From www.bloodjournal.org by guest on January 20, 2015. For personal use only.
Characterization
mRNA
of Glucocorticoid
Receptors
and
in Human
Leukemia
Cells:
Stabilization
by Diisopropylfluorophosphate
By Clark W. Distelhorst
We
have
shown
cells
frequently
that
have
a mol
demonstrate
derived
by
the
wt
intact
action
of
samples.
present
phate
(DFP).
added
to the
In the
in cytosol
proteolysis
was
doacetamide,
or mercuric
digests
T
the
which
receptor
at
the
molecule
to
steroid-receptor
form
kilobase
serine
for
important
mas.
therapeutic
Although
corticoids
The
is unknown,
site
tor defects
Because
the
than
on cells
steroid
specific
leukemias
of cell
and
killing
lymphoma
cell
receptors
killing
and
cell
process
is
that a variety
can result in glucocorticoid
resistance.3
of the importance
of glucocorticoids
apy of human
able interest
glucocorticoid
cells. Recent
leukemias
and
in the structural
Hematology/Oncology,
Department
of
Medicine,
©
I 987
this fact.
by Grune
& Stratton,
0006-4971/87/6903-0007$3.00/0
750
Inc.
receptor
was
mRNA.
are
size
which
6.5-
receptor
all
of
tran-
the
human
52.000-receptor
frag-
abnormalities
may
receptors
cells.
in
M
gluco-
5.0-kilobase
normal
size
present
S49
small
a
the
A normal
Therefore,
of foreshortened
S
by Grune
1987
human
properties
The
or
by glucoand
have
mediated
of recep-
give
apparently
absent
& Stratton,
Inc.
of glucocor-
rise
in certain
ther-
lymphoma
receptors
University
leukemia
cells
and
to the
mutant
from
synthesis
mouse
human
abnormal
ability
lym-
leukemia
receptor
fragments
corticoid
receptors
are
normal
being
both
studied
by
if not
to digestion
several
receptor
in
20,000
to 25,000)
extent
of receptor
with
the
activity
specific
the
that
kidney
cytosol
from
acute
by a factor
lymphocytic
et aht found
from
receptor
influenced
not
et
gluco-
cytosol
to
receptor
binding
site.
to correlate
for receptor
have
high
Both
proteases
cell cytosol
by the
Vol
identified.
of peptidases
cell
cytosols
isolated
the
cells
presence
could
be
or absence
No 3 (March),
stabi-
leukemia
cells.
form
level
of factors
proteolysis.8
1987:
is
of receptor
cytosol
from human
leukemia
52,000-receptor
fragment.”
69,
rat
to a merorereceptors
meroreceptor
by the
receptor
and
from
glucocorticoid
lymphocytic
to the
but also
that inhibit
We recently
found that
frequently
contains
a M,
Blood,
leukemia
prescells.
degradation
been
activities
leukemia
degradation
only
not
leukemia
cells
that glucocorticoid
chronic
the
fragment
the steroid
appeared
of converting
nonlymphocytic
Therefore
Sherman
kidney
endopeptidase
is capable
lized
in
is currently
endopeptidases.9
in human
a lysine-specific
receptor
in chronic
ceptor.’#{176}Holbrook
pro-
of the intact
and
cytosol
et a17 found
specificities
showed
cells
degradation
investigators.
responsible
cell
Sherman
leukemia
cells
smallest
of
gluco-
endogenous
receptor
that retains
degradation
protease(s)
of various
degrading
leukemia
liver
is
that
Holbrook
et a15 demonstrated
the
in cytosol
from human
leukemia
leukemia
However,
by
of “lysine-specific”
Sherman
et a17 and
ence of meroreceptors
human
human
degradation
rat
and
leukemia
of
of glucocortipresence
the
cytosol
all,
and
groups
which
(Mr
The
The
cells
proteolytic
meroreceptor,
cell
prior
normal
aI9 demonstrated
cellulose
Glucocorticoid
abnormalities
be due
to
of glucocorticoid
from
corticoid
described
could
in most,
in size
of their
to DNA
human
leukemia
cells are subject
to
proteases.8
Thus
it has been sug-
in leukemia
teases.5’7
The mechanisms
in terms
to bind
cellulose.4’5
in cytosol
from
by endogenous
gested
that previously
coid
receptor
function
in
are
their
diethylaminoethyl(DEAE)
receptors
digestion
cytosol
Hospitals,
Case
Western
Reserve
University,
Cleveland,
OH
44106.
The publication
costs ofthis article were defrayed
in part by page
charge payment.
This article
must therefore
be hereby
marked
“advertisement”
in accordance
with
18 U.S.C.
§1 734 solely
to
indicate
ticoid
to
with
Mutant
make
48.000)
or not
present.
physical
From
the Department
of Pediatrics,
Washington
University
School of Medicine,
St. Louis; the Department
of Medicine,
Case
Western Reserve University,
School ofMedicine.
Cleveland;
and the
Department
ofBiochemistry
and Biophysics,
University
of California San Francisco.
San Francisco.
Submitted
June 2, 1986; accepted
September
27, 1986.
Supported
by a grantfrom
the Children ‘s United Research
Effort
(CWD), by Grant CA42755from
the National
Institutes
of Health
(CWD), and by afellowshipfrom
the Jane Coffin Childs Memorial
Fundfor
Medical
Research
(RM).
