[CANCER RESEARCH (SUPPL.) 49, 2266s-2274s, April 15, 1989]

Oncogene Mediated Repression of Glucocorticoid Hormone Response Elements andGlucocorticoid Receptor Levels1

Rolf Jaggi,2'3 Wolfgang Hock,4 Andrew Ziemiecki,3 Roman Kleinen// Robert Friis,3 and Bernd Groner4

Ludwig Institute for Cancer Research, Bern Branch, Inselspital, CH-3010 Bern, Switzerland

Abstract

We have previously described the inhibition of glucocorticoid-depend-

ent transcription from the mouse mammary tumor virus long terminalrepeat promoter by products of the H-ros and \-mos oncogenes. We havestudied the effects of conditional oncogenes on expression of glucocorti-coid-dependent indicator genes. Expression of the glucocorticoid-depend-

ent transcription of the tyrosine aminotransferase gene was monitored inFTO-2B rat hepatoma cells during M, 21,000 protein (p21) H-ras

induction. A strong transcriptional repression of the tyrosine aminotransferase gene followed p21 I I-ruv expression. The sequences in a glucocor-ticoid-dependent promoter which are responsible for the oncogene-me-

diated repression could be localized to the glucocorticoid response element; a construct in which a 15-base pair glucocorticoid response elementwas inserted 5' of the thymidine kinase promoter exhibited the oncogene-

mediated repression of transcription. We observed a strong repression ofglucocorticoid-dependent promoters and promoter constructs not only inthe presence of p21 H-ras and p37 \-mos but also with p60 v-src. p57 v-

myc, however, had no effect. Oncogene expression is not a sufficientprerequisite for an initial repression of glucocorticoid hormone-dependentgene transcription, since even in the presence of constitutive!}' high levels

of oncogene product a transient stimulation of glucocorticoid-dependent

gene expression was found. Protein synthesis inhibition experimentsrevealed that no hormonal!) induced cellular protein is needed for theoncogene-mediated repression. It seemed reasonable that this phenome

non might reflect oncogene effects on the glucocorticoid receptor. We,therefore, made measurements of the glucocorticoid receptor protein. Inthe presence of glucocorticoid hormone the receptor translocated rapidlyfrom the cytoplasm to the nucleus. In normal NIH 3T3 cells, after 24-h

treatments the nuclear receptor levels had declined to about 50% of thosedetermined at 2 h and in the presence of p21 H-ras they declined to 15%.The levels of cytoplasmic receptor were not affected by p21 Il-ruv

expression.

Introduction

The identification of oncogenes as the genetic basis of cellulartransformation has spurred the hope of understanding thedetermining biochemical and cell biological events of cancer.Oncogenes might be considered as switches in the hierarchy ofgenetic pathways which govern growth and differentiation programs. The description of the biochemical actions of oncogenesand the merging of signals into common pathways will determine whether a generalized description of transformation willbecome possible. The pleiotropic effects of oncogenes on thecell phenotype are most likely due to changes in gene expression. Oncoproteins might modulate promoter activities of genesimportant for growth control (1). Examples of the interferenceby oncogene products or signals emanating from them withtranscriptional activity of specific genes have been described,e.g., for \-src (2), SV40 T antigen (3), the adenovirus Eia andElb genes (4, 5), the c-myc gene (6), and the H-ras (7, 8) and

1Presented at the Symposium on "Glueocorticoid Receptors: Evolution, Structure, Function and Abnormalities," July 14 and IS, 1988, Osaka, Japan.

3To whom requests for reprints should be addressed.3 Present address: Institut fürklin.-exp. Tumorforschung, Universität Bern,

Tiefenaustrasse 120, Tiefenauspital, CH-3004 Bern, Switzerland.' Present address: Friedrich Miescher Institut, P. O. Box 2543, CH-4002

Basel, Switzerland." Present address: Pathologisches Institut der Universität Zürich,Schmelz-

bergstrasse 12, CH-8006 Zürich,Switzerland.

\-mos (9) oncogenes. Insights into the components constitutingthe transcriptional machinery make it likely that regulatoryelements such as enhancer sequences and specific proteinsinteracting with these sequences are responsible for the efficiency with which genes are transcribed (10). These componentscould also be the targets which mediate the action of oncogenes.

We have investigated the proviral promoter of MMTV6 andpreviously reported a negative effect of the \-mos and H-rasoncogene expression on the transcriptional efficiency withwhich the MMTV promoter is utilized (7, 11). The MMTVLTR contains a promoter element, the hormone response element, which makes the transcription dependent on glucocorticoid hormone (12, 13). The hormonal effect is mediated via ahigh affinity receptor molecule which interacts with specificDNA sequences in the vicinity of the transcriptional start inthe MMTV LTR. Hormonal regulation can therefore be considered as the activation of specific gene transcription by receptor interaction with a hormone response element (14-16). TheHRE was shown to contain four glucocorticoid receptor bindingregions comprising the hexanucleotide sequence 5'-TGTTCT-3' (17) and a sequence homology to the binding domain fornuclear factor 1 at position -80 to -60 (18, 19). Combination

of the HRE with heterologous promoters (e.g., the promoter ofthe thymidine kinase gene or the mouse a-globin gene) showedthat the hormonal inducibility can be conferred, i.e., the constitutive level of tk or a-globin gene transcription can be enhancedin the presence of glucocorticoid hormone (12). Deletion ormutation of individual hormone-binding sites or the recognitionsignal for nuclear factor 1 leads to a strong reduction of theinducibility by glucocorticoid hormone /// vivo, indicating asynergistic rather than an additive cooperativity between individual elements within the HRE (12, 13).

Jantzen et al. (20) have shown that a glucocorticoid hormoneresponse element is present in the tyrosine aminotransferasegene which is located within a 2.5-kilobase sequence flankingthe coding region to its 5' side. These authors could show that

the glucocorticoid inducibility can be conferred to a chimericTAT-CAT gene construct containing the regulatory sequencesof the TAT gene and the coding sequences of the bacterialchloramphenicol acetyltransferase gene.

