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(Received for publication, September 26, 1995; and in revised form, November 30, 1995) From the
Treatment of mouse teratocarcinoma F9 cells with
all-trans-retinoic acid (RA) causes a 9-fold increase in
steady-state levels of mRNA for UDP-Gal:
Retinoids, which are natural and synthetic analogs of vitamin A,
alter in vitro growth and differentiation of a variety of
normal and neoplastic cell
lines(1, 2, 3, 4, 5) .
Profound changes in gene expression and pattern formation during
embryogenesis are regulated by retinoic acid (RA) ( F9 cells contain many surface carbohydrate antigens that are shared
with those in early mouse
embryos(17, 18, 19, 20, 21, 22) .
Surface glycoconjugates in F9 cells contain the repeating disaccharide
[3Gal Cell extracts from both F9 and RA-treated F9 cells (RA/F9 cells)
contain a UDP-Gal: We have now examined the steady-state levels of the
Reaction mixtures for the
Figure 1:
Kinetics of
It has been reported that
dbcAMP enhances the transcriptional activation of genes by
RA(44) . Therefore, we tested the combined effects of RA and
dbcAMP on the changes induced in the transcript for the
Figure 2:
Expression of
Figure 3:
Nuclear run-on analysis of
Figure 4:
Time course for RA-dependent induction of
In preliminary experiments, we found a considerable amount of
activity of the The To assess whether the increase in
Figure 5:
Effects of RA on the production of laminin
in the F9 cell culture media. F9 cells were grown in the absence of RA (open circles) or in media containing 10
Figure 6:
GS
I-B
Differentiation of F9 cells induced
by RA is also accompanied by a large increase in the terminal
To test for the possibility of masking of
Le
Figure 7:
Flow-cytometric analysis of F9 and RA/F9
cells for surface expression of Le
After treatment of F9 cells with To gain a full understanding of the roles of glycoconjugates
in development and differentiation and in disease conditions, it is
important to define the mechanisms regulating expression of
glycosyltransferases and their cognate glycoconjugate structures. In
many ways, F9 cells are ideal for studying these changes, since new
glycoconjugate expression is inducible by RA within 3 days in the
irreversibly differentiated cells. We previously reported that F9 cells
have an Although it is
appreciated that expression of glycosyltransferases in some cases is
regulated during cellular differentiation and transformation, little is
understood about the mechanisms controlling enzyme
expression(66, 67, 68) . Two of the best
studied glycosyltransferases in this respect are the sialyltransferases (69) and the There are several cases in
vitro where treatments of cells with various agents appears to
alter the levels of glycosyltransferases (76, 77, 78, 79, 80, 81, 82, 83) .
In regard to the The activity of some enzymes appears to
decline in F9 cells after RA treatment. The N-acetylgalactosaminyltransferase activity that converts
globoside to Forssman glycosphingolipid declines by approximately 70%
in F9 cells within 3 days following RA treatment (87) .
Treatment of F9 cells for 5 days with RA and dibutyryl cAMP results in
an approximately 80% decline in the activity of an The time course of induction of
We observed that there is significant
Although the
full-length transcript for the murine There
are many studies demonstrating that expression of cell surface
carbohydrates is developmentally regulated, but the mechanisms of
regulation are largely unknown. Our finding that Le The observed masking of Le The results of this study demonstrate that
differentiation of F9 cells into parietal endoderm-like cells is
accompanied by a profound increase in
Volume 271,
Number 6,
Issue of February 9, 1996 pp. 3238-3246
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
1,3-Galactosyltransferase in Embryonal Carcinoma
Cells by Retinoic Acid
MASKING OF LEWIS X ANTIGENS BY 
-GALACTOSYLATION (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-D-Gal
1,3-galactosyltransferase (1,3GT) beginning at 36 h. Enzyme
activity rises in a similar fashion, which also parallels the induction
of laminin and type IV collagen. Nuclear run-on assays indicate that
this increase in 1,3GT in RA-treated F9 cells, like that of type
IV collagen, is transcriptionally regulated. Differentiation also
results in increased secretion of soluble 1,3GT activity into the
growth media. The major -galactosylated glycoprotein present in
the media of RA-treated F9 cells, but not of untreated cells, was
identified as laminin. Differentiation of F9 cells is accompanied by an
increase in -galactosylation of membrane glycoproteins and a
decrease in expression of the stage-specific embryonic antigen, SSEA-1
(also known as the Lewis X antigen or Le
), which has the
structure Gal1-4(Fuc1-3)GlcNAc1-R. However,
flow cytometric analyses with specific antibodies and lectins,
following treatment of cells with -galactosidase, demonstrate that
differentiated cells contain Le antigens that are masked by
-galactosylation. Thus, RA induces 1,3GT at the
transcriptional level, resulting in major alterations in the surface
phenotype of the cells and masking of Le antigens.
)via its
interactions with retinoic acid binding proteins and transcription
factors(6, 7, 8, 9, 10) . A
model system for studying such changes related to cellular
differentiation and development has been the mouse teratocarcinoma cell
line F9(11) . These cells exhibit characteristics of embryonic
cells from the inner cell mass of the blastula stage. When F9 cells are
treated with all-trans-retinoic acid, within 3 days they
differentiate into primitive endoderm-like cells, which begin to
synthesize basement membrane proteins, including type IV collagen and
laminin(11, 12, 13, 14, 15, 16) . 1-4GlcNAc1]
or
poly-N-acetyllactosamine sequence (23, 24, 25) and the stage-specific embryonic
antigen SSEA-1 (also known as the Lewis X antigen or Le),
which has the structure
Gal1-4(Fuc1-3)GlcNAc1-R(17, 26) .
