[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 (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 . 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 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, 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 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. Debris was removed by fraction was pelleted at 10,000 x g for 10 min. The pellet was dissolved centrifugation at 1000 x g for 10 min. The supernatant, a solubilized in 0.01 M Tris-saline, pH 7.4, containing 0.5% Nonidet P-40, 1 mM total cell membrane preparation, was used for the assays. Aliquots (20 Mg2*and Mn2*. Solubilized glycoprotein solution (50 u\) was incubated M!)of this supernatant (typically containing 40-60 pg of protein) were with 10 ííCiCMP-['"C]sialicacid diluted as above and 0.05 milliunit of incubated with 10 p\ of 0.05 M Tris-HCI (pH 7.2), 10 n\ of 0.5 mM purified placenta! 2-3-sialyltransferase for l h at 37°C.The reaction CMP-[4-'"C]NeuAc (0.06 ^Ci, 25 mCi/mmol; New England Nuclear), was terminated by addition of 25 Ml(x5) SDS-PAGE gel sample buffer. and 10 M' of the substrate Gal/3l-3GalNAc-a-ONP (2 mM final con The reaction tubes were boiled and the products were resolved by SDS- centration). The Km for CMP-[4-'4C]NeuAc was previously shown to PAGE and fluorography (22). Similar experiments were performed be 0.18 mM, and that for Gal/3l-3GalNAc-«-ONP was 0.3 mM (16). using CML granulocytes. Reaction mixtures were incubated at 37°Cfor 1 h, and the reaction was terminated by freezing. In some experiments, a radioactive low molec ular weight product was separated at 4°Con columns (16.5 x 0.5 cm) RESULTS of Dowex AG 2-X8, Cl~ form, 200-400 mesh (Bio-Rad Laboratories) as described previously (16). Briefly, the Dowex columns were poured In the first series of experiments, levels of a(2-3)-sialyltrans- with 3 M NaCl and extensively washed with 15 mM Tris-HCI, pH 7.0, ferase activity were measured in granulocytes from 6 normal at 4°C.The first 5 ml of eluate contained all the labeled product. volunteers, granulocytes from 6 patients with CML in chronic Products were counted by liquid scintillation using ACS (Amersham) phase, and myeloblasts from 5 patients with CML in myeloid as scintillation fluid. Endogenous acceptor controls showed less than blast crisis (Table 1). Using the synthetic substrate Gal/31- 5% incorporation relative to exogenous acceptor assays; this incorpo 3GalNAc-ONP, which is a specific acceptor for this Sialyltrans ration was subtracted in calculation of enzyme activities. In other experiments where either Gal/31-3GalNAc-«-ONP or Gal/31- ferase, enzyme activities were 1.5 ±0.7 (SD) nmol/mg/h in 3GalNAc-a-PNP were used as substrates, radioactive products were normal granulocytes, 4.2 ±1.6 nmol/mg/h in CML granulo separated by high voltage electrophoresis. The reaction mixtures were cytes, and 6.7 ±2.2 nmol/mg/h in CML myeloblasts, suggest spotted in parallel lanes onto Whatman No. 3M paper (18 x 22 inches), ing a hierarchy in which leukemic blast cells have higher enzyme premoistened with sodium tetraborate, pH 9.1. Electrophoresis was levels than leukemic granulocytes, which in turn have higher performed at 1.5 kV and 170 mA for 90 min. The paper was air dried enzyme levels than normal granulocytes. In 2 patients with and individual "lanes" were cut into strips. The strips were cut into 1- CML blast crisis, individual myeloblast and granulocyte prep inch pieces and counted in Beckman Redi-Solv E.P. scintillation fluid. arations were obtained. Higher Sialyltransferase activity was Control samples lacking synthetic acceptor were performed in duplicate observed in the patients' blasts than their granulocytes in both along with each test set. The results of each assay are expressed as the cases (Table 1). net difference in counts between the mean test and the control. To study the distribution of this enzyme in