Differential Effects of Amsacrine and Epipodophyllotoxins on … · solutions were made in dimethyl sulfoxide at 10 mM immediately before use. Further dilutions were made in deionized

[CANCER RESEARCH 52. 3125-3130, June I, 1992)

Differential Effects of Amsacrine and Epipodophyllotoxins on Topoisomerase IICleavage in the Human c-myc Protooncogene

Yves Pommier,1 Ann Orr, Kurt W. Rollii, and Jean-Francois Riou

laboratory of Molecular Pharmacology, Division of Cancer Treatment, National Cancer Institute, National Institutes of Health, Bethesda. Maryland 20892 [Y. P.,A. O., K. W. K.], and RhÃ´ne-Poulenc Rarer. Centre de Recherche de Vitry-Alfortville, 94403 Vitry-sur-Seine Cedex, France Â¡J-F.R.]

ABSTRACT

Amsacrine and demethylepipodophyllotoxins (etoposide and tenipo-side) are potent topoisomerase II inhibitors which have optimum activityin different cancers. To investigate whether these differences are due todifferent activity on cellular oncogenes, drug-induced topoisomerase IIcleavage sites were mapped and sequenced in the human i -mir protooncogene. In the presence of purified murine 1 1210 topoisomerase II,amsacrine induces prominent cleavage in the P2 promoter (site 2499/2502). Footprinting experiments indicate that topoisomerase II binds tothe entire promoter region (-20 base pairs on the sides of the P2 site).In the case of teniposide or etoposide, cleavage is more diffuse andmarkedly less at the P2 site. Mapping of cleavage sites in human smallcell lung carcinoma cells (NCI N417) also shows that cleavage in the P2promoter region is induced preferentially by amsacrine but not by demethylepipodophyllotoxins. Thus, selective gene damage among topoisomerase II inhibitors may contribute to differential anticancer activity.

INTRODUCTION

The c-myc protooncogene appears to play a central role inthe control of cell proliferation and differentiation (1-3). c-mycexpression is controlled by several mechanisms, includingchange in transcription initiation and elongation, RNA turnover, and translation (3-6). The deregulation of c-myc expression by amplification, chromosomal translocation, retroviralinsertion, or point mutation causes transformation and immortalization of normal cells (1, 3, 7, 8). The human c-myc protooncogene contains two promoters (PI and P2) separated byabout 165 base pairs and located near the 5' end of the first

exon (Fig. 1) (9). The transcription of c-myc is regulated by acomposite of positive and negative elements located both upstream and downstream from these promoters (5, 10-17). However, many of the cellular factors involved in the c-myc expression have not been characterized.

Eukaryotic DNA topoisomerase II is the cellular target of avariety of anticancer drugs including m-AMSA2 and the epi-podophyllotoxin derivatives, VP-16 and VM-26. These drugspoison topoisomerase II by stabilizing enzyme-DNA cleavagecomplexes (18,19). Amsacrine and epipodophyllotoxins inducedifferent cleavage patterns (20, 21), while VM-26 and VP-16induce similar patterns; VM-26 is more potent than VP-16(22-24). Previous studies have indicated that the c-myc oncogene may be 20-fold more sensitive to amsacrine-induced topoisomerase II inhibition than the overall genome, suggestingthat cleavage complexes induced in the c-myc oncogene may beimportant for antitumor activity (25).

Received 12/27/91: accepted 3/24/92.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

' To whom requests for reprints should be addressed, at Bldg. 37, Rm. 5C27.

National Institutes of Health. Bethesda. M D 20892.2The abbreviations used are: m-AMSA, amsacrine. 4'-(9-acridinylamino)-

methancsulfon-m-anisidide: VP-16, etoposide; VM-26, teniposide; SDS. sodiumdodecyl sulfate; 1 x TBE, 89 HIMTris-89 mM boric acid-2 mivi EDTA. pH 8.

