Download Cytoplasmic Glucocorticoid-binding Proteins in

[CANCER RESEARCH 34, 1572-1576,July 1974]
Cytoplasmic Glucocorticoid-binding Proteins in
Glucocorticoid-unresponsive Human and Mouse Leukemic Cell
Lines
Marc E. Lippman, Seymour Perry, and E. Brad Thompson
Office ofDirectorfor
Program EM. E. L., S. P.], Division ofCancer Treatment, and Laboratory ofBiochemistry
NIH, Bethesda, Maryland 20014
SUMMARY
[E. B. T.], National Cancer Institute,
containing receptor but not killed by glucocorticoid
has
been described (4). This is of considerable importance in
light of recent studies suggesting that the presence or
absence of SBP may be used to predict steroid responsive
ness clinically (7, 8, 10). We now present data for 3
malignant cell lines which are shown to contain SBP that
appears normal in quantity, specificity, affinity, and nuclear
uptake but which are apparently steroid unresponsive by a
variety of criteria.
Cytoplasmic glucocorticoid-binding
proteins of high af
finity and specificity were identified, quantified, and par
tially characterized
in three steroid-unresponsive
tissue
culture lines, one derived from an AKR mouse leukemia
and two derived from human lymphoblastic leukemia cells.
The number of receptors per cell appeared to be comparable
to that found in many steroid-responsive
cells. Tempera
ture-dependent
entry of cytoplasmic
receptor into the
nucleus did not appear to be abnormal as determined in an
in vitro nuclear binding assay. Cell growth, nucleoside and MATERIALS AND METHODS
amino acid incorporation into macromolecules, amino acid
Cells and Culture Techniques. AKR-A cells, a tissue cul
pools, and glucose uptake were found to be unaffected by
ture line derived from a spontaneously arising leukemia in
added glucocorticoid. The presence of specific cytoplasmic
receptor proteins for glucocorticoids
in malignant tissue AKR mice, were grown in suspension or monolayer culture
by established techniques (23). Two cell lines, CCL I 19
does not appear to guarantee steroid responsiveness.
(CCRF-CEM)
and CCL 120 (CCRF-SB),
derived from
patients with acute lymphoblastic leukemia, were obtained
INTRODUCTION
from American Type Culture Collection (Rockville, Md.)
and were grown by established methods (3). Normal mouse
Much recent work on the mechanism of action of steroid thymocytes were obtained from 7-week-old BALB/c mice.
hormones has focused on the initial association between the
Nucleoside and Amino Acid Incorporation. Cells growing
hormone and a SBP' (14, 18, 22). In fact, these high-affin
logarithmically
in suspension or monolayer were treated
ity, high-specificity “receptor―
molecules have been found with various concentrations of dexamethasone phosphate or
in all glucocorticoid-responsive
tissues examined. For ex other glucocorticoids
in the presence of a radiolabeled
ample, we have shown that glucocorticoid-responsive
acute precursor molecule for at least 12 hr. Following incubation,
human leukemic cells contain SBP (7, 8). In cells that have the incorporation
of precursor into trichloroacetic
acid
become steroid resistant, several laboratories have reported
precipitable material was assayed as previously described
a marked quantitative loss of SBP activity as the mecha
(7).
nism of resistance (5, 8, 15, 17). On the other hand, it is now
Amino Acid Pool Size. Following treatment of one-half of
clear that there are many steps that follow the initial binding
1000 ml of a logarithmically
growing cell culture with
reaction between glucocorticoid and cytoplasmic receptor
10- 6 M dexamethasone
phosphate for 18 hr. the cells were
and precede the phenotypic expression of steroid response collected and washed 3 times in ice-cold phosphate-buffered
characteristic of a given cell type (2, 12, 22). These include isotonic saline (0. 138 M NaCI; 2.7 mM KC1; 8. 1 mM
temperature-dependent
transformation
of the steroid-recep
Na2HPO4; and 1.47 M KH2PO4), pH 7.4. Cytosol was pre
tor complex with subsequent nuclear entry, association with pared by homogenizing the cells in water (1 : 1 v/v) in a
chromatin, presumed alterations in transcription of genetic tightly fitting Dounce homogenizer. The homogenate was
material, and finally the synthesis of specific proteins or
centrifuged at 100,000 x g for 30 mm, and a small aliquot
RNA's mediating phenotypic effects. A priori, one would was reserved for estimation of protein. The bulk of the su
expect that a defect in any of these other steps could lead to
pernatant fluid was precipitated with 10% trichloroacetic
a lack of hormone responsiveness. One lymphoid cell type acid at 0—4°.
