Transcriptional Consequences of Topoisomerase Inhibition

MOLECULAR AND CELLULAR BIOLOGY,0270-7306/01/$04.00�0 DOI: 10.1128/MCB.21.24.8437–8451.2001

Dec. 2001, p. 8437–8451 Vol. 21, No. 24

Transcriptional Consequences of Topoisomerase InhibitionIRENE COLLINS, ACHIM WEBER,† AND DAVID LEVENS*

Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland 20892-1500

Received 14 June 2001/Returned for modification 17 July 2001/Accepted 17 September 2001

In principle, the generation, transmission, and dissipation of supercoiling forces are determined by thearrangement of the physical barriers defining topological boundaries and the disposition of enzymes creating(polymerases and helicases, etc.) or releasing (topoisomerases) torsional strain in DNA. These features arelikely to be characteristic for individual genes. By using topoisomerase inhibitors to alter the balance betweensupercoiling forces in vivo, we monitored changes in the basal transcriptional activity and DNA conformationfor several genes. Every gene examined displayed an individualized profile in response to inhibition oftopoisomerase I or II. The expression changes elicited by camptothecin (topoisomerase I inhibitor) or adria-mycin (topoisomerase II inhibitor) were not equivalent. Camptothecin generally caused transcription com-plexes to stall in the midst of transcription units, while provoking little response at promoters. Adriamycin, incontrast, caused dramatic changes at or near promoters and prevented transcription. The response to topo-isomerase inhibition was also context dependent, differing between chromosomal or episomal c-myc promoters.In addition to being well-characterized DNA-damaging agents, topoisomerase inhibitors may evoke a biologicalresponse determined in part from transcriptional effects. The results have ramifications for the use of thesedrugs as antineoplastic agents.

Transcription, replication, recombination, DNA repair, andDNA compaction generate torsional stress in prokaryotic andeukaryotic chromosomes and episomes. This stress must eitherbe accommodated by conformational changes in DNA struc-ture (e.g., supercoils) or else dissipated. If not dissipated, highlevels of torsional stress can halt RNA polymerase and deformchromosomal structure (4). Torsional stress may be dissipatedby rotation of a free DNA end, i.e., chromosome termini orstrand breaks. Alternatively, stresses accumulating within to-pological domains may be dissipated by topoisomerases. Atopological domain is formed whenever both ends of an intactDNA segment are restricted from rotating relative to eachother. The boundaries of these domains may be delimited byDNA loops via protein-protein interactions or tethering ofDNA to an immobile matrix or scaffold. The energy requiredto rotate a large, free-ended DNA segment with bound pro-teins through a viscous medium may become so great thattorsional strain accumulates within a pseudo-domain boundedat one end by a kinetic barrier (40). Topological microdomainsmay be nested within larger and larger macrodomains (24, 70).These domains may be short-lived or stable, depending on thenature of the particular protein-protein and protein-nucleicacid interactions creating their boundaries. A loop formedbetween a DNA-bound factor and a complex tracking alongand around the double helix, such as RNA polymerase II,creates a mobile boundary. Little is known about the arrange-ment, interlinks, and transmission of torsional stress betweentopological domains in vivo. It is likely that the influence ofDNA topology on genetic transactions may be determined by

the architecture and arrangement of cis elements and factorsgoverning the distribution of torsional stress.

Topoisomerases I and II relieve torsional strain incremen-tally within a domain by using controlled breakage of one orboth strands, respectively; passage of DNA through the strandbreak; and reunion (73). Adjacent domains are not affected.The efficiency of topoisomerase is modified by domain size,binding site preference, and site accessibility. The intranucleardistribution of topoisomerase is not known. A large fraction oftopoisomerase II is bound by the nuclear matrix and so isavailable only to local DNA sequences (13, 46). The packagingof DNA into chromatin restrains approximately one negative-supercoil on the surface of each nucleosome (51). This pack-aging may hinder the operation of topoisomerases and delaythe relief or transmission of torsional strain (55). Inhibitors oftopoisomerases I and II freeze these enzymes as protein-DNAcomplexes at various steps in their reaction pathways (31, 49).Topoisomerase-DNA-inhibitor complexes (cleavable com-plexes) are poisoned and are unable to execute a completeenzymatic cycle. Topoisomerase-DNA covalent adducts areconverted into DNA strand breaks upon protein removal. Thetopological state of the domains encompassing these frozencomplexes remains fixed; even in cleavable complexes torsionalstrain is not liberated until the topoisomerase subunits co-valently coupled to the DNA ends dissociate, allowing the endsto rotate independently. Topoisomerase inhibitors haveproven to be potent antineoplastic agents. The efficacy of theseagents for cancer therapy is explained only in part by theirability to damage DNA. The response of individual genes totopoisomerase inhibitors may result directly from enzyme in-hibition or may arise through secondary mechanisms.

Structural considerations dictate that no global generaliza-tions summarize the role of DNA topology for the regulationof any given gene. The microarchitecture of matrix attach-ments, protein-protein-mediated loops, the arrangement ofpromoter sites, and the disposition of topoisomerases and nu-

* Corresponding author. Mailing address: Laboratory of Pathology,Center for Cancer Research, National Cancer Institute, Bldg. 10, Rm.2N106, Bethesda, MD 20892-1500. Phone: (301) 496-2176. Fax: (301)594-5227. E-mail: [email protected].

† Present address: Institute for Molecular Pathology, Eberhard-Karls-Universitat, 72076 Tubingen, Germany.

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cleosomes all mold the physiological or pathological responseof a transcription unit. The expression of the c-myc gene isparticularly sensitive to perturbations of its normal chromo-somal milieu. Translocations, regional amplifications, and viralinsertions and mutations, sometimes at vast distances either 5�or 3� from the c-myc promoters, all deregulate c-myc transcrip-tion (28, 36, 50). Although topoisomerase inhibitors influencec-myc expression (2, 6, 16, 19, 43, 48, 56, 57, 58), it is unknownwhether this results from perturbation of c-myc DNA andchromatin structure driven by changes in the localization andlevels of torsional strain or whether this results from indirecteffects. If c-myc transcription is sensitive to torsional strain,then changes in c-myc mRNA levels, promoter structure, andthe distribution of RNA polymerase II molecules along thegene should all respond to topoisomerase inhibitors. More-over, because every gene is subject to unique structural andphysical constraints, the features controlling the expressionof a particular transcription unit should be characteristicallyaltered in response to manipulations perturbing supercoil-ing.

To explore the relationship between torsional strain andgene activity, the features of c-myc, c-fos, hsp70, gapdh, andrRNA transcription units were examined in response to theinhibition of topoisomerase I and/or II. A polymorphous pat-tern of activation or repression was observed for these genes.Redistribution of transcriptionally engaged RNA polymerasesand structural changes in and around promoters were alsonoted. Notably, the response of the c-myc promoter was mod-ified when carried as a stable episome. These results indicatethat the interplay between torsional strain and chromatin ar-chitecture helps to define the expression profiles of manygenes.

MATERIALS AND METHODS

Plasmids. The vector pREP9/CAT (Invitrogen) encoding chloramphenicolacetyltransferase (CAT) was cut with XbaI and NotI (blunted); an XbaI-HindIIIfragment containing five GAL4 DNA binding sites as well as the human c-mycpromoter from the HindIII site at nucleotide 1 to SexAI (blunted) at position2882 (accession number X00364) was inserted. This resulting plasmid, pMYC/CAT, contains the Epstein-Barr virus (EBV) DNA replication origin P1 and theEBNA1 gene and so is maintained as a circular episome.

