Download Immunocytochemical Localization of the

(CANCER RESEARCH 50. 1337-1345. February 15. I990|
Immunocytochemical Localization of the Glucocorticoid Receptor in Steroidsensitive and -resistant Human Leukemic Cells1
Tony Antakly,2 Dajan O' Donnell, and E. Brad Thompson
Department of Anatomy, McGill L'niversity, 3640 L'nirersily Street, Montreal, Quebec, Canada H3A 2B2 [T. A..D. OJ; and Department of Human Biological Chemistryana Genetics, t 'niversity of Texas Medical Rranch. Galveston, Texas [E. B. T.J
glucocorticoid receptor levels assayed by ligand binding was
found (15, 17). The variability in absolute number of binding
Because of their lympholytic action, glucocorticoids are included in sites per cell from patient-to-patient
has precluded, however,
most therapeutic regimens for the treatment of lymphomas and leukemuch practical clinical use of such data.
mias. I In presence of functional glucocorticoid receptor may be predictive
One problem may be the fact that in many instances the cells
of the response to hormonal therapy in these diseases. \Ve have developed
assayed are a mixture of normal and malignant cells. In addi
an ¡imminiii
\nn lii'iniial procedure for human glucocorticoid receptor
(GR) in order to assess its level and subcellular distribution in a well- tion, recent flow cytometry data using monoclonal antibodies
studied system of childhood lymphoblastic leukemia cells (GEM), »here to the human glucocorticoid receptor suggests that even in a
sensitive and resistant subclones have been established. Several Fixation clonal line of steroid-sensitive, receptor-containing cells, there
may be considerable cell-to-cell variation in receptor content
and cell permeabilization protocols were compared. The most sensitive
and reproducible one for light microscopy was prefixation in Bouin's
(18).1 Thus, it would appear that combined ligand-binding and
ABSTRACT
solution followed by cytocentrifugation. L'sing various polyclonal and
monoclonal antibodies, the GR was consistently localized predominantly
in the cell cytoplasm in the absence of steroid. We compared localization
of GR following glucocorticoid treatment in the glucocorticoid-sensitive
clone CEM C7 with resistant subclones (4R4, 3R43, ICR27). Upon
incubation with glucocorticoid, an increase in nuclear staining was clearly
observed in the steroid-sensitive C7 cells. Although the resistant cell
lines contain immunoreactive GR, they failed to show nuclear transloca
tion following glucocorticoid treatment. This outlines the importance of
the immunocytochemical procedure to distinguish between sensitive and
resistant leukcmic cells. Whether this test could be used prospectively
to help select glucocorticoid therapy in human leukemias and lymphomas
can now be examined.
INTRODUCTION
Because of their lympholytic actions, glucocorticoids are
included in many therapeutic regimens for the treatment of
various forms of leukemias and lymphomas (1,2). These actions
may be both direct on the malignant cell and indirect, through
the down-regulation of lymphokines upon which some, but not
all such malignancies depend (3). The exact cause of the direct
lympholytic action of glucocorticoids is unknown; it may in
volve induction of a lethal substance or deinduction of a critical
gene driving cell replication (4-6). In any case it is clear from
studies done in tissue culture that the presence of adequate
quantities of properly functioning intracellular glucocorticoid
receptors is a necessary though not sufficient component for
lymphocytolysis (7-9). These facts, plus the success in predict
ing response to endocrine therapy in breast cancer simply by
evaluating the level of estrogen receptors (10-14) led to several
studies attempting to do so in various leukemias and lympho
mas (15, 16). The diversity of these diseases, coupled with their
relative rarity, resulted in no simple, overall correlation being
found. However, in chronic lymphocytic leukemia, in certain
types of childhood acute lymphoblastic leukemia and in malig
nant lymphomas, a correlation between response to glucocorticoid-containing or single agent glucocorticoid therapy and
Received 6/13/88; revised 9/7/89; accepted 10/26/89.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
' This work was supported by grants from the National Cancer Institute of
Canada (to T. A.) and The National Cancer Institute of the United States (to E.
B. T.). This paper is dedicated to the memory of Howard J. Eisen (7/31/42-2/
7/87).
2To whom requests for reprints should be addressed.
immunocytochemical analysis might provide a much better
evaluation for patient material in those leukemias/lymphomas
potentially able to respond to glucocorticoids than biochemical
radioligand assays alone.
In addition to the above, a second issue, basic to the action
of steroids, is addressed herein. Recent studies on estrogen and
progesterone receptors have localized them almost exclusively
in nuclei, challenging the classic biochemical model proposing
that these receptors are located in the cytoplasm and only
associate with nuclei after binding ligand (19, 20). Yet accu
mulating immunocytochemical reports on glucocorticoid recep
tors in several tissues indicate a cytoplasmic localization of
unliganded receptor (21-23). Since specific antibodies to the
human GR4 have been prepared (24, 25), we have developed a
sensitive immunocytochemical procedure requiring minimal
amounts of human cells to assess the level and subcellular
distribution of GR in normal and neoplastic human tissues and
subsequently evaluate its usefulness as a predictive tool for
glucocorticoid response. The present paper describes the eval
uations that lead to that immunocytochemical procedure, and
show the localization of the human GR in a well-studied cell
culture system of human childhood leukemia (8, 26-28, and
reviews in refs. 7, 15). We demonstrate cytoplasmic localization
of the human GR in these lymphoid cells and show that
sensitive, but not resistant cell lines show a degree of nuclear
translocation following dexamethasone treatment correspond
ing to that determined by biochemical assays.
MATERIALS
AND
METHODS
Cell Culture
The isolation and characterization of the glucocorticoid-sensitive,
receptor-positive (r*) CEM-C7 clonal cell line and two types of glucocorticoid-lysis-resistant cell lines, activation labile (r""") and receptor
deficient (r~) derived from it have been already described in detail (8,
15, 26-28). Two r"cl/lclones were employed, 4R4 and 3R43; the r~
clone used was ICR27. Each of these cell lines was grown as stationary
suspension cultures in RPMI 1640 medium (GIBCO, NY) supple
mented with 5% fetal calf serum (GIBCO). Where indicated, sera were
previously treated with dextran-coated charcoal (DCC) (Pharmacia and
Fisher Scientific) to remove endogenous steroids; this procedure re
moved over 99% of added ['Hjdexamethasone. Cells were maintained
3 D. Marchetti, B. Barlogie. and E. B. Thompson, unpublished data.
4 The abbreviations used are: GR, glucocorticoid receptor; DCC, dextrancoated charcoal; PBS, phosphate buffered saline.
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GLUCOCORTICOID
RECEPTOR IN HUMAN LEUKEM1C CELLS
in a tissue culture incubator at 37°Cwith a humidified atmosphere of
94% air and 6% CO2 at a density of 0.5-2 x IO6cells/ml.
