glucocorticoid receptors in subpopulations of childhood ... · useful in designing therapeutic...

[CANCER RESEARCH 37. 2688-2695, August 1977]

these patients, in general, have a more aggressive form ofdisease (2, 28, 29). Most patients have lymphoblasts thatlack identifiable surface markers. Thus, the subcategoriesof acute Iymphocytic leukemia in humans probably represent proliferation of different clones of lymphoblasts. Thereare known differences in glucocorticoid susceptibilityamong functional subcategories of lymphocytes in mice(24, 27, 31, 32). We therefore assayed lymphoblasts for avariety of surface markers and then examined lymphoblast populations with known characteristics for differences in specific cyhoplasmic GA activity and responsiveness as a function of these various surface markers.

MATERIALS AND METHODS

Patients. Forty-five patients with acute lymphocytic leukemia were studied . Diagnoses were established in all casesby standard morphological diagnostic criteria (20). The agemangewas from 1 to 20 years. There were 16 females and 29males. Four of the males were black. Lymphoblasts wereobtained from the peripheral blood of 40 patients prior toinitial therapy and from 5 patients in relapse and storedfrozen in a viable state, as described below. No treatedpatients had received any glucocorticoid therapy within 2weeks prior to their inclusion in this study.

Storage of Malignant Cells. Leukemia blast cells werecollected from the peripheral blood of patients by conventional leukophonesis (22), by use of the IBM-NCI continuouscell separator (7) or by phlebotomy, at the time of diagnosisor relapse before chemotherapy was given. Peripheralblood specimens were collected in 5 units of hepanin per ml(Upjohn Co. , Kalamazoo, Mich.). The cells were frozen in aviable shale at a concentration of 1 to 2 x 10@cells/mi with10% dimethyl sulfoxide and stored in the vapor phase ofliquid nitrogen by a method previously described (8). At thetime of the study, the cells were thawed rapidly and thenplaced on a Ficoll-Hypaque density gradient (Ficoll-Paque;Pharmacia Fine Chemicals, Piscalaway, N. J.). The resulting interface cell suspension was >95% mononuclear cellsand >95% viable as measured by hrypan blue dye exclusion.

Lymphocyte Surface Markers. Lymphocyteswere exammed for their ability to form E-rosethes by a previouslydescribed (19) modification of the method of Weiner et a!.(30), using neuraminidase-pretreated sheep ABC. At theend of the procedure, the cells were spun in a cytocentrifuge (Cytospin; Shandon Elliott Co. , Surrey, England), andsmears were made and stained with Giemsa stain so thatdifferential counts could be performed; the cells at the

2688 CANCERRESEARCHVOL. 37

Glucocorticoid Receptors in Subpopulations of ChildhoodAcute Lymphocytic Leukemia1

Geraldine S. Konior Yarbro,2 Marc E. Lippman,3 Gloria E. Johnson, and Brigid G. Leventhal

The Oncology Center, The Johns Hopkins University School of Medicine, Baltimore 21205 (G. S. K. V., B. G. L.], and Medicine fM. E. L.] and PediatricOncologyBranches(G. E. J.], Divisionof CancerTreatment,NationalCancerInstitute,NIH, Bethesda,Maryland20014

SUMMARY

In view of the known differences in sensitivity to glucocorticoids among functional subcategories of lymphocytes inthe mouse and the awareness that different subcategoriesof acute lymphocytic leukemia may represent proliferationof different clones of lymphoblasts, we examined lymphoblast populations with known surface markers for differencesin specific cyhopiasmic glucocorticoid receptor activity.Lymphoblasts were divided into those that formed sheeperythrocyte rosettes (T-lymphoblasts) and those that lackedall surface markems (null lymphoblasts). The latter werefurther subdivided by their ability to stimulate allogeneicdonors in mixed lymphocyte culture (MLC). Lack of stimulalion in MLC is a characteristic of mature T-cells. Eighteenpatients had null lymphoblasts that did stimulate in MLC,and glucocorticoid binding sites in these ranged from 4,096to 21 869 sites/cell (median, 7,571). Eighteen patients withT-lymphoblasts had binding sites ranging from 0 to 5,887sites/cell (median, 2,173). This difference in specific glucocorticoid receptor levels is highly significant (p < 0.001).Nine patients whose lymphoblasts lacked identifiable sunface markers but failed to stimulate in MLC had intermediate values for receptor sites. Thus it appears that null lymphoblasts are more likely to have higher numbers of specificglucocorticoid receptor than T-lymphoblasts. The diffenential capacity of lymphobiasts to bind steroid may proveuseful in designing therapeutic regimens for the differentsubcategories of acute lymphocytic leukemia.

INTRODUCTION

Acute lymphocytic leukemia is a heterogeneous form ofcancer. Approximately one-fifth of patients have lymphoblasts that form spontaneous rosettes with E-nosettes4 and

I Presented in part at the Annual Meeting of the American Federation for

Clinical Research, Atlantic City, N. J.. May 1 and 2, 1976 (10), and in part atThe 16th International Congress of Hematology. Kyoto, Japan. September 5to 11, 1976 (11).