Differentiation of F9 cells induced by RA causes changes in expression
of surface glycoconjugates, and within 3 days following RA treatment
there is a marked decrease in expression of the Le antigen(14, 19, 27) . However, the
mechanism by which RA/F9 cells express less Le antigen is
unclear. Although it has been shown that 5 days following RA treatment
of F9 cells there is an 80% decrease in the level of an
1,3-fucosyltransferase activity that can synthesize the Le antigen(28) , there is little change in this enzyme
activity following 3 days of RA treatment(29) . These changes
in Le expression in differentiated F9 cells are relevant to
events occurring during early embryogenesis, since there are notable
changes in expression of Le antigens during normal
differentiation of mouse embryos, and some studies have suggested that
Le antigens play important roles in cell-cell adhesion and
development(30, 31, 32, 33) . -D-Gal 1,3-galactosyltransferase
(1,3GT) activity that synthesizes the terminal sequence
Gal1-3Gal-R(29, 34) . The presence of
this enzyme is consistent with studies suggesting that glycoproteins
from F9 and RA/F9 cells contain terminal -galactosyl
residues(29, 35, 36) . However, the activity
of the 1,3GT is higher in RA/F9 cells, and there is an apparent
increase in -galactosylation of surface glycoconjugates induced by
RA(29) . Little information is available about the induction of
the 1,3GT by RA, the glycosylation of proteins accompanying this
induction, and whether there is a relationship between changes in the
expression of terminal -galactosyl residues on cellular
glycoproteins and the apparent decrease in expression of Le antigens.1,3GT transcript during differentiation by RA and the enzymatic
activities in both cell extracts and cell culture media following RA
treatment. Our findings demonstrate that (i) 1,3GT is
transcriptionally regulated by RA treatment; (ii) differentiation of F9
cells results in increased activity of the enzyme and increased
expression of terminal -galactosyl residues on selected cellular
glycoproteins; and (iii) the apparent loss of the Le antigens on cell surface glycoconjugates in RA/F9 cells is in
part due to masking by terminal -galactosyl residues.
Materials
Restriction enzymes, Escherichia
coli tRNA, RNase-free DNase I, proteinase K, and green coffee bean
-galactosidase were purchased from Boehringer Mannheim. Sonicated
salmon sperm DNA, Bluescript phagemid pBSIISK(
) and
murine -actin cDNA cloned in Bluescript SK()
phagemid were obtained from Stratagene. RNase inhibitor was purchased
from Promega. UDP-[6-
H]galactose (15 Ci/mmol) was
purchased from American Radiolabeled Chemicals Inc. (St. Louis, MO). 
P-Labeled radioisotopes were from DuPont NEN. Murine type
IV procollagen (1) in pBluescript (37) was obtained from
American Type Culture Collection. Bovine serum albumin, UDP-Gal,
raffinose, EDTA, N-acetyllactosamine, GlcNAc, ATP, D-galactono-1,4-lactone, rabbit anti-mouse EHS laminin, p-nitrophenyl phosphate, Tween 20, phenylmethylsulfonyl
fluoride, pepstatin, aprotinin, leupeptin, streptavidin-agarose, and
protein A-Sepharose were obtained from Sigma. Mouse EHS laminin was
obtained from Upstate Biotechnology Inc. Alkaline
phosphatase-conjugated goat anti-rabbit IgG, Sulfo-NHS biotin, and BCA
protein assay kit were obtained from Pierce. Alkaline phosphatase color
developing kit, gelatin, and Triton X-100 were obtained from Bio-Rad.
ECL Western blotting kit was purchased from Amersham Corp. Griffonia simplicifolia I-B
(GS I-B)
was obtained from Vector Laboratories, Inc. (Burlingame, CA).
Phycoerythrin-streptavidin and fluorescein isothiocyanate-conjugated
anti-Le monoclonal antibody (CD15) were purchased from
Becton Dickinson (San Jose, CA). Tissue culture reagents were obtained
from Life Technologies, Inc. All-trans-retinoic acid was
purchased from Eastman Kodak Co. All other chemicals used were of the
highest grade available.Cell Culture
Mouse teratocarcinoma F9 cells were
cultured in Dulbecco's modified Eagle's medium containing
15% fetal calf serum on gelatin-coated tissue culture plates as
described(12) . For induction of differentiation, cells were
grown under identical conditions in media supplemented with either
10
M RA alone or 10M RA plus 10
M dibutyryl
cyclic AMP (dbcAMP) for 3 days. Differentiation was assessed by
measuring the amount of laminin secreted in the cell culture media
using a solid-phase assay described below. Chinese hamster ovary (CHO)
cells were cultured in -minimal essential media containing 10%
fetal calf serum.RNA Blot Analysis
Total cellular RNA was isolated
from cultured cells essentially as described(38) . For Northern
blot analysis, RNA samples were transferred to nylon membranes
(Schleicher & Schuell) by capillary action in 20 
SSPE for
16 h and immobilized by UV cross-linking (Autocrosslink on Stratalinker
2400). The filters were prehybridized for at least 4 h in a solution
containing 50% formamide, 5 SSPE (0.18 M NaCl, 10
mM sodium phosphate, pH 7.7, 1 mM EDTA),
phycoerythrin (250 mM Tris, pH 7.5, 25 mM EDTA, 0.5%
sodium pyrophosphate, 5% SDS, 1% polyvinylpyrrolidone, 1% Ficoll 400,
and 1% bovine serum albumin), and 150 µg/ml denatured salmon sperm
DNA. RNA bound to nylon membrane was hybridized with P-labeled cDNA inserts from murine 1,3GT clone
PCDM7-GT(34) , murine -actin cDNA, and rat tubulin
cDNA (a gift from Dr. Kelley Moremen, University of Georgia). The RNA
blots were hybridized separately with the individual probes.
