Cheng, Stanley

Friedman, Susan J.

Skehan, Philip

Elsevier - Publisher Connector 

Volume 152, number 2 FEBS LETTERS February 1983 
The ~ce~rrence of polysialogangliosides in a human trophublast 
cell line 
Susan J. Friedman, StanIey Cheng and Philip Skehan 
Department of Pharmacology/Oncology Research Group, University of Calgary, Calgary, Alberta, 
Canada T2N 4NI 
Received 25 October 1982; revision received 22 December 1982 
Abstract not supplied 
~~~y~~o~~g~~g~~o~jde Vernon Chor~o~ar~~noma BeWo Cefl Chromotogro~hy 
1. INTRODUCTION 
Highly sialylated gangliosides containing 4 or 
more sialic acid residues per lipid backbone are pri- 
marily found in brains of teleost fishes and amphi- 
bians [l-&J where they contribute 40-50% of total 
ga~glioside-bound sialic acid, Tetrasi~oga~~iosi- 
des are present in mammalian brain [2,5-71, retina 
(51, and several established cell lines [f&9), but are 
relatively minor components. Comparisons of 
adult brain ganglioside composition in lower and 
higher vertebrates have led to the conclusion that 
polysialoganglioside content decreases relative to 
other gangliosides during vertebrate evolution 
[2,3]. The discovery of structural differences bet- 
ween tetrasialogangliosides of fish and mammalian 
brains [ i&l l] led to the proposal of a novel bio- 
synthetic pathway for polysialoga~gliosides in fish 
brain [12j, and provided a plausible biosynthetic 
rationale for their abundance in brains of fishes 
and perhaps other lower vertebrates. 
In 1980, Rosner presented evidence for the 
stage-specific accumulation of polysialoganglio- 
sides in developing optic lobes and cerebrum of the 
* A portion of this work was presented at the 12th Inter- 
national Congress of Biochemistry, Perth 1281 
+ Ganglosides are named according to Svennerholm 
f291 
chick, and suggested that these gangliosides might 
play a role in cellular differentiation and morpho- 
genesis of the brain [13]. Recent studies [14] sug- 
gest that chick brain polysialogangliosides are pro- 
bably synthesized by the ‘b’ pathway 111,15,16] 
which is ubiquitously found in neural and extra- 
neural tissues of higher vertebrates. 
Here, we report the occurrence of a high propor- 
tion of polysialogangliosides in BeWo, a human 
cell line of trophoblastic origin. Although BeWo 
was established from a gestational trophoblastic 
tumor [ 171, its morphological and biochemical 
properties closely resemble those of normal early 
placental trophoblast, making it a useful in vitro 
model for the study of selected aspects of tropho- 
blastic behavior [ 181. 
This is the first report of polysialogangliosides 
present in high proportions in a mammalian cell 
type. Trophoblast is the earliest cell lineage to 
emerge during mammalian development and it is 
possible that polysialogangliosides may play a role 
in implantation of the blastocyst and/or subse- 
quent events leading to the formation of the fetal 
placenta. 
2. MATERIALS AND METHODS 
2. I. Ceil clrlrure 
BeWo choriocarcinoma cells were cultured at 
37°C in RPM1 1640 (Gibco) + 10% fetal calf se- 
Volume 152, number 2 FEBS LETTERS February 1983 
rum (Flow labs) in a 95% air-50lo CO2 atmosphe- 
re. Near-confluent monolayers were dissociated 
with 0.25% trypsin-1 mM EDTA in NKT buffer 
(138 mM NaCI, 5.3 mM KCl, 1 mM Tris, pH 7.2) 
and plated for experiments at 8 x 10’ cells/75 cm’ 
tissue culture flask. Medium was changed daily to 
avoid nutrient depletion artifacts. 
2.2. Ganglioside labeling 
14C-Labeled gangliosides were prepared by 
adding D-(l-14C]galactose (Amersham, 
56.6 mCi/mmol, 0.5-1.0 &i/ml) to cultures in 
complete growth medium for periods of 24-96 h. 
Steady state labeling (cpm incorporated/mg cell 
protein) was achieved for most of the gangliosides 
by 48-72 h. 
2.3. Extraction and fractionation of gangliosides 
Cell monolayers were washed 3 times with NKT, 
scraped with a rubber policeman, and pelleted in a 
low speed centrifuge. When necessary, pellets were 
frozen at - 80°C prior to extraction of total cell li- 
pids with CHClj/MeOH (20 vol. CHClJMeOH, 
2: 1 (v/v); 10 vol. CHCl3/MeOH, 1:2 (v/v) + 5% 
HzO; 20 vol. CHClJMeOH, 2: 1 (v/v) [19]. Ex- 
tracted pellets were solubilized in alkali and assay- 
ed for protein content as in [20] using BSA as a 
standard. Ganglioside fractions were obtained by 
fractionating lipid extracts on silicic acid columns 
as in [21]. These were lyophilized and redissolved 
in CHCl3/MeOH for chromatographic separation 
on silica gel hptlc plates (Merck) in the following 
solvent systems: 
(i) I. CHCl3; II. CHC13/MeOH/Hz0 (70: 30: 4 
by vol.); III. CHCl3/MeOH/0.25% KC1 
(60:35:8 by vol.) 1221; 
(ii) I. CHC13/MeOH/aqueous 0.02010 CaClz 
(55:45: 10 by vol.); II. CHCl3/MeOH/2.5 N 
NHdOH (60 : 35 : 8 by vol.) (modified from ref. 