Tissue culture. Raji cells were grown in RPMI with 10% fetal calf serum at adensity of �106/ml. Raji (5 � 106 cells) were electroporated with the pMYC/CAT (190 V and 1180 �F; BRL Cell Porator). The transfected cells wereselected with 1 mg of G418 (BRL)/ml. The pool of stable transfectants, calledRaji pMYC/CAT, was maintained in medium containing 0.5 mg of G418/ml.

Chemicals. G418 (BRL) was dissolved in 100 mM HEPES (pH 7.3) at 100mg/ml and stored at 4°C. Camptothecin (Sigma) was dissolved and diluted indimethyl sulfoxide (DMSO) at 1, 5, or 10 mM. Adriamycin, also called doxoru-bicin, (Sigma) was dissolved and diluted in H2O at 1, 5, or 10 mM. Sodiumbutyrate (Sigma) was dissolved in H2O at 3 M. Trichostatin A (Sigma)was dissolved in DMSO at 500 ng/�l. �-Amanitin (CalBiochem) was dis-solved in H2O at 1 mg/ml. All solutions except G418 were stored at –20°C.For these experiments, 1 �l of the camptothecin or trichostatin A stock wasadded per ml of medium. When appropriate, 0.1% DMSO was added tocontrol samples and to the adriamycin and sodium butyrate samples to ensurecomparability.

RNase protection. A total of 3 � 107 cells in 60 ml of medium were incubatedwith DMSO, camptothecin, adriamycin, sodium butyrate, or trichostatin A at theconcentrations indicated. After 4 h of drug treatment, cells were pelleted andresuspended in 3 ml of Trizol (BRL), and RNA was isolated according to themanufacturer’s protocol (BRL). The RNA was treated with RNase-free DNaseI, phenol-chloroform extracted, ethanol precipitated, washed with 75% ethanol,air dried, resuspended in 100 �l of H2O, and stored at –80°C. Then, 10 �g of totalRNA was resuspended in Hybridization Buffer (PharMingen RPA Kit) and

hybridized overnight with gel-purified RNA probes. After RNase and proteinaseK treatments, the samples were separated on 5% acrylamide–bisacrylamide(29:1) gels with 50% urea, dried, and autoradiographed.

The gapdh template for the RNA probe was purchased from PharMingen. ThepSP72/hsp70RNPA template for the hsp70 RNA probe contains the 543-bp SmaIfragment at the 3� end of the hsp70 coding sequence. The CAT, c-myc exon 2,c-fos, and rRNA templates used to synthesize RNA probes were created byinserting gene-specific PCR fragments into the pGEM-T Easy vector (Promega).The CAT and c-myc exon 2 probes were specific for episomal or endogenousc-myc promoter-driven transcripts, respectively. For a complete list of the oligo-nucleotides used, see supplemental material (www-dcs.nih.gov/branches/lop/Research/genreg/levens/html). To obtain signals of comparable intensity, theRNA probes were transcribed by T7 RNA polymerase by using different ratios ofradioactive and cold UTP as follows: CAT and c-fos 1 radioactive UTP out of 10total UTPs, hsp70 and c-myc 1 radioactive UTP out of 100 total UTPs, and rRNAand gapdh 1 radioactive UTP out of 500 total UTPs.

Nuclear run-on. A total of 4 � 107 cells in 80 ml of medium were incubatedwith DMSO, camptothecin, adriamycin, sodium butyrate, or trichostatin A at theindicated concentrations. These chemicals were not included in phosphate-buff-ered saline (PBS), Lysis Buffer, or Glycerol Buffer. Nuclear isolation and nuclearrun-on were as described previously (11). Nuclei were resuspended in GlycerolBuffer (80 �l) and frozen in liquid nitrogen. For run-on experiments, 150 �Ci of[�-32P]UTP (800 Ci/mmol) was included in a 200-�l reaction volume. After a10-min incubation at 30°C, 10 �l of a 10-U/�l solution of DNase I (Roche) wasadded for another 10 min; then, 20 �l of the sodium dodecyl sulfate-ProteinaseK Stop Solution was added, followed by incubation at 42°C for 30 min. Afterphenol-chloroform and ethanol precipitation, the DNase treatment was re-peated. The RNA was passed over a G25 Sephadex spin column (Roche) andthen hybridized in Church Buffer for 36 to 48 h at 65°C with Hybond N�

membranes (Amersham) loaded with 1 �g each of antisense DNA oligonucle-otides corresponding to the genes of interest. For a complete list of the oligo-nucleotides used, see supplemental material (www-dcs.nih.gov/branches/lop/Research/genreg/levens/html). Exposures at –80°C of Kodak BioMax MS filmwith the transcreen HE are shown (Fig. 2A is 14 days; Fig. 2B is 7 days; Fig. 3Ais 9 h for rRNA and 10 days for the Raji cells and the Raji pMYC/CAT cells; Fig.3B is 1 day for rRNA, 12 days for RAJI cells, and 14 days for the Raji pMYC/CAT cells).

Ligation-mediated PCR (LM-PCR). The in vivo footprinting was done asdescribed previously (17, 27, 38) with the following modifications. A total of 2 �

107 cells were treated with 25 mM KMnO4 for 2 min at room temperature. Forthe PCRs, 2 �g of KMnO4-treated template DNA was used with NEB Ther-moPol Buffer, including a total of 4, 5, or 6 mM MgSO4 as needed for thedifferent oligonucleotide sets. For a complete list of the oligonucleotides used,see supplemental material available online (www-dcs.nih.gov/branches/lop/Research/genreg/levens/html). The samples were run on a denaturing 8%acrylamide–bisacrylamide (29:1) gel with 50% urea and then compared with aDNA sequencing ladder.

Southern blot analysis. A total of 2 � 107 cells in 40 ml of medium wereincubated with the following chemicals: DMSO, camptothecin, or adriamycin for4 h and 16 h. When cells were harvested, these chemicals were added to both thePBS (without Ca or Mg) and the Digestion Buffer (67). Cells were washed twicein 10 ml of cold PBS and then resuspended in 0.2 ml of PBS. Next, 1.8 ml ofDigestion Buffer containing 0.4 mg of proteinase K/ml was added, mixed bygentle inversion, and incubated at 37°C overnight. Using large-bore pipette tips,the samples were extracted once with phenol, extracted twice with phenol-chloroform, ethanol precipitated, and resuspended in Tris-EDTA. After RNaseA treatment, the samples were phenol-chloroform extracted, ethanol precipi-tated, and resuspended in 1 ml of H2O. DNA was digested with the restrictionenzyme NheI and separated electrophoretically on a 20-by-25-cm 0.5% agarosegel in 1.5� Tris-borate-EDTA at 50 V for 48 h. The gels were transferred toAmersham Hybond N� membranes, and the DNA was cross-linked to themembrane with a Stratagene Stratalinker.

The c-myc probe was a 1.7-kb PstI fragment containing exon 2. The CAT probewas the 3.1-kb NotI-to-BglII fragment from the pREP9/CAT plasmid (Invitro-gen). The gel eluted DNA fragments were labeled with [�-32P]dATP (3,000Ci/mmol) by using the RadPrime DNA Labeling System (BRL). The membraneswere hybridized overnight at 65°C. A 2-day exposure at –80°C of Kodak BioMaxMS film with the transcreen HE is shown (Fig. 9A and C). As a reference,samples were compared with the plasmid pMYC/CAT before and after topo-isomerase I (BRL) treatment for 1 h at 37°C.

