Download Glucocorticoid Receptor Determinations in

[CANCER RESEARCH 41, 3979-3984,
0008-5472/81
/0041-OOOOS02.00
October 1981]
Glucocorticoid Receptor Determinations in Leukemia Patients Using Cytosol
and Whole-Cell Assays1
Stefano lacobelli,2 Vittoria Natoli, Paola Longo, Franco O. Ranelletti, Giulio De Rossi, Daniela Pasqualetti,
Franco Mandelli, and Renato Mastrangelo
Laboratory of Molecular Endocrinology [S. I., V. N.. P. L.]. Departments of Histology
Department of Hematology [G. D., D. P., F. M.j, University of Rome, 00100 Rome. Italy
[F. O. P.] and Pediatrics [R. M.], Sacro Cuore Catholic
University;
and
ABSTRACT
al. (13, 14) demonstrated that cells of 22 ALL patients with
favorable prognoses contained high GR levels while the cells
Parallel determinations of glucocorticoid
receptors in the of 6 other patients in relapse who were refractory to treatment
cells of patients with various forms of leukemia were made by contained barely detectable GR levels. In a subsequent study,
two assay methods, one using cell-free cytosolic extracts and
Lippman et al. (15) reported that the cells of AML patients had
the other using whole-cell preparations. Both assays revealed
appreciable GR levels in only 3 of 16 cases. In independent
saturable binding of triamcinolone acetonide in all cases. The studies, Gailani ef al. (4) found that the cells of 4 of 4 lymphomean equilibrium dissociation constant for the interaction of
triamcinolone acetonide with the cytoplasmic receptor at 2° sarcoma patients, 3 of 3 ALL patients, 2 of 6 AML patients, 0
of 8 CLL patients, and 0 of 2 CML patients contained appre
was 9.45 ±6.33 (S.D.) nM while that for the whole-cell binding
ciable
GR levels. Furthermore, Terenius ef al. (28) reported
at 37°was 6.13 ±3.25 nM, suggesting an increase in receptor
that the lymphocytes of only 17 of 27 patients with CLL
contained significant levels of GR's, while Homo et al. (8) were
affinity at physiological temperatures. Competition experiments
with various unlabeled steroids revealed a higher degree of
able to demonstrate the presence of specific receptors in all of
glucocorticoid specificity at 37°in whole-cell suspensions than
the patients with CLL and ALL as well as in normal lymphocytes.
at 2°in cytosol. In a comparative analysis of 41 leukemic cell
Finally, more recent reports have shown the presence of GR's
specimens, it was found that determinations carried out by
whole-cell assay, calculated as number of sites per cell, cor
related well with those performed by cytosol assay, calculated
as fmol/mg protein, independent of the type of leukemia.
However, for cells with low receptor content, the two assay
methods were more difficult to compare. In agreement with
previous reports, the cytosol assay consistently underesti
mated the number of receptors with respect to the whole-cell
assay, particularly in cases of lymphatic leukemia. Further
more, the underestimation decreased for increasing levels of
total cellular receptor. These results suggest that, in addition
to possible defects in the cytoplasm-to-nucleus
translocation
process, the acceptor-binding capacity of the nucleus may also
represent one of the factors which determines the levels of
assayable cytoplasmic receptors. Moreover, they indicate that
the two assay methods furnish nonequivalent information.
INTRODUCTION
The possibility of using steroid receptor analysis to predict
the response of a potentially hormone-dependent
tumor to
endocrine therapy has attracted considerable interest, espe
cially in the area of breast cancer (20). On the basis of en
couraging results obtained with this disease, many laboratories
have attempted analogous studies on GR's3 in human leukemia
cells in the hope of finding a correlation with the clinical
response to glucocorticoids.
So far, however, these studies
have produced conflicting results. In early reports, Lippman ef
' Supported by Grant 800156696 of the Finalized Project "Control of Tumor
Growth."
2 To whom requests for reprints should be addressed.
3 The abbreviations used are: GR, glucocorticoid receptor; ALL, acute lymphoblastic leukemia; AML. acute myelogenous leukemia; CLL, chronic lympho
cyte leukemia; CML, chronic myelogenous leukemia; ANLL, acute nonlymphocytic leukemia; TA, triamcinolone acetonide.