Address
reprint requests
to Clark W. Distelhorst,
MD, Division
of
were
in many
lymphomas,
there
is considerand functional
properties
of
receptors
in human
leukemia
and
studies
have found that glucocorticoid
whether
are
hor-
lympho-
in the
kilobases
human
hybridization
abnormally
addition
RNA
in
cells.
a
regions
lines
6.5
and
receptor.
have
transcript.
cells
ments
phoma
chymo-
complex.’
with
pro-
produce
of glucocorticoid-sensitive
mouse
demonstrated
that
through
glucocorticoid
cell
to
the
io-
transcription.2
Glucocorticells and, therefore,
are an
for
studies
DFP.
leukemia
receptor
mechanism
glucocorticoid-resistant
preparation.
aprotinin.
of
leukemia
was
by hydrolyzed
interacts
modality
the exact
script
proteases.
cytosol
receptor
by Northern
that
receptors
a different
in the genome
to regulate
gene
coids kill leukemia
and lymphoma
cells
messenger
cells
glucocorticoid
glucocorticoid
intact
then
analyzed
normal
(Mr
a steroid-receptor
complex
lymphoma
52,000-receptor
HE
EFFECTS
of glucocorticosteroids
mediated
through
receptors
that bind
mone
was
the
in
chloride.
digests
cells
receptors
inhibited
phenylmethylsulfonylfluoride.
trypsin.
mouse
if diisopropylfiuorophos-
benzamidine.
tease
for
transcript
used
not
leukemia
a cDNA
Receptor
lymphoma
receptor
of
buffer
we
are
97.000)
fragment.
mouse
S49
corticoid
samples
hypotonic
receptor
in
of 52 leukemia
M
from
inhibitor
40.000
Mr
24
protease.
size
study
(Mr
Miesfeld
(mRNA)
fragments
receptors
in cytosol
normal
leukemia
fragments
present
52.000-receptor
present
a potent
human
receptor
glucocorticoid
Only
were
from
of -52,000.
M
a serine
were
samples
glucocorticoid
the
from
Receptor
cytosol
(Mr)
that
fragments
cell
that
contain
and Roger
Glucocorticoid
Receptor
of the Receptor
in
cells
The
pp 750-756
From www.bloodjournal.org by guest on January 20, 2015. For personal use only.
GLUCOCORTICOID
RECEPTORS
52,000-receptor
type
of leukemia
Mr
lar
previously
tor fragment
corresponds
)
(nt
and
fragment
previously
is considerably
mutant-glucocorticoid
These
reduced
in
size
contain
and
in
therefore
for
the
in
52,000-fragment.
in over
size
if diisopropylfluorophosphate,
inc
proteases,
with
this
human
50 different
is used
that
fragment
to stabilize
cells
samples
has
the
that
a normal
recep-
are
normal
inhibitor
of
receptor.
the
in
demonstrate
for
contain
a serine
receptor
to a
glucocorticoid
cell
we find
a
other
a potent
observation,
leukemia
find
leukemia
is
of
the mech-
52,000-receptor
We
tors
that
synthesis
to determine
human
leukemia
cell cytosol.
Our studies
the first time
that human
leukemia
cells
protease
that digests
the intact
glucocorticoid
M,
lymphoma
mRNA
results
M,
This
above)
and
increased
mouse
a receptor
in
ser-
Consistent
receptor
mRNA
in
size.
METHODS
Materials.
tors,
and
Chemical
ide,
and
Louis.
Coomassie
Richmond,
was
St.
blue
CA.
The
purchased
F6,7-3H]
Acryhamide,
N,N,’methylenebisacryham-
R-250
from
low mol
from
Pharmacia
Dexamethasone-2
Nuclear,
were
wt standards
Fine
I -mesylate
Bio-Rad
inhibiSigma
was
Laboratories,
Piscataway,
from
New
Cell
St.
line)
was
Mouse
were described
Leukemia
given
human
leukemia
by Dr Robert
lymphoma
cell
lines
Allen
and
cell line (a B hympho(American
methods
Red
of cell
cell
isolation.
Human
leukemia
cells
Cross,
transferred
RNA
were
-
to
sodium
dodecyl
sulfate-polyacrylamide
leukemia
bone
marrow
mesylate
analysis.
Total
Cytosol
determine
gel
electrophoresis
(SDS-PAGE)
as detailed
previously.”
Gels were
analyzed
by autoradiography.”
Molecular
weight standards
were
phosphorylase
b (97,400),
bovine serum albumin
(67,000),
ovalbumm (45,000),
carbonic
anhydrase
(30,000),
trypsin
inhibitor
and
dilution
of 1 :1000.
sample
buffer
cellular
RNA
then
isolated
A 100
and
boiled
was
to
isolated
incubated
label
the
cell
receptors.
from
peripheral
in cells
bind
with
[3H]dexamethasone
presence
binding
with
or absence
The
not
mesylate
compete
mesylate
excess
for
binding
for
binding
nonreceptor
bands
because
in the absence
of competing
apparent
on
the
markedly
differences
two
We
are
For
designated
the
clarity,
by
mol
the
binding
but
sites
on
band(s)
that
can
be distinthe
receptor
bands
steroid
(lane
-)
of competing
in the amount
on gels could
unlabeled
of cytosol
give rise to
of corresponding
bands
only those
bands
that
differ
and + lanes are considered
between
bands.
determined
intensity
Therefore,
in intensity
to be receptor
diograms
in the
lanes.
with
to receptors
to nonsaturable
and are not seen in the presence
steroid
(lane
+ ). Small
differences
applied
to the - lane and + lane
dexametha-
competes
other
proteins.