Preincubation with Dexamethasone
In order to study the effect of incubation with glucocorticoid on the
localization of the glucocorticoid receptor, cells were first collected by
low speed centrifugation (100 x g. clinical centrifuge) washed twice
with serum-free medium and finally incubated with or without DCCtreated serum in the presence of IO"6 M dexamethasone (Sigma, St.
Louis, MO) or the vehicle used to dissolve the steroid (ethanol. final
concentration 0.1%) for 15, 30, or 60 min at 37°C.Subsequently the
cells were collected by centrifugation as above and processed for fixation
and immunocytochemistry as described below.
Fixation and Cell Preparation for Immunocytochemistry
In order to obtain reliable and reproducible immunocytochemical
results, cells must be fixed to cross-link the proteins and cellular
elements in situ and to allow access to the immunocytochemical re
agents. However, results may vary' depending upon the tissue prepara
tion and type of fixative used and subsequent procedures followed for
sectioning the cells and enhancing their permeability to antibodies. In
all these situations, a compromise must be reached for a maximum
preservation of the ultrastructure of the cells close to the native state
and minimum denaturation of the antigenic sites. With these issues in
mind we have previously developed an immunocytochemical procedure
for the intracellular and intranuclear localization of a variety of antigens
in whole cells in culture (30). This procedure was adapted to the present
study as described below. We also evaluated detergent permeabilization
of cell membranes by treating the prefixed cells with either Triton X100, Nonidet-P40, or Saponin at a concentration of 0.1, 0.2%, 0.5, and
1% for 2-30 min. Control experiments (see below) were also system
atically performed to insure proper penetration of the antibodies to the
cellular compartments, the nucleus in particular and to avoid artefacts
caused by poor fixation, diffusion, and antigen denaturation. The
fixation protocols and types of cell preparation are shown in Table 1.
Briefly, four methods for fixation and cell preparation were compared
as follows.
Preparation of Cell Suspensions. The cells were collected by centrif
ugation and resuspended in the indicated fixative as shown in the legend
for various periods of time. Following three quick rinses in isotonic
PBS, the cells were washed overnight in PBS (18-20 h) at 4°C.
Subsequently, the cells were collected by centrifugation, incubated in
suspension with the glucocorticoid receptor antibody with or without
prior detergent permeabilization as above (see "Fixation").
Preparation of Cytospins. Two methods were tested: cells were cytospun with or without prefixation. In the first method, following fixation
of the cells as indicated above (see "Preparation of Cell Suspensions"),
the cells were resuspended in 49i bovine serum albumin. Subsequently,
cells were adhered onto gelatin-coated glass slides using a ShandonEliott cytocentrifuge at 1100 rpm for 5 min (1 x IO5 cells per slide).
Following cytospinning, the cells were air dried. In the second method,
the cells were cytospun without prefixation and finally postfixed in one
of the fixatives indicated in Table 1 for 30 min. In all cases cytospins
were air dried and immediately processed for immunocytochemistry or
alternatively, stored at -20'C for up to 3 months.
Preparation of Paraffin-embedded Sections. The cells prefixed as
indicated in Table 1 were subsequently collected as a compact cell pellet
before being embedded in a thin layer of 1% agarose and finally
routinely processed for paraffin embedding. These embedded pellets
were subsequently cut at 5 urn, affixed onto glass slides, and stored at
room temperature until processed for the localization of the glucocor
ticoid receptor.
Frozen Sections. The cultured cells were collected by centrifugation
as a compact cell pellet and immediately frozen in liquid N2 without
prefixation. The frozen pellets were covered with OCT embedding
compound (Tissue-Tek, Miles Scientific catalog no. 4583) and sec
tioned in a cryotome (Reichert, Austria) at —¿30°C.
Sections S-j/m each
Immunocytochemical Procedure
The tissue slides were processed for immunocytochemistry as de
scribed earlier (21) with one of the glucocorticoid receptor antibodies
listed below. Briefly, the slides were incubated with the primary anti
bodies for 18-24 h at 4°Cthen washed three times in PBS (15 min
with agitation). The reacted antibodies were revealed using biotinlabeled antimouse or antirabbit IgG and the avidin-biotin complex
(Vector Labs, Bringhame, CA). Four washes in PBS were done as
above. Alternatively the antibodies were revealed either by the peroxidase-anti-peroxidase method using previously documented second an
tibodies (30) or by fluorescein-labeled antibodies (Cappel, PA).
Peroxidase activity was demonstrated by the diaminobenzidine
(Sigma) cytochemical reaction (31). In this report, "staining" and
"stain" refer to the dark brown reaction products resulting from the
peroxidative polymerization of diaminobenzidine. The slides were ob
served by at least two observers who were not aware of the slide's
identity. This double-blind procedure is necessary to insure objective
recording of results. The staining of the preparation was scored on a
scale of + (low) to 5+ (very high). This is a widely accepted method of
evaluating immunocytochemical staining. In order to identify cell mor
phology, slides were weakly counterstained with 0.12% méthylène
blue.
Glucocorticoid Receptor Antibodies
In order to demonstrate the validity of the immunocytochemical
reaction, two sets of independently developed antibodies were used.
First, three polyclonal antibodies (882, 884, and 202) to the human
glucocorticoid receptor purified from IM9 cells (24) were used, they
contained approximately 20-25 mg protein/ml. Second, a monoclonal
antibody to the glucocorticoid receptor purified from human Hela cells
(32) used in our earlier studies (33) contained 21 mg protein/ml. Data
shown in Tables I and 2 are based on studies using all the above
antibodies.
Immunocytochemical Controls
To confirm the specificity of the immunocytochemical staining, the
reaction was shown to be eliminated by absorbing the antibody with
purified receptor as indicated in Fig. \B. Additional controls were
performed by substituting the receptor antibodies with preimmune
serum. For the monoclonal antibody, culture media nonimmune hybridoma cells or nonimmune mouse immunoglobulin (Sigma, MO)
were used. The data summarized in this paper represents approximately
3000 tissue slide preparations. Most experiments were repeated 5-10
times.
RESULTS
Effect of Fixation. Table 1 summarizes the pattern of subcel
lular localization of the glucocorticoid receptor using different
fixation and preparation procedures. With all fixation proce
dures that gave a satisfactory staining and morphological pres
ervation, a predominantly cytoplasmic localization was ob
served in the absence of dexamethasone or endogenous gluco
corticoid. A small percentage of cells showed mixed nuclear
and cytoplasmic labeling but no exclusively nuclear labeled cells
were seen either in absence (Figs. 1-3) or presence (Fig. 4) of
dexamethasone.