Received February 23, 1981 : accepted June 15. 1981.
OCTOBER
1981
in virtually all patients with various forms of hematological
cancer (1, 2, 11, 19).
Some of these discrepancies could be related to the different
methods used to measure the receptors. Many of the studies
cited above utilized either a whole-cell or a cytosol assay
indifferently. Although cytosol preparations have been used to
quantify a variety of steroid receptors, including those for
glucocorticoids, several reports claim that this method is un
reliable for the measurement of GR content in leukemia (1, 2,
7) since both normal and neoplastic lymphocytes contain a
very thin rim of cytoplasm and one must be aware of the
efficiency of cell breakage during homogenization. Besides,
unbound GR's may be preferentially localized in the nuclei of
lymphocytes (23); therefore, cytoplasmic assay may result in
underestimation of cellular receptor content. By using a wholecell procedure, some of these problems can be overcome and
appropriate measurement of total (cytoplasmic plus nuclear)
receptors can be obtained. However, a major technical problem
associated with this assay technique is the difficulty of distin
guishing the proportion of nonspecifically bound steroid, often
quite elevated, from free steroid after cell washes. It appears
obvious that a thorough comparison of GR measurement using
both the whole-cell assay and the cytosol assay could be
useful. This paper presents the results of such a comparison
performed on the cells from a group of patients with various
types of leukemia.
MATERIALS
AND METHODS
Patients
The study was done on 41 patients with various forms of leukemia
(14 patients with CLL, 15 patients with ALL, and 12 patients with
ANLL). Diagnoses were established by means of conventional morpho
logical and cytochemical criteria. None of the ALL and ANLL patients
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S. lacobelli et al.
had received antileukemic therapy of any kind at the time of study.
None of the CLL patients under treatment had received any glucocor-
dissociation
ticoid therapy within a week of their inclusion in the study.
calculated as the difference between the values obtained with and
without the excess of radioinert steroid. Binding capacity was ex
pressed as number of sites per cell (whole-cell assay) or fmol/mg
Isolation
of Leukocytes
constant (K :) and the concentration
of [3H]TA binding with
the aid of an Olivetti P-6060 desk-top computer.
Specific binding was
Heparinized peripheral blood was diluted 2 to 3 times with 0.9%
NaCI solution. Oextran T 500 (Pharmacia, Uppsala, Sweden) was
added to a final concentration of 1% (w/v). After gravity sedimentation
for 30 min at room temperature, the supernatant containing leukocytes
was centrifugea at 400 x g for 6 min. Hypotonie lysis of contaminating
erythrocytes was achieved by treating the cells with 0.84% ammonium
chloride for 15 min. After 2 washes with phosphate-buffered
saline
protein (cytosol assay). Repeated determinations of numbers of sites
per cell on the same fresh cell suspensions gave an average range of
±7% around the mean value. Repeated determinations on the same
group of frozen cells (cytosol assay) showed a similar variability.
(0.136 M NaCI: 2.6 mw KCI: 6.4 mw Na2HPO4: 1.4 mM KH2PO4), the
cell pellets were resuspended in Roswell Park Memorial Institute Tissue
Culture Medium 1640 (Grand Island Biological, Co., Grand Island, N.
Y.) supplemented with 25 mM 4-(2-hydroxyethyl)-1-piperazinethane-
GR Assays. Since the amountof cells recovered from most
of the patients was not sufficient to furnish high enough cytosol
protein concentrations to make a complete binding curve, we
decided to use the DE 81 ion-exchange filter assay which
accurately measures GR levels at very low protein concentra
tions (25). Using this procedure, appreciable levels of receptor
were measured in all the patients studied. Furthermore, in
some patients with a high peripheral white blood count, recep
tor determinations were made by using dextran-coated char
coal and filter assays simultaneously. The 2 procedures gave
comparable results (data not shown).