Here we report that the interference of glucocorticoid-regu-lated MMTV LTR transcription by oncoproteins is a processwhich is observable in different cell types, extends to otherglucocorticoid-regulated genes and gene constructs, and is exerted by different classes of oncogenes. The role of the glucocorticoid hormone receptor and its binding site in the promoterregion at the GRE in bringing about the observed repression oftranscription by oncogenes is emphasized by experiments withGRE-gene constructs. The glucocorticoid receptor is involvedin the oncogene-mediated repression. We report that normalNIH 3T3 cells exhibit a 50% reduction in amounts of immu-

' The abbreviations used are: MMTV, mouse mammary tumor virus; HRE,

hormone response element; LTR, long terminal repeat; TAT, tyrosine aminotransferase gene; CAT, chloramphenicol acetyltransferase gene; GRE, glucocorticoid response element; HSP, heat shock protein; p21, M, 21,000 protein (otherproteins are similarly designated); Tk, thymidine kinase; GR, glucocorticoidreceptor; SDS, sodium dodecyl sulfate.

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nologically detectable nuclear glucocorticoid receptor 24 h afterglucocorticoid hormone addition. NIH 3T3 cultures expressingp21 H-ras show a 85-90% loss of nuclear receptor.

Materials and Methods

Plasmids and Cells. Construction of MMTV LTR H-ras (A), LTRH-ras (N), LTR v-mos (7), and the HRE a-globin plasmid (12) has beendescribed. The TAT-CAT plasmid consisting of a 3-kilobase Sa/I-fis/N 1fragment which contains the hormone-responsive element and thetranscriptional start site (up to +65) and the coding sequence of theCAT gene has been described by Jantzen et al. (20). GRE-Tk-CAT and4xGRE(-37)Tk-CAT constructs have been described by Strähleet al.(21). The HSP H-ras (A) construct consisting of a 1.35-kilobase Smal-BamHl promoter region of the human HSP 70 gene (22) was recom-bined with the 4.8-kilobase BamHl fragment containing the codingregion of the H-ras (A) gene (7) at position +155 with respect to thestart site of transcription. NIH 3T3 cells or FTO-2B cells (23) werecotransfected with 5 Mgplasmid DNA and 500 ng pSV2neo (24) or500 ng pY3 (25) according to the protocol of Wigler et al. (26).Transfected cell lines were grown in Dulbecco's medium containing

10% fetal calf serum and 500 Mg/ml G418 or 200 Mg/ml hygromycin Band single cell clones were isolated. The glucocorticoid response element in the MMTV LTR or the HRE was induced with 10"' M

dexamethasone for the times indicated. Heat shock treatment of thecells was for l h at 42°C.Protein synthesis was blocked by the addition

of 3 Mg/ml cycloheximide to the growth medium. In transient transfec-tion experiments IO6 cells were transfected with 5 Mgplasmid DNA

(26) 48 h before isolating the RNA.Analysis of RNA. Total cytoplasmic RNA was isolated from cells (7)

stimulated with 10 " M dexamethasone or after heat shock treatment

as indicated in the text. Total RNA was analyzed in a RNase protectionassay (7, 27) using the following SP6 probes: A 261-base pair Smal-Xbal fragment of the H-ras (A) gene (positions 1644-1905) spanningthe H-ras exon 1 (133 base pairs), the 1.3-kilobase MMTV LTRcontaining 112 nucleotides from the RNA initiation site to the BamHl-site and the 347-base pair EcoRl-Pvull Tk-CAT promoter fragment

(indicative fragments 185 and 210 N).Retroviral Infection. Transfected cells (as indicated in the text) were

infected with retroviruses expressing the following oncogenes: Harveyv-ras; \-mos; avian O K-10 v-myc, and \-src. For this purpose the naturalretrovirus isolates of Harvey murine sarcoma virus (28) and Moloneymurine sarcoma virus (29) were used, as well as retroviral constructsbased on murine leukemia virus for expression of avian OK-10 v-myc(30) and v-src.7 All infections were performed using 8 jig/ml final

concentration of Polybrene (Aldrich), and single transformed cloneswere isolated from soft agar suspension culture for the studies describedhere.

Nuclear Run-on Transcription. In vitro transcription assays in isolatednuclei were performed essentially as described (7, 31): 0.5 x IO6nucleiwere incubated for 20 min at 26°Cin 50 ¿d25 IHMTris (pH 7.4); 150

mM KC1; 5 mM MgCl2; 1 mM ATP, CTP, GTP each; 5 HIMdithiothre-itol; 20 units of the RNase inhibitor RNasin (Promega Biotec); and 50/iCi [32P]UTP (400 Ci/mmol; Amersham). After DNase I and protein-

ase K digestion, the samples were phenol/chloroform extracted andethanol precipitated. Unincorporated [32P]UTP was separated from

RNA by the spin column procedure (32). The RNA was hybridizedovernight to 0.5 ^g electrophoretically separated DNA restriction fragments transferred to Gene Screen Plus filters (DuPont) in a final volumeof 1 ml in the presence of 10% dextran sulfate at 65°C(Fig. 4; Table

1). In the case of RNA made in nuclei from FTO-2B cells, nitrocellulosefilters containing the gene-specific DNA restriction fragments [6.6-kilobase BamHl H-ras (33) 1.55-kilobase Rl/Hindlll 5' TAT (34),0.95-kilobase Hindlll 3' TAT, 2-kilobase BamHl genomic fragments

of the H2-L? gene (35), and a 0.6-kilobase Pstl actin complementaryDNA fragment (36)] were prepared and hybridized at 40°Cfor 3 daysin a total volume of 1 ml containing 50% formamide and 3-5 x IO6cpm [32P]RNA, 4 x standard saline-citrate, 5 x Denhardt's solution,

0.2% SDS, 0.1% sodium pyrophosphate, 100 Mg/ml tRNA (yeast), and300 mg/ml calf thymus DNA. All the filters were washed 5 times for20 min in 0.1 x standard saline-citrate 0.2% SDS at 65°C.The

transcriptional rates were quantitated by densitometric scanning of theautoradiograms and determination of the signals of the hormone-inducible gene relative to the signals obtained from actin (Fig. 1, A andB) or H-2 transcription (Fig. 1C; Fig. 4; Table 1).

