Proc. Nati. Acad. Sci. USAVol. 82, pp. 7076-7080, October 1985Medical Sciences

Site-specific carcinogen binding to DNA in polytene chromosomes(immunofluorescence/autoradiography/benzo[alpyrene/transcription)

PAUL D. KURTH AND MICHAEL BUSTINLaboratory of Molecular Carcinogenesis, National Cancer Institute, National Institutes of Health, Bethesda, MD 20205

Communicated by 0. L. Miller, Jr., June 17, 1985

ABSTRACT Treatment of Chironomus polytene chromo-somes with the ultimate carcinogen benzo[a]pyrene diol epox-ide I or in vivo administration of the parent hydrocarbon tolarvae indicates that the carcinogen interacts with the genomein a nonrandom manner. Visualization of the carcinogen-DNAbinding sites by immunofluorescence reveals that, in vivo, somesites are preferentially modified. The combined effects ofDNAsequence, chromatin structure, and gene localization may leadto selective targeting ofcarcinogens to specific genomic regions.In polytene chromosomes the targeting effect is amplified,thereby making these chromosomes a uniquely suitable systemfor visualizing and studying site-specific interactions of carcin-ogens with the genome.

The critical step in the interaction of a chemical carcinogenwith the genome of a target cell, which may lead to trans-formation, is the formation of covalent carcinogen-DNAadducts. The generation of these adducts can be correlatedwith cellular mutation and transformation (1-3) and activa-tion of cellular oncogenes (4, 5). Since many ultimate car-cinogens are mutagens (1-3) and since the activation ofoncogenes is often associated with point mutations in theDNA sequence (6, 7), it could be expected that, in apopulation of cells, the frequency of mutation should beapproximately equal to the frequency of transformation.However, measurement of the frequencies of mutation andtransformation revealed that in some cases the frequency oftransformation was 10-1000 times higher than the frequencyof mutation (3, 8, 9). These results are especially puzzlingbecause cellular transformation is a multistep process thatmay require activation of more than one gene (10-12). Thepossibility that the high frequency of transformation is due tomany target genes has been ruled out by experiments thatrevealed that, in several independently derived chemicallytransformed mouse cell lines, the same gene was responsiblefor transformation (13).The unexpected difference between the frequency of

mutation and transformation could be explained by assumingthat the binding of a carcinogen to DNA is not random andthat, in fact, particular DNA sequences in the genomeconstitute preferential targets for carcinogen binding. Tar-geting of carcinogens to the DNA in the cell nucleus could bedue to DNA sequence effects (14, 15) or to various nucleo-protein structures found in chromatin. Indeed, it has beenreported that the linkerDNA between adjacent core particlesis more susceptible to carcinogen binding than the coreparticle DNA (16-20) and that transcribing genes are pref-erentially modified as compared to nontranscribing genes (14,21).We previously have demonstrated that serological tech-

niques can be used to study the distribution of the ultimatecarcinogen benzo[a]pyrene diol epoxide I (BPDE-1) in boththe cellular chromatin and in simian virus 40 minichromo-

somes (16, 22, 23). In the present manuscript we use polytenechromosomes from Chironomus thummi to study the distri-bution of this carcinogen in the genome after both in vitro andin vivo exposures. Polytene chromosomes are giantinterphase chromosomes active in transcription and replica-tion, consisting of >1000 individual chromatids, which areprecisely aligned. The size and structure of these chromo-somes provide an excellent system for studying the effect ofgene activity and chromatin structure on the distribution of acarcinogen in the genome. Furthermore, we have found thatChironomus thummi larvae can metabolize benzo[a]pyrene,thereby allowing examination of the carcinogen bindingunder in vivo conditions. The distribution of the radioactivecarcinogen along the chromosome can be detected by auto-radiography or by indirect immunofluorescence using anti-bodies specific for DNA modified by BPDE-1.

