Download Human Leukemic Myeloblasts and Myeloblastoid Cells Contain the

[CANCER RESEARCH 50. 5003-5007. August 15. 1990]
Human Leukemic Myeloblasts and Myeloblastoid Cells Contain the Enzyme
Cytidine 5 -Monophosphate-jV-acetylneuraminic Acid:Galßl3GalNAca(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
We have examined the role of CMP-NeuAc:Gal£I-3GalNAc-R o(23)-sialyltransferase in fresh leukemia cells and leukemia-derived cell
lines. Enzyme activity in normal granulocytes using Gal$l-3GalNAcao-nitrophenyl as substrate was 1.5 ±0.7 nmol/mg/h whereas activity in
morphologically mature granulocytes from 6 patients with chronic myelogenous leukemia (CML) was 4.2 ±1.6 nmol/mg/h (/' < 0.05). Myelo
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
and granulocytes were obtained, with higher enzyme activity in the
patients' blasts (7.1 nmol/mg/h) than in their granulocytes (4.9 nmol/mg/
h) in both cases, suggesting that the increase in enzyme activity is related
to the differentiation or proliferation status of the CML cells. However,
similarly high enzyme levels were also seen in myeloblasts from acute
myeloblastic leukemia patients (5.6 ±1.4nmol/mg/h) and in some acute
myeloblastic leukemia-derived cell lines (KGla and 111,60). suggesting
that increased levels of this enzyme are not directly correlated with the
presence of the Ph1 chromosome. This a(2-3)-sialyltransferase activity
can also be detected in normal peripheral blood lymphocytes and exhibits
increased activity in chronic lymphocytic leukemia cells and acute lyinphoblastic leukemia. These data suggest that the level of enzyme activity
may vary with growth rate and maturation status in myeloid and lymphoid
hemopoietic cells. Finally, we have identified a glycoprotein in acute
myeloblastic leukemia cells that serves as a substrate for the a(2-3)sialyltransferase. The desialylated form of the glycoprotein was resialylated in vitro by the purified placenta! form of this a(2-3)-sialyltransferase and exhibits a molecular weight of about 150,000.
INTRODUCTION
CML3 is characterized by early release of myeloid cells from
bone marrow into the peripheral blood and a marked increase
in the circulation time of the leukemic granulocytes (1). We
and others have shown that CML cell membranes are more
highly sialylated than normal granulocyte membranes (2-4).
Consistent with these data is the observation that the binding
of the galactose-specific lectin of Ricinus communis (RCA 1) to
CML granulocytes is significantly increased after neuraminidase treatment (5). In vitro studies of granulocyte function in
Received 11/7/89; revised 3/16/90.
The costs of publication of this article were defrayed in part by the payment
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.
1Supported by the Medical Research Council of Canada; The National Cancer
Institute of Canada; National Cancer Institute Grants CA31762 and CA35329;
the William J. Matheson Foundation; and the Canadian Cystic Fibrosis Foun
dation.
2 To whom requests for reprints should be addressed, at Toronto General
Hospital, Mulock Larkin Wing 1-005, 200 Elizabeth Street. Toronto. Ontario,
M5G 2C4, Canada.
3The abbreviations used are: CML, chronic myelogenous leukemia; GalNAc,
/V-acetylgalactosamine; NeuAc, yV-acetylneuraminic acid; GlcNAc, iV-acetylglucosamine; CMP-NeuAc, cytidine monophosphate A'-acetylneuraminic acid; LJDPGal, uridine diphosphate galactose; ONP, o-nitrophenyl; PNP, p-nitrophenyl;
AML, acute myeloblastic leukemia; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; PNA, peanut agglutinin.