Immunoreactivity was stronger using Bouin's fixative but
quite satisfactory results were obtained using Zamboni and 4%
paraformaldehyde; the latter resulted, as expected, in a good
morphological preservation. The addition of glutaraldehyde to
the paraformaldehyde fixative, even in minute amounts, did not
improve the staining intensity and resulted in an artefactual
staining of the cell periphery (not shown) presumably reflecting
poor penetration of the antibodies. As expected, cells prefixed
were collected on warm glass slides and air dried for 15 min before
and cytospun resulted in a good morphological resolution at
fixation as described in Table 1.
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GLUCOCORTICOID
RECEPTOR IN HUMAN LEUKEMIC CELLS
procedure (Figs. 1-3). However, whereas permeabilization is
needed when cells are fixed with paraformaldehyde, this pro
cedure is not needed in Bouin-fixed cells (Fig. 4).
In order to further demonstrate that our experimental con
Cell suspension
ditions allow penetration of antibodies to the nucleus, Cos cells
2% Parai?
+++
E
Mostly cyto. & low nucÃ-.
2% Paraf. + 0.01% Glut.
++
E
Mostly cyto. & low
transfected with SV40 which express the T-antigen in their
nucÃ-.,artefact on cell
nucleus, were processed simultaneously with C7 cells for imperiphery
4% Paraf.
+++
¡
munocytochemistry. The C7 cells were stained with a glucocor
Mostly cyto. & low nucÃ-.
Mostly cyto. & low nucÃ-.
Zamboni
+++
E
ticoid receptor antibody, whereas Cos cells were stained with
Bouin
++++
E-G
Cyto. & nucÃ-.
an antibody to the T-antigen (34). Fig. 5 demonstrates that the
T-antigen is solely localized in the cell nuclei.
Cytospin
With prefixation
Effect of pH on the Staining Pattern: Demonstration of an
1% or 0.4% Glut.
+
E
Nonspecific staining on
Artefact. Immunocytochemistry is normally performed at pH
cell periphery
2% Paraf.+ 0.1% Glut.
++
G
Nonspecific staining on
7-7.6. Unless otherwise indicated, the staining reported in this
cell periphery
paper
is performed at pH 7.6. Accidentally, we found that when
2% Paraf.
+++
G
Mostly cyto. & low nucÃ-.
the pH was dropped below 5, even for short periods of time (5
4% Paraf.
++++
G
Mostly cyto. & low nucÃ-.
Mostly cyto. & low nucÃ-.
Bouin
+++++
G
min), the staining of the glucocorticoid receptor became nu
Zamboni
++++
G
Mostly cyto. &.low nucÃ-.
clear. This staining at low pH was considered artefactual be
Carnoy
P
Without prefixation
cause the preimmune control serum also showed significant
100% methanol
+
G
Cyto. periphery only.
staining of nuclei in cells stained at pH < 5. This intriguing
nucÃ-,negative
Ethanol + acetic acid'
+
G
finding is currently under further investigation and will be
Cyto. periphery only.
nucÃ-,negative
reported elsewhere. Fig. 3, A and B, contrasts the staining at
pH 7.6 and 4. This observation highlights the need to keep the
Paraffin-embedded pellet then
pH close to 7.6 during staining to avoid artefactual localization.
sectioned
4% Paraf.
Mostly cyto., some nu
Glucocorticoid Receptor Immunoreactivity using Various An
clear staining variable
tibodies.
All antibodies mentioned above gave a reproducible
among cells
4% Paraf. + 0.05% Glut.
and specific staining, there were, however, quantitative differ
Bouin
Cyto. & nucÃ-.
ences with respect to staining intensity and titer. Monoclonal
antibody 11 gave an optimal signal at 1/50 to 1/100. Polyclonal
Unfixed frozen cell pellei, sec
thenpostfixedNo
tioned at -30°C,
antibodies 882, 884, and 201 gave a strong specific staining at
dilution range from 1/100 to 1/4000 depending upon the
fixative4%
Paraf.BouinFormalin
P+
weak nucÃ-.
fixation procedure. Control experiments in which the glucocor
PPPPArtifactsCyto..Cyto.,
ticoid receptor antibody was preabsorbed with purified gluco
vapor100%
corticoid
receptor resulted in a decrease and in some cases a
ethanolAcetoneP+
near complete absence of immunoreactivity (Fig. IE). Other
* Morphological preservation: E, excellent; G. good: P. poor.
controls mentioned above were devoid of staining (Fig. l, D
'Abbreviations: Glut., glutaraldehyde; Paraf.. paraformaldehyde: cyto., cytoand £;Fig. 2, insert; 3C; Fig. 4£).
plasmic staining: nucÃ-.,nuclear staining: Dcx, dexamethasone.
Difference between the GR Localization in Steroid-sensitive
' Ethanol-acetic acid = 95% ethanol + 5% acetic acid.
and Steroid-resistant Cell Lines: Nuclear Translocation. Table 2
summarizes the results of the relative staining intensity with
the light microscope only but not at the electron microscope;5
glucocorticoid receptor antibody in various cell lines. It is clear
however the quality of these preparations may vary depending
that all cell lines studied contain glucocorticoid receptor im
on the cytocentrifugation parameters (speed, time, handling).
munoreactivity, although some of them are resistant to gluco
Finally, unlike other systems, e.g., cell surface markers, stain
corticoid action. In general 4R4 and 3R43 cells showed weaker
ing of the glucocorticoid receptor requires prefixing of all cells
staining than C7 cells. Although ICR27 cells contain very few
before centrifugation in order to avoid loss of antigen and
glucocorticoid
binding sites, they were clearly immunologically
deformation of cell morphology. When unfixed cells were cypositive. Next, we studied whether glucocorticoid has an effect
tospun then postfixed, neither the staining nor the morphology
on the subcellular distribution of GR. We had previously re
was satisfactory (Table 1 and Fig. 3).
