Mithramycin inhibits SP1 binding and selectively inhibits transcriptional activity of the dihydrofolate reductase gene in vitro and in vivo

Mithramycin Inhibits SP1 Binding and Selectively Inhibits Transcriptional Activityof thq Dihydrofolate Reductase Gene In Vitro and In VivoScott W. Blume,* Richard C. Snyder,* Ratna Ray,* Shelia Thomas,* Charles A. Koller,* and Donald M. Miller**Departments of Internal Medicine, Biochemistry, and Comprehensive Cancer Center, University ofAlabama at Birminghamand Birmingham Veterans Administration Medical Center, Birmingham, Alabama 35294;and tLeukemia Branch, M. D. Anderson Hospital, Houston, Texas 77030

Abstract

The promoter of the human dihydrofolate reductase (DHFR)gene contains two consensus binding sites for the DNAbindingprotein Spl. DNAse protection and gel mobility shift assaysdemonstrate binding of recombinant Spl to both decanucleo-tide Spl binding sequences which are located 49 and 14 basepairs upstream of the transcription start site. The more distal ofthe two binding sites exhibits a somewhat higher affinity forSpl. The G-C specific DNAbinding drug, mithramycin, bindsto both consensus sequences and prevents subsequent Spl bind-ing. Promoter-dependent in vitro transcription of a DHFRtem-plate is selectively inhibited by mithramycin when compared tothe human H2b histone gene. A similar effect is also noted invivo. Mithramycin treatment of MCF-7 human breast carci-noma cells containing an amplified DHFRgene induces selec-tive inhibition of DHFRtranscription initiation, resulting in adecline in DHFRmRNAlevel and enzyme activity. This selec-tive inhibition of DHFRexpression suggests that it is possibleto modulate the overexpression of the DHFRgene in metho-trexate resistant cells. (J. Clin. Invest. 1991. 88:1613-1621.)Key words: DNAbinding drug - methotrexate resistance * geneamplification

Introduction

Dihydrofolate reductase (DHFR)' is the product of a "house-keeping" gene, which is transcribed constitutively (1), althoughits expression is modulated by a number of physiologic andenvironmental factors (2). DHFRgene amplification and asso-ciated overexpression is an important mode of in vitro and invivo methotrexate resistance (3). The human DHFRgene pro-moter contains two "G/C box" sequences (4), consensus deca-nucleotide binding sites for the transcriptional regulatory fac-tor Sp 1 (5). Mutagenesis studies of the hamster DHFRpro-moter demonstrate that the Sp 1 binding sequences arenecessary for transcriptional activity (6). In vitro transcriptionstudies have confirmed the necessity of Spl for DHFRpro-moter activity (7).

Mithramycin (Plicamycin) is a G-C specific DNAbindingdrug (8, 9) which selectively inhibits transcription of genes,

Address correspondence to Donald M. Miller, M. D., Department ofInternal Medicine, Division of Hematology/Oncology, University ofAlabama, 1918 University Blvd., BHSB288, Birmingham, AL 35294.

Received for publication 25 January 1990 and in revised form11 July 1991.

1. Abbreviation used in this paper: DHFR, dihydrofolate reductase.

The Journal of Clinical Investigation, Inc.Volume 88, November 1991, 1613-1621

such as the human c-myc gene, which have G-C rich promotersequences (10, 1 1). Mithramycin induces myeloid differentia-tion of leukemic blasts, both in vivo (in leukemic cells of cer-tain patients with blast phase chronic granulocytic leukemia)and in vitro (12). This differentiation is accompanied by a dra-matic decrease in the level of expression of the c-myc and c-ablprotooncogenes (13), both of which have G-C rich promoters.Mithramycin prevents the binding of Spl and other proteins bythe c-myc P1 and P2 promoters (14). It also blocks Spl bindingto its consensus binding sites in the SV40 early promoter, pre-venting promoter activity of this sequence (15).

The present work was undertaken to determine whethermithramycin binding to the G-C rich regulatory sequences ofthe DHFRpromoter prevents Spl binding to the 5' flankingregion of this gene, resulting in inhibition of its expression incells with DHFRgene amplification. In vitro experiments indi-cate that both mithramycin and the transcriptional regulatoryfactor Spl bind specifically to the two G/C box sequences inthe human DHFRpromoter. Mithramycin binding preventssubsequent Sp I binding to these sequences resulting in inhibi-tion of promoter-dependent in vitro transcription. Mithramy-cin treatment of methotrexate resistant human breast carci-noma cells in culture induces a decrease in DHFRtranscrip-tion resulting in a fall in DHFRmRNAlevels and DHFRenzyme activity. These experiments indicate that Spl bindingis necessary for DHFRtranscription initiation and that interfer-ence with Spl binding to the DHFRpromoter results in selec-tive inhibition of DHFRtranscription.

MethodsPreparation of the labelled DHFRpromoter fragment. The 367-bpAvall restriction fragment containing the DHFRtranscription startsite and the sequence immediately upstream was isolated from the1.8-kb EcoRI human DHFRclone (pDHFRI.8) (16). This DHFRpromoter fragment sequence was subcloned into the SmaI site in vectorpGEM3Zto create pDHFR.37. The 186-bp Not I-EcoRl digestionproduct of the AvaII subclone was 3' 32P end labelled on the DHFRcoding strand for use in DNAseprotection and gel mobility shift assays.A 1 18-bp human H2b promoter fragment was used as a control (17).This fragment contains no G-C rich protein binding sites and has fullpromoter activity.

