Annals of Oncology 2: 541-557, 1991.O 1991 Kluwer Academic Publishers. Printed in the Netherlands.

Review

Glucocorticoid receptors in human malignancies: A review

P. Nergaard & H. Skovgaard PoulsenDepartment of Oncology, Rigshospitalet, Copenhagen, Denmark

Summary. The present knowledge of the human glucocorticoid receptor (hGCR) in primary malignancies isreviewed. It is concluded that hGCR is present in a large number of these tissues; in all tissue specimens oflymphoid malignancies and in varying fractions of the different solid tumors. The hGCR functions as a hormonedependent, specific enhancer interacting protein in mediating the considerable effects of glucocorticoids ongrowth regulation, both through stimulation and inhibition of expression of the target genes, including other trans-cription regulation systems. The processes of receptor activation and regulation, as well as the effects of glucocor-ticoids, are tissue-specific. Subjects for future research are proposed: Establishment of more cell lines and animalmodels to extend investigation beyond the present concentration on only a few cell lines, especially CEM-C7,application of 'dynamic' assays to cells obtained from patients, in an attempt to predict development of glucocor-ticoid resistance, and further investigation of the relationships among GCR and growth factors and oncogenes.

Key words: cell lines, glucocorticoid receptor, glucocorticoid resistance, human malignancies, receptor activation

Introduction

The effect of glucocorticoids, both physiological andtherapeutic, have been the subject of research for morethan three decades, and a vast amount of informationhas been generated.

Human and non-human, normal and malignant tis-sues have been studied, and there appears to be onlypartial consistency between the different tissues andspecies with respect to the properties and effects ofglucocorticoids and the glucocorticoid receptor(GCR).

The aim of this review is to describe the presentknowledge of the human glucocorticoid receptor(hGCR) in primary human malignancies.

General features of the hGCR will be considered inthe first part of this review, and hGCR and glucocor-ticoid effects in the various malignant tissues will be re-viewed in detail in the second part. Clinical reports ofvarious treatment schedules which include glucocorti-coids will be described only briefly.

I. The human glucocorticoid receptor

Physico-chemical properties

Human GCR was first described in leukemic lympho-blasts in 1972 by Lippman et al. [1]. The purifiedhGCR is a phosphorylated peptide monomer, with aMW of about 94 Kda [2-6].

Cortisol and various synthetic glucocorticoids, e.g.,

dexamethasone (DEX), bind to one specific, high-affinity binding site per receptor molecule (dissocia-tion constant in the nM range, corresponding to normalphysiological levels of glucocorticoid) [4, 7-9]. Re-cently it has been shown that Cortivazol (CVZ), asynthetic, high-potency glucocorticoid, binds two siteson the human GCR, or on a protein very similar to it[10,11].

Using either affinity chromatography or immuno-adsorbtion with monoclonal anti-receptor antibodies(MoAbs), several in vitro reports have shown thatmolybdate-stabilized, unactivated (i.e., in the absenceof ligand) GCR is part of a heteromer complex with aMW of about 310 Kda [6, 9, 12-15], containing (asmentioned above) a single steroid, binding unit, and amonomer or dimer of Mr 90,000 heat shock protein -HSP-90. In this form the GCR is incapable of bindingtoDNA[16].

Recently Pratt et al. [12] demonstrated that thisheteromer complex is associated with microtubules ofthe cell cytoskeleton.

Structure

By means of limited proteolysis, the rat GCR has beenshown to consist of three domains; steroid-binding,DNA-binding and the immunogenic domain [2,17, 18].In 1985 the gene coding for the hGCR was sequencedby the cloning of GCR cDNA [19], which revealed asimilar domain structure. By somatic cell hybridiza-tion (between GCR-positive human leukemic T-cells,CEM-C7 and GCR-deficient mouse T-cells, EL4) the

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gene was assigned to chromosome 5 [20], and recentlyfurther localized to 5q31 on this chromosome [21].

Hollenberg et al. [19] described two hGCR forms of777 (alpha) and 742 (beta) aminoacids which differ attheir carboxy termini. The alpha-hGCR was found tobe the major form identified in various human celllines, i.e., the GM 637 fibroblast cell line, the IM-9lymphoid cell line and HeLa cells. However the pres-ence of beta-hGCR has not been confirmed by othergroups.

The conclusions from these and other reports [22-24] were that the hGCR consists of a centrally locatedDNA-binding domain, a steroid-binding domain locat-ed in the carboxy terminal and an immunogenic do-main in the amino terminal half of the peptide, thelatter containing antigenic sites most frequently recog-nized by antibodies [2, 3, 7]. A schematic diagram ofthe hGCR is shown in Figure 1.

421 486

KH -2 L

Fig. I. Schematic diagram of human glucocorticoid receptor.Numbers refer to amino acids. A: Steroid-binding domain. B: DNA-binding domain. C: Immunogenic domain.

active transport mechanisms have been described insome systems [2,8,38, 39].

Binding of glucocorticoids to the GCR is non-covalent, and triggers a process through which theGCR converts from its non-DNA-binding to its DNA-binding form. Two models for this process, calledtransformation, have been proposed.

According to the heteromer dissociation model, theabove mentioned HSP-90-GCR-complex dissociateswhen exposed to glucocorticoid, yielding the freeDNA-binding form of the GCR [9, 12-14]. The asso-ciation between GCR and newly synthesized HSP-90has been shown to take place in intact mouse lympho-ma cells incubated in hormone-free medium but not incells exposed to DEX [14, 25]. The observations byGroyer et al. [40] also support this model of transfor-mation, and evidence for association between HSP-90and the steroid-binding domain of non-human GCR,has recently been published [6, 41, 42]. A schematicdiagram of the heteromer dissociation model is shownin figure 2.

L Jc

B

nni i

A

Two regions that block receptor activation havebeen described recently as being located within thesteroid-binding domain [25]. In the immunogenic do-main, two regions were found which are necessary fortranscription activation, but which are not specificallyinvolved in either the steroid- or the DNA-binding[22]. In the rat GRC two signals mediating hormone-dependent nuclear localization of the receptor havebeen mapped and found to be closely associated to theDNA- and steroid-binding domains [26].