Hybridization was carried out at 42 °C overnight in the
prehybridization solution using a hybridization incubator (Robbins
Scientific, Sunnyvale, CA). The filters were washed twice in 6
SSPE, 0.5% SDS at room temperature for 15 min, twice in 1 SSPE,
0.1% SDS at 42 °C for 15 min, and three times in 0.1 SSPE,
0.1% SDS at 65 °C for 15 min. Hybridization was visualized by
autoradiography, using Kodak XAR-5 film (Kodak). After development,
exposed areas of film were quantified by densitometry (Molecular
Dynamics model 3008 computing densitometer). The signals were
normalized for variations in the amount and quality of RNA using actin
mRNA or tubulin mRNA as internal controls.Nuclear Run-on Assays
Nuclei were isolated from F9
or RA/F9 cells as described (39) and run-on transcription
reactions were carried out as described (40) but with the
following modifications. Transcription was initiated by incubating 0.2
ml of nuclei (2-3 10
nuclei) with 12.5 µl
of [-P]UTP (800 Ci/mmol, 10 µCi/µl)
in a 0.4-ml reaction volume containing 126 µl of reaction buffer
(10 mM Tris-Cl, pH 7.5, containing 35% glycerol, 5 mM MgCl
, and 300 mM KCl), 20 µl of 10 mM GTP, CTP, ATP, respectively, 1 µl of 0.5 M dithiothreitol, and 80 units of RNase inhibitor. After mixing the
components, the reactions were incubated at 30 °C for 40 min with
shaking. RNase-free DNase I (50 units) was added with 22.5 µl of 20
mM CaCl. After 30 min incubation at 37 °C, 5
µl of 16 mg/ml proteinase K and 47.25 µl of proteinase K buffer
(5% SDS, 50 mM EDTA, 100 mM Tris, pH 7.4) was added
along with the 10 µl of 10 mg/ml yeast tRNA, and the samples were
incubated at 37 °C for 30 min. The P-labeled
transcripts were resuspended in 0.5% SDS and hybridized onto DNA
fragments immobilized on nylon membranes at 65 °C for 36 h.Glycosyltransferase Assays
Cell extracts were
prepared as described (29) in the presence of protease
inhibitors, phenylmethylsulfonyl fluoride (1 mM), pepstatin (1
µg/ml), aprotinin (10 µg/ml), and leupeptin (10 µg/ml).
Concentrated cell culture media were prepared by centrifugation at
100,000 g for 60 min, and the supernatant was
concentrated using centriprep-10 concentrators (Amicon Inc.). 1,3GT routinely contained 30 mMN-acetyllactosamine, 20 mM MnCl, 0.5
mM UDP-[H]Gal (35,000 cpm/nmol), 5
mM ATP, 50 mMD-galactono-1,4-lactone, and
about 80 µg of cell extracts or 12.5 µl of 10-fold concentrated
cell culture media in a final volume of 25 µl. The reactions were
carried out at 37 °C for 3 h. The product of the enzyme reaction
was analyzed by Dionex high performance anion-exchange chromatography
using a Carbopac PA1 column (4 250 mm). The separation was
carried out in 160 mM sodium hydroxide for 15 min with a flow
rate of 1 ml/min. The fractions were collected and monitored by
scintillation counting. The radioactivity corresponding to the position
of standard Gal1-3Gal1-4GlcNAc was taken as the
product of 1,3GT. The values obtained from assays performed in the
absence of the acceptor were used as background. Assays for
UDP-Gal:GlcNAc 1,4-galactosyltransferase (1,4GT) were
performed as described for 1,3GT, except that 20 mM GlcNAc was used as an acceptor, and cell culture media centrifuged
at 100,000 g for 1 h were used as an enzyme source
without concentration. The product N-acetyllactosamine was
identified by high performance anion-exchange chromatography, as
described above.Detection of Laminin by Enzyme-linked Immunosorbent
Assay
The wells of Immulon 4 microtiter plates (Dynatech
Laboratories, Chantilly, VA) were coated at 4 °C overnight with 100
µl of either F9 or RA/F9 cell culture media or 100 µl of
varying concentrations of mouse EHS laminin in carbonate coating buffer
(150 mM NaCO, 348 mM NaHCO, and 0.02% NaN, pH 9.6). After
blocking with 5% BSA in PBS/NaN (PBS containing 0.02%
NaN), laminin was detected by incubating the wells with
rabbit anti-mouse EHS laminin (100 µl of 1:2,000 dilution) followed
by alkaline phosphatase-conjugated goat anti-rabbit IgG (100 µl of
1:4,000 dilution). Antibody solutions were prepared in dilution buffer
(PBS/NaN containing 0.1% BSA and 0.05% Tween 20), and
incubation was performed at room temperature for 1 h. The wells were
treated with 100 µl of a freshly prepared 1 mg/ml alkaline
phosphatase substrate p-nitrophenylphosphate in coating buffer
containing 1 mM MgCl at 37 °C. The optical
density at 405 nm of each well was recorded by V-Max kinetic microplate
reader (Molecular Devices Corp., Menlo Park, CA). Each assay was
performed in triplicate.Lectin Blotting
GS I-B was
biotinylated with Sulfo-NHS biotin according to the
manufacturer's instructions. Microsomes were prepared as
described (41) in the presence of protease inhibitors,
phenylmethylsulfonyl fluoride (1 mM), pepstatin (1 µg/ml),
aprotinin (10 µg/ml), and leupeptin (10 µg/ml).
Streptavidin-agarose was added to the microsomes to remove cellular
proteins that nonspecifically interact with streptavidin, and the
supernatant fraction was recovered following centrifugation at 5,000
g for 15 s. Total protein was determined by the BCA
protein assay. For the preparation of serum-free cell culture media, F9
and RA/F9 cells were grown in Dulbecco's modified Eagle's
medium containing 15% fetal calf serum for 3 days, as described above,
and the culture media were replaced with serum-free media. Media were
collected after 1 day and centrifuged at 100,000 g for
60 min. The supernatant (soluble proteins) was concentrated about
10-fold using a centriprep-10 concentrator. The microsomal fraction and
soluble proteins in culture media were subjected to electrophoresis on
10% SDS/polyacrylamide gels(42) . Proteins were transferred
electrophoretically onto nitrocellulose membranes (Hybond-ECL Western,
Amersham)(43) . Blots were blocked with TBS (20 mM Tris, 500 mM NaCl, pH 7.5), 5% BSA at room temperature
for 3 h, then incubated with 10 µg/ml of biotinylated GS I-B in dilution buffer (TBS, 0.1% BSA, 0.3% Tween 20) for 1 h.