Protein and drug binding assays. The recombinant Sp l polypeptidewas prepared from pSp 1-5 16C (a gift of R. Tjian, Department of Bio-chemistry, University of California, Berkeley, CA) as described (18)after expression in BL-21 Escherichia coli. For DNAse 1 protectionassays, varying concentrations of the Sp 1 preparation were allowed toequilibrate with 40-100,000 cpm of the 186-bp labelled promoter frag-ment in the presence of nonspecific competitor DNA(poly d[l-C], 100,qg/ml) for 30 min on ice. Other reaction conditions were: Tris-Cl pH8.0,25 mM; MgCI2, 6.5 mM;EDTA, 0.5 mM;DTT, 0.5 mM;KCl, 50mM;ZnCl2, 0.5 mM;and glycerol, 10%. After the incubation, DNAse I(Boehringer-Mannheim Biochemicals, Indianapolis, IN), 20 U/ml in10 mMMgCl2, 5 mMCaC12, was added and limited digestion allowed

Mithramycin Prevents Spi Binding to the Dihydrofolate Reductase Promoter 1613

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to proceed for 30 s on ice. Samples were then analyzed on an 8 Murea,8%polyacrylamide sequencing gel.

For drug binding experiments, mithramycin (Sigma Chemical Co.,St. Louis, MO)was allowed to equilibrate with the labelled DNAfrag-ment in the presence of 3 mMMgCl2 at 370 for 30 min before digestionwith 5-7.5 U/ml DNAse 1. For gel mobility shift assays, SpI bindingreactions were carried out as above; Ficoll dye was added, and sampleswere analyzed on a nondenaturing 5%polyacrylamide gel. For samplesin which DNAwas exposed to both mithramycin and Spl, drug bind-ing conditions were achieved first, after which buffer, competitorDNA, and Spl were added and samples incubated under the appro-priate conditions for protein binding.

In vitro promoter-dependent transcription. The 1.8-kb EcoRl hu-manDHFRfragment or its appropriate restriction fragment was usedas a template for in vitro transcription assays. The 1 18-bp H2b pro-moter fragment was used as a control. HeLa whole cell extract wasprepared according to Manley et al. (19). Template (0.5-1.0 pmol) wasfirst incubated in the presence or absence of mithramycin and 3 mMMgCl2 at 370C for 30 min. The standard 20-,ul transcription reactioncontained 12-!d whole cell extract, template, 0.5 mMof each ATP,CTP, and GTP, 0.05 mMUTP, 2 mMcreatine phosphate, and 4 ,Ci[a-32P]UTP (400 Ci/mmol; Amersham Corp., Arlington Heights, IL).Transcription reactions were allowed to proceed at 30'C for 90 min,then were terminated by the addition of 200 gl of a solution of 8 Murea, 0.5% SDS, and 10 mMEDTA. After extraction with phenol/ch-loroform, 120 Ml of a solution of 7 Murea, 0.35 MNaCl, 10 mMTris-HCl (pH 7.9), 10 mMEDTA, and 0.5% SDSwas added along with20 g carrier tRNA. Samples were again extracted with phenol/chloro-form, precipitated with ethanol, and resuspended in 240 Ml of 0.2%SDS, 0.3 Msodium acetate (pH 5.2), ethanol precipitated, and ana-lyzed on an 8 Murea, 5%polyacrylamide sequencing gel. The HaeIIIrestriction fragments of PX174 (Bethesda Research Laboratories,Gaithersburg, MD) and the BamHl/Sspl restriction fragments of the1.8-kb EcoRl DHFRclone were labelled and used as molecular weightmarkers.

Inhibition of DHFRtranscription in vivo. A methotrexate-resistantderivative of the MCF-7 (20) human breast carcinoma cell line, was agenerous gift of K. Cowan, Clinical Pharmacology Branch, NationalCancer Institute, Bethesda, MD. For nuclear runon transcription ex-periments, cells were plated at low density (2.25 X 106/75 cm2-flask)and allowed overnight incubation for adherence. Media was changed

and mithramycin (Miles Laboratories, Inc., Elkhart, IN) added at theappropriate concentration for the desired duration of treatment. Afterdrug exposure, cells were collected by centrifugation, resuspended in400 Ml lysis buffer (10 mMTris-Hcl pH 7.4, 10 mMNaCl, 3 mMMgCl2, and 0.5% NP-40), and incubated on ice for 5 min. Nuclei werecollected by low speed centrifugation at 4C. Each pellet was then resus-pended in 32 Ml of transcription buffer (240 mMKCG, 8 mMMgCl2, 64mMTris-HCI pH 7.9, 8 mMDTT, 22%glycerol). The following wereadded: ATP, CTP, and GTP, 2 Ml each of 25 mMstocks; creatinephosphate, 1 Ml of 200 mMstock; creatine phosphokinase, 1.2 M1 of 3mg/ml stock; and 4Ml (40 MCi) [a-32P]UTP. Runon transcription wasallowed to proceed at 30°C for 30 min.