The DNA-binding domain is rich in cysteine, lysineand arginine, sequenced in two repeat units that havebeen suggested to fold into loop-structures containing ametal ion, e.g. Zn++. Such spatial structures are consid-ered to intercalate in the DNA [3, 7, 27, 28].

Moreover, the DNA-binding domain shows greathomology to the DNA-binding regions of other humanand non-human steroid hormone receptors [18, 29].Homology is also shown between these DNA-bindingregions and the v-erbA proto-oncogene product [23,29, 30], which is known to be the thyroid hormonereceptor [31, 32].

Receptor activation

The hGCR (as in other species) is located predomi-nantly in the cell cytoplasm in the absence of steroidhormone, as determined by immunocytochemicalmethods using various monoclonal and polyclonal anti-bodies [5, 33, 34-37].

Glucocorticoids probably enter the cell by simplediffusion through the membrane lipid bilayer, although

cB

nn A

Fig. 2 Schematic diagram of the heteromer dissociation model ofGCR transformation. A: Steroid-binding domain. B: DNA-bindingdomain. C: Immunogenic domain. GC: Glucocorticoid. For detailssee text.

The conformational model proposes that the recep-tor transformation is an intramolecular phenomenon:In its non-activated form, the steroid-binding domainrepresses the function of the DNA-binding site. Bind-ing of hormone induces a conformational change whichresults in exposition of the DNA-binding site [2, 25]. Aschematic diagram of the conformational model isshown in Figure 3.

nn

cB

nn1 c c i —

A

Fig. 3. Schematic diagram of the conformational model of GCRtransformation. A: Steroid-binding domain. B: DNA-bindingdomain. C: Immunogenic domain. GC: Glucocorticoid. For detailssee text.

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Gruol and Wolfe propose GCR transformation to bea multistep process [43]. By modified anion-exchangechromatography they identified a variety of (non-human) receptor subspecies with different DNA-bind-ing capacities, following binding of glucocorticoid toGCR.

In vivo, but not in vitro, activation and subsequentnuclear translocation of GCR is induced by both gluco-corticoid agonists and antagonist RU38486 in thehuman leukemic cell line CEM-C7 [44], whereasRU38486 cannot activate GCR in vivo in the humanlymphoblastoid cell line IM-9 [44,45].

These somewhat divergent data imply that the proc-esses of hGCR activation, and of hGCR-DNA bindingdescribed below are not yet completely understood.

Factors other than HSP-90 are involved in regula-tion of GCR activation [12]. Recently two aminophos-phoglycerides with inhibitory action, i.e. 'modulators',were purified from rat liver cytosol [46, 47]. Increasedcellular calcium content [8, 48], elevated temperature[49, 50], and exogenous ATP [50, 51] have been report-ed to stimulate GCR activation. It has been proposedfrom studies of non-human GCR, that the GCR under-go a cycling process modulated by changes in phos-phorylation [51,52].

Upon activation the glucocorticoid-GCR-complextranslocates to the nucleus, traversing the nuclearmembrane probably by a facilitated transport mech-anism [7,26, 53].

Receptor-DNA binding

The hGCR regulates the rate of transcription initiationfrom promoter-sites by binding to specific sequences inthe genome, known as hormone-responsive elements(HRE) or glucocorticoid-responsive elements (GRE)[4 (for a detailed review), 22]. The designations 'posi-tive' GRE and 'negative' GRE are used for GRE'sstimulating and inhibiting expression of target genesrespectively.

The relation between GRE and the promoter seemsto be relatively independent of distance and orienta-tion within the gene [4]. Induction of conformationalchanges in the DNA following GRE activation hasbeen proposed as a mechanism for GRE-promoter as-sociation [54-56]. Sequences thought to modulate theefficiency of the GCR-GRE binding have recently beendiscovered on the two sides of a GRE [57].

GCR-GRE-binding has been shown to be depend-ent upon hormone binding in vivo [58], and in vitro[33]. In contrast, as reported by Willmann and Beato[59] using heat-activated rat GCR, and Rajpert et al.[45] using the human lymphoblastoid cell line IM-9,GCR-GRE-binding takes place in vitro irrespective ofthe presence of hormone, and when the GCR is ligand-ed with the glucocorticoid antagonist RU38486. Thiscontroversy has been explained by the effect of bindingof HSP90 to GCR (described above), which is present

in vivo but not in vitro [29, 40, 45]. Further evidencefor accordance between in vitro GCR-GRE-bindingand in vivo hormone responsiveness is supplied byChandler etal. [54].

The GCR thus function as a hormone dependent,specific enhancer interacting protein. Different GRE'swere all shown to comprise the hexanucleotide se-quence 5'-TGTTCT-3' [4,60].

In particular the long terminal repeat promoter ofthe mouse mammary tumor virus (MMTV-LTR) hasbeen used as a well described glucocorticoid respon-sive enhancer in investigations of expression of gluco-corticoid regulated genes [54, 61-64], f.ex. by recom-bination of various oncogenes and MMTV-LTR andsubsequent transfecn'on of cell lines (mostly non-human). Transcription from the MMTV is stimulatedby glucocorticoids. Also the human metallothioneinHA (Mt-IIa) gene contains a positive GRE [65, 66].

In the non-human cell lines NIH3T3 (mouse fibro-blast), 6m2 (rat kidney) and FTO-2B (rat liver) theexpression from MMTV-LTR and Mt-IIa was shown tobe repressed in the presence of H-ras, v-mos and v-srconcogene products [65-69]. Expression of the normalhuman protooncogene H-ras had no effect on GCRfunction [66]. In normal NIH3T3 cells the level ofnuclear GCR, but not cytoplasmic -, declined in thepresence of p21 H-ras membrane associated oncopro-tein [67], and in 6m2 cells the nuclear retention oftranslocated GCR was reduced in cells expressing thev-mos oncoprotein [70, 71], which is known to becytoplasmic [72]. Very recently Touray et al. [65] re-ported that this effect of v-mos probably is mediated bythe expression of fos nuclear oncoprotein directlyaffecting the translocated GCR. Fos expression hasbeen shown to be stimulated by expression of H-rasand v-mos oncogenes, and evidence for reciprocalrepression between GCR and fos oncoprotein [73, 74]and between GCR and c-Jun oncoprotein [73, 75] hasbeen published. The v-myc oncoprotein was found tohave no inhibitory effect on GCR [67].