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RESULTS

This study was undertaken to assess the influence of DNAsupercoiling on transcription in vivo. Eukaryotic topoisomer-ases I and II remove both positive and negative supercoils,resulting in relaxed DNA (7, 22). When these enzymes areinhibited by drugs, the superhelical density of the affectedDNA is altered. To examine the transcriptional response totopoisomerase inhibition, cells were incubated with camptoth-ecin, a topoisomerase I inhibitor, or adriamycin (doxorubicin)to inhibit topoisomerase II. The transcriptional response ofspecific genes to drug treatment was monitored by RNaseprotection and by nuclear run-on, and conformational and/ortopological changes were visualized by in vivo footprinting andSouthern blotting.

Two cell lines were used for these experiments. Raji cells area B-lymphocyte Burkitt lymphoma cell line in which one c-mycgene is translocated near an immunoglobulin gene [chromo-some, t(8, 14)] (20). In these cells the translocated myc allele ishighly transcribed, while the wild-type copy is repressed (41).The translocation breakpoint is at position �1398 relative tothe c-myc P2 promoter start site (9). The translocated c-mycallele also has scattered mutations within exon 1, intron 1, andexon 2 (53).

The second cell line, Raji pMYC/CAT, was a Raji cell linestably transfected with the plasmid pREP9/GAL45 MYC/CAT(pMYC/CAT). In this EBV-based autonomously replicating,

neomycin-selectable plasmid, 2.9 kb of c-myc genomic DNA,starting 2.3 kb upstream of promoter P1, was fused with theCAT coding sequence (CAT). Similar MYC EBV-based plas-mids have previously been shown to bear nucleosomes posi-tioned identically as seen for the endogenous c-myc gene (38,52). Raji pMYC/CAT was used to follow myc promoter func-tion. Driven by neighboring c-myc upstream and downstreamsequences, and properly assembled into chromatin, the episo-mal c-myc promoter was expected to recapitulate many fea-tures of c-myc regulation; the high-copy-number plasmid wasexpected to yield an amplified signal.

Polymorphous response of RNA synthesis to inhibition oftopoisomerases I and II. To assess the effect of topoisomeraseinhibitors on mRNA abundance, the levels of the transcripts ofseveral genes were directly measured by using RNase protec-tion (Fig. 1). Cells were treated with camptothecin and adria-mycin separately or together for 4 h. (Camptothecin inhibitstopoisomerase I enzyme, and adriamycin inhibits topoisomer-ase II.) The influence of the histone deacetylase inhibitorsbutyrate and trichostatin A was also observed (35, 60). Typi-cally, histone deacetylase inhibition leads to increased chroma-tin acetylation and conditions conducive for increased geneactivity. Each of the mRNAs tested displayed a distinctiveprofile in response to topoisomerase inhibition:

hsp70 mRNA, which was high in all control samples (Fig. 1,lanes 7 to 9), was decreased by camptothecin (Fig. 1, lanes 10

FIG. 1. The steady-state level of each mRNA displays a distinctive profile in response to inhibition of topoisomerase I or II, by monitoringRNase protection. The c-myc exon 2 versus CAT probes used in this experiment distinguish between the c-myc RNAs encoded by the endogenousc-myc gene and the hybrid MYC/CAT mRNA encoded by the episome pMYC/CAT. Cpt, camptothecin; Adr, adriamycin; TSA, trichostatin A. Thecells were incubated with inhibitors for 4 h. The duplicate lanes are each from different independent experiments. A 3-day exposure made withKodak XAR film is shown, along with a 13-h exposure for the full-length probes and the GAPDH (glyceraldehyde-3-phosphate dehydrogenase)inset.

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to 12) but increased by adriamycin (Fig. 1, lanes 13 to 15),butyrate (Fig. 1, lane 19), and trichostatin A (Fig. 1, lane 20).The hsp70 mRNA half-life is 50 min (69).

The c-fos message, which was quite low in the control cells(Fig. 1, lanes 7 to 9), was strongly increased by 5 and 10 �Mcamptothecin (Fig. 1, lanes 11 and 12) and was raised by bothbutyrate (Fig. 1, lane 19) and trichostatin A (Fig. 1, lane 20).Induction of c-fos transcription by these concentrations ofcamptothecin has been reported previously (66). In contrast,adriamycin decreased c-fos mRNA (Fig. 1, lanes 13 to 15).

Unexpectedly, endogenous c-myc and the episomal pMYC/CAT respond differently to the drugs tested. Endogenous c-myc levels were high in the controls (Fig. 1, lanes 7 to 9) butwere lowered by all of the drug treatments (camptothecin[lanes 10 to 12], adriamycin [lanes 13 to 15], butyrate [lane 19],and trichostatin A [lane 20]). In contrast, the c-myc promoterdriven CAT mRNA was dramatically increased by camptoth-ecin (Fig. 1, lanes 10 to 12), butyrate (lane 19), and trichostatinA (lane 20) but decreased by adriamycin (lanes 13 to 15). CATmRNA levels generally paralleled c-fos. It is important to notethat, although c-fos and c-myc messages both have short half-lives (54, 65), these genes responded very differently to drugtreatment. Moreover, the half-life of CAT mRNA is also short(3). Because some of these RNAs increased, while others de-creased during the 4-h drug treatment (long relative to the

half-lives of these molecules), differential kinetics of RNAdegradation do not explain these results unless these topo-isomerase inhibitors differentially modify mRNA half-life.

Of all of the mRNAs analyzed, gapdh was least affected bythe drug treatments. It is clear from the behavior of thesegenes that there is no stereotypical response of mRNA levelsafter topoisomerase inhibition. In contrast, butyrate and tri-chostatin A increased hsp70, c-fos, and MYC/CAT, as expectedfor histone deacetylase inhibition; only endogenous c-myc de-creased after histone deacetylase inhibition, as reported pre-viously by others (45).

The steady-state mRNA levels assayed by RNase protectionindicate the net effect on RNA synthesis and degradation. If,for example, a message is increased, this may be due to anincrease in synthesis or a decrease in degradation. One way todiscriminate between these possibilities is to measure RNAsynthesis directly with nuclear run-on assays. These assays al-low a limited extension of RNA polymerases transcriptionallyengaged in vivo. A battery of genes transcribed by RNA poly-merase I, II, or III was analyzed with nuclear run-on assays. Toassess structural changes at promoters after topoisomeraseinhibition, several genes were examined by in vivo footprintingwith the conformation-sensitive DNA reagent potassium per-manganate.

FIG. 2. Heterogeneous response of promoter activity to topoisomerase inhibition. (A) Nuclear run-ons of Raji cells. (B) Nuclear run-ons ofRaji cells with pMYC/CAT. c-myc exon 2 (slot 10) and CAT (slot 9) distinguish between the c-myc RNAs encoded by the endogenous c-myc geneand the hybrid MYC/CAT mRNA encoded by the episome pMYC/CAT. The arrow indicates the direction of transcription. Cpt, camptothecin;Adr, adriamycin; TSA, trichostatin A. The cells were incubated with inhibitors for 4 h. A 10-min run-on reaction was performed.