Production of Polyclonal Antibodies Directed against the GR. A partialcDNA clone representing amino acids 440-795 of the GR was expressed in Escherichia coli. It contains the domains for DNA-bindingand hormone interaction. In this construct, the partial GR-codingregion was recombined with the bacterial T7 promoter (a gift of S.Rusconi, Zurich, Switzerland) and introduced into E. coli strainBL21(DE3) (37). Addition of isopropyl-/3-D-thiogalactopyranidose tothe bacteria led to the synthesis of T7 RNA polymerase and as aconsequence to the synthesis of a M, 40,000 truncated GR polypeptide.The M, 40,000 protein was purified by SDS-polyacrylamide gel electro-phoresis and electroelution from the gel and used for immunizingrabbits. The antibodies obtained reacted with human, rat, and mouseglucocorticoid receptor in immunoprecipitations and immunoblots.8

Cells were homogenized in 10 mM NaCl- 1 mM EDTA-10 mM Hepes,pH 7.8 (38), and the nuclei were prepared by centrifugation at 800 x gfor 5 min. Cytoplasm was cleared by centrifugation at 100,000 x g for60 min. The nuclear protein was extracted in the same buffer supplemented with 0.5 M NaCl for 60 min at 0°Cand centrifugation at

100,000 x g for 60 min. Cytoplasmic protein or nuclear protein wasseparated by polyacrylamide gel electrophoresis, transferred to a nitrocellulose filter, and probed with the anti-GR antibody.

Results

The p21 H-ras (A) and p37 \-mos Oncoproteins Cause aRepression of the Hormonal Enhancement of Tyrosine Amino-transferase Gene Transcription. We have shown that the activated human H-ras [li-ras (A)] and the viral mos [v-mos] oncogenes exert a negative effect on the glucocorticoid hormone-dependent transcription of the MMTV LTR (7). In order toinvestigate whether the transcriptional repression effect is apeculiarity of the MMTV promoter in NIH 3T3 cells or if itextends to other glucocorticoid hormone-regulated genes andother cell types, we analyzed the hormonal regulation of theTAT gene in the presence and absence of oncoproteins. TheTAT gene is expressed in parenchymal liver cells and cyclicAMP and glucocorticoid hormone cause an increase in thetranscriptional rate (34). We have analyzed the effects of oncogenes on the glucocorticoid hormone induction of the TATgene in the rat hepatoma cell line FTO-2B. This cell lineexpresses TAT and responds to glucocorticoid hormone induction (23).

LTR H-ras (A) DNA was transfected and stably integratedinto FTO-2B cells. The transcriptional rates of the LTR H-ras(A) and the endogenous TAT gene were measured in isolatednuclei after glucocorticoid hormone treatment of the cells. Fig.\A shows an autoradiogram of nuclear RNA labeled in an invitro run-on experiment after hybridization to filter-bound H-ras, TAT, H2, and actin gene sequences. Since the filter-boundDNA is present in great excess the intensity of the hybridizationsignal is proportional to the transcriptional rates. FTO-2B cellstransfected with LTR H-ras (A) DNA were induced for 0 (Lane1), 1 (Lane 2), 3 (Lane 3), 8 (Lane 4), and 24 (Lane 5) h withdexamethasone and the transcriptional rates of the LTR H-ras(A), the TAT (present as a 5' and a 3' specific fragment), theH2, and the actin genes were determined. Induction of LTR H-ras and TAT transcripts was observed from a very low orundetectable level in the absence of hormone (Lane 1) to a

7 B. Vennström, unpublished observations. *W. Hock et al., manuscript in preparation.

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Fig. l. Transcriptional rates of transfected and endogenous genes in FTO-2B and in NIH 3T3 cells. Transcriptional rates were measured in FTO-2B cells stablytransfected with a MMTV LTR H-ras (A) construct (A), in untransfeeted FTO-2B cells (B), and in NIH 3T3 cells containing a stably integrated MMTV LTR v-mosconstruct and a transiently transfected TAT-CAT construct (C). FTO-2B cells were stimulated with dexamethasone for 0, 1, 3, 8, and 24 h (Lanes 1-5, in A and B)and the 32P-labeled RNA synthesized from isolated nuclei in vitro was hybridized to nitrocellulose filters to which were bound the complete H-ras gene, 5'- and 3'-specific genomic TAT sequences, the H-2L1 gene, and an actin-specific complementary DNA fragment (for details see "Materials and Methods"). Transfected NIH

3T3 cells were stimulated for 0, 1, 2, and 6 h with dexamethasone and the rate of transcription for the moj gene (Lanes I, 4, 7, and 10 in Q, the CAT gene (Lanes 2,5, 8, and //) and the H-2 gene (Lanes 3, 6, 9, and ¡2)was measured in isolated nuclei. At the bottom of each autoradiogram the densitometric quantitation of the ras-(left columns, A) and TAT-specific transcription (A and B) relative to the actin-specific transcription are shown. The mos-specific (left columns in C) and the CAT-specific signals (right columns in C) are shown relative to the constant H-2 signal.

maximal level after 3 h (Lane 3). The transcriptional rates ofthe LTR H-ras (A) and the TAT gene decreased 8 and 24 hafter hormone treatment (Lanes 4 and 5). The expression ofthe H2 and the actin genes was rather constant over the timecourse observed. In Fig. IB the transcriptional rates of theendogenous TAT, H2, and actin genes were measured in untransfeeted FTO-2B cells. Three h after hormone administration a maximal rate of transcription of the endogenous TATgene was seen (Lane 3). After 8 and 24 h only a moderatedecrease in the rate of TAT gene transcription was observed(Lanes 4 and 5). The maximal level of TAT transcription waslower in the untransfeeted FTO-2B cell population than in theLTR H-ras (A)-containing cells. The analysis of individualFTO-2B cell clones transfected with MMTV LTR constructsalso revealed variations in the maximal rate of LTR transcription 3 h after hormone administration. The transcription ratesof the LTR H-ras (A) and the 5' region of the TAT genes

relative to the actin gene were quantitated by densitometricscanning of the autoradiograms and are shown as a percentageof maximal transcription occurring after 3 h of hormone stimulation. The transcriptional rate of the TAT gene in the presence of the p21 H-ras (A) oncoprotein was repressed to about5-10% of the maximal level (Fig. IA). The nontransfected FTO-2B cells showed a reduction to only about 70% of the maximallevel. A similar decrease in the rate of MMTV LTR transcription, independent of oncogene action, was observed in NIH3T3 cells after prolonged hormone stimulation (7).