Glucocorticoid stimulation of malignant transforma-tion of human cells has been reported. The humanpapilloma viruses HPV 16, 18, 31, 33 and 35 are asso-ciated with squamous cell carcinomas of the humananogenital tract [76]. HPV 16 contains a positive GRE[77] and various authors have presented evidence forcooperativity between these HPV's and activated rasoncogene in inducing malignant transformation andgrowth, only in the presence of GC [78-83]. Theinvolvement of growth factors was investigated in thehuman cervical cancer cell line C4-1 [76], and inras-transformed mouse mammary epithelium cells(NOG-8) [84]. In the latter, DEX induced an increasedexpression of transforming growth factor alpha (TGF-alpha) whereas epidermal growth factor receptor(EGF-r) was not found to be enhanced in either cellline. Ebstein-Barr virus (EBV) located in the genomeof a human lymphoma cell line was reported to contain

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a positive GRE [85]. Expression of EBV was also stim-ulated by TGF-beta alone and additively together withDEX [86].

Glucocorticoids also modulate the expression ofc-myc and c-myb protooncogenes coding for nuclearproteins. Vedeckis et al. [87] showed that TA treatmentof CEM-C7 cells resulted in decrease of c-myc mRNAlevel followed by decrease in the levels of c-myb - andc-Ki-ras mRNAs. A similar effect of DEX on c-mycexpression in correlation with growth arrest in Gl-phase in CEM-C7, but not in glucocorticoid-resistantsubclones of this cell line, was reported in a studywhere the expression of 13 different oncogenes/growthgenes were unaffected by DEX [88]. Maroder et al. [89]recently provided evidence for this effect to be medi-ated at posttranscriptional level through the synthesisof hormone-induced regulatory proteins controllingc-myc transcript stability. Similar results was obtainedthrough studies of non-human cell lines [87, 90-93].

Glucocorticoids exert considerable effects ongrowth regulation of human and non-human malignantcells via the GCR, stimulating transcription of the tar-get genes. Recent investigations of glucocorticoid inhi-bition of gene expression point towards this beeingmediated through secondary regulation as describedabove.

Regulation of GCR and GCR-mRNA levels

Together with reports of GCR autoregulation in non-human malignant tissue [94-97], several studies ofhGCR autoregulation have been published.

Glucocorticoids have been shown to down-regulatethe GCR level in normal human lymphocytes from vol-unteers treated with glucocorticoid [98], in humanleukemia and lymphoma cells from patients treatedwith glucocorticoid [99], in HeLa cells [100] and in vivoin IM-9 cells [45]. In the latter, the effect was obtainedas a consequence of a decreasing GCR half-life.Rosewicz et al. [101] found that DEX treatment re-sulted in decreased glucocorticoid gene transcriptionand GCR-mRNA in IM-9 cells. Similar results wereobtained by Burnstein et al. [102], using COS-1 cellstransfected with an hGCR cDNA expression vector. Inthe latter cells the glucocorticoid antagonist RU 486was also capable of inducing receptor down-regulation,whereas with neither of the ligands was any effect ob-served in cells transfected with cDNAs encodingmutant hGCR, which were defective in either steroid-or DNA binding.

These results indicate that functional steroid- andDNA-binding domains are required for down-regula-tion, in agreement with the observations describedabove concerning oncogene transformed cells.

In contrast, recent studies using the human leukemicT-cell line CEM-C7 demonstrated up-regulation ofGCR and GCR-mRNA initiated as early as 1 h afterDEX treatment, suggesting an additional posttranscrip-tional effect on mRNA and/or translation [35, 103].

Mouse GCR [104], and glucocorticoid-GCR com-plexes of HeLa cells [105, 106] have been shown tobind to small RNA in vitro.

The discrepancies described above have led variousauthors to suggest that GCR regulation is to someextent tissue-specific. Methodological considerationshave been taken into account since recent informationwas obtained using hybridization techniques and im-munocytochemistry instead of a competitive ligand-binding assay.

The GCR level may be influenced by other factorssuch as the cell cycle phase [107] and stage of differen-tiation [108]. In addition measurements of GCR levelsare influenced by several methodological factors [49,109,110], and tumor heterogeneity.

II. GCR in human malignant tissue

Cell lines

Table 1 shows a list of the established human malignantcell lines which have been used for in vitro studies ofglucocorticoid effects and hGCR in the reports re-viewed, all of which involve specific GCRs with highaffinity.

Table 2 lists the human malignant, GCR-positive celllines used for in vivo studies as xenografts on nudemice, some of which are established cell lines andothers tumor specimens from cancer patients trans-planted to the animals.

The effects of glucocorticoids on these cells, listed inTables 4 and 5, will be dealt with below.

Tissue specimens

Table 3 lists hGCR detected in biopsies of variousmalignancies. In melanoma, lung cancer, primary CNS-tumors and sarcoma the most conspicious variations in'receptor-positivity' are seen, whereas lymphoid malig-nancies show consistency, since almost all specimensare receptor-positive.

Breast cancer

The presence of GCR in human breast cancer cells wasfirst described in the MCF-cell line in 1975 by Horwitzetal.[119].

Later, GCR were found in other cell lines (Table 1)as well as in tumor specimens (Table 3). In most reportsof GCR determination, one-half of the tumors werefound to be receptor-positive.