2 Deceased May 17, 1977.

3 To whom requests for reprints should be addressed, at Medicine Branch,

Division of Cancer Treatment, National Cancer Institute, NIH, Building 10,Room 6B02, Bethesda. Md. 20014.

4 The abbreviations used are: E-rosettes, sheep erythrocyte rosettes; GA,specific glucocorticoid receptor; EAC rosettes, erythrocyte-antibody-complement rosettes; MLC, mixed leukocyte culture; SR. stimulation ratio; PBS,phosphate-buffered saline (NaCI, 8.5 g/liter; Na,HPO4, 0.79 g/liter; andKH,P04, 0.14 g/litar); N+ blasts, null blasts that stimulated in MLC; Nâ€”blasts, null blasts that did not stimulate in MLC; T-Iymphoblasts, E-rosetteforming lymphoblasts.

Received January 18, 1977; accepted May 16, 1977.

Research. on December 10, 2020. © 1977 American Association for Cancercancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

Glucocorticoid Receptors in Childhood Leukemia

center of the rosettes were confirmed to be blasts. EACrosettes were measured with the use of a commerciallyavailable reagent (EAC; Cordis Labs, Miami, Fla.) by apreviously reported method (3).

Receptors for the Fc portion of the immunoglobulin molecule were measured by a previously described method (21).Cells were incubated initially with an antigen-antibody compIex consisting of keyhole limpet hemocyanin and an aff inity column-purified rabbit anti-keyhole limpet hemocyaninlgG antibody. They were then washed and incubated withfluorescein-labeled goat anti-human immunoglobulin to seewhether intact immmunoglobulin molecules were coatingtheirsurface.

MLC. Hepaninizedwhole blood (Heparin; Upjohn Co.; 5 to10 units/mI of blood) was spun at 225 x g, and cell-richplasma aspirate was placed on a Ficoli-Hypaque gradientand spun for 15 mm at 20Â°at 2000 x g . Cells at the interface,which were 95 to 100% lymphocytes, were used as responding cells. Leukemic lymphoblasts and normal lymphocytesfrom both patients and unrelated donors were used asstimulating cells. Stimulating cells were irradiated with 5000A in a 137Cesource (Gammator M; Kewaunee ScientificEngineering, Adrian, Mich.). Triplicate cultures were set upwith 10@responding lymphocytes and 10@stimulating leukemic blasts on normal lymphocytes in a 0.2-mi volume inTC 199 (Flow Labs, Rockville, Md.). Details of the culturemethod have been described previously (25). Cultures wereincubated for 6 and 7 days in 5% CO2. Eighteen hr prior toharvest, 0.8 @Ciof Initiated thymidine, specific activity, 6.7mCi/mmole (New England Nuclear, Boston, Mass.), wereadded to each well in a 2O-pi volume. Cultures were hamvested and dpm/cultune were calculated . SR are expressedas the dpm in the sample containing the responding lymphocytes plus the X-inradiated blast preparation divided bythe dpm in the sample containing the responding lymphocytes plus X-imnadiahedlymphocytes. A technically satisfactory study was one in which the normal allogeneic donorresponded to the remission lymphocytes of the patient, orto the cells of another individual. Stoned blast cells wereconsidered stimuiatory if there was a vigorous response (SR> 5) to them even on 1 occasion. They were considered

nonstimulatory if they had failed to stimulahe at least 3different allogeneic normal donors on 3 separate occasions, or if the median ratio of multiple determinations wasless than 2 even though 1 on2 ratios might be above that.

Cytoplasmic Glucocorticoid Receptor Determination.Cell populations obtained as noted above were suspendedat a density varying between 0.5 and 10 x 106 cells/mI inEagles minimal essential medium supplemented with 25mM N-tnis(hydroxymethyl)methylglycine and 0.6 g glutamine per liter. Most measurements of binding were made atcell concentrations between 1 and 3 x 106cells/mI Aliquotsof 0.8 ml of the cell suspension were delivered into 12- x 75-mm glass tubes. [3H]Dexamethasone (28 Ci/mmole; Amensham/Searle Corp., Evanston, Ill.) in ethanol was diluted inEagle's minimal essential medium, supplemented as descnibed above, to yield a concentration 5 times the finalconcentration desired. Final ethanol concentration was always less than 0.1%, a concentration that has no effect onbinding. Dexamethasone was used rather than cortisol,