This result indicates that the H-ras-mediated repression oftranscription is not restricted to genes under the control of theMMTV LTR. Transcriptional effects are also found for theglucocorticoid-regulated, endogenous tyrosine aminotrans-ferase gene. The oncoprotein effect is not restricted to fibro-blasts since the TAT gene and the transfected MMTV LTRsequences are affected in the hepatoma cell line.

In order to show the effect of p37 v-mos on the TAT promoterwe used a chimeric TAT-CAT gene construct and transiently

transfected it into NIH 3T3 cells which contained a stablytransfected LTR \-mos gene (7). The cells were induced withdexamethasone for 0, 1,2, and 6 h and nuclei were prepared.Nuclear run-on RNA was hybridized to filter-immobilized v-mos, CAT, and H2 DNA fragments. Fig. 1C (Lanes 1, 4, 7, and10) shows that LTR v-mos transcripts were induced and repressed as described earlier (7). H2 transcripts (Lanes 3, 6, 9,and 12) were not affected by the accumulation of p37 v-mos.TAT-CAT transcription (Lanes 2, 5, 8, and 11) followed apattern very similar to that of LTR v-mos transcription. Afterthe initial induction (1 and 2 h, Lanes 5 and 8) the transcriptionrate was much reduced after 6 h of hormonal stimulation (Lane11). We suggest that this reduction is caused by the concomitantaccumulation of p37 v-mos and mediated by a mechanismsimilar to that of the repression of the MMTV LTR. NormalNIH 3T3 cells expressing no oncoproteins showed a similarinducibility of a transiently transfected TAT-CAT construct asLTR v-/nos-containing cells but no apparent reduction of thehormone-induced TAT-CAT transcription after 6 h of treatment (data not shown). These experiments show that the transcriptional repression of the TAT gene is not restricted to theH-ras (A) gene but that it can also be exerted by the v-mos gene.They also show that transient transfection procedures intomouse fibroblasts are suitable to visualize the oncogene effect.

p21 H-ras (A) Modulates the Expression of MMTV LTRmRNA but Not the Initial Stimulation by Glucocorticoid Hormone. Transcriptional repression of the glucocorticoid hormone-regulated MMTV LTR was studied in a situation wherethe expression of the transforming p21 H-ras (A) was uncoupledfrom glucocorticoid regulation. For this purpose we transfectedNIH 3T3 cells containing a MMTV LTR indicator gene withthe HSP H-ras (A) gene construct. In this construct the codingregion of the activated H-ras gene is subjected to the transcriptional control of the promoter of the human heat shock protein70 (22). In these cells it was possible to accumulate p21 H-ras(A) either before or after the stimulation of MMTV LTR

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transcription by glucocorticoid hormone. As an indicator geneconstruct we used a MMTV LTR H-ras (N) gene. We haveshown previously that the nontransforming p21 H-ras (N)protooncogene product does not inhibit the glucocorticoid hormone-dependent transcription (7). When the cells were culturedat 37°Cin the absence of glucocorticoid hormone the hsp 70

promoter and the MMTV LTR are transcribed at a very lowlevel (Fig. 2, Lane 1). The cells showed a normal, nontrans-formed phenotype. We measured the concentration of H-ras(exon 1 probe) and LTR H-ras (N) mRNA (LTR probe) in aRNase protection assay. Heat shock treatment (42°C,1 h) led

to a transient accumulation of HSP H-ras (A) mRNA after 3 h(Fig. 2A, Lane 2, probed with H-ras exon 1). Twelve h after theheat shock treatment no HSP H-ras (A) mRNA could bedetected in the cells; however, high levels of p21 H-ras (A)persisted for at least 24 h after a single heat shock treatmentand the cells showed a transformed phenotype for 5 to 7 days.Heat shock treatment of the cells at 48-h intervals maintainedthe transformed phenotype and allowed the cells to form colonies in soft agar (data not shown). The half-life of p21 H-rashas been determined to be about 20 h (39).

Sixteen h after p21 H-ras (A) induction by heat shock treatment the MMTV LTR was stimulated for 0, 6, 12, and 24 h

(Fig. 2A, Lanes 3-6) by the addition of glucocorticoid hormoneto the cells. In both the presence (Lane 4) and the absence (Lane7) of p21 H-ras (A) a similar level of MMTV LTR mRNA wasmeasured 6 h after hormone addition. Twelve and 24 h afterhormone stimulation a rapid decrease in the amount of MMTVLTR mRNA was observed in p21 H-ras (A) containing cells(Lanes 5 and 6) whereas no or only a slight decrease of MMTVLTR mRNA was found 24 h after hormone stimulation in thesame cells but without heat shock treatment (Lane 8). Heatshock treatment of NIH 3T3 cells containing a MMTV LTRgene construct but no HSP H-ras (A) construct had no effecton the inducibility and steady state level of MMTV LTRmRNA (data not shown).