other cell types Galactosyltransferase Assay. An aliquot of the same solubilized cell of the hematopoietic system, a number of lymphoid and mye- membrane preparation as used for the Sialyltransferase assay was used loid-derived cell lines were collected and assayed for «(2-3)- to measure the activity of the enzyme UDP-Gal:GalNAc /33-gaIactosyl- transferase (EC 2.4.1.122). This transferase will catalyze the addition sialyltransferase activity. In these preliminary experiments, a of galactose (in /31-3 linkage) from UDP-Gal to the terminal GalNAc Table 1 Sialyltransferase activity in granulocytes from CML in chronic phase, residue of either high or low molecular weight substrates. The acceptor blast crisis, and normal donors used for this assay was asialo-ovine submaxillary (20). Enzyme incubation mixtures comprised 20 ^1 cell lysate; 10 pi 0.2 M 1-(N- Sialyltransferase Source of cells No. of samples activity (nmol/mg/hy morpholino)ethanesulfonic acid (pH 6.0), containing 40 mM MnCl2, 10 i/1 2 mM UDP-[14C]galactose (0.05 ßC\,337.0 mCi/mmol; New Normal granulocytes CML granulocytes 6 4.2 ±1.6 England Nuclear); 40 mM MnCl2; and 20 ^1 asialo-ovine submaxillary CMLblastsCML 5Granulocytes 6.7 ±2.25.5 mucin (equivalent to 3.2 mM GalNAc). Control incubations lacked acceptor. Reaction mixtures were incubated at 37°Cfor l h and the O.CMLpatient M. BlastsGranulocytes reaction was terminated by the addition of 15% (w/v) trichloroacetic 8.94.25.2 acid and 5% (w/v) phosphotungstic acid and the resulting precipitate patient F. A.6* was pelleted by centrifugation. The pellet was dissolved in 0.5 ml of Blasts1.5±0.7CJ' NCS (Amersham) for l h and the solubilized proteins were finally " Substrate used was Gal/3l-3GalNAc-o-O-nitrophenyl at 2 mM. * Granulocytes were tested from 12 normal donors pooled in pairs to obtain incorporated into organic counting scintillation fluid. Glacial acetic sufficient cells for assay. acid (7 n\) was added to the contents of each scintillation vial. After ' P < 0.05 for normal granulocytes compared to CML untreated or CML incubation in the dark overnight to reduce chemiluminescence, the blasts. samples were counted in a /3-scintillation counter. Enzyme activity was " Mean ±SD. 5004

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1990 American Association for Cancer Research. SIALYLTRANSFERASES IN HUMAN LEUKEMIC CELLS range of enzyme activity levels was detected ranging from 4.5 Table 3 ß3-galactosyltransferaseactivity in leukemic cells nmol/mg/h for KG la cells to only 0.3 nmol/mg/h for the B- transferase activity (nmol/mg/h) lymphoma-derived Daudi cell line. Significantly, however, there CellsNormal no.°6 usingasialo-OSM*14.2 were no differences in c*(2-3)-sialyltransferase activity between granulocytes ±3.0C those cell lines which contained the Ph1 chromosome (K562, CML granulocytes 6 10.3 ±4.0 EM2, and EM3) and those which did not (KG1, KG la, HL60, CML blasts 55/33-Galactosyl 14.2 ±6.0 U937, Nalm-6, HOON, Daudi, GH-1, HSB-2, and Jurkat) AML blastsSample 20.5 ±9.2 " Normal granulocytes were tested from 12 normal donors pooled in pairs to (data not shown). obtain sufficient cells for assay. In the next series of experiments, a large number of chronic 4 Substrate used was asialo-ovine submaxillary mucin (20) at 3.2 itiM (GalNAc) concentration. and acute leukemia samples of both lymphoid and myeloid c Mean ±SD. types were collected and assayed for a(2-3)-sialyltransferase activity. As shown in Table 2A, myeloblasts from 17 untreated MrixlO"3) AML patients contained relatively high sialyltransferase levels (5.6 ±3.7 nmol/mg/h), similar to the levels detected in mye loblasts of 8 CML blast crisis samples (6.2 ±2.2 nmol/mg/h). 