In vitro MCF-7 [118] and ZR-75 [121] cell lines arereversibly growth-inhibited by DEX, reflecting inMCF-7 an inhibition of thymidine incorporation. Inthese and other GCR-positive human breast cancer celllines, DEX was shown to stimulate the binding ofepidermal growth factor (EGF) to its receptor [120].

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Table 1. Established glucocorticoid receptor-positive human malig-nant cell lines, used for in vitro studies.

Table 2. Glucocorticoid receptor-positive human malignant celllines, used for in vivo studies as xenografts on nude mice.

Type Cell line Ref.

Melanoma

Uterine cervix cancer

Breast cancer

Lung cancerSmall cell

SarcomaOsteosarcomaFibrosarcoma

G.-l. cancerColon carcinomaColon carcinoma

Leukemia

ALL

CMLAML

Myeloma

Lymphoma

NKI-26SK-Mel-25Mel-IPC-48M-5 (M-5A)C8146cNEL

NHIK 3025HeLa

C4-1

MCF-7ZR-75-1MDA-231MDA-330G-llT-47DHBL 100

A549COR L103

HT-1080

LoVoH81-4

MOLT-4-FCEM-C7"

Bel3K562KG-1HL-60BRM

HMy2GM4672AOPM-1OPM-2RPMI 8226

Daudi

JijoyeHD-MarEB1-9019RAJIEB4-6161U937U266-B1NAMALVA

111111111111112113, 114, 115

34, 11642, 74, 77, 83, 100, 105,106, 11776

34, 77, 118, 119, 120118, 121118, 120120118120120

122123

124125, 126

120, 127120, 127

110, 1285, 11,20,24,33,36,37,44,50,87,88,89, 103,128, 129, 130, 131, 132,133, 134, 135, 136, 137,139, 140, 141, 142, 143,144, 145, 146147147110, 148149143

150150, 151152152152

86, 110, 146, 147, 153,15488147128, 14686, 128, 146, 153128155128, 146128, 146

1 Including subclones; Cl, ICR 27, 4R4, 3R43, 3R7.

Type Cell line Ref.

Melanoma

Lung cancerAdenocarcinomaLarge cell carcinomaEpidermoid carcinoma

SarcomaLeiomyosarcomaFibrosarcoma

G.-I. cancerColon carcinomaColon carcinoma

Head and Neck cancerSalivary glandadenocarcinoma

NEL 115

SK-LMS-1HT-1080

LoVoH81-4

156156156

157158

127, 156127, 156

HSG 160

EGF is known to stimulate growth of these cell lines[202]. These results therefore suggest that the inhib-itory effect of DEX is not mediated via EGE

Considering the clinical relevance of 'GCR positiv-ity' and glucocorticoid treatment in breast cancer,glucocorticoid treatment in an early autopsy study wasfound to enhance development of metastases [203]. Incontrast, glucocorticoids included in multi-drug regi-mens on an empirical basis enhance response rate in aminority of patients [204]. The correlation betweenGCR content and tumor size, location, clinical stage,menopausal status [180] and disease-free interval [178],was investigated and found to be insignificant in allrespects, in contrast to what is known concerning theestrogen receptor. Correlation has been shown betweenlevels of the different steroid receptors [180,182].

At present, determination of GCR status as a prog-nostic parameter therefore seems to have no relevanceto the treatment of breast cancer.

Lung cancer

Little information has been published about glucocorti-coid, GCR and lung cancer. Specific, high-affinity GCRswere found to be present in the human alveolar car-cinoma cell line A549 [122]. In vitro DEX treatmentcaused growth retardation with inhibition of DNA syn-thesis. In vivo treatment of GCR-positive human ade-nocarcinoma xenografts on nude mice with hydrocorti-sone induced a decrease in tumor size, which was alsoseen following treatment with the potent antiglucocor-ticoid RU 38486 or medroxy-progesterone acetate.Clark et al. [123] investigated the effect of glucocorti-coid on human GCR containing small-cell lung cancer(COR L103), and found that hydrocortisone had noeffect on the proopiomelanocortin (POMC) geneexpression. The latter is frequently used as an indicatorof glucocorticoid-dependent ACTH secretion of thesecells.

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Table 3. Glucocorticoidmalignancies.

receptors in biopsy samples of human Table 4. Reports of glucocorticoid effects on glucocorticoid recep-tor-positive human malignant cells.

Tissue type

Breast carcinoma

Lung carcinomaAdenocarcinomaSquamous cellSmall cell

Colon carcinoma

Gastric carcinoma

Hepatic carcinoma

Malignant melanoma

Renal cell carcinoma

CNS-tumorMeningioma

Glioma

Acoustic neuroma

Sarcoma

Leiomyosarcoma

LeukemiaALLANLL

CLLSezary syndrome

Lymphoma

No. total

11312

329122

8790

67

144

7

25

10

296512

7

195

66

1028

1265

726

8

27364352

7

4642

No.

576

2046

3510

6

19

7

251812

4

154

66710765

498

27364346

5

4641

Positive

50%50%52%23%23%5 1 %

50%7 1 %

7%0%

89%

76%

70%

86%28%

100%

57%

79%80%

100%100%

70%50%

0%58%

100%100%

57%35%

100%

100%100%100%

88%7 1 %

100%98%

Ref.

178*179"180"181"182"183"

179"184*184"184'

127

185'

186"

187-188'189"

179*

190"161b

191"1921

1931

161b

194"+c

161*192"192"

195'196"189"

197b

168"198b

199"166"

200b

201b

Tissue type

Breast

LungAdenocarcinomaAlveolarcarcinoma

Colon

Salivary glandAdenocarcinoma

Melanoma

CNS tumor

SarcomaLeiomyosarcoma

Fibrosarcoma

Glucocorticoid effects

Thymidine incorporationinhibition

Decreased cell divisionProliferation inhibitionEGF - EGF-r bindingstimulation

Decrease in tumor sizeGrowth inhibitionThymidine incorporationinhibition

Proliferation inhibitionThymidine incorporationinhibition

Growth inhibition

Decrease in tumor sizeGrowth inhibition

Arrest in Gl phaseArrest in G2/M phaseDNA synthesis stimulation(4 h incubation)

DNA synthesis inhibition(18 h incubation)

Melanogenesis stimulationTyrosinase activity stimula-tion

L-DOPA incorporationstimnlfltinnolllllUltlllUlJ

Growth inhibition(high dose DEX)

Growth stimulation(low dose DEX)

Growth inhibition (in vivo)Growth stimulation (in vitro)Growth inhibition (in vivoand in vitro, high dose)

Growth stimulation (in vivo,low dose)

Fibronectin biosynthesis

Ret

118

121121120

156122122

127, 159127, 159

160

115111, 112,113,115111111113

113

111114

114

157

157

9797158

158

126

All detected by the radioreceptor method, unless * by electro-phoresis.1 cytosol assay.b whole cell assay.c nuclear assay.

