From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
Ricin
Binding
and
in Human
By
Previous
studies
varying
sensitivities
these
showed
differences.
cell
lines
was
characterized.
Ricin
and
binding
ricin
analyses
and
unlabeled
minimum
2.5
x iO
the
with
0.1
surface
increased
37CC
at
levels
L
linearly
face
from
some
tinguishes
was
binding
Differences
to
not
binding
1 .5
in the
37’C,
x
1 5 to 30
minutes
and
to 29%
of the
amount
cells
and
isolate
murine
binding
marrow
carbohydrate
content
or structure.
lectin
ricin is a protein
heterodimer
The
beans
(B
binding
and composed
of a catalytic
chain)
subunit.5’6
The A
protein
synthesis
from 28S ribosomal
inhibits
residue
A chain
ized
The
cells.
molecules
B chain
galactose
and
toxin
inhibit
ricin
may
lactose.
and
decreased
used
Preliminary
cell
tions
in
ricin
from
conjugate
cells
studies
resistance
surface
ricin
treatment
and
sensitivity
ricin also
toxicity.
in
from
a
adenine
internaleukaryotic
attachment
aids
to
in
competitively
form
Because
with
of human
the ability
the
basis
that increased
associated
with
ricin
monocbonal
has
been
antibod-
bone marrow,
variaof lactose
to protect
for nonspecific
immuno-
surface
Blood,
Vol
72,
major
and
are
with
as
parent
For
(r
ricin
varying
in the
carbohydrate
ricin
and
major
=
all
+ .9)
sensitivity.
widely
the
ricin
lactose
correlation
glycoproteins
probably
of ricin
ricinin
sensitive
in vitro
show
of surface
by Grune
1988
a close
cell
cells.
reduction
as
and
with
immunoconjugate.
differences
toxicity
were
bindcon-
glycolipids.
determinant
of
immunoconjugates.
& Stratton.
Inc.
MATERIALS
AND
serum
METHODS
Ricin agglutinin
and electrophoretically
pure ricin
from E.Y. Laboratories
(San Mateo,
CA). Bovine
(BSA)4
was purchased
from Sigma
(St Louis).
obtained
albumin
was
purchased
from
Cabbiochem
Behring
(La
(NTG)
was
obtained
from Aldrich
Chemicals
(Milwaukee).
Concanavalin
A
(Con A), Phaseolus
vulgaris
leukoagglutinin
(PHA-L),
and wheat
germ agglutinin
(WGA)
were obtained
from
Vector
Laboratories
(Burbingame,
CA). Fluorescein-conjugated
swine anti-goat
antibody
was from TAGO
(Burlingame,
CA). L-[U-3H]Leucine
and carrierfree
‘25I-sodium
iodide were obtained from Amersham
(Arlington
Heights,
IL).
Established
cell lines.
The cell lines used have been described
previously.’2”3
CEM and 8402 are T cell acute lymphocytic
leukemia (ALL)
lines and line 8392 is a human
B-lymphocyte
line derived
from
the same patient as cell line 8402.IA HEL is a human erythroleukemia
line and HL-60 was derived
from a patient
with
promyeloCA).
N-methyb-N’-nitro-N-nitrosoguanidine
leukemia.’9’2#{176} Other
cell
and the Burkitt
lines
used
were
line NAMAL
the
non-T-ALL
line
WA.2’
ricin
binding,
in
intracellular
No 4 (October),
From
UCSan
alterations
toxin
1988:
in toxin
processing.’7
pp
1357-1363
internalization,
Diego
Cancer
Center
T-O1 1, University
of Califor-
nia, La Jolla; and The Departments
of Pathology
and
Divisions
ofLaboratory
Medicine
and Hematology/Oncology
versity
Medical
ofCalifornia
Submitted
November
Supported
by
37497
and
Research
with
or variations
per
CEM
binding
were
at 4#{176}C
lines
incubation
parent
ricin
cells
variations
NALM-6
and without
lactose
and ricin uptake.
Ricin
and ricin/bactose
resistance
in human
cells is similar
to that
in other mammalian
cells,’5”6
and may be caused
by reduced
cell
bound
structure
nonspecific
cytic
In the present
study,
we investigated
the basis for variable
cellular
sensitivity
to ricin.
We examined
cellular
ricin
binding
with and without
lactose,
and correlated
ricin binding
with in vitro
toxicity.
In addition,
we measured
ricin
dissociation
or
Jolla,
cells.3’4
presumably
suggested
was
binding.
immunoconjugates
for ex vivo
and
ricin
by
detectable
was
cell
a >95%
cells
there
binding
we described
variable
sensitivities
of
cell lines to ricin
with and without
ricin/bactose
to prepare
ies’24
residues
ricin
these
Lactoperoxidase
lectin
chain)
and
catalytically
to kill
surface
no
hematopoietic
tent
with
showed
an anti-T-cell
Materials.