Therefore,
on autoradiograms,
correspond
to affinity-labeled
receptor(s)
guished
from
are seen only
and
cell suspensions
mesylate
in the
unlabled
dexamethasone
[3H]dexamethasone
receptors.
autoradiograms
of gels. To
proteins,
or receptors,
from
proteins,
duplicate
[3H]dexamethasone
of I 00-fold
unlabeled
Huor
to SDS-PAGE
to
receptors.
Multi-
[3H]dexamethasone
appear
as bands
on
the specific
binding
blood
glucocorticoid
was then prepared
and subjected
the mol wt of the affinity-labeled
the nonspecific
were
incubated
does
leukemia
were
affinity
proteins
sone.
human
cells
to
isolated
blood or bond marrow
by Ficoll-Hypaque
previously.”
Only freshly isolated cells were
labeling.
subjected
and
labeling
man
ple
for experiments.
and
at a final
to the gel electrophoresis
isolation
therefore
distinguish
The method
of labeling
receptors
in intact
cells with [3H]dexamethasone
mesylate
was described
in detail
previously.”
Briefly, cells were washed with PBS and resuspended
in
PBS in Eppendorf
microcentrifuge
tubes so that each tube contained
10 to 20 million cells. The cell suspensions
were incubated
for four
hours at 4 #{176}C
with 200 nmol/L
[3H]dexamethasone
mcsylate.
Duplicate
cell suspensions
contained
either the radiolabeled
steroid
alone (labeled
in figures)
or the radiolabeled
steroid
plus 100-fold
excess unlabeled
dexamethasone
(labeled
+ in figures).
At the end
of the incubation
period, the cells were pelleted and the cell pellets
were frozen
at - 80 #{176}C
for I 5 minutes and then thawed in hypotonic
buffer at 4 #{176}C
for 15 minutes
to prepare
cytosol. The cytosol was
then boiled for two minutes in the sample buffer for gel electrophoresis
buffer
stop the reaction.
culture
previously.20
from either peripheral
separation
as described
Affinity
to the hypotonic
RESULTS
Na2MoO4.
The Victor
Lines.
cell
Louis).
used
added
mmoL/L
stock solution of iodoacetamide
was added to the hypotonic buffer to give a final concentration
of 1 mmoL/L.
A stock
solution of0.Ol moL/L
HgCI2 was added to the hypotonic
buffer to
give a final concentration
of 0.01 mM. The hypotonic
buffer was
prepared
without EDTA when HgCI2 was used.
Limited
protease
digestion.
A concentrated
solution of chymotrypsin in deionized
water was prepared
fresh for each experiment.
An aliquot
of the chymotrypsin
solution
was added
to affinitylabeled cytosoh and incubated
at 25 #{176}C
for 1 5 minutes.
The cytosol
Affinity
20 mmoL/L
of phenylmethylsulfonylfluoride
Ni.
Phosphate
buffered
saline
(PBS)
was prepared
as
described
previously.”
The hypotonic
buffer used in cytosol preparation consisted
of 25 mmoL/L
Tris p1-I 8.2, 1 mmoL/L
EDTA,
10%
and
solution
England
Buffers.
blast
was
Boston.
glycerol,
a stock
from leukemia cells by sedimentation
through cesium chloride.23 The
RNA
was analyzed
by Northern
blot hybridization
using a cDNA
for the rat liver glucocorticoid
receptor as described
previously.’9
kit for gel electrophoresis
Chemicals,
experiments
(PMSF)
was prepared
in absolute
ethanol and added to the hypotonic buffer to give a final concentration
of 10 mmoL/L.
Benzamidine was added to the hypotonic
buffer to give a final concentration
of5 mmoL/L.
Aprotinin
(2 mg/mL;
14.5 trypsin inhibitory
U/mg)
was
All chemicals,
including
proteases,
protease
unlabeled
dexamethasone,
were purchased
from
Co.
and hactalbumin
(14,400),
References
for the mol wt
were given previously.”
Protease
inhibitors.
To inhibit proteolysis
of the affinity-labeled
receptor
during cytosol preparation,
protease
inhibitors
were added
to the hypotonic
buffer immediately
prior to use. Diisopropylfluorophosphate
(DFP) was added to the hypotonic
buffer to give a final
concentration
of 5 mmoL/L.
To exclude
the possibility
that the
inhibitory
effect of DFP was due to an impurity
present in certain
commercial
preparations
of DFP, samples of DFP were treated with
cupric ion and imidazole
to catalyze
the hydrolysis
of DFP.2”22 In
(20,100),
values
the merorecep-
by others
(see
nuclear
transfer
receptor
study was undertaken
responsible
patients.”
than
receptor
mutants
foreshortened
The present
anism
treated
larger
described
previously
closely
in size to the
cells.’2’8
751
CELLS
fragment
was not unique
to a particuand
was present
in samples
from
untreated
receptor
IN LEUKEMIA
an
receptor
bands
on autora-
arrowhead.
wt of glucocorticoid
receptors
in
From www.bloodjournal.org by guest on January 20, 2015. For personal use only.
752
DISTELHORST
cytosol
from 52 different
human
leukemia
samples.
Cytosol
from
all of the samples
contained
intact
receptors
that
migrated
opposite
the phospohorylase
b marker
and therefore
have
been
receptor
assigned
fragment
the ovalbumin
cytosol
from
that
find
along
the
(mean
fragment
consistently
assigned
now
with
SE,
was
N
the
24).
major
we cannot
exclude
receptor
fragments
may
cells.