200- In cells of the lymphoid lineage, unstimulated peripheral blood lymphocytes contained relatively low levels of the «(2- 3)-sialyltransferase activity (1.2 ±0.3 nmol/mg/h), similar to those found in normal granulocytes (1.3 ±0.4 nmol/mg/h). Interestingly, lymphocytes from 4 untreated chronic lympho- cytic leukemia patients contained increased levels of this trans- 92- ferase activity (mean, 3.76 nmol/mg/h). Lymphoblasts from 4 acute lymphoblastic leukemia patients contained even higher levels of a(2-3)-sialyltransferase activity (mean, 4.7 nmol/mg/ h), once again indicating a hierarchy in which leukemic lym- 68- 0 phoblasts have the highest levels, followed by leukemic lympho cytes, and finally normal peripheral blood lymphocytes. Samples taken from CML patients before or after chemo therapy were assayed for sialyltransferase activity. Higher levels of enzyme activity were found in the samples preceding a period 43- of chemotherapy (Table 2A) than after treatment (Table 2B). Enzyme activities in granulocytes from treated patients were almost the same as those in normal control granulocytes. In a smaller series of CLL patients, a similar phenomenon was A s B C s D observed (Table 2B). Fig. 1. Fluorograph of desialylated PNA-4b-affinity-purified glycoproteins /33-Galactosyltransferase activity was determined in normal after resialylation in vitro with CMP-[14C]sialic acid and placenta! a(2-3)-sialyl- and leukemic granulocytes and leukemic myeloblasts using the transferase. Track A, PNA-bound/eluted glycoproteins from AML cells, lysed after sialidase treatment; Track B, PNA unbound fraction from same AML cell substrate asialo-ovine submaxillary mucin (Table 3). Normal lysate; Track C, PNA-bound/eluted glycoproteins from CML cells lysed after granulocytes and myeloblasts from CML patients in blast crisis neuraminidase treatment; Track D, PNA unbound fraction from same CML cell exhibited very similar ß-3-galactosyltransferase activities (14.2 lysate. Track s, radiolabeled molecular weight standards. Arrow, position of resialylated natural substrate. ±3.0 nmol/mg/h). CML granulocytes showed lower activities (10.3 ±4 nmol/mg/h) and AML blasts from 4 patients were AML cell lysate contained a glycoprotein which could be sia- higher on the average than normal granulocytes and CML lylated in vitro by the placental <*(2-3)-sialyltransferase. This blasts. Moreover, wide variations were observed among AML sialylated product was resolved in SDS-PAGE at about M, patients (20.5 ±9.2 nmol/mg/h). These differences were not 150,000 (Fig. 1, Track A). This structure was not identified in statistically significant. the PNA unbound fraction from the same AML cell lysate (Fig. Natural Substrates of the a2-3-Sialyltransferase. The peanut agglutinin bound/eluted fraction of a neuraminidase-treated 1, Track B). When a CML cell lysate was used, a resialylated product was not detected in either the PNA bound fraction Table 2 a(2-3)-Sialyltransferase activity" in leukemia cells (Fig. 1, Track C) or the PNA unbound fraction (Fig. 1, Track D). of patient samples12 activity" (nmol/mg/h)4.1 A. Untreated patients DISCUSSION CML granulocytes CML myeloblasts 817 6.25.6 We have shown previously that CMP-NeuAc:Gal/31- AML myeloblasts 3GalNAc-R a(2-3)-sialyltransferase is present in human leu CLL lymphocytes 44Sialyltransferase 3.76 ALL lymphoblastsNo. 4.4 kocytes and has increased activity in granulocytes from patients with chronic myelogenous leukemia (16). The human placental B. Treated patients CML granulocytes 6 1.76 form of this enzyme which ¡ssimilar, if not identical to the CLL lymphocytes 4 2.2 leukocyte form,4 exhibits relatively restricted substrate specific ity and will efficiently catalyze the addition of NeuAc in a(2- C. Normal controls Granulocytes 3 1.3 3) linkage to unsubstituted Gal01-3GalNAc-R structures (14). Peripheral blood lymphocytes 3 1.2 In our previous studies (16), evidence was not found for the * Mean sialyltransferase activity expressed as nmol/mg/h using the substrates Gal/3l-3GalNAc-ONP or Gal01-3GalNAc-PNP. ' I. Brockhausen et al., manuscript in preparation. 5005