Reactions were terminated by the addition of 0.5 ml buffer A (10mMTris-HCl pH 7.4, 1 mMEDTA, 0.3% SDS) and 20 MgtRNA, afterwhich 0.5 ml buffer B (100 mMsodium acetate ph 5.2, 20 mMEDTA)was added. The reaction mixtures were extracted with phenol/chloro-form and extracted with 0.7 ml buffer C (100 mMsodium acetate pH5.2, 20 mMEDTA, 0.4% SDS). After precipitation with ethanol thepellets were resuspended in 150 Ml Tris-EDTA buffer, brought to 5 mMMgCI2, 0.1 mMCaCI2, 1 mMDTT, and digested with 1.5 URNAse-free DNAse 1 at 37°C for 20 min. After hybridization, filters werewashed four times with 2x SSC, 0.1% SDS, air dried, and loaded forautoradiography.

For DHFRenzyme activity assays, 1.1 X 10' cells were plated in175-cm2 flask and allowed - 30 h for adherence and initiation ofgrowth. The drugs readded every 48 h, as indicated. Cells were har-vested using trypsin/EDTA, washed three times in saline, and frozen at-70°C. Cell pellets were resuspended in 200 Ml buffer I (50 mMTris-HCl, pH 7.4; 100 mMKCl; 10%glycerol; and 10 mM2-mercaptoeth-anol) and sonicated on ice. After centrifugation, the supernatants wereassayed for DHFRactivity in 100 mMTris-HC1; pH 8.0; 150 mMKCI;0.1 mMNADPH; 0.05 mMdihydrofolate; and 0.5 mM2-mercap-toethanol. The rate of decrease in absorbance at 340 nmwas comparedto a standard curve obtained with purified DHFR(Sigma).

ResultsStructure of the human DHFRpromoter. Figure 1 illustratesthe primary structure of the humanDHFRgene promoter. The1.8-kb EcoRI fragment which contains the 5' end ofthe humanDHFRgene (16) is shown in panel A (pDHFR1.8). The first

dhfr transcript Figure 1. The human DHFRA G A I S 2 PUCS

promoter contains two G/C boxA puc8 l | J o; i ' | S sequence elements. (A) Diagram

E divergent / N \ E of the 1.8-kb EcoRl fragment1-transcript / \ 1780 containing the 5' end of the hu-

man DHFRgene. The positionsof DHFRexon 1 and the 5' por-tion of exon 2 are indicated by

/ dhtr \ rectangles. The DHFRtranscrip-B pGEM3Z ;NG 0 \1-OWpEM3Ztion start site, as well as that for

941 a divergent transcript, are shown1140 1252 1308 by arrows. The two G/C box se-

quence elements near the DHFRtranscription start site are repre-sented by ellipses. NumberingC proceeds 5' to 3' relative to the

...GACCCTGCGTGCGCCGGGGCGGGGGGGCGGGGCCTCGCCTGCACAAATGGGGACGAGGGGGGCGGGGCGGCCACAATTTCGCGCCAAACTT...DHFRcoding strand, beginning* ** with the first nucleotide of the

1204 1238 1252 recognition sequence of the firstEcoRI site. (B) Diagram of the

367-bp AvaIl fragment subclone. The double lined segment represents the 186-bp NotI-EcoRI fragment used for DNAseprotection and gelmobility shift assays. Restriction sites: E, EcoRi; A, AvaIl; S, SstI; N, NotI; B, BamH1. (C) Sequence of the DHFRcoding strand in the regionsurrounding the G/C box sequences and transcription start site. The transcript is indicated by an arrow. The G/C box decanucleotides are un-derlined. Nucleotides at which the sequence of the fragment used differs from the published sequence are marked by asterisks: *A to G, **inser-tion of G.

1614 S. WBlume, R. C. Snyder, R. Ray, S. Thomas, C. A. Koller, and D. M. Miller


exon, first intron, and 5' end of the second exon, as well as 1.3kb of 5' flanking sequence, are included in this fragment (4).The DHFRtranscription start site is located 71 bp upstream ofthe translation initiation codon. A divergent (non-DHFR)transcription start site is located several hundred bp furtherupstream. A 367-bp Avall restriction fragment containing thetranscription start site and the sequence immediately upstreamwas isolated and subcloned (panel B; pDHFR.37) to facilitateDNAse protection and gel mobility shift studies of the proxi-mal promoter region. The NotI restriction site, located 1 12 bpupstream of the transcription start site, was used as a5' limit forthe proximal promoter region. The two decanucleotide Splbinding sequences (5'-GGGGCGGGGC-3) begin 14 and 49 bpupstream of the transcription start site, and each is part of alarger region of G-C rich DNA(panel C).

Binding of recombinant Spi to the DHFRpromoter se-quence. Partially purified recombinant Spl was prepared fromthe portion of the Spl gene, pSpl-5 16C, cloned by Kadonagaand coworkers (18) after expression of this plasmid in a pro-tease deficient bacterial host. The expressed protein productcontains the three zinc fingers responsible for DNAsequence-specific binding, the polypeptide region responsible for the af-finity characteristics of Spl binding, as well as the domain re-sponsible for activation of transcription (21).