For
example,
appeared
samples,
Mr
to
be
studied
detected
in the
untreated
produced
during
cytosol
cytosol
in
1). These
35%)
is present
detected
intact
by proteolytic
digestion
of M
cytosol
preparation,
5 mmoL/L
with
types
fragments
from
treated
if the
cells
of
were
previously
previously
To determine
within
leukemia
different
cells
and
minor
of leukemia
three
receptor
of leukemia
(8/23,
500
fragment
of
the
and
±
other
30,000-receptor
the
(16/29,
55%).
ofprotease
inhibitors.
52,000-receptor
that
in the cytosol
frequency
(Table
patients
possibility
M,
in
+
52,000-receptor
detected
in our
is shown
in Fig 2.
fragments
were
equal
leukemia
patients
Effect
a
present
one of which
52,000-receptor
approximately
the
be present
-
+
in
in
here
to be 52,000
the M
fragment
C
-
reported
reported
fragment
+
a M, of
previously
experiments
B
-
above
fragment
the
Although
receptor
studies,
to just
was
detected
as illustrated
receptor
MIESFELD
1). A major
migrated
this
receptor
=
(Fig
45,000)
samples,
reanalyzed
additional
of this
Mr
±
of 97,000
standard
(Mr
24 of the 52 leukemia
Fig 1 . We previously
55,000.” We have
results
a M,
A
AND
or
M,
if
it
is
97,000-receptor
diisopropylfluoro-
Fig 2.
Effect
of chymotrypsin
on glucocorticoid
receptors.
Same as Fig 3 except
that leukemia
cells from a different
patient
(chronic
lymphocytic
leukemia)
were studied.
(A) No chymotrypsin. (B) 5 tg/mL
chymotrypsin.
(C) 10
zg/mL
chymotrypsin.
Arrowheads
point to bands that represent
affinity-labeled
receptor fragments.
An additional
receptor
fragment
appears
to be
located
above
phosphate
for cytosol
only
Mr
band
on
the
30k
marker
(DFP)
was
preparation.
protein.
included
in the hypotonic
buffer
used
In cytosol
samples
that contained
97,000-receptors,
the
autoradiograms
was
intensity
not
of the
affected
by
receptor
inclusion
DFP in the hypotonic
buffer
(not shown).
In samples
contained
both Mr
97,000
and Mr
52,000-receptors,
inclusion
of DFP in the hypotonic
buffer
eliminated
the
52,000-receptor
intensity
band
of
finding
the
indicates
sents
that
a receptor
receptor
The
and
increased
97,000-receptor
Mr
the
by proteolytic
ability
band
of DFP
was
leukemia
no experiment
from
digestion
in
from
did
produced
during
to eliminate
confirmed
cells
52,000-receptor
Mr
fragment
proportionately
band
(Fig
the
ten
a different
DFP
fail to prevent
the type
of protease
M
cytosol
M,
separate
patient
the
1). This
band
the
of
that
the
M
repre97,000
preparation.
52,000-receptor
experiments
in each
using
experiment.
formation
In
of the
M
52,000-receptor.
To determine
Table
1 . Glucocorticoi
d Receptor
responsible
s in Human
Type of Leukemia
DFP on glucocorticoid
lymphocytic
leukemia)
receptors.
Human
were incubated
with
)
rH ]dexamethasone
mesylate
in the absence
(or presence
( +)
of excess
unlabeled
dexamethasone
to affinity
label the glucocorticoid receptors.
Cytosol was prepared
using hypotonic
buffer that
either
did not contain
DFP (A) or did contain
DFP (B). The cytosol
was subjected
to SDS-PAGE.
and the gels were
analyzed
by
autoradiography.
as shown
here. Arrowheads
point to bands that
represent
affinity-labeled
receptors.
Leukemia
Receptor
97,000
Fig 1 .
Effect
of
leukemia
cells (acute
97.000
for generat-
Cells
M,
+
52.000
Total
Acute
lymphoblastic
9
8
17
Acute
nonlymphoblastic
9
8
17
Chronic
lymphocytic
8
7
15
Chronic
myelogenous,
2
1
3
blast
Values
either
M,
receptors.
crisis
represent
97,000
the number
receptors
of leukemia
or both
M,
cell samples
97,000
that
contained
and Mr
52,000
From www.bloodjournal.org by guest on January 20, 2015. For personal use only.
GLUCOCORTICOID
ing
M,
of
were
with
in the
hypotonic
Inhibitors
(PMSF,
that
have
factor
that
inhibits
protease
inhibitory
sis, but the cysteine
hydrolysis.2122
DFP
serine
inhibitory
protease
tion
of Mr
studies
digestion
are
by
the
protease
responsible
receptor
to a Mr
serine
class
and
cells
the
digests
human
(Fig
serine
shown).
protease
for digestion
of the
generareceptor
intact
leukemia
to belong
Limited
digestion
with chymotrypsin
domain
the
from
of the
endogenous
protease
glucocorticoid
receptor
leukemia
of
cells
chymotrypsin
was
incubated
prior
that contained
only Mr
digestion
produced
a M,
3).
In cytosol
that
at the
contained
to
both
M,
chymotrypsin
intact
M,
97,000
cells
for
To further
producing
of the
Victor
substantiate
the M,
human
that proteolysis
52,000
receptor
leukemia
with
[3Hjdexamethasone
unlabeled
fresh human
cell
line
were
mesylate
and
then
leukemia
cells immedi-
the
site
prevent
methasone
their
mesylate.
experiment
employed
Victor
addition
cells
of
the
affinity-labeled
as
a range
of
receptors
generated
Mr
affinity-labeled
The
tamed
leukemia
variable
from
A typical
sample
that was employed
In the absence
of added
the
To
electrophoresis.