since the former will not bind ho transcortin (corticosteroidbinding globulin) (18), and small amounts of serum proteinsmight conceivably have remained adherent to cellsthroughout the purification procedure. To one-half of thissteroid solution, a 100-fold excess of unlabeled competingdexamethasone was added. Aliquols of 0.2 ml of this 5-foldconcentration were added to the 0.8-mi cell suspensions,and the tubes were gently mixed. In some experiments,complete binding curves were constructed by using multipie concentrations (3 to 7) of radioactive dexamethasone,with and without unlabeled dexamethasone, run in tniplicate. In other experiments, because of limited cell numbers,tniplicahe or quadruplicate determinations of binding at asingle steroid concenhrahion (2 x 10@ M) on 2 steroid concenlrations (2 and 1 x 10@ M) estimated to be about 5 and2.5 times the dissociation constant, respectively, wereused. Every 15 mm, the cells were gently resuspended bybrief mixing or were incubated continuously using a mockerplatform (Bellco Glass, Inc., Vineland, N. J.). Incubahionswere performed at 21Â°.At the end of 2 hr, 2 ml of cold PBS,pH 7.4, were rapidly added to each tube. This time intervalexceedsthatrequiredformaximal binding,as determinedby kinehic studies described below. Viability, as determinedby trypan blue exclusion, invariably exceeds 90% underthese assay conditions (15). Cell pellets were collected bycentnifugation at 1100 to 1200 rpm for 5 mm in a ModelPR-6 centrifuge (International Equipment Co. , NeedhamHeights, Mass.) and then resuspended and washed twicemore in ice-cold PBS. The cell pellets were then suspendedin 0.2 ml PBS and 1 ml of ethanol to assist in extraction ofprotein-bound steroid. This suspension was transferred toliquid scintillation vials and counted in 12 ml of Aquasol(New England Nuclear, Boston, Mass.) at an efficiency of40%. Simultaneously, 0.2-mi samples of the 5-fold concentrations of radioactive steroid solutions, with 1 ml of ethanoladded , were counted in 12 ml of Aquasol. Cytoplasmicbinding sites pen cell and equilibrium dissociation conslants were obtained from Scatchard analyses (26) preparedby computer-assisted analysis of the binding data (1). Binding sites per cell reported are, in all cases, those obtained atthe highest steroid concentrations and are, therefore, 15 to20% below the total receptor capacity.

RESULTS

After the surface marker studies were analyzed, all thelymphoblasts could be classified (Table 1) as either thosethat did make E-nosettes and were called T-blasls, onthose that had no markers and were called null blasts. Nolymphoblashs that formed EAC-rosetles on had Fc receptorswere seen in this group of patients. We have previouslyshown (13) that lymphoblasts that make E-rosettes usuallyshare with normal human E-nosette-fonming cells the inability ho stimulate aliogeneic responders in the MLC. We havepostulated that the inability on loss of ability to stimulate inMLC may represent maturation along the pathway of T-celidifferentiation. We therefore subdivided the null lymphoblasts into 2 categories by whether or not they stimulated inMLC forthepurposesofanalysisinthispaper.The 3 groups

AUGUST 1977 2689



Patientswere said to havenull cellor T-cell leukemia basedonsurfacemarkerstudies asshown.Surface

marker NullTâ€” +

Patient and lymphoblastcharacteristicsPatientswere divided into 3groups,onthe basis of lymphoblast characteristics. Patientcharacteristicsat the time ofdiagnosisareshown

for each of thegroups.MarkersSRWBC

x 103/cu mmb% blastsâ€•Age atdiagnosis(NulIÂ°/MLCNo.ofSexGroup

T) (+ or â€”)MedianRange patients Median RangeMedian RangeMedian Range(M/F)N +

1@

G. S. K. Yarbro et a!.

of patients are described in Table 2. Group 1 (18 patients)had lymphoblasts that lacked identifiable surface markersbut did stimulahe in MLC. These were called N+ blasts.These patients ranged in age from 3 ho 18 with a median of7.3 years at diagnosis. There were 9 males and 9 females.The WBC ranged from 2,800 to 200,000/cu mm with amedian of 15,000. Group 2 (9 patients) had lymphoblashsthat also lacked idenhifiable surface markers, but these cellsfailedtostimulateinMLC and were calledNâ€”blasts.Thesepatients ranged from 1 to 18.5 years of age with a median of4.5 ahdiagnosis. Themewere 6 males and 3 females, and theWBC ranged from 2,400 to 1,000,000/cu mm with a medianof 17,200. Group 3 (18 patients) had E-rosehhe-forming onTlymphoblashs. Sixteen of these failed to stimulate allogeneicdonors in MLC and 2 did stimulate. These patients ranged inage from 3 to 20 years with a median of 12.7 years at thetime of diagnosis. There were 14 males and 4 females.Median WBC was 22,000 with a range from 1,900 to415,000/cu mm. There were differences in the age and sexpattern within the patient groups, with Group 3 patientstending to be olden and more often male, as has beenreported for E-rosette-forming leukemia (29). These patienhs were selected for study because they had a highpercentage of circulating blasts at the time the cells werecollected.

Effect of Cell Concentration on Glucocorticoid Binding.In order to determine the effect of cell concentration in thisassay, tests were performed at varying cell concentrations,ranging from 0.5 to 10 x 106cells/mI. In Chart 1, it can beseen that this assay is linear within a broad mangeof cellconcentrations. Results are identical at concentrations of 1

x 106 cells/mI and higher, for both null and T-lymphoblasts,

and all determinations were done well within this range,most at 3 x 10@cells/mi.

At concentrations less than 1 x 10@cells/mI, results werevariable, presumably due to loss of cells in washing at theselow concentrations.

