Vol. 186, No. 1, 1992
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS
July 15, 1992
Pages 102-113
Novel G l y c o s y l a t i o n - D e f e c t i v e
James Ripkal
Baby Hamster Kidney Cells
and Michael Pierce2
I D e p a r t m e n t of Medicine U n i v e r s i t y of Miami School of M e d i c i n e Miami, FL 33101 2 D e p a r t m e n t of Cell B i o l o g y U n i v e r s i t y of Georgia Athens, Georgia
Received May 18, 1992
The
plant lectin wheat germ a g g l u t i n i n
(WGA) has p r e v i o u s l y
been used to select more than ten d i f f e r e n t
glycosylation-defective
phenotypes
somatic
in
a
variety
of
mammalian
W G A - r e s i s t a n t phenotypes have now been obtained baby
hamster
p a t t e r n of lectins,
kidney
cross
resistance
suggesting
cells are altered. to
(BHK) cells.
WGA-resistant
that
the
and
cells
BHK
cell
sensitivity
to
multiple
plant
cell surface c a r b o h y d r a t e s of these
that
phenotype
W G A - r e s i s t a n t m a m m a l i a n cells.
from
These mutant BHK cells exhibit a
lack
galactose residues on their cell surface WGA-resistant
Three
spontaneously
Two W G A - r e s i s t a n t BHK phenotypes CHO
cells.
terminal
similar
sialic acid and
carbohydrates.
The
third
has not p r e v i o u s l y been seen in
©1992AcademicPress,
The plasma m e m b r a n e of animal
appear
cells
Inc.
contains
carbohydrate
m o l e c u l e s c o v a l e n t l y linked to a variety of proteins and lipids. way
to
study
the
structure
c a r b o h y d r a t e s has been
to
and
isolate
ooo6-291x/92 $4.00 Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
102
function
One
of these cell surface
glycosylation-defective
mutant
Vol, 186, No, 1, 1992
cells.
These
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
somatic
cell
mutants
have been used to examine the
pathways of c a r b o h y d r a t e b i o s y n t h e s i s molecular
cloning
of
Glycosylation-defective
(I),
metastasis
(2,3) and the
glycosyltransferases
cell lines have p r e d o m i n a n t l y
been
by selection for resistance to cytotoxic plant lectins Multiple lines
have
glycosylation-defective
been
recognition The
mammalian
derived
(I). somatic
cell
selected for resistance to the plant lectin wheat
germ a g g l u t i n i n WGA particularly
(4,5).
(i).
useful (6),
These W G A - r e s i s t a n t
in
studies
on
natural
insulin receptor activity
possibility
of
selecting
a
(WGA R) lines have been killer
(NK)
cell
(7) and m e t a s t a s i s
large
number
of
(8).
different
glycosylation-defective
mutants using a single lectin suggested the
utility
a
of
WGA
as
glycosylation-defective In this
cytotoxic
agent
to
select
novel
BHK cells.
paper,
we
glycosylation-defective
report
BHK
the
lines.
selection
Three
of
mutant
spontaneous
phenotypes were
obtained that appear to be affected in cellular glycosylation.
M A T E R I A L S AND METHODS Cell Lines and kidney
Cell
Culture.
The
parental
baby
(BHK) cell line BHK 21/c13 was a gift from Dr.
Parental
Chinese
hamster
ovary
hamster
Clayton Buck.
(CHO) cells were obtained from Dr.
Pamela Stanley.
Cells were cultured in m o n o l a y e r
containing
10%
fetal
Island, NY)
at 37°C in a h u m i d i f i e d atmosphere w i t h 5% CO 2 .
Lectins. vulgaris);
calf
The lectins:
LCA
from P. vulgaris);
RCA-I
biflorus);
WGA
and
antibiotics
BSL- I B4
(from L. culinaris);
max);
UAE-I 103
medium
(GIBCO,
ensiformis); E-PHA
simplicifolia);
(from U.
Grand
from T__a.
Ricin
(toxin
(erythroagglutinin
(agglutinin from R. communis);
(isolectin from B.
SBA (from G.
alpha
(wheat germ agglutinin,
Con A (Concanavilin A, from C.
from R. communis);
sativum);
serum
in
PSA
(from P__a.
DBA
(from D__~.
europaeus);
PNA
(from
Vol. 186, No. 1, 1992
A. hypogaea); and
SuWGA
BIOCHEMICAL AND 81OPHYSICALRESEARCH COMMUNICATIONS
BSL-I
(from B. simplicifolia);
(succinylated
Burlingame,
WGA)
were
CA; The lectin L-PHA
Louis, NaCI,
B.
simplicifolia) Missouri.