97,000-receptors,
40,000-receptor
the
the
to
cytosol
with
gel
in
sone
leukemia
same
contains
present
site
receptor
52,000
cell
to the
mesylate-labeled
at a different
Mr
leukemia
cells
dexametha-
of normal
is known
to
human
cytosol
is
2).
cell
prior to cytosol
preparation.
The human
preincubated
with 20 smoL/L
unlabeled
molecule.24’25
in
receptor
the
(Fig
leukemia
ately
were
the steroid-binding
receptor
that
Mixing
studies.
responsible
This
digestion.
receptors
fragment
endogenous
that
elimi-
52,000-receptors
‘
the
Therefore,
appears
leukemia
site
in
inhibit
papain.
in
and
digestion
Mr
receptor.
In addition,
not
fragment
sensitive
the
the glucocorticoid
chymotrypsin
incubated
mixed
with
the
did
digests
does
fragment,
eliminate
DFP
40,000-receptor
that
its
to
the
a Mr
was
block
not
97,000
indicate
by hydrolyis stable
upon
did
[3H]dexamethasone
concentrations
ment
The
and
Mr
results
than
(not
with
chymotrpytic
the
produced
protease
inhibi-
52,000-receptors,
both
These
(HgC12
hydrolyzed
(not
shown,
domains
the
chymotrypsin,
In cytosol
motryptic
and
proteases.21’22
was
immunoactive
if
from
and
serine
proteases
M,
cytosol
of proteases.
DNA-binding
determine
cysteine
52,000-fragment
Limited
protease
size glucocorticoid
separate
benzamidine)
activity
cysteine
for
and
nated
for
of DFP is eliminated
inhibitory
activity
that
not
used
specificity
a
specifi-
inhibit
receptor
digestion
of DFP are contaminated
52,000-receptors
that
buffer
cysteine
activity
protease
cytosol,
protease
major
for
did not
preparations
that
in leukemia
and
specificity
753
CELLS
different
have
aprotinin,
major
and iodoacetamide)
shown).
Certain
a
inhibitors
included
proteases
LEUKEMIA
fragments
protease
preparation.
tors
IN
-52,000-receptor
variety
cities
RECEPTORS
the
only
unlabeled
cell
human
M,
cells
samples
of red
to
employed
to
the
preparation
from
in this
cells
The
cells
cytosol
fragments
blood
cell
shown
in Fig 2.
cells, cytosol
from
leukemia
prior
in Fig 4.
leukemia
-97,000-receptors.
human
52,000-receptor
receptors.
numbers
same
[3H]dexa-
is shown
in the experiment
human
leukemia
contained
Victor
binding
experiment
and
the
intact
study
con-
platelets.
To
chyfrag97,000
97
6
4
3
2C
Fig 3.
Effect
of chymotrypsin
on glucocorticoid
receptors.
Receptors
in human leukemia
cells (chronic
lymphocytic
leukemia)
were
affinity
labeled
as described
in Fig 1 . The cytosol
was
incubated
at 25 ‘C for 1 5 minutes
with
chymotrypsin
prior to
SDS-PAGE.
(A)
No chymotrypsin.
(B) 5 g/mL
chymotrypsin. (C) 10 ig/mL
chymotrypsin.
Arrowheads
point to bands that
represent
affinity-labeled
receptors
or receptor
fragments.
Fig 4.
Mixing
studies.
Glucocorticoid
receptors
in Victor
cells
were affinity-labeled
as described
in Fig 1 . Leukemia
cells (chronic
Iymphocytic
leukemia)
from the same sample
used in Fig 2 were
incubated
for four hours with 20 imoL/L
unlabeled
dexamethasone.
The unlabeled
cells were
then
mixed
with
the affinitylabeled
Victor
cells in a ratio of 3:1 immediately
prior to cytosol
preparation.
Cytosol
was
then
analyzed
by SDS-PAGE
as
described
in Fig 1 . (A) Victor
cells with no addition.
(B) Victor
cells plus unlabeled
leukemia
cells. Arrowheads
point to bands
that represent
affinity-labeled
receptors
or receptor
fragments.
From www.bloodjournal.org by guest on January 20, 2015. For personal use only.
754
DISTELHORST
determine
cells,
if red blood
be the
could
tors to the M,
formed
in which
were
added
52,000-fragment,
human
red blood
(data
not shown).
lymphoma
human
Northern
blot
Receptor
to
lines.