Glucocorticoid hormone stimulation of HSP H-ras (A)- andLTR H-ras (N)-transfected cells resulted in a high level ofMMTV LTR mRNA after 24 and 30 h (Fig. 2B, Lanes 2 and3), compared to a very low level in untreated cells (Fig. IB,Lane 1). Heat shock treatment 18 h after hormone administration induced a dramatic decrease of MMTV LTR mRNA whichbecame apparent 12 h after p21 H-ras (A) induction (Fig. 2B,Lane 5). Hormone-treated cells and cells cultured in the absenceof hormone accumulated similar amounts of HSP H-ros mRNAafter heat shock treatment (data not shown); i.e., no effect of

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heat shock- 3 1622 2840 - - - - - 6 12

hrs dex -6 12 24 6 24 - 24 30 24 30Fig. 2. Glucocorticoid hormone and p21 H-ras (A) modulate the level of MMTV LTR mRNA. NIH 3T3 cells were stably transfected with a MMTV LTR H-ras

(N) and a HSP H-ras (A) gene construct. The effect of p21 H-ras (A) accumulation on MMTV LTR initiated RNA was measured in response to dexamethasone (dex)and the heat shock induction of HSP I l-ra.v (A). Ten fig total cytoplasmic RNA were analyzed in a RNase protection assay using probes specific for 11-ra.\. exon 1(133 N, A, Lanes 1 and 2) or the MMTV LTR (112 N; A, Lanes 3-8, and B, Lanes ¡-5).In A, RNA was analyzed before (Lane 1) and 3 h after heat shock treatment(Lane 2) for the presence of H-ras mRNA. Sixteen h after p21 H-ras (A) induction by heat shock treatment (Lanes 3-6) or in the absence of heat shock treatment(Lanes 7 and 8) the cells were treated with dexamethasone for 0 (Lane 3), 6 (Lanes 4 and 7), 12 (Lane 5), and 24 h (Lanes 6 and 8) before the RNA was isolated andprobed for LTR-specific transcripts. In A, 18 h after the addition of dexamethasone to the culture medium the synthesis of p21 H-ras (A) was induced by heat shocktreatment. Six h (Lane 4) and 12 h (Lane 5) later the RNA was analyzed. Hormone-treated cells kept at 37°Cfor 24 (Lane 2) and 30 h (Lane 3) were analyzed forLTR-specific transcripts, //pall-digested pBR322 DNA was end-labeled and used as marker fragments (sizes in base pairs).

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dexamethasone on the heat shock response was found. In theseexperiments we have measured the levels of mRNA by RNaseprotection assays. We have shown previously that the level ofMMTV LTR mRNA in NIH 3T3 cells is modulated by onco-genes at the transcriptional level (7).

In NIH 3T3 cells expressing the transforming H-ras (A)oncogene under the control of the human heat shock promoterHSP 70, the expression of p21 H-ras (A) and the MMTV LTRcan be stimulated independently either by culturing the cells at42°Cfor l h or by stimulation with glucocorticoid hormone,

respectively. We have shown that initial stimulation of theMMTV LTR promoter by glucocorticoid hormone is unaffected by the presence or absence of p21 H-ras (A) in the cells.Accumulation of transforming p21 H-ras (A) either before orafter stimulation of the LTR by hormones has the same effect.The amount of MMTV LTR-derived transcripts decreases to avery low level in the cells within 12-24 h.

The Hormone Receptor-binding Site Mediates OncogeneRepression of Hormonal Induction. A combination of glucocorticoid receptor binding experiments to the MMTV LTR or theTAT promoter and functional assays of artificial promoter geneconstructs has led to the precise definition of the recognitionsequence for the receptor and the sequence requirements fortranscriptional induction. A minimal GRE 15 base pairs longhas been described (21). A gene construct comprising a GREplaced upstream of the thymidine kinase (Tk) promoter anddirecting the synthesis of CAT RNA was used to correlate thetranscriptional repression effect of oncogene products on theglucocorticoid receptor binding site.

To evaluate the effect of the v-mos oncogene on the GRE-Tk-CAT gene, we used NIH 3T3 cells stably transfected withthe MMTV LTR \-mos construct and introduced the GRE-Tk-CAT gene by transient transfection. The transcription of theMMTV LTR \-mos and the GRE-Tk-CAT genes was simultaneously induced by the addition of glucocorticoid hormone,and the levels of MMTV LTR v-mos and GRE-Tk-CAT mRNAwere quantitated 0, 2, and 16 h after hormone addition. Fig.3A, Lanes 1-3, shows the transcriptional induction and repression of the MMTV LTR-initiated RNA. Fig. 3B, Lanes 1-3,shows that the transiently transfected GRE-Tk-CAT RNA appeared and declined with very similar kinetics. A strong signalof 185 nucleotides, indicative of the GRE-Tk-CAT transcript,was observed only 2 h after hormonal stimulation. The sametranscriptional pattern of induction and repression of MMTVLTR (Fig. 3C, Lanes 1-3) and Tk-CAT RNA (Fig. W, Lanes1-3) was exhibited by cells which were transfected with aconstruct containing 4 instead of 1 GRE located 5' of a trun

cated Tk promoter. In this construct the Spl binding sequenceof the Tk promoter was deleted.

NIH 3T3 cells stably transfected with a HRE a-globin geneconstruct and no oncogene construct were transiently transfected with the GRE-Tk-CAT. Addition of hormone to thesecells resulted in an induction of globin mRNA after 2 h (Fig.3E, Lane 2) and a sustained expression after 16 h hormonetreatment (Fig. 3E, Lane 3). Tk-CAT transcripts showed asimilar pattern of expression (Fig. 3F, Lanes 1-3); i.e., thedecrease of mRNA in the absence of oncogene expression ismuch slighter than in its presence. Actin mRNA levels werenot affected by oncogene expression and hormone treatment(Fig. 3, lower panels).

The delimitation of glucocorticoid receptor binding to theGRE and the conferrai of hormonal inducibility to heterologouspromoters have led to "minimal" constructs such as the GRE-Tk-CAT gene. The effect of the \-mos oncogene on the tran-

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Fig. 3. The transcriptional repression effect of p37 v-mos is mediated by theGRE. NIH 3T3 cells stably transfected with a LTR v-mos (A-D) or a HRE a-globin construct (E and F) were transiently transfected with a GRE-Tk-CATconstruct (A, B, E, and F) or a 4xGRE(-37)Tk-CAT construct (C and D). Thecells were stimulated with dexamethasone for 0 (Lanes /), 2 (Lanes 2), and 16 h(Lanes 3) and total cellular RNA was prepared. RNA (5 ¿tg)was probed with aSP-LTR probe (A and C, indicative fragment 112 N), a SP-a-globin probe (E,indicative fragment 133 N). RNA (40 Mg)(B, D, and F) was probed with a SP-Tk-CAT probe (indicative fragments 185 and 210 N). RNA (5 fig) was separatedon a 1% agarose gel, blotted to a nitrocellulose filter, and hybridized with anactin-specific probe (lower panels). P, SP-Tk-CAT probe (347 nucleotides); M,//pall-digested pBR322 marker.

scription of this construct makes it likely that the glucocorticoidreceptor itself plays a role in the oncogene-induced pathway.