10mM
from
Vector Labs,
(leukoagglutinin from P. England;
vulgaris)
and BSL-II
(isolectin
was p u r c h a s e d from Sigma Chemical Co.,
Lyophilized
Na2HPO4,
(from S. japonica);
purchased
was obtained from Burroughs Wellcome, from
SJA
pH7.2
lectins were
(PBS),
rehydrolyzed
in
St. 0.15M
filter sterilized and stored at
4°C. Selection
Protocols.
Cells were aliquoted at
t i s s u e - c u l t u r e dish into alpha m e d i u m with 10% FCS overnight day,
and
antibiotics
at 37°C in a h u m i d i f i e d atmosphere with 5% CO 2.
the cells
had
lectin was added. picked
5xl05/100-mm
doubled
(106 c e l l s / 1 0 0 - m m dish)
After 8 days at 37°C,
and
The next selective
the surviving colonies were
into sterile 12 x 75-mm tubes in n o n s e l e c t i v e medium.
6-7 days at 37°C,
cells were
transferred
to
T-25
After
tissue-culture
flasks and grown in monolayer. Determination
of
Lectin-Resistance.
assays were used to d e t e r m i n e surviving
colonies.
Colonies
the
Two
rapid
lectin-resistance
picked
into
12
properties
aliquot
of cells
(107 cells) were
plate in the presence or absence of volume
of
Iml.
An
alternative
cells)
in 96-well m i c r o t i t e r dishes
lectin
in a final volume of 0.2ml.
(Fisher Scientific Co.,
Pittsburgh,
for growth compared to parental BHK the selective
lectin
37°C.
A
plated
a
total
0.1ml cells
(2x10 Z
in the presence After
from both screening assays were stained
at
t r a n s f e r r e d to a 24-well
selective assay
of
x 75-mm tubes were
e x a m i n e d after growth in n o n s e l e c t i v e media for 4 days 0.Sml
screening
in
or
absence
4-5 days at 37°C,
with
0.2%
of
plates
methylene
blue
PA) in 50% m e t h a n o l and scored cells.
Colonies
resistant
lectin were then tested by the s e m i q u a n t i t a t i v e
to
P test
(9) against several lectins. For the P test, plate and grown at 37°C
500 cells were plated in 96-well m i c r o t i t e r overnight. 104
The
presence
of
a
range
of
Vol. 186, No. 1, 1992
lectin
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
c o n c e n t r a t i o n s were then added to individual wells.
days at 37°C,
plates were stained
with
methylene
blue.
After 4 Colonies
e x h i b i t i n g novel l e c t i n - r e s i s t a n t phenotypes were cloned by limiting d i l u t i o n in 96-well m i c r o t i t e r plates. Lectin-resistant
clones
were tested for their q u a n t i t a t i v e
resistance or s e n s i t i v i t y to lectins by P test and DI0 analysis c o n c e n t r a t i o n of lectin that reduces cell survival to 10%). analysis,
30 or 300 cells were
plates overnight at 37°C.
plated
in
The next day,
a range of lectin c o n c e n t r a t i o n s were
24-well
(the
For DI0
tissue-culture
the presence or absence of
added
to
individual
wells.
Cells were grown for 8 days at 37°C and the plates were then stained with
methylene
blue.
Colony
growth
plating e f f i c i e n c y was c o r r e c t e d for the
was
scored and the relative
growth
of
cells
in
the
absence of lectin. Galactosyltransferase
Activity Assay.
incubated with U D P - [ 3 - 3 H ] - g a l a c t o s e , ADP, (i0)
MnCI 2
and
a trisaccharide
for 1 hour at
37°C.
sonicates
were
N-acetyl-D-glucopyranosylamine,
acceptor,
Radiolabeled
counted by liquid s c i n t i l l a t i o n
Cell
as p r e v i o u s l y d e s c r i b e d
product
was
isolated
and
(ii).
RESULTS Lectin-Resistance different
wheat
germ
glycosylation-defective (i).
Properties of Hamster Cells.
expressed
on
the
agglutinin-resistant
In
L-PHA, the
addition,
order
to
determine
if
the
carbohydrates
cell surfaces of CHO and BHK cells were similar.
cells
(Table
BHK cells are also more resistant to the lectins
LCA and Ricin
hamster
(WG~)
(Lec R) phenotype of parental CHO and BHK
BHK cells are twofold resistant to WGA c o m p a r e d to CHO cells i).
i0
cell lines have been isolated from CHO cells
The l e c t i n - r e s i s t a n c e
cells were compared in
At least
(Table i).