Uterine cervix

stimulation

Growth inhibition 116Growth stimulation 117Arrest in Gl phase 116GCR 'down-regulation' 100Expression of HPV 16 gene 77products

Induction of ras oncogene 83expression

EGF-r level increase 117, 162

LeukemiaALL Cell lysis

Proliferation inhibitionAnest in Gl phaseCholesterol synthesisinhibition

Nucleoside uptake inhibitionGlucose uptake inhibition

103, 139, 140141, 147, 163164133, 138138, 139138

1, 163, 165163

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Table 4. (Continued) Table 5. Reports of glucocorticoid effects on human malignantcells, not assayed for glucocorticoid receptor.

Tissue type

ANLL

Lymphoma

Myeloma

Glucocorticoid effects

Protein synthesis inhibitionGluthamine synthetase induc-tion

Increased GCR-mRNA levelDecreased GCR-mRNA levelGCR 'up-regulation'GCR 'down-regulation'Interleukin-2 productioninhibition

C-myc mRNA suppressionC-myb mRNA suppressionC-Ki-ras mRNA suppression

Colony formation inhibitionGlucose transport stimula-tion/inhibition

Amino acid incorporationstimulation/inhibition

Cell viability/increase/decrease

Cell lysisNucleoside uptake inhibitionExpression of EBVInhibition of interleukin-1expression

Cell killGrowth inhibitionPlasminogen activatorinhibition

Immunoglobulin secretioninhihitionLJLlULMLlwll

Increased GCR-mRNA level

Ref.

138130, 140, 166,137103101

35, 10399138

87, 88, 898787

148168

168

168

146, 169125, 17085,86155

152150, 152150

152

152

Tissue type

LeukemiaALALLCLL

CML

Sarcoma

CNS-lumorAstrocytoma

Uterine cervix

Hepatoma

Colon

Glucocorticoid effects

Mitochondria degenerationDNA fragmentationCell lysisDNA fragmentationNAD- and ATP depletionDNA fragmentation

Increase in size of cell, nucleusand mitochondria

Decrease in number of mitochondria

Growth arrestIncreased differentiationCell surface alteration

Expression of HPV 18 gene productsEnhancement of malignant transformation

ras oncogene

Enhancement of cytochrome P450IA1gene expression

Enhancement of malignant transformationby ras oncogene

Ref.

171172173174174172

124

124

175175175

8079

176

177

AL: acute undifferentiated leukemia. ALL: acute lymphocyticleukemia. CLL: chronic lymphocytic leukemia. HPV: Humanloma virus. CML: chronic myelocytic leukemia.

i papil-

DEX in GCR-positive human colon tumor xenograftson nude mice has also been demonstrated [127,but the clinical implications of these data have m

159],r>t vet

Abbreviations: ALL: acute lymphocytic leukemia. ANLL: acute been investigated,nonlymphocytic leukemia. DEX: Dexamethasone. GCR: Glucocor-ticoid-receptor. EGF: Epidermal growth factor. EGF-r Epidermalgrowth factor receptor. EBV: Ebstein-Barr virus. HPV: Humanpapilloma virus.

Head and neck cancer

The reports of the presence of GCR in lung cancerbiopsy and surgical specimens vary from 0-74%, beinghighest in adenocarcinoma (71%) and lowest in small-cell carcinoma (0%) (Table 3). Bearing in mind thesmall number of specimens evaluated, it is possible thatthese data, together with the above mentioned in vitrodata, could reflect a role for glucocorticoids in the cells,which are thought to be progenitors of adenocarcinomaand alveolar cell carcinoma, but further investigationsare needed.

Gastro-intestinal cancer

Reports of GCR and glucocorticoid effects on humangastro-intestinal cancer are limited to single accounts ofthe demonstration of GCR in specimens of colorectalcancer [127], hepatocellular carcinoma [186] and gas-tric cancer [185].

In vivo, GCR level-dependent growth inhibition by

DEX has been shown to inhibit the growth of humansalivary gland adenocarcinoma cells both in vitro andin vivo on nude mice [160]. A shift of these cells to-wards a more differentiated state, morphological by theformation of duct - like structures and functional bythe expression of epithelial membrane antigen, is alsoinduced by DEX. These effects are probably mediatedvia GCRs, whose presence was established earlier byassaying the cytosol of the cells [160]. Apparently, noclinical reports of hormonal treatment of human headand neck cancer have been published.

Melanoma

Hormonal responsiveness of human melanoma hasbeen predicted on the basis of early clinical data andstudies of non-human melanomas, mostly concerninggonadal steroids reviewed by Neifeld and Lippman[188].

As shown in table 3, different estimates of GCR-positivity have been obtained in biopsy samples ofhuman melanomas.

Various in vitro studies of glucocorticoid effects on

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Table 6. Determinations of glucocorticoid binding sites in humanlymphoid malignancies.