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Context-dependent response of the c-myc promoter to topo-isomerase inhibition. c-myc expression is very context depen-dent. In Burkitt lymphoma cells such as Raji cells, the trans-located c-myc allele is deregulated, while the unrearrangedallele is underexpressed. How immunoglobulin regulatory se-quences project their influence over vast stretches of DNA(sometimes exceeding hundreds of kilobases) to activate thetranslocated allele is unknown. Disturbances of c-myc expres-sion are also associated with far-3�-genetic irregularities in cisat the PVT locus in some tumors (62). To explore directlyc-myc promoter activity in different chromosomal contexts,chromosomal or episomal transcription was compared be-tween Raji cells and Raji pMYC/CAT by using nuclear run-onassays. After 4 h of drug treatment, nuclei were harvested, andRNA labeled in a brief run-on reaction was hybridized with apanel of oligonucleotides derived from the genes of interest.The run-on findings showed that all of the drug treatmentsrepressed endogenous c-myc in agreement with the RNaseprotection results.

Labeled nascent transcripts from untreated Raji cells andcells treated with the vehicle DMSO hybridized similarly withantisense myc oligonucleotides extending from the P1 pro-moter through the P2 promoter and into exon 2 (Fig. 2A, lanes1 and 2). Little evidence of holdback of RNA polymerase atthe P2 promoter was noted. (Holdback would be indicated bystrong hybridization with slot 5, with declining signals in slots 6to 10.) The loss of RNA polymerase holdback in Burkitt lym-phoma has been previously described (10). Camptothecin,even at the lowest dose, caused dramatic holdback of the RNApolymerase at the endogenous c-myc P2 promoter (Fig. 2A,lanes 3 to 5). The RNA labeled during the 10-min run-on

reaction hybridized only with the first P2 sequence (slot 5),indicating that the RNA polymerase is loaded at the promoterbut progresses less than 50 nucleotides. Hybridization with P1oligonucleotides was lost (slot 4), suggesting that this promoteris vacant after camptothecin treatment. Similar results wereobserved with nuclei from camptothecin-treated BJAB cells(that possess only untranslocated c-myc), showing that thisdrug-induced holdback requires no immunoglobulin sequences(data not shown). Adriamycin (Fig. 2A, lanes 6 to 8), butyrate(lane 12), and trichostatin A (lane 13) all yielded uniformlyweaker myc signals than the DMSO control. With 5 �M adria-mycin, only weak holdback at P2 was noted (Fig. 2A, lane 7).

Transcription from the episome in Raji pMYC/CAT wasalso analyzed by using nuclear run-on (Fig. 2B). The intensityof the hybridization to sequences downstream of c-myc P2(slots 6 and 7) was increased in Raji pMYC/CAT comparedwith Raji cells (Fig. 2B, lanes 2 and 3 versus lane 1) as ex-pected, due to the increased copy number of this template.Overexpression from the episome was confirmed by comparingthe relative intensities of the hybridization with CAT (slot 9)versus c-myc exon 2 (slot 10); CAT is specific for the episomewhereas c-myc exon 2 is specific for the endogenous gene. Therelative transcription of sequences shortly downstream of P2(Fig. 2B, lanes 1 to 3, slots 5 to 7) was increased in RajipMYC/CAT compared with Raji cells, a finding indicative ofgreater utilization of P2 from the episome, whereas P1 usagewas not increased (slot 4). The intensity of the c-myc run-onsignal from pMYC/CAT cells declined progressively further 3�of the start site, suggesting that on the episome there is hold-back of RNA polymerase near the c-myc P2 promoter, andthus fewer polymerases are located distally. P2 utilization with

FIG. 2—Continued.



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promoter proximal holdback of episomal c-myc has been notedpreviously (1, 34).

Although RNase protection of Raji pMYC/CAT cellsshowed simply that camptothecin induced CAT, the run-ontranscription from pMYC/CAT in the presence of increasingcamptothecin gave a more complex pattern (Fig. 2B, lanes 4 to6). The first P2 oligonucleotide (slot 5) hybridized morestrongly than the 3� sequences, indicating more RNA polymer-ase holdback, but the P2 distal sequences and CAT were alsomore intensely transcribed, suggesting that transcription pen-etrated further into the gene with less holdback. These twocontradictory observations are reconciled by superimpositionof the transcription profiles of the episomal and endogenousc-myc promoters. As described above, camptothecin causedstrong holdback of the endogenous gene at the first P2 oligo-nucleotide (Fig. 2A, lanes 3 to 5, slot 5). Therefore, increasedholdback of the endogenous c-myc gene overlying increaseddownstream transcription from the plasmid pMYC/CAT rec-onciles these results with the RNase protection data. Adria-mycin strongly repressed the episomal c-myc promoter just asit did the endogenous gene, abolishing hybridization with all

probes (Fig. 2B, lanes 7 to 9). Interestingly, the run-ons ob-tained with both camptothecin and adriamycin most closelyresembled those obtained with adriamycin alone (Fig. 2A,lanes 9 to 11, and B, lanes 10 to 12). Butyrate and trichostatinA caused upregulated transcription of all MYC/CAT se-quences without altering their relative intensities (Fig. 2B,lanes 13 and 14). Adriamycin inhibition of transcription wasnot mitigated by butyrate (data not shown). If immobilizedtopoisomerase II recruited histone deacetylase to inhibit tran-scription (23, 71), then these drugs should have increased tran-scription in the presence of adriamycin; this did not occur.

Importantly, inclusion of adriamycin or camptothecin di-rectly in the nuclear run-on reactions neither inhibited norstimulated transcription of c-myc or other genes (data notshown); thus, the changes observed in these experiments re-flect conditions set up in vivo. Run-on analysis of nuclei har-vested during a time course of adriamycin drug treatmentdemonstrated full promoter responses in less than 2 h (Fig.3B).

RNase protection and nuclear run-on experiments indicatedthat different functional states of the c-myc promoter, directly

FIG. 3. Rapid response of promoter activity to topoisomerase inhibition as shown by nuclear run-on. (A) Time course of camptothecininhibition. In lane 1, slots 5 and 23 show the strong 28S rRNA signal spreading from the slot above. (B) Time course of adriamycin inhibition. c-mycexon 2 (slot 10) and CAT (slot 9) distinguish between the c-myc RNAs encoded by the endogenous c-myc gene and the hybrid MYC/CAT mRNAencoded by the episome pMYC/CAT. The arrow indicates the direction of transcription. Cpt, camptothecin; Adr, adriamycin. Lane 1 shows cellstreated with DMSO for 4 h. Lanes 2 to 6 were incubated with either camptothecin or adriamycin for 0.5, 1, 2, 3, or 4 h before nuclei were harvested.A 10-min run-on reaction was performed.



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affecting transcriptional levels, exist in the chromosome versusthe epsiome and in response to topoisomerase I versus topo-isomerase II inhibition.

Context-dependent KMnO4 sensitivity of the c-myc pro-moter to topoisomerase inhibition. Topoisomerase I and IIinhibitors modify transcription of the endogenous and episo-mal c-myc genes. What structural alterations of the templateDNA occur concomitant with topoisomerase inhibition? Invivo footprinting was performed with KMnO4 to see whetherthe changes in transcription due to drug treatments are re-flected in the conformational state of promoter DNA se-quences. KMnO4 is most reactive with pyrimidines in single-stranded or otherwise conformationally distorted DNA (17, 27,38). Concomitant alterations or adjustments of protein con-tacts with melted or strained DNA would further modify thechemical reactivity of promoter and regulatory DNA. Aftertreatment with permanganate, cellular DNA was extracted,and the phosphodiester backbone at the modified bases wascleaved with piperidine; ligation-mediated PCR was then per-formed with gene specific primers to amplify and display thesites of altered KMnO4 reactivity.