The acid-soluble fraction was collected, to
gether with a single wash of the acid precipitate with 5% tn
chloroacetic acid at 4°,and amino acid analysis was per
Received June 14, 1973; accepted March 21, 1974.
formed on an aliquot on a Beckman amino acid analyzer
I The
1572
abbreviation
used
is:
SBP,
cytoplasmic
steroid-binding
protein.
CANCER
RESEARCH
VOL.
Downloaded from cancerres.aacrjournals.org on May 2, 2017. © 1974 American Association for Cancer Research.
34
Steroid Receptors in Steroid-resistant
(21). The results were then normalized for protein content
presented by the respective aliquots.
Protein Determination.
Protein content was determined
by means of the Lowry technique (9).
Cytoplasmic Receptor Assay. SBP was estimated with the
use of a nadiolabeled steroid competitive-binding
assay in
which unbound steroid was removed either by charcoal
adsorption (7) or by filtration through DEAE filter papers
(19).
Nuclear Binding of Steroid-Receptor Complexes. Temper
ature-dependent
transformation
of the cytoplasmic recep
ton-steroid complex followed by uptake into isolated nuclei
was measured in an in vitro incubation system. Cytosol, the
100,000 x g supernatant
@
@
from cell homogenate(s), was
prepared as previously described (7, 19) in Buffer 1 (20 mM
Tnis, pH 7.4; 1 mM MgCl2; and 2 mM CaCl2) and incubated
for 2 hr at 0—2°
with 8 x 108 M dexamethasone-3H
in the
presence or absence of 10 M unlabeled dexamethasone.
The nuclei from the homogenization
of the cells were
obtained by collecting the nuclear pellet by centrifugation at
800 x g for 5 mm. This pellet was suspended and washed 3
times in 10 ml Buffer 1 and then was incubated at 0—2°
for 2
hr with 8 x 10-8 M dexamethasone-3H
in the presence or
absence of l0@ M unlabeled dexamethasone
to compete
with the labeled steroid. After the preliminary incubation of
both nuclei and cytosol with steroid, they were mixed in a
ratio of I :4, v/v. in a final volume of 100 @tl,uncompeted
cytosol with uncompeted nuclei and competed cytosol with
competed nuclei. After varying time periods, the nuclei were
harvested by centrifugation
and washed 3 times in 2 ml
Buffer 1. The washed nuclei were sonically dispersed in a
small amount of water in a Bronwill sonicator (10 sec, Set
ting 15), and a protein determination by the Lowry method
(9) was done on an aliquot. An additional aliquot was
codnted in a liquid scintillation counter in 8 volumes of
NCS to which were added 10 ml of Liquifluor:toluene
(both NCS and Liquifluor were from New England Nu
clear, Boston, Mass.). Cytoplasmic receptor concentrations
were also measured in both original cytosol and, where de
sired, in the cytosol recovered from the nuclear incubation.
Specifically bound nuclear steroid is taken to represent the
difference between the total nuclear bound counts and the
counts bound in the competed sample containing unlabeled
steroid as well as dexamethasone-3H.
Glucocorticoid-induced
Inhibition of Glucose Uptake. Al
terations in glucose uptake induced by glucocorticoids were
measured by the technique of Kattwinkel and Munck (6).