Control lanes were incubated with a lectin solution containing 200
mM hapten sugar raffinose. Following three 15-min washes in
TTBS (TBS, 0.3% Tween 20), the lectin blots were incubated at room
temperature for 1 h with horseradish peroxidase-conjugated streptavidin
diluted 1:5,000 in dilution buffer. Blots were washed three times (15
min each) in TTBS followed by one wash in TBS and developed using the
enhanced chemiluminescence (ECL) Western blotting kit following the
manufacturer's instructions. Molecular weight determinations were
made using prestained high molecular mass standards (14.3-200
kDa, Life Technologies, Inc.).Immunoprecipitation of Laminin in RA/F9 Culture
Media
Serum-free RA/F9 cell culture media were prepared, as
described above, and 84 µl from each of these (corresponding to 400
µg of soluble protein) was incubated with 50 µl of protein
A-Sepharose at 4 °C for 1 h to remove components that react
nonspecifically with protein A. The supernatant was recovered by
centrifugation at 10,000 g for 15 min and incubated
with 5 µl of rabbit anti-mouse laminin antibody at 4 °C for 2
h. The mixture was subsequently incubated with 50 µl of protein
A-Sepharose for 1 h and centrifuged to pellet immunoprecipitated
complexes. The laminin-antibody complexes immobilized on the protein
A-Sepharose were washed five times with washing buffer (1% BSA, 0.05%
Tween 20, 1% SDS in PBS) and finally boiled in Laemmli sample buffer (42) for 5 min to dissociate the immune complexes. The samples
were subjected to electrophoresis on 6% SDS-PAGE followed by GS
I-B lectin blotting, as described above.Immunoblotting of Laminin in RA/F9 Media
Mouse EHS
laminin and RA/F9 serum-free culture media were separated by SDS-PAGE
on 6% polyacrylamide gel under reducing conditions and transferred
electrophoretically onto nitrocellulose sheets as described above.
Immunoblotting was performed as described for the GS I-B blotting, except that rabbit anti-mouse laminin antibody
(1:2,000) and anti-rabbit-horseradish peroxidase conjugate (1:5,000)
were used as first and second antibodies, respectively.Flow Cytometry Analysis
Cells were harvested and
washed once with Hanks' balanced salt solution. Approximately 5
10
cells were incubated with 100 milliunits of
green coffee bean -galactosidase in HEPES buffer (10 mM HEPES, pH 6.5, 0.15 M NaCl, 5 mM CaCl) at 37 °C for 1 h. Enzymes boiled for 20 min
were used as controls. The cells were washed four times with staining
buffer (Hanks' balanced salt solution, 1% heat inactivated goat
serum) and incubated with 20 µl of fluorescein
isothiocyanate-conjugated anti-Le monoclonal antibody
without dilution or 50 µl of biotinylated GS I-B (22
µg/ml), followed by incubation with 8 µl of
phycoerythrin-streptavidin (1 mg/ml) in 100 µl of staining buffer
on ice for 30 min, respectively. Flow cytometry was performed with a
FACStar-Plus flow cytometer (Becton Dickinson Immunocytometry, San
Jose, CA).
To address the mechanism(s) underlying the apparent
induction of 1,3GT mRNA Is Increased upon RA Treatment of F9
Cells1,3GT in F9 cells, we first examined the effect of 72
h of RA treatment on the induction of 1,3GT transcripts. A
representative time course of the Northern blot analysis using cells
treated with RA is shown in Fig. 1A. The steady-state
levels of 1,3GT mRNA begin to increase at 36 h following RA
treatment and reached a maximum level at 48 h. This result was
reproducible in four separate experiments, and the average of all the
results obtained from densitometric scans is shown in Fig. 1B. The results demonstrate that RA treatment
causes a 9-fold induction of the 1,3GT mRNA within 48 h. As a
control, we analyzed total RNA from CHO cells and COS7 cells. These
cells lack -galactosyltransferase activity (34, 45) and lack any detectable transcript for the
1,3GT (Fig. 1A).
1,3GT mRNA induction in F9 cells by RA. A, total RNA was
isolated from F9 cells (0 h) and F9 cells exposed to RA for increasing
periods of time. 40 µg of total RNA from each time point was
isolated from the cells, electrophoretically separated on a 1% agarose
gel containing 7% formaldehyde, and transferred to nylon filters. The
immobilized RNAs were then hybridized with a P-labeled
1,3GT cDNA probe, followed by stripping and reprobing with a P-labeled mouse actin cDNA probe as a control. As negative
controls, RNAs isolated from CHO and COS7 cells were included. B, the magnitude of expression of 1,3GT was quantified by
densitometry, and the relative signal was normalized to the level of
actin message. The levels of expression of the 1,3GT transcript in
F9 cells were arbitrarily taken as 1 unit.
1,3GT.
About 15 µg of total cellular RNA from untreated and treated cells
was analyzed by Northern blot analysis using a P-radiolabeled murine 1,3GT cDNA in pCDM7. The
results demonstrate that after 3 days of treatment the inclusion of
dbcAMP and RA only slightly enhances the level of transcript over that
of RA alone (Fig. 2, A and B). Since the
levels of 1,3GT mRNA message of RA/F9 and that of RA/dbcAMP/F9 are
rather similar, we focused on the effect of RA alone in all other
experiments.