The endogenous c-myc promoter region demonstrated dra-matic changes at the P2 promoter in response to topoisomer-ase II inhibition with adriamycin. Enhanced reactivity at mul-tiple specific bases on both strands extended 87 bases, from�34 upstream of the P2 promoter transcription start site to�53 downstream (positions 2456 to 2542, with the mRNA startat position 2490 [accession number X00364]) (Fig. 4A and B,lanes 3). Sequences further 3� displayed generalized hypore-activity, presumably reflecting the depletion of elongationcomplexes and consequently fewer transcription bubbles togenerate permanganate targets. Decreased sensitivity to per-manganate was also seen at the c-myc P1 promoter start site inresponse to adriamycin. Similar adriamycin sensitivity was ob-served on both the endogenous and episomal c-myc sequenceswithin the P2 promoter proximal region (Fig. 4A and B, lanes3 and 10). These data indicate that adriamycin freezes a hyper-open complex at c-myc P2 transcription start site but depressesopen DNA downstream. The promoter-frozen complexes arenot activated during nuclear run-on experiments and so musteither be inactivated or else have encountered an insurmount-able barrier.

Camptothecin, in contrast to adriamycin, elicited only subtlechanges in permanganate sensitivity from the chromosomalc-myc in Raji. Reactivity at several bases from positions 2450 to2501 surrounding the P2 start site was slightly reduced afterdrug treatment (Fig. 4B, lane 2). Opposite to the endogenousgene, camptothecin enhanced the KMnO4 sensitivity of down-stream-transcribed episomal template, indicating more elon-gating complexes (Fig. 4A, lane 9), a finding consistent with theincrease of CAT mRNA (Fig. 1, lanes 10 to 12). Butyrate (Fig.4B, lane 12) and trichostatin A (lane 13) augmented the per-manganate reactivity of residue �58 relative to residue �53 onthe nontemplate strand (positions 2547 and 2542, respectively)and increased the overall reactivity farther downstream on thetemplate strand (Fig. 4A, lanes 12 and 13); this pattern oftencorrelates with high-output states of c-myc. Thus, unlike theendogenous gene, histone deacetylase inhibition correlatedwith increased output and openness of episomal c-myc DNA.The footprints obtained upon combination of camptothecin

and adriamycin most closely resembled those obtained with thelatter alone (Fig. 4A and B, compare lanes 2, 3, and 4). Foot-print changes after topoisomerase I or II inhibition were notsimply a secondary result of transcription inhibition, sincetreatment with �-amanitin, which freezes transcription com-plexes in place, generated no specific response to KMnO4 atpromoters (data not shown).

c-myc upstream CT element becomes conformationallystressed after topoisomerase II inhibition. When stressed bytorsional strain, particular DNA segments adopt altered DNAconformation or structure. FUSE and CT are two such ele-ments upstream of active c-myc genes that are peculiarly sen-sitive to KMnO4 (38). If topoisomerase inhibition perturbs thedegree or distribution of torsional strain, then altered reactivityof FUSE and CT should follow. The permanganate footprintof the CT element, located 300 bp 5� of the MYC P2 start site,was dramatically altered after adriamycin treatment. The en-dogenous c-myc gene seen in the Raji footprint (Fig. 5A, lane3) shows several dramatically darker DNA bands correspond-ing to the T nucleotides of the three CT repeats closest to thepromoter; these same repeats are the preferred targets forhnRNP K binding in vitro. The two most upstream elementsexhibited sharply reduced reactivity; these sites are the pre-ferred binding sites for the transcription factor Sp1. Competi-tion between hnRNP K and Sp1 for binding in this region haspreviously been reported (38). Raji pMYC/CAT cells (Fig. 5A,lanes 7 to 9) showed only minor changes in this same region,due either to reduced reactivity in all plasmids or to heteroge-neous CT reactivity between plasmids; the number of activeepisomes at any instant is unknown. (By comparing the chro-mosomal and episomal exposure times, it was estimated thatthese cells have at least 20 copies of the episome [Fig. 5].) Theincreased reactivity of the CT element after adriamycin treat-ment might indicate a greater propensity toward melting, per-haps due to the failure of topoisomerase II to remove accu-mulated strain at the promoter and at the CT element 250 to300 bp upstream of the promoter.

In addition the AT-rich FUSE element, found 1,700 bp 5� ofthe MYC P2 start site, was examined in the Raji pMYC/CATcell line (data not shown). Here adriamycin treatment showedsubtle changes in the footprint. Several DNA bands with de-creased sensitivity to permanganate were seen within theFUSE element, indicating a more closed, double-strandedcharacter. The FUSE element in the parental Raji cell line wasnot examined because in the expressed c-myc allele the FUSEelement is translocated away from the myc coding sequence.The decreased reactivity of the FUSE element after adriamy-cin treatment might be due to repression of the episomalMYC/CAT gene, since a closed FUSE sequence is associatedwith a repressed c-myc gene (38). Camptothecin failed to mod-ify the permanganate footprints of both the FUSE and the CTupstream elements, indicating that topoisomerase I was notactive in these topological domains (Fig. 5A, lanes 2 and 6, anddata not shown).

Adriamycin alters the structure of the c-fos and hsp70 pro-moters. c-fos transcription was also examined by using nuclearrun-on assays. Promoter proximal segments of c-fos were un-dertranscribed relative to sequences that are more distal (Fig.2A, lanes 1 and 2, and Fig. 2B, lanes 1 to 3, slots 12 to 14 versusslots 15 and 16), a finding consistent with the transcriptional



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pause reported in murine c-fos intron 1 (44). In fact, the signalsarising from the 5�-most sequences of the transcript were dif-ficult to discern above the background. Adriamycin induced abiphasic response of the c-fos promoter. Within the first hourof treatment, transcription was increased, most noticeably atthe 5� end (Fig. 3B, lanes 2 and 3). Subsequently, transcriptionfrom the entire gene was shut down (Fig. 3B, lanes 4 to 6).Transient augmentation of c-fos transcription by camptothecinwas noted (Fig. 3A, lanes 2 to 6). Butyrate and trichostatin Aslightly elevated transcription at the c-fos start site (Fig. 2A,lane 12, and Fig. 2B, lanes 13 and 14).

The dramatic shutoff of c-fos due to adriamycin treatmentwas explored further by using KMnO4-LM-PCR in vivo foot-printing. Dramatic alterations in the promoter conformationwere caused by topoisomerase II inhibition. Increased reactiv-ity within the immediate vicinity of the start site was promi-nent, while just 3� of the start site on the bottom strand reac-

tivity was diminished (Fig. 6A, lanes 4 and 9). (The DNAregion shown in the c-fos footprint Fig. 6 maps to slots 11 and12 of the run-on transcription reactions shown in Fig. 2 and 3.)Farther downstream, the same intron 1 segments holdingpaused RNA polymerases detected with nuclear run-on (Fig. 2,slots 15 and 16, and Fig. 3, slots 13 and 14), showed bases withaltered reactivity (both increased and diminished), perhapsindicating that the drug treatment thwarted downstream tran-sit of complexes (data not shown). Consistent with this inter-pretation, the DNA from adriamycin-treated cells became ex-clusively hyporeactive distal to the downstream pause region(data not shown). As with c-myc, topoisomerase II inhibitionfreezes complexes at the c-fos promoter and prevents RNAsynthesis either by inactivating the transcription machinery orby imposing an insurmountable barrier such as accumulatedtorsional strain or drug-frozen topoisomerase II bound to theDNA template. It is also likely that unrelieved torsional strainperturbs the reactivity of downstream segments.