Affinity of Glucocorticoid Binding to Cytoplasmic Receptor. in order to characterize the affinity of the binding,uptake of glucocorticoid was measured at varying concentrations of Initiated dexamethasone, ranging from 3 x 10 â€˜Â°to 2 x 108 M. The binding curves obtained for representative null and T-lymphoblasts are shown in Chart 2. Thelower curve represents T-lymphoblasls, while the uppercurve represents null lymphoblasts. As can be seen, although the total number of sites per cell differs markedly,the shape of the curves is the same, with both approximately 70% saturated at a concentration of 0.8 x 10 8 Mdexamethasone. The inset shows the data replotted foreach of the curves by the Scatchand technique (26). Parallelstraight lines are obtained, indicating an identical dissociation constant of 3.9 x 10@ M for both null and T-blasts. Thelinear relationship obtained, fomeach cell type, is consistentwith radioactive dexamethasone binding hoa single class of

24000â€”

â€œNuIr'LymphOblaStS

Table1Surface markers

. â€˜T'

E-rosettesÂ°EAC-rosettesâ€•Fc receptorsr

a Spontaneous rosettes with sheep erythrocytes.

b Erythrocyte-antibody-complement test for complement recep

hors.C Receptor for the Fc portion of the immunoglobulin molecule.

.@

uJz

@1200O@aa

hoo.

â€” 2x10' 4x10' SxlO'

CELL CONCENTRATION

Chart 1. Dexamethasone binding at varying cell concentrations in reprasentative T- and null lymphoblasts of childhood acute lymphocyteleukemia. Specifically bound [3Hjdexamethasone was determined with asteroid concentration of 2 x 10' M.

Table2

123.53.8-21118152.8-20080%9-957.33-189/92Nâ€”1.00.7-1.6917.22.4-100080%10-984.51-18.56/33Tâ€”1.10.7-2.916221.9-41575%11-10012.73-2014/4

>18+ 6.4, 10.3 2 ____________

a Null lymphoblasts are defined as those lacking the ability to form either E- or EAC-rosettes and lack of receptor for the Fc fragment of

immunoglobulin.b WBC and percentage blast obtained from peripheral blood of patients on the day cells were collected, prior to concentration and

purification with Ficoll-Hypaque gradient.




â€œTâ€•Lymphoblasts TIMI m,nu@es)

120

100

80


60

2.632 x 10@ M can be calculated for the binding reaction,which is in good agreement with 3.89 x 10@ M which wasobtained from the equilibrium data for dexamethasoneshown in Chart 2.

Specificity of Glucocorticold Binding to Cytoplasmic Receptor. Evidence that dexamethasone is binding to a specific glucocorticoid receptor is shown in studies graphicallyillustrated in Charts 5 and 6. The ability of various unlabeledsteroids,presentin molar excess,to compete with 10@ MInitiated dexamethasone is shown for both null and T-lym

40

0.5 1 1.5 2 3 4 5 6 7 8

IncubaflonTime lhoursl

E

@100

[email protected]

@40

z0

@ 20â€¢>--J-J

U

@ 5 20 40 60

@ INCUBATION TIME (minutes)

Chart 3. Kinetics of association between dexamethasone and T- and nulllymphoblast GA at a steroid concentration of 2 x 10' M. The reaction approaches equilibrium after approximately 1 hr. Inset, plot of log,,, (unboundsteroid concentration/unbound receptor concentration) versus time for earlytime points of the association curve for null lymphoblasts.

E

0E

E

aI

C

2

Chart 4. Kineticsof dissociationof the lymphoblastGA-dexamethasonecomplex. After 90 mm of incubation with radioactive dexamethasone, a100-fold excess of nonradioactive dexamethasone was added to the incubation mix, and specific binding was determined at the times shown. Inset,plot of log,, (bound steroid) versus time for the data shown. Curve is forrepresentative null lymphoblasts.

Concentration [3H] Dexamethuone lM)

II

Chart 2. Specific binding of dexamethasone to GA in representative Tand null lymphoblasts at varying steroid concentrations. The free steroidconcentration is determined by subtracting the bound steroid from theinitial amount added to the incubation. Inset, Scatchard plot of thespecific binding curves.

receptor sites of uniform affinity. Similar results were obtamed in all 14 cases where sufficient cells were available toobtaincompletebindingcurves.

Kinetics of the Specific Glucocorticold Binding Reaction. The rates of association and dissociation betweendexamethasone and specific glucocorticoid bindi ng siteswere studied. Chart 3 shows the time course of specificbinding of dexamethasone to GR at 21Â°.It can be seen that,at a concentration of steroid sufficient to saturate the GR,the reaction has approached equilibrium after about 1 hr.Although not shown in this chart, themewas no difference inbinding at 2, 4, 6, or 8 hr. In the binding reaction

Dexamethasone + GR@ )(dexamethasone

specific GA complex)

2nd-order kinetics may apply. If this is the case, a plot oftime versus log@ (unbound dexamethasone concentration/unbound specific GA concentration) should give a straightline. Results are seen in Chart 3, inset, and are consistentwith 2nd-order kinetics in the binding reaction (23). Fromthe slope of the line (r = 0.94) the association rate constant(k) may be calculated to be 2.025 x 106t@ M â€˜.