Radioactive Compounds. These were dexamethasone- 1, 23H, 22 Ci/mmole
(Amersham/Searle
Corp., Arlington
Heights, Ill.), unidine-5-3H, 25 Ci/mmole (Schwarz/Mann,
Orangeburg,
N. Y.), and thymidine-methyl-3H,
18.5 Ci/
mmole (Schwarz/ Mann). Radioactivity was estimated by
counting in a liquid scintillation spectrophotometer
at an
efficiency of 20%.
saturated with dexamethasone-3H
at a concentration
of
about 1 to 2 x l0@ M (Chart I). From the Scatchard plot of
the data (Chart 1), the dissociation constant for this re
ceptor may be estimated to be 6 x lO_8 M/liter. This value
is similar to what we have reported for steroid-responsive
human leukemic cells (7) and higher than that found in liver.
The total specifically bound steroid was about 0.2 pmole
dexamethasone per mg of cytoplasmic protein. The steroid
binding curve of the 2 human leukemic lines, CCL 119 and
CCL 120, was extremely similar to that of AKR in terms of
affinity
for dexamethasone.
However,
at saturating
amounts ofdexamethasone,
CCL I 19 and CCL 120 yielded,
respectively, 0.67 and 0.88 pmole of dexamethasone
bound
per mg of cytoplasmic protein. For comparative purposes,
Chart 1 also shows a binding curve for normal mouse
thymocytes (a glucocorticoid-responsive
tissue). The affin
ity constant determined from the Scatchard data (Chart I,
inset) is 4.2 x l08 M/liter, similar to that of AKR-A cells
or steroid-responsive
human lymphoblastic leukemic cells
(8). The total amount ofdexamethasone
bound at saturating
levels of steroid to normal thymocytes was 0.30 pmole/mg
of cytoplasmic protein. Thus, we conclude that these 3
leukemic cell lines contain quantities of high-affinity recep
tor protein in the same range as those found in both
steroid-sensitive leukemic cells and thymocytes.
If the substance measured by these assays is to be
implicated in glucocorticoid action, one would predict that
it would show preferential affinity for glucocorticoids. The
specificity of binding for AKR SBP is shown in Table I.
Potent glucocorticoids
and mineralocorticoids
which can
function as glucocorticoids compete well with dexametha
sone for the binding site, whereas cortisol metabolites,
androgens, and other steroids show little ability to displace
dexamethasone in the binding reaction. Slight differences in
specificity of glucocorticoid
receptors from differing ste
roid-responsive cells have been previously described (6, 8).
We do not interpret our data as showing that receptors from
sensitive and resistant cells are necessarily identical.
Other workers have shown that, in addition to the
quantitative
defect in SBP apparent in steroid-resistant
cells, there appears to be a defect in the remaining receptor,
@;@1@:@/―
!
I
C
5
0
5
DEXAMETHASONE
RESULTS
Leukem ias
Chart
I
I
20
25
CONCENTRATION
8O@fr@0 ‘ /L
4080
(M,iO8i
I . Binding of dexamethasone-3H to cytoplasmic receptors from
AKR-A cells (0) or cytoplasmic receptorsfrom BALB/c mousethymo
The cytosol of AKR-A leukemic cells contains a high
affinity glucocorticoid
receptor molecule which becomes
JULY
cytes (•).Techniques are given in “Materialsand Methods.― Inset,
Scatchard plot of the binding data.O, AKR-A cells; •,
mouse thymocytes.
1974
Downloaded from cancerres.aacrjournals.org on May 2, 2017. © 1974 American Association for Cancer Research.
I 573
M. E. Lippman
et a!.
present in diminished amount. This defect does not permit it
to translocate to the nucleus after the initial binding step
takes place in the cytoplasm (1 1, 17). We therefore exam
med the ability of the cytoplasmic receptor to undergo
temperature-dependent
alteration and uptake by the nucleus
in an in vitro nuclear binding assay previously developed
(17). Results are shown in Chart 2 for the human leukemic
cell line CCL 120. As can be seen, at 0°cytoplasmic binding
is apparent, but there is no significant nuclear uptake of
steroid-receptor complexes. At 20°there is an accumulation
of radioactivity in the nucleus associated with a concomitant
decrease in steroid-receptor
activity in the cytosol. Very
similar results were obtained with AKR-A and CCL I 19.