(ie,
dis-
extracted
‘ Lactose
and gabactose
binding
to most cells.’2
In a previous
study,’2
human
hematopoietic
ricin
cell
galactosamine
B chain
added
be sufficient
mediates
to
sur-
variations
by removing
a single
RNA7;
as few as two
compared
showed
Ricin
tenfold
variants
However,
indicating
S
cellular
stem
(A
chain
and
These
at
surface
castor
CEM
Human
cell
eg,
reflect
When
resistant
line.
hematopoietic
of ricin
others;
presumably
lactose.
ing,
cell
among
approximately
between
plateaued
varying
from
reduced
ricin
was
on the
to fivefold
iO
to growth
stimulation
to toxicity.2
Lectin
binding
cells
two-
examined.
uptake
through
cause
at 4#{176}C.
depending
cells
dissociated
Ricin
bound
with
ricin
Taetle
varied
cells
of
Lines
Raymond
CEM
presence
ricin
and
added.
albumin;
sites,
at
Grothaus,
binding
time-
Scatchard
for
hematopoietic
used
but
kinetics.
to mammalian
in lectin
ricin,
and
Inhibition
Cell
was
ricin
rapid,
surface-bound
At
moieties
for
membrane-bound
biphasic
agglutination’
PHA),
phytohemagglutinin,
isolated
12%
carbohydrate
reactions,
in vitro
also
was
surface
lactose.
with
BIND
cell
of the
mol/I
with
estimated
At 4nC,
>95%
representing
ECTINS
cell
K,, values
the
basis
hematopoietic
cells
of
D.
exhibited
the
at 3 zmol/L.
classes
mol/I.
but
cell
CEM
by unlabeled
from
ricin.
removed
to
multiple
slowly
were
Cyrene
cells
human
correlated
saturation
and
dissociated
from
blocked
approached
maximum
to
mutants
binding
showed
mol/L
and
blood
investigate
binding
assessed
Resistant
To
Synthesis
Hematopoietic
E. Leonard,
human
ricin.
ricin
sensitivities.
dependent,
that
to
John
Protein
Institute,
US
Public
Institutes
reprint
maceuticals,
requests
Health
1 1099
N
Torrey
costs ofthis
charge
This
payment.
“advertisement”
article
in accordance
San
Pines
article
must
with
Diego.
June
Service
15,
No.
Suite
18 U.S.C.
CA
MD.
PhD.
160,
were defrayed
therefore
I P01
ofNew
Investigator
the National
Cancer
Bethesda,
E. Leonard,
Rd.
1988.
Grants
recipient
from
ofHealth,
to John
92037.
The publication
indicate
H-720,
accepted
CA 35692.
J.E.L.
was the
Award
1 R23
CA 35692
National
Address
Center
20, 1987;
Medicine,
Uni-
be
section
IDEC
La
Phar-
Jolla.
CA
in part by page
hereby
marked
1 734 solely
to
this fact.
© 1 988 by Grune
& Stratton.
Inc.
0006-4971/88/7204-0035$3.00/0
1357
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
1358
LEONARD,
Cell
lines
were
with
glutamine
with
viabilities
maintained
in RPM!
1640
medium
supplemented
10% fetal bovine serum (FBS).
Log-phase
>90% were used in all studies. Cell viabibities
and
determined
by trypan
blue exclusion.
of ricin.
bactoperoxidase
mL azide-free
hydrogen
peroxide
and
10 oL
(1
mCi)
carrier-free
sodium
25I
reaction
was allowed
to proceed
for 45 minutes
at 23#{176}C
and stopped
with 10 iL 1% sodium
azide. Iodinated
ricin was
separated
from free sodium iodide using Sephadex
G-25 equilibrated
iodide.
with
The
PBS
containing
0.02%
sodium
azide
and 0.1%
BSA
and
then
to remove residual azide. Specific activity of labeled ricin
varied
from 0.4 to 1.0 x 106 cpm/ig
protein.
Iodinated
ricin was
shown to be functionally
and structurally
intact by sodium
dodecyl
sulfate-polyacrylamide
gel ebectrophoresis
(SDS-PAGE)
and autoradiography,
competitive
binding with equimolar
unlabeled
ricin,22
and in vitro inhibition
of protein
synthesis.’2
Ricin
binding
assay.
Test tubes for binding
assays were precoated with 4 mL PBS containing
1% BSA (Fraction V. Sigma) and
0.025%
sodium
azide and allowed to stand for 30 minutes at 4#{176}C.
This precoat
buffer was then discarded.
Mid-log-phase
cells were
washed once with fresh growth
medium
containing
0.01% sodium
azide
(wash
medium)
and resuspended
to 4 x 106 cebls/mL
in wash
medium.
Twenty-five
microbiters
of cells was then added to each
tube, followed
by 50 L ‘25I-ricin in wash medium.
Binding
was
allowed to proceed for 0 to 90 minutes
at 4#{176}C,
and the reaction was
dialyzed
by addition
stopped
The cells
were
of 4 mL
collected
PBS
containing
by centrifugation
0.01%
sodium
at 4#{176}C,
and the pellet
azide.
was
azide buffer. Following
centrifugation
at
4#{176}C,the buffer was removed
and the radioactivity
associated
with
the cell pellet was determined.
Nonspecific
binding was determined
washed
with
with
lOOx
PBS/sodium
excess
unlabeled
ricin
added.