The dissociation reaction of the dexamethasone-GR complex was also studied. After the GR was allowed to becomesaturated with nadiolabeled dexamethasone, a 100-fold excess of nonnadioactive dexamethasone was added as achase, and specifically bound counts, at 21Â°(Chart 4), demonstrate the reversibility of the cytoplasmic GA-binding meaction. If it is assumed that the dissociation reaction follows1st-order kinetics, than a plot of log11,(bound steroid concentration) versus time should be linear (20), as shown inthe inset of Chart 4. From the slope of this line (r = 0.91),the dissociation rate constant (k2) of 5.33 x 10@ t â€˜isobtained. From the dissociation and association rate conslants, an overall equilibrium (dissociation) constant (k,,) of

160

140

20

AUGUST 1977 2691



E

10C',

@ 60@

4)<

@ 45*I

az

0

>.

4U

U

U,

RATIO OF COMPETITIVE STEROIDS TO H DEXAME1HASONE

Chart5. SpecificityofglucocorticoidbindingtoGAinnulllymphoblasts.The ability of various unlabeled steroids, present in molar excess, to compete with 10' M tritiated dexamethasone is shown.

E

0E

10C',4II-

1000

22,500

20.000

17,500

-J 15,000-JwUU) 12500UiI-U)

@ 10,000

aza 7500-

5000W

2500

0

G. S. K. Yarbro et a!.

â€”@ â€” â€” Tetrahydrocortisol

lla.Cortisol

. â€˜@ -@ -;@A@_@1@@

EsCradiol-@ Tetrahydrocou,sol-â€˜.1

Progesle,one

75@

.#

30@CortisoI

15S..

10 100

RATIO OF COMPETITIVE STEROIDS TO [ â€˜HIDEXAMETHASONE

Chart 6. specificity of glucocorticoid binding to GA in T-lymphoblasts.Theabilityof variousunlabeledsteroids,presentin molarexcess,to cornpete with 10 M tritiatad dexamethasone is shown.

Med@n4,484 â€¢.

. Mean2,538@ Median2173

â€œTâ€•

Chart 7. Correlation between surface markers, ability to stimulate allogeneic lymphocytes in MLC, and specific GA-binding sites in subpopulations ofleukemic lymphoblasts. Mean, median, and range of GA sites per cell areshown for the 3 groups of patients. Steroid concentrations used were 5 timesthe k,, and approached saturation.

p<O.001

Mean10,117

Median7,571 --Mean6,729

phoblasts. Biologically active glucocorticoids, such as dexamethasone and 11@-hydmoxycortisoI, compete with Iabeled dexamethasone. Biologically inactive steroids, suchas 11(EhydnOXycOrhisOl,or the reduced metabolite, tehnahydrocortisol, and the sex steroids, compete little, if at all.

In these experiments, the various unlabeled steroids wereadded to the incubation mixture before the Inhaled dexamethasone was added. While this exaggerates the competition of biologically active glucocorticoids at the variousmolar ratios, it also emphasizes the failure of biologicallyinactive steroids to compete for these binding sites. Thus,binding appears specific for known biologically active glucocorticoids. The curves shown here for null and T-lymphoblasts are virtually identical.

Correlation between Surface Markers and Specific Cytoplasmic GR Binding Sites. Chart 7 compares GA activityin the lymphoblasts from the patients, as described in Table

In the 18 patients in Group 1 with N+ lymphoblasts, GAsites ranged from 4,096 to 21 869 sites/cell, with a mean of10,117 and a median of 7,571 sites/cell. In the 9 Group 2patients with Nâ€”lymphoblasts, GR sites ranged from 2,936to 16,469, with a mean of 6,729 and a median of 4,484 sites/cell. In the 18 Group 3 patients with T-lymphoblasts, GAsites ranged from 0 to 5,887 with a mean of 2,538 and amedian of 2,173 sites/cell.

While there is overlap of the intermediate Group 2 withGroups 1 and 3, there is very little overlap between Groups 1and 3. Fourteen of the 18 patients with T-lymphoblasts hadless than 4000 sites/cell, while none of the 18 patients withN+ blastsand only 3 of 9 patientswith Nâ€”blastshad lessthan 4000 sites/cell.

Using the 2-tailed Wilcoxon rank sum test, each of thegroups is different from the others. Group 1 is different fromGroup2atp - 0.1, Gnoup2 isdifferentfnom Group3atp <0.01 , and Group 1 is different from Group 3 at p < 0.001.