Assuming 1 steroid molecule bound per cytoplasmic recep
ton, a concentration of SBP of about 2 nM was sufficient to
saturate homologous nuclei, a concentration comparable to
that needed for a rat hepatoma and mouse fibroblasts in
culture (unpublished results). We interpret these results as
being consistent with the presence of a viable cytoplasmic
receptor in these cells.
We next sought some evidence of a phenotypic response
to glucocorticoids.
Although not necessarily the primary
cause of glucocorticoid
inhibition of lymphoid cells, a
pronounced decrease of macromolecular
synthesis has been
uniformly described (I, 7, 22) following exposure of sensi
tive cells to such steroids. As shown in Chart 3, glucocorti
coids fail to show any effect on nucleoside incorporation in
Table I
CCL 120, even at concentrationsof glucocorticoidfar in
Inhibition
of specific binding by various competing steroids
excess ofthose required to saturate the SBP. Similar results
The various steroids listed were compared as to their ability to compete
were obtained repeatedly with the CCL I 19 and AKR-A
with dexamethasone-3H for specific binding sites in AKR cell cytosol
cell
lines. None ofthe 3 lines displayed inhibition of growth,
fraction. A
100-fold excess of each steroid was added to a saturating
incorporation
into RNA, or of amino acid
concentration of dexamethasone-3H in a series of duplicate tubes; “% of uridine
inhibition― refers to the relative ability of each steroid to prevent
incorporation
into protein following exposure to SBP
dexamethasone-3H binding. Non radioactive dexamethasone itself in 100- saturating
amounts of corticosteroids.
Representative
re
fold excess did so virtually completely and was arbitrarily set at 100% for
suIts for CCL I 19 are shown in Table 2. No inhibitory
reference.
effects on the growth of AKR-A cells could be elicited by
inhibitionDexamethasone100Prednisolone100Cortisol83Aldosterone77Deoxycorticosterone56Corticosterone4717$-Estradiol26Etiocholanolone19I
the use of alternate glucocorticoids
such as cortisol and
Competing steroid%
prednisolone (results not shown). Effects of alternate gluco
corticoids were not measured on CCL 119 or CCL 120.
Inhibitory effects on glucose metabolism have also been
widely reported as an early response of lymphoid cells to
glucocorticoid
(13, 22). We found no glucocorticoid
mediated effect on glucose uptake for CCL I 19 and CCL
120 (Chart 4). The differences shown between CCL I 19 and
7a-MethyltestosteroneIS19
Nortestosterone14Prednisone13Tetrahydrocortisol10
‘0
x3
w
CCL 120 reflectincubationof differingcell concentrations
and suggest that the results do not reflect an artifact of cell
density. Similar results were obtained for AKR-A cells
using dexamethasone,
cortisol,
and prednisolone
(not
shown).
Glucocorticoid-provoked
alterations in amino acid poo1
sizes are seen commonly in responsive tissues whether
inhibited or stimulated by glucocorticoids
(16, 20). We
estimated intracellular
amino acid pool sizes for equal
numbers of AKR cells in the presence or absence of l06 M
dexamethasone
(Table 3). Glucocorticoids
do not induce
0.
b
C')
-J
4
0
C-
C
0
z
Lu
40
TIME ( minutes)
Chart 2. Temperature dependence of nuclear binding of cytoplasmic
receptor-steroid complexes. Approximately 4 x I0@cells were homoge
nized for this experiment, and nuclear and cytosol fractions were prepared
as described in “Materialsand Methods.―Cytosol preincubated at 0°with
dexamethasone-3H in the presence or absence of a 100-fold excess of
unlabeled dexamethasone was mixed with nuclei that had been similarly
preincubated with steroid. At Time 0, nuclei and cytosol were mixed (nuclei
without unlabeled steroid mixed with cytosol without unlabeled steroid).
The mixes were incubated at 0°or 20°and, at various times, pairs of
aliquots were taken, nuclei and cytosol were separated by centrifugation,
and the specifically bound counts in nuclei at 0°(•)or 20°(•)or cytosol
at 0°(0) or 20°(0) were estimated.
l574
Cl,
4
I
C-
2
4
‘Lu
C')
0
0
z
g
-C
4
0
@Lu
0@
z
0
0.
