GlucocorticoidReceptors and Glutamine - Cancer Research

GlucocorticoidReceptors and Glutamine - Cancer Research

ICANCER RESEARCH 39,376-382,February1979] GlucocorticoidReceptors and Glutamine Synthetase in Leukemic SézaryCells1 Thomas J. Schmidt and E. Brad Th...

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ICANCER RESEARCH 39,376-382,February1979]

GlucocorticoidReceptors and Glutamine Synthetase in Leukemic SézaryCells1 Thomas J. Schmidt and E. Brad Thompson2 Laboratory of Biochemistry, National Cancer Institute, NIH, Bethesda, Maryland 20914

ABSTRACT Using a competitive binding assay, we have detected cytoplasmic glucocorticoid receptors in the leukemic cells of several patients with Sezary syndrome, while cells from other patients appear to contain very low or nondetectable receptor levels. The receptors are saturated at approxi mately 4 x 10-s M rHldexamethasone, and Scatchard analyses of the binding data indicate a high affinity ([email protected]= 6.9 x 10-i M). The cytoplasmic receptors are inactivated at 370,

but

at

4°

they

are

relatively

stable,

even

in

the

absence

of glucocorticoids. Competition studies have established the specificity of these receptors. Active glucocorticoids such as dexamethasone, prednisolone, and cortisol (50fold molar excess) completely block rH]dexamethasone binding, while nonglucocorticoids such as 17f3-estradiol and 5a-dihydrotestosterone have no effect at equivalent concentrations. The antiinducer progesterone is also an effective competitor of rHidexamethasone binding. The [3H]dexamethasone-receptor complex migrates in linear 5 to 20% sucrose gradients (0.4 M KCI) with a sedimentation coefficient of [email protected] In those cells which contained recep tors, the specific activity of glutamine synthetase was in creased 2-fold after an 18-hr exposure to dexamethasone ([email protected]), whereas no such increase occurred in cells lacking receptor. The presumptive induction of glutamine synthe tase activity may thus serve as a marker for functional receptors in leukemic Sézarycells. Retrospective studies (4 patients) suggest a possible correlation between receptor levels and clinical responsiveness to glucocorticoid then apy.

INTRODUCTION Sezary syndrome is a rare and frequently lethal chronic leukemia characterized by atypical circulating malignant lymphocytes (10), cutaneous infiltration, and an intensely pnuritic enythrodermia with the formation of plaques (36). The distinguishing abnormal circulating lymphocyte seen in this leukemia is typically a large cell with a voluminous convoluted nucleus surrounded by a thin rim of cytoplasm (11, 19). Several investigators (6, 7, 19) have shown that the membrane characteristics of Sezary cells are those of T lymphocytes. Broder et al. (4) have also presented data which suggest that the neoplastic lymphocytes from the majority of patients with Sezary syndrome originate from a subset of T-cells programmed exclusively for helper-like interactions with B-cells in their production of immunoglob ulins. I This

work

was

supported

in

part

by

NIH

Postdoctoral

CA05447-01 from the National Cancer Institute. 5 To

whom

requests

for

reprints

should

be

addressed.

Received June 26, 1978; accepted October 25, 1978.

376

Fellowship

1

F32

Much of the edema and erythrodermia associated with the Sezary syndrome can be improved by local glucocorti coid therapy, and often the systemic use of glucocorticoids further

reduces

these

symptoms

as well

as prunitus.