These results were quite reproducible from day to day atvarious cell concentrations within the optimal range, asshown in Table 3 for 4 of the patients in whom tests were

â€œNâ€•.

performed on more than one occasion.There was, within the different groups, some variation in

GA levels with age and with sex. However, these variationswere not consistent and, in most instances, not statisticallysignificant. In Table 4 ihcan be seen that, in the N+ group,patients over 10 years of age had higher levels of receptor(median, 12,480 sites/cell) than those under 10 years (median, 7,571 sites/cell), although not significantly different (p= 0.8). In the Nâ€” group, just the opposite is seen. Those

under 10 years had higher receptor levels (median, 6783sites/cell) than those oven 10 years (median, 3265 sites/




Reproducibility ofresultsGAsites per cell values obtained for the same patient's lympho

blasts when tested on different days and using different cell concentrations between 1 and 3 x 106cells/mI were quite reproducible, as shownhere.Patient

No. of tests GAsites/cellT.P.31,7062,0951,834MA.

21,7962,181J.

B. 22,7462,676C.

M. 5 20,93322,61621,66622,24221,555

Lymphoblastswere isolatedand assayedfor specific cytoplasmicglucocorticoid receptorlevels.Variationof GA levelswith age, within each group, areshown.Total

GR sites/cellno. of No. of

Cell pa- paGroup type tients Age (yr) tients MedianRangeSignificanceâ€•1

N+ <10 12 7,571 4,096-16,23618@ 6 12,480 4,748-21,869NSâ€•

(p0.8)2

Nâ€” <10 6 6,783 3,582-16,4599 >10 3 3,265 2,936â€”4,463p

=0.0483

T <10 4 2,601 160-4,42018@ 14 1,995 0-5,887NS

(r = 0.64)

Glucocorticoid receptor levelsand sexLymphoblasts were isolated and assayed for specific cytoplasmic glucocort

Variation of GA levels with sex, within eac@group, are shown.icoidreceptorlevels.Total

GA sites/cellno.of No.of

Cell pa- paGroup type tients Sex tients MedianRangeSignificanceâ€•1

N+ 18 M 9 5,721 4,906-21,869F 9 8,836 6,365-21548o.os2

Nâ€”@ M 6 4,033 2,936-16,459F 3 5,5704,463-7,995NS'@(p=0.714)3

T 18 M 14 2,174 160-5,672F 4 2,869 0-5,887NS(p>0.56)


cell), and in this group the difference is significant (p =0.048). In Table 5 it can be seen that females had somewhathigher levels of GA than males within each group. However,only in the N+ patients was this difference significant.

Table 3

Glucocorticoid receptor levels and age

a Two-tailed Wilcoxon rank sum test.

b NS, not significant.

Table 5

a Two-tailed Wilcoxon rank sum test.b NS, not significant.

AUGUST 1977 2693

DISCUSSION

From Scatchard analysis of our concentration curves(Chart 2) we conclude that the cytoplasmic receptors we arestudying are of high affinity, and from the competition studies (Charts 5 and 6) we conclude that they are specific forglucocorticoids. While binding capacity differed, there appeaned to be no difference in binding affinity on specificitybetween receptors in T- and null lymphoblasts. Thus, specific cytoplasmic glucocorticoid receptors are present inlarger quantities in null than in T-lymphoblasts, and lymphoblasts with intermediate characteristics have intermediatevalues.

Animal models might lead us to predict whether thisdetermination will be clinically useful for prediching in vivotumor response to steroid. Lampkin and Potter (12) andBaxter et al. (2) have studied steroid-sensitive and steroidresistant lymphoma cell lines from identical parent humors.In such pairs, the resistant sublines bound quantitativelyless steroid than the sensitive sublines; in 1 pair of suchhumors, studied by Baxter (2), the sensitive and the resistantsublines from the same parent tumor had the same numberof steroid receptors. A â€œtransitorysensitive cell line wasalso described (2) in which the cells were transiently inhibiled but nol killed in the presence of giucocorticoid.

Table 4



G. S. K. Yarbro4et a!.

Gehning et a!. (6) described glucocorticoid action on hybnid clones derived from cultured mouse myeloma and lymphoma cell lines. Cells of a mouse myeloma line containedspecific cyhoplasmic glucocorticoid receptors and werekilled by dexamethasone. Cells from a mouse lymphomaline also containing specific glucocorticoid receptors wereresistant to glucocorticoids. Two different hybrid clonesderived by fusion of the resistant lymphoma and the sensilive myeloma contained specific receptors, and both hybnids were killed by dexamethasone.

Thus we might predict that a critical number of cytoplasmic receptors may be required by a tumor line for a steroidresponse to occur. However, since many steps distal to theinitial binding of steroid to receptor may well be defective inresistant cells, it is certainly possible that cells may containapparently adequate, or even increased, amounts of cytoplasmic receptor activity and yet fail to respond to glucocorticoids. We have previously reported this result in bothrodent and human leukemic cells in culture (17).