Chart 1 illustrates the time course of [ 'H]TA binding to
sulfonic acid buffer (Sigma Chemical Co., St. Louis, Mo.), penicillin
(100 units/ml), and streptomycin (100 fig/ml). Viability was always
greater than 90%, as determined by the trypan blue exclusion proce
dure (24). A portion of the cell suspension was used immediately for
GR determination by whole-cell assay while the remaining part was
centrifuged again and the cell pellet stored frozen at -85° for subse
quent analysis of GR content by cytosol assay. Under these storage
conditions, no loss of receptor activity was noted for up to 3 months.
Binding Studies
Whole-Cell Assay. Duplicate 0.1-ml aliquots of the leukocyte sus
pensions (10 to 20 x 10'v ml) were pipeted into 10 x 85 plastic tubes
which already contained 0.15 ml of Roswell Park Memorial Institute
Tissue Culture Medium 1640 with [3H]TA (25 Ci/mmol; the Radiochemical Centre, Amersham, England) at 10 different concentrations varying
from 0.8 to 25 RM, with or without a 100-fold excess of radioinert TA.
Tubes were incubated under 5% CO.. in air at 37° for 60 min except
for experiments examining the time course of binding. After incubation,
1.5 ml of phosphate-buffered
saline were added to each tube; the
tubes were kept in ice for 5 min and then centrifuged at 200 x g for 6
min. The washing process was repeated twice. The extent of nonspe
cific bound counts was reduced to a minimum, and less than 7% of
specifically bound counts were lost in this washing procedure. Cell
pellets were suspended in 1 ml of 80% ethanol, and the suspensions
were transferred to counting vials. In order to determine the exact
number of cells recovered at the end of the washing procedure, 4
additional tubes containing the same amounts of cells were prepared
and processed in the same manner as the tubes used for binding
determinations.
Cytosol Assay. Cell pellets were rapidly thawed and resuspended
in approximately 4 volumes (v/v) of 10 mM Tris:1.5 mw EDTA:12 mM
monothioglycerol:20%
glycerol, pH 7.4 at 2°. After homogenization
with 20 strokes in an all-glass homogenizer, the cytosol fraction was
obtained by centrifugation at 145,000 x g for 45 min at 2°.Duplicate
0.06-ml aliquots were transferred into 10 x 75 glass tubes which
already contained 0.06 ml of 10 mM Tris:1.5 mM EDTA:12 mM monothioglycerol:20%
glycerol, pH 7.4 at 2°, with [3H]TA at 7 different
RESULTS
receptor in whole cells as well as in cytosol preparations. In
the cells, the highest level of specific binding is achieved
between 20 and 30 min of incubation at 37°and remains
stable for at least 60 min thereafter. At 2°,specific steroid
binding in cytosol reaches a maximum after 120 min of incu
bation and remains constant between 120 and 240 min.
The affinity of various steroids for GR sites in the cells of
some patients was evaluated by both assay methods. A rep
resentative experiment carried out in one case of CLL is illus
trated in Table 1 where the results are expressed as relative
binding affinity. In both assays, the most efficient competitor
was TA itself (relative binding affinity defined as 100) followed
by dexamethasone, prednisolone, and cortisol. Corticosterone
and aldosterone displayed an intermediate level of competition
which was not dissimilar in both intact cells and cytosol prep
arations. 17/¿-Estradici,testosterone, and biologically inactive
glucocorticoids such as 21-deoxycortisone and prednisone did
not compete. However, there are definite differences in the
100
o
5
m
concentrations varying from 1 to 20 nM, with or without a 100-fold
excess of radioinert TA. Tubes were incubated at 2°for 3 hr except for
experiments examining the time course of binding. After incubation,
duplicate 0.05-ml aliquots were deposited onto DEAE-cellulose filter
paper discs, DE 81 (2.5-cm diameter; Whatman Co., W. & R. Baiston,
Ltd., Maidstone, England); filters were washed as described (25) and
transferred to scintillation vials. The protein content of cytosols was
determined by the method of Lowry ef al. (17) using bovine serum
albumin as standard.