Com

bined therapy including chlorambucil and prednisolone3 has also been shown to result in freedom from pruritus, followed by partial resolution of the erythrodenmia and histological disappearance of Sezary cells in many patients (35). Considering that most Sezary patients live an average of 5 years from the onset of symptoms (4), it would be beneficial to determine at which specific stage(s) the malig nant lymphocytes are most sensitive to systemic glucocor ticoids. Omission of glucocorticoids from the therapy of resistant patients would reduce immunosuppression and thus render these patients less susceptible to infections. Although glucocorticoid hormones elicit a series of pro found intracellular responses which ultimately result in the cytolysis of lymphoid cells (31), the exact mechanism of this induced lysis is unknown. However, most, if not all, of these effects are mediated through specific cytoplasmic receptors which, when complexed with active glucocorticoids, are translocated to the nucleus where they bind to chromatin (23). Glucocorticoid receptors have been characterized and quantified in acute lymphoblastic leukemia (15, 16), which is also thought to be often of T-cell origin. These studies suggested that in acute lymphoblastic leukemia the pres ence of functional glucocorticoid receptors may reflect the responsiveness of patients to glucocorticoid therapy. How ever, in other leukemias, particularly chronic lymphocytic leukemia, in which the level of lymphocyte receptors is frequently comparable to that found in normal lymphocytes, receptor measurements alone appear insufficient to deter mine glucocorticoid sensitivity (14). The glucocorticoid sensitivity of various subclasses of T cells has also been studied using the mouse thymus as a model system. Cantor and Boyse (8) have shown that specific Ly alloantigens are expressed on lymphoid cells undergoing thymus-dependent differentiation . Although helper (Ly 1 @2iand killer/suppressor (Ly 1 23k) medullary cells are generally considered relatively resistant to gluco corticoids, their immature immunoincompetent precursors in the cortex are very sensitive (2). Other data suggest that certain mature immunocompetent T-Iymphocytes can retain their glucocorticoid sensitivity. Waldmann and Broden (34) have found that in a subset of hypogammaglobulinemia patients, the abnormality of immunoglobulin synthesis is 3 The

trivial

names

used

are:

prednisolone,

1,4-pregnadien-11f3,

17a,

21-

triol-3,20-dione; cortisol, 4-pregnen-11/3, 17a, 21-triol-3,20-dione; predni sone, 1,4-pregnadien-1 7a, 21-diol-3,1 1,20-trione; estradiol, 1 3,5(101-estra trien-3,1 [email protected]; dihydrotestosterone, 5a-androstan-1 7f3-oI-3-one; cortexo lone, 4-pregnen-1 7a, 21-diol-3, 20-dione; progesterone, 4-pregnen-3,20dione.

CANCER RESEARCHVOL. 39

Downloaded from cancerres.aacrjournals.org on February 7, 2018. © 1979 American Association for Cancer Research.

Glucocorticoid Receptors in Sézary Cells associated with a disorder of regulatory suppressor T-cells which may be very sensitive to glucocorticoids. Since the majority of Sezanycells are helper lymphocytes, these cells afforded us the opportunity to study the gluco corticoid

receptors

in another

subset

of immunocompetent

T-cells. The present study was thus undertaken to deter mine whether leukemic Sezary cells contain cytoplasmic glucocorticoid receptors and, if so, to determine the affinity and specificity of these receptors. Since several lymphoid mutants have been described which possess cytoplasmic glucocorticoid receptors but are resistant to glucocorti coids (29, 30), we were also interested in establishing an assay which would reflect the presence of functional recep tons in Sezary cells. Since the induction of glutamine synthetase by glucocorticoids is strictly dependent on the presence of functional cytoplasmic receptors (32), we have studied the possible induction of this enzyme in Sezany cells.

MATERIALS AND METHODS Source of SézaryCells. All patients used in this study (NIH Clinical Center and Georgetown University Hospital) were diagnosed as Sezany syndrome patients by standard clinical and cytological criteria. All suffered from prunitic erythrodermia and had atypical circulating Sézarylympho cytes, and skin biopsies revealed Sezary cells infiltrating the dermis. None of the patients had received chemother apy of any type which included glucocorticoids within 2 weeks of their inclusion in this study. Leukocytes were obtained from Patients 1, 2, 3, 4, and 6 by leukapheresis using an IBM-National Cancer Institute continuous cell separator (12). Following leukapheresis, the cells were collected by centnifugation, and the serum was aspirated. Peripheral blood was obtained from Patients 5 and 7. Hypotonic lysis of RBC was achieved by pipeting cells repeatedly in cold distilled water for 75 sec and was termi nated by the addition of an equal volume of 2x phosphate buffered saline (KCI, 0.4 g/liter; KH2PO4,0.4 g/Iiten; NaCI, 16.0 g/Iiter; and Na2HPO4•7 H2O, 4.32 g/Iiter). The washed leukocytes were used immediately or were resuspended in RPMI4 (prepared by NIH Media Unit) supplemented with 20% heat-inactivated fetal calf serum, 4 mM glutamine, and 10% dimethyl sulfoxide (Fisher Scientific Co., Fair Lawn, N. J.) and were then frozen in an acetone-dry ice bath and transferred to liquid nitrogen for storage. At the time of an experiment, the cells were quickly thawed at 37°,washed in the above medium devoid of dimethyl sulfoxide, and then washed twice in phosphate-buffered saline (one-half the concentrations listed for 2x phosphate-buffered saline). Viability of the cells was generally about 75% as determined by trypan blue exclusion. Glucocorticoid Receptor BIndIng Assay. Cytoplasmic binding assays for glucocorticoid receptors were performed using the method of Baxter and Tomkins (1) and Rousseau et al. (25) with certain modifications. The binding buffer (0.01 M Tnis-0.0015 mM EDTA-0.5 [email protected], pH 7.4) was supplemented with 20% glycerol, which is thought to increase the stability of glucocorticoid receptors (27). After 4 The