In the present study, we have mapped and sequenced thecleavage sites of mammalian DNA topoisomerase II in the 5'

region of the human c-myc protooncogene. The data suggestthat topoisomerase II sites are critically positioned relative toc-myc transcription regulatory sequences and that differencesin DNA sequence selectivity of drug action may contribute tothe selective antitumor activity of topoisomerase II inhibitors.

MATERIALS AND METHODS

DNA, Enzymes, and Chemicals, c-myc DNA (plasmid pJB327) (Fig.1) was a gift from Dr. Jim Battey (Laboratory of Neurochemistry,National Institute of Neurological Disorders and Stroke, NationalInstitutes of Health, Bethesda, MD). Restriction endonucleases, DNaseI, T4 polynucleotide kinase, calf intestine phosphatase, agarose, andpolyacrylamide/bis were purchased from GIBCO BRL (Gaithersburg,MD) or from New England Biolabs (Beverly, MA). Â¡7-"P]ATP was

purchased from New England Nuclear Research Products (Boston,MA). DNA topoisomerase II was purified from mouse leukemia L1210cell nuclei as described previously and was stored at -70Â°Cin 40% (v/

v) glycerol-0.35 M NaCl-5 mM MgCl2-l mM ethyleneglycol bis(ÃŸ-aminoethyl ether)-./V,A',A'',A"-tetraacetic acid-l mM KH2PO4-0.2 mM

dithiothreitol-0.1 mM phenylmethanesulfonyl fluoride, pH 6.4. Thepurified enzyme yielded a single M, 170,000 band after silver stainingof SDS-polyacrylamide gels (26 27).

m-AMSA was obtained from the Drug Synthesis and ChemistryBranch, National Cancer Institute, Bethesda, MD. VM-26 and VP-16were obtained from Bristol-Myers Co. (Wallingford, CT). Drug stocksolutions were made in dimethyl sulfoxide at 10 mM immediately beforeuse. Further dilutions were made in deionized water. Doxorubicin and4-demethoxydaunorubicin (Idarubicin) were obtained from FarmitaliaCarlo Erba (Milan, Italy). Stock solutions were made in deionized waterat 0.2 mM and kept frozen at -20"C.

Preparation of End-labeled DNA Fragments. DNA fragments were5' end labeled as described previously (28, 29). Briefly, pJB327 DNA

was first linearized with one of the restriction enzymes shown on Fig.1, and then the 5'-DNA termini were dephosphorylated with calfalkaline phosphatase and labeled with Â¡7-12P]ATPusing T4 polynucle

otide kinase. Next, labeled DNA was cleaved with a second restrictionenzyme in order to generate fragments of different lengths, which wereseparated by agarose gel electrophoresis and isolated by electroelution.DNA fragments were purified by phenol-chloroform extraction andethanol precipitation between each step and at the end of the labelingprocedure.

Topoisomerase H-induced DNA Cleavage Reactions. DNA fragmentswere equilibrated with or without drug in 0.01 M Tris-HCl, pH 7.5-0.05 M KC1-5 mM MgCI2-0.1 mM EDTA-1 HIMATP-15 ^g/ml bovineserum albumin for 5 min before addition of topoisomerase II (40-70ng in 20 n\ final reaction volume). Reactions were performed at 37'C.

They were stopped by adding SDS to a final concentration of 1% andproti-illusi- K to 0.1 mg/ml, followed by incubation for l h at 42Â°C.

For agarose gel analysis, 3 u\ (10-fold) loading buffer (0.3% bromo-phenol blue-16% Ficoll-O.OI M Na2HPO<) was added directly to eachsample, which was then heated at 65"C for 1-2 min before loading into

1% agarose gels made in 1 x TBE buffer (28-30). Electrophoresis wasat 2 V/cm overnight.

For DNA sequence analysis, samples were precipitated with ethanol

3125

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c-myc GENE CLEAVAGE BY TOPOISOMERASE II INHIBITORS

and resuspended in 2.5 /ul loading buffer (80% formamide-10 mMNaOH-1 mM EDTA-0.1% xylene cyanol-0.1% bromophenol blue).Samples were heated to 90Â°Cand immediately loaded into DNA

sequencing gels [8% polyacrylamide-acrylamide:bis (29:1)] containing7 M urea in 1x TBE buffer. Electrophoresis was at 1500 V (60 W) for2-3 h.

