-4GlcNAc-R product. These sialyl trans- phoblastic leukemia. These data suggest that the level of enzyme activity ferases will also use the gangliosides GMiaand GDibas substrates may vary with growth rate and maturation status in myeloid and lymphoid (13, 14) as well as asialo-GMi, since these glycolipids contain hemopoietic cells. Finally, we have identified a glycoprotein in acute the required unsubstituted Gal/3l-3GalNAc-R sequence (re myeloblastic leukemia cells that serves as a substrate for the a(2-3)- sialyltransferase. The desialylated form of the glycoprotein was resialy- viewed in Ref. 15). lated in vitro by the purified placenta! form of this a(2-3)-sialyltransfer- Lectin studies utilizing peanut agglutinin, which binds most avidly to Gal01-3GalNAc moieties, have suggested that the ase and exhibits a molecular weight of about 150,000. aberrant sialylation in CML cell membranes occurs on O-linked glycans (6). We have shown previously that an enzyme which INTRODUCTION specifically catalyzes the synthesis of NeuAca2-3 Gal/31-3 GalNAc-R is present in human granulocytes and has increased CML3 is characterized by early release of myeloid cells from activity in CML granulocytes, possibly accounting for the aber bone marrow into the peripheral blood and a marked increase rant sialylation and playing a pathophysiological role in CML in the circulation time of the leukemic granulocytes (1). We (16). and others have shown that CML cell membranes are more CML granulocytes may represent a population of cells less highly sialylated than normal granulocyte membranes (2-4). mature than normal granulocytes, in which case the increased Consistent with these data is the observation that the binding a(2-3)-sialytransferase would reflect relative immaturity of the of the galactose-specific lectin of Ricinus communis (RCA 1) to leukemic cells. Myeloid cells exhibiting a less differentiated CML granulocytes is significantly increased after neuramini- phenotype are readily available from both CML patients (in dase treatment (5). In vitro studies of granulocyte function in myeloid blast crisis) as well as from patients with acute myelo blastic leukemia. Relatively undifferentiated cells are also avail Received 11/7/89; revised 3/16/90. able in the form of leukemia-derived cell lines, some of which, The costs of publication of this article were defrayed in part by the payment e.g., K562 (17), EM2, and EM3 (18) were derived from Ph1- of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. positive leukemic blasts. Thus we have studied the levels of 1Supported by the Medical Research Council of Canada; The National Cancer sialyltransferase activity in fresh leukemia samples as well as Institute of Canada; National Cancer Institute Grants CA31762 and CA35329; the William J. Matheson Foundation; and the Canadian Cystic Fibrosis Foun leukemia-derived cell lines of both myeloid and lymphoid line dation. ages. Finally, we have attempted to identify some of the glyco 2To whom requests for reprints should be addressed, at Toronto General Hospital, Mulock Larkin Wing 1-005, 200 Elizabeth Street. Toronto. Ontario, proteins which serve as the natural substrates for this enzyme M5G 2C4, Canada. in intact cells. 3The abbreviations used are: CML, chronic myelogenous leukemia; GalNAc, /V-acetylgalactosamine; NeuAc, yV-acetylneuraminic acid; GlcNAc, iV-acetylglu- cosamine; CMP-NeuAc, cytidine monophosphate A'-acetylneuraminic acid; LJDP- MATERIALS AND METHODS Gal, uridine diphosphate galactose; ONP, o-nitrophenyl; PNP, p-nitrophenyl; AML, acute myeloblastic leukemia; SDS, sodium dodecyl sulfate; PAGE, poly- Cells and Cell Lines. More than 95% morphologically mature gran acrylamide gel electrophoresis; PNA, peanut agglutinin. ulocytes from both CML and normal samples were obtained from 5003 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1990 American Association for Cancer Research. SIALYLTRANSFERASES IN HUMAN LEUKEMIC CELLS EDTA-anticoagulated peripheral venous blood after dextran sedimen expressed as nmol of ['4C]galactose transferred per mg of protein per h tation, ammonium chloride lysis, and layering of leukocytes onto a of incubation as described previously (19). Endogenous acceptor con double gradient of Hypaque and dextran, as described previously (6, trols showed less than 5% incorporation relative to exogenous acceptor 19). Leukemic blast cells (as defined by hematological and phenotypic assays: this incorporation was subtracted in calculation of enzyme criteria) were obtained from patients with acute leukemia and CM L in activities. myeloid blast crisis having greater than 70% blasts in the peripheral In Vitro Sialylation of Natural Substrates. AML cells (2 x 10s) were blood. Mononuclear cell fractions were prepared from heparinized washed with phosphate-buffered saline, resuspended in 2 ml, and peripheral venous blood by Ficoll-Hypaque density gradient centrifu- treated with 100 units of neuraminidase (BDH Chemicals, Poole, gation as reported previously (19). Hematopoietic cell lines were ob United Kingdom) for 30 min at 37°C.Thecells were washed twice with tained from the American Tissue Culture Collection (Rockville, MD) ice-cold phosphate-buffered saline and lysed in 1% Nonidet P-40 as or from colleagues in Toronto. Cell lines were maintained in RPMI described previously (21, 22). The clarified cell lysate was passed 1640 with 10% heat-inactivated fetal calf serum in a 5% CO2 atmos through a 2-ml column of peanut agglutinin agarose (Pharmacia). After phere at 37°C.Allcells and cell lines were washed 3 times in saline and extensive washing away of unbound material, the bound glycoproteins stored at -70°Cas pellets of 2 x 10s cells. were slowly eluted with cell lysis buffer supplemented with 4% D- Sialyltransferase Assays. Enzyme assays were set up as previously galactose. The eluted fraction (10 ml) was dialyzed and concentrated described (8, 19). Briefly, frozen pellets of 2 x 10* cells were resus- against 3- x 1-liter volumes of 0.01 M Tris-saline, pH 7.4. The glyco- pended in 2 ml of 0.2 M NaCl, washed in saline, treated with 10 units protein solution was brought to 70% with ethanol and the proteins of DNase (Sigma, St. Louis, MO), washed again in saline, and solubi- were precipitated overnight at -70°C. The precipitated glycoprotein lized in 1% Triton X-100 at 4°Cfor 20 min.