Type

LeukemiaALLALLALLALLALLALLALLT-ALLPre-B-ALLUndiff. ALL

. Common ALL' Null-ALL

Pre-B-ALLT-ALL

- B-ALL' ALL(non-B)

B-ALLAML

- CMLCMLCMLANLLANLLANLLANLLCLLCLLCLL

Lymphoma

Hodgkin'sNon-Hodgkin'sBurkitt's

Source

CEM-C7*PBPB + BMBel3c

PBBMBMBM

BM

BM

K562C

PBBMPB + BMPBPBPBPBPB

PB + BM + LNb

PB + LNHD-Marc

PBDaudic

Bd.-sites per cell

15,000-20,00017,000" (3000-44,000)

7500" (1350-18,700)28,000'+/-1500

4600* (1000-7000)800

45008300" (0-21,900)

19,600" (7000-64,700)23,100" (1100-47,800)21,900" (1200-187,000)

9700" (0-82,500)8100" (0-38,300)4000" (300-26,400)3200" (100-17,100)

18,125" (2250-79,400)5900" (1100-8900)

18,100"(2150-81,300)13,145" (6500-27,450)33,500" + / - 2600

5000" (2600-6300)18,100'(2150-81,300

8800" (4300-28,400)4800" (2000-13,000)7250" (0-15,250)4500"4000-60002100" (500-4500)

3850" (750-13,000)14,500-56,000

4250-0-17,20031,400"+/-2100

3500"30,100'+/- 1800

Ref

129239240147163143143237237237237235235235235223223223223147163198168163243243245163

200128201147243147

All detected by whole cell assay.* Median values.b Five different cell lines.' Cell lines, see Table 1 and text.PB: peripheral blood. BM: bone marrow. LN: lymph node.

established human melanoma cell lines (Table 1) havebeen made. Osman et al. [Ill] measured GCR contentin six cell lines using a whole cell assay, and reportedfour of them to contain specific, high affinity GCR.M-5A cells contained 170,000 binding sites per cell,which is more than is normally found in leukemic cellsknown to be highly sensitive to glucocorticoids [205].Following a 1-h treatment with a pulse dose of DEX,growth inhibition was seen in all GCR-positive celllines, and it was irreversible in M-5A cells with a 40%reduction in cell survival. By flow cytometric analysiscells were shown to be blocked in the Gl phase. Noevidence of cell lysis was seen. Cell differentiationduring growth inhibition was proposed since melano-genesis was stimulated. Largely consistent data, thoughless extensive, have been reported using the NEL [113,114] and C8146c [112] cell lines. In a recent report,DiSorbo described in vivo growth inhibition induced

by the glucocorticoid triamcinolone acetate (TA), onNEL cells injected into nude mice [115]. He also inves-tigated the effect of in vitro treatment with TA onsecretion of the autocrine growth factor of these cells,and glucocorticoid-sensitive secretory proteins regulat-ing the activity of this growth factor. No evidence forsuch an effect was observed.

Clinical reports of glucocorticoid therapy of malig-nant melanoma are sparse. An interesting case reporthas been published, which describes a patient withcerebral melanoma metastases who was treated withDEX together with brain radiation, which resulted inexacerbation of cutaneous metastases. Biopsy speci-mens were assayed for GCR and found to be positive]205'].

CNS tumor

The GCR level has been measured in surgical speci-mens of intracranial tumors by several authors. As re-vealed in Table 3, the results concerning meningiomasare more homogenous (70-100% GCR-positive tu-mors) than those obtained from studies of various typesof gliomas. A source of error which has to be taken intoaccount when assaying the GCR in surgical specimensof brain tumors is the fact that the patients usually aregiven glucocorticoid treatment prior to surgery in orderto reduce intracerebral oedema. Together with theendogenous glucocorticoids released because of anxi-ety and the stress of anesthesia and surgery, the exo-genous glucocorticoids will bind to, and cause trans-location of the receptors to the nucleus. This interfer-ence with the labeled glucocorticoids used in the com-petitive binding assays results in detection of lowerlevels of cytoplasmic GCR [190,194,206].

Glucocorticoids have been known for some years toinhibit growth rate and transition time through the Glphase of non-human glioma cell lines [207-209]. Re-cently similar results have been obtained from studiesof human meningioma [161] and neuroepithelial [175]cell lines. Gibelli et al. [161] examined the effects of invitro DEX treatment on meningioma- and astrocytomacells from tumor specimens grown in culture and foundthat GCR-positive cells responded to high-dose DEXtreatment with growth inhibition. In contrast, low-doseDEX resulted in a significant enhancement of prolifer-ation in some of the cell cultures. Mackie et al. [175]studied cultures of cells established from biopsies ofhuman anaplastic astrocytoma, and reported thatDEX-induced cytostasis of these cells was accompa-nied by cell surface alteration, i.e., a change of pheno-type, in this case towards a higher degree of differentia-tion.

Glucocorticoids are often used to reduce clinicalsymptoms of intracranial tumors and to prevent acutereactions during radiation therapy for these tumors.In order to elucidate the possible interference ofthese agents in the radiosensitivity of the tumors,Benediktsson et al. [210] exposed two human malig-

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nant glioma cell lines to betamethasone prior to radia-tion and found no significant change in radiosensitivity.The above mentioned effects are thought to be due tovascular changes, which decrease the development ofoedemas.

Sarcoma

In tissue samples of human soft tissue sarcomas [189,196] and skeletal sarcomas [195], GCR has been de-tected in cytosols with incidences varying from 35 to100%. The total number of specimens evaluated wasonly 41, which precludes conclusions about the relativeincidences between the various sarcomas.

The human fibrosarcoma cell line HT-1080 hasbeen studied in vitro [125, 126, 158] and in vivo innude mice [158]. In vitro treatment with DEX and TAresulted in growth inhibition and stimulation of fibro-nectin production, a glycoprotein affecting cell adhe-sion known to be decreased in neoplastic cells, f.ex.after transformation by carcinogens and oncogenicviruses [125, 126, 211]. However, it was recentlyshown, using oncogene transformed NIH3T3 mousefibroblasts, that this reversion of morphology inducedby DEX was specific to certain oncogene transformedcells (neu, ras, src and sis) and was independent offibronectin production [212].