DEXAMETHASONECONcENTRATIONI M x C0@I
Chart 3. Comparison of
glucocorticoid concentration
chloroacetic acid-precipitable
ration. Techniques are given
glucocorticoid binding to SBP and effects of
on incorporation of thymidine-3H into tn
material. •,binding; 0, thymidine incorpo
in “
Materials and Methods.―
CANCER
RESEARCH
VOL. 34
Downloaded from cancerres.aacrjournals.org on May 2, 2017. © 1974 American Association for Cancer Research.
Steroid Receptors in Steroid-resistant
@
Effrct of 10
Table 2
M dexamethasone phosphate on cell growth and uridine incorporation
lymphoblastoid leukemic cells in suspension culture
Leuk emias
in CCL 119
A growing culture of cellswasdivided. Half wasmade 10 6Min dexamethasone.To both halves.
unidine-3H was added. At the stated intervals, aliquots were removed from the cultures for counting
of cells and for estimation of unidine incorporation into tnichloroacetic acid-precipitable material.
ControlDexamethasoneUnidineNo.
ofincorporation5No.
ofUnidineTime
(hr)cellsa(cpm)cells'incorporation―0220,000574200,0004846215,000155,203220,000146,47922425,000309,362415,000286,78828460,000297,04
a Cell
counts
were
p Into tnichloroacetic
cells.
performed
in triplicate
acid-precipitable
in a hemocytometer.
material
800
1.
@6oo
@400
N
-C
@
200
Cl,
Lu
8°
30
(8
@
25
50
75
TIME
00
25
(nanut.s)
50
75
200
Chart 4. Effects of lO-@M dexamethasone on glucose incorporation in
human lymphoblastoid leukemic cells. CCL I 19: 0, control: S. + 10' M
dexamethasone phosphate. CCL 120: 0, control: •,+ 10'
dexametha
sone phosphate.
any gross alterations in acid-soluble amino acid pools. We
obtained similar results for CCL I 19 and CCL 120.
DISCUSSION
A variety of lymphoid cells and fibroblasts have been
shown to respond to glucocorticoids by inhibition of macro
molecular synthesis, of glucose uptake, and of growth, and
in some cases by eventual lysis. We here describe 3
lymphoid cell lines in tissue culture, 2 human and 1 mouse,
which show none of these responses. We assessed the SBP's
of these lines by 4 criteria: quantity, affinity for steroids,
specificity for glucocorticoids,
and nuclear entry. By all
these criteria, the 3 steroid-resistant
lines possess steroid
binding proteins equivalent to normally steroid-sensitive
cells. Although Gehning et a!. (4) have described a cell line
which contained a SBP and which was not lysed by gluco
corticoids, we believe that the present paper is the first
report of steroid-resistant cell lines containing SBP in which
the receptor has been shown to be equivalent to that in
normally responsive cells by the same criteria used to define
specific glucoconticoid receptors (1, 14, 15, 18, 22). Whether
JULY
per 2 x l0@control
or dexamethasone-treated
the SBP of the unresponsive cells differs from normal in
some subtle way, we cannot tell.
Finding equivalent concentrations ofhigh-affinity
SBP in
unresponsive cells to those shown in a variety of steroid
responsive cells is not a surprising result when the compli
cated and poorly understood
events which follow the
association
of steroid and receptor molecule and their
translocation
into the nucleus are considered. Many sites
subsequent to SBP binding of steroid could be responsible
for the failure of the cells to respond. Indeed, more
surprising is the great regularity with which the appearance
of steroid resistance in both normal and malignant tissue
has been associated with a quantitative defect in receptor
activity (5, 7, 8, 15, 17, 22). There is at present little evidence
to suggest whether this observed defect in binding activity
reflects decreased amounts of SBP per se or whether a
defective SBP is present. Equally unclear is the reason for
the relative frequency ofloss ofthe cytoplasmic binding step
in steroid-resistant
cells.