To assess ricin uptake by CEM cells, cells were
.cmol/L labeled ricin at 4#{176}C
for 30 minutes
in
PBS alone or PBS containing
0.01% sodium azide. Cells incubated in
PBS without
azide were washed once in medium
and then warmed
for periods of up to 24 hours at 37#{176}C
in Dulbeceo’s modified Eagle’s
medium
(DMEM)
(Grand
Island Biobogicals,
NY). An aliquot of
cells was washed,
and total cell-associated
ricin was determined.
A
separate
aliquot
of cells was washed
in medium
containing
0.1
mol/L lactose and incubated
with
lactose
for 30 minutes
at 4#{176}C
to
remove residual surface-bound
ricin. Non-bactose-dissociable
ricin
was then determined
by ‘y counting.
A parallel
set of cells
was
incubated
at 4#{176}C
in azide for identical
periods of time and washed in
medium.
Total cell-associated
or lactose nondissociabbe
ricin was
then
determined.
Wash medium
containing
0.1 mob/L galactose
Ricin
uptake.
incubated
could
with
1.0
be substituted
for lactose
medium
with
similar
results.
Log-phase
CEM cells were
cultured
for 16 hours at 37#{176}C
with 0, 0.1, 0.5, 1.0, 2.0, or 5.0 Mg/mL
NTG. Mutagenized
cells were washed
with fresh growth medium
and cultured for five days to eliminate
auxotrophs.
Surviving
cells
were plated in 0.8% methylcebbulose
containing
growth medium and
0, 0.01,
or 0.1 nmob/L
ricin and cultured
for four days at 37#{176}C.
Large colonies were picked, washed, and plated in fresh methylcellulose containing
ricin.
Large
colonies
were again
picked,
and their
ricin sensitivity
was determined.
Ricin-resistant
clones
were subcloned and expanded,
and their ricin sensitivity
was tested once
Isolation
ofricin-resistant
AND
TAETLE
synthesis.
Preparation
of T101-ricin
was performed
previously’2”3
using the reducible
cross-linking
reagent
N-succinimidyl
3(2-pyridyldithio)propionate
(SPDP).
Ten milligrams of antibody
was derivatized
with
a tenfold
molar excess of
SPDP during a 30-minute
incubation
at 23#{176}C.
Approximately
3 mob
accessible
subfhydryl
groups was introduced
per mob of antibody
as
determined
by measuring the formation
of pyridine-2-thione
at 343
nm following
reduction
of an aliquot of derivatized
antibody.
Separate 20-mg abiquots of ricin were similarly
derivatized
with SPDP
and then reduced
with
25 mmol/L
dithiothreitol.
Following
extenConjugate
cells
were
Ten microliters
of a l-mg/mL
solution of
in azide-free
PBS was added to 1 mg ricin also in 1
PBS. This was followed
with 10 ML 4.9 mmol/L
lodination
GROTHAUS,
variants.
more.
Monoclonal
antibodies.
The pan-T-celb
antibodies
Tb 12324
and 3A1 ,25 the antitransferrin
antibody
L22,26 and the irrelevant
IgG2b antibody
MPC-l 1 (Sigma)
were purified from mouse ascites
fluids as described.’3
Antibodies
OKT9 and Leu3 were purchased
from Ortho
Diagnostics
(Westwood,
MA) and Litton
Bionetics
(Kensington,
MD), respectively.
as described
sive dialysis
to remove
derivatives
were
the
mixed
excess
with
reducing
the reduced
reagent,
the pyridyldithiopropionyl
ricin
derivative
of
the antibody
and the conjugate
was allowed
to form
during an
overnight
incubation
at 4#{176}C.
Unreacted
ricin was removed
by
chromatography
on protein
A-Sepharose,
and the conjugate
was
purified
from
free
antibody
by chromatography
on Sepharose
4B.
Approximately
1 to 2 mol ricin was coupled
per mob of antibody.
In vitro treatment
of cells.
Cells were incubated
with various
of whole
concentrations
ricin
or immunotoxin
with
mob/L
lactose for one hour at 37#{176}C,
washed three
medium,
and cultured overnight at 37#{176}C.
Treated
washed
into beucine-free
DMEM
and 0.25 j.iCi
added.
The cells were incubated
for four more
harvested
onto
glass-fiber
or without
0.1
times with culture
cultures were then
of 3H-leucine
was
hours at 37#{176}C
and
filters.
Surface
antigens
were detected
staining
using fluorescein-conjugated,
goat anti-mouse
immunogbobulin
as described
2,3
Determination
oflectin
sensitivity.
To determine
sensitivities
of
CEM and its ricin-resistant
variants
to Con A, PHA-L,
and WGA,
the lectins were dissolved
in PBS at a concentration
of 5 mg/mL
and
filter-sterilized.
Log-phase
cells at a concentration
of 5 x i0
Indirect
immunofluorescence.
indirect
by
cebls/mL
were
incubated
with
final
lectin
concentrations
of 0.1
to
100 og/mL.
The cells were then washed
three
times
with
fresh
growth medium,
cultured
overnight
at 37#{176}C,
and washed three times
in leucine-free
DMEM.
Protein
synthesis
was assessed
using a
four-hour
pulse
Protein
the method
Statistical
the
with
3H-beucine
as described
above.