abbreviation

used

is:

RPMI,

Roswell

Park

Memorial

Institute.

homogenization with 20 strokes in a Ten Broeck glass homogenizer, the cytosol fraction was obtained by centnif ugation at 100,000 x g for 30 mm in a refrigerated Spinco Model L centrifuge. The cytoplasmic extract was collected with a Pasteur pipet and aliquoted into bonosilicate glass tubes (Corning Glass Co.). All subsequent incubations were performed at 4°in a Knyorack (Streck Laboratories Inc., Omaha, Nebr.) for 6 hn, except for experiments examining the time course of binding and the effects of elevated temperatures. Experiments designed to study the time course of specific binding indicated that an incubation period of 6 hr was required to reach equilibrium. In the standard binding assay, [email protected] aliquots of the cytosol were incubated with increasing concentrations of the synthetic glucocorticoid [3Hjdexamethasone (26 Ci/mmol; Amen sham/Searle Corp., Arlington Heights, Ill.) in the presence on absence of a 500-fold excess of nonlabeled dexametha sone. Following the incubation, 25 @lof dextran-coated charcoal suspension were added; each tube was agitated on a Vortex mixer for 10 sec and then centrifuged at 3000 rpm for 10 mm in a refrigerated Sorvall RC-3 centrifuge. Three 50-liter aliquots of the resultant clear supernatant were then added to 10 ml of Aquasol Scintillation Cocktail (New England Nuclear, Boston, Mass.) and were counted in a Beckman LS-255 scintillation spectrometer (Beckman Instruments, Inc. , Fullerton, Calif.) with an average count ing efficiency of 45% for tnitium. Total counts minus non competable counts were then taken to represent specifi cally bound dexamethasone (1). Final results are expressed as pmol [3H]dexamethasone specifically bound per mg of cytoplasmic protein. Protein determinations were per formed according to the method of Lowry et al. (18) using, as the standard, bovine serum albumin (Sigma Chemical Co., St. Louis, Mo.) dissolved in the binding buffer. It is unlikely that endogenous glucocorticoids would interfere to any extent in this standard binding assay, since small amounts of free endogenous glucocorticoids would be removed during the washing process and any specifically bound steroid would be displaced from the receptor by the large excess of competing [3Hjdexamethasone. Although it is possible that the cytosols were contaminated by very small amounts of serum proteins, [3Hjdexamethasone binds only to intracellular receptors and not to serum corticoste roid-binding globulin (transcortin) (17). Nonradioactive steroids used in the competition and induction experiments were obtained from the following sources: cortisol, prednisone, prednisolone, 17/3-estradiol, and 5a-dihydrotestosterone from Sigma Chemical Co. ; dex amethasone

from

Merck,

Sharp

and

Dohme

(West

Point,

Pa.); and cortexolone from Aldrich Chemical Co. (Milwau kee, Wis.). Sucrose Gradients. Linear 5 to 20% sucrose gradients were prepared using binding buffer (10% glycerol; 0.4 M KCI) in cellulose nitrate tubes (Beckman Instruments, Inc., Palo Alto, Calif.) with a gradient-former (Hoefer Scientific Instruments, San Francisco, Calif.) and a penistaltic pump (LKB Instruments, Inc., Rockville, Md.). Following adsonp tion of free [3H]dexamethasone with dextran-coated char coal, 0.3-mI aliquots of the labeled cytosols were layered onto gradients which were then centrifuged for 18 hr at 149,000 x g in the SW 50.1 rotor of a refrigerated L5-65

FEBRUARY1979

Downloaded from cancerres.aacrjournals.org on February 7, 2018. © 1979 American Association for Cancer Research.