CML compared to normals have demonstrated decreased ad
hesiveness (6), decreased chemotaxis (7), and reduced mem
brane binding of the chemotactic peptide /V-formylmethionylleucylphenylalanine (8). These altered functions are partially
reversible by removal of membrane sialic acid with neuraminidase, suggesting a role for aberrant sialylation in the abnormal
cell behavior (8). Sialic acids are common constituents of both
the O- and /V-linked glycan chains of glycoproteins (9), as well
as of many glycolipids (10). They are found in a variety of
linkage patterns to galactose, GalNAc, GlcNAc, or other
NeuAc moieties. Nevertheless, there seem to be many more
sialyltransferases than there are sialic acid linkages, thus sup
porting the contention (11) that the activity of these enzymes
in various tissues is probably largely regulated by the strict
substrate specificity of each sialyltransferase. For example, the
a(2-3)-sialyltransferases
(EC 2.4.99.4) which have been puri
fied from porcine submaxillary gland (12, 13) and human
placenta (14), specifically sialylate the galactosyl residue of
Gal01-3GalNAc-R via an «2-3linkage but cannot synthesize
the NeuAca2-3Gal/31-4GlcNAc-R
product. These sialyl transferases will also use the gangliosides GMiaand GDibas substrates
(13, 14) as well as asialo-GMi, since these glycolipids contain
the required unsubstituted Gal/3l-3GalNAc-R
sequence (re
viewed in Ref. 15).
Lectin studies utilizing peanut agglutinin, which binds most
avidly to Gal01-3GalNAc moieties, have suggested that the
aberrant sialylation in CML cell membranes occurs on O-linked
glycans (6). We have shown previously that an enzyme which
specifically catalyzes the synthesis of NeuAca2-3 Gal/31-3
GalNAc-R is present in human granulocytes and has increased
activity in CML granulocytes, possibly accounting for the aber
rant sialylation and playing a pathophysiological role in CML
(16).
CML granulocytes may represent a population of cells less
mature than normal granulocytes, in which case the increased
a(2-3)-sialytransferase would reflect relative immaturity of the
leukemic cells. Myeloid cells exhibiting a less differentiated
phenotype are readily available from both CML patients (in
myeloid blast crisis) as well as from patients with acute myelo
blastic leukemia. Relatively undifferentiated cells are also avail
able in the form of leukemia-derived cell lines, some of which,
e.g., K562 (17), EM2, and EM3 (18) were derived from Ph1positive leukemic blasts. Thus we have studied the levels of
sialyltransferase activity in fresh leukemia samples as well as
leukemia-derived cell lines of both myeloid and lymphoid line
ages. Finally, we have attempted to identify some of the glyco
proteins which serve as the natural substrates for this enzyme
in intact cells.
MATERIALS
AND
METHODS
Cells and Cell Lines. More than 95% morphologically mature gran
ulocytes from both CML and normal samples were obtained from
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SIALYLTRANSFERASES
IN HUMAN LEUKEMIC CELLS
EDTA-anticoagulated peripheral venous blood after dextran sedimen
tation, ammonium chloride lysis, and layering of leukocytes onto a
double gradient of Hypaque and dextran, as described previously (6,
19). Leukemic blast cells (as defined by hematological and phenotypic
criteria) were obtained from patients with acute leukemia and CM L in
myeloid blast crisis having greater than 70% blasts in the peripheral
blood. Mononuclear cell fractions were prepared from heparinized
peripheral venous blood by Ficoll-Hypaque density gradient centrifugation as reported previously (19). Hematopoietic cell lines were ob
tained from the American Tissue Culture Collection (Rockville, MD)
or from colleagues in Toronto. Cell lines were maintained in RPMI
1640 with 10% heat-inactivated fetal calf serum in a 5% CO2 atmos
phere at 37°C.All cells and cell lines were washed 3 times in saline and
stored at -70°Cas pellets of 2 x 10s cells.
Sialyltransferase Assays. Enzyme assays were set up as previously
described (8, 19). Briefly, frozen pellets of 2 x 10* cells were resuspended in 2 ml of 0.2 M NaCl, washed in saline, treated with 10 units
of DNase (Sigma, St. Louis, MO), washed again in saline, and solubilized in 1% Triton X-100 at 4°Cfor 20 min. Debris was removed by
centrifugation at 1000 x g for 10 min. The supernatant, a solubilized
total cell membrane preparation, was used for the assays. Aliquots (20
M!)of this supernatant (typically containing 40-60 pg of protein) were
incubated with 10 p\ of 0.05 M Tris-HCI (pH 7.2), 10 n\ of 0.5 mM
CMP-[4-'"C]NeuAc (0.06 ^Ci, 25 mCi/mmol; New England Nuclear),
and 10 M' of the substrate Gal/3l-3GalNAc-a-ONP
(2 mM final con
centration). The Km for CMP-[4-'4C]NeuAc was previously shown to
be 0.18 mM, and that for Gal/3l-3GalNAc-«-ONP was 0.3 mM (16).