]71); 
(iii) I. CHCl3/MeOH/l2 mM MgCl2/15 N 
NH40H (60: 35:7.5:3 by vol., at 37°C); II. 
CHClj/MeOH/l2 mM MgCl2 (58 :40: 9 by 
vol., at 37°C) [23]. 
All chromatographic solvents were of analytical 
reagent grade and were redistilled before use. 
Ganglioside standards were purchased from Sigma 
(bovine brain gangliosides, type Ii) and Supelco 
(GTlb i- GDlb, GDla, GMl). GM2 was provided 
by courtesy of Dr Robert Yu. 
176 
2.4. Detection and quantitation of gangliosides 
Radioactive gangliosides fractionated on thin 
layers were detected by autoradiography, using 
Kodak Blue Brand film. The radioactive bands 
were scraped into scintillation vials, hydrated with 
1 ml H20, and counted in 10 ml of Triton-toluene 
PPO-POPOP liquid scintillation mix (1 :4 v/v). 
Unlabeled gangliosides were stained with resor- 
cinol reagent [24] as in [7], and scanned on a Beck- 
man DU-8 spectrophotometer (absorbance mode) 
at 580 and 450 nm. After correcting for 450 nm 
absorbance of interfering sugars [25] the relative 
proportions of sialic acid in each peak were calcu- 
lated from the integrated scans. 
2.5. digestion of ceils with neur~~~nidase 
Cells labeled for 3 days with [r4C]galactose 
(0.5 &&‘ml) were dissociated with trypsin-EDTA, 
replated at a subconfluent density in 60 mm tissue 
culture dishes in growth medium minus isotope, 
and allowed to attach for 2.5 h. Medium was re- 
moved and the monolayers were washed 3-times 
with NKT buffer adjusted to pH 5, then incubated 
for 1 h at 37°C in 2 ml NKT, 10 mg/ml BSA, 
1 mM CaC12, pH 5 (control) or the same solution 
containing 5 units of C~ost~idiu~ perfringens neu- 
raminidase (Sigma Type VI). Cells remained atta- 
ched to the dishes during this treatment and cell ly- 
sis was not detected. Cells were then harvested for 
extraction and fractionation of gangliosides in sol- 
vent system C as previously described. 
3. RESULTS 
BeWo cultures incorporated [‘4C]galactose into 
a broad spectrum of gangliosides both at early and 
later times after plating (fig. 1). Of particular inte- 
rest is a cluster of gangliosides whose mobility is 
greatly retarded on silica gel 60 thin layers chroma- 
tographed in commonly used solvent systems. In 
solvent system A, shown here, these gangliosides 
separate into two peaks of radioactivity which tra- 
vel between the origin and the trisialoganglioside 
GTlb. 25% of total ganglioside radioactivity was 
present in this fraction at the 27-70-h labeling pe- 
riod. When cultures were labeled continuously af- 
ter plating and analyzed at 48, 72 and 96 h for 
[‘4C]galactose incorporation into each of the gang- 
lioside fractions, the low mobility gangliosides in- 
corporated label at a faster rate and to a greater ex- 
Volume 152, number 2 FEBS LETTERS 
GTI GDlbGDla GM1 GM2 
I I I I I 
I 1 1 1 1 1 1 1 ’ ’ ’ 
DISTANCE (mm) 
Fig.1. Densitometric scan of autoradiogram of 
[14C]galactose-labeled BeWo gangliosides. Cultures were 
labeled with D-[l-14C]galactose from 27-70 (lower 
trace) or 70-I 17 (upper trace) hours after plating. 
Cangliosides were extracted as described in section 2 and 
chromatographed on silica gel 60 thin layer plates in 
solvent system A. Autoradiogr~s were scanned at zero 
wavelength in the absorbance mode. Peak areas are not 
linearly proportional to radioactivity on the scan shown 
here. Peaks were identified by comparison with co- 
chromatographed standard gangliosides stained with 
resorcinol. 
February 1983 
tent than all of the other species. By using solvent 
system C, which is capable of resolving highly sia- 
lylated gangliosides from fish and chick brain [23], 
it was possible to separate the low mobility fracti- 
on into 7 discrete peaks of radioactivity (fig.2A). 