ported a striking increase in nuclear staining in vivo of rat liver
Effect of Cell Permeabilization. Aldehyde fixation is known
and pituitary cells following adrenalectomy and treatment with
to result in a tight cross-linking of cellular proteins; conse
glucocorticoid (21 ). We observed a slight but detectable increase
quently, antibody penetration must be facilitated by opening
in nuclear staining of glucocorticoid-treated
CEM-C7 cells,
"holes" into the cellular membranes. We have previously estab
particularly in paraformaldehyde-fixed
cell suspensions and
lished an experimental protocol for performing immunocytoBouin-fixed, paraffin-embedded preparations (not shown). In
chemistry on paraformaldehyde-fixed
whole monolayer cell
preliminary
experiments, the striking glucocorticoid effect ob
cultures using ethanol as a permeabilizing agent (30). In con
served
in
the
rat in vivo was not as dramatic in CEM cells in
trast to permeabilization using detergents such as Triton, the
vitro. At first, we attributed this difference to the in vitro model
ultrastructure and cell surface morphology (scanning electron
microscopy) of ethanol-permeabilized cells is remarkably well system. However, after attempting numerous types of cell prep
arations, fixatives and permeabilization
agents, discussed
preserved. In the present study we compared permeabilization
above,
we
were
able
to
detect
a
significant
increase
in nuclear
by ethanol (50% 2 min, 70% 2 min); 0.1, 0.2, 0.5%, 1% Triton
staining of glucocorticoid treated CEM C7 cytospun BouinX-100; 0.5% saponin; or 0.2% Nonidet P-40. The most con
fixed cells (Fig. 4). This nuclear "translocation" phenomenon
sistent and reproducible staining was obtained with the ethanol
seen in C7 (glucocorticoid-sensitive) cells but not in the gluco5T. Antakly et al., unpublished data.
corticoid-resistant cells 4R4, 3R43, or ICR27 (Fig. 4). Cl cells
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Table I Effect affixation on immunocvtochemical staining of(}R in CEM C7
cells
Stain
Morpholintensity
ogy"
Fixation
Localization
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GLUCOCORTICOID RECEPTOR IN HUMAN LEUKEMIC CELLS
Fig. 1. Immunoperoxidase localization of glucocorticoid
receptor in CEM-C7 (A and and B) and CEM-C1 (C) cells
prefixed with 4% paraformaldehyde and stained in suspen
sion with antiserum 882 (1/100) (A and C) or with the
monoclonal antibody (0.2 mg protein per ml). D. control
preparation where the antiscrum was preabsorbed with pu
rified glucocorticoid receptor extracted from C7 cells (24).
Preabsorbed conditions were as described previously (21).
/.. control in which the monoclonal antibody was replaced
by culture medium from nonimmune hybridoma cells (0.2
mg protein/ml). These intact cells were photographed in
suspension. In A, B, and C, note strong staining in the
cytoplasm and a weaker one in the nucleus. Some hetero
geneity in staining intensity among cells is observed, a few
cells are weakly or even unstained. No staining is observed
in D and E. Bar. lOjim.
also showed nuclear translocation (Table 2). In addition, it is
interesting to note that in general, at high glucocorticoid recep
tor antibody concentrations from 1/100 to 1/200, where the
staining intensity is very high it was sometimes difficult to see
difference between cells incubated in the presence or absence of
dexamethasone, cortisone, or cortisol acetate at either of the
concentrations tested.
Heterogeneity of Glucocorticoid Receptor Expression. Within
any given cell preparation, the intensity of staining was variable
among individual cells. Although heterogeneity is a character
istic of a cell population and of neoplastic cells in particular
(18, 35) we are investigating glucocorticoid receptor gene
expression in individual cells using /// situ hybridization to a
cloned glucocorticoid receptor cRNA probe.6 Our preliminary
results show that the GR mRNA labeling intensity was variable
among individual cells. In another set of experiments we subcloned wild type CEM-C7 using soft agar technique described
earlier (36). Four subclones, generated from individual cells,
were processed for immunocytochemistry or in situ hybridiza
tion of the glucocorticoid receptor as described above. Results
(not shown) demonstrated a heterogeneity of the labeling com
parable to the C7 wild type (Figs. 1-4).
DISCUSSION
Technical Aspects of GR Localization. In the present study,
we developed a reliable method for the immunocytochemical
6 T. Antakly and D. O'Donnell, unpublished data.
staining of the glucocorticoid receptor in a well-established
model system of human leukemic cells. The difficulty of proc
essing such cells for immunocytochemistry lies in the fact that
they grow as suspensions. In the present experiments, we dem
onstrated that the most suitable and reproducible method for
light microscopy is fixation of the cells in Bouin's solution
while in suspension; then adhering them to glass slides by
cytocentrifugation. Such slides can be easily processed through
out the immunocytochemical procedure. This procedure would
be readily adaptable to cells obtained from peripheral blood or
marrow. As contrasted to formaldehyde fixation, the use of
Bouin's solution avoids the need for permeabilization agents.
If electron microscopic localization is desired, formaldehyde
fixation is necessary to preserve the ultrastructural morphol
ogy.5 In the case of formaldehyde fixation our results herein
and in monolayer cultures of epithelial cells (30) show that
permeabilization with ethanol is sufficient for antibody pene
tration to various cell compartments, including the nucleus.
Furthermore, this mild ethanol treatment produced minimal
artifacts in cell morphology. A number of immunocytochemical
studies, particularly on surface antigens, used unfixed cells for
cytocentrifugation followed by postfixation in ethanol-acetic
acid. When this method was followed for the GR, staining
artifacts were observed, presumably due to the extreme flatten
ing of the unfixed cells which resulted in a thin rim of cytoplasm
pushed to the periphery. We observed a narrow band of cyto
plasm which was stained moderately, plus little if any nuclear
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GLUCOCORTICOID
RECEPTOR IN HUMAN LEUKEMIC CELLS
Fig. 2. Immunofiuorcscence localization of the glucocorticoid receptor in C7
cells cultured in the presence of 5*7 fetal bovine serum, prefixed in 2rr paraformaldehyde and stained in suspension with antibody 202. 1:200. The cell cytoplasm
of most but not all cells is labeled, some nuclei are also labeled. Insert, a control
preparation stained with preimmune serum at an identical dilution. Bar. 10 .in.
staining (presumably due to poor penetration of antibody) (Fig.
3D).
A general conclusion from this study was that, in lymphoid
cells, fixation is necessary to maintain native cell morphology
and probably to stabilize antigens. Such a conclusion regarding
the need for fixation has been reached in a number of studies
on the GR and estrogen receptor in a variety of cell types (13,
14, 21, 22, 33, 37, 38). In previous studies we demonstrated
GR staining in histológica! sections of Bouin-fixed paraffin-
embedded rat tissues (21 ). This method was successfully applied
herein to the CEM cell line. However, we noted two drawbacks;
the signal is weaker and the morphology is less well defined,
particularly the cytoplasm. The disadvantage of paraffin sec
tions as compared to unembedded cells (suspensions or cytospins) is probably due to the physical difficulty in obtaining
good quality sections of tissue pellet from dispersed (8-10 /^m)
lymphoid cells and to antigen denaturation during paraffin
embedding. The main advantage of paraffin sections remains
that lymphoid cells previously obtained from leukemia or lymphoma patients and processed for pathology, can be used for
the immunocytochemical staining of GR. Thus, the patients
can be studied retrospectively.