The gels were dried on 3MM paper sheets and autoradiographedwith Kodak XAR-5 film. In some instances, dried gels were used toquantify DNA cleavage using a Betascope 603 blot analyzer (BetagenInc.). Computer analysis was performed using the Excell spread-sheetprogram.

DNase I Digestion. The reaction buffer was the same as that used inthe topoisomerase II reactions. For footprinting experiments, DNase Iwas added 15 min after the beginning of the topoisomerase II reactions.DNase I digestion was allowed to proceed for the indicated times, andreactions were terminated by adding 25 mM EDTA-1% SDS-0.1 mg/ml proteinase K (final concentrations). DNA was precipitated, andsamples were processed for DNA sequencing as described in the abovesection.

Analysis of in Vivo Topoisomerase II Cleavage Site. Human smallcell lung carcinoma NCI-N417 cells, which contain 40-50 copies of thec-myc protooncogene, were grown as floating aggregates in RPMI 1640

medium containing 10% fetal calf serum and antibiotics (31). AboutIO6cells in exponential growth phase were exposed to drugs for l h at37Â°Cin fresh culture medium. Cells were washed with 50 mM Tris â€¢¿�

HC1, pH 7.5-1 mM EDTA and immediately lysed with 1% SDS-50 mMTris â€¢¿�HC1, pH 7.5-25 mM EDTA. Proteinase K was added to a finalconcentration of 0.5 mg/ml for 12 h at 50Â°C.Lysates were then treated

with phenol, phenol-chloroform, and chloroform, and DNA was precipitated with ethanol, dried, and resuspended in 10 mM Tris â€¢¿�HC1, pH7.5-0.5 mM EDTA at a concentration of 2 mg/ml. DNA samples (10iig) were digested with Bglli restriction endonuclease and electropho-resed in 1.2% agarose gel. DNA fragments were transferred onto aHybond N membrane (Amersham). Hybridization and washing wereperformed in stringent conditions as described previously (25). Theprobe used was the 414-base pair Pstl fragment encompassing thegreatest part of the second exon (see Fig. 1). The probe was labeledwith [Â«-"PjdCTP and [a-"P]dATP (3000 Ci/mmol; Amersham) to aspecific activity of 3-7 â€¢¿�IO8dpm//jg using the nick translation technique(25). Hybrids were revealed by autoradiography on Amersham hyper-films MP.

Genomic Mapping of DNA Breaks. The genomic localization of thein vitro and in vivo drug-induced topoisomerase II-mediated DNAbreaks was determined as described previously (28-30, 32). Briefly,autoradiography films were scanned with a DU-8B Beckman spectro-photometer set at 555 nm, and the position and absorbance values weretransmitted to a computer. 32P-end-labeled DNA fragments were run

as markers and were used to determine the regression lines of thelogarithm of the fragment size (in base pair) versus the migrationdistance of each fragment. Regression coefficients were consistentlynear 0.99. Each autoradiography lane was analyzed by using the samereference line, and the size of each DNA fragment induced by topoisomerase II was computed. The reproducibility of fragment size determinations in different gels was usually within 50 base pairs. A finalcorrection was made to convert DNA fragment size to genomic positionby taking the Hindlll restriction site as position 1 (Fig. 1; 33, 34).

RESULTS

m-AMSA Induces a Major DNA Double-Strand Cleavage Sitein the P2 Promoter of c-myc DNA. DNA double-strand breaksinduced by purified murine leukemia topoisomerase II in thepresence or in the absence of m-AMSA were mapped in the c-myc plasmid pJB327 (Figs. 1 and 2). Fig. 2, A and B, showsthat m-AMSA induces cleavage in several discrete regions. Thecleavage pattern induced by m-AMSA differs from that induced

by topoisomerase II alone, implying that m-AMSA attackscertain topoisomerase II sites (20). Densitometer scanning ofthe autoradiographies was used to determine the position of thecleavage sites (20, 28-30, 32). A composite picture of the m-AMSA-induced cleavage sites was obtained by computer analysis to normalize the intensity distribution relative to a lineargenomic position scale (Fig. 2C). Cleavage is most intensearound position 2500, in the vicinity of the P2 promoter. Aseries of cleavage sites is also observed in the first intron, witha strong cleavage site around position 3200.