Most of the label was removed from the polysialo- 
ganglioside region of the chromatogram following 
treatment of intact cells with Cl. perfringens neu- 
raminidase suggesting that these gangliosides are 
primarily cell surface-localized (fig.2B). 
Although BeWo cells synthesize multiple species 
of less complex gangliosides from radioactive pre- 
cursors (galactose, D-glucosamine), SO-75070 of 
ganglioside-bound sialic acid is contained in the 
polysialoganglioside fraction, as determined by 
quantitative scanning densitometry of resorcinol- 
Fig.2. (A) Autoradiogram of biosynthetically-labeled 
BeWo polysialogangliosides. Gangliosides were ex- 
tracted from cultures which were grown to confluency in 
[‘4C]galactose. Following chromatography on silica gel 
60 hptlc plates in solvent system C, the thin layer was 
autoradiographed on Kodak Blue Brand X-ray film at 
- 80°C. Numbered bands correspond to those referred 
to in the text. 
177 
VaIume 152, number 2 FBBS LETTERS February 1983 
Fig-J. (B) Autoradiogram of biosynthetically-labeled 
BeWo polysialogangliosides before and after cell treat- 
ment with neuraminidase. Subconfluent cultures 1abeIed 
for 3 days with [‘4C]gaiactose were dissociated with 
try&r-EDTA, repiated in growth medium without 
radioactive isotope, and incubated for I It at 37°C in the 
presence (lane t) or absence Qane 2) or ~e~~~n~dase as
described in section 2+ Gangfiosides were extracted and 
~hromat~graphed in solvent system C. 
stained gangliosides separated in solvent system B. 
It is unlikely that these results can be ascribed to 
the presence of sialoglycoproteins or free siahc 
acid since simiIar results were obtained with ex- 
haustively trypsinized gangfioside extracts and 
with ganghosides prepared by chromatography on 
DEAE Sephadex, Sephadex LH20, and silicic acid 
columns 1X]_ 
In sofvent system C, the pol~si~~~~g~~~s~d~ 
fraction separates into 4 peaks which are detected 
by resorcinol staining (fig.3) and correspond in Rf 
DISTANCE hn f 
Fig& ~ensito~ne~i~ scan af resorsjnol-staj~ed BeWo 
po~ysialo~aRgliosides. About 7 gg gang~~~side~~A~A 
was spotted on silica gel hptte $&es, ~~rornat~g~~~hed 
in solvent system C, and r~~orc~nol*stai~e~. The 
~o~ys~al~~~~~~ios~de region of the scan is shown here. 
Peaks are numbered to correspond with those in the text, 
to peaks 1 f 4, 5, and 6 of the biosynthetically labe- 
led polysialogangliosides shown in fig.2A, A com- 
parison of the migration rates of BeWo poiysido- 
ganghosides with those of chick embryo optic 
lobes with known sir&c ac~d~sphjn~osi~e molar 
rntios /27f suggests &at peaks 4 and 3 are tetra- 
s~~o~a~~~iosjdes, peak2 Is a pent~siaiog~~gIioside 
and peak i may contain more than 7 siafic acid 
residues/molecule. 
4. DISCUSSION 
BeWo, a human trophoblast cell line, is the first 
mammalian cell line found to contain a high pro- 
portion of ~~iysi~~ogangliosides. In this respect it 
is atypical of otker established cell tines of either 
norrnaf or t~morigen~c origin. Thus far the two 
examples of ~o~ysi~og~ng~~osjde abundance in 
higher vertebrates - embryunic chick brain and 
BeWo - are developing tissues. Adult mammalian 
tissues and nvian brain have not been found to 
contain significant amounts of polysialoganglio- 
sides. Recent studies from our laboratory have 
found that polysialoganglioside abundance in 6 
mammalian cell lines correlates with the ability of 
cells to form multicelh&r clusters at subconfluent 
densities iPj* fn chick embryo optic lobes, poIysia- 
~ogan~Iios~~es ar  preferentiaIly accumulated du- 
ring a specific developments period whew neuro- 
naf rn~~r~t~~~ and d~ff~rent~~t~on take pface and 
new contacts are made between optic nerve axons 
f 131. We suggest that polysialoganghosides may 
178 
Volume 152, number 2 FEBS LETTERS February 1983 
mediate specific types of cell contact interactions 
during avian and mammalian development. 
The availability of mammalian cell lines such as 
BeWo that contain a high proportion of polysialo- 
gangliosides, and lines which totally lack these 
gangliosides, should prove useful for further stu- 
dies on polysialoganglioside synthesis and function 
in mammalian tissues. 
ACKNOWLEDGEMENTS 
These studies were supported by grants from the 
Medical Research Council of Canada, the Alberta 
Heritage Foundation for Medical Research, and 
the Alberta Heritage Savings Trust Fund: Applied 
Research-Cancer. 
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The occurrence of polysialogangliosides in a human trophoblast cell line 

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The occurrence of polysialogangliosides in a human trophoblast cell line

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