377

T. J. Schmidt and E. B. Thompson Beckman ultracentnifuge. [‘4C]Bovmne serum albumin (4.6 5) was used as a marker protein. Following centnifugation, the cellulose nitrate tubes were pierced using a Beckman fraction recovery system, and 30 fractions (0.18 ml each) were collected from the top of the gradient by displacement with 50% glycerol. After addition of 10 ml of Aquasol (80 ml H2Oper liter, 50 ml glacial acetic acid per liter), the fractions were counted in a Beckman LS-255 scintillation spectrom eter with a counting efficiency of approximately 30%. Glutamine Synthetase Assay. The glutamyltransferase activity of glutamine synthetase was measured spectropho tometnically (at 500 nm) according to the assay described by Thorndike and Reif-Lehrer (33), except that each reac tion mixture (800 @I) contained 0.1 mM ADP and the [email protected] concentration was increased to 10 [email protected] obtain maximal activity (13). The specific activity of the enzyme is expressed as @moI y-glutamylhydroxamate produced pen mm pen mg protein. A standard calibration curve was prepared using L glutamic acid-'y-monohydroxamate (Sigma) dissolved in 25 mM citrate buffer. Since preliminary experiments showed that the assay was linear for 1 hn, the standard incubation was for 1 hr. Viable lymphocytes used in these experiments were first isolated on Ficoll-Hypaque density gradients (Pharmacia Fine Chemicals, Piscataway, N. J.) (3) and were then equilibrated in RPMI tissue culture medium (supple mented with 20% heat-inactivated fetal calf serum and 4 mM glutammne) for 24 hr in a humidified incubator (5% CO2). Following this equilibration , dexamethasone d issolved in absolute ethanol was added at the approximate concentra tion and the cells were incubated for an additional 18 hr. At the end of the incubation, the cells were harvested by

patientsPatientSexAgeHighest

Clinica!

leu kocyte count (cu mm)% trol)1M(64)80,00085

informationand

centrifugation, washed once in 5 ml of RPMI medium devoid of serum, resuspended in 0.5 ml of 25 [email protected] buffer, frozen in dry ice, and transferred to a Revco freezer (—70°). Within 1 week, samples for assay were thawed and sonicated with two 10-sec bursts using a Biosonik probe (Bronwell Scientific, Rochester, N. V.) at an intensity of 15. Visible particulate matter was removed by centnifugation (5 mm; iooo x g), and the supennatants were assayed for specific enzyme activity. Freezing for 1 week had no effect on the specific activity of the enzyme. RESULTS Affinity of Cytoplasmic Glucocorticoid Receptors. Using the competitive binding assay described, we detected spe cific glucocorticoid receptors in the cells from several Sézarypatients, while the cells from other patients con tamed very low or undetectable receptor levels (Table 1). Lymphocytes from Patients 1 and 6 showed significant binding of [3H]dexamethasone and were available in large numbers; therefore, these were used in subsequent ex periments designed to characterize cytoplasm ic glucocor ticoid receptors. As mentioned earlier, preliminary expeni ments revealed that an incubation of 6 hr was required for maximal binding of [3H]dexamethasone. Specific binding of [3H]dexamethasone was also shown to be linearly related (r =

0.99)

to

the

protein

concentration

Table 1 glucocorticoid receptorcontent

(Kd

erythrodermia1.92M(46)12,00050

a

given

cytoplasmic

in Sézary

Lympho content (pmol cytes (% Se rH]dexamethasone bound/ zany)Receptormg cytoplasmic protein)aResponsiveness (100)0.42

for

extract under the conditions of the assay. Chant 1A shows a binding curve for increasing concentrations of [3H]dexa methasone at a cytoplasmic protein concentration of 3.4 mg/mI (Patient 6). At 4°,these cytoplasmic receptors be

of glu tamine synthe tase (EsA'@In duced/ESA con

to glucocorticoid therapyInduction

agent (prednisone) dramati 6.9 x 10-s M)Single cally reduced Sézary count; corn bined chemotherapy reduced edema and

+ prednisone had no (20)0.04Chlorambucil

effect

(75)0.08No

systemic glucocorticoids given; triamcinolone (topical) improved skin slightlyNot

on Sézary count

or skin le

sions1.03F(57)7,50033 assayed4M(57)8,50050

detectableNo

assayed5M(47)35,00092 (100)None

chemotherapy esisNot

detectableRefractory

.16M(46)258,00099

(85)None

prior to leukapher

to combined chemother

apy including

glucocorticoids1

chemotherapy at time of leuka (100)0.13

(K,1= 6.9 x [email protected] M)No

pheresis;

developed

malignant

lym

phoma2.27F(46)46,00060

(100)0.07Refractory

to combined chemother apy including glucocorticoidsNot

assayed

a Determined using a saturating concentration of [3Hjdexamethasone. b ESA,specific activity of the enzyme (induced and control) expressed as mean of duplicate determinations

378

(range < 15% of mean).