1,3GT mRNA in F9 cells
upon induced differentiation by RA or RA combined with dbcAMP. A, F9 cells were treated for 3 days with either
10M RA alone or 10M retinoic acid plus 10M dibutyryl cyclic AMP (RA/dbcAMP). 15 µg of total RNA was
isolated from the cells and analyzed as in Fig. 1. B,
densitometry scans were used to quantify the relative signal in each
lane. Tubulin mRNAs were used to normalize for variations in the amount
and quality of RNA loaded in each lane. The level of expression of
1,3GT transcript in undifferentiated F9 cells was arbitrarily
taken as 1 unit.
RA Increases the Transcriptional Rate of
Nuclear run-on assays were conducted to address the
question of whether the change in 1,3GT1,3GT mRNA induced by RA
reflects an effect at the transcriptional level. For these nuclear
run-on assays, we also analyzed expression of type IV collagen
(1), which is known to have a higher rate of transcription in
RA/F9 cells compared to F9 cells (46) . RNA
``run-on'' transcripts were synthesized by incubating the
nuclei isolated from both F9 and RA/F9 cells in the presence of
[-p]UTP, and similar amounts of
radiolabeled RNA (
2 10
cpm/ml) were then
hybridized to DNA fragments immobilized on nylon filters. The
transcriptional rate of the type IV collagen (1) gene increases
during F9 cell differentiation (Fig. 3), which is consistent
with earlier reports(39, 46) . Likewise, the
transcriptional rate of the 1,3GT gene is much higher in RA/F9
cells than in F9 cells (Fig. 3). As controls, there was no
significant change in run-on transcripts for actin, and no transcripts
were detected for the control pBluescript vector DNA. These results
demonstrate that elevated transcripts for the 1,3GT in RA/F9 cells
results from an increase in the transcriptional rate for the gene.
1,3GT
transcripts in F9 cells and RA/F9 cells. Nuclei were isolated from F9
and RA/F9 cells, and transcription was performed in the presence of
[-P]UTP as described under
``Experimental Procedures.'' The labeled RNAs were hybridized
onto nylon filters containing pBluescript and 1,3GT, murine
-actin, and type IV collagen (1) cDNA
fragments.
Time Course of Induction of
To understand the relationship between 1,3GT Enzyme Activity in
F9 Cell Extracts and Cell Culture Media Following RA
Treatment1,3GT mRNA
level and enzymatic activity during differentiation, the 1,3GT
activity was determined in total cell extracts and concentrated cell
culture media at varying times after RA treatment. The specific
activity of 1,3GT in cell extracts starts to increase at 36 h
after RA treatment, and the specific activity in 72-h RA/F9 cell
extracts is approximately 5-fold higher than that in F9 extracts (Fig. 4A). This agrees with the previous findings when N-acetyllactosamine was used as an acceptor(29) .
1,3GT in F9 cells. F9 cells were exposed to RA for increasing
periods of time. A, approximately 80 µg of cell extracts
was assayed for 1,3GT activity by incubation at 37 °C for 3 h
using N-acetyllactosamine as an acceptor. The product was
isolated as described under ``Experimental Procedures.'' B, The culture media were concentrated 10-fold, and 12.5
µl portions were assayed for 1,3GT
activity.
1,3GT in the culture media from RA/F9 cells. We
therefore conducted a careful study of the enzymatic activity present
in the culture media at various times during differentiation. The total
enzyme activity was normalized to the amount of total cellular protein
on the dish. The 1,3GT activity in the cell culture media begins
to rise after 36 h of RA treatment and is approximately 4-fold higher
after 72 h of RA treatment (Fig. 4B). It can be seen
from the results in Fig. 4B that approximately of the
total 1,3GT enzyme activity detectable in the cultures of the
differentiated F9 cells is present in the culture media, and the
remaining is recoverable in cell extracts. These results demonstrate
that the 1,3GT is efficiently secreted by cells and that a
majority of the enzyme activity detected is present in a soluble form.
The increase in 1,3GT activity in media following RA addition
appears to rise with a time course slightly lagging behind the rise
observed in the cell-associated enzyme activity (Fig. 4, A and B).1,3GT activity in culture media
arises by secretion from cells, since the growth media prior to
addition to the cultured cells has no detectable activity (data not
shown). In addition, the 1,3GT activity recoverable in the media
of RA/F9 cells cannot be pelleted by prolonged centrifugation at
100,000 g, although the cell-associated activity is
clearly microsomal (data not shown). This indicates that the enzyme in
culture media is soluble and is an active and probably a
proteolytically cleaved form, as has been observed for some other
glycosyltransferases(47, 48, 49, 50, 51, 52) . 1,3GT in F9 cells in response
to RA treatment is specific for this enzyme, we also measured the
activity of 1,4GT. It has been reported that 1,4GT activity
decreases to approximately one-third of control values during the first
3 days of differentiation of F9 cells following RA addition and then
begins to rise slowly on day 4 following RA treatment(53) .
Consistent with this, we found that the activity of the 1,4GT in
extracts of both F9 and RA/F9 cells is similar during the first 3 days
following RA treatment (data not shown). Our results are also
consistent with another recent study showing that there is little
change in either the 1,4GT activity or transcript levels within 3
days of treatment of F9 cells with RA(54) . The 1,4GT
activity is detectable in the cell culture media of both cell types,
but RA treatment causes only a small change (<2-fold increase) in
1,4GT activity in media after 72 h (data not shown). These results
demonstrate that RA treatment of F9 cells does not cause a general rise
in the activities of all galactosyltransferases but results in a
pronounced change in the expression and activity of the 1,3GT.Laminin Is Secreted into Culture Media Following RA
Treatment of F9 Cells
To determine the temporal relationship
between induction of the 1,3GT compared by RA to other known
markers and to assess the efficiency of differentiation of F9 cells in
response to 10M RA in our system, we
determined the amount of laminin secreted into the cell culture media.
It has been shown that the steady-state levels of mRNAs for laminin B1
and B2 increase dramatically following differentiation of F9 cells, and
laminin has been defined as a differentiation marker, along with other
basement membrane components, such as type IV collagen
(1)(39, 55, 56, 57, 58, 59, 60) .