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1990 American Association for Cancer Research. SIALYLTRANSFERASES IN HUMAN LEUKEMIC CELLS presence of either the GalNAc a-protein a(2-6)-sialyltransfer- glycoproteins contain a common polypeptide which can be ase (EC 2.4.99.3) or the (NeuAca2-3Gal/31-3)GalNAca-R differentially O-glycosylated in the many different cell types a(2-6)-sialyltransferase (EC 2.4.99.7), neither of which can which constitute the hemopoietic system. Studies of both leu sialylate the Gal/3l-3GalNAc-a-ONP (23, 24) in CML or nor kemic and normal myeloid cells indicate that leukosialins con mal granulocytes. Although the trisaccharide product generated tain several potential substrate structures for the a(2-3)-sialyl- by the action of the a(2-3)-sialyltransferase can theoretically transferase (3). Compared to those of normal granulocytes, the be further sialylated by the subsequent action of the latter a(2- leukosialins isolated from immature myeloblasts contained 6)-siaIyltransferase (EC 2.4.99.7) (23-25), it was concluded that mainly short O-linked oligosaccharides, including NeuAca2-3 the <*(2-6) enzymes are unlikely to be responsible for the Gal,81-3GalNAc and NeuAccv2-3Gal01-3(Neu Ac«2-6)Gal- apparent hypersialylation of CML plasma membrane glycopro- NAc. The detection of those structures in immature myeloid teins compared to those of normal granulocytes (16). Since cells correlates well with our observations that such cells contain circulating granulocytes in CML are thought to be of immature significantly elevated levels of the a(2-3)-sialyltransferase. phenotype (26), we have hypothesized that the hypersialylation Since AML cells and myeloblasts from CML patients in blast of CML cell membranes may reflect their relative immaturity crisis contained the highest levels of this a(2-3)-sialyltransfer- (8). In support of this notion, we have shown here that this ase, we tried to isolate natural substrates from those cells, i.e., a(2-3)-sialyltransferase activity is further increased in the my- glycoproteins containing the substrate sequence Gal/31- eloblasts from CML blast crisis patients, compared to granu 3GalNAc-R. Thus, a neuraminidase-treated, PNA-agarose locytes isolated from the same or other CML patients. In bound glycoprotein fraction was prepared and was shown to contrast, the 03-galactosyl (EC 2.4.1.122) transferase activity contain a substrate molecule for the purified placenta! form of synthesizing the Gal0(l-3)GalNAc a-R acceptor substrate this a(2-3)-sialyltransferase (14). After in vitro resialylation, structure for the a(2-3)-sialyltransferase did not differ signifi the product of this catalysis was resolved as a broad band both cantly between normal granulocytes, CML granulocytes, and a molecular weight of approximately 150,000 in reduce SDS- CML blasts. These data initially indicated a potential correla PAGE. Since many of the leukosialin molecules exhibit a tion between cell maturation or growth rate and the activity of similar apparent size in SDS-PAGE (32), particularly after this a(2-3)-sialyltransferase in CML. desialylation (22), it is possible that this M, 150,000 structure The cells of over 90% of CML patients contain a specific may be a member of the leukosialin family. No substrate chromosomal abnormality, the Philadelphia or Ph1 chromo structures could be isolated from CML granulocytes using some; therefore it was of interest to assess its role, if any, in similar procedures. A possible explanation for this is that the modulating enzyme activities. However, several pieces of data lysis of granulocytes under the conditions we used here results presented herein indicate that the increase in sialyltransferase in the release of proteases from lysosomal vesicles which destroy activity in CML granulocytes and myeloblasts is unlikely to be the substrate molecule(s) before they can be isolated on the due solely to the presence of a Ph1 