Calculations
Radioactivity was measured with a 38% mean efficiency. Binding
data were analyzed by Scatchard plot (26) to determine the equilibrium
3980
9«
60
120
MINUTES
180
240
Chart 1. Time course of specific |'H |TA binding to GR in intact cells ( ) or in
cytosol (•)
in one case of ALL. Aliquots of cell suspensions (0.1 ml; 18 x 10*
cells/ml) or cytosol (0.06 ml) were incubated at 37°and 2°,respectively, for the
times indicated with 20 HM[ 'H]TA alone (total binding) or in the presence of 2
/IM radioinert TA (nonspecific binding). Specific binding was calculated as the
difference between total binding and nonspecific binding. The maximum binding
attainable during the time of the kinetic experiment was taken as 100% and the
other values were expressed as a percentage thereof. The maximum binding for
whole cells and cytosol was 4200 sites/cell and 318 fmol/mg protein, respec
tively.
CANCtR
RESEARCH
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VOL. 41
Gf? 's in Leukemia
whole-cell assay are given in Chart 3. The values of receptors
are: mean, 570 ±540 fmol/mg protein; median, 325 fmol/mg
Standard competition curves were obtained by incubating replicate aliquota of
protein (N = 14) in CLL; mean, 1492 ±1521 fmol/mg protein;
cytosol or cell suspensions with 20 nM |H|TA in the presence of varying
median,
706 fmol/mg protein (N = 15) in ALL; mean, 1239
concentrations (1 nM to 1 /IM) of different radioinert competitors. Incubation was
done at 2°for 3 hr (for cytosol) or 37°for 60 min (for cells). Bound radioactivity
±1156
fmol/mg
protein; median, 720 fmol/mg protein (N =
was measured as described in "Materials and Methods." The ratio of the
12)
in
ANLL.
The
Kd
values are: mean, 12.8 ±8.2 nM; median,
concentration of ligand to the concentration of competitor required to displace
9.18 nM (N = 14) in CLL; mean, 8.34 ±4.65 nM; median, 7.2
the radioligand by 50% (relative binding affinity) was determined. The relative
binding affinity of TA was taken to be 100.
OM (A/ = 15) in ALL; mean, 9.1 ±7.44 nM; median, 7.3 nM (N
Relative binding affinity
= 12) in ANLL. The values of receptor sites are significantly
different
among the various types of leukemia (P < 0.05,
CompetitorTADexamethasonePrednisoloneCortisolCorticosteroneAldosteroneProgesteroneCortexolonePrednisone21
Kruskal-Wallis test). Analogous to previous findings (18), the
Kd value measured by cytosol assay in all types of leukemia
(9.45 ±6.33 nM) is significantly higher (p < 0.01, Student's
Table 1
for [3HJTA binding in one case of CLL
Competition
f test) compared to that found in intact cells (6.13 ±3.25 nM).
Comparison of GR Measurement: Whole-Cell versus Cy
tosol Assay. In Chart 4, the numbers of GR sites per cell
measured by whole-cell assay are plotted against the amounts
-Deoxycortisone1
of fmol per mg protein determined by cytosol assay. There
7/3-EstradiolTestosteroneCytosol100"92858028235349<1<1<1<1Cells1008886772229141B5<1<1<1
appears to be a good linear correlation between the 2 series of
measurements (r = 0.91 ) independent of the type of leukemia
Mean of 2 determinations.
(CLL, r = 0.78; ALL, r = 0.92; ANLL, r = 0.90). However, for
low binding site concentrations (less than 10,000 sites/cell or
1000 fmol/mg protein), the points are less aligned along the
50.00040000y
ia°
regression line.
In some of the previous reports on the levels of GR sites in
o0
leukemic
cells, the binding studies were done on cytosol prep
30.000UÕS
arations at 0-4° (4, 13). It has been shown that this procedure
;o
*8
Ì
*^^
20.00010,000i
o
Õ—
*^ST
»r-
~*~
:H *»
•¿
CLL
0
-K
S
i!
J
8
cfla2010
ALL
ANLL
Chart 2. QR levels O and steroid-binding affinities (•)in leukocytes isolated
from the blood of 41 patients with various types of leukemia. Receptor determi
nations were made using whole-cell assay as described in "Materials and
Methods."
specificity of the binding sites in the cytosol as compared to
those in whole cells. In particular, cortexolone and progester
one have a lower affinity for receptor sites in whole cells at 37°
than in cytosol at 2° which is in agreement with previous
observations made on rat splenic lymphocytes (18).