The d i f f e r e n t Lec R
phenotypes
of
suggested the p o s s i b i l i t y that WGA might select 105
Vol. 186, No. 1, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE ] LECTIN-RESITANCE PROPERTIES OF HAMSTER CELL LINES DI0 (~g/ml) Cell Line
WGA
L-PHA
BHK
4.2
10.2
CHO
2.2
2.1
Con A
Ricin
LCA
14
0.072
45
18
0.007
16
FOLD-RESISTANCE BHK
2
5
WT
i0
3
The lectin-resistance properties of BHK and CHO
cells
were
examined by DI0 analysis as described in Materials and Methods. fold-resistance of BHK cells to lectin are compared
to
CHO
The
cells.
WT, wild type level of resistance.
novel
lectin-resistant
BHK cell mutants
not previously
isolated
from
C H O cells. Selection were
isolated
selection
of W G A - r e s i s t a n t
in
at a
selection frequency
cloned
by
found
to be g r e a t e r
was
limiting
obtained
(Table
2).
using
than
tenfold
showed of
BHK
resistance using
the
sensitivity
and
and and
W G A R.
BHK
mutants
Lec R
sensitivity
against
semiquantitative to a lectin,
P
a panel
test.
compared
106
the
Two colonies
were
and
of W G A
2-fold
of
cells cells
Lectins.
lectins
rapidly
of (i).
screened
plant with may
2
to WGA.
phenotypes
plant
Mutants
to p a r e n t a l
selection
Plant
sixteen
were
WGA R phenotype
in
unique
were
WI.4,
resistance
to
various
BHK clones
survived
slightly
Cells
to
WGA R phenotypes
Wl.l
exhibit
sensitivity the
10-6.
A
approximately
Two
Colonies
concentration
Lec R
Glycosylation-defeetive
2).
and two clones
a decreased
Sensitivity
Parental
(Table
of a p p r o x i m a t e l y
dilution,
Clone W4.2
cross-resistance
1
B H K Cells.
for
lectins increased
indicate
a
Vol. 186, No. 1, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE 2 SELECTION OF LECTIN-RESISTANT BHK CELLS WGA-Resistant
Selection
WGA
Number
Cells
1
(~g/ml)
BHK
Frequency
Phenotype of
Surviving
Clones
Colonies
20
WGA
8.9xi0-7
Clone
R>I0
WI.I
R>I0
WI.4
2
BHK
15
7.5x10-6
R2
W4.2
3
BHK
25
30
>90
6-9
L-PHA
10-20
>90
>90
10-20
Ricin
0.1-0.3
Con A
20-30
WGA
LCA
>160
BSL-II
>200
E-PHA
0.12 30-40 >160 I-I0
I-i0
PSA
0.05-0.25
>200
50-200
0.05-0.25
30
10-20
80
40
ND
>200
"
I-i0
50-200
"
50-200
50-200
"
>200
BSL-I B4
>200
DBA
>200
>200
"
>200
SBA
>200
>200
"
>200
UAE-I
>200
>200
"
>200
PNA
>200
>200
"
>200
BSL-I
>200
>200
"
>200
SJA
>200
>200
"
>200
SuWGA
>200
>200
"
>200
Parental
BHK
and
W G A - r e s i s t a n t BHK clones were tested for
s e n s i t i v i t y to plant lectins by the P test as d e s c r i b e d in M a t e r i a l s and Methods.
suggest
ND, not determined.
that
carbohydrates
the and
Lec R are
The
Lec R BHK
six
lectins
cells
of
or
hypersensitivity.
BSL-II-sensitive.
cells
therefore,
panel
L-PHA-resistant,
BHK
in
order
WI.I
is
altered
cell
glycosylation-defective
were
examined
to
quantitate
Clones
2-fold
express
Wl.l
Con
by
DI0
the and
108
of
WI.4
A-resistant
slightly
mutants.