Protein
concentrations
were determined
using
of Lowry27; bovine ‘y-globubin was used as the standard.
methods.
Correlation
analyses were performed
using
assays.
standard
product-moment
formula.
RESULTS
Binding
surface
of
ricin
binding,
ricin
peroxidase-catalyzed
dure
to
target
cells.
procedure.
migrated
To
was iodinated
as a single
using
Ricin
species
in
an
characterize
ricin
a modified
bacto-
labeled
by this proce8% SDS pobyacryb-
gel under nonreducing
conditions;
a minor
representing
self-aggregated
toxin,
detected.
The IC50 values
for inhibition
of protein
amide
(<5%),
component
was
also
synthesis
in
CEM
cells were
similar
for native and iodinated
toxin
pmob/L).
Incubation
oflabeled
ricin
at 37#{176}C
for 36 hours
not cause degradation
of the labeled
toxin (not shown).
Ricin binding
to CEM
and 8392 cells was time-dependent
at
4#{176}C,
with
minutes
Ricin
ranging
and
-50%
of ricin
binding
occurring
90% occurring
within
1 5 minutes
binding
from
to CEM
0.2 to
cells
106/mL
from
cultures
was
identical.
with
(not
1
(.-
did
within
10
shown).
cell densities
Labeled
ricin
binding
was inhibited
by unlabeled
ricin but not by a lOOx
molar
excess
of BSA. Whereas
a 1 : 1 mixture
of labeled
and
unlabeled
ricin produced
a 50% reduction
in binding
relative
to the control,
effect
on binding
a 100 x molar
of labeled
excess
toxin.
of serum
albumin
had
no
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
RICIN
SENSITIVITY
1359
Table
1 . Perce ntage
of Protein
of Control
Sy nthesis
of Lectin-Mediated
in CEM
Percentage
of Control
Variants
of Protein
Synthesis
Cell Line
U)
.J
-J
Ui
U
Lectin
(0
0
lO
Ui
Q.
0
with
0
74
88
79
ConA
86
16
22
and its ricin-resistant
lectins
0.
produced
at
in the
g/mL
3.0
2.0
9.0
Fig I .
Ricin binding
to CEM and the ricin-resistant
variants
El 2B and G2B.
Cells were
incubated
with
iodinated
ricin at
concentrations
shown for 90 minutes
as described
in the Materials
and Methods
section.
The cells were then washed.
and the level of
cell-associated
radioactivity
was determined.
Data are the means
of triplicate
determinations
from four separate
experiments;
SEM
values ranged from 0.01 to 2.1 . CEM (#{149});
G2B (0); and E12B (A).
profiles
Concentration-dependence
ricin.
ricin)
labeled
Nonspecific
represented
(described
below).
to CEM
cells
8392,
and
other
binding
was
near
binding
(at
1.1% (mean
±
to
Ricin binding
of 3 mob/L.
concentration
binding
binding
ricin
8.9%
of ricin
bound
to CEM,
but -28%
of total
ricin
ricin
to
x
8392 and
Scatchard
shown
data points ranged from 0.8%
of binding
sites/cell
Concentration-dependent
HL6O
plots
cells
(data
lectin
synthesis
from
incubated
1 00
g/mL
as
Only the data obtained
lower
of protein
determinations
were
and
1 0,
section.
because
no inhibition
number
l0.
E 1 2B and G2B
0. 1 #{149}
1 .0.
(<20%).
separate
two
at
concentrations
Data are
experiments;
to 9%.
were
1 .5 x l0
binding
profiles
showed
similar
saturation
not shown),
but maximum
and
for
profiles
and
binding
to
8392
was approximately
one-half
that of CEM
1).
Because
of the demonstrated
ability
of whole-ricin
or
HL6O
(Table
notoxins
to produce
investigated
to promote
CEM
cells
variable
nonspecific
the effectiveness
the displacement
(Fig
3). When
cellular
of lactose
(gabactose)
of ricin bound
to the
CEM
cells
were
immu-
toxicity,
we
and ricin
surface
of
incubated
for 30
variants
are shown
in Fig
for nonspecific
ricin bindin with a 100-fold
excess of
CEM
cells and two ricin-resistant
1 . Specific
binding
was corrected
ing to tubes and cells determined
unlabeled
for
are
of triplicate
respective
M
of
Materials and Methods
essentially
SE for individual
variants
concentrations
lectin
the means
RICIN,
100
27
1 00
1.0
G2B
33
0
0
E12B
PHA-L
described
-J
CEM
WGA
CEM
z
ricin
Inhibition
and Ricin-Resistant
cell
lines
ricin-resistant
approached
As shown
linear
1 mol/L
± SE, n =
17)
tested,
clones
0
z
saturation
at a
in Fig 1 (inset),
at low ricin
0
z
concentraz
tions.
Figure
2 shows
a representative
ricin binding
to CEM
cells.28 With
of 1 .5 x
b08
and
2.5
x
l0
0
(3
Scatchard28
analysis
of
the CEM
data, K,, values
mob/L
were
estimated.
z
Ui
U
The
4
3
0
“a
60
“a
20
TIME,
mm
‘I.
Fig 3.
incubated
washed.