Differential Induction of DNA Double-Strand Breaks by m-AMSA and VM-26 in Purified DNA. Fig. 3 shows that VM-26and m-AMSA induce very different cleavage patterns. Insteadof inducing the P2 site as the case of m-AMSA (A), VM-26(lane V) or VP-16 (cleavage pattern similar to VM-26, data notshown) induce a large number of breaks that result in a smearof fragments smaller than full-size DNA. This was also the casefor topoisomerase II in the absence of drug (lane E), althoughcleavage was globally less intense than in the presence of drugs.Therefore, m-AMSA and VM-26 produce very different cleavage patterns in the 1500 base pairs corresponding to the 5'flank of the c-myc protooncogene.

Sequencing of the m-AMSA Cleavage Sites in the P2 Promoter. In order to sequence topoisomerase II cleavage sites inthe P2 promoter, the DNA was labeled at the Xhol site of thecoding strand (Fig. 1). Fig. 4 shows that the major m-AMSAcleavage site is at position 2499 of the DNA-coding strand, 10nucleotides downstream from the promoter start (lane A). Aweaker site is visible at position 2486, three bases upstreamfrom the promoter start. Another cluster of sites is foundimmediately upstream from the TATAA box sequence andadditional sites at specific locations (Fig. 4, lane A). Additionalexperiments using DNA labeled on the complementary strandat the Rsrll restriction site (see Fig. 1) showed that the mostintense cleavage site is at position 2502 on the transcribed

RM I2035

Pat IXb. I 48423507

i Pat I; 5058

Bgl II5385

Exon 1 Exon 2

Fig. 1. Schematic representation of the 5' region of the human c-myc protoon

cogene. Open boxes, exons 1 and 2; PI and P2, two promoters. The plasmid(pJB327) used for DNA cleavage reactions with purified DNA topoisomerase IIis shown. Filled boxes, polylinker used to clone the Rsal-Xbal c-myc fragmentinto pBR327 (dashed lines). Restriction sites are indicated and numbers correspond to their genomic positions (Hindlll restriction site is 1). Solid line (topright), probe used for in vivocleavage reactions.

3126




Fig. 2. DNA double-strand breaks inducedby DNA topoisomerase II in the presence andabsence of m-AMSA. EcoRl/Xbal DNA fragments (see Fig. 1) that had been uniquely 3:P-end labeled at one of their 5' termini (A,

EcoR\; B, Xbal) were reacted as indicated tothe left for 30 min at 37"C. Reactions werestopped by adding SDS and proteinase K (l'i

and O.I mg/ml final concentrations, respectively). Samples were run into 1% agarose gelsin TBE buffer, and autoradiography was performed. Marker sizes are, from left to right,1857, 1060, 929, 383, and 121 base pairs.Autoradiographies were scanned and genomicpositions of the breaks computed (numbers inA and B). Actual cleavage sites are usuallywithin 50 nucleotides of the computed values.C, composite graph of A and B in which intensity distribution is plotted against a linear genomic position scale.

B

TOPO II * m-AMSA

TOPO II no drugCONTROL

MARKERS

8

2000

n ' i r

2328P1

2490P2

EXON 1

2500 3000

GENOMIC POSITION

3500

Strand (data not shown). The 4-base pair staggering of sites2499 on the coding strand and 2502 on the transcribed strandindicates that these break sites correspond to the two cleavagesites of the DNA double-strand break that is detected undernondenaturing conditions around position 2500 (see Fig. 2).Thus, the major /w-AMSA double-strand break site in the P2promoter (Figs. 2 and 3) corresponds to a unique double-strandbreak at positions 2499 and 2502 on the coding and thetranscribed strands, respectively.