Oncogene Repression Is Exerted by the H-ras, v-mos, and v-src but Not the \-myc Proteins. Sequence delimitation of thefunctionally relevant region within the MMTV LTR responsible for glucocorticoid hormone induction of transcription hasshown that a purely regulatory element (HRE) is located atposition -236 to -59 with respect to the LTR RNA initiation

site (12, 13). We have investigated the effects of four differentoncogenes on the hormone-dependent transcription of a functionally neutral HRE a-globin gene construct stably transfectedinto NIH 3T3 cells. Transcriptional rates of the HRE a-globingene were measured in cells infected with the \-ras or v-mosoncogene-expressing retroviruses at various times after hormone addition to the growth medium. The transcriptional ratesof the H-2 gene family which is independent of oncogeneexpression were also determined and used as an internal standard. The HRE a-globin transcription was expressed as percentage of maximal expression (Table 1). The transcriptional ratesof the HRE a-globin construct were initially induced and returned to a basal level 16 h after hormone addition. We haveextended our analysis to \-src, a member of the tyrosine kinasegene family, and \-myc, which codes for a protein localizing tothe nucleus. Hormonal inducibility and transcription of theHRE a-globin gene were measured in cells infected with achimeric murine leukemia retrovirus containing and expressingthe \-src gene of the chicken Rous sarcoma virus or the v-mycgene of the chicken OK-10 virus. In a p60 v-src-expressing cell

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ONCOGENE MODULATION OF HORMONE ACTION

Table 1 NIH 3T3 cells stably Iransfected with a HRE a-globin gene constructand infected with retroviruses expressing Harvey v-ras, v-mos, v-src, or v-myc

oncogene!The cells were stimulated with dexamethasone and the nuclei were processed

in an in vitro run-on analysis. The RNA was hybridized against a-globin- and H-2-specific DNA. The intensity of the hybridized signal was determined by densi-tometric scanning of the bands. The numbers (%) are given as the ratio ofmaximum stimulation of HRE a-globin versus stable H-2 expression.

% of maximum expression(HRE o-globin/tf-2) at

following times afterstimulation with dexamethasone

Virusmos

rassrcmycOh7.1

6.31.49.33h100

100100

48.78h14.5

59.821.362.016

h5.3

6.12.5

100

clone, the hormonal induction of the HRE a-globin gene wasalso observed 3 h after dexamethasone addition. Eight and 16h after hormone stimulation it declined to a low level. In p57v-wyc-expressing cells hormonal induction but no transcrip-tional repression was observed (Table 1). H-2 gene expressionwas rather constant during the course of hormonal induction.

The results shown in Table 1 distinguish the effect which theproducts of the v-ras, the v-mos, and the v-src oncogenes exerton HRE a-globin transcription from the effects of the v-myconcogene. These results were confirmed by the analysis of NIH3T3 cells transfected with the M MTV LTR H-ras (N) geneconstruct. In these cells the coding region of the nonactivatedp21 H-ras protooncogene was subjected to the control of thecomplete M MTV LTR promoter. Expression of p21 H-ras (N)only weakly interferes with LTR transcription (7). When thesecells were infected with the v-ras-, v-mos-, v-src-, or v-myc-expressing retroviruses a similar time course of induction andrepression was observed for the MMTV LTR as described inTable 1 for the HRE a-globin gene (data not shown).

The experiments shown in Table 1 confirm the observationsmade in Fig. 2,4. A high level of oncogene expression is not asufficient requirement to repress the hormonal induction. Thetranscriptional repression effect on the enhancement of HREa-globin transcription was observed only after an initial induction. This induction occurred in the continuous presence of thev-ras, the v-mos, and the v-src proteins.

Oncogene Expression Predisposes NIH 3T3 Cells to the Transcriptional Repression of the Hormone Response Element. Thetranscriptional repression effect caused by the expression of thev-ras, the v-mos, and the v-src oncogenes acts on the MMTVLTR and on the HRE or GRE gene constructs. This observationmakes it likely that components and steps of the hormonalinduction mechanism are affected. The subcellular locations ofthe p21 v-ras, the p37 v-mos, and the p60 v-src oncogeneproducts make it unlikely that a direct interaction between theHRE DNA sequence and these proteins occurs but that theoncogene effect is mediated via cellular components. Proteinsynthesis inhibition experiments were used to gain additionalinsights. We have shown previously that oncogene expressionis required for the repression effect (40). If protein synthesiswas inhibited by cycloheximide simultaneously with the induction of the MMTV LTR v-mos construct by dexamethasone,the rate of LTR initiated v-mos transcription increased and norepression of transcription was observed after 5 h when theprotein synthesis was blocked (40). The accumulation of p37 v-mos in the absence of cycloheximide resulted in the transcriptional repression of the LTR (7). This experiment confirmedthe role of p37 v-mos in the repression process. It left open thepossibility of additional cellular gene products induced as a

consequence of p37 v-mos expression or glucocorticoid treatment.

In cells highly expressing the p21 v-ras gene product we showthat the hormonal induction and the transcriptional repressionoccur both in the absence or presence of protein synthesisinhibitors. For this purpose NIH 3T3 cells transfected with theHRE a-globin construct were infected with a p21 v-ras-express-ing retrovirus (Fig. 4). Stimulation of the cells for 1 or 2 h withdexamethasone led to an increased transcription of the HREa-globin in the absence (Lanes 3 and 7) and presence of cycloheximide (Lanes 5 and 9). Eight h after hormone administrationthe transcription of the HRE a-globin was repressed in theabsence (Lane 11) and in the presence (Lane 13) of cycloheximide. The rate of H-2 gene expression (Fig. 4, even numbers)was rather constant during the hormonal stimulation and cycloheximide treatment.