Dissociation
of bound
ricin from CEM cells. Cells were
with I Mmol/I
iodinated
ricin for 30 mintues
at 4’C.
and resuspended
in PBS. PBS containing
100 zmol/I
unlabeled
ricin.
or PBS containing
0.1 mol/L
D-galactose.
Cells
were
further
incubated
at 4#{176}C
(open
symbols)
or 37#{176}C
(closed
symbols).
RICIN
Fig 2.
Scatchard
analysis
binding
data from Fig I were
Scatchard.
BOUND,
mol psr Iltar
of ricin
plotted
binding
according
X
to CEM cells. Ricin
to the method
of
aliquots
were
removed
at the indicated
times.
and the
amount
of cell-associated
ricin was determined.
Data
are the
means
of triplicate
determinations
from three
to six separate
experiments;
SEM ranged from 0.4% to 1 .3%. PBS 4#{176}C
(0); PBS +
100 gsmol/I
ricin 4#{176}C(t-);
PBS + 0.1 mol/L
galactose.
4#{176}C
(0-0);
PBS 37#{176}C
(#{149}-#{149});and PBS + 0.1 mol/I
galactose
37#{176}C
(-).
Similar
results
w#{149}reobtained
when
galactos.
was
replaced
with lactose.
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
1360
LEONARD,
minutes
at
material
assessed
slow
loss
4#{176}C
with
labeled
after
of ricin
ricin,
varying
the
from
washed,
and
bound
of time,
there
was
periods
cell
surface.
When
cells
incubated
with labeled
ricin and then with a lOOx
tration
of unlabeled
ricin, there was an accelerated,
loss
of labeled
roughly
material
60%
of the
from
ricin
the
was
cells.
Within
displaced
toxin
within
1 5 minutes
1 5% of the original
the
Thus,
cells.
slowly
from
added
but
bound
at 4#{176}C
and,
material
cell
the
was
surface
rapidly
when
removed
the
At 37#{176}C,
the dissociation
biphasic
and more rapid
galactose
the dissociation
to that
determined
when galactose
was
Ricin
CEM
8392
or labeled
ricin
azide
were
surface-bound
with
azide
the
added.
at 37#{176}C
were
4#{176}C
performed
with azide
linearly
4) increased
cells.
obtained
levels of ricin uptake,
at 4#{176}C
with labeled
ricin
Cells
incubated
compared
with
incubations
60 minutes
After
1O molecules
of ricin
at 37#{176}C,
-4.9
had
been
x iO and
incorporated
per
of
for
2.2 x
for
CEM
and 8392 cells,
respectively.
These
one-hour
values
represent
for CEM
cells 16.3%
of the total cell-associated
ricin at 4#{176}C
and 6.8% uptake
of bound ricin; respective
values
for 8392 cells are 24.9% and 1 1.4%.
Characterization
ricin-resistant
of
ricin-resistant
G2B
variants,
CEM
cells by mutagenesis
selection
in methylcellulose
trations
used
were
GEM
and
E12B,
with NTG
containing
, , , ,
in the
100
range
of 0.1
mutants.
were
Similarly,
isolated
binding
variants
However,
than
increased
antibodies
and
the
mannose
120,000-Dalton
agglutinin
to
(data
not
CEM
were markedly
more sensitive
line (Table
1), suggesting
an
the variants
parental
content
of cell-surface
and
glycoproteins
were
more
galactose
of cell-surface
Mechanism
shown in Fig
glycoproteins
and glycobipids.
of ricin resistance
in resistant
5, panel A, the E12B and G2B
suggest-
N-acetyl-gabactosamine
content
mutants.
As
CEM
variants
cells
clones
when
are shown
incubated
cells
showed
a decrease
findings
bound
<2 pmob/
of
a one
suggest
to decreased
<0.001
binding
that
ricin
>
nmol/L
profiles
in Fig 3. CEM
with
3 jmol/L
resistant
approximately
was
ricin
bound
ricin,
1 06 cells.
100-fold
in
for
Concen-
the
resistant
-15 pmol/106
whereas
the
Thus,
cells.
ricinthe
ricin.
these
cells
ricinvariants
ricin
sensitivity
ricin
binding.
These
at least
in part,
log
reduction
in
ricin
resistance
was due,
and
binding.
Although
ricin binding
was reduced
in the ricin-resistant
CEM
variants,
other
forms
of ricin
resistance
were
not
completely
ruled out. To determine
if ribosomal
resistance
to
A chain
or alterations
in ricin
A chain
processing
might
The
from
followed
by clonal
ricin. NTG
concento 5 g/mL,
and
resistant
and PHA-L,
ing a reduced
for CEM
Leu3
receptor).
ricin
from
In contrast,
the variants
cells to the effects of WGA
glycolipids.29
than CEM
and
3Al,
the
cells.
By
exhibited
(transferrin
indistinguishable
two
clones
displayed
as CEM
cell lines
TiOl,
OKT9
the
ricin.
and G2B
phenotype
all three
L22
TAETLE
shown).
Of
ricin-resistant
nmol/L
EI2B
with
of the
was
A
IC50 value
at
cell
and
tration-dependent
added.