To characterize binding of the recombinant Spl to theDHFRpromoter sequence, DNAse I protection (footprinting)experiments were performed (Fig. 2). The position of the twoSpl binding sequences and the DHFRtranscription start siteare indicated by the Maxam-Gilbert G-specific sequence reac-

Sp 1-5 16CG 0 2 3 5 10

1204-.-- 5

1238--G/C _

UA-

] DistalFootprint

I ProximalFootprint

DHFRTranscript

No

a b c d e f

Figure 2. SpI binds tothe two G/C box se-quences of the humanDHFRpromoter.DNAse 1 protection wasused to assay bindingof Spl-516C to the cod-ing strand of the humanDHFRpromoter. AMaxam-Gilbert G-spe-cific sequence reaction(lane a) indicates theposition of the G/C boxsequences and the tran-scription start site. Acontrol (no Spl added)DNAse 1 digestion ofthe promoter fragmentis shown in lane b. Inlanes c-f increasingconcentrations of theSpl-516C preparationwere incubated with thepromoter fiagment be-fore digestion. The nu-meral over each laneindicates the gg of totalprotein of the recombi-nant SpI preparationadded in each 20-,g to-tal binding reaction.Regions of DNAse pro-tection (footprinting)are bracketed.

tion (lane a). A control sample without added Spl (lane b) andfour samples incubated with increasing concentrations of Splbefore digestion (lanes c-f ) are shown. Two specific regions ofprotection coinciding with the two Spl binding site sequenceswere seen. The position and extent of the two protected se-quences indicate that each represents the binding of one mole-cule of Spl to one of the Spl binding site sequence elements.The distal (relative to the DHFRcoding sequence) footprintbecame evident at a lower Sp 1 concentration than that atwhich the proximal footprint was seen, indicating a somewhathigher affinity of the distal binding site for Spl binding. Thesequences surrounding the two Sp 1 binding sites or the second-ary structure of the DNAin these regions may be important indetermining Spl binding affinity since the decanucleotide se-quences are identical (21).

Binding of mithramycin to the DHFRpromoter sequence.Mithramycin binds to DNAsequences which contain a mini-mumof two contiguous dG-dC base pairs (9). DNAse I protec-tion was used to compare the sites of mithramycin binding onthe human DHFRpromoter to those of Spl (Fig. 3). AMaxam-Gilbert G-specific sequence reaction (lane a), controlDNAsedigestion (lane b), and DNAse I digested samples pre-treated with increasing concentrations of mithramycin beforedigestion (lanes c-e) are shown. Two specific areas of mithra-mycin binding were revealed, each in the region of one of thetwo Spl binding sites (lanes d and e). The distal mithramycinfootprint was slightly more 5', and the proximal mithramycinfootprint was slightly more 3', relative to the Spl footprints.DNAsehypersensitivity was seen in the nucleotides adjacent toeach of the two mithramycin footprints. These may representdrug-induced alterations in DNAconformation which facili-tate endonuclease attack. The degree of DNAse protection af-forded by mithramycin was relatively comparable at each ofthe two Spl binding site sequences.

Effect of mithramycin binding on Spi binding to the DHFRpromoter. Gel mobility shift assays were used to test whethermithramycin binding interferes with the binding of Spl to thehuman DHFRpromoter fragment (Fig. 4). In the control sam-ple (untreated, radiolabelled fragment), a single intense bandwas seen near the bottom of the gel representing the normalmigration of the 1 86-bp DNApromoter fragment with no pro-tein bound (lane a). At a low concentration of Spl (lane b), twoclosely spaced bands (s-x, s-y) which migrate more slowly thanthe unbound DNAwere seen. At a higher Spl concentration(lane c) a third, more slowly migrating band (s-z)appeared. Asthe Sp I concentration was increased further (lane d) the mostslowly migrating shifted band (s-z) increased in intensity whilethe shifted doublet (s-x, s-y) faded. At the same time, the un-shifted band disappeared as progressively more DNA wasbound by Spl .

Correlation of these results with those of the DNAseprotec-tion experiments, which were carried out under identical bind-ing conditions, suggests that the shifted bands s-x and s-y repre-sent binding of a single Spl molecule to one of the Spl bindingsequences. The consistent relative intensities of the s-x and s-ybands correlate with the relative affinities of the proximal anddistal Spl binding sites respectively. At the highest Spl concen-tration, both Spl binding sites were completely protected fromDNAseactivity, and s-z became the predominant shifted band.It is likely, therefore, that this band represents the complex oftwo molecules of Spl bound simultaneously to the two Splbinding site sequences on the DHFRpromoter.

Mithramycin Prevents Spl Binding to the Dihydrofolate Reductase Promoter 1615


PA 1, h r a ro.y {- ir,

.Z n _1

i,_ 0 L) L.2 -in

A_ rIs.i_ .......s_ A. .w

*..* X!

-he:;s': _

.. ffi

*3iiCI' .*

*Bj: w< w

AS*: 41W IT

And :::. 'S'a: k

..... :.: As, _I'm'I

_ A.4_ at v

a b c d e

Figure 3. Mithramycinbinds to the two G/C

Distal box sequences of theFootprint human DHFRpro-

moter. DNAse 1 protec-tion was used to assaybinding of mithramycinto the coding strand ofthe human DHFRpro-moter. A Maxam-Gil-bert G-specific sequencereaction (lane a) indi-

Prox imrl cates the position of theFootp}rint G/C box sequences and

the transcription startsite. A control (nomithramycin added)DNAse 1 digestion ofthe promoter fragmentis shown in lane b. Inlanes c-e, increasingconcentrations ofmithramycin were incu-bated with the promoterfragment before diges-tion. Regions of DNAseprotection (footprint-ing) are bracketed.