Physiological Relevance of GR Localization. Previous immunochemical studies have shown that in several nonlymphoid
cells and tissues, the level of the GR in the nucleus increases
following glucocorticoid treatment (22, 25, 39,40). In addition,
using a monoclonal antibody, Robertson et al., showed a similar
localization of glucocorticoid receptor in CEM-C7 cells (25).
The present study used a variety of fixation and cell preparation
procedures, as well as monoclonal and polyclonal antibodies,
to demonstrate that the glucocorticoid receptor in the human
lymphoid CEM-C7 cells is localized largely in the cytoplasm
and that following glucocorticoid treatment the GR level is
increased in the nucleus. This partial nuclear translocation by
immunocytochemistry
corresponds well to the biochemical
data: CEM-C7 cells show only 40% nuclear "translocation" by
the classical method of physically separating nuclei and cyto
plasm after administration of radioligand to whole cells (7, 8,
43). Moreover, we obtained identical localization using anti
bodies to a synthetic peptide in the amino terminus of the GR
(23). Thus, the present results are in full agreement with pre
vious studies on rat liver and pituitary paraffin sections (30),
rat brain vibratome sections (40), rat monolayer pituitary cells,6
pH
Fig. 3. Effect of changing no. of fixation
conditions and the changing the pH of the
staining media on the immunoperoxidase lo
calization of the glucocorticoid receptor in C7
cells. The cells were adhered to glass slides
using a cytocentrifuge (cytospun). The cells
were either prefixed in 2ri paraformaldehyde
(A-C). or unfixed (D) prior to cytospin. Only
in £>.
the cells were postfixed in 95r¿methanol5% acetic acid following cytospin. Cell prepa
rations were stained with antiserum 882 1/200
(A. B, 1)) or preimmune serum 1/200 (C). In
B. the cells were stained in diaminobenzidine
at pH 4.0 instead of the usual pH 7.6. this
resulted in an almost exclusive nuclear stain
ing. ßar(forall micrographs), 10 ¿im.
pH 4-0
7.6
©
©
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GLUCOCORTICOID RECEPTOR IN HUMAN LEUKEMIC CELLS
C7
dex
©
©
control
©
Fig. 4. Effect of dexamethasone on the immunoperoxidase staining glucocorticoid receptor in the steroid-sensitive C7 cells (I, B) or in the steroid-resistant 4R4
cells (C, D). The cells were incubated for 30 min at 37'C with: A, C. the vehicle or B. D dexamethasone. The cells were subsequently fixed in Bouin and cytocentrifuged
before being processed for immunocytochemistry with antibody 882 1:4000. The immunocytochemical staining is brown. The cells were finally counterstained with
méthylène
blue in order to distinguish cell morphology and enhance color contrast. Note an increase in the brown staining of the nuclei of cells exposed to
dexamethasone in C7 but not 4R4 cells. E. is a control preparation in which the primary antibody was substituted by nonimmune immunoglobulin (0.2 mg/ml). F. is
an immunocytochemical control in which preimmune serum 1:4000 was used. Bar, 10 ,<m.
1342
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GLUCOCORTICOID RECEPTOR IN HUMAN LEl'KEMIC CELLS
glucocorticoid receptor is present in two types of steroid resist
ant subclones act1 (4R4 and 3R43) and r~ cells, but show no
nuclear translocation. Biochemically, this seems to be due to a
defect whereby the receptor loses its steroid-binding capability
under activating conditions (8, 9). It is also noteworthy that the
act1 clone 4R4, shown in Fig. 5, displays a lower intensity of
staining, corresponding to its lower level of binding sites and
GR mRNA (8, 45). In the case of the "receptorless" (r~) cells,
Fig. 5. Immunocytochemical localization of the T-antigen in Cos cells previ
ously infected with SV40 virus. Cells were fixed as in Fig. 1. Immunocytochemistry was done as in the case of GR except that antibody to T-antigen was used.
This is a phase-contrast micrograph. Note that the staining is exclusively in
nuclei. Bar. 10 pm.
Table 2 Subcellular localization and (jR staining intensity in CEM cell lines
of [3H1Cell
dexamethasom
sites"14,0001
lineC7Cl4R43R43ICR27No.
localizationintensity
steroid++++ (—)
Largely
cyto-plasmic
weaknuclear+++
2.0005.0003,000±Same++
Same++
Same+++
or<
none
1000Subccllular
Same(+)
steroidCytoplasmic,
increasednuclearCytoplasmic,
increasednuclearCytoplasmic.
increasein no
nuclearCytoplasmic,
increasein no
nuclearCytoplasmic.
increasein no
nuclear
" Values taken from Ref. 7.
rat hepatoma and human carcinoma monolayers (22). All these
studies converge to the conclusion that in most cell types in the
absence of steroid, the glucocorticoid receptor is largely cytoplasmic, and that at least part of it translocates to the nucleus
following exposure to glucocorticoid. Our study shows that it
is specifically so in leukemic cells. This conclusion is further
reinforced by molecular genetic studies obtained from transfected cells which transiently express high levels of GR; in this
case, GR translocation was observed following glucocorticoid
treatment (41). The localization of the glucocorticoid receptor
is therefore different from the reported exclusive nuclear local
ization of the estrogen and progesterone receptors even in the
absence of steroid (42).
The nuclear translocation shown here in CEM-C1 cells which
though in full agreement with biochemical data (7, 43) may
seem unexpected since CEM-C1 cells are in part resistant to
glucocorticoid lysis. However, the CEM-C1 cells show glutamine synthetase induction by glucocorticoid (7). Since glutamine synthetase induction occurs at the transcriptional level,
one would expect to find glucocorticoid receptors in the nucleus.
The observation that CEM-C1 cells are not lysed at short-term
interval (24 h) treatment with dexamethasone strongly sug
gested that these cells are altered in one or more nonreceptor
steps of the lysis pathway (7). Recent studies6 have shown that
Cl are growth arrested after longer periods of incubation with
dexamethasone (>4 days).