Ricin uptake
in CEM cells
with time for -30 minutes
at 37#{176}C
period
lower
0.01
AND
were at beast 3 logs more
resistant
to ricin
than
CEM
Although
both variants
exhibited
an IC50 value
for
mediated
inhibition
of protein
synthesis
of
1 nmol/L,
without
and reached
a plateau
after
-.70 minutes.
The
linear
incorporation
was shorter
and the plateau
8392
similar
then warmed
for varying
periods,
and nonricin was detected
by washing
cells in lactose
at 4#{176}C.
Results
(Fig
To determine
were incubated
cells
were
reactivity
to Con
of ricin from the
than at 4#{176}C.
With
at 37#{176}C
was
positive
shown).
by 0.1
with
variants
(limited)
(pan-T-cebl),
was
ligand
surface
at 4#{176}C.
Similar
results
replaced
with lactose.
uptake.
and
of ricin
cell
with
cultured
indirect
the
dissociated
unlabeled
from
mol/L
gabactose.
cell surface
was
added,
ricin
cells
ricin-resistant
cell surface
immunofluorescence,
same
90 minutes,
associated
bound
from
The
incuof the
-50%
cell kill (not
used,
most of the
concentrations
arose
15 minutes,
after
remained
at 4#{176}C,
previously
were
the cell surface;
from
producing
ricin
concenbiphasic
thereafter,
little more toxin was released.
In contrast,
bation
with 0. 1 mol/L
D-gabactose
released
>75%
bound
g/mL
a
GROTHAUS,
with
.5
C,
3
z
-4
C,
z
I,-
0
z
z
-4
0
0
.5
80
0
0
.60
0
0,
C
(3
40
-4
z
Cs
-C
z
-4
x
z
,_0.-o------,.,
20
0
8392
0
a
U
0
0.5
1.0
Time,
1.5
Hours
‘
2.0
24
at 37#{176}C
Fig 4.
Lactose-resistant
ricin uptake
in CEM and 8392 cells.
Cells were
incubated
for 30 minutes
at 4#{176}C
with iodinated
ricin
(300 nmol/I
final concentration)
in the
presence
or absence
of
sodium azide. Cells incubated
without
azide were
washed
twice
and then incubated
at 37#{176}C
for varying
periods.
Nonsurface-bound
ricin was determined
after cells were washed
in medium
containing 0.1 mel/I
lactose.
Data
are the means
of two separate
experiments;
SEM values
ranged
from 4.6 to 1 3.1 for 8392 cells
and 16.5 to 30.5 for CEM cells. CEM (0); 8392 (-).
I.0
RICIN, nM
TIOI’RICIN,
sq/mi
Fig 5.
Inhibition
of protein
synthesis
by ricin or TiOl-ricin
in
CEM and ricin-resistant
variants
E12B and G2B. Cells were incubated with ricin (panel A) or TiOl-ricin
(plus lactose)
(panel B) at
the concentrations
shown for one hour at 37#{176}C.
Treated
cells were
washed
and cultured.
and their
protein
synthesis
activity
was
measured
as described.’3
Data
shown
in each
panel
are the
means
of triplicate
determinations
from at least two
separate
experiments;
SEM ranged
from 0.3 to 5.1 . CEM (O); CEM plus 0.1
M lactose
(0); G2B (A); and E12B (#{149}).
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
RICIN
1361
SENSITIVITY
contribute
to
CEM
resistant
ricin
ricin
clones
studies
mediated
were
showed
killed
conjugate
tested
that,
for
indirect
processing
in
to
CEM
intracellular
in the
presence
target
three
the
to ricin
lines
with
the IC50 values
CEM.
to 1 ng/mL
Thus,
these
of ricin
mechanisms
variants,
the
toxicity
suggested
were
to
Pretreatment
of
chloride
reduced
ammonium
for both
studies
variants
and
provided
other
resistance
-10
by altering
3,30
10 mmol/L
no
than
evidence
ricin
ty.
lines
ricin
binding
and
Quantitative
ricin binding
to human
and the ricin-resistant
CEM variants
Specific
ricin binding
was determined
labeled
ricin and corrected
for nonspecific
using
1 tmol/L
binding
in the
of 100 mol/L
unlabeled
bigand.
These
data conour previous
indirect
findings’2
and showed
differ-
presence
firmed
ences
in ricin
analysis
showed
a close relationship
bound
with
and without
the
lactose
P
<
binding
between
and the IC50
.005).
The B-cell
when
binding
various
cell lines.
between
addition
values
for protein
line 8392 showed
lactose
was
added
CEM
as the ricin-resistant
and
Correlation
synthesis
(r
no detectable
was
as resistant
=
.90
ricin
to ricin
variants.
immunofluorescence,
suggested
that these differences
mine, in part, the nonspecific
and
jugates
showed
that
had to be chosen
characterized
cells.
data
for intrinsic
In the present
binding,
were
in ricin binding
toxicities
of ricin
controls
carefully.