CANCER RESEARCH VOL. 39

Downloaded from cancerres.aacrjournals.org on February 7, 2018. © 1979 American Association for Cancer Research.

@

£

@[email protected]

I

I

Glucocorticoid

@ @

z

I

.12

9

specific binding activity. At 4°,100% of the specific binding was preserved after 24 hr if [3Hldexamethasone was present throughout the incubation period. If the cytosol was stoned

.

.

A

0

@

at

@

@

g

@

j:

@ @

@ @

24

hr

in

the

absence

of

dexamethasone

followed

by

[email protected]) 77% of the specific binding activity was detected. _______

x

____

‘.—‘

O((3HJOursaIIi,SSWI. 50 [email protected],@s,sdFrom

[email protected]@

@. .90

[email protected]

[email protected]

2

3

4

5

6

7

of

these

Glucocorticoid

cytoplasmic

Receptors.

receptors

was

The

specificity

demonstrated

by

mea

ability of dexamethasone, prednisolone, and cortisol at a

Lv,vç[email protected]

_ 1

Specificity of

suning theability ofcompeting steroid analogs todisplace @I 5X10's M[3H]dexamethasone inthebinding reaction. The

[email protected] @

4° for

addition of [3H]dexamethasone and an incubation of 6 hr,

I

@ @

Receptors in SézaryCells

8

9

(3HJDE2CAMETHASONE (moVIIw s [email protected]

50-fold

molar

excess

to completely

displace

the specifically

bound [3H]dexamethasone (Chart 2,4) agrees with the known cytolytic activity of these steroids in lymphoid sys tems (21). Prednisone (Chart 2B) and cortexolone (Chart

24), which in rodent thymocytes is an antiglucocorticoid (22), also competed for binding when present at higher molar excesses. Progesterone (Chart 2B), an antiinducer in human lymphoid tissues (15), was also an effective com petitor, while 17/3-estradiol and 5a-dihydrotestostenone showed weak competition (Chart 2B). The specificity of this competition for receptor sites is also reflected in sucrose gradient profiles of cytoplasmic extracts. As seen in Chart 3, the [3H]dexamethasone-necep ton complex migrated with a sedimentation coefficient of approximately [email protected],,, in linear 5 to 20% sucrose gradients containing 0.4 M KCI. A 500-fold molar excess of unlabeled dexamethasone completely abolished this 4S binding peak,

[email protected] [email protected]

a

A A Duronsthisons @

0

.e @

[email protected] [email protected] .os .10 .12 .14

1$ .11

I

I I

[email protected]

. Crowd U [email protected]

SOUNDPH) [email protected]@)

Chart 1. A, specific and nonspecific binding of (3Hjdexamethasone to cytoplasmic extract of Sezary lymphocytes (3.4 mg cytoplasmic protein/mI);

B, Scatchardplot of specificbindingdata presentedin A. NATIO OF cOMPETITIVE [email protected] TO l3Nl [email protected])[email protected]

came saturated at a dexamethasone concentration of ap proximately 4 x 10-i M. Chart lB shows a Scatchard plot (25) of the specific binding data in Chart 1A. Although there appeared to be some nonlinearity at the lower concentra tions of [3H]dexamethasone, this was probably artifactual and may have resulted from failure to achieve equilibrium binding after 6 hr. The straight line, which was drawn by hand for the remaining points, is consistent with a single class of receptors of uniform affinity. The equilibrium dis sociation constant (K,,@) calculated from the slope of the Scatchard plot was 6.9 x [email protected] M, which agrees with the reported affinity of cytoplasmic glucocorticoid receptors in acute lymphoblastic leukemia (16). Thermal Lability of Receptors. The cytoplasmic gluco conticoid receptors in Sezary cells were found to be then molabile. A 30-mm preincubation at 37°left only 5.4% of the specific cytoplasmic binding activity relative to a control incubated for 6 hr at 4°,while 30 mm at 22°left 67% of the

I RATiO OF

@[email protected]@OIO$ TO ([email protected]@[email protected]@

Chart 2. A and B, displacement of specific (3Hjdexamethasone (5 x 10' PA) binding

by

various

competing

steroids

present

at

a

1-

to

500-fold

molar

excess.