Doxorubicin (lane D) and its more potent analogue, 4-de-methoxydaunorubicin (Idarubicin, lane I) (32) induce prominent cleavage at the 2486 site and no cleavage at the 2499position. These two drugs also stimulate cleavage near theTATAA box. In agreement with the results shown in Fig. 3,VM-26 (lane V) induces many more sites than the other drugs.Topoisomerase II induces several weak cleavage sites (lane E)that coincide with those induced in the presence of drugs. The2499 site is the most intense site in the case of topoisomeraseII in the absence of drug, indicating that the enzyme binds ator near the promoter and that drug-induced cleavage resultsfrom the stabilization of enzyme cleavage complexes.

Relative Intensity of the m-AMSA-induced Cleavage Sites inthe P2 Promoter Region. The autoradiography shown in Fig. 4had been overexposed in order to show all the cleavage sitesinduced in the presence and absence of drugs. In order toquantify cleavage at the m-AMSA-induced sites, a dried gel

similar to that used to generate the autoradiography shown inFig. 4 was processed by betascope analyzer. Such an analysisshows that cleavage intensity is markedly more at site 2499 (atleast 10-fold) than at any other site within the 140 nucleotidesanalyzed (Fig. 5).

DNase I Footprinting of the 2499 wi-AMSA Cleavage Site inthe P2 Promoter. Fig. 6 shows that in the absence of topoisomerase II, DNase I produces a large number of cleavage sites ofdifferent intensities (lanes 1 and 2). Analysis of the protectionconferred by topoisomerase II in the presence of w-AMSA,upstream from the 2499 cleavage site shows a 20-nucleotideprotected region (lanes 3 and 4). This result is in agreementwith an independent determination in another DNA fragmentusing Drosophila topoisomerase II (35). If we assume thatsimilar protection would be seen on the other strand, then the

3127




C E V A M

-1060- 929

treated N417 cells reveals the presence of additional DNAbands of lower molecular weight that are not observed inuntreated cells and presumably correspond to topoisomerase IIcleavage sites. m-AMSA stimulates the formation of a strongcleavage site in the P2 promoter region in a concentration-dependent manner. By contrast, in the case of VP-16 (secondlane from left) and VM-26 (not shown), cleavage is more diffuseand markedly less in the P2 promoter region (Fig. 7).

P2 >- 383

-121

Fig. 3. Comparison of topoisomerase Il-mediated DNA double-strand breaksinduced in the absence of drug (lane E, enzyme alone) or in the presence of VM-26 (lane V) or m-AMSA (lane A). The EcoRl/Xbal fragment uniquely end labeledat the EcoR\ site was used (lane C, control). Reactions were performed asdescribed in Fig. 2. Lane M, molecular size markers (their size in base pairs isindicated at far right). Pi, position of the P2 promoter which corresponds to themajor m-AMSA-induced double-strand break site.

total binding region of topoisomerase II would be approximately 40 base pairs and would cover most of the P2 promoter.

In Vivo Cleavage Induced by m-AMSA and VP-16 in the c-myc Protooncogene of Human Small Cell Lung Carcinoma NCI-N417 Cells. NCI-N417 cells have 40-50 copies of the c-mycprotooncogene/cell (31). In previous studies, the c-myc genefrom cells treated with m-AMSA or epipodophyllotoxins hasbeen found to be cleaved at specific positions (25, 36, 37). Mostof these in vivo cleavage sites are located in the 5'-noncoding

region of the gene and close to DNase I-hypersensitive sites.However, the mapping strategy previously used did not permitthe localization of cleavage sites with a sufficient precision inthe region encompassing the first exon of c-myc. Here, we useda strategy that enabled us to analyze this region (Figs. 1 and 7).DNA double-strand breaks are measured after cell lysis atneutral pH. When DNA from untreated NCI-N417 cells isdigested with Bglll and probed with the 0.4-kilobase Pstl fragment of c-myc, a DNA band of 6.5 kilobases is detectable (Fig.7, left lane) that corresponds to the size predicted by thepublished sequence data (33, 34). The DNA pattern from drug-