While the presence of glucocorticoid receptor is a prerequisite
for glucocorticoid response (44), we find that immunoreactive
ICR 27, our finding that they contain immunoreactive receptor
[reported here and published as an abstract (46)] was subse
quently confirmed in two papers published while this manu
script was in revision (45, 47). In addition, ICR 27 cells were
shown to contain glucocorticoid receptor mRNA in comparable
quantity to wild type C7 cells (47). Thus, the data herein again
agree with and support the biochemical findings. Although
somatic cell hybrids have shown this is not a dominant phenotype (48) there is still a possibility that the abnormal receptor
behavior may be due to abnormal association with Cytoplasmic
factors (49-55) causing sequestration there. In this context,
there is evidence that, in the cytoplasm, GR can bind to the
heat-shock protein (hsp 90) from which it presumably disso
ciates before entering the nucleus (49-54). Furthermore, the
glucocorticoid antagonist RU 486 was shown to block gluco
corticoid action by preventing the hsp 90 from dissociating
from the GR (53, 54). Other cystolic factors could also inhibit
the activation of GR. In recent studies (55), an inhibitor of GR
activation which stabilizes the steroid binding ability of the
unoccupied GR has been purified. Interestingly, the lack of
expression of immunoglobulin K chain was recently shown to
be associated with sequestering of a specific nuclear /raws-acting
factor in the cytoplasm of nonexpressing cells (56). It is also
possible that loss of steroid binding causes failure of the GR to
interact with nuclear pores. Transport of nuclear proteins, such
as nucleoplasmin and SV40 large-T involve two steps; binding
to nuclear pore elements by specific amino acid signal sequences
and translocation through nuclear pores (57, 58). One of us
have demonstrated glucocorticoid-dependent
presence of GR
on nuclear envelope preparations in vivo (59) suggesting that
translocation of GR to the nucleus also involves interaction
with nuclear envelope elements. Interestingly, the GR and other
steroid receptors bear homology to the plasminogen and largeT nuclear location signals (60, 6 1). Mutations in the GR nuclear
location signal region were shown to result in an absence of GR
nuclear translocation (41).
Clone ICR-27 represents a different case. It is grossly defi
cient in glucocorticoid receptor binding (9), but contains unex
pectedly high levels of GR immunoreactive protein and GR
mRNA (45, 47). This is in agreement with the high level of
Cytoplasmic immunocytochemical reaction in ICR-27 cells seen
in this study, in an earlier communication (46) and on flow
cytometry (18). Taken together, the present data strongly sup
port the view that bound ligand is involved in the process
whereby the GR enters the nucleus. Our data are consistent
with the studies of Becker et al. who showed the necessity of
ligand in vivo for the effective binding of GR to GR element
sites, and consequent gene induction (62).
ACKNOWLEDGMENTS
We thank Jeff Harmon for helpful discussions and for supplying
additional amounts of the rabbit polyclonal antibody. We also thank
John Cidlowski for a generous supply of human monoclonal antibody,
Mike Gannon for initial technical help, and Donna Keays for typing
the manuscript.
1343
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GLUCOCORTICOID
RECEPTOR IN HUMAN LEL'KEMIC CELLS
Note Added in Proof
Using a newly developed antibody to a synthetic GR peptide (Antakly
et al.. Endocrinology, March 1990 issue, in press), we further examined
GR localization in the CEM cell lines. Results identical to those
reported herein were obtained.
REFERENCES
1. Goldin. A., Sandberg. J. S., Henderson. E. S.. and Newman, J. W. The
chemotherapy of human and animal acute leukemia. Cancer Chemotherapy
Rep., 55: 309-507, 1971.
2. DeVita, V. I., Jr., Hubbard, S. M., and Longo. D. L. The chemotherapy of
lymphomas: looking back, moving forward. Cancer Res.. 47: 5810-5824.
1987.
Õ. Araya, S. K., Wong-Staal, F., and Gallo, R. C. Dexamethasone-mediated
inhibition of human T-cell growth factor and gamma interferon messenger
RNA. J. Immunol.. 133: 273-276. 1984.
4. Compton. M. M., and Cidlowski. J. A. Identification of a glucocorticoidinduccd nuclcas in thymocytes: a potential "lysis gene" product. J. Biol.
Chem., 262: 8288-8292, 1987.
5. Forsthoefel. A. M.. and Thompson, E. A. Glucocorticoid regulation of
transcription of the c-myc cellular protooncogene in P1798 cells. Mol. Endo..
/; 899-907. 1987.
6. Eastman-Reks, S. B., and Vedeckis, W. V. Glucocorticoid inhibition of cmyc, c-myb, and C-Ki-ros expression in a mouse lymphoma cell line. Cancer
Res.. 46: 2457-2462, 1986.
7. Thompson, E. B., Zawydiwski. R.. Brower, S. T., Eisen, H. J.. Simons, S.
S.. Schmidt, T. J., Schlechte, J. A., Moore. D. E.. Norman, M. R., and
Harmon, J. M. Properties and function of human glucocorticoid receptors
in steroid-sensitive and -resistant leukemic cells. In: H. Eriksson and J. A.
Gustafsson (eds.). Steroid Hormone Receptors: Structure and Function, pp.
171-194. Amsterdam: Elsevier Science Publishers, 1983.
8. Schmidt. T. J.. Harmon, J. M., and Thompson, E. B. Activation-labile
glucocorticoid receptor complexes of a steroid-resistant variant of CEM-C7
human lymphoid cells. Nature (Lond.). 286: 507-509. 1980.
9. Harmon, J. M.. and Thompson, E. B. Isolation and characterization of
dexamethasone-resistant mutants from human lymphoid cell line CEM-C7.
Mol. Cell. Biol., /: 512-521, 1981.
10. DeSombre. E. R., Greene. G. L., and Jensen. E. V. Estrogen receptors and
the hormone dependence of breast cancer. In: M. J. Brennan, C. M. McGrath.
and M. A. Rich (eds.). Breast Cancer: New Concepts in Etiology and Control,
pp. 69-87. New York: Academic Press, 1980.
11. McGuire, W. L. Steroid receptors and breast cancer. In: H. Aldercrentz. R.
D. Bulbrook, H. J. Van der Molen, A. Vermeulen, and G. Sciana (eds.).
Research on Steroids, Vol. 9, pp. 11-17. Amsterdam: Exerpta Medica, 1981.
12. Thorpe, S. M., Rose, C., Rasmussen, B. B., Mouridsen, H. T., Vayer, T.,
and Keiding. N. on behalf of the Danish Breast Cancer Cooperative Group.
Prognostic value of steroid hormone receptors: multivariate analysis of
systematically untreated patients with node negative primary breast cancer.
Cancer Res., 47: 6126-6133, 1987.
13. King, W. J., DeSombre, E. R., Jensen, E. V., and Greene, G. L. Comparison
of immunocytochemical and steroid-binding assays for estrogen receptor in
human breast tumors. Cancer Res., 45: 293-304, 1985.