Also like c-myc, camptothecin evoked no clear changes inthe pattern of KMnO4 reactivity for c-fos (Fig. 6A, lanes 3 and

FIG. 4. Response of c-myc promoter structure to topoisomeraseinhibition. In vivo potassium permanganate footprint. (A) BottomDNA strand. (B) Top DNA strand. Cpt, camptothecin; Adr, adriamy-cin; TSA, trichostatin A. The cells were incubated with inhibitors for4 h. The arrow indicates the transcription start site. Bases made par-tially hyposensitive by camptothecin are present but are not annotated(panel B, lane 2). (C) Nucleotides with altered intensities in the foot-print are identified. Symbols: Š, hypersensitivity; �, hyposensitivity.The asterisk indicates reactivity at bases that were not exactly mappeddue to DNA compression in this region of the gel. The duplicate lanesare each from different independent experiments.



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8). Camptothecin perturbs c-fos transcription by elevatingbasal expression (as detected with RNase protection [Fig. 1,lanes 10 to 12] and with run-on assays within 3 h of drugtreatment [Fig. 3A, lanes 2 to 5]), while delaying and damp-ening induced expression (66). These effects occur withoutdisturbing promoter-DNA conformation. Therefore, it is likelythat the primary effect of this drug is exerted at the level ofelongation throughout the body of the gene.

Basal (non-heat shocked) hsp70 RNA levels were depressedby camptothecin (Fig. 1, lanes 10 to 12) but increased byadriamycin (Fig. 1, lanes 13 to 15) and HDAC inhibitors (Fig.1, lanes 19 and 20). hsp70 nuclear run-on transcription in-creased transiently within the first hour of camptothecin oradriamycin treatment and then declined by 4 h (Fig. 3, lanes 2to 6). Adriamycin developed an alternating hyphenated pat-tern of augmented and diminished permanganate reactivitythroughout the region of the paused polymerase (Fig. 7A, lane3). While on the opposite DNA strand, a single downstreamresidue (�9) (position 282, accession number M11717) be-came intensely reactive after adriamycin treatment (Fig. 7A,

lane 10). Thus, as with c-myc and c-fos, adriamycin seemed tocause increased hsp70 promoter occupancy after a transientincrease in promoter activity.

gapdh is often employed as a normalization standard. Con-sidering the relative stability of gapdh mRNA levels (Fig. 1)and the modest response of gapdh nuclear run-on activity (Fig.2 and 3) in the face of assorted pharmacological challenges, itmay be likely that several molecular devices cooperate to en-force homeostasis on this gene.

Topoisomerase I inhibition forces downstream stalling inrRNA transcription units. Humans have 200 to 300 copies ofthe 43-kb ribosomal DNA repeat unit, each transcribed from asingle promoter. Each 13-kb primary transcript consists of a3.6-kb 5� leader sequence, followed by a 1.9-kb 18S gene, a1-kb spacer, a 0.15-kb 5.8S gene, a 1.1-kb spacer, and a 5-kb

FIG. 5. Topoisomerase II inhibition provokes conformationalchanges at the CT-element of the endogenus c-myc gene. (A) In vivopotassium permanganate footprint. Cpt, camptothecin; Adr, adriamy-cin. The cells were incubated with inhibitors for 4 h. (B) Nucleotideswith altered intensities in the footprint are identified. Symbols are asdefined for Fig. 4. The duplicate lanes are each from different inde-pendent experiments.

FIG. 6. Topoisomerase II inhibition alters the structure of the c-fospromoter. (A) In vivo potassium permanganate footprint. Cpt, camp-tothecin; Adr, adriamycin. The cells were incubated with inhibitors for4 h. (B) Nucleotides with altered intensities in the footprint are iden-tified. Symbols are as defined for Fig. 4. The duplicate lanes are eachfrom different independent experiments.



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28S gene; the transcript encoding segments are separated by a30-kb spacer (accession number U13369). The long half-lifeand large pool of ribosomes and rRNA precursors bufferrRNA levels from rapid fluctuation. Therefore, failure ofcamptothecin to depress rRNA levels as measured with RNaseprotection (by using a probe at the rRNA transcription start)was not surprising (Fig. 8A, lanes 5 to 7), despite the well-established requirement of proper topoisomerase function forrRNA transcription (12, 75). More perplexing was the dramaticincrease of the rRNA nuclear run-on signal seen in response tocamptothecin (Fig. 2A, lanes 3 to 5, and Fig. 2B, lanes 4 to 6).Nascent rRNA synthesized by nuclear run-on was detected by

hybridization with sequences at the proximal segment of the18S rRNA, ca. 4 kb downstream of the start site. The confor-mation of the rRNA promoter before and after camptothecintreatment was assessed by using LM-PCR after permanganateoxidation of intact cells. Camptothecin induced no significantchanges of the rRNA promoter, suggesting that topoisomeraseI inhibition provoked no alteration of transcription complexesat most of the rRNA promoters (Fig. 8B, lanes 2 to 4). Themost straightforward explanation of these data requires thestalling of elongation complexes within the proximal one-thirdof the rRNA transcription unit after topoisomerase I inhibition.To test this possibility, nuclear run-on assays were performedby using nuclei from cells treated with camptothecin for vari-ous times; the nascent rRNA transcripts elongated in vitrowere hybridized with a battery of oligonucleotide sequencesderived from segments throughout the rRNA transcriptionunit. Indeed, the predicted holdback was observed in the prox-

FIG. 7. Topoisomerase II inhibition alters the structure of thehsp70 promoter. (A) In vivo potassium permanganate footprint. Analternating pattern of increased and decreased intensity near the hsp70transcription start site is seen. Cpt, camptothecin; Adr, adriamycin;TSA, trichostatin A. The cells were incubated with inhibitors for 4 h.(B) Nucleotides with altered intensities in the footprint are identified.Symbols are as in Fig. 4. The duplicate lanes are each from differentindependent experiments.

FIG. 8. Adriamycin, but not camptothecin, depresses rRNA pro-moter activity and alters promoter structure. (A) RNase Protectionwith a probe for the rRNA transcription start site. A 13-h Kodak XARfilm exposure and a 2-h exposure (inset) are shown. (B) In vivo po-tassium permanganate footprint. Cpt, camptothecin; Adr, adriamycin.The cells were incubated with inhibitors for 4 h. Symbols are as in Fig.4. (C) Nucleotides with altered intensities in the footprint are identi-fied. The duplicate lanes are each from different independent experi-ments.



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imal one third of the transcribed region (Fig. 3A, lanes 2 to 6).Similar over-representation of proximal rRNA gene sequencesin nuclear run-on assays has been noted previously (75). Initi-ated RNA polymerase I fails to penetrate very far into the bodyof the gene, perhaps due to accumulated torsional strain.

Adriamycin depressed rRNA levels in RNase protectionstudies (Fig. 8A, lane 8) and diminished rRNA synthesis asdetected by nuclear run-on (Fig. 2A, lanes 6 to 8, Fig. 2B, lanes7 to 9, and Fig. 3B, lanes 2 to 6). In contrast to topoisomeraseI inhibition, this topoisomerase II inhibitor augmentedKMnO4 reactivity at some nucleotides at the promoter whiledepressing oxidation at others, implying considerable reorga-nization of the template at the transcription start site (Fig. 8B,lane 5).

Transcription by RNA polymerase III of 7SK RNA in Rajicells was fully resistant to camptothecin and partly resistant toadriamycin as measured by nuclear run-on (Fig. 2A, lanes 3 to8, and Fig. 2B, lanes 4 to 9).