DISCUSSION

The present study demonstrates that m-AMSA and epipodophyllotoxins (VP-16 and VM-26) produce very differentcleavage patterns in the human c-myc protooncogene. Both incell-free systems and in NCI-N417 cells, m-AMSA selectivelyinhibits topoisomerase II at the c-myc P2 promoter site, whileVM-26 or VP-16, but not m-AMSA, block topoisomerase II inmany other regions. Hence, selective gene damage by m-AMSAand demethylepipodophyllotoxins may contribute to the differential activity of these drugs in different tumors.

DNA sequence analysis and comparative compilation of alarge number of drug-induced cleavage sites in simian virus 40DNA has provided some insight into the molecular mechanismsresponsible for differential topoisomerase II inhibition by different drugs at different sites (21, 38). m-AMSA preferentiallyenhances topoisomerase II sites that have an adenine at one ofthe 5' termini of a topoisomerase II double-strand break, while

VM-26 or VP-16 generally enhance the sites which have acytosine at one of the 3' termini of the breaks (21). Preference

for one of the bases immediately flanking the drug-inducedcleavage site is also found for anthracyclines, in which caseadenine is always present at one of the 3' termini of the

topoisomerase II indiiceli double-strand breaks (38), and forellipticines, in which case there is a strong preference forthymine at one of the 3'-DNA termini (39). We have suggested

that the drugs bind inside one of the cleavage sites of a topoisomerase Il-mediated DNA double-strand break by stacking(21). Therefore, local base sequence selectivity of drugs togetherwith chromatin structure (29) probably contribute to cleavagespecificities of m-AMSA and epipodophyllotoxins in the humanc-myc protooncogene.

The strong m-AMSA-induced cleavage and binding site oftopoisomerase II in the P2 promoter was found both in purifiedenzyme-DNA systems and in drug-treated cells. This indicatesthat the enzyme binds to the P2 promoter region in chromatin.At this site, topoisomerase II may act as a negative regulatorof transcription by blocking the accessibility of the promoterregion. Another negative regulatory factor (/wye-binding protein1, Fig. 4) has been hypothesized to act in this way (16).Alternatively, topoisomerase II binding may facilitate transcrip-

Fig. 4. Sequencing of the topoisomerase IIcleavage sites induced in the P2 promoter. TheDNA was "P-end labeled at the Xhol site

(position 2394 of the coding strand). Reactionswere performed in the presence of the following drugs: A, + m-AMSA (10 MM);D, + dox-orubicin (0.5 ^M); /, + Idarubicin (4-deme-thoxydaunorubicin) (0.5 /IM); V, + VM-26 (10JIM); E, enzyme without drug. Dashed line,binding site of the /nyc-binding protein 1,which is a negative regulatory' factor for thehuman c-myc (16).

I I

MM I \MO 2477 (24UI I \ t I \ I

25112492 2499II I I I '111' CGCGCroAC6aaad^GCCGCTTTTCC<MXXTrTATCTAACTCGCTGTAGTAATTCCAGCGAGAGCCAGAGCCAGCGAGC GGGCGGCCGG

2452 MBP-1 24S2 n2490




5000

4000-

3000 â€¢¿�

2000-

1000-

2499

2440

2458

2461

JLi.

24862485

2565 261Â°

2680i . I

100 200 300 400 500 600

DISTANCE FROM THE BOTTOM OF THE GEL (mm)

Fig. 5. Quantitative analysis of m-AMSA-induced cleavage in the P2 promoterregion of the c-myc gene. A dried gel similar to that used to generate theautoradiography shown in Fig. 4 was scanned with a betascope analyzer, and thedata were computed with the Excell spread-sheet program. Numbers above eachpeak, cleavage sites indicated in Fig. 4 (sites 2565 and 2610 correspond to thetwo bands at the right end of lane A in Fig. 4). Positions 2440 and 2680 do notcorrespond to cleavage sites but to the boundaries of the examined region.