MATERIALS AND METHODS

Autoradiography and Determination of Grain Density.Polytene chromosome squashes prepared from isolated mid-fourth instar Chironomus thummi salivary glands as de-scribed (24) were overlaid with 50 A.l of [3H]BPDE-1 (ob-tained from the National Cancer Institute standard chemicalcarcinogen repository at a concentration of 1.66 ,umol/mlwith a specific activity of 220 p.Ci/,umol; 1 Ci = 37 GBq) in30% tetrahydrofuran. The incubation was stopped by im-mersing the slides in 100%o ethanol. Unbound reagent wasremoved by several washes of ethanol, and the slides wereprocessed for autoradiography using Kodak NTB-2 emul-sion. After 8 days, the slides were developed and photo-graphed under phase optics in a Zeiss photomicroscope III.The grain density was determined semiautomatically byobserving the slides in a Leitz Dialux 20 at x40 magnificationunder bright-field optics. The image was projected onto aHitachi television monitor connected to an Artek model 880micro/macro counting system. The electronic aperture wasadjusted to bracket a desired area on the chromosome. Therelative surface area and the number of grains in this area aredigitally displayed. In this manner it is possible to "travel"along the chromosome axis and count the number of grainsover various regions. The number of grains divided by thesurface area counted equals the grain density.Immunofluorescence. Squashes of polytene chromosomes

were treated for 1 hr at 24°C with 100 y1 of 0.15 M NaCI/10mM Na phosphate, pH 7, containing 0.3 mg of RNase per mlthat was devoid of DNase activity. After removal of RNase,the slides were processed for immunofluorescence as de-scribed elsewhere (24).Antibody Preparation. The preparation and characteriza-

tion of the antibodies against BPDE-1-DNA has been de-scribed (16, 22). The antibodies react with modified DNA andRNA but not with modified proteins or free benzo[a]pyrene.

Abbreviation: BPDE-1, benzo[a]pyrene diol epoxide I.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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RESULTS

In Vitro [3H]BPDE 1 Binding to Proteins and Nucleic Acidsin Bands. Initially, [3H]BPDE-1 was allowed to react withpolytene chromosome spreads, and the location of the boundcarcinogen was visualized by autoradiography (Fig. 1 A andB). Because BPDE-1 binds to a variety of cellular molecules(25, 26), the distribution of the autoradiographic grainsrepresents binding to both the nucleic acid and the proteincomponents of the chromosomes. The number of autoradio-graphic grains found over the chromosomes was dependenton both the concentration of [3H]BPDE-1 and on the lengthof time that the chromosomes were exposed to the carcino-gen. The kinetics ofthe interaction and the relative density ofthe autoradiographic grains over a particular region of thechromosomes were measured semiautomatically with an

Artek counter. Significant binding of BPDE-1 to chromo-somes occurred within 1 min after the addition of a 100 nMcarcinogen solution to the chromosome preparation. At thisconcentration of carcinogen, saturation of all binding siteswas reached within 60 min. Examination of the distributionof the autoradiographic grains along the axis of the variouschromosomes revealed that the grains did not tend to cluster

20-1

It~*-e-'n7

11~ccCoNZ 10L\

0 5 10 15GRAINS PER BAND OR INTERBAND

FIG. 1. Localization of carcinogen binding in polytene chromo-some squashes. Polytene chromosome squashes prepared fromisolated mid-fourth instar Chironomus thummi salivary glands asdescribed (24) were overlaid with 3H]BPDE-1 and processed forautoradiography. (A) Chromosome I treated with 0.10 mM BPDE-1for I niin #atO24°CABChooom raedwt .50fmMBDE-11 for30 min at 24C. (C) Distribution ofBPDE-1 grains over 64 bands and65 interband regions. The graph depicts a plot ofthe number ofbands(0) or interbands (o) containing a certain number of grains.