analysis
level
LCA-resistant,
surface
and
against
a
resistance are
4-fold >200-fold
while
WI.4
is
Vol. 186, No. 1, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE 4 L E C T I N - R E S I S T A N C E PROPERTIES OF PARENTAL AND W G A - R E S I S T A N T BHK CELLS DI0 (~g/ml) Cell Line
WGA
L-PHA
Ricin
Con A
LCA
BSL-II
WI.I
>150
45
0.102
25.0
66
1.5
WI.4
127
44
0.095
23.0
26
1.7
W4.2
6.3
7.2
0.036
13.0
49
ND
Parental BHK
4.2
10.2
0.072
14.0
45
>400
FOLD-RESISTANCE WGA
L-PHA
Ricin
Con A
LCA
BSL-II
WI.I
R>35
R4
(R)
R2
(R)
S>250
WI.4
R30
R4
-
R2
$2
S>235
W4.2
(R)
(S)
$2
The
lectin-resistance
WGA-resistant
BHK
is shown (top panel).
each
lectin
sensitivity;
ND
properties
of
parental
BHK
and
cells were examined by DI0 analysis as described
in Materials and Methods.
WGA-resistant
-
BHK
The quantitative DI0 value for each lectin
The fold resistance or
cell
(bottom
sensitivity
of
each
line is compared to parental BHK cells for
panel).
R,
-fold
resistance;
S,
-fold
( ), less than twofold resistant or sensitive;
-, not
significantly different from parental cells; ND, not determined.
2-fold WI.I
LCA-sensitive. is
greater
And whereas
than
35-fold
WGA R and BSL-II S phenotype that
of
Lec8
CHO
parental
CHO
Stanley,
unpublished The
the
same
(i)
final time
L-PHA-sensitive,
is
30-fold
WGA-resistant
of W I . I
cells.
and
WI.4
Lec8
and
CHO
approximately
(Table
WI.4
are
4).
appears
100-fold
12-fold
WGA-resistant, The
highly
similar
to
WGA R compared
to
(J. R i p k a
P.
BSL-II S
and
observation).
Lec R BHK W4.2 and
cell
isolate,
W4.2,
is
2-fold
Ricin-sensitive,
exhibits
parental I09
is
slightly
sensitivity
W G A R.
At
slightly
for both
Con A
Vol. 186, No. 1, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE 5 GALACTOSYLTRANSFERASE
ACTIVITY OF WGA-RESISTANT BHK CELLS
Specific Activity (pmol/mg*h)
Cell Line Parental BHK
8.53
i00.0
WI.I
10.38
121.7
WI.4
8.19
96.0
W4.2
9.14
107.2
Cell synthetic
sonicates
were
trisaccharide
galactosyltransferase
4).
incubated
(Ref.
(Table
similar
to any p r e v i o u s l y
isolated
Galactosyltransferase
terminated
phenotype
similar
to
exhibited parental
also u n a f f e c t e d
WI.4
cells
revertants BSL-II
to BSL-II
(Selection
In order
with
were utilized. recovered (Selection
in 3,
vitro cells
I,
Table
6).
This
5).
Small numbers presence
Table
6).
Lines.
The
these
cells
could be due to the indicated
The W4.2
activity
BHK cells
of BSL-II
BHK phenotype,
a
This
110
large
survive
5).
hand,
to isolate
WI.I
do
2,
phenotype BHK
that
WI.I
in
is suitable
quantity
of
selection
cell m i x i n g
suggested
line was
sensitivity
(Selection
selection
of p a r e n t a l
(Table
that
activity
cell
experiment
On the other
of
BHK
assays
The
to be
lines.
suggested
a possible
if the BSL-II
the p a r e n t a l
not appear
cell
Lec R
Enzyme
(Table
Parental
in the p r e s e n c e
the
a for
galactosyltransferase
suggested
lines.
to d e t e r m i n e
revertants
residues.
of G l y c o s y l a t i o n - D e f e c t .
of these
not p r o l i f e r a t e
of
in g a l a c t o s y l t r a n s f e r a s e
Revertion
and
specific
does
and WI.4
activity.
in
BHK
of W4.2
Lec R hamster
of WI.I
in N - a c e t y l g u c o s a m i n e
and WI.4
UDP-[3H]-Gal
acceptor
Activity
lack of g l a c t o s y l t r a n s f e r a s e Wl.l
10)
The Lec R p h e n o t y p e
lectin-resistance
with
activity.
and LCA
and
Relative Activity (%)
cells
Table
6).