Laminin was detected by an enzyme-linked immunosorbent assay using cell
culture media immobilized in microtiter wells and anti-laminin antibody
as the detecting reagent. Laminin levels in the media of F9 cells
treated with RA starts to increase at 36 h after seeding the cells and
adding RA and continues to increase during the entire 72-h period of RA
treatment (Fig. 5). In contrast, there is only a modest amount
of immunoreactive material in media from F9 cells not treated with RA.
These studies show that induction of the 1,3GT parallels that of
laminin in response to 10M RA.
M RA for 3 days (closed circles), and 100
µl of media was removed at each time indicated and coated onto
microtiter plates. The amount of laminin adsorbed onto the wells was
detected by anti-mouse laminin antibody, as described under
``Experimental Procedures.'' An arbitrary unit was
established by dividing the OD value (405 nm) by total cellular protein
amount in one plate of cells at different periods of RA treatment. The
results are the mean ± S.E. of triplicate
analyses.
Soluble and Membrane-associated Glycoproteins Express
Increased Amounts of
To determine the effects of induced -Galactosyl Residues After RA
Treatment1,3GT
expression on glycosylation of proteins in the cells, we utilized the
plant lectin, GS I-B, which binds specifically to terminal
-linked galactosyl residues(61) . Previous studies
indicated that RA/F9 cells have more binding sites for GS I-B than F9 cells(29) , but the nature of the bound material
was not studied. Microsomes from both F9 and RA/F9 cells were analyzed
by SDS-PAGE on 10% polyacrylamide gel and blotted with GS
I-B. There is a large increase in the amount of GS
I-B-reactive glycoproteins in RA/F9 cells compared to F9
cells, especially in the high molecular mass (>200 kDa) range and
the 68-200 kDa range (Fig. 6A). The binding of GS
I-B to glycoproteins is specific, since inclusion of the
hapten sugar raffinose (200 mM) reduces binding (Fig. 6A). In other control experiments, we found no
specific binding to microsomal glycoproteins from CHO cells, whereas
there was significant specific binding of the lectin to a commercial
preparation of EHS laminin, as expected and as discussed below (Fig. 6A). These results confirm that
-galactosylation of glycoproteins is dramatically increased upon
differentiation of F9 cells. This evidence, coupled with flow
cytometric data described below, demonstrate that increased binding of
GS I-B may be considered as a marker for F9 cell
differentiation induced by RA.
blotting of glycoproteins from F9 and RA/F9 cells. A, approximately 10 µg of microsomes from F9 and RA/F9
cells was separated by SDS-PAGE on a 10% polyacrylamide gel. The
proteins were transferred to nitrocellulose and incubated with 10
µg/ml of biotinylated GS I-B in the presence or absence
of 200 mM raffinose followed by incubation with horseradish
peroxidase-conjugated streptavidin. The blots were developed using the
ECL chemiluminescence Western blotting kit. Mouse EHS laminin was
included as a positive control. B, serum-free culture media from each
cell line was also collected, and approximately 40 µg of soluble
protein was blotted with biotinylated GS I-B. C,
RA/F9 media were prepared as described under ``Experimental
Procedures.'' Following immunoprecipitation with anti-laminin
antibody, bound proteins were subjected to SDS-PAGE on a 6%
polyacrylamide gel and blotted with biotinylated GS I-B. D,
RA/F9 serum-free culture media and mouse EHS laminin were analyzed by
SDS-PAGE and immunoblotted with rabbit anti-mouse laminin antibody
followed by goat anti-rabbit IgG-peroxidase
conjugate.
-galactosylation of high molecular weight soluble glycoproteins
secreted into the cell media (Fig. 6B). A significant
band of the high molecular weight soluble glycoprotein in the range of
200-400 kDa reactive with GS I-B is detectable in
RA/F9 serum-free cell culture media, whereas very little material in F9
serum-free cell culture media is reactive with GS I-B. The
molecular weight of this GS I-B-reactive material in the
growth media of RA/F9 cells suggested that it might be laminin. To
confirm the identity of this material, the RA/F9 culture media were
immunoprecipitated with anti-mouse laminin antibody, analyzed by
SDS-PAGE on 6% acrylamide gel, and then blotted with GS I-B in the presence or absence of 200 mM raffinose. The
anti-mouse laminin antibody immunoprecipitates the major
-galactosylated components from RA/F9 cell culture media (Fig. 6C). As a control, the same RA/F9 culture media
and commercially purchased murine laminin were subjected to 6% SDS-PAGE
and analyzed by immunoblotting directly with rabbit anti-mouse laminin
antibody. The results obtained from immunoblotting the media of RA/F9
cells are similar to those obtained with commercial EHS laminin (Fig. 6D). Murine EHS laminin is known to contain
terminal -1,3-galactosyl residues and to be reactive with GS
I-B(62, 63) . Taken together, these data
demonstrate that the major -galactosylated glycoprotein secreted
by RA/F9 cells is laminin.Flow Cytometric Analysis of Cells for Altered Expression
of Le
Treatment
of F9 cells with RA reportedly causes a decrease in expression of the
Le Antigens in Response to RA Treatment antigen within 2-3 days(14) , but the
cause of this change is not clear. Although this decrease might be due
to loss of a GDP-Fuc:-D-GlcNAc
1,3-fucosyltransferase (1,3FT) (28) , F9 cells
treated with RA for 3 days have approximately the same amount of
1,3FT as untreated cells(29) . Thus, there is a temporal
difference between loss of Le and decrease in the
1,3FT activity. We considered the possibility that the Le antigen might be apparently lost because it could be
``masked'' by the addition of terminal -galactosyl
residues upon RA-induced differentiation. This might generate the
structure Gal1-3Gal1-4(Fuc1-3)GlcNAc-R
(termed -galactosylated Le). It has been shown that at
least one type of 1,3FT can utilize the
Gal1-3Gal1-4GlcNAc-R to generate
-galactosylated Le(64) , and
-galactosylated Le occurs naturally in mucins of cobra
venom(65) . determinants, the levels of Le antigen on F9
and RA/F9 cells were analyzed by flow cytometry using the anti-Le monoclonal antibody (anti-CD15 monoclonal antibody). As expected,
F9 cells express much higher levels of Le antigen than
RA/F9 cells (Fig. 7, A and B). When F9 cells
are treated with -galactosidase, there is no significant change in
the expression of the Le antigen (Fig. 7A).