Glucocorticoid
leukemia
mRNA
was
both
normal
size
The
patient
samples
observed
receptors
receptors
5 and
human
mRNA
was
a receptor
in human
and
containing
7 may
M
cDNA
The
reflect
cells
that
by
mRNA
sample
faintness
lower
levels
receptor
lymphoma
mRNA
regardless
in human
of the
mol
unlike
the
present
in nt mutant
highly
frag-
6.5
kb
of receptor
type
cells
of
mouse
the
leukemia
cells
5.0
kb
lymphoma
have
48,000),
that
the
is the expected
in cytosol
characterized
lym-
which
receptors
(M,
We conclude
wt of receptors
mouse
S49
(S49.l43r),
normal
glucocorticoid
kb receptor
mRNA.
size
preparations,
receptor
mRNA
cells.’9
DISCUSSION
We
demonstrate
that
frequently
contains
have a Mr of -.52,000
that
looks
containing
of the
mouse
wild
degradation
cells, which have normal
size (M,
97,000)
glucocorreceptors,
have a 6.5 kb receptor
mRNA
(lane 4). The
mutant
The
or
phoma
ticoid
of
(kb)
contain
samples
preparation.
for the
four
52,000-fragments.
receptor
the patient
2).
in six
analyzed
52,000-receptor
Mr
have
from
(lane
the
mouse
leukemia
during
nt
cell
from
in these
MIESFELD
RNA
smaller
than
have
a 5.0
produce
receptor,
and results
from
in Fig 5. A normal
6.5-kilobase
(lanes
5, 6, 7)
to the mRNA
in lanes
using
were perplatelets
to cytosol
from
mRNA
samples
recep-
not
receptor
cell
hybridization
rat liver glucocorticoid
these samples
are shown
receptor
receptor
did
mRNA
leukemia
digests
prior
cells
mRNA
compared
cell
different
bands
cells
degradation
was
intact
leukemia
of these
mRNA.
than
that
experiments
cells or human
admixture
samples
only
rather
protease
The
Receptor
ments
identical
or platelets,
of the
to affinity-labeled
preparation.
receptor
cells
source
AND
a Mr of -97,000.
two
types
of
receptor
by
Several
30,000-receptor
Mr
samples
evidence
proteolytic
that
arises
human
also
appeared
prove
that
the intact
during
resulted
receptors
previously
This led
in
reported
us to the
not responsible
fragments
in
digestion
to M,
mixture
of fresh
receptor
preparation.
First,
was
blocked
inhibitor
preparation.
DFP
in the
Second,
leukemia
line prior
of
the
cells to an
to cytosol
intact
affinity-
fragments.
We
mixing
studies
to be negative.”
conclusion
that proteolysis
was
involved
stored
at
intact
52,000
size
fragment
for the formation
of
the cytosol
of human
mixing
studies
that
had been
that
have
provides
Mr
52,000-receptor
similar
incorrect
previous
samples
the
cytosol
52,000-receptor
admixture
of fresh
unlabeled
human
affinity-labeled
human
leukemia
cell
labeled
cells
to contain
study
normal
of the potent
protease
buffer
used
for cytosol
preparation
leukemia
The present
from
digestion
of the Mr
by inclusion
hypotonic
from
fragments.
fragment
formation
cytosol
steroid
binding
receptor
fragments
in addition
to intact
receptors
that
cells
M, -52,000-receptor
leukemia
cells.
the mixture
80 #{176}C
rather
as in the
present
of
The
cytosol
than
the
study.
There-
fore the failure
of the earlier
mixing
studies
to demonstrate
receptor
proteolysis
suggests
that the leukemia
cell protease
responsible
for receptor
digestion
may not be stable
upon
storage
at low temperatures.
Limited
chymotrypsin
digestion
of the normal
is known
to separate
of the receptor
from the
domains.24’25
The leukemia
digest
the
intact
different
digestion
M,
Fig 5.
Northern
blot analysis
of leukemia
cell receptor
RNA.
Total cellular
RNA from five human
samples
(lanes 1 . 2. 5-7) and
two
murine
samples
(lanes 3. 4) were
blotted
with a probe from
the 5’ region of the rat glucocorticoid
receptor
eDNA”
(Miesfeld
et
al, unpublished
data). Lane 1 (HoLe cells). lane 2 (human
leukemia
cells).
and lane 4 (mouse
lymphoma
cells S4.9.A2wt)
show the
6.5-kb
receptor
RNA present
in cells containing
only M,
97.000
receptors.
Lanes
5. 6. 7 (human
leukemia)
and lane 3 (mouse
lymphoma
S49.143r;nt’)
contain
RNA from cells that also possess
a smeller
receptor
protein
of M,
48.000
to 52.000.
The ntspecific
receptor
RNA is shown as a 5.0-kb transcript
(lane 3). The
nature
of the smear around 4 kb is unclear,
but it appears
to be due
to partially
degraded
RNA.
97,000
the
trypsin
Mr
sensitive
and DNA-binding
described
here must
glucocorticoid
fragment
receptor
that
52,000-receptor
could
be digested
by chymotrypsin,
52,000-receptor
site
receptor
by
domain
at a
chymotrypsin,
since
chymotryptic
receptor
produced
a M,
40,000
3). The Mr
cell cytosol
40,000-receptor
Mr
that
steroid-binding
cell protease
site than
does
of the intact
receptor
fragment
(Fig
ment
in human
leukemia
glucocorticoid
the immunoactive
fragment
is present
indicating
retains
in normal
fragto a
the chymoreceptors
(Fig
2).
The
leukemia
Mr
cell
patients.
The
was consistent
52,000-receptor
samples
obtained
presence
when
in
some
24
was
of
detected
in
52 different
of the M,
52,000-receptor
leukemia
cell samples
were
again from the same patient
We previously
demonstrated
occur
fragment
from
leukemia
fragment
obtained
at time intervals
of up to 1 year.
that receptor
digestion
does not
cell
cytosol
samples
even
when
From www.bloodjournal.org by guest on January 20, 2015. For personal use only.