to recover experiments cells
were
of
WI.I
cells
even
a
single
Vol. 186, No. 1, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE 6 REVERSION OF WGA-RESISTANT BHK CELLS Experiment
WI.I Cells
BHK Cells
Plates
Colonies
1
0
5
2
8
2
I0 6
0
25
0
3
106
5
2
i0
WGA-resistant separately
or
BHK
and
together,
as
parental noted
BHK
0.8
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS
July 15, 1992
Pages 102-113
Novel G l y c o s y l a t i o n - D e f e c t i v e
James Ripkal
Baby Hamster Kidney Cells
and Michael Pierce2
I D e p a r t m e n t of Medicine U n i v e r s i t y of Miami School of M e d i c i n e Miami, FL 33101 2 D e p a r t m e n t of Cell B i o l o g y U n i v e r s i t y of Georgia Athens, Georgia
Received May 18, 1992
The
plant lectin wheat germ a g g l u t i n i n
(WGA) has p r e v i o u s l y
been used to select more than ten d i f f e r e n t
glycosylation-defective
phenotypes
somatic
in
a
variety
of
mammalian
W G A - r e s i s t a n t phenotypes have now been obtained baby
hamster
p a t t e r n of lectins,
kidney
cross
resistance
suggesting
cells are altered. to
(BHK) cells.
WGA-resistant
that
the
and
cells
BHK
cell
sensitivity
to
multiple
plant
cell surface c a r b o h y d r a t e s of these
that
phenotype
W G A - r e s i s t a n t m a m m a l i a n cells.
from
These mutant BHK cells exhibit a
lack
galactose residues on their cell surface WGA-resistant
Three
spontaneously
Two W G A - r e s i s t a n t BHK phenotypes CHO
cells.
terminal
similar
sialic acid and
carbohydrates.
The
third
has not p r e v i o u s l y been seen in
©1992AcademicPress,
The plasma m e m b r a n e of animal
appear
cells
Inc.
contains
carbohydrate
m o l e c u l e s c o v a l e n t l y linked to a variety of proteins and lipids. way
to
study
the
structure
c a r b o h y d r a t e s has been
to
and
isolate
ooo6-291x/92 $4.00 Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
102
function
One
of these cell surface
glycosylation-defective
mutant
Vol, 186, No, 1, 1992
cells.
These
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
somatic
cell
mutants
have been used to examine the
pathways of c a r b o h y d r a t e b i o s y n t h e s i s molecular
cloning
of
Glycosylation-defective
(I),
metastasis
(2,3) and the
glycosyltransferases
cell lines have p r e d o m i n a n t l y
been
by selection for resistance to cytotoxic plant lectins Multiple lines
have
glycosylation-defective
been
recognition The
mammalian
derived
(I). somatic
cell
selected for resistance to the plant lectin wheat
germ a g g l u t i n i n WGA particularly
(4,5).
(i).
useful (6),
These W G A - r e s i s t a n t
in
studies
on
natural
insulin receptor activity
possibility
of
selecting
a
(WGA R) lines have been killer
(NK)
cell
(7) and m e t a s t a s i s
large
number
of
(8).
different
glycosylation-defective
mutants using a single lectin suggested the
utility
a
of
WGA
as
glycosylation-defective In this
cytotoxic
agent
to
select
novel
BHK cells.
paper,
we
glycosylation-defective
report
BHK
the
lines.
selection
Three
of
mutant
spontaneous
phenotypes were
obtained that appear to be affected in cellular glycosylation.
M A T E R I A L S AND METHODS Cell Lines and kidney
Cell
Culture.
The
parental
baby
(BHK) cell line BHK 21/c13 was a gift from Dr.
Parental
Chinese
hamster
ovary
hamster
Clayton Buck.
(CHO) cells were obtained from Dr.
Pamela Stanley.
Cells were cultured in m o n o l a y e r
containing
10%
fetal
Island, NY)
at 37°C in a h u m i d i f i e d atmosphere w i t h 5% CO 2 .
Lectins. vulgaris);
calf
The lectins:
LCA
from P. vulgaris);
RCA-I
biflorus);
WGA
and
antibiotics
BSL- I B4
(from L. culinaris);
max);
UAE-I 103
medium
(GIBCO,
ensiformis); E-PHA
simplicifolia);
(from U.
Grand
from T__a.
Ricin
(toxin
(erythroagglutinin
(agglutinin from R. communis);
(isolectin from B.
SBA (from G.
alpha
(wheat germ agglutinin,
Con A (Concanavilin A, from C.
from R. communis);
sativum);
serum
in
PSA
(from P__a.
DBA
(from D__~.
europaeus);
PNA
(from
Vol. 186, No. 1, 1992
A. hypogaea); and
SuWGA
BIOCHEMICAL AND 81OPHYSICALRESEARCH COMMUNICATIONS
BSL-I
(from B. simplicifolia);
(succinylated
Burlingame,
WGA)
were
CA; The lectin L-PHA
Louis, NaCI,
B.
simplicifolia) Missouri.