In vivo studies revealed a biphasic response to DEXtreatment (as in human meningioma cells, describedabove), with low-dose DEX resulting in an increase oftumor, and high-dose DEX resulting in a significantinhibition of tumor growth. In contrast in vivo treat-ment with low-dose DEX of nude mice bearing thehuman leiomyosarcoma cell line SK-LMS-1 resulted ininhibition of tumor growth in a recent study [157]. Invitro growth of this cell line was stimulated by DEX.

In an early report, in vitro treatment of human-osteosarcoma cells with hydrocortisone was shown toalter mithochondria morphology and number [124].

GCR are thus present in human sarcomas. Gluco-corticoids can alter the cell biology of these tumors invitro and in vivo, but present information points invarious directions. Glucocorticoids are not included inthe established treatment schedules of sarcoma andpresent information is too scarce for any conclusions tobe drawn about their future role.

Thymoma

Non-human thymoma cell lines have been used in stud-ies of GCR function and glucocorticoid resistance[213-216], and glucocorticoids are known to causelysis of non-human thymoma cells. As mentionedbelow, lysis of rat thymocytes has been shown to in-volve extensive DNA fragmentation by an endonuc-lease activated by elevated Ca++ concentration [217,218].

To our knowledge there have been no reports ofGCR determinations in human thymoma.

Dex and prednisone are included in treatmentschedules for human thymoma in which response ratesof up to 50% have been reported [219]. They are alsoapplied as single-agent therapy, and complete remis-sions have been reported [149, 220, 221] in patientswith recurrent thymoma.

Leukemia

A great deal of our present knowledge about the hGCRhas been attained by studying the T-cell-derived acutelymphoblastic leukemia [ALL] cell line CEM-C7 andits glucocorticoid-resistant subclones. CEM-C7 wasfirst characterized in 1977 by Norman and Thompson[141] as being sensitive to glucocorticoid treatmentwhich results in lysis. CEM-C7 cells are arrested in Glphase and protein and cholesterol synthesis is inhib-ited, prior to lysis [138, 139]. Evidence that this cyto-lytic action is a GCR-mediated process has been sup-plied by studies using glucocorticoid-resistant mutantsof CEM-C7 [132, 137] and hybrids of these cell lines[142, 222]. In a recent report, lysis of CEM-C7 isshown to be dependent upon the presence of a func-tional GCR-DNA-binding region [140].

As described above CVZ binds to two sites on theGCR (or a protein immunologically indistinguishablefrom it) in CEM-C7 and IM-9 cells, one with a relative-ly lower affinity which recognizes DEX, and one with a20- to 50-fold greater affinity. In mutant clones ofCEM-C7 resistant to the lytic effects of DEX, CVZrecognizes the high affinity site and causes lysis of thecells. The anti-glucocorticoid RU 38486 is able toblock both binding sites [10, 11].

Another established human T-ALL cell line, Bel3,responds to glucocorticoid with lysis, but in a more'direct' way than CEM-C7, i.e., more rapidly and with-out cell cycle alteration [147]. The cytolytic response inBel3 also seems to be dependent on GCR activation.

As discussed below, lysis of cells of all types ofleukemia isolated from blood samples following invitro DEX treatment, have been shown to be withoutcorrelation to GCR content [163]. Generally T-cells areregarded as being more susceptible to the cytolyticeffect of glucocorticoids than B-cells [223].

The biochemical events leading to cytolysis are notfully understood. Glucocorticoid may act both directlyon the cell and indirectly as, for example, by down-regulation of growth factors. T-cell growth factor(TCGF), also termed interleukin 2, (IL-2) is producedby both glucocorticoid-sensitive and -resistant CEMclones, but this production can only be inhibited byglucocorticoid in cells susceptible to this hormone, i.e.,CEM-C7 [138] and normal human lymphocytes [224].Studies of murine T-cells suggested that DEX inhibitsIL-2 gene transcription rather than the stability of IL-2mRNA [225]. In contrast, Yuh et al. found no effect ofDEX on the expression of an IL-2 gene transfectedinto CEM-C7 [88]. Concerning the direct action,various reports have established the presence of endo-

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nuclease activity. DEX induced DNA fragmentation invitro, in cells isolated from patients suffering fromALL, CML [172] and CLL [174], and in CEM-C7 cells[144,145], in a manner similar to what has been shownin rat- and mouse lymhoid cells, both in vitro [174] andin vivo [226, 227]. Present knowledge of the enzymesinvolved is incomplete and contradictory concerninghuman [144, 145] and non-human cells [217, 218].DNA cleavage and cytolysis in CEM-C7 cells are inde-pendent of cellular calcium concentration, in contrastto what has been shown in rat thymocytes where eleva-tion of cellular calcium activates endonuclease activity.

Other direct glucocorticoid effects have been stud-ied. As described in the first part of this review, DEXcauses suppression of c-myc protooncogene expressionin vitro in correlation with arrest in the Gl phase inglucocorticoid-sensitive CEM cells (i.e., containingfunctional GCR), suggesting that growth arrest in thesecells is regulated in part via suppression of c-mycexpression [87-89]. Similar results have been obtainedstudying mouse lymphoma cells [87,91-93].

In studies of CEM and BRM (myeloid leukemia)cells and resistant subclones, DEX was shown to de-crease membrane fluidity in a dose-dependent mannerin cells containing functional GCR [143]. On the basisof knowledge gained from studies of non-human cells,this effect was suggested to contribute to the cytolyriceffect of glucocorticoids on human leukemic cells.

For many years glucocorticoids have been used inthe treatment of lymphoid leukemia, previously assingle agent therapy, but now always in multi-drug regi-mens [129, 205, 228-230]. Remission rates followingglucocorticoid treatment alone have been relativelyhigh in ALL, especially in childhood ALL, whileANLL is considered not to respond significantly tosteroids.

In vitro several DEX-resistant subclones of CEM-C7 have been identified as a result of both clonal selec-tion and chemical mutagenesis [129, 130, 231, 232].Most of these clones contain immunologically detect-able GCRs, some defective and some functionallyintact, but some are deficient in GCRs. The molecularbasis of glucocorticoid resistance in leukemic cells hasbeen discussed in detail by Stevens et al. [223].