GLUCOCORTICOID
proteolysis
RECEPTORS
IN LEUKEMIA
is encouraged
is
that
possible
degradation
some
may
leukemia
protease
cells
leukemia
cells
corticoid
receptors
by the
protease
but
some
We
clear
cell
here.”
samples
(data
may
be
cells
similar
may
present
time.
human
the
have
assay
when
cell cytosol
analyzed
normal
tions
mononu-
earlier
digestion
human
is incubated
digestion
employed
receptor
peripheral
The
similar
blood
under
protease
mononuclear
52,000-receptor
in size to the defective
these
responsible
from
nt
mutant
mouse
for
cells but
activity,
cells.
fragment
receptors
Mr
lymphoma
there is some evidence
nt mutant
cells
are
degradation
during
here
is
in cytosol
cell
This
receptor
abnormalities
mutant
mouse
a variety
DFP,
do not
cytosol
from
inhibit
nt
the
phoma
trypsin’7
the
cells
of different
the
of defective
lymphoma
receptors
mRNA
in nt
mutant
mutant
mouse
receptor
mRNA
normal
whether
observed.
mRNA,
receptors
human
or not
Therefore
leukemia
the
cells
M, -52,000-receptor
abnormalities
absent
from
human
leukemia
form
having
a Stokes
of Sherman
expected
possible
radius
According
fragment
of this
is
to
be
is
to have
that
by us is the
the
same
a Mr
Mr
as the
described
previously
Holbrook
et al5’8 found
40,000
to 60,000.
52,000-receptor
lower mol
by Stevens
that
are
in
to a
to the work
would
be
it is
fragment
described
wt form of the receptor
et al.27 Sherman
cytosol
from
human
conditions
intermediate
in
condi-
a128 demonstrated
denaturing
for only
periods
a brief
of time,
Although
that
gel
is a more
sensi-
receptor
frag-
size
period
of time
(-30
whereas
the
required
cytosol
thus
we tested
permitting
a variety
specificities
inhibited
contain
an
serine
minutes)
assay
methods
incubations
for
more
extensive
of protease
for serine
receptor
impurity
protease
hydrolysis,
and
digestion
that
recep-
is resistant
the
cysteine
the
Mr
protease
to
M,
the
with
only
52,000
proteases.2”22
of
DFP
protease
Since
of the
proteases,
cysteine
activity
to hydrolysis.2t’22
generation
inhibitors
cysteine
effect
of DFP
is to inhibit
preparations
of DFP
often
inhibits
inhibitory
whereas
The
is sensitive
inhibitory
hydrolyzed
for
to
activity
DFP
-52,000-receptor
responsible
did
receptor
it
digestion
leukemia
cell
fragment
et a17 and
leukemia
from the protease
responsible
in leukemia
cytosol,
since
receptor
is prevented
activated
receptor
cysteine
proteolysis
a
the cytosol
of the rat
by calpastatin,
calcium-activated
the
cysteine
protease
receptor
following
Second,
for merorelatter
has
is incubated
at 3 #{176}C
for
thymus
glucocorticoid
an inhibitor
of calcium
the
protease.28’29
is responsible
to a M,
reasons.
inhibitor
of calcium-activated
han cells,3#{176}did not inhibit
digestion.
the
proteases.29
Therefore
it appears
that
in rat thymus
cells is due to the activity
unlikely
that the same
the human
leukemia
for
cysteine
human
hypotonic
First,
It
idoacetamide,
proteases
leukemia
buffer
used
for
Third,
under
in the
study,
when
intact
cytosol
et
al, unpublished
assay
conditions
employed
of
an
in mammacell-receptor
EDTA,
receptors
is incubated
seems
for digestion
52,000-receptor
preparation
in the present
study
contained
would be expected
to inactivate
calcium-activated
the
not
fragment,
class of proteases.
The
the M,
52,000-receptor
receptor
fragment
when
two hours.
Proteolysis
fragment
Therefore
receptors
tor degradation.
of
receptor
size
of
nondenaturing
been reported
to be a “lysine
specific”
protease.7.’#{176}
Mendel
et al2t showed
that intact
glucocorticoid
receptors
in rat thymus
cytosol
are converted
to a M,
52,000
cells.
of 3.5 nm.
et al,9 a receptor
size
we
condi-
lym-
cells
receptors
degraded
the
under
First,
denaturing
may be different
ceptor
formation
fragment
Stevens
et a127 showed
that
glucocorticoid
chronic
lymphocytic
leukemia
cells are often
whereas
fragments
studies.
in
site,
mRNA
in
the size of
of glucocorticoid
and
gel electrophoresis,
in earlier
studies
to the serine
that generates
in
which
may give rise to the synthesis
of foreshortened
in certain
mutant
mouse
lymphoma
cells,
apparently
incubated
belongs
protease
appears
reducing
explanations
receptor
than is gel filtration
under nondenaturSecond,
in the present
study,
cytosol
samples
that
lymphoma
earlier
for detecting
appears
to digestion
by chymoobservations).
Third,
in size compared
to the size of receptor
cells.’9 In the present
study we show that
and
receptor
significantly
potential
study
et
major
was
50,000)
including
mouse
two
between
Mendel
inhibit
(Distelhorst
decreased
wild type
in
filtration.
under
prior
to
employed
are
the
study
size
measured
Although
similar
were
detected
in
here,
fragment.
Although
the major
serine
proteases,
commercial
in nt
receptors
cells
in nt
was
respects.
data).