10mM
from
Vector Labs,
(leukoagglutinin from P. England;
vulgaris)
and BSL-II
(isolectin
was p u r c h a s e d from Sigma Chemical Co.,
Lyophilized
Na2HPO4,
(from S. japonica);
purchased
was obtained from Burroughs Wellcome, from
SJA
pH7.2
lectins were
(PBS),
rehydrolyzed
in
St. 0.15M
filter sterilized and stored at
4°C. Selection
Protocols.
Cells were aliquoted at
t i s s u e - c u l t u r e dish into alpha m e d i u m with 10% FCS overnight day,
and
antibiotics
at 37°C in a h u m i d i f i e d atmosphere with 5% CO 2.
the cells
had
lectin was added. picked
5xl05/100-mm
doubled
(106 c e l l s / 1 0 0 - m m dish)
After 8 days at 37°C,
and
The next selective
the surviving colonies were
into sterile 12 x 75-mm tubes in n o n s e l e c t i v e medium.
6-7 days at 37°C,
cells were
transferred
to
T-25
After
tissue-culture
flasks and grown in monolayer. Determination
of
Lectin-Resistance.
assays were used to d e t e r m i n e surviving
colonies.
Colonies
the
Two
rapid
lectin-resistance
picked
into
12
properties
aliquot
of cells
(107 cells) were
plate in the presence or absence of volume
of
Iml.
An
alternative
cells)
in 96-well m i c r o t i t e r dishes
lectin
in a final volume of 0.2ml.
(Fisher Scientific Co.,
Pittsburgh,
for growth compared to parental BHK the selective
lectin
37°C.
A
plated
a
total
0.1ml cells
(2x10 Z
in the presence After
from both screening assays were stained
at
t r a n s f e r r e d to a 24-well
selective assay
of
x 75-mm tubes were
e x a m i n e d after growth in n o n s e l e c t i v e media for 4 days 0.Sml
screening
in
or
absence
4-5 days at 37°C,
with
0.2%
of
plates
methylene
blue
PA) in 50% m e t h a n o l and scored cells.
Colonies
resistant
lectin were then tested by the s e m i q u a n t i t a t i v e
to
P test
(9) against several lectins. For the P test, plate and grown at 37°C
500 cells were plated in 96-well m i c r o t i t e r overnight. 104
The
presence
of
a
range
of
Vol. 186, No. 1, 1992
lectin
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
c o n c e n t r a t i o n s were then added to individual wells.
days at 37°C,
plates were stained
with
methylene
blue.
After 4 Colonies
e x h i b i t i n g novel l e c t i n - r e s i s t a n t phenotypes were cloned by limiting d i l u t i o n in 96-well m i c r o t i t e r plates. Lectin-resistant
clones
were tested for their q u a n t i t a t i v e
resistance or s e n s i t i v i t y to lectins by P test and DI0 analysis c o n c e n t r a t i o n of lectin that reduces cell survival to 10%). analysis,
30 or 300 cells were
plates overnight at 37°C.
plated
in
The next day,
a range of lectin c o n c e n t r a t i o n s were
24-well
(the
For DI0
tissue-culture
the presence or absence of
added
to
individual
wells.
Cells were grown for 8 days at 37°C and the plates were then stained with
methylene
blue.
Colony
growth
plating e f f i c i e n c y was c o r r e c t e d for the
was
scored and the relative
growth
of
cells
in
the
absence of lectin. Galactosyltransferase
Activity Assay.
incubated with U D P - [ 3 - 3 H ] - g a l a c t o s e , ADP, (i0)
MnCI 2
and
a trisaccharide
for 1 hour at
37°C.
sonicates
were
N-acetyl-D-glucopyranosylamine,
acceptor,
Radiolabeled
counted by liquid s c i n t i l l a t i o n
Cell
as p r e v i o u s l y d e s c r i b e d
product
was
isolated
and
(ii).
RESULTS Lectin-Resistance different
wheat
germ
glycosylation-defective (i).
Properties of Hamster Cells.
expressed
on
the
agglutinin-resistant
In
L-PHA, the
addition,
order
to
determine
if
the
carbohydrates
cell surfaces of CHO and BHK cells were similar.
cells
(Table
BHK cells are also more resistant to the lectins
LCA and Ricin
hamster
(WG~)
(Lec R) phenotype of parental CHO and BHK
BHK cells are twofold resistant to WGA c o m p a r e d to CHO cells i).
i0
cell lines have been isolated from CHO cells
The l e c t i n - r e s i s t a n c e
cells were compared in
At least
(Table i).