FEBRUARY1979

Downloaded from cancerres.aacrjournals.org on February 7, 2018. © 1979 American Association for Cancer Research.

379

T. J. Schmidt and E. B. Thompson ficity of the increase in specific activity in Sezary cells is demonstrated in Chart 6. Although dexamethasone ([email protected] M) induced enzyme activity maximally, other active glucocor ticoids such as prednisolone ([email protected] M) and cortisol ([email protected] M) also resulted in increased enzyme activity. Nonglucocorti coids such as progesterone and 17f3-estradiol failed to increase the specific activity of glutamine synthetase.

a

I

DISCUSSION The data presented demonstrate that lymphocytes from some Sezary patients contain significant quantities of cyto @90

FRACTIONNUMBER

Chart 3. Linear 5 to 2O%sucrose gradient (containing 0.4 M KCI) profiles of cytoplasmic [‘H]dexamethasonebinding in the presence of various corn peting steroids (500-fold molar excess). Arrow, location of [‘@C]bovine serum albumin (4.6s). •,control; A, [email protected]; 0, 5a-dihydrotestosterone; 0, dexamethasone.

@70 t60

124

whereas an equivalent excess of either 17f3-estradiol or 5adihydnotestosterone resulted in only partial inhibition.

Increase In SpecificActivityof GlutamineSynthetase. As seen in Table 1, dexamethasone ([email protected] M) increased the specific activity of glutamine synthetase approximately 2fold in 2 Sezary cell populations (Patients 1 and 6) which contained cytoplasmic glucocorticoid receptors. However, dexamethasone failed to increase specific enzyme activity in 2 populations of Sezany cells (Patients 2 and 5) which apparently lacked significant quantities of glucocorticoid receptors. Because a large number of cells were available from Patient 6, these cells were used to study the kinetics of the glucocorticoid mediated increase in enzyme activity. The time course (Chart 4) indicates that maximal enzyme activity was achieved after 18 hr. Chart 5 depicts the dose response relationship and indicates that maximal enzyme activity was achieved with approximately [email protected] M dexameth asone. Half-maximal enzyme activity was achieved at ap proximately 2.4 x 10-s M dexamethasone, which is higher than the experimentally calculated concentration required for half-maximal saturation of cytoplasmic receptors ([email protected]= 6.9 x 10-i M). The explanation for this apparent discrep ancy is unclear. Using several munine T-celI lines which are sensitive to glucocorticoids, we have also found that the equilibrium dissociation constant determined using a whole-cell binding assay is higher than that determined using a cytosol assay ft. J. Schmidt and E. B. Thompson, unpublished data). It is possible that the glycerol added to the homogenization buffer used in the cytoplasmic assay slows the dissociation of rHldexamethasone from recep tons and thus lowers the equilibrium dissociation constant

@

The kinetics of this increase in specific activity of glutamine synthetase in Sezary cells are identical to those seen in an established human leukemic cell line (CEM) which contains specific glucocorticoid receptors.5 It has been shown with the latter cell line, that the basal specific activity of glutamine synthetase is inversely related to the concentration of glutamine in the medium and that dexa methasone always produces a 2.5-fold increase in specific activity regardless of the basal enzyme activity. The speci 5 J.

380

M.

Harmon

and

E.

B.

Thompson,

manuscript

in

preparation.

1

3

6

9 12 LENGTH OF INCUBATION (SOIIISP

18

2436

Chart 4. Time course of glutamine synthetase induction by dexametha sone (10' M) in Sezary lymphocytes. Points, mean of duplicate determina tions (range < 15% of mean). The basal enzyme activity increases with time presumably due to gradual utilization of glutamine in the medium. There is a known reciprocal relationship between the enzyme activity and available glutamine concentration in several cell lines. The steroid effect, however, is independent of the glutamine effect. When the specific activities of the induced (18 hr) and noninduced (incubated 18 hr) cells were compared, the induction in this experiment was approximately 1.6-fold. 100 90 B ;eo @70

f 90 4 350

20 10

10'

5,10―

10―

5m10

10'

CONC DEXAMETHASONE

2,10

[email protected]'

Chart 5. Dose-response relationship for the increase in specific activity of glutamine synthetase by dexamethasone in Sezary lymphocytes. Points, mean of duplicate determinations (range < 15% of mean).

a,

E C E I

I HORMONE

Chart 6. Increase in specific activity of glutamine synthetase in Sezary lymphocytes by different steroids (1O N) (single determinations).