To determine the effect of mithramycin on Spl binding,the DHFRpromoter fragment was also incubated with mithra-mycin before the SpI binding reaction. Although the same

concentration of Sp was used in lanes e-g as in lane d, thepattern of shifted bands was altered. Preincubation of the DNAwith a high concentration of mithramycin (lane e) preventednearly all Spl binding; band s-z was absent, the doublet (s-x,s-y) was greatly decreased in intensity, and the majority of theDNAwas unshifted. This mithramycin effect was concentra-tion dependent (lanesf-g) and corresponds to the footprintingeffects of the drug.

Effect ofmithramycin on in vitro transcription of the DHFRpromoter. To determine the functional consequences ofmithramycin binding to the DHFRpromoter, we investigatedthe effect of this agent on promoter-dependent in vitro tran-scription. Whenthe 1.8-kb EcoRl DHFRfragment was incu-bated with ribonucleotides and a HeLa whole cell extract con-

taining RNApolymerase II, the 529-nt DHFRtranscript as

well as an - 650-nt divergent transcript generated severalhundred bp upstream of DHFRwere synthesized (data notshown). The direction of each transcript was confirmed whenrestriction digests which removed sequence either from the 5'(Sspl) or 3' (Sstl) end of the 1.8-kb template appropriatelyaltered the size of the divergent or DHFRtranscripts, respec-tively.

A series of template molecules was prepared in which pro-gressively more 5' sequence was removed from the original 1.8-

kb fragment. Templates with 821 bp, 565 bp, 322 bp, and 112bp of 5' flanking sequence (relative to the DHFRtranscriptionstart site) were tested, each retaining the ability to be efficientlytranscribed to produce the 529-nt DHFRtranscript (data notshown). The functional template produced by Notl restrictionof the 1.8-kb EcoRl fragment contains the same upstream pro-moter sequence (1 12 bp) as the 1 86-bp fragment of the Avallsubclone used in DNAse protection and gel mobility studies,although additional 3' sequence is used to facilitate detection ofthe transcript on autoradiography.

In Fig. 5, the effect of mithramycin on in vitro transcriptionof the Notl restricted template is shown. A reaction with noadded template DNA(lane a) showed only nonspecific bands.In the control DHFRtranscription reaction (lane b), the 529-ntDHFRtranscript was seen. This band was decreased in inten-sity by preincubation of the template with a low concentrationof mithramycin (lane c). Synthesis of the DHFRtranscript wascompletely inhibited by the higher mithramycin concentra-tions (lanes d-e). This concentration-dependent interferenceby mithramycin with DHFRpromoter function correlateswith the binding of the drug to the promoter and its inhibitionof Sp I binding as measured in DNAseprotection and gel mobil-ity shift experiments. Inhibition of H2b transcription was signif-icantly less sensitive to mithramycin than DHFRat all concen-trations tested. At 1 X l0-5 Mmithramycin DHFRtranscrip-tion was inhibited by 36% while H2b transcription was notsignificantly inhibited.

Sp 1 + Mithrarnycin

)iSp1-5166C -

O'1 1 2 5 ,u

UnshiftedDNA

a b c d e f g

Figure 4. Mithramycin interferes with Spl binding to the DHFRpromoter. The gel mobility shift technique was used to measure theeffect of mithramycin on Spl binding to the DHFRpromoter. Thecontrol (no Spl added) migration of the promoter fragment is shownin lane a. In lanes b-d, increasing amounts (1-5 fg) of partially puri-fied recombinant Spl were incubated with the promoter fragment.The shifted bands s-x, s-y, and s-z represent DNA-protein complexeswith altered electrophoretic migration relative to the unbound DNA.In lanes e-g, DNAwas preincubated with decreasing concentrationsof mithramycin before incubating with the highest concentration ofSpI (5 gg).

1616 S. W. Blume, R. C. Snyder, R. Ray, S. Thomas, C. A. Koller, and D. M. Miller

1204 -

G/CI

1238

G/C

I11252

DHFRTranscriptl

.-, WIVA.. - S-Z10 i- W

1,-, ",-I

MI''W.I21.ANAML

Al,'' 4 "I,. 4* , .: 'I - S-Y-x'p ,'I- 0

- s


Figure 5. Mithramycininhibits in vitro pro-moter-dependent tran-

DHFR scription of DHFR. TheTemplate NotI restricted DHFR

Ci) + template was incubateda Mithramycin with an RNApolymer-E r ase II containing HeLa

< - whole cell extract in thecx cc I", _11 I_, presence of ribonucleo-0 I x x x tides and [a-32P]UTP toZ 0 to) to LO allow for in vitro pro-

moter-dependent syn-thesis of the DHFRtranscript. A transcrip-tion reaction with noadded template isshown in lane a. In laneb, the result of the con-trol DHFRtranscrip-tion reaction is seen. Inlanes c-e, the DHFRtemplate DNAwas

529 nt preincubated with in-v t AdDDHFR creasing concentrations

transcript of mithramycin beforethe transcription reac-tion. The number overeach lane indicates the

^sixX mithramycin concen-tration in mmol/ml of

a b c d e each binding reaction.