ricin
These
we previously
in vitro sensitivity
to ricin
in human
hematopoietic
that ricin binding
and
lactose
differed
widely
with
showed
and ricin/
cells.’2
We
may deterimmunocon-
ricin
studies,
dissociation,
correlated
and
uptake
observed
ricin
of ricin
cells
to CEM
half-maximum
Scatchard
and
binding
occurred
rapidly,
occurring
within
of binding
sites. A similar
conclusion
the data were analyzed
by the method
Wablach.29’3’
that
G2B
0.4
0
0.2
0
2.1
8392
4.7
0
2.8
0
0.2
0.01
6.6
0.01
0.3
2.4
0.2
0.8
9.5
0.2
0.1
ND
3.4
3.8
ND
0.1
0.04
ND
ND
0.02
0.2
ND
0.5
0.2
HEL
11
HL-60
3.9
NALM-6
NAMALWA
CFU-GM
5.6
6.2
ND
labeled
corrected
ricin
was
SEM
ricin
erythrocytes32
These
and
abrin
with
data
differ
bound
uniform
studies
showed
from
other
to sites
on
affinities.
that
ranged
galactose
tMeans
nonspecific
ten
from
0.01
was
three
reports
HeLa
binding
of ricin
However,
and
Sandvig
et
each
ricin
B chain
contains
Similar
concentration
of
experiments
are
two
G2B
and E 1 2B are
results
were
obtained
lactose.
SEM
determinations;
ranged
from
0.01
to
12
dialysis
and
added
lactose
sites3336
and equilibrium
B chain
with or without
sites
two
sites.
However,
drate
demonstrating
analyses
detectable
amounts
of ricin
sites.
ricin dissociation
release
process
and
B-chain.
toxin
bound
material
cannot
determine
to CEM
binding
in the
cells,
presence
which
data (Fig
of toxin from
and
similar
differ-
data
galactose
competed
binding
at both
3) showed that at 4#{176}C
a
the cell surface
occurred;
accelerated
may again
was
by the presence
of
reflect
the apparent
binding
sites
on the
more effectively
than unbasites, releasing
--85%
of the
4#{176}
and
whether
37#{176}C.From
all of the
ricin
our
data,
dissociating
we
from
surface
was surface-bound
or whether
it was interinto cellular
compartments
accessible
to lactose.
the released
ricin was intact,
suggesting
that if it
internalized
it was
not
findings
are consistent
with
tose) to reduce
substantially
cells)2
Finally,
cell-associated
processed
the ability
or block
our data
indicate
that
for considerable
periods
by
the
cells.
of bound
ricin.
After
one hour
These
of lactose
(or galacricin toxicity
in most
some
ricin remains
of time, even in the
presence of high lactose (gabactose)
concentrations,
be capable
of mediating
continued
cytotoxicity.
We exploited
this property
of lactose
to examine
uptake
bind
of lactose,
to ricin in the presence of lactose
were
elimination
of high-affinity
lactose
low-affinity
Lactose
for ricin
affinities
for carbohycould also exist.33
no ricin with lactose,
showed
and low-affinity
sites.
Thus,
These
ricin.
equal
cooperativity
of ricin
ences in the sensitivity
not due to selective
binding
Our
with
negative
Scatchard
was
showing
cells29
ND
suggested
that these exist as high-affinity
and low-affinity
binding
sites.33’37 In the present studies, cellular
ricin binding
also showed
apparent
high-affinity
and bow-affinity
binding
the cell
nalized
However,
and
Final
of at least
CEM.
with
Data from Leonard et
ricin
obtained
of Steck
ND
to 1 .6. Cell lines
from
was replaced
high-affinity
mm-
binding.
The means
derived
of at least
biphasic
at
9.0
20
5.3.
unlabeled
a129 also noted biphasic
ricin binding
to erythrocytes
at both
0#{176}
and 37#{176}C.
The reasons for disparities
among these studies
are unclear.
Previous
for
1 zmol/L.
variants
this
even
0.25
21
ND, not determined.
sensitivity
analyses
of ricin binding
to CEM
and 8392
to the ricin-resistant
variants
showed
curvilinear
relationships
(Fig 2) consistent
with heterogeneous
populautes.
cells
when
2.0
bebed
4#{176}C,
with
tions
0.01
0
we further
in these
Gal
+
0.3
biphasic
sensitiv-
Gal
0
slow
ities.
Binding
-
8392 cells, which
bind
apparent
high-affinity
DISCUSSION
indirect
With
+ Gal
0.5
two lactose
iodination
the amount
of
of 0.1 mol/L
-Gal
0.4
when
toxici-
ICR, (nmol/L)t
+GaI
5.6
reported;
hematopoietic
cell
is shown
in Table
2.
ricin
ricin
Bound
106 CeIIs
Synthesis
Inhibition
0.6
ricin-resistant
between
Molecules
Bound
per Cell x 1O
9.2
Values
for
decreased
of Prote in Synthesis
CEM
E12B
to 3 ng/mL
binding.
Relationship
and Inhibition
- Gal
IC50
of
immunoconjugates
immunotoxin
the three
pmol
pa’
Cell Line
immunotoxin
T101-ricin
has
been
Binding
Protein
by TiOl-
lactose
ricin-resistant
Rlcin
this
exhibited
of intracellular
2.
to a TiOl(Fig 5B).
only
lines
Table
ricin-
of lactose,
plus
chloride
on
chloride
the
lactose
cells
T10l-ricin
and
sensitivity
and
sensitivity
mob/L
comparison
effects
of ammonium
examined.