Effect of topoisomerase I and II inhibition on the state ofc-myc sequences in vivo. To assess more directly the influenceof adriamycin and camptothecin on DNA topology in vivo, theepisome from drug-treated Raji pMYC/CAT was recoveredand analyzed for linear, relaxed and/or nicked (relaxed/nicked), and supercoiled forms by Southern blotting. PlasmidpMYC/CAT extracted from bacteria provided the referencefor this analysis. As expected, the plasmid linearized with NotImigrated as a single band in the middle of the gel (Fig. 9B, lane12). The untreated plasmid revealed three bands: the fastest-migrating band represented supercoiled plasmid; the band ofintermediate mobility was the linearized plasmid, while the

slowest species was the relaxed/nicked plasmid (Fig. 9B, lane13). Figure 9C shows the DNA recovered from the RajipMYC/CAT cell line electrophoretically separated, blotted toa membrane and probed with the plasmid-specific CAT se-quence. DNA from untreated or DMSO-only treated cellsdisplayed a mixture of supercoiled and relaxed/nicked episo-mal DNA with minimal linearization (Fig. 9C, lanes 15 and16). Four hours of camptothecin treatment yielded the ex-pected conversion from supercoiled to nicked plasmid (Fig.9C, lane 17). Linear forms accrued only slowly because of thelow probability that two closely spaced single-stranded nicksoccurred on opposite DNA strands. In contrast to camptoth-ecin, adriamycin had the opposite effect. Adriamycin treatmentyielded more supercoiled and less relaxed episomal DNA (Fig.9C, lane 18). These data suggest that under the conditions usedin this study, adriamycin inhibited topoisomerase II prior toformation of the protein-DNA adduct cleavable complexes.(DNA fragmentation passes through a maximum and thendeclines as the concentration of adriamycin is increased [15,64].) Such inhibition would result in hypersupercoiled episo-mal DNA in contrast to the linearized DNA expected frompoisoned topoisomerase II-DNA complexes. The minimal lin-earization promoted by adriamycin on the endogenous c-mycin Raji (Fig. 9A, lane 4) or on the episome recovered from RajipMYC/CAT (Fig. 9C, lane 18) indicated that the transcrip-tional holdback and promoter remodeling occurring in re-sponse to this drug did not result from local DNA damage orrepair complexes. Moreover, the rapidity of these changesmight suggest direct involvement of topoisomerase II in main-taining a transcriptionally conducive chromatin environment.

FIG. 9. DNA damage is unlikely to account fully for the transcriptional response to topoisomerase inhibition. Southern blots showed that briefcamptothecin treatment yielded relaxed, but not linear plasmid. Adriamycin yielded hypersupercoiled episomal DNA. (A) Raji cells probed forc-myc exon 2. (B) Pure preparation of pMYC/CAT and the effects of topoisomerase I treatment. (C) Raji pMYC/CAT cell line probed with CATsequences. Cpt, camptothecin; Adr, adriamycin. The cells were incubated with inhibitors for 4 and 16 h.



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DISCUSSION

The experiments reported here reveal a protean and pleo-morphic response of transcription to topoisomerase inhibition.Increased or decreased torsional strain may alter transcriptionand may antagonize or synergize with chromatin modificationand remodeling. Nucleosomes with acetylated histones restrainand stabilize negative supercoils less tightly than when unmod-ified (39, 42). Chromatin remodeling machines generate andare influenced by torsional strain (14, 21). Thus, there is con-siderable potential for mechanical linkage and cross-regulationof all of these processes by using DNA as a force-bearing cable.The pattern of protein-tethering and DNA-looping coupledwith the distinctive properties of individual genes are likely todetermine the effect of topoisomerases and their inhibition ongene activity. RNA levels for particular genes may rise or fallin response to camptothecin and adriamycin treatment. Howmight topoisomerase inhibition differently influence the ex-pression of diverse genes? Several nonexclusive possibilitiesinclude: (i) disturbance of the distribution and transmission oftorsional strain; (ii) direct protein-protein interaction of topo-isomerases with transcription factors, the basal transcriptionapparatus or chromatin modifying or remodeling machineryaltering the expression of specific genes; (iii) immobilized to-poisomerase-inhibitor-DNA complexes forming a roadblockhindering the movement of transcription elongation complex-es; and (iv) topoisomerase inhibitors as DNA-damaging agentsindirectly modifying gene expression pursuant to the activationof the particular signal transduction pathways regulating DNArepair.

When constrained by physical barriers, transcriptionally gen-erated torsional strain diffusing behind and in front of a mov-ing transcription complex accumulates within topological do-mains unless dissipated by topoisomerases I and/or II (32). Sothe consequences of camptothecin and/or adriamycin treat-ment on gene expression should relate to the rate of transcrip-tion and the specific architecture of the affected targets. Theresults described here clearly show that the changes producedby these two drugs on mRNA levels, nuclear run-on rates, andon the conformation of promoters and upstream regulatorysequences are not equivalent (Fig. 10).

Camptothecin produces few if any detectable changes inpromoter architecture despite activating the transcription ofsome genes while repressing others. Therefore, topoisomeraseI more likely influences RNA synthesis away from the pro-moter, probably at the level of elongation. Topoisomerase Ihas been implicated in the penetration of elongation com-plexes into the body of a gene; c-fos, Drosphila hsp70, dhfr, andrRNA transcription are all accompanied by recruitment of to-poisomerase I to downstream sequences (25, 33, 66, 75). Fail-ure of topoisomerase I to remove the positive supercoils ac-cruing in front of RNA polymerase may lead to transcriptionarrest. Superhelical densities of ca. 0.1 generate sufficienttorque to oppose RNA polymerase translocation (74). Tran-scription within a large topological domain may proceed for aconsiderable distance before arresting forces are achieved; inthe absence of any release of torsional strain, elongationthrough 10% of the transcription unit would be required.Therefore, camptothecin inhibition would be predicted to stallpolymerases away from the promoter, within the body of the

gene, just as occurred with rRNA transcription. This explana-tion conceivably may explain the opposite response of thenative and the episomal c-myc promoters to camptothecin. If atopological boundary were imposed on the native gene withinexon I or at the 5� end of intron I, but not on the episome(which lacks these sequences), then minimal transcription ofthe cellular gene would generate sufficient force to hold backtranscription; without this barrier, episomal transcriptionwould progress further. Indeed, low levels of positive torsionmight even destabilize the wrapping of negative supercoils onnucleosomes to facilitate elongation transiently.

Adriamycin, in contrast, caused clear changes at all promot-ers analyzed. In addition, in the case of c-myc, topoisomeraseII inhibition increased the permanganate sensitivity of the up-stream CT element (Raji and Raji pMYC/CAT), in a regionlikely to experience strong negative supercoiling forces. In thecase of c-fos dramatic changes were present downstream of thetranscription start site, at the region of strongest run-on basalc-fos transcription. Thus, topoisomerase II inhibition altersDNA conformation most dramatically at and near promoters,at sites likely to experience strong unwinding stresses. South-ern blot analysis, in fact, visualizes the accumulation of nega-tive supercoils within the episomal DNA. Conformationalchanges at elements such as CT or FUSE may change thespectrum of bound factors at these elements, thus altering geneactivity.

Thus, topoisomerase II operates predominantly within a do-main embracing the promoter and nearby regulatory se-quences. Simultaneous inhibition of topoisomerase I and IIneither abrogated nor exacerbated permanganate reactivity,nor did it modify the nuclear run-on pattern, suggesting thateach enzyme operates in an architecturally separate topologi-cal domain during transcription. If topoisomerase I and IIenzymes have the same biological activity, then incubating cellswith both topoisomerase I and II inhibitors together shouldgive an additive or synergistic effect, rather than the adriamy-cin dominance seen in the run-ons and footprints or the com-promise between the two drugs seen with RNase protection.