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)
of Dowex AG 2-X8, Cl~ form, 200-400 mesh (Bio-Rad Laboratories)
as described previously (16). Briefly, the Dowex columns were poured
with 3 M NaCl and extensively washed with 15 mM Tris-HCI, pH 7.0,
at 4°C.The first 5 ml of eluate contained all the labeled product.
Products were counted by liquid scintillation using ACS (Amersham)
as scintillation fluid. Endogenous acceptor controls showed less than
5% incorporation relative to exogenous acceptor assays; this incorpo
ration was subtracted in calculation of enzyme activities. In other
experiments where either Gal/31-3GalNAc-«-ONP
or Gal/313GalNAc-a-PNP were used as substrates, radioactive products were
separated by high voltage electrophoresis. The reaction mixtures were
spotted in parallel lanes onto Whatman No. 3M paper (18 x 22 inches),
premoistened with sodium tetraborate, pH 9.1. Electrophoresis was
performed at 1.5 kV and 170 mA for 90 min. The paper was air dried
and individual "lanes" were cut into strips. The strips were cut into 1inch pieces and counted in Beckman Redi-Solv E.P. scintillation fluid.
Control samples lacking synthetic acceptor were performed in duplicate
along with each test set. The results of each assay are expressed as the
net difference in counts between the mean test and the control.
Galactosyltransferase Assay. An aliquot of the same solubilized cell
membrane preparation as used for the Sialyltransferase assay was used
to measure the activity of the enzyme UDP-Gal:GalNAc /33-gaIactosyltransferase (EC 2.4.1.122). This transferase will catalyze the addition
of galactose (in /31-3 linkage) from UDP-Gal to the terminal GalNAc
residue of either high or low molecular weight substrates. The acceptor
used for this assay was asialo-ovine submaxillary mucin (20). Enzyme
incubation mixtures comprised 20 ^1 cell lysate; 10 pi 0.2 M 1-(Nmorpholino)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
England Nuclear); 40 mM MnCl2; and 20 ^1 asialo-ovine submaxillary
mucin (equivalent to 3.2 mM GalNAc). Control incubations lacked
acceptor. Reaction mixtures were incubated at 37°Cfor l h and the
reaction was terminated by the addition of 15% (w/v) trichloroacetic
acid and 5% (w/v) phosphotungstic acid and the resulting precipitate
was pelleted by centrifugation. The pellet was dissolved in 0.5 ml of
NCS (Amersham) for l h and the solubilized proteins were finally
incorporated into organic counting scintillation fluid. Glacial acetic
acid (7 n\) was added to the contents of each scintillation vial. After
incubation in the dark overnight to reduce chemiluminescence, the
samples were counted in a /3-scintillation counter. Enzyme activity was
expressed as nmol of ['4C]galactose transferred per mg of protein per h
of incubation as described previously (19). Endogenous acceptor con
trols showed less than 5% incorporation relative to exogenous acceptor
assays: this incorporation was subtracted in calculation of enzyme
activities.
In Vitro Sialylation of Natural Substrates. AML cells (2 x 10s) were
washed with phosphate-buffered saline, resuspended in 2 ml, and
treated with 100 units of neuraminidase (BDH Chemicals, Poole,
United Kingdom) for 30 min at 37°C.The cells were washed twice with
ice-cold phosphate-buffered saline and lysed in 1% Nonidet P-40 as
described previously (21, 22). The clarified cell lysate was passed
through a 2-ml column of peanut agglutinin agarose (Pharmacia). After
extensive washing away of unbound material, the bound glycoproteins
were slowly eluted with cell lysis buffer supplemented with 4% Dgalactose. The eluted fraction (10 ml) was dialyzed and concentrated
against 3- x 1-liter volumes of 0.01 M Tris-saline, pH 7.4. The glycoprotein solution was brought to 70% with ethanol and the proteins
were precipitated overnight at -70°C. The precipitated glycoprotein
fraction was pelleted at 10,000 x g for 10 min. The pellet was dissolved
in 0.01 M Tris-saline, pH 7.4, containing 0.5% Nonidet P-40, 1 mM
Mg2*and Mn2*. Solubilized glycoprotein solution (50 u\) was incubated
with 10 Ã-Ã-Ci
CMP-['"C]sialic acid diluted as above and 0.05 milliunit of
purified placenta! 2-3-sialyltransferase for l h at 37°C.The reaction
was terminated by addition of 25 Ml(x5) SDS-PAGE gel sample buffer.