Chart 2 shows the values of GR sites determined by wholecell assay in the cells of 41 patients with various forms of
leukemia. The number of sites varies widely from one type of
leukemia to another and from one patient to another within
each type as well. The values of receptors are: mean, 8061
±4491 sites/cell; median, 6501 sites/cell (N = 14) in CLL;
mean, 17,106 ± 13,737 (S.D.) sites/cell;
median, 15,070
sites/cell (N = 15) in ALL; mean, 14,954 ±10,083 sites/cell;
median, 11,287 sites/cell (N = 12) in ANLL. The Kd values
obtained by the slopes of Scatchard plots are: mean, 3.97 ±
2.28 nM; median, 3.7 nw (N = 14) in CLL; mean, 6.57 ±2.98
nM; median, 2.7 OM (N = 15) in ALL; mean, 8.1 ±3.25 nM;
median, 7.2 nw (N = 12) in ANLL. The K«values appear similar
to those determined for the receptor-dexamethasone
interac
tion in the leukemic cells of previous studies (1, 6). However,
they are significantly different among the various types of
leukemia (p < 0.01, Kruskal-Wallis test).
The results of GR determinations by cytosol assay using
cytosol obtained from the same leukocyte specimens used for
OCTOBER
1981
leads to an underestimation of the total number of binding sites
present in the cell. To verify this, the number of GR sites per
cell determined by whole-cell assay has been compared with
the number of sites per cell obtained by cytosol assay. This
latter parameter was calculated by dividing the fmol bound/mg
of cytosol protein by the number of cells per mg protein [based
on cell count and protein determination by the method of Lowry
ef a/. (17)] and multiplying by Avogadro's number. The results
of this comparison indicate that the relationship between these
2 estimates is not linear and may be well represented by a
parabolic equation (Chart 5). Specifically, in cells with low GR
content (less than 10,000 sites/cell), the amount of receptor
measured by the cytosol assay does not vary greatly as a
function of the variations in the number of sites per cell,
whereas it increases rapidly in cells with higher receptor levels.
Furthermore, it is interesting to note that, even though all 3
types of leukemia follow the same behavior, ANLL is predomi
nantly distributed above the theoretical equation curve with
respect to the others. This behavior pattern is further illustrated
5.0004.0003.0002,0001.000•
e••
¡8^115
0o
:I:
ars
i
g>3jl
^frT'- s
_^_o
"T-
r"* *a"
«,8
CLL
° ^..
t*
ALL
r30
ANLL
Chart 3. GR levels O and steroid-binding affinities (•)in leukocytes isolated
from the blood of 41 patients with various types of leukemia. Receptor determi
nations were made using cytosol assay as described in "Materials and Methods."
3981
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S. lacobelli et al.
Table 2
5.000
Underestimation of GR content (cytosol assay versus whole-cell assay) in
leukocytes from patients with various types of leukemia
Sites/cellLeukemia
4.000
typeCLL
cell8,061
± 4,491"
(%)78.7
±18.4°
±1,201
73.1 ±14.4o
ALL (15)
17,106 ±13,737
5,434 ±6,81 7
57.8 ±22.3e
ANLL (1 2)Whole 14,964 ±10,083Cytosol1,551
6.536 ±6,822Underestimation
(14)"
1000
Numbers in parentheses, number of cases.
6 Mean ±S.D.
c' ' * Mean difference (Student's f test): c versus e, p < 0.05; d versus e, p
2.000
< 0.02; c versus d, not significant.
Table 3
1.000.
Stability of GR in the cytosol of leukocytes from patients with various types of
leukemia
10.000
30.000
50,000
SITES/CELL
Chart 4. Correlation between QR levels measured by whole-cell assay and
cytosol assay in leukocytes from patients with CLL (•).
ALL (A), and ANLL, (O).
Receptor determinations were carried out as described in "Materials and Meth
ods."