Ammonium
increase
for
All
sensitivity
ng/mL.
As an
cells
ofO.1
antigen-positive
internalization.’2”3
values
for
CEM
in the presence
conjugate
Previous
resistance,
at 37#{176}C
only
and
may
cellular
a small
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
LEONARD.
1362
amount
of ricin
uptake
8392
cells)
was
have
may
been
alter
reported,8’38
membrane
prevent
(-7%
detectable.
significant
bectin
et al suggested
mechanisms
may
uptake.2
that
actually
important
than measured
lar uptake.
The binding
and protein
relationship
Finally,
binding
form
rates
synthesis
the
lactose
such
may
report
by
not be able
to protect
may
surface-bound
differences
in
and HL-60
cells exhibited
binding
(Table
2), they
Similar
results
studies,”
these
ited
increased
to WGA
not
result
from
growth
make
have
been
may
sensitivities.
roughly
displayed
cells,
such
as
the
also
to ricin
(CEM)
mutant
that
these
cells
alterations.45
This
exotoxin
does
is an impor-
conjugate.48
cells
by ammonium
process
Similar
in the
chloride
also
immunoconjugates
in
parent
suggests
a
similar
13.30
The
present
structure.
or
intrinsic
cell
As
other
include
cessing.
reflect
with
structures
sensitivity;
tivity
studies
in hematopoietic
slow
of ricin
determinants
the
and
of ricin
cell
clinical
of specificity
whole-ricin
these
effective
sensipro-
described
binding
from
dissociation
cells,
variations
in ricin
and intracellular
translocation
characteristics
its
primary
con-
carbohydrate
eukaryotic
governing
membrane
The
as
other
are
factors
and mutation-induced
surface
mination
diptheria
by
mutants
toxicity
contribute
to
gaof the
Lyall et al reported
that ribosomal
KB cells treated
with an epidermab
of lactose
of cellular
implications
CEM
sensitivities
internalized
in these
of TlOl-ricin/bactose
and
exhib-
sensitivities
resistance
factor/Pseudomonas
CEM
As in
also
decreased
incomplete
removal
in the presence
may also be important
determinants
These characteristics
have important
Although
for
reduced
reflecting
such
equivalent
bevels of ricin
vastly
different
sensitividescribed
A and
2
intrinsically
to the presence
of lactose.
in which
cells internalize
ricin
ricin
some
by
in
to Con
ribosomal
enhancements
tent
ricin
variants
ricin
exhibit
sites)7’
CEM
comparable
TAETLE
which
binding
stable
line
that
AND
variants
ricin
The
because
in human
tant conclusion
variants
arose
carbohydrate
high
sites.2’47
suggest
Lecr
presumably
and the parental
with
toxicity
differences
PHA-L,
binding
differences
cells
sensitivity
and
or glycolipid
of these
differ-
cells,
from
nonspecific
On the other
hand,
structure
process
previous
T101-ricin
data are in complete
conet al.#{176}
Variations
in ricin
granulocyte/macrophage
to ricin and indifferent
variations
in the manner
observed
of cell-surface
variants
in Table
the
numbers
lactose
be more
of cellu-
data
variants:
reduced
nonspecific
toxicities
of wholein the
presence
of lactose
and,
as CEM
conjugates.
carbohydrate
ties.
inhibition
reflect
differences
in glycoprotein
content
or structure.
Because
binding,
whole-ricin
and
ricin
thus
ricin-resistant
for
basis
immunoconjugates
presumably,
carbohydrate
human
resistant
Finally,
that
and
a recent
for
uptake
binding
may actually
or total percentages
cellular
protein
synthesis.
These
trast to those reported
by Vallera
ences,
1 1.5%
our previous
indirect
data and demonstrate
a close
between
ricin binding
and in vitro IC50 values for
2 confirm
ricin
and
of ricin
ricin internalized
by nonendoaccount
for cellular
intoxica-
Thus, the extent of toxin
tion.39
cells
low levels
suggesting
the possibility
fluidity
following
binding
Braham
cytic
for CEM
Similar
GROTHAUS.
above,
surface
and
(galactose),
sensitivity.
for deterapplication
of
immunoconjugates.
toxACKNOWLEDGMENT
in.’’
The
1 and
ricin-resistant
2 are
described
variants
similar
to the
msot
common
in Fig 1 and Tables
class of eukaryotic
We
assistance
thank
Michael
and Marsha
Mullen
Dodds
and
Carolyn
for administrative
technical
Bailey
for
support.
REFERENCES
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K, Christensen
TB, Pihl A: Studies on the
structure
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Ricinius
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2. Stanley
P: Surface
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WJ
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York,
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1983
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JGJ, Wagemaker
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1986
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K, Refsnes
K, PihI A: Rates of different
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1976
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JW:
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Immunol
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7. Endo Y, Mitsui K, Motizuki
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The
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1987
8. Youle
lular
RJ, Colombatti
anti-ricin
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antibody
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entering
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1988 72: 1357-1363
Ricin binding and protein synthesis inhibition in human hematopoietic cell
lines
JE Leonard, CD Grothaus and R Taetle
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