14. Charpin, C., Martin, P. M., DeVictor, B.. Lavaut, M. N., Habib, M. C.,
Andrac. L., and Toga. M. Multiparametric study (SAMBA 200) of estrogen
receptor immunocytochemical assay in 400 human breast carcinomas: analy
sis of estrogen receptor distribution heterogeneity in tissues and correlations
with dextran coated charcoal assays and morphological data. Cancer Res.,
48: 1578-1586. 1988.
15. Thompson. E. B.. and Harmone, J. M. Glucocorticoid receptors and gluco
corticoid resistance in human leukemia in vivo and in vitro. In: G. P.
Chrousos, D. L. Loriaux, and M. B. Lipsett (eds.). Advances in Experimental
Medicine in Biology. Vol. 196. pp. 111-127. New York: Plenum Publishing
Corp., 1986.
16. Stevens. J.. and Stevens, Y-W. Glucocorticoid receptors in human leukemia
and lymphoma: quantitation and clinical significance. In: V. P. Hollander
(ed.), Hormonally Responsive Tumors, pp. 155-181. New York: Academic
Press, 1985.
17. Bloomfield, C. D., Munck, A. U.. and Smith, K. A. Glucocorticoid receptor
levels predict response to treatment in human lymphoma. In: E. Gurpide
(ed.). Hormones and Cancer, pp. 223-233. New York: Alan R. Liss, Inc.,
1984.
18. Marchetti, D.. Van, N. T., Gametchu. B., Thompson, E. B.. Kobayashi, Y.,
Walanabe. F., and Barlogie. B. Flow cytometric analysis of glucocorticoid
receptor using monoclonal antibody and fluoresceinated ligand probes. Can
cer Res., 49: 863-869, 1989.
19. Walters, M. R. Steroid hormone receptors and the nucleus. Endo. Rev., 6:
512-543. 1985.
20. Welshons, W. V., Cormier, E. M.. Jordan, V. C., and Gorski, J. Biochemical
evidence for the exclusive nuclear localization of the estrogen receptor. In:
A. K. Roy and J. H. Clock (eds.). Gene Regulation by Steroid Hormones,
pp. 1-20. New York: Springer-Verlag, 1987.
21. Antakly, T., and Eisen, H. J. Immunocytochemical localization of glucocor
ticoid receptor in target cells. Endocrinology, 115: 1984-1989, 1984.
22. Wikstrom, A-N., Vakke, O., Okret, S., Bronnegard, M., and Gustafsson. J-
A. Intracellular localization of the glucocorticoid receptor: evidence for
cytoplasmicand nuclear localization. Endocrinology, 720: 1232-1242, 1987.
Antakly.
T.. O'Donnell. D., Shustick, C., and Zhang. C. Localization and
23.
quantitation of glucocorticoid receptor and its mRNA in human lymphoid
cells (Abstract). Blood, 70(Suppl.).- 250a. 1987.
24. Harmon, J. M., Eisen. H. J., Brower, S. T.. Simons, S. S., Langley, C. L.,
and Thompson. E. B. Identification of human leukemic glucocorticoid recep
tors using affinity labeling and anti-human glucocorticoid receptor antibod
ies. Cancer Res., 44: 4540-4547, 1984.
25. Robertson. N. M., Kusmik, W. F., Grove, B. F., Miller-Diener. A.. Webb.
M. L.. and Litwack, G. Characterization of monoclonal antibody that probes
the functional domains of the glucocorticoid receptor. Biochem. J., 246: 5565. 1987.
26. Foley, G. E., Lazarus, H.. Farber, S., Uzman, G. B., Boonc, B. A., and
McCarthy. R. E. Continuous culture of human lymphoblasts from peripheral
blood of a child with acute leukemia. Cancer (Phila.). IS: 522-592, 1965.
27. Harmon, J. M., Norman. M. R., Fowlkes, B. J.. and Thompson, E. B.
Dexamethasone induces irreversible Gl arrest and death of a human lymph
oid cell line. J. Cell Physiol.. 98: 267-278. 1979.
28. Harmon. J. M.. and Thompson. E. B. Glutamine synthetase induction by
glucocorticoids in the glucocorticoid-sensilive human leukemic cell line
CEM-C7. J. Cell Physiol., 110: 155-160, 1982.
29. Harmon, J. M.. Schmidt. T. J.. and Thompson, E. B. Defective steroid
receptors in a glucocorticoid resistant clone of human leukemic cell line. In:
W. W. Leavitt (ed.). Advances in Experimental Medicine and Biology. Vol.
138, pp. 301-313. New York: Plenum Publishing Corp.. 1982.
30. Antakly. T., Pelletier. G., Zeytinoglu. F.. and Labrie, F. Changes of cell
morphology and prolactin secretion induced by 2-Br-Alpha-Ergocryptine.
estradiol. and thyrotropin-releasing hormone in rat anterior pituitary cells in
culture. J. Cell Biol.. 86: 377-387, 1980.
31. Graham, R. C.. and Karnovsky. M. J. The early stages of absorbtion of
injected horseradish peroxidase in the proximal tubules of mouse kidney:
ultrastructural cytochemistry by a new technique. J. Histochem. Cytochem..
14: 291-302, 1966.
32. Cidlowski, J.. and Richon, V. Evidence for microheterogeneity in the struc
ture of human glucocorticoid receptors. Endocrinology, 115: 158-1597.
1984.
33. Antakly, T.. Sasaki, A., Liotta, A. S., Palkovitz, M., and Krieger. D. T.
Induced expression of the glucocorticoid receptor in the rat intermediate
pituitary lobe. Science (Wash. DC), 229: 277-279, 1985.
34. Harlow. E., Crawford, L. V., Pirn, D. C., and Williamson. N. M. Monoclonal
antibodies specific for simian virus 40 tumor antigens. J. Virol., 39: 861869. 1981.
35. Schnipper, L. E. Clinical implications of tumor-cell heterogeneity. New Engl.
J. Med.. 314: 1423-1431. 1986.
36. Norman. M. R.. Harmon. J. M., and Thompson, E. B. Use of a human
lymphoid line to evaluate interactions between prednisolone and other chcmothcrapeutic agents. Cancer Res., 38: 4273-4278, 1978.
37. Cheng, L., Binder, S. W., Fu, Y. S., and Lewin. K. J. Methods in laboratory
investigation: demonstration of estrogen receptors by monoclonal antibody
in formalin-fixed breast tumors. Lab. Invest.. 58: 346, 1988.
38. Andersen, J., Bentzen, S. M., and Poulsen, H. S. Relationship between
radioligand binding assay, immunoenzyme assay and immunohistochemical
assay for estrogen receptors in human breast cancer and association with
tumor differentiation. Eur. J. Clin. Oncol., 24: 377-384, 1988.