Direct interaction of topoisomerases with the transcriptionfactors, the transcription apparatus or chromatin modifica-tion and/or remodeling machinery. How topoisomerases arerecruited to genes is unknown. Topoisomerase II is a compo-nent of nuclear matrix (13, 46). Topoisomerase I is a promoter-specific repressor of basal transcription operating throughTATA binding protein in vitro (37). Activators abolish thisrepression. This repression does not demand the camptoth-ecin-sensitive topoisomerase I catalytic activity, indicating thattopoisomerase I most likely plays a structural role at promot-ers. The drug, nevertheless, may interfere with the architectureof preinitiation complexes as topoisomerase I enhances assem-bly of the transcription apparatus at some promoters (63).Furthermore, topoisomerase activity might facilitate subse-quent stages of transcription, such as via interaction with RNApolymerase II (61). Overlying any general influence on basaltranscription is the pattern of recruitment of topoisomerase viaDNA-bound, sequence-specific transcription and chromatinmodifying and/or remodeling factors. A variety of factors re-ported to bind with topoisomerases I or II, including p53, jun,CREB, HTLV1-Tax, simian virus 40 T antigen, histonedeacetylase, Drosophila CHRAC, and pRb, are recruited to



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particular genes depending upon the particular assortment ofcis elements at a given promoter (5, 18, 23, 26, 47, 49, 68, 71,72). Interaction of these factors with topoisomerases may aug-ment or diminish topoisomerase activity. Reciprocally, mask-ing of transcription factor effector domains by interaction withtopoisomerases, although unreported, would seem possible.

In this scheme, if topoisomerases served solely as architec-tural components of transcription complexes, then topoisom-erase inhibitors might not alter transcription. Alternatively,topoisomerase inhibitors might lock the enzyme onto theDNA, providing a stable platform for recruiting factors. Forexample, increased recruitment of histone deacetylase by to-poisomerase II frozen on the DNA could repress gene activity.(The opposite actions of the deacetylase inhibitors butyrate

and trichostatin A on episomal versus chromosomal c-mycpromoters argue against this alternative.) Topoisomerase en-zymatic activity might help dissipate the strain generated by thewrapping of DNA in topological microdomains created bylooping of chromatin bound factors during transcription com-plex assembly or during chromatin remodeling (14, 21, 24, 70);topoisomerase inhibitors might then lessen the activity of thetranscription complexes dependent on enzyme activity for as-sembly. These conditions impose no a priori requirement atany particular gene for topoisomerases I or II to be recruitedupstream or downstream.

Drug-immobilized topoisomerases in principle might im-pose a downstream impediment to the passage of elongationcomplexes. However, such a mechanism does not provide a

FIG. 10. Summary of response to camptothecin or adriamycin treatment. Symbols: 1, increased transcription; 2, decreased transcription. A1 then2 combination indicates that in the run-on time course experiment, transcription was induced at the early time point but then repressedby the late time point. The dash (—) reflects no change in transcription. The black triangle (Š) indicates hypersensitivity, while the open triangle(�) indicates hyposensitivity in the in vivo potassium permanganate footprint. The combination “Š and �” indicates a mixed hypersensitivity andhyposensitivity in the in vivo potassium permanganate footprint.



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general explanation for drug effects because (i) drug-inducedupregulation of transcription is not explained and (ii) thecamptothecin-induced holdback at the promoter of the endog-enous c-myc gene and loss of nascent RNAs hybridizing withdownstream sequences indicates that distal elongating com-plexes cleared the gene (drug removal during the run-on assayshould have allowed measurable downstream activity).

Although topoisomerase inhibitors damage DNA, severalfactors argue that such damage is not directly responsible forthe transcriptional effects observed here. (i) In the case ofadriamycin, under the chosen conditions minimal damage ofeither the endogenous c-myc gene or episomal sequences oc-curred (15, 64). (ii) Drug treatments were quite brief; aftersuch brief treatment with topoisomerase inhibitors, cleavablecomplex formation is fully reversible upon drug removal (29,31). Cleavable complexes are converted into a site of DNAdamage after dissociation of the topoiosmerases frozen for aprotracted period at the strand breaks. During the brief inter-val of drug treatment, most cells would not transit S phase, andso replication forks would convert cleavable complexes intonicks or breaks (30) in only a minority of cells. (iii) Raji andRaji pMYC/CAT express only mutant p53 and so would notmount an effective apoptotic response (8). (iv) Microarrayclassification of the global gene response of 60 cell lines testedwith 118 compounds revealed that topoisomerase I inhibitors(including campthothecin) cluster together; likewise, varioustopoisomerase II inhibitors (including adriamycin) all clustertogether (59). These two clusters together form a superclusterdistinct from DNA-damaging agents. The data indicate thatthese drugs, including camptothecin and adriamycin, targettopoisomerase I or II to produce the observed biological re-sponse by mechanisms distinct from or in addition to second-ary DNA damage.

In addition to the implications for mechanisms for transcrip-tional regulation, these results have important implications forthe utilization of topoisomerase inhibitors as antineoplasticagents. When acting as topoisomerase poisons, topoisomeraseinhibitors damage DNA. This damage, in the face of normalhomeostatic mechanisms, leads to cell cycle arrest or apoptosisvia standard pathways. Acting as topoisomerase inhibitors,these drugs produce a protean genetic response, modifying theexpression of many genes. Chromosomal c-myc expression isparticularly susceptible to downregulation by topoisomeraseinhibition. Combinations of drugs acting in concert to up- ordownregulate special targets, such as c-myc, may allow themanipulation of genetic programs to enhance or retard theperformance and selectivity of a pharmacologic regimen. Mod-ification of the expression profiles of various genes would beexpected to alter cell growth and proliferation and hence drugsensitivity. For example, camptothecin kills cells in S phase; soif c-myc shutoff in some cells prevents entry into S phase, thenthese cells may be less susceptible to killing by this drug. Uti-lization of microarray data may help to identify and classifytranscriptional targets responsive to topoisomerase inhibitors,alone or in combination with other agents. One caveat to thisapproach is the relative insensitivity of microarray analysis torapid changes in the regulation of stable and/or abundant tran-scripts. Dramatic effects at the level of nuclear run-on or DNAconformation may precede by many hours correlative changesat the RNA level. Elucidation of the mechanisms generating

the regulatory diversity conferred by topoisomerase inhibitorswill emphasize the importance of the topological architectureof genes for physiological expression and afford opportunitiesfor pharmacological intervention to mediate the recruitmentand activity of the machinery controlling transcription andchromatin through protein-protein interactions.

ACKNOWLEDGMENTS

We thank L. Liotta, S. Mackem, and Y. Pommier for helpful com-ments, critical review, and fruitful discussions.

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2. Aller, P., C. Rius, F. Mata, A. Zorrilla, C. Cabanas, T. Bellon, and C.Bernabeu. 1992. Camptothecin induces differentiation and stimulates theexpression of differentiation-related genes in U-937 human promonocyticleukemia cells. Cancer Res. 52:1245–1251.

3. Amara, F. M., J. Entwistle, T. I. Kuschak, E. A. Turley, and J. A. Wright.1996. Transforming growth factor-�1 stimulates multiple protein interactionsat a unique cis-element in the 3�-untranslated region of the hyaluronanreceptor RHAMM mRNA. J. Biol. Chem. 271:15279–15284.

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Transcriptional Consequences of Topoisomerase Inhibition

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