Cytosols were prepared and maintainedat 2°,and at hourly intervals thereafter
up to 6 hr, aliquots were incubated with 20 HM| H|TA alone (total binding) or in
the presence of 2 «M
radioinert TA (nonspecific binding) for 3 hr at 2°.The loss
of binding capacity referred to specific binding which was calculated as the
difference between total binding and nonspecific binding. One-half of the maxi
mum binding capacity was calculated as 50% of the initial (zero time) amount of
specific binding.
Loss of one-half of the maximum binding
capacity
Leukemia
typeCLL
ALL
ANLLRapid
hr)7A«3
(87.5)"
(>3
hr)'A
% (67)
%(50)Slow
%
%
Numbers in parentheses, percentage.
were arbitrarily established as less or more than 3 hr as shown
in Table 3. A rapid loss (less than 3 hr) was observed in 87%
(7 of 8) OfCLL, 67% (4 of 6) of ALL, and 50% (3 of 6) of ANLL.
DISCUSSION
10000
SITES/CELL
30,000
[whole cell assay]
50,000
Chart 5. Correlation between the number of QR sites measured by whole-cell
assay and cytosol assay in leukocytes from patients with CLL (•),
ALL (A), and
ANLL (O). The number of sites per cell relative to the cytosol assay were
calculated from the binding data as described in "Results." The best fit for the
regression curve was represented by a parabolic equation (y - a + ox + ex2);
r, the correlation coefficient for this curve.
The results of this study demonstrate that the 2 GR assay
methods furnish correlated values, independent of the type of
leukemia studied. The correlation is less evident for cells with
low receptor content in which the 2 series of measurements
are more difficult to compare. Moreover, our data indicate that
the cytosol assay consistently leads to an underestimation of
the number of binding sites as compared to the values obtained
by whole-cell assay. There may be several factors accounting
for this.
First, the whole-cell assay procedure might ensure the de
termination of the total (cytoplasmic and nuclear) receptor
content. It has long been reported that in rat thymocytes
exposed to glucocorticoids at 37°the majority (approximately
80%) of the receptor is found to be associated with the nucleus
in the form of receptor-hormone complex (22). Similar obser
vations have recently been made on human thymocytes (10).
Interestingly enough, a similar percentage of receptor under
estimation was seen in the cytosol of leukemic cells of lym
phatic origin (Table 2). Thus, a simple explanation which could
account for the low number of binding sites measured by the
Loss of Binding Capacity. For these experiments, the cy
cytosol assay is that in intact cells at physiological tempera
tosol was prepared and maintained at 2°,and at hourly intervals tures the majority of receptor is localized in the nucleus. On
aliquots were incubated with saturating concentrations (20 HM) the other hand, the higher number of binding sites detected in
of [3H]TA in the absence or presence of radioinert TA to the cytoplasm of ANLL could reflect an altered distribution of
examine the binding during the subsequent 3-hr incubation receptor between the cytoplasmic and nuclear compartments
period. The rate of loss of one-half of the binding capacity in possibly due to a variable rate of receptor translocation as
20 different cytosols fell into 2 basic time categories which reported elsewhere (1).
by the data in Table 2 which compares the means of values for
receptor sites obtained by the 2 assay methods. The percent
age of underestimation (cytosol assay/whole-cell assay) is
significantly lower in ANLL than in CLL and ALL (p < 0.05 and
0.02, respectively).
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RESEARCH
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VOL. 41
GR 's in Leukemia
Second, possible breakage of lysosomes and release of
proteolytic enzymes that partially inactivate the cytoplasmic
receptor must also be considered. Our data show that the
receptor-binding capacity in cytosol from certain types of leukemic cells, notably those of CLL, decreases rather rapidly,
suggesting that substances capable of inactivating the receptor
molecule (which could account for the generally lower binding
capacity of these particular cells) are active in these cells.
Although these substances (possibly enzymes) have not yet
been identified, it is interesting that Sloman and Bell (27) have
shown that the cytosol of CML contains receptor-inactivating
factor(s) which could be activated during the homogenization
process. The presence of similar factors in leukemic myeloblasts of AML has also been proposed by other authors (1).
Third, it is possible that some cells are particularly resistant
to the breaking process during homogenization. Although this
possibility has been postulated to explain the negative results
obtained by the cytosol assay (1 ), it seems to be unlikely since,
at least in our hands, complete cell breakage was observed in
all cases.