The d i f f e r e n t Lec R
phenotypes
of
suggested the p o s s i b i l i t y that WGA might select 105
Vol. 186, No. 1, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE ] LECTIN-RESITANCE PROPERTIES OF HAMSTER CELL LINES DI0 (~g/ml) Cell Line
WGA
L-PHA
BHK
4.2
10.2
CHO
2.2
2.1
Con A
Ricin
LCA
14
0.072
45
18
0.007
16
FOLD-RESISTANCE BHK
2
5
WT
i0
3
The lectin-resistance properties of BHK and CHO
cells
were
examined by DI0 analysis as described in Materials and Methods. fold-resistance of BHK cells to lectin are compared
to
CHO
The
cells.
WT, wild type level of resistance.
novel
lectin-resistant
BHK cell mutants
not previously
isolated
from
C H O cells. Selection were
isolated
selection
of W G A - r e s i s t a n t
in
at a
selection frequency
cloned
by
found
to be g r e a t e r
was
limiting
obtained
(Table
2).
using
than
tenfold
showed of
BHK
resistance using
the
sensitivity
and
and and
W G A R.
BHK
mutants
Lec R
sensitivity
against
semiquantitative to a lectin,
P
a panel
test.
compared
106
the
Two colonies
were
and
of W G A
2-fold
of
cells cells
Lectins.
lectins
rapidly
of (i).
screened
plant with may
2
to WGA.
phenotypes
plant
Mutants
to p a r e n t a l
selection
Plant
sixteen
were
WGA R phenotype
in
unique
were
WI.4,
resistance
to
various
BHK clones
survived
slightly
Cells
to
WGA R phenotypes
Wl.l
exhibit
sensitivity the
10-6.
A
approximately
Two
Colonies
concentration
Lec R
Glycosylation-defeetive
2).
and two clones
a decreased
Sensitivity
Parental
(Table
of a p p r o x i m a t e l y
dilution,
Clone W4.2
cross-resistance
1
B H K Cells.
for
lectins increased
indicate
a
Vol. 186, No. 1, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE 2 SELECTION OF LECTIN-RESISTANT BHK CELLS WGA-Resistant
Selection
WGA
Number
Cells
1
(~g/ml)
BHK
Frequency
Phenotype of
Surviving
Clones
Colonies
20
WGA
8.9xi0-7
Clone
R>I0
WI.I
R>I0
WI.4
2
BHK
15
7.5x10-6
R2
W4.2
3
BHK
25
30
>90
6-9
L-PHA
10-20
>90
>90
10-20
Ricin
0.1-0.3
Con A
20-30
WGA
LCA
>160
BSL-II
>200
E-PHA
0.12 30-40 >160 I-I0
I-i0
PSA
0.05-0.25
>200
50-200
0.05-0.25
30
10-20
80
40
ND
>200
"
I-i0
50-200
"
50-200
50-200
"
>200
BSL-I B4
>200
DBA
>200
>200
"
>200
SBA
>200
>200
"
>200
UAE-I
>200
>200
"
>200
PNA
>200
>200
"
>200
BSL-I
>200
>200
"
>200
SJA
>200
>200
"
>200
SuWGA
>200
>200
"
>200
Parental
BHK
and
W G A - r e s i s t a n t BHK clones were tested for
s e n s i t i v i t y to plant lectins by the P test as d e s c r i b e d in M a t e r i a l s and Methods.
suggest
ND, not determined.
that
carbohydrates
the and
Lec R are
The
Lec R BHK
six
lectins
cells
of
or
hypersensitivity.
BSL-II-sensitive.
cells
therefore,
panel
L-PHA-resistant,
BHK
in
order
WI.I
is
altered
cell
glycosylation-defective
were
examined
to
quantitate
Clones
2-fold
express
Wl.l
Con
by
DI0
the and
108
of
WI.4
A-resistant
slightly
mutants.