We have also previously shown that glucocorticoid inhibilion of DNA synthesis in leukemic cells in vitro correlateswith quantitative differences in receptor activity (16). Inlymphoblasts sensitive to glucocorticoid, glucocorticoid meceptor binding increases with increasing dexamethasoneconcentration, and then plateaus. Dexamethasone inhibitshhymidine incorporation at concentrations that closely conrespond to those that saturate receptor sites. A very different pattern of binding and biological effect was seen inlymphoblasts that lacked significant glucocorticoid recepton activity. No concentration of glucocorticoid tested wasinhibitory of thymidine incorporation. All of 28 samplescontaining significant glucocorticoid receptor were inhibited by dexamethasone, and none of 6 lacking significantreceptor activity were so inhibited. Thus, lack of receptoractivity and lack of biological response were correlated.

Cline and Rosenbaum (4, 5) studied unidine incorporationin acute lymphocytic leukemia cells incubated for 24 hr inthe presence or absence of glucocorticoids. They concludedthat in vitro cytotoxicity was partially successful in predicting in vivo steroid effect, defined as 50% reduction in circulating or bone marrow blasts or onganomegaly during a 14-day treatment period, but was not successful in predictingremission induction.

In general, patienhs with T-cell leukemia have a poorerprognosis than those with null cell disease (9, 28, 29), andperhaps the inability of their lymphoblasts to bind steroid tothe same extent as null lymphoblasts is one factor in thispoorer prognosis. However, while poor steroid bindingwithin a general class of lymphoblasls may be partiallyrelated ho the poor prognosis of the group as a whole, a'high level of cytoplasmic giucocorticoid receptors probablydoes not guarantee a good response for any individualpatient,sincestepsdistaltothe initialbindingofsteroidtoreceptor may be defective (14).

It seems clear, however, that specific cytoplasmic glucocorticoid receptors can be conveniently detected on smallnumbers of leukemic lymphoblasts. It appears, from thesestudies, that null lymphoblasts are more likely to have largernumbers of these receptors than T-lymphoblasls. If onebeans in mind that the presence of these cytoplasmic recep

tons may be required, although not sufficient alone, forsteroid effect, the differential capacity of lymphoblasts tobind glucoconticoid may prove useful in designing therapeuhic regimens for the different subcategories of acuhelymphocytic leukemia, particularly if it is possible to show,in studies currently underway, hhat these quanhihativedifferences in receptor activity are correlated with differences inglucocorticoid effect in vivo.

ACKNOWLEDGMENTS

The authors wish to thank Dr. Larry Norton for his assistance with thestatistical analysis.

REFERENCES

1. Aitken, S. C.. and Lippman, M. E. A Simple Computer Program forQuantitation and Scatchard Analysis of Steroid Receptor Proteins. J.Steroid Biochem., 8: 77-93, 1977.

2. Baxter, J. D., Harris, A. w., and Tomkins, G. M. Glucocorticoid Aeceptors in Lymphoma Cells in Culture: Relationship to Glucocorticoid KillingActivity. Science, 171: 189â€”191. 1971.

3. Bowles, C. A., White, G. T., and Lucas, B. Rosette Formation by CaninePeripheral Blood Lymphocytes. J. Immunol., 114: 399â€”402,1975.

4. Cline, M. J. Prediction of In vivo Cytotoxicity of ChemotherapeuticAgents by Their Effect on Malignant Leukocytes In vitro. Blood, 30: 176-188, 1967.

5. Cline, M. J., and Rosenbaum, E. Prediction of in Vivo Cytotoxicity ofChemotherapeutic Agents by Their in Vitro Effect on Leukocytes fromPatients with Acute Leukemia. Cancer Aes., 28: 2516-2521 , 1968.

6. Gehring, u., Mohit, B., and Tomkins, G. M. Glucocorticoid Action onHybrid Clones Derived from Cultured Myeloma and Lymphoma CellLines. Proc. NatI. Acad. Sci. U. 5., 69: 3124â€”3127,1972.

7. Graw, A. G., Herzig. G. P., Eisel, A. J., and Perry, 5. Leukocyte andPlatelet Collection from Normal Donors with the Continuous Flow BloodCell Separator. Transfusion, 11: 94-191, 1971.

8. Halterman, A. H., Johnson, G. E., and Leventhal, B. G. Storage Methodfor Leukemia Blast Cells. Nat). Cancer Inst. Monograph, 37: 149-151,1973.

9. Kersey, J., Nesbit, M.. Hallgren, H., Sabad, A., Yunis, E.. and GajIPeczalsak, K. Evidence for Origin of Certain Childhood Acute Lymphoblastic Leukemias and Lymphomas in Thymus-derived Lymphocytes.Cancer, 36: 1348-1352, 1975.

10. Konior, G. S., Lippman, M., Johnson, G., and Leventhal, B. G. Glucocorticoid Receptors in Childhood Acute Lymphocytic Leukemia (ALL). Clin.Res., 24: 337. 1976.

11. Konior, G. S., Lipprnan, M., Johnson, G., and Leventhal, B. G. Glucocorticoid Receptors in Functional Subpopulations of Acute LymphocyticLeukemia (ALL) Cells. In: Proceedings of the 16th International Congress of Hematology. Tokyo, Japan, 1976.

12. Lampkin, J., and Potter, M. Response to Cortisone and Development ofCortisone Aesistance in a Cortisone-sensitive Lymphosarcoma of theMouse. J. NatI. Cancer Inst., 20: 1091-1111, 1958.