Interestingly, mithramycin also inhibited in vitro transcrip-tion of the divergent promoter sequence (observed when the1.8-kb template was used). This transcription start site, likethat of DHFR, is preceded by very G-C rich sequences, includ-ing one exact G/C box decanucleotide. On the other hand,transcription of a human H2b template was 10-fold less sensi-tive to inhibition by mithramycin in the same system (data notshown).

Effect of mithramycin on intracellular DHFRtranscription.To detect the in vivo effect of mithramycin on intracellularDHFRtranscriptional activity, runoff transcription of DHFRby nuclei isolated from whole cells exposed to mithramycinwas measured (Fig. 6). A methotrexate-resistant derivative ofthe human breast carcinoma MCF-7 was used for these experi-ments. This cell line, developed by Cowan and coworkers, ischaracterized by DHFRgene amplification and overexpres-sion (20). Hybridization of radiolabelled transcript RNAto the1.8-kb EcoRl DHFRfragment indicated that DHFRtran-scription by the cells exposed to higher mithramycin concen-trations was almost completely inhibited within 9 h ofthe onsetof drug exposure (panel A). A lower concentration of mithra-mycin caused a slower, but similarly effective, inhibition ofDHFRtranscription initiation (panel B). Transcriptional activ-ity detected by a human tubulin probe followed a pattern simi-lar to that of DHFR, as expected since the tubulin promotercontains Sp 1 binding sequence. However, transcriptional activ-ity of the human H2b gene was considerably less sensitive toinhibition by mithramycin (data not shown). The total tran-scriptional activity of nuclei isolated from mithramycin-treated cells was identical to that of nuclei from untreated con-trol cells.

The transcriptional inhibition of DHFRby mithramycin isreflected in the concentration of DHFRmRNAin cells thathave been exposed to mithramycin. As shown in Fig. 7, treat-ment of the methotrexate-resistant MCF-7 cells with mithra-mycin resulted in a significant decrease in the level of DHFRmRNAin both control, and methotrexate treated cells. In ad-dition, mithramycin completely abrogated the increase inDHFRmRNAin response to exposure to methotrexate, al-though there is no evidence that this increase is transcription-ally mediated.

Effect of mithramycin on the intracellular level of DHFRgene product. To measure the end result of the transcriptionalinhibition of DHFRby mithramycin, DHFRenzyme activitywas assayed in the homogenate of cells treated by continuousexposure to the drug (Fig. 8). Use of the DHFRgene amplifiedcells facilitates measurement of the normally low level of activ-ity of the housekeeping enzyme. Panel A illustrates the effect of

A

1o0

C,

0 CD UWI I0 0 0

X X XLO) ' U)

_ _ +~~~~'avie DHFR

__gW Tubulin

9 Hours Exposure to Mithramycin

B (a (aL- L.

o 0

o 0)

CO COL- -

U) ('Jo o0 CU

CM

DHFR

_- Tubulin

5 x 10-8M MithramycinFigure 6. Mithramycin inhibits intracellular transcription initiation ofDHFR. The technique of nuclear runoff transcription was used todetermine the effect of mithramycin on intracellular DHF`R tran-scriptional activity. DHFRgene amplified cells were treated withmithramycin. Nuclei were isolated and runoff transcription allowedto proceed in the presence of [a-32P]UTP. Labelled RNAwas thenallowed to hybridize to the 1.8-kb EcoRl human DHFRfragment inorder to measure the drug effect on the level of intracellular DHFRtranscription. A human tubulin probe was used as a reference. (A)Mithramycin concentration was varied with a 9-h duration of drugexposure. (B) The duration of drug exposure was varied with a con-stant concentration of mithramycin.

Mithramycin Prevents SpJ Binding to the Dihydrofolate Reductase Promoter 1617


Methotrexate

0 12 24

MethotrexateMithramycin

12 24

Mithramycin

12 24

Discussion

Regulation ofDHFR gene expression. Although DHFRis con-stitutively expressed in all cells, a number of physiologic andenvironmental stimuli influence the rate of transcription ofthis gene. DHFRexpression increases in early S phase of thecell cycle (1, 22, 24). The addition of fresh serum to cells in

A

1 2 3

lb

0.2

0co

4 5 6 7Figure 7. Mithramycin causes a decline in intracellular DHFRmRNA. Northern analysis of RNAisolated from methotrexate resis-tant MCF-7 cells treated with mithramycin and/or methotrexate wasused to determine drug effects on DHFRmRNAlevels. Drug con-centrations used were: methotrexate, 5 x lo-7 M; mithramycin, 5x I O-8 M. The number over each lane represents the duration of drugexposure in hours.

mithramycin on cellular proliferation. Untreated cells supple-mented with fresh medium divided rapidly until they began toapproach confluence - 48 h into the experiment. Mithramy-cin-treated cells divided normally for 24 h but reached aplateau at a cell density below confluence. Panel B shows totalDHFRenzyme levels assayed for each sample in panel A. Stim-ulation of control cells with fresh serum (arrow at 15 h) induceda transcription-mediated increase in DHFRenzyme activity.Mithramycin-treated cells showed a much lower level of seruminduction of DHFR. Control cells maintained the elevatedDHFRactivity as they proliferated. In mithramycin-treatedcells, DHFRactivity leveled off (48 h) and then began to de-cline (72 h). Cellular depletion of enzyme activity depends notonly on inhibition of transcription by mithramycin but also onthe decay of pretreatment mRNA(t,/2 = 7.5 h) (22) and en-zyme (t,/2 = 28 h) (23) levels.