The reaction tubes were boiled and the products were resolved by SDSPAGE and fluorography (22). Similar experiments were performed
using CML granulocytes.
RESULTS
In the first series of experiments, levels of a(2-3)-sialyltransferase activity were measured in granulocytes from 6 normal
volunteers, granulocytes from 6 patients with CML in chronic
phase, and myeloblasts from 5 patients with CML in myeloid
blast crisis (Table 1). Using the synthetic substrate Gal/313GalNAc-ONP, which is a specific acceptor for this Sialyltrans
ferase, enzyme activities were 1.5 ±0.7 (SD) nmol/mg/h in
normal granulocytes, 4.2 ±1.6 nmol/mg/h in CML granulo
cytes, and 6.7 ±2.2 nmol/mg/h in CML myeloblasts, suggest
ing a hierarchy in which leukemic blast cells have higher enzyme
levels than leukemic granulocytes, which in turn have higher
enzyme levels than normal granulocytes. In 2 patients with
CML blast crisis, individual myeloblast and granulocyte prep
arations were obtained. Higher Sialyltransferase activity was
observed in the patients' blasts than their granulocytes in both
cases (Table 1).
To study the distribution of this enzyme in other cell types
of the hematopoietic system, a number of lymphoid and myeloid-derived cell lines were collected and assayed for «(2-3)sialyltransferase activity. In these preliminary experiments, a
Table 1 Sialyltransferase activity in granulocytes from CML in chronic phase,
blast crisis, and normal donors
Source of cells
Normal granulocytes
CML granulocytes
CML
blastsCML
No. of samples
Sialyltransferase
activity (nmol/mg/hy
6
5Granulocytes
4.2 ±1.6
6.7 ±
2.25.5
O.CMLpatient M.
BlastsGranulocytes
8.94.25.2
patient F. A.6*
Blasts1.5±0.7CJ'
" Substrate used was Gal/3l-3GalNAc-o-O-nitrophenyl
at 2 mM.
* Granulocytes were tested from 12 normal donors pooled in pairs to obtain
sufficient cells for assay.
' P < 0.05 for normal granulocytes compared to CML untreated or CML
blasts.
" Mean ±SD.
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SIALYLTRANSFERASES
IN HUMAN LEUKEMIC CELLS
range of enzyme activity levels was detected ranging from 4.5
nmol/mg/h for KG la cells to only 0.3 nmol/mg/h for the Blymphoma-derived Daudi cell line. Significantly, however, there
were no differences in c*(2-3)-sialyltransferase activity between
those cell lines which contained the Ph1 chromosome (K562,
Table 3 ß3-galactosyltransferaseactivity in leukemic cells
CellsNormal
no.°6
transferase
activity (nmol/mg/h)
usingasialo-OSM*14.2
±3.0C
granulocytes
10.3 ±4.0
CML granulocytes
6
CML blasts
55/33-Galactosyl 14.2 ±6.0
20.5 ±9.2
AML blastsSample
" Normal granulocytes were tested from 12 normal donors pooled in pairs to
obtain sufficient cells for assay.
4 Substrate used was asialo-ovine submaxillary mucin (20) at 3.2 itiM (GalNAc)
concentration.
c Mean ±SD.
EM2, and EM3) and those which did not (KG1, KG la, HL60,
U937, Nalm-6, HOON, Daudi, GH-1, HSB-2, and Jurkat)
(data not shown).
In the next series of experiments, a large number of chronic
and acute leukemia samples of both lymphoid and myeloid
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
200loblasts of 8 CML blast crisis samples (6.2 ±2.2 nmol/mg/h).
In cells of the lymphoid lineage, unstimulated peripheral
blood lymphocytes contained relatively low levels of the «(23)-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 lympho92cytic leukemia patients contained increased levels of this transferase 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/
680
h), once again indicating a hierarchy in which leukemic lymphoblasts 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
43of enzyme activity were found in the samples preceding a period
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
C s D
A s B
observed (Table 2B).