39. Svec, F.. and Williams. J. Characterization of the glucocorticoid receptor
translocated to the nucleus in intact AtT-20 mouse pituitary tumor cells at
low temperatures. Endocrinology, 113: 1528-1530, 1983.
40. Fuxe, K., Wikstrom, A.. Okret. S., Agnati. L. F., Harfstrand. A.. Yu, Z.,
Granholm, L., Zoli, M., Vale. W., and Gustafsson, J. A. Mapping of
glucocorticoid receptor immunoreactive neurons in the rat tel- and diencephalon using a monoclonal antibody against rat liver glucocorticoid receptor.
Endocrinology. 117: 1803-1812, 1985.
41. Picard, D., and Yamamoto, K. R. Two signals mediate hormone-dependent
localization of the glucocorticoid receptor. EMBO J., 6: 3333-3340, 1987.
42. Antakly. T. The localization of the glucocorticoid receptor: nuclear or cytoplasmic? (Abstract.) Anat. Ree., 211: 11A, 1985.
43. Zawydiwski, R., Harmon, J. M.. and Thompson, E. B. Glucocorticoid
resistant Human Acute Lymphoblastic Leukemic Cell Line with Functional
Receptor. Cancer Res.. 43: 3865-3873, 1983.
44. Munck. A., and Leung. K. Glucocorticoid receptors and mechanisms of
actions. In: J. R. Pasqualini (ed.). Receptors and Mechanisms of Action of
Steroid Hormones, Part 2. p. 311. New York: Marcel Dekker. 1977.
45. Thompson. E. B., Yuh, Y. S., Ashraf, J., Gametchu. B., Linder, M., and
Harmon, J. M. Molecular genetic analysis of glucocorticoid action in human
leukemic cells. J. Cell. Biochem., 74: 221-237, 1988.
46. Antakly, T.. and Thompson, E. B. Localization of the glucocorticoid receptor
in steroid-sensitive and steroid-resistant human leukemic cell lines. In: Ad
vances in Gene Technology: Molecular Biology of the Endocrine System,
ICSU Short Reports, Vol. 4, pp. 258-259. Cambridge. MA: Cambridge
University Press, 1986.
47. Harmon, J. M., Elsasser, M. S., Eisen, L. P., Urda, L. A., Ashraf, J., and
Thompson, E. B. Glucocorticoid receptor expression in receptorless mutants
isolated from the human leukemic cell line CEM-C7. Mol. Endocrino!., 3:
734-743, 1989.
48. Harmon, J. M.. Thompson, E. B., and Baione, K. A. Analysis of glucocorticoid-resistant human leukemic cells by somatic cell hybridization. Cancer
Res., 45: 1587-1593. 1985.
1344
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1990 American Association for Cancer Research.
GLUCOCORTICOID
RECEPTOR IN HUMAN LEUKEMIC CELLS
49. Mendel, D. B.. Bodwell. J. E., Gametchu, B.. Harrison, R. W., and Munck.
A. Molybdale-stabilized nonactivaied glucocorticoid-receptor complexes
contain a 90-kDa non-steroid-binding phosphoprotein that is lost on activa
tion. J. Biol. Chem.. 267: 3758-3763. 1986.
50. Sanchez. E. R., Meshinchi. S.. Tienrungroj, W.. Schlesinger. M. J.. Toft. D.
O., and Pratt. W. B. Relationship of the 90-kDa murine heat shock protein
to the untransformed and transformed states of the L cell glucocorticoid
receptor. J. Biol. Chem., 262: 6986-6991. 1987.
51. Howard. K. J., and Distelhorst. C. W. Evidence for intracellular association
of the glucocorticoid receptor with the 90-kDa heat shock protein. J. Biol.
Chem.. 263: 3474-3481, 1988.
52. Howard, K. J.. and Distelhorst. C. W. Effect of the 90 kDa heat shock
protein. HSP90. on glucocorticoid receptor binding to DNA-cellulose.
Biochem. Biophys. Res. Commun., 151: 1226-1232, 1988.
53. Groyer, A., Schweizer-Groyer, G., Cadepond, F., Mariller, M., and Baulieu.
E-E. Antiglucocorticosteroid effects suggest why steroid hormone is required
for receptors to bind DNA in vivo but not in vitro. Nature (Lond.). 328:624626, 1987.
54. Lefebvre, P., Formstecher, P., Richard. C., and Dautrevaux, M. Ru 486
stabilizes a high molecular weight form of the glucocorticoid receptor con
taining the 90 K non-steroid binding protein in intact thymus cells. Biochem.
Biophys. Res. Commun.. ISO: 1221-1229. 1988.
55. Bodinc. P. V.. and Litwack. G. Purification and structural analysis of the
56.
57.
58.
59.
60.
61.
62.
modulator of the glucocorticoid-receptor complex. J. Biol. Chem..26.?: 35013512. 1988.
Baeuerle. P. A., and Baltimore. D. 1KB: a specific inhibitor of the NF-KB
transcription factor. Science (Wash. DC), 242: 540-546, 1988.
Newmeyer, D. D.. and Forbes. D. J. Nuclear import can be separated into
distinct steps in vitro: nuclear pore binding and translocation. Cell, 52: 641653. 1988.
Richardson, W. D., Mills, A. D., Dilworth. S. M., Laskey, R. A., and
Dingwall, C. Nuclear protein migration involves two steps: rapid binding at
the nuclear envelope followed by slower translocation through nuclear pores.
Cell, 52: 655-664, 1988.
Lebebvre, Y. A.. Howell. G. M.. and Antakly. T. Localization of the gluco
corticoid receptor to the nuclear periphery. The Endocrine Society. Endocrine
Society Annual Meeting, no. 1470. 1989.
Wolff. B., Dickson, R. B.. and Hanover. J. A. Current Awareness: a nuclear
localization signal in steroid hormone receptors? TIPS, 8: 119-121, 1987.
Hollcnberg. S., Weinberger, C., Ong, E., Cerelli. G.. Oro, A., Lebo, R.,
Thompson, E.. Rosenfeld. M., and Evans. R. Primary structure and expres
sion of a functional human glucocorticoid receptor cDNA. Nature (Lond.),
318: 635-641, 1985.
Becker, P. B., Gloss. B., Schmid, W.. Strahle. U., and Schutz. G. In viro
protein-DNA interactions in a glucocorticoid response element require the
presence of the hormone. Nature (Lond.), 324: 686-688. 1986.
1345
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Immunocytochemical Localization of the Glucocorticoid
Receptor in Steroidsensitive and -resistant Human Leukemic
Cells
Tony Antakly, Dajan O'Donnell and E. Brad Thompson
Cancer Res 1990;50:1337-1345.
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