The experiment shows that no hormonally induced cellulargene functions are required to exert the repression effect. Because the half-life of the p21 H-ras protein is about 20 h (39),

8 h after blocking protein synthesis by the addition of cycloheximide to the growth medium about 75% of the p21 shouldremain in the cells. This is sufficient to mediate the observedrepression effect. The ras oncogene product causes a predisposition to repression which takes effect only after the activationof a HRE by the activated glucocorticoid receptor.

Oncogene Expression Leads to a Reduction of GlucocorticoidReceptor Concentration after Hormone Treatment. Our experiments with different glucocorticoid-regulated genes and promoter constructs indicate that the effects of oncogenes on thehormonal regulation of transcription are mediated by the receptor-binding sites. It is therefore likely that oncogene effectsconcern the glucocorticoid receptor.

We have analyzed the glucocorticoid receptor levels in NIH3T3 cells transfected with the HSP H-ras (A) construct as afunction of p21 H-ras (A) expression. For this purpose we haveraised antibodies against a glucocorticoid receptor fragment.The carboxy-terminal fragment of the rat glucocorticoid receptor (amino acid positions 440-795) was expressed in bacteriaand the truncated receptor protein was used as an antigen forimmunizing rabbits. The monospecific antiserum recognizedcytoplasmic and nuclear forms of the glucocorticoid receptorin NIH 3T3 cells in immunoblot experiments (Fig. 5). Similaramounts of GR were detected in the cytoplasm of NIH 3T3cells before and after heat shock, i.e., in the absence andpresence of p21 H-ras (A) (Fig. 5/4, Lanes 1 and 2). Little GRwas detected in the nucleus of these cells (lanes 3 and 6). Twoh after hormone addition the GR was translocated to thenucleus (Lanes 4 and 7). The amount of GR detected in thenuclei of p21 H-ras-expressing cells (Lane 7) was reduced toabout 50% when compared to the nontransformed cells (Lane4). Twenty-four h after hormone addition, in nontransformedNIH 3T3 cells the GR concentration dropped to about 50% ascompared to the level observed after 2-h hormone treatment.

12 3456 7 8 9 10 11 12 13 14

Fig. 4. v-ras oncogene expression predisposes NIH 3T3 cells to the transcriptional repression of the hormone response element. HRE a-globin-containingNIH 3T3 cells infected with a raj-expressing retrovirus were stimulated withdexamethasone for 0 (Lanes I and 2), l h (Lanes 3-6), 2 (Lanes 7-10) and 8 h(Lanes 11-14) in the absence (Lanes 3, 4, 7, 8, II, 12) or presence of cycloheximide(Lanes 5, 6, 9, 10, 13, and 14) and the in vitro synthesized RNA from isolatednuclei was hybridized to 0.5 >ig filter-immobilized a-globin and H-2L''-specificDNA fragments. Uneven numbers, hybridization to a-globin-specific DNA; evennumbers, hybridization to H-2Li-specific DNA.

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ONCOGENE MODULATION OF HORMONE ACTION

1 2 345678

III+ HS

171}I^02 24 0 2 24HD

HS

kd 180 -

116 -84 -

48 -

36 -

BV. 100

80

60

40

20

0

Fig. 5. p2l H-ras causes a reduction of nuclear GR concentration. NIH 3T3cells stably transfected with a HSP H-ras (A) and a LTR H-ras (N) constructwere stimulated at 42'C for l h (Lanes 2, 6-8) or grown at 37'C (Lanes I, 3-5).

A, 16 h after heat shock treatment the cells were stimulated with dexamethasonefor 0 (Lanes 1-3, 6), 2 (Lanes 4 and 7) and 24 h (Lanes 5 and Õ).Cytoplasmicprotein (Lanes I and 2) and nuclear protein were prepared and 25 *ig proteinwere separated electrophoretically, transferred to nitrocellulose, and probed withan anti-GR antibody. In B, the densitometric quantitation of the nuclear GRbands from three individual experiments is shown.

This ligand-induced down-modulation of the GR has beendescribed in several systems (41-44) and was also found innontransfected NIH 3T3 cells. It will be described in greaterdetail elsewhere.7 It is probably the reason for the reduced

transcriptional stimulation observed in cells after prolongedhormone treatment (see Fig. IB, Lane 5; Fig. 3, E and F, Lane3). The ligand-induced down-modulation was enhanced in p21H-ras (A)-expressing cells (Fig. 5, Lane 8). Only about 10% ofthe GR found in non-heat-shocked cell nuclei after 2 h ofhormone treatment was present in nuclei of heat-shocked cells24 h after hormone addition. Heat shock treatment of NIH3T3 cells containing no oncogene construct did not affect thetranslocation of GR to the nucleus (data not shown). It hasbeen shown earlier that intracellular receptor concentrationslimit the glucocorticoid-dependent enhancer activity (45). It istherefore reasonable to conclude that the combined effects ofligand-induced down-regulation of the GR concentration andoncogene enhancement of this process result in a GR levelwhich is no longer capable of mediating transcriptional induction.

Discussion

Modulation of transcriptional efficiency of the TAT gene,the MMTV LTR, and HRE or GRE promoter constructs canbe achieved in two ways. Addition of glucocorticoid hormoneto the growth medium of rat hepatoma FTO-2B cells or NIH3T3 cells transfected with the chimeric construct results in anenhanced utilization of the promoters and an increased rate ofspecific RNA synthesis. Expression of the ras, mas, and srconcoproteins in the same cells reverses the effect of glucocor

ticoid hormones and reduces the promoter utilization to a lowlevel, while the myc oncogene is without effect. The transcriptional repression is accompanied by a reduction in GR levels.

One gene and three chimeric gene constructs are shown hereto be subject to the oncogene effect. The expressions of theTAT gene and the MMTV LTR in FTO-2B cells were testedin the absence and the presence of the I I-ra.voncogene product.A TAT-CAT chimeric construct and a GRE-Tk-CAT constructwere tested in the presence of the \-mos oncogene product inNIH 3T3 cells. The MMTV LTR and a HRE a-globin constructwere studied in the presence of the \-ras, v-mos, and \-srconcogenes in NIH 3T3 cells.