In panel C the effect of mithramycin and/or methotrexatetreatment is measured in terms of DHFRactivity per cell. After24 h of exposure to mithramycin or mithramycin plus metho-trexate, treated cells had roughly 50% the DHFRactivity ofuntreated cells. After 4 d of continuous drug exposure, mithra-mycin-treated cells contained 15% of the DHFRactivity percell of their untreated counterparts. This level of DHFRactiv-ity approaches that of the parent MCF-7 cells in which theDHFRgene is not amplified or overexpressed. This decrease inDHFRactivity per cell represents a considerable lowering ofthe parameter responsible for methotrexate resistance in thesecells. There was no change in the total protein concentration ofthe mithramycin treated cells.

12rB WENc0

E'a0

0

.0

0-

N

C w

- "-

0 0

E

* Controlo Mith5xlO-8M

3 days of treatment

* Controlo Mith5x1O-8M

3 days of treatment

o ControlMtx5xlO-7MMith 5x1O-8M or

2x10-8 MMtx + Mith

* Untreated MCF-7 cels(dhfr not amplified)

i Day 4 Days *

Figure 8. Mithramycin causes a decline in intracellular DHFRen-zyme activity. DHFRenzyme activity was assayed spectrophotomet-rically on the homogenate of cells treated by continuous exposure tomithramycin in order to determine the effect of the drug on intracel-lular DHFRgene product levels. (A) Effect on cellular proliferation.Cell number is expressed relative to the onset of treatment. The dottedhorizontal line approximates cellular confluence. Arrows along thehorizontal axis indicate points at which media was changed and drugadded. (B) Effect on DHFRenzyme synthesis. The samples from (A)above were lysed and the cell homogenate assayed for DHFRenzymeactivity. Results are expressed as total activity per sample, relative tothe start of treatment. (C) Cellular depletion of DHFR. DHFRactiv-ity per cell after varying treatments is shown. Results are expressedas a percent of untreated cell values. For reference, the relative cellularDHFRactivity of non-gene amplified parent MCF-7 cells harvestedunder growth conditions comparable to 4-d samples is shown in thebar marked with an asterisk.

1618 S. W. Blume, R. C. Snyder, R. Ray, S. Thomas, C. A. Koller, and D. M. Miller

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culture stimulates DHFRtranscription, while cellular con-fluence is associated with a decrease in expression (2, 25).Mouse erythroleukemia cells exhibit a dramatic decrease inDHFRtranscription when chemically induced to differenti-ate (26).

Although the extent of regulatory sequences which controlexpression of the DHFRgene is unknown, our data indicatethat 1 12 bp 5' of the transcription start site is sufficient for invitro transcription. This is consistent with the results obtainedby other workers in experiments in which a DHFRminigenelimited to the same 112 bp of promoter sequence was func-tional in transfected DHFR-deficient Chinese hamster ovarycells (16). This promoter segment includes both the distal andproximal Spl binding site sequences, and is located within anopen chromatin (nonnucleosomal), CpGhypomethylated re-gion of DNA(7).

A number of genes which bind Spl have now been identi-fied (27-31). From the known Spl binding sites, a consensusdecanucleotide sequence has been proposed; within this con-sensus, the sequence 5'GGGGCGGGGC3'(sometimes re-ferred to as the heptanucleotide 5'GGGGCGG3'), of whicheach of the human DHFRSpl binding site sequences consist,represents the most preferred, highest affinity Spl binding site(5). Spl binding sites vary in number, relation to the transcrip-tion start site, and to each other as well as among heterologousregulatory elements in the different promoters in which theyare found. The most proximal Spl binding site is frequentlypositioned 40-60 bp upstream of the transcription start site (5,32-34). Activation of transcription by Spl has been demon-strated in vitro and in vivo for several of these genes.

Our data demonstrate that the human DHFRpromoterbinds two molecules of Spl at the two Spl binding site se-quences. Our in vitro data suggest that Spl binding is necessaryfor DHFRtranscription. The distal Spl binding site sequencebegins 49 bp upstream of the transcription start site. It is asso-ciated 6 bp downstream with the sequence 5'-TGCACAAAT-GGGG-3', a "CAA element" homologous among the human,mouse, and hamster DHFRgenes (6, 33, 34). The associationof a G/C box and a CAAelement has been found to precede by

- 50 bp the transcription start sites of these three mammalianDHFRgenes. Both Spl and a SpI binding site have beenshown to be essential for DHFRtranscription in the mouse (6,33). Because of its high affinity for Spl, its position relative tothe transcription start site, and its homologous association withthe CAAelement, the distal Spl binding site of human DHFRgene would be expected to be critically involved in transcrip-tion initiation of the DHFRgene.

The proximal G/C box of the human DHFRpromoter islocated only 4 bp upstream of the transcription start site. Thedecanucleotide sequence comprises a strong SpI binding site,and the DNAse protection assay shows clear binding albeitwith lower affinity than the distal G/C box. It is conceivablethat Spl binding to the proximal binding site could either stim-ulate, inhibit, or be independent of DHFRtranscription.