chromosome: (a) myeloblas- PNA lectin column. More likely explanations are that since toid and lymphoblastoid cell lines exhibited a range of increased leukosialins in CML cells contain longer, more complex, and/ enzyme activities irrespective of whether they were CML de or branched O-glycans than those in AML cells (3), the desialy- rived (EM-2, EM-3, and K562) or not; (b) myeloblasts of all lated forms of these leukosialins would not bind to the Gal/31- untreated AML patients contained elevated levels of sialyltrans 3GalNAc-R-specific PNA. Furthermore, these desialylated ferase activity. Interestingly, these levels were similar to those structures would not be good substrates for the a(2-3)-sialyl- exhibited by myeloblasts from CML patients in myeloid blast transferase. The biochemical nature of O-linked sialylated sur crisis with which they share significant hematological and phe- face glycoproteins, leukosialins, or sialophorins may be impor notypic characteristics. Subsequently, a lower level of a(2-3)- tant in the physiology of leukocyte recirculation (33). The sialyltransferase activity was also detected in normal peripheral increase in expression of a(2-3)-sialyltransferase in myeloid blood lymphocytes, with increased activity in lymphocytic and blasts and the decrease in activity with maturity to normal lymphoblastic leukemias; (c) the presence of a Ph1 chromosome granulocytes may reflect a physiological role for O-linked sia- in CML cells and in CML-derived cell lines is overwhelmingly lylation in adhesion and receptor binding. The permanence of correlated with the expression of the protein called this otherwise transient change in leukemic myeloid cells may p210bcr~ablwhichexhibits tyrosine-specific kinase activity in vitro contribute to this abnormal pathophysiology (8). It may be of (27, 28). However, we did not find the p210bcrabl)kinase from interest that the a(2-3)-sialyltransferase enzyme is encoded by K.562 cells to be capable of phosphorylating the purified pla- a on chromosome 11 suggesting a role for this chromo cental form of the (2-3)-sialyltransferase in vitro (data not some site in normal myeloid differentiation and leukemic shown). pathophysiology (34). Several structures including N- and O-glycosylated glycopro- teins, and glycolipids have been shown to be more highly ACKNOWLEDGMENTS sialylated in CML cell membranes compared to normal gran- ulocyte membranes. Fukuda et al. (29, 30) have described a The authors thank Professor H. Shachter of the Hospital for Sick series of TV-linkedpolylactosaminoglycans in both normal and Children, Toronto, for his helpful suggestions and encouragement, and Claire Guiver-Bond, for her infinite patience throughout the revisions. CML cells and concluded that those from leukemic cells were both shorter and more highly sialylated. Fukuda et al. (31) also showed that the glycolipids of granulocytes and CML cells REFERENCES contained polylactosamine structures and, additionally, isolated 1. Athens, J. W., Raab, S. O., Haab, O. P., Boggs, D. R., Ashenbrucker, H., a novel sialylated fucosyl glycolipid from CML cells. However, Cartwright, G. E., and Wintrobe, M. M. Leukokinetic studies: blood granu- none of the glycans in these structures contained the key sub locyte kinetics in chronic myelocytic leukemia. J. Clin. Invest., 44: 765-777, strate sequence Gal/31-3GalNAc for the a(2-3)-sialyltransfer- 1965. 2. Baker, M. A., Taub, R. N., Whelton, C. H., and Hindenburg, A. 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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1990 American Association for Cancer Research. Human Leukemic Myeloblasts and Myeloblastoid Cells Contain the Enzyme Cytidine 5 ′-Monophosphate-N-acetylneuraminic Acid:Gal β1-3GalNacα(2−3)-sialyltransferase

Amita Kanani, D. Robert Sutherland, Eitan Fibach, et al.

Cancer Res 1990;50:5003-5007.

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