The cell lines discussed here, which contain receptor but
do not respond to glucocorticoids, are of particular interest
for 2 additional reasons. First, we feel that they may be of
great value in investigating the more distal steps in steroid
action, for example in complementation
analysis of somatic
cell hybrids. Second, the results obtained in our investiga
tions of these 3 cell lines should serve as a caution against
the complete acceptance of the presence of SBP as a
guarantor of steroid response. While there is no evidence to
suggest that a SBP-negative cell will respond specifically to
glucocorticoids,
the converse, i.e., a SBP-positive
cell
responding to glucocorticoids, can no longer be considered
absolutely valid. While admittedly these are cells in culture
which have had considerable time to undergo alterations in
phenotype typical of tissue culture cell lines, we feel that it is
likely that spontaneously occurring human leukemias which
are SBP positive and which are uninhibited by glucocorti
coids will be found. On the other hand, our previous work
(7, 8, 22) has suggested that this will be considerably less
common than leukemias with alterations in SBP activity
mediating the control of steroid response.
1974
Downloaded from cancerres.aacrjournals.org on May 2, 2017. © 1974 American Association for Cancer Research.
I 575
M. E. Lippman
el a!.
Table 3
Amino acid concentrations in trichloroacetic acid-soluble fraction, from control and JO “
M
dexamethasone phosphate-treated A KR-A cells
A growing culture of AKR cells wasdivided, and one-halfwastreatedwith dexamethasone.The
results shown give the amino acid content of equal aliquots of the acid-soluble fraction of the
hormone-treated and control cells, normalized for their slight differences in protein content. Details
of procedure are given in ‘Matenialsand Methods.―
I .0 ml aliquotof
acid-soluble fractionDexamethasone/
cellsbControl'DexamethasoneaAspartic
Amino acidMmoles/
control
acid0.2560.2140.85Glutamicacid0.2900.3161.10Proline0.2260.2621.16Glycine0.5720.6151.07Alanine0.3430.3731.09Half-cystineNone
detectedValine0.02870.03
detectedNone
21.08Methionine0.01780.01480.84Isoleucine0.03160.03261.03Leucine0.03530.03621.02Tyrosine0.02880.02420.84Phenylalanine0.03320.03100
1
(C Total
volume,
2.5
ml.
p Ratio of amino acid concentration in acid-soluble fractions
compared with equivalent amount from control cells.
REFERENCES
I. Baxter, J. D., and Forsham, P. H. Tissue Effects of Glucocorticoids.
Am. J. Med., 53. 573-589, 1972.
2. Baxter, J. D., Rousseau, G. G., Benson, M. C., Garcea, R. L., Ito, J.,
and Tomkins, G. M. Role of DNA and Specific Cytoplasmic
Receptors in Glucocorticoid Action. Proc. NatI. Acad. Sci. U. S.. 69:
1892-1896, 1972.
3. Foley. G. E., Lazarus, H., Farber, S., Uzman, B. G., Boone, B. A.,
and McCarthy. R. E. Continuous Culture of Human Lymphoblasts
from Peripheral Blood of a Child with Acute Leukemia. Cancer, 18:
522-529,1965.
4. Gehning. U.. Mohit, B., and Tomkins, G. M. Glucoconticoid Action on
Hybrid Clones Derived from Cultured Myeloma and Lymphoma Cell
Lines. Proc. NatI. Acad. Sci. U. S., 69: 3124-3127, 1972.
5. Hollander, N., and Chiu, Y. W. In Vitro Binding of Cortisol-l ,2-3H
by a Substance in the Supernatant Fraction of P1798 Mouse
Lymphosarcoma. Biochem. Biophys. Res. Commun., 25: 291-297,
1966.
6. Kattwinkel, J., and Munck, A. Activities In Vitro of Glucocorticoids
and Related Steroids on Glucose Uptake by Rat Thymus Cell
Suspensions. Endocrinology, 79: 387-390, 1966.
7. Lippman. M. E., Halterman, R. H., Leventhal, B. G., Perry, S., and
Thompson, E. B. Glucocorticoid Binding Proteins in Human Acute
Lymphoblastic Leukemic Blast Cells. J. Clin. Invest., 52: 1715—1725,
1973.
8. Lippman, M. E., Halterman, R. H., Leventhal, B. G., Perry, S., and
Thompson, E. B. Glucocorticoid Binding Proteins in Human Leu
kemic Blast Cells. Nature New Biol., 242: 157-158, 1973.