The positive transcriptional activity of a relatively weakSp I binding site in the LDL receptor promoter can be down-regulated by the action of an adjacent sterol-dependent re-sponse element. Whenthe Spl binding sequence was changedto a high affinity Spl binding sequence by site directed muta-genesis, the promoter became constitutive, unresponsive to theadjacent element (35). A similar significance of the Spl affinity

of multiple Spl binding sequences can be found in the herpessimplex virus thymidine kinase promoter (29). These varia-tions in multiplicity, spatial arrangement, and affinities of tran-scription factor binding sites may be responsible for differentialpatterns of gene expression (36).

Methotrexate resistance and modulation ofDHFR gene ex-pression. DHFRgene amplification has been demonstrated ina number of methotrexate-resistant human (37-39) and ani-mal (25, 33, 40-43) cell lines, and documented clinically in anumber of cases of methotrexate resistant neoplasms (44-46).The methotrexate resistant cells used in this work have an

25-fold increase in DHFRgene copy number, mRNA,andenzyme activity, but a disproportionately high (l0,OOOX) in-crease in methotrexate resistance (20). The association of asmall increase in gene dosage with a large increase in drugresistance is a consistent finding. No other mode of methotrex-ate resistance has been found for these cells.

Spl binding appears to be critical for DHFRtranscription.Because of this, the G-C specific DNAbinding drug mithramy-cin represents an initial candidate for an attempt to selectivelyinhibit the transcriptional activity of the human DHFRpro-moter. This molecule binds G-C rich DNAand inhibits DNA-dependent RNAsynthesis in vitro; in vivo data has shown thatmithramycin selectively inhibits expression of genes with G-Crich promoters.

DNAse I footprinting experiments demonstrated thatmithramycin binds specifically to the same two G/C box re-gions of the human DHFRpromoter as Sp 1. The drug concen-trations that result in mithramycin binding to the DHFRpro-moter are comparable to those used in the original studies onmithramycin binding specificity. In contrast to Spl, mithra-mycin shows no difference in binding affinity between the twosites. The specific DNAse enhancements which are seen at thehighest mithramycin concentration suggest that mithramycinbinding induces a different type or greater degree of conforma-tional alteration on the DNAthan Spl binding (23).

The abrogation of the Spl-induced gel mobility shifts indi-cates that mithramycin binding prevents subsequent Spl bind-ing to the DHFRpromoter in vitro. This suggests that mithra-mycin interferes with Spl binding and the formation of theDHFRtranscription initiation complex. The concentrations ofmithramycin that result in binding of the DHFRpromoter andprevention of Spl binding also effectively inhibit promoter-de-pendent in vitro transcription of DHFR. The selective inhibi-tion of DHFR, as compared to histone H2b, suggest that inhibi-tion of Spl binding is causally related to inhibition of DHFRtranscription.

The nuclear runoff, Northern analysis, and enzyme assayexperiments confirm that mithramycin also inhibits DHFRtranscription initiation in whole cells and induces a selectivedecrease in DHFRmRNAand enzyme activity. Drug treat-ment significantly lessened the increase in DHFRactivity inresponse to serum stimulation. After 72 h of mithramycin ex-posure, DHFRtranscription was essentially abolished. Therate of decrease in DHFRenzyme activity was consistent withfirst order decay and the estimated half-life of the enzyme.After 96 h of mithramycin treatment, DHFRgene amplifiedcells contained only slightly greater DHFRactivity than thatpresent in non-gene amplified cells under comparable growthconditions. Since mithramycin inhibits DHFRtranscriptionand causes the overexpressed enzyme to be depleted, the sensi-



tivity to methotrexate should be returned to near normal. Thelack of the expected mithramycin-methotrexate synergismsuggests that expression of other genes, presumably genes im-portant in the control of cellular proliferation, are also beinginhibited. In fact, it has recently been shown that mithramycininhibits protein binding and transcriptional activity of the c-myc gene, as well (14). This gene, which plays an importantrole in the regulation of cellular proliferation, also has a G-Crich promoter.

With a recognition sequence of three base pairs limiting itsspecificity, mithramycin undoubtedly affects the expression ofmultiple genes with G-C rich promoter elements. This allowsfor the possibility that mithramycin inhibits the expression ofone or more genes other than DHFRwhich may: (a) be lethalto the cell (primary mithramycin toxicity) (47), which wouldsupercede synergism; (b) alter the program for the cell cycle(antiproliferative effect of mithramycin) which could obscureany mithramycin-induced increase in methotrexate sensitivity(48, 49); or (c) eliminate a gene product which is necessary forthe mechanisms of toxicity of methotrexate (50, 51). An agentwith greater sequence specificity than mithramycin might notexhibit such effects.

Acknowledgments

Wethank Dr. K. Cowan and Dr. Merrill Goldsmith for helpful discus-sion of this project as well as the MR100cells and DHFRprobes. Weare grateful to Ms. Barbara Wilson for her expert assistance in prepara-tion of this manuscript.

S. W. Blume is a postdoctoral fellow of the American Cancer Soci-ety (PRTF-94). This work was supported by grants from the NationalInstitutes of Health (CA 42664 and CA42337), Leukemia Society ofAmerica, and the Veterans Administration Research Program to Don-ald M. Miller.

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Mithramycin inhibits SP1 binding and selectively inhibits transcriptional activity of the dihydrofolate reductase gene in vitro and in vivo