Second,
the Mr ‘ 52,000-receptor
cells contains
a chymotryspin-sensitive
defective
were
DFP
the
and
inhibitors,
cells are relatively
resistant
(Distelhorst
et al, unpublished
receptor
cells
in several
protease
mouse
However,
leukemia
differ
formation
mutant
et al, unpublished
human
leukemia
whereas
in human
electrophoresis,
gel
different
receptors
in
and undergo
preparation.26
lymphoma
First,
that glucocorticoid
normal
in size
cytosol
gel
by
30,000)
under
studies
electrophoresis
longer
described
present
observation
raised the possibility
that the receptor
abnormality in human
leukemia
cells and in the nt mutant
mouse
lymphoma
cells may involve
a common
mechanism.”
In this
regard
intact
by
blood
tive technique
at 25 #{176}C ments
(Mr
ing conditions.
conditions
hours
digestion
may not be unique
to leukemia
to be present,
although
probably
at a lesser
in normal
receptors
a degraded
(Mr
There
present
represents
to 25,000.
present
in
by Sherman
cytosol,
the
to nor-
described
20,000
studied
in
difference
in the
tions
to a
kidney
samples
peripheral
for two
not shown).
Receptor
digestion
by DFP. Therefore,
the
between
blood
and
52,000).
the
in addition
meroreceptor,
has a Mr
fragments
(Mr
for
detected
Receptor
liver
observed
larger
to digestion
conditions
observed
gluco-
meroreceptors
The
leukemia
fragment
in all
contain
peripheral
several
proteolysis.
to distinguish
is inhibited
receptor
appears
from
attempting
under
we
receptor
contained
receptor
that
size receptor
occurring
sensitive
the
52,000-fragment
-
mononuclear
the
naturally
frequently
size receptors.
et a19 in rat
for receptor
cells or that
are abnormally
in normal
However,
a
activity
are
at
occur
mal
contain
leukemia
that
possibilities
not
cells
incubation
responsible
in all leukemia
protects
the
protease.
these
does
may
that
Alternatively,
M,
the protease
not be present
inhibitor
or incuba-
receptor
digestion
occurs
cell samples
is unknown.
by prolonged
tion at 25 #{176}C
to 37 OCI
The reason
in some but not all human
leukemia
It
755
CELLS
cytosol
which
proteases.
present
in rat thymus
cells are stable,
even
for two hours at 4 #{176}C
(Distelhorst
observations).
From www.bloodjournal.org by guest on January 20, 2015. For personal use only.
DISTELHORST
756
Protease
receptor
inhibitors
binding,
are
inactivation,
known
to
degradation,
affect
and
glucocorticoid
protease
affects
transforma-
receptors
in vivo.
tion in vitro.3’
However,
the effect of protease
inhibitors
on
glucocorticoid
receptor
function
in vivo is unknown.
We are
currently
isolating
and characterizing
the serine
protease
from human
leukemia
cells that produces
the M,
52,000
receptor
fragment.
Also,
we are
trying
to determine
if this
the
function
or turnover
AND
MIESFELD
of glucocorticoid
ACKNOWLEDGMENT
The
authors
technical
thank
ion
Wasson
and
Laurie
Kullman
for expert
assistance.
REFERENCES
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aspects of steroid hormone-receptor
interactions, in Pasqualini
JR (ed): Receptors
and Mechanisms
of Action
ofSteroid
Hormones
- Part I. New York Marcel Dekker, 1976, p 1
2. Rousseau
GG: Control
of gene expression
by glucocorticoid
hormones.
Biochem i 224:1, 1983
3. Yamamoto
KR, Gehring
U, Stampfer
MR, Sibley
CH:
Genetic
approaches
to steroid hormone
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Res32:3,
1976
4. McCaffrey
R, Lillquist
A, Bell R: Abnormal
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receptors
in acute leukemia
cells. Blood 59:393, 1982
5. Holbrook
NJ, Bboomfield CD, Munck A: Analysis of activated
and nonactivated
cytoplasmic
glucocorticoid-receptor
complexes
from human leukemia
cells by rapid DNA-diethylaminoethyl
minicolumn chromatography.
Cancer Res 43:4478,
1983
6. Foster CM, Eisen Hi, Bloomfield
CD: Covalent
labeling of rat
thymocyte
and human
lymphoid
glucocorticoid
receptor.
Cancer
Res43:5273,
1983
7. Sherman
MR, Stevens Y-W, Tuazon
FB: Multiple
forms and
fragments
of cytosolic
glucocorticoid
receptors
from human
leukemic cells and normal lymphocytes.
Cancer Res 44:3783,
1984
8. Holbrook
NJ, Bloomfield
CD, Munck
A: Stabilization
of
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from acute nonlymphocytic
leukemia
cells by a factor from chronic lymphocytic
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cells.
Cancer
Res 44:407,
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1984
MR. Moran MC, Tuazon F, Stevens YW: Structure,
dissociation,
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of mammalian
steroid receptors.
Multiphicity of glucocorticoid
receptor
forms and proteolytic
enzymes
in
rat liver and kidney cytosols. i Biol Chem 258:10366,
1983
10. Sherman
MR. Tuazon
FB, Stevens Y-W, Niu E-M: Oligomeric steroid receptor
forms and the products
of their dissociation
and proteolysis,
in Eriksson
H, and Gustafsson
i-A (eds): Steroid
Hormone
Receptors:
Structure
and Function.
Amsterdam,
Elsevier
North Holland,
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From www.bloodjournal.org by guest on January 20, 2015. For personal use only.
1987 69: 750-756
Characterization of glucocorticoid receptors and glucocorticoid receptor
mRNA in human leukemia cells: stabilization of the receptor by
diisopropylfluorophosphate
CW Distelhorst and R Miesfeld
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