9. Lowry, 0. H., Rosebrough, N. J., Farn, A. L., and Randall, R. J.
Protein Measurement with the Folin Phenol Reagent. J. Biol. Chem.,
193.265-275,1951.
10. McGuire, W. L. Estrogen Receptors in Human Breast Cancer. J. Clin.
Invest., 52: 73-77, 1973.
I I. McGuire, W. L., Huff, K., Jennings, A., and Chamness, G. C.
Mammary Carcinoma: A Specific Biochemical Defect in Autonomous
Tumors. Science, 175: 335-336, 1972.
I576
from dexamethasone-treated
cells,
12. Mosher, K. M., Young, D. A., and Munck, A. Evidence for
Irreversible, Actinomycin
D-sensitive, and Temperature-sensitive
Steps following Binding of Cortisol to Glucocorticoid Receptors and
Preceding Effects on Glucose Metabolism in Rat Thymus Cells. J.
Biol. Chem., 246: 654-659, 1971.
13. Munck, A. Glucocorticoid Inhibition ofGlucose Uptake by Peripheral
Tissues: Old and New Evidence, Molecular Mechanisms and Physio
logical Significance. Perspectives Biol. Med., 14. 265—289,1971.
14. Munck, A., Wira, C., Young, D. A., Moshen, K. M., Hallahan, C.,
and Bell, P. A. Glucocorticoid-Receptor
Complexes and the Earliest
Steps in the Actions of Glucocorticoids on Thymus Cells. J. Steroid
Biochem., 3: 567-578, 1972.
15. Pratt, W. B., and Ishii, D. N. Specific Binding of Glucocorticoids In
Vitro in the Soluble Fraction of Mouse Fibroblasts. Biochemistry, 11:
1401-1410,1972.
16. Risser, W. L., and Gelehrter, T. D. Hormonal Modulation of Amino
Acid Transport in Rat Hepatoma Cells in Tissue Culture. J. Biol.
Chem., 248: 1248-1254,1973.
17. Rosenau, W., Baxter, J. D., Rousseau, G. G., and Tomkins, G. M.
Mechanism of Resistance to Steroids: Glucocorticoid Receptor Defect
in Lymphoma Cells. Nature New Biol., 237: 20-24, 1972.
18. Rousseau, G. G., Baxter, J. D., and Tomkins, G. M. Glucoconticoid
Receptors: Relation between Steroid Binding and Biologic Effects. J.
Mol. Biol., 67:99-115, 1972.
19. Santi, D. V., Sibley, C., Perniard, E., Tomkins, G. M., and Baxter, J.
D. A Filter Assay for Steroid Hormone Receptor. Biochemistry, 12:
20.
21.
22.
23.
2412-2416,1973.
Segal, S. Hormones, Amino-Acid Transport and Protein Synthesis.
Nature, 203: 17-19, 1964.
Spackman, D. H., Stein, W. H., and Moore, S. Automatic Recording
Apparatus for Use in the Chromatography of Amino Acids. Anal.
Chem., 30: 1190-1206,1958.
Thompson, E. B., and Lippman, M. E. The Mechanism of Action of
Glucocorticoids. Metabolism, 23: 159-202, 1974.
Woods, W. A., Wivell, N. A., Massicot, J. G., and Chinigos, M. A.
Characterization of a Rapidly Growing AKR Lymphoblastic Cell
Line Maintaining Gross Antigens and Viral Replication. Cancer Res.,
30:2147-2155,1970.
CANCER
RESEARCH
Downloaded from cancerres.aacrjournals.org on May 2, 2017. © 1974 American Association for Cancer Research.
VOL.
34
Cytoplasmic Glucocorticoid-binding Proteins in
Glucocorticoid-unresponsive Human and Mouse Leukemic Cell
Lines
Marc E. Lippman, Seymour Perry and E. Brad Thompson
Cancer Res 1974;34:1572-1576.
Updated version
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/34/7/1572
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from cancerres.aacrjournals.org on May 2, 2017. © 1974 American Association for Cancer Research.