The level of p21 H-ras was determined in the various ras-expressing cells; 16 h after heat shock treatment or dexamethasone stimulation the cells contained similar levels of p21 H-ras- as v-ras-transformed cells (data not shown). The level ofH-ras protein is still sufficient to mediate the repression effect

8 h after the inhibition of protein synthesis by cycloheximideand 30 h after a single heat shock treatment. The transcriptionalrepression is specific for the regulation exerted by the glucocorticoid receptor. The a-globin transcription is not dependentupon HRE function and glucocorticoid hormone action, as isthe MMTV LTR. A low constitutive expression can be observedin the absence of hormone. The return to the uninduced levelof transcription of the HRE a-globin construct upon hormoneaddition and oncogene expression argues that the transcriptionmachinery is not affected in a global manner (Table 1). Otherunaffected genes (e.g., 11-2, actin) allow a similar conclusion.

The glucocorticoid receptor undergoes a hormone binding andan activation step before it assumes the role of a transcriptionfactor (14). These steps might include a conformational alteration or a loss of interaction with proteins masking the DNA-binding ability, possibly HSP 90 (46). The glucocorticoid receptor may have to be activated before it can become the substrateof a modifying enzyme, e.g., a protein kinase which mightinactivate its transcriptional role (47). This possibility is supported by our observation that the high expression of oncoproteins is not sufficient to repress the initial hormonal inducibilityof the MMTV LTR. Shortly after hormone addition to H-ras-, v-mos-, and v-sre-expressing cells, the transcriptional rate

of the MMTV LTR is enhanced and MMTV LTR mRNAaccumulates in the cells. The oncogene effect occurs late in thehormonal response; i.e., the transcriptional repression is complete 8 to 16 h after hormone stimulation. In LTR H-ras-transfected cells the synthesis of H-ras mRNA and p21 H-rasbegins very soon after stimulation, with the result that there isno apparent delay in the process of MMTV LTR repression inthese cells when compared to cells constitutively expressingp21 H-ras. In nontransfected FTO-2B cells and in NIH 3T3cells transfected with a nontransforming LTR H-ras (N) construct, a reduction of hormone-induced transcription to 60-70% was observed after 24 h. This process has been termed"down-modulation." It is possible that the oncogene-mediated

repression (to 5-10%) is functionally related to the down-

modulation process. Modification and inactivation by an oncogene generated signal could precede the increase in degradation of the GR.

An important conclusion which can be drawn from the experiments reported here concerns the specificity of oncogeneaction. Oncogenes from four different classes were investigatedwith respect to their effect upon glucocorticoid hormone action.The raíoncogenes, usually activated by point mutations, encodeA/r 21,000 proteins which bind guanine nucleotides, haveGTPase activity, share sequence homology with G proteins,

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ONCOGENE MODULATION OF HORMONE ACTION

and are associated with the plasma membrane. These propertiesindicate that the ras family of proteins might participate insignal transduction across the membrane (48). The \-ras andthe H-raî(A) oncogenes have the same repression effectswhereas the nonactivated form [H-raî(N)] does not (7). The v-mos product is a M, 37,000 protein with cytoplasmic localization (49). It can act as a cyclic AMP-independent serine/threonine protein kinase (50). The product of the v-src gene isa M, 60,000 phosphoprotein which has intrinsic tyrosine kinaseactivity and is associated with the plasma membrane. Phospho-rylation sites and states of phosphorylation might be crucial inthe activation process (51). Although of different origins (rat,mouse, and chicken), different biochemical properties, and different cellular locations, all three oncogenes are capable ofefficiently transforming mouse fibroblasts in culture. The signals generated by these oncogenes might be different at theirpoints of departure but could feed into common pathways. Oneof the consequences of these signal chains is the cellular transformation; another is the transcriptional interference with glucocorticoid hormone action. It is conceivable that both eventsare related. The absence of the repression effect in FTO-2Bcells and the repression of hormonal responsiveness in H-ras-expressing FTO-2B cells might indicate that the process of livercell transformation yielding the FTO-2B cell line does notinvolve an oncogene of \-ras-, v-mos-, and v-src-like activity.

The oncogene which does not influence the hormonal regulation is the v-myc oncogene. For these experiments retroviralvector constructs were used expressing the v-myc oncogene(30). It codes for a A/r 57,000 protein localizing to the nucleusand is associated with small nuclear ribonuclear protein particles (52). Less well defined than for H-ras, v-mos, and v-srconcogene expression are the phenotypic consequences of mycgene activation. The NIH 3T3 cells infected with murine leukemia retrovirus encoding v-myc in this study had a morphologically transformed phenotype. Our experiments indicate thatthe influence on gene expression and on the correspondingcellular phenotype exerted by the myc oncogene is differentfrom those exerted by the H-raî,v-mos, and v-îrconcogenes. Areflection of this observation might be the noninterference ofmyc protein with the hormonal regulation of MMTV LTRtranscription.

Acknowledgments

We thank Dr. G. Schützand Dr. W. Schmid (Heidelberg, Germany)for the TAT-CAT and GRE Tk-CAT plasmids, Dr. G. U. Ryffel(Karlsruhe, Germany), for FTO-2B cells, Dr. H. Ponta (Karlsruhe, Germany), for the HRE a-globin gene, and Dr. B. Wu (Evans-ton, IL), for the human HSP 70 gene. Dr. W. Ostertag (Hamburg, Germany) provided a stock of Harvey murine sarcoma virus andDr. B. Vennström(Heidelberg, Germany), provided the murine leukemia virus constructs containing the avian OK-10 v-myc and \-src genes.We thank Dr. F. W. Studier (Brookhaven, NY), for the T7 RNApolymerase expression vectors and bacteria and Dr. S. Rusconi (Zurich, Switzerland), for the plasmid T7 (440-795). We thank A. Schläfliand R. G. Müllerfor excellent technical assistance. We are grateful forthe editorial assistance of M. T. Diabatéand C. Wiedmer and thegraphical presentation by J. Grünigand A. Rohner.

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