analysis
level
LCA-resistant,
surface
and
against
a
resistance are
4-fold >200-fold
while
WI.4
is
Vol. 186, No. 1, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE 4 L E C T I N - R E S I S T A N C E PROPERTIES OF PARENTAL AND W G A - R E S I S T A N T BHK CELLS DI0 (~g/ml) Cell Line
WGA
L-PHA
Ricin
Con A
LCA
BSL-II
WI.I
>150
45
0.102
25.0
66
1.5
WI.4
127
44
0.095
23.0
26
1.7
W4.2
6.3
7.2
0.036
13.0
49
ND
Parental BHK
4.2
10.2
0.072
14.0
45
>400
FOLD-RESISTANCE WGA
L-PHA
Ricin
Con A
LCA
BSL-II
WI.I
R>35
R4
(R)
R2
(R)
S>250
WI.4
R30
R4
-
R2
$2
S>235
W4.2
(R)
(S)
$2
The
lectin-resistance
WGA-resistant
BHK
is shown (top panel).
each
lectin
sensitivity;
ND
properties
of
parental
BHK
and
cells were examined by DI0 analysis as described
in Materials and Methods.
WGA-resistant
-
BHK
The quantitative DI0 value for each lectin
The fold resistance or
cell
(bottom
sensitivity
of
each
line is compared to parental BHK cells for
panel).
R,
-fold
resistance;
S,
-fold
( ), less than twofold resistant or sensitive;
-, not
significantly different from parental cells; ND, not determined.
2-fold WI.I
LCA-sensitive. is
greater
And whereas
than
35-fold
WGA R and BSL-II S phenotype that
of
Lec8
CHO
parental
CHO
Stanley,
unpublished The
the
same
(i)
final time
L-PHA-sensitive,
is
30-fold
WGA-resistant
of W I . I
cells.
and
WI.4
Lec8
and
CHO
approximately
(Table
WI.4
are
4).
appears
100-fold
12-fold
WGA-resistant, The
highly
similar
to
WGA R compared
to
(J. R i p k a
P.
BSL-II S
and
observation).
Lec R BHK W4.2 and
cell
isolate,
W4.2,
is
2-fold
Ricin-sensitive,
exhibits
parental I09
is
slightly
sensitivity
W G A R.
At
slightly
for both
Con A
Vol. 186, No. 1, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE 5 GALACTOSYLTRANSFERASE
ACTIVITY OF WGA-RESISTANT BHK CELLS
Specific Activity (pmol/mg*h)
Cell Line Parental BHK
8.53
i00.0
WI.I
10.38
121.7
WI.4
8.19
96.0
W4.2
9.14
107.2
Cell synthetic
sonicates
were
trisaccharide
galactosyltransferase
4).
incubated
(Ref.
(Table
similar
to any p r e v i o u s l y
isolated
Galactosyltransferase
terminated
phenotype
similar
to
exhibited parental
also u n a f f e c t e d
WI.4
cells
revertants BSL-II
to BSL-II
(Selection
In order
with
were utilized. recovered (Selection
in 3,
vitro cells
I,
Table
6).
This
5).
Small numbers presence
Table
6).
Lines.
The
these
cells
could be due to the indicated
The W4.2
activity
BHK cells
of BSL-II
BHK phenotype,
a
This
110
large
survive
5).
hand,
to isolate
WI.I
do
2,
phenotype BHK
that
WI.I
in
is suitable
quantity
of
selection
cell m i x i n g
suggested
line was
sensitivity
(Selection
selection
of p a r e n t a l
(Table
that
activity
cell
experiment
On the other
of
BHK
assays
The
to be
lines.
suggested
a possible
if the BSL-II
the p a r e n t a l
not appear
cell
Lec R
Enzyme
(Table
Parental
in the p r e s e n c e
the
a for
galactosyltransferase
suggested
lines.
to d e t e r m i n e
revertants
residues.
of G l y c o s y l a t i o n - D e f e c t .
of these
not p r o l i f e r a t e
of
in g a l a c t o s y l t r a n s f e r a s e
Revertion
and
specific
does
and WI.4
activity.
in
BHK
of W4.2
Lec R hamster
of WI.I
in N - a c e t y l g u c o s a m i n e
and WI.4
UDP-[3H]-Gal
acceptor
Activity
lack of g l a c t o s y l t r a n s f e r a s e Wl.l
10)
The Lec R p h e n o t y p e
lectin-resistance
with
activity.
and LCA
and
Relative Activity (%)
cells
Table
6).
to recover experiments cells
were
of
WI.I
cells
even
a
single
Vol. 186, No. 1, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE 6 REVERSION OF WGA-RESISTANT BHK CELLS Experiment
WI.I Cells
BHK Cells
Plates
Colonies
1
0
5
2
8
2
I0 6
0
25
0
3
106
5
2
i0
WGA-resistant separately
or
BHK
and
together,
as
parental noted
BHK
0.8