In contrast, -galactosidase treatment of RA/F9 cells causes a
clear enhancement in the amount of surface Le antigen (Fig. 7B). These results demonstrate that terminal
-galactosyl residues in differentiated F9 cells mask Le determinants on RA/F9 cells generated by 3 days of RA treatment.
antigens and terminal
-galactose residues. F9 cells (panels A and C)
and RA/F9 cells (panels B and D) were treated with
either -galactosidase(+) or heat inactivated
-galactosidase(-) as described under ``Experimental
Procedures.'' The washed cells (5 10) were
analyzed for surface Le expression by direct immunostaining
with anti-CD15 monoclonal antibody (panels A and B)
and for surface expression of the terminal -galactose residues by
indirect staining with biotinylated GS I-B and
streptavidin-phycoerythrin conjugate (panels C and D). The numbers in each panel refer to the
experimental treatments as indicated on the right.
-galactosidase, there is only
a slight decrease in GS I-B staining (Fig. 7C). In contrast, treatment of RA/F9 cells with
-galactosidase causes a significant decrease in staining by GS
I-B (Fig. 7D). These results demonstrate
that RA/F9 cells express more surface -galactosyl residues than F9
cells and that many of the terminal -galactosyl residues bound by
GS I-B on RA/F9 cells are accessible to
-galactosidase. Taken together, these results support the
conclusion that RA treatment of F9 cells causes an increase in levels
of surface glycoconjugates containing -galactosyl residues and
that these residues mask Le determinants in RA/F9 cells.
1,3GT that appears inducible by
RA(29, 34) . Our studies now extend these findings to
show that differentiation of F9 cells is accompanied by a 9-fold
increase in steady-state levels of 1,3GT mRNA and that this
increase is transcriptionally regulated. This induction is associated
with secretion of the 1,3GT and enhanced -galactosylation of
surface glycoproteins and secretion of an -galactosylated
glycoprotein identified as laminin. Upon induced differentiation, there
is a marked decrease in expression of surface Le determinants, but this decrease is due in part to masking of the
Le determinants by -galactosylation.1,4GT. Within the 2,6-sialyltransferase
(ST6N) gene are at least four promoters, one of which is responsive to
liver-restricted transcription factors (70) and another which
appears to be B-cell specific and is regulated during B-cell
development(71, 72) . The 1,4GT is known to have
a regulatory element that controls transcript initiation (73) and may be related to hormone-dependent stimulation of the
enzyme in mammary glands(74) . Although the complete sequence
of the murine 1,3GT gene is known(75) , the cis-
and/or trans-acting elements that regulate the transcription
of 1,3GT are not yet understood.1,3GT, it has been observed that enzyme activity
is higher in mouse peritoneal macrophages elicited with
thioglycollate(84) , which may correlate with increased binding
to GS I-B observed in stimulated macrophages compared to
resident mouse macrophages(85) . Other studies have examined
the effects of RA on glycosyltransferase activities in F9 cells, but
the results obtained are clearly different from those observed here for
the 1,3GT. Prolonged treatment of F9 cells with RA causes an
increase in activity for
1,6-N-acetylglucosaminyltransferase V and the core 2
1,6-N-acetylglucosaminyltransferase(86) . There
is little change of activity in these enzymes, however, after 3 days of
differentiation, but an increase in activity begins to occur after 4
days of differentiation.1,3FT (28) . However, using a different assay for fucosyltransferase
activity another group observed a slight increase in the activity of
fucosyltransferase upon RA treatment of F9 cells(88) . In the
case of 1,4GT, it was shown that 1,4GT activity in F9 cells
declines after 3 days of differentiation and then begins to rise around
5-6 days of differentiation, which correlates with increased
levels of transcript observed after 5-6 days of treatment with
RA(53) . Recently, it was reported that neither the 1,4GT
enzyme nor transcript levels change significantly in F9 cells treated
with RA alone, but treatment with RA and cAMP causes a 6.5-fold
induction of the transcript in 8 days(54) . Nuclear run-on
experiments demonstrated that this increase in 1,4GT was not due
to an increase in transcriptional rate, but it was due to
post-transcriptional regulation (54) . Induction of the
1,3GT by RA is clearly different from those other
glycosyltransferases cited above, in that elevated transcripts and
activity of the 1,3GT clearly begin to rise after 24-36 h
and are maximal at 3 days.1,3GT transcripts by RA in F9 cells indicates that this gene is
induced like other known late or secondary genes in contrast to RA
induction of early or primary genes(44) . Examples of the early
inducible genes, which are transcriptionally activated within hours,
are ERA-1/Hox-1.6 (58) and the Hox 1.3(59) . Their
activation results from interaction of the RA-RA receptor complex with
their promoters(44, 89) . Examples of the late
inducible genes, which exhibit a delayed induction (24-48 h), are
laminin B1 and type IV collagen (1)(55, 90) .