The concentrations of GR sites in the different types of
leukemic cells used in our study are analogous to those ob
served elsewhere (1, 10). The receptor content of these leu
kemic cells greatly exceeds that found in normal leukocytes of
both lymphatic and myelomonocytic origin (12, 23). This ca
pacity of the leukemic cell to express a greater quantity of
receptor protein with respect to its normal counterpart seems
to be a more general characteristic of the neoplastic phenotype. Thus, for example, the normal mammary cell does not
seem to possess appreciable amounts of estrogen receptor
while as a result of the neoplastic transformation process the
quantity of receptor molecules can reach abnormally high
levels (3). Although the mechanisms which regulate receptor
levels in the cell are only now beginning to be clarified, it is
possible to hypothesize that the redundance of GR's in leu
kemic cells, especially in ALL and ANLL as indicated in this
study, represents a class of heterogeneous receptor molecules
with functional defects such as wide ranges of steroid-binding
affinity (this paper and Ref. 1) as well as variable capacities of
cytoplasm-to-nucleus
translocation and of interaction with ac
ceptor sites in the chromatin (5). In the presence of this
redundance, the nuclear acceptor-binding capacity could be
come the limiting step in the process, independent of any
translocation defect. Therefore, in cells with low receptor con
tent (i.e., closer to normal cell values), the quantity of binding
sites measured in the cytoplasm could represent a minimal part
of the total cellular receptor sites which in the presence of
endogenous hormone would be preferentially localized in the
nucleus. This could explain the high frequency of negative
results in GR determinations made by cytosol assay in leukemic
cells with low receptor levels, particularly in CLL (4, 28).
However, when the cellular receptor levels exceed the nuclear
acceptor capacity, as in the case of cells with extremely high
receptor content, high proportions of binding sites are found in
the cytoplasmic compartment. This hypothesis could explain
the nonlinear correlation between the quantity of receptor sites
measured by the 2 assay methods.
It has been suggested that steroid receptors are not re
stricted to the cytoplasm alone and that in some cases they
can be localized exclusively in the nucleus (9). As a conse
quence, the measurement of cytoplasmic binding sites alone
OCTOBER
1981
may lead to falsely negative results if nuclear receptors are not
considered as well. On the other hand, demonstration of total
cellular receptor sites in whole-cell preparations does not give
information about their localization, i.e., if in the cytoplasm
and/or in the nucleus. Moreover, binding to receptor in intact
cells may depend on such factors as the metabolic state of the
cells (21) and the rate of steroid transport through the cell
membrane.
While the conclusions drawn from this study may be used to
partly explain the contrasting data on GR determinations in
leukemic cells so far reported, at least in cases of low receptor
content, they indicate that receptor assessments by whole-cell
and cytosol assays provide nonequivalent but possibly comple
mentary information. Interestingly, each of the only 2 studies
correlating GR levels in leukemic cells with clinical aspects
furnished entirely different prognostic indications and each was
based on one of the 2 assay procedures herein discussed (13,
16). Therefore, it seems reasonable not to depend exclusively
on only one of these procedures, at least until their precise
prognostic significance is more fully clarified by further correl
ative clinical studies.
REFERENCES
1. Crabtree, G. R . Smith, K. A., and Munck. A. Glucocorticoid receptors and
sensitivity of isolated human leukemia and lymphoma cells. Cancer Res..
38. 4268-4272.
1978.
2. Duval, D., and Homo, F. Prognostic value of steroid receptor determination
in leukemia. Cancer Res.. 38 4263-4267.
1978.
3. Feherty, P., Ferrer-Brown, G.. and Kellie, A. E. Oestriol receptors in carci
noma and benign disease of the breast: an in vitro assay. Br. J. Cancer, 25.
697-710, 1972.
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CANCER
RESEARCH
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VOL. 41
Glucocorticoid Receptor Determinations in Leukemia Patients
Using Cytosol and Whole-Cell Assays
Stefano Iacobelli, Vittoria Natoli, Paola Longo, et al.
Cancer Res 1981;41:3979-3984.
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