Fig. 1. Fluorograph of desialylated PNA-4b-affinity-purified glycoproteins
after resialylation in vitro with CMP-[14C]sialic acid and placenta! a(2-3)-sialyl/33-Galactosyltransferase activity was determined in normal
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
neuraminidase treatment; Track D, PNA unbound fraction from same CML cell
granulocytes and myeloblasts from CML patients in blast crisis
lysate. Track s, radiolabeled molecular weight standards. Arrow, position of
exhibited very similar ß-3-galactosyltransferase activities (14.2
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 siahigher 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
1, Track B). When a CML cell lysate was used, a resialylated
agglutinin bound/eluted fraction of a neuraminidase-treated
product was not detected in either the PNA bound fraction
(Fig. 1, Track C) or the PNA unbound fraction (Fig. 1, Track
Table 2 a(2-3)-Sialyltransferase activity" in leukemia cells
D).
A. Untreated patients
CML granulocytes
CML myeloblasts
AML myeloblasts
CLL lymphocytes
ALL lymphoblastsNo.
B. Treated patients
CML granulocytes
CLL lymphocytes
of patient
samples12
activity" (nmol/mg/h)4.1
817
6.25.6
DISCUSSION
44Sialyltransferase 3.76
4.4
6
4
1.76
2.2
C. Normal controls
Granulocytes
3
1.3
Peripheral blood lymphocytes
3
1.2
* Mean sialyltransferase activity expressed as nmol/mg/h using the substrates
Gal/3l-3GalNAc-ONP or Gal01-3GalNAc-PNP.
We have shown previously that CMP-NeuAc:Gal/313GalNAc-R a(2-3)-sialyltransferase
is present in human leu
kocytes and has increased activity in granulocytes from patients
with chronic myelogenous leukemia (16). The human placental
form of this enzyme which ¡ssimilar, if not identical to the
leukocyte form,4 exhibits relatively restricted substrate specific
ity and will efficiently catalyze the addition of NeuAc in a(23) linkage to unsubstituted Gal01-3GalNAc-R structures (14).
In our previous studies (16), evidence was not found for the
' I. Brockhausen et al., manuscript in preparation.
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SIALYLTRANSFERASES
IN HUMAN LEUKEMIC CELLS
presence of either the GalNAc a-protein a(2-6)-sialyltransferase (EC 2.4.99.3) or the (NeuAca2-3Gal/31-3)GalNAca-R
a(2-6)-sialyltransferase
(EC 2.4.99.7), neither of which can
sialylate the Gal/3l-3GalNAc-a-ONP
(23, 24) in CML or nor
mal granulocytes. Although the trisaccharide product generated
by the action of the a(2-3)-sialyltransferase
can theoretically
be further sialylated by the subsequent action of the latter a(26)-siaIyltransferase (EC 2.4.99.7) (23-25), it was concluded that
the <*(2-6) enzymes are unlikely to be responsible for the
apparent hypersialylation of CML plasma membrane glycoproteins compared to those of normal granulocytes (16). Since
circulating granulocytes in CML are thought to be of immature
phenotype (26), we have hypothesized that the hypersialylation
of CML cell membranes may reflect their relative immaturity
(8). In support of this notion, we have shown here that this
a(2-3)-sialyltransferase activity is further increased in the myeloblasts from CML blast crisis patients, compared to granu
locytes isolated from the same or other CML patients. In
contrast, the 03-galactosyl (EC 2.4.1.122) transferase activity
synthesizing the Gal0(l-3)GalNAc
a-R acceptor substrate
structure for the a(2-3)-sialyltransferase
did not differ signifi
cantly between normal granulocytes, CML granulocytes, and
CML blasts. These data initially indicated a potential correla
tion between cell maturation or growth rate and the activity of
this a(2-3)-sialyltransferase in CML.
The cells of over 90% of CML patients contain a specific
chromosomal abnormality, the Philadelphia or Ph1 chromo
some; therefore it was of interest to assess its role, if any, in
modulating enzyme activities. However, several pieces of data
presented herein indicate that the increase in sialyltransferase
activity in CML granulocytes and myeloblasts is unlikely to be
due solely to the presence of a Ph1 chromosome: (a) myeloblastoid and lymphoblastoid cell lines exhibited a range of increased
enzyme activities irrespective of whether they were CML de
rived (EM-2, EM-3, and K562) or not; (b) myeloblasts of all
untreated AML patients contained elevated levels of sialyltrans
ferase activity. Interestingly, these levels were similar to those
exhibited by myeloblasts from CML patients in myeloid blast
crisis with which they share significant hematological and phenotypic characteristics. Subsequently, a lower level of a(2-3)sialyltransferase activity was also detected in normal peripheral
blood lymphocytes, with increased activity in lymphocytic and
lymphoblastic leukemias; (c) the presence of a Ph1 chromosome
in CML cells and in CML-derived cell lines is overwhelmingly
correlated
with the expression
of the protein called
p210bcr~abl
which exhibits tyrosine-specific kinase activity in vitro
(27, 28). However, we did not find the p210bcrabl) kinase from
K.562 cells to be capable of phosphorylating the purified placental form of the (2-3)-sialyltransferase
in vitro (data not
shown).