In order to develop a method for predicting re-sponse to glucocorticoids, as in the case of estrogenreceptor determination in breast cancer, several at-tempts to correlate GCR concentration in leukemiccells with glucocorticoid response have been made[147, 148, 163, 168, 197-199, 223, 234-245]. The re-sults of these investigations are contradictory, evenwhen methodological differences and heterogeneity ofspecimens are taken into account [129, 205 (detailedreview)]. The fact that the method is not admitted toclinical use demonstrates its deficiency. In Table 6, a listof glucocorticoid binding site determinations in humanmalignant lymphoid cells from established cell linesand from biopsy specimens is shown, to illustrate thevariations.

In two prospective studies [197, 230], a correlationhas been found between in vivo prednisone response inchildhood ALL and event-free survival following mul-ti-drug induction therapy in patients stratified accord-ing to risk factor.

The value of in vitro glucocorticoid sensitivity deter-mination has also been investigated in childhood ALL,using various assays [246, 247]. The results have beencontradictory. Recently Antakly et al. [33] described anin vitro assay, using CEM-C7 and resistant subclones,where only cells containing functional GCR show in-creased nuclear staining upon incubation with DEX.This method remains to be tested on cells isolated fromleukemia patients.

Myeloma

Glucocorticoids are often included in multi-drug regi-mens for aggressive multiple myeloma [152].

GCR has been identified in myeloma cells in culture[150, 152] and in cells isolated from patients [152].DEX treatment has also been shown to inhibit growthand plasminogen activator produced by myeloma cellsin vitro [150]. However, it was not possible in this studyto completely attribute these effects to GCR. Gomi etal. [152] recently studied 3 myeloma cell lines, one sen-sitive, one resistant and one slightly sensitive to DEX.Specific, high-affinity GCR, responding to heat activa-tion with nuclear transfer, was found to be present in allthree cell lines. In the sensitive cell line OPM-2, Ig(alpha) secretion was reduced, and GCR-mRNA in-duced by DEX, suggesting that the relative resistanceof the other two is caused by post-receptor mech-anisms.

Little is known about the in vivo effect of glucocorti-coids on myeloma cells.

Lymphoma

Most lymphoma types are sensitive to glucocorticoidswhich therefore is used in the primary combinationchemotherapy (reviewed in [248]).

Much of the information about GCR comes fromstudies of non-human lymphoma cells, and the mech-anisms involved in lysis of leukemic cells, as describedabove, have been shown to play a part in lymphoma celllysis [169].

As described above a positive GRE was identified inthe EBV-positive lymphoma cell line Jijoye [85], andDEX stimulates expression of EBV early antigen (EA)in Daudi cells but not in Raji cells [86]. TGF-beta stim-ulates EBV-EA expression in both of these cell linesalone and additively together with DEX.

In the U937 cell line DEX suppress interleukin-1synthesis both on transcriptional and post-transcrip-tional levels [155].

As shown in Table 3, all biopsy specimens from lym-phoma patients in two reports are GCR-positive.

In an in vitro study using cells taken from lymph

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nodes in patients with B-cell lymphomas, the responseto single-agent glucocorticoid therapy was compared tothe GCR concentration, showing significantly higherGCR levels in responders than in non-responders.Nucleoside uptake in lymphoma cells treated withDEX was significantly lower than in lymphocytes fromnormal volunteers. In addition the GCR level wasshown to decrease following glucocorticoid treatmentin vivo [200]. In another report [215], the GCR level inlymphoma cells showed a similar correlation to the re-sponse to in vitro DEX treatment. However, GCR leveldetermination has at present no clinical application,presumably for the same reason mentioned above withrespect to leukemia.

m. Conclusion and future outlook

(1) GCR is present in a large number of human malig-nancies (see Table 3), reflecting its wide distribution innormal human tissue. Likewise, several establishedhuman malignant cell lines are GCR-positive (see Table1). Within the different types of tissue the reports tendto concentrate on the use of relatively few cell lines,especially the lymphoid malignant cell line CEM-C7.There is therefore a need for establishment of more celllines and animal models for in vivo studies, to confirmthe vast amount of information obtained from studiesof these cells.

(2) The GCR functions as a hormone dependent,specific enhancer interacting protein in mediating theconsiderable effects of glucocorticoids on growth regu-lation, both through stimulation and inhibition ofexpression of the target genes, which include othertranscription regulation systems. The processes ofreceptor activation, receptor-DNA-binding and theregulation of GCR levels and transcription are not yetfully understood, and seem to a certain extent to betissue-specific. Both pre- and post-transcriptional regu-lation takes place, and both through direct effects onGCR and on transcription products, e.g., RNA. Still, alot of in vitro data seems not to correlate with in vivoobservations.

(3) The ultimate effects of activation of the recep-tors obviously also reveals a tissue-specific pattern (seeTable 4). In all the cell lines reviewed, glucocorticoidinduces growth inhibition and, in the lymphoid cells,cytolysis. Considering the biochemical events leadingto growth arrest and lysis, evidence for GCR-depend-ent activation of endonuclease activity has been sup-ported by some reports.

(4) Glucocorticoids form part of established treat-ment of the lymphoid malignancies. Prior attempts tocorrelate cellular GCR level with the risk of develop-ment of glucocorticoid resistance have been ambig-uous. An interesting future area is application of 'dy-namic' assays, i.e., measuring only functional GCR,using immunohistochemical methods, on cells obtained

from patients, in an attempt to predict development ofglucocorticoid resistance.

(5) The fact that glucocorticoids induce growth ar-rest in Gl -phase in other malignancies may suggest abeneficial therapeutic result of glucocorticoids giveneither alone or together with other cytostatic agents.

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Received 12 April 1991; accepted 12 June 1991.

Correspondence to:Peter Nergaard, M.D.Department of OncologyRigshopitaletBlegdamsvej 9DK-2100 Copenhagen 0 , Denmark

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