13. Leventhal, B. G., Leung, E., Johnson, G. E., and Poplack, D. E. ERosette Forming Lymphoblasts Fail to Stimulate Allogeneic Donors inMixed Leukocyte Culture (MLC). Cancer Immunol. Immunotherapy. 2:21-25,1977.

14. Lippman, M. E. Steroid Hormone Receptors in Human Malignancy. LifeSci., 18: 143-152, 1976.

15. Lippman, M. E., and Barr, A. Glucocorticoid Receptors in Purified Subpopulationsof Human Peripheral Blood Lymphocytes. J. Immunol., 118:1977-1981, 1977.

16. Lippman, M. E., Halterman, A. H., Leventhal, B. G., Perry, S., andThompson, E. B. Glucocorticoid-binding Proteins in Human Acute Lymphoblastic Leukemic Blast Cells. J. Clin. Invest., 52: 1715-1725, 1973.

17. Lippman, M. E., Perry, S., and Thompson, E. B. Cytoplasmic Glucocorticoid Binding Proteins in Glucocorticoid Unresponsive Human andMouse Leukemia Cell Lines. Cancer Res., 34: 1572-1576, 1974.

18. Lippman, M. E., and Thompson, E. B. The Role of Transcortin in Glucocorticoid Mediated Enzyme Induction: Tyrosine Aminotransferase Induction in Hepatoma Tissue Culture Cells. J. Steroid Biochem.. 5: 461-465,1974.

19. Lohrmann, H. P., Novikovs, L., and Graw, A. G., Jr. Stimulatory Capacityof Human T and B Lymphocytes in the Mixed Leukocyte Culture. Nature,250: 144-146, 1974.

CANCER RESEARCH VOL. 372694



20. Nelson, D. A. Cytomorphologic Diagnosis of the Acute Leukemias. Seminars Oncol., 3: 201-208, 1976.

21. Nelson, D. L., Bundy, B. M., Pitchon, H. E., Blaese, A. M., and Strober,W. The Effector Cells in Human Peripheral Blood Mediating MitogenInduced Cellular Cytotoxicity and Antibody-dependent Cellular Cytotoxcity. J. lmmunol., 117: 1472-1481, 1976.

22. Perry, S., and Yankee, A. A. Transfusion and Preservation. In: S. A.Johnson (ed), The Circulating Platelet, pp. 541-563. New York: Acedernic Press, Inc., 1971.

23. Prutton, C. F., and Maron, S. G. Fundamental Principles of PhysicalChemistry. New York: Macmillan Co., 1944.

24. Raft, M., and Cantor, H. Subpopulations of Thymus Cells and ThymusDerived Lymphocytes, In: B. Amos (ed), Progress in Immunology, p. 83.New York: Academic Press, Inc., 1971.

25. Sacks, K. L., Olweny, C., Mann, D. L.,Simon, A., Johnson, G. E., Poplack, D. G., and Leventhal, B. G. A Clinical Trial of Chemotherapy andRAJI Immunotherapy in Advanced Acute Lymphatic Leukemia. CancerRes., 35: 3715-3720, 1975.

26. 5catchard, G. The Attractions of Proteins for Small Molecules and Ions.Ann. N. Y. Acad. Sci., 51: 660-675, 1949.

27. Segal, S., Cohen, I. A., and Feldman, M. Thymus-derived Lymphocytes:Hurnoral and Cellular Reactions Distinguished by Hydrocortisone. Science,175:1126-1128,1972.

28. Sen. L. , and Borella, L. Clinical Importance of Lymphoblasts with TMarkers in Childhood Acute Leukemia. New EngI. J. Med., 292: 828-832,1975.

29. Tsukimoto, I., Wong, K. Y., and Lampkin, B. C. 5urface Markers andPrognostic Factors in Acute Lymphoblastic Leukemia. New EngI. J.Med.,294:245-248,1976.

30. Weiner, M. S., Bianco, C., and Nussenzweig. v. Enhanced Binding ofNeuraminidase-treated Sheep Erythrocytes to Human T Lymphocytes.Blood, 42: 939-946, 1973.

31. Weissman, I. L. Thymus Cell Maturation: Studies on the Origin of Cortisone-resistant Thymic Lymphocytes. J. Exptl. Med. . 137: 504-510. 1973.

32. Weston, W. L. , Claman, H. N. , and Krueger, G. G. 5ite of Action ofCortisol in Cellular Immunity. J. Immunol.. 110: 880-883. 1973.

2695


AUGUST 1977



1977;37:2688-2695. Cancer Res Geraldine S. Konior Yarbro, Marc E. Lippman, Gloria E. Johnson, et al. Acute Lymphocytic LeukemiaGlucocorticoid Receptors in Subpopulations of Childhood

Updated version

http://cancerres.aacrjournals.org/content/37/8_Part_1/2688

Access the most recent version of this article at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/37/8_Part_1/2688To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]



glucocorticoid receptors in subpopulations of childhood ... · useful in designing therapeutic...

Documents