Several structures including N- and O-glycosylated glycoproteins, and glycolipids have been shown to be more highly
sialylated in CML cell membranes compared to normal granulocyte membranes. Fukuda et al. (29, 30) have described a
series of TV-linked polylactosaminoglycans in both normal and
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
contained polylactosamine structures and, additionally, isolated
a novel sialylated fucosyl glycolipid from CML cells. However,
none of the glycans in these structures contained the key sub
strate sequence Gal/31-3GalNAc for the a(2-3)-sialyltransferase of this study. Recently, the same group has described a
family of glycoproteins, called leukosialins/CD43 (32). These
glycoproteins contain a common polypeptide which can be
differentially O-glycosylated in the many different cell types
which constitute the hemopoietic system. Studies of both leu
kemic and normal myeloid cells indicate that leukosialins con
tain several potential substrate structures for the a(2-3)-sialyltransferase (3). Compared to those of normal granulocytes, the
leukosialins isolated from immature myeloblasts contained
mainly short O-linked oligosaccharides, including NeuAca2-3
Gal,81-3GalNAc and NeuAccv2-3Gal01-3(Neu
Ac«2-6)GalNAc. The detection of those structures in immature myeloid
cells correlates well with our observations that such cells contain
significantly elevated levels of the a(2-3)-sialyltransferase.
Since AML cells and myeloblasts from CML patients in blast
crisis contained the highest levels of this a(2-3)-sialyltransferase, we tried to isolate natural substrates from those cells, i.e.,
glycoproteins containing the substrate sequence Gal/313GalNAc-R. Thus, a neuraminidase-treated,
PNA-agarose
bound glycoprotein fraction was prepared and was shown to
contain a substrate molecule for the purified placenta! form of
this a(2-3)-sialyltransferase
(14). After in vitro resialylation,
the product of this catalysis was resolved as a broad band both
a molecular weight of approximately 150,000 in reduce SDSPAGE. Since many of the leukosialin molecules exhibit a
similar apparent size in SDS-PAGE (32), particularly after
desialylation (22), it is possible that this M, 150,000 structure
may be a member of the leukosialin family. No substrate
structures could be isolated from CML granulocytes using
similar procedures. A possible explanation for this is that the
lysis of granulocytes under the conditions we used here results
in the release of proteases from lysosomal vesicles which destroy
the substrate molecule(s) before they can be isolated on the
PNA lectin column. More likely explanations are that since
leukosialins in CML cells contain longer, more complex, and/
or branched O-glycans than those in AML cells (3), the desialylated forms of these leukosialins would not bind to the Gal/313GalNAc-R-specific PNA. Furthermore, these desialylated
structures would not be good substrates for the a(2-3)-sialyltransferase. The biochemical nature of O-linked sialylated sur
face glycoproteins, leukosialins, or sialophorins may be impor
tant in the physiology of leukocyte recirculation (33). The
increase in expression of a(2-3)-sialyltransferase
in myeloid
blasts and the decrease in activity with maturity to normal
granulocytes may reflect a physiological role for O-linked sialylation in adhesion and receptor binding. The permanence of
this otherwise transient change in leukemic myeloid cells may
contribute to this abnormal pathophysiology (8). It may be of
interest that the a(2-3)-sialyltransferase enzyme is encoded by
a gene on chromosome 11 suggesting a role for this chromo
some site in normal myeloid differentiation and leukemic
pathophysiology (34).
ACKNOWLEDGMENTS
The authors thank Professor H. Shachter of the Hospital for Sick
Children, Toronto, for his helpful suggestions and encouragement, and
Claire Guiver-Bond, for her infinite patience throughout the revisions.
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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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