P2 >

-Â« 2.32-* 2.03-Â« 1.8

tion. This possibility is attractive because topoisomerase IIcleavage sites in the /3-globin gene locus coincide with DNaseI-hypersensitive sites. During chicken erythrocyte maturation,these two types of sites appear and disappear at the same time

2499

PROTECTION

2479

Fig. 6. Footprinting of the major m-AMSA cleavage site in the P2 promoter.DNA was 32P-end labeled at the Xhol site (same as in Fig. 4). Topoisomerase IIreactions were for 30 min at 37"C prior to DNase I addition (0.01 fig/ml for I,

3, or 10 min). Reactions were stopped by adding simultaneously 25 min EDTAand 1% SDS (final concentrations). Autoradiography lanes with comparableDNase I activity are as follows: lane I, DNase I alone, 0.01 Mg/ml, l min; lane 2,DNase I -I-topoisomerase II (no drug), 0.01 Mg/ml, 10 min; lane 3, DNase I +topoisomerase II + m-AMSA, 0.01 MI;ml, IO min; lane 4, DNase I + topoisomerase II + m-AMSA, 0.01 pg/ml, 3 min; lane 5, topoisomerase II + m-AMSA(no DNase).

Fig. 7. In vivo cleavage sites induced by m-AMSA and VP-16 in the c-mycprotooncogene of human small cell lung carcinoma NCI-N417. Cells were treatedfor 60 min at 37'C as indicated. The DNA was extracted, electrophoresed,

transferred to nylon filter, and hybridized to the second exon probe shown in Fig.1. The size (in kilobases) of DNA markers is indicated to the far right. The full-size DNA band at the gel origin is 6.5 kilobases.

upstream from transcribed genes (40). In addition, topoisomerase II preferentially forms cleavage complexes in the nuclearmatrix-associated region of simian virus 40 (30) and has been

shown to be a major component of the nuclear matrix (41)where actively transcribing genes are concentrated. In the caseof the human c-myc promoter, its binding to topoisomerase IImay facilitate its association with transcription factors. Enzymeactivity at this site may play a critical role in relaxing thenegative superhelical tension that is associated with transcription (42-45). Further experiments will be necessary to elucidatewhether topoisomerase II forms a multiprotein complex atpromoter sites and the exact role of the enzyme in transcription.

In conclusion, genetic machinery involved in cell proliferationcontrol is regulated by various DNA-binding proteins whichare often deregulated in cancer cells, some of them identifiedas oncogenes. Their sequence recognition requirements areunique, and it is now accepted that heterogeneity among varioustypes of tumors is associated with different patterns of deregulation of these DNA-binding proteins. The functional significance of the cleavage site differences among topoisomerase II

3129




inhibitors at the gene level may contribute to differential anti-cancer activity.

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14. Postel, E. H.. Mango. S. E., and Flint. S. J. A nuclease hypersensitiveelement of the human i- mir promoter interacts with a transcription initiationfactor. Mol. Cell. Biol., 9: 5123-5133, 1989.

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17. Saksela. K.. Koshinen, P. J., and Alitalo, K. Binding of a nuclear factor tothe upstream region of the c-myc gene. Oncogene Res., 6: 73-76, 1991.

18. Liu, L. F. DNA topoisomerase poisons as antitumor drugs. Annu. Rev.Biochem., 58: 351-375, 1989.

19. Pommier. Y., and Kolin. K. W. Topoisomerase II inhibition by antitumorintercalators and demethylepipodophyllotoxins. In: Glazer, R. I. (ed.), Developments in Cancer Chemotherapy, pp. 175-196. Boca Raton, FL: CRCPress, Inc., 1989.

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1992;52:3125-3130. Cancer Res Yves Pommier, Ann Orr, Kurt W. Kohn, et al.

ProtooncogenemycTopoisomerase II Cleavage in the Human c-Differential Effects of Amsacrine and Epipodophyllotoxins on

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Differential Effects of Amsacrine and Epipodophyllotoxins on … · solutions were made in dimethyl sulfoxide at 10 mM immediately before use. Further dilutions were made in deionized