CANCER RESEARCHVOL. 39

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Glucocorticoid plasmic glucocorticoid receptors, whereas cells from other Sezary patients appear to contain low or undetectable levels of such receptors. This continuum of glucocorticoid receptor levels is analogous to the situation seen with estrogen receptors in human breast cancer. Obviously, it is not possible to identify precisely the glucocorticoid-recep ton concentration which demarcates a receptor-positive from a receptor-negative cytosol. Those glucocorticoid re ceptors detected in these helper T-lymphocytes appeared very similar in their saturability, affinity, thermolability, and specificity to glucocorticoid receptors described in other human leukemic and lymphoid cells. Considering that only a small number of patients were studied, it is not possible to draw conclusions concerning the value of receptor determinations in predicting clinical responsiveness to glu cocorticoid

therapy.

Patient

1 , whose

cells

contained

levels

Receptors in SézaryCells

ability of dexamethasone to increase the specific activity of glutamine synthetase through a presumed inductive mech anism might serve as such a functional test in Sezary cells. This enzyme has been studied extensively in chick embryo retinal tissue where it can be induced prematurely by dexamethasone (21, 24). Induction in this tissue requires new protein and RNA synthesis (20) and increased levels of specific translatable mRNA (28). Induction of glutamine synthetase by glucocorticoids has also been reported in L cells (32) and rat hepatoma tissue culture cells (9). Since we have found a number of mouse and human B- and T-cell lines which are lysed by glucocorticoids but show no induction of glutamine synthetase, it is clean that induction of this enzyme is not required for glucocorticoid killing of lymphoid cells (unpublished data). As previously men tioned, glutamine synthetase is inducible in a human leu kemic cell line (CEM).4 Approximately 40 glucocorticoid resistant mutants of this cell line have been analyzed and found to be noninducible for glutamine synthetase. These data suggest that the induction of glutamine synthetase in this cell line is a marker for functional receptors and glucocorticoid-mediated killing. The data presented here suggest that induction of glutamine synthetase may also serve as a marker for functional glucocorticoid receptors in Sézarycells.

of receptor several times greater than those found in any other patient, did in fact respond to prednisolone alone and in combination with other drugs; and Patient 5, in whose cells no receptor was detected, was refractory to combined therapy. However, Patients 2 and 7 were found to have measurable receptor levels and yet were refractory to glu cocorticoid therapy. Obviously, further prospective studies with additional untreated patients would be required to ascertain whether receptor levels can provide an accurate prediction of glucocorticoid-mediated cytolysis of Sezary ACKNOWLEDGMENTS cells. Inasmuch as the levels of cytoplasmic receptor in the We wish to thank Billie Wagner for her technical assistance with the cells from some Sézarypatients were very low on undetect protein and enzyme assays and Jean Regan for her secretarial assistance. We also thank Dr. Philip S. Schein, Department of Oncology, Georgetown able, it is possible that the majority of the unbound recep University Hospital, and Dr. Paul Bunn, NCI-VA Medical Oncology Branch, tons in these cells were inactivated during homogenization, Veterans Administration Hospital, for generously supplying cells from several perhaps due to the release of proteolytic enzymes or other Sezary patients. inactivators. However, mixing experiments in which recep REFERENCES ton-negative and receptor-positive cytosols were utilized indicated that the receptor-negative cytosols lacked factors I . Baxter, J. D., and Tomkins, G. M. Specific Cytoplasmic Glucocorticoid which could inactivate receptors in the receptive-positive Hormone Receptors in Hepatoma Tissue Culture Cells. Proc. NatI. Acad. Sci. U. S., 68: 932-937, 1971. cytosols. In interpreting the receptor data for each patient 2. Blomgren, H., and Anderson, B. 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Glucocorticoid Receptors and Glutamine Synthetase in Leukemic Sézary Cells Thomas J. Schmidt and E. Brad Thompson Cancer Res 1979;39:376-382.

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