Whether the molecular mechanism for induction of the 1,3GT is
related to those for these other late inducible genes is currently
under investigation.1,3GT activity in the cell culture media and that 
of the
total activity detectable in the cultured cells is recoverable in the
media. The amount of activity in the media increases in response to RA
treatment of the cells and is similar to the increase in activity
observed in the cell extracts. Soluble forms of some other
glycosyltransferases, such as 2,6-sialyltransferase and
1,4GT, have been found in various secretions and body fluids
including milk(91) , colostrum(92) , and
serum(48, 52, 93, 94) , and some
soluble glycosyltransferases have been purified from these
sources(91, 92, 95) . These soluble forms
appear to result from the proteolytic cleavage of the membrane-bound
forms of the enzymes(47, 48, 49) , and the
levels of some soluble enzymes are affected by disease status and
inflammation(50, 51) . Although the proteases
responsible for the solubilization of glycosyltransferases have not
been identified, there are suggestions that cathepsin D-like
proteases within the acidic trans-Golgi might be
involved(52) . The mechanism by which 1,3GT is secreted
into the culture media is not known, although we are currently
attempting to define the N terminus of the secreted 1,3GT to
identify the cleavage site. It would be expected that the cleavage
site(s) occur in the so-called ``stem-region,'' proximal to
the C-terminal catalytic domain(67) . The biological functions,
if any, for secreted glycosyltransferases are not known. It is
conceivable that the secretion of 1,3GT could be regulated and
purposeful and that the soluble enzyme could have a function during
differentiation of F9 cells. In preliminary studies, we have detected a
soluble and active form of the 1,3GT in mouse serum and media from
other cultured murine cells, indicating the secretion of soluble forms
of this enzyme may be a common occurrence in cells.1,3GT is 3.7 kilobases, four
different mRNA transcripts that differ in the length of the sequences
encoding the putative stem region have been detected by RNA-polymerase
chain reaction analysis(75) . It is thought that these
transcripts arise by alternative splicing of a pre-mRNA according to a
cassette model. Interestingly, although all four transcripts are
present in both F9 and RA/F9 cells, only a single transcript for bovine
1,3GT was observed in bovine thymus and MDBK cells(75) .
The splice variants generate enzymes predicted to vary particularly in
the stem region, which is predicted to contain the putative cleavage
site for proteases. Future studies are required to understand the
functions of different 1,3GT splice variants and whether these
isoforms exhibit differential cell and/or tissue expression. antigens may be masked in differentiated F9 cells is consistent
with some studies performed on mouse embryos. Both 8-cell mouse embryos
and embryonic ectoderm of 5- and 6-day-old embryos express Le antigens, and -galactosidase treatment enhances expression
of the antigen(96) . Although there is scant information about
antigen masking in vivo, such masking of carbohydrate
determinants has been performed in vitro using recombinant
glycosyltransferases. For example, transfection of cDNA for
2,6-sialyltransferase in Xenopus oocytes inhibits the
formation of polysialic acid onto neural cell adhesion
molecule(97) , and transfection of 2,3-sialyltransferase
into T lymphoblastoid cells converts the cells from a peanut agglutinin
positive (PNA
) phenotype to PNA,
possibly caused by masking of the peanut agglutinin positive receptor,
Gal1-3GalNAc1-Ser/Thr, with sialic acid(98) .
Our study demonstrates, however, that antigen masking by
-galactosylation is a biochemical response of the embryonal
carcinoma cells to RA. antigens on RA/F9 cells suggests that differentiated cells
synthesize the new sequence
Gal1-3Gal1-4(Fuc1-3)GlcNAc1-R
(-galactosylated Le). This possibility is supported by
recent evidence that Gal1-3Gal1-4GlcNAc can be
fucosylated in vitro using a partially purified
1,3-fucosyltransferase from human milk(64) . In contrast,
fucosylated oligosaccharides, such as
Gal1-4(Fuc1-3)GlcNAc1-2Man, do not
serve as acceptors for 1,3GT(99) , indicating that there
must be an ordered addition of terminal sugars. Interestingly,
-galactosylated Le structures occur normally in
oligosaccharides of mucins from cobra venom(65) . It is
conceivable that the carbohydrate phenotypes of F9 and RA/F9 cells are
reflective of a balance between competing glycosyltransferases for
available substrates. Competitive enzyme reactions are known to be
important in influencing the structures of newly synthesized
glycoconjugates(100-103). We previously demonstrated that
introduction of the murine 1,3GT into CHO cells results in
synthesis of glycoconjugates containing terminal -galactosyl
residues and decreased levels of terminal sialic acid (45) .
Enzyme competition has been shown to be involved in the biosynthesis of
mucin oligosaccharides in porcine and ovine submaxillary glands (103, 104) and other mucin chains and blood group
antigens(100) .1,3GT transcripts and enzyme
activity, resulting in changes in the surface carbohydrate phenotype
and masking of antigens. These changes may result in the synthesis of
carbohydrate structures containing -galactosyl residues, which
could be functionally important for cellular interactions during murine
embryogenesis (30, 31, 32, 33) .
There is particular interest in Le antigens or masked
Le antigens because of evidence that they may participate
in intercellular adhesion events during early embryogenesis via direct
interactions with endogenous lectins or via direct
carbohydrate-carbohydrate interactions(33) . Although no
lectins have yet been found that exclusively prefer Le itself or -galactosylated Le as a carbohydrate
ligand, many different animal lectins bind to oligosaccharides
containing the underlying Le structure, such as sialyl
Le and sulfated sialyl Le(105) . Future
studies will be aimed at defining the mechanism by which the 1,3GT
gene is responsive to RA, the detailed structures of the masked
Le antigens, and the possibility that murine cells express
lectins interactive with Le-containing oligosaccharides.
)1,3GT,
UDP-Gal:-D-Gal 1,3-galactosyltransferase;
1,4GT, UDP-Gal:-D-GlcNAc
1,4-galactosyltransferase; 1,3FT,
GDP-Fuc:-D-GlcNAc 1,3-fucosyltransferase; GS
I-B, G. simplicifolia I-B isolectin;
dbcAMP, dibutyryl cyclic adenosine monophosphate; PAGE, polyacrylamide
gel electrophoresis.
-We thank Dr. Kwame Nyame, Russell DeBose-Boyd,
and Patricia Wilkins for critical reading and Judy Gaar for assistance
in the preparing the manuscript.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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