FIG. 2. Specific visualization of BPDE-1-DNA adducts by im-munofluorescence. (A and B) Chromosomes treated with 0.20 mMBPDE-1 for 10 min and stained with 1:100 dilution of anti-BPDE-1-DNA, which was preadsorbed with BPDE-1-DNA (gift from M.Poirier). (C and D) Chromosomes not treated with BPDE-1 stainedwith 1:100 dilution of anti-BPDE-1-DNA. (E and F) Chromosomestreated with BPDE-1 as in A and B and stained with 1:150 dilution ofanti-BPDE-1-DNA. (G and H) Chromosomes treated with 0.10 mMBPDE-1 for 2 min and stained with 1:200 dilution of anti-BPDE-1-DNA. All chromosomes were treated with RNase. The dot in F andarrow in G and H identify region Bic/e in chromosome III, which ispreferentially modified.

over any particular region. This was most apparent at earlyreaction times, when very few molecules of the carcinogenwere bound to the chromosomes (Fig. 1A). Examination ofhighly modified chromosomes (Fig. 1B) also revealed that thecarcinogen did not have preferential affinity for any identi-fiable locus in the genome.To determine whether the carcinogen preferentially binds

to the bands or interbands in the chromosomes, the numberof autoradiographic grains was measured in 64 bands and 65interbands present in 10 different chromosomes that wereexposed for 5-15 min to a 0.25 mM solution of I3PDE-1. Thedata presented in Fig. 1C indicate that the distribution of thegrains over the bands and interbands could be approximatedby Poisson distributions because they deviate from this typeof curve with g values of 0.01-0.02 and 0.02-0.05, respec-tively. Thus, the distribution of grains over these bands andinterbands was essentially random. A modal count of sixgrains for the bands and one for the interbands suggests thatthere is a significantly higher probability that the carcinogenwill interact with the bands. However, since it is known that

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in polytene chromosomes about 95% of the chromosomalmass resides in the bands (27), it can be concluded that thebinding is not strictly dependent on the chromosomal masspresent in these regions. It is possible that the binding of thecarcinogen is also dependent on the total surface area withwhich it can interact.

Specific Binding toDNA Visualized by Immunofluorescence.The autoradiographic studies visualized the binding of thecarcinogen to the proteins, RNA, and DNA of the chromo-some. The binding to nucleic acids could be distinguishedfrom the binding to proteins by using anti-BPDE-1-DNAantibodies in the indirect immunofluorescence technique.The antibodies used were those previously characterized byus (22, 23) and also preparations kindly provided by M.Poirier (28). The sera do not react with proteins modified withBPDE-1 or with the ligand itself. Binding to RNA is elimi-nated by RNase digestion prior to antibody treatment (24).The suitability of these antibodies for detecting carcinogen-modified regions in polytene chromosomes was furtherdemonstrated by the fact that BPDE-1-treated chromosomesdid not react with anti-BPDE-1 adsorbed on BPDE-1-DNA(Fig. 2 A and B) and that untreated chromosomes did notreact with anti-BPDE-1 (Fig. 2 C and D). Positive immuno-fluorescence was obtained only when BPDE-1-treated chro-mosomes were treated with specific antiserum (Fig. 2 E-H).Comparison of the fluorescence and phase-contrast patternof these chromosomes showed that generally the fluores-cence intensity was proportional to the density of the bandobserved under phase-contrast optics; however, the fluores-cence intensity of the various regions depended on theconcentration ofthe carcinogen and on the sera dilution used.At low carcinogen concentrations and high sera dilutions, thebands became more discrete, and differences in the intensityof fluorescence between various regions were obvious. Wenoted a particularly prominent band in region Bic/e ofchromosome III (Fig. 2 G and H). We conclude that thebinding in vitro of the carcinogen to the DNA is not randomand that region Bic/e of chromosome III is a preferentialtarget for carcinogen binding.

Site-Speciflic Binding in Vivo. The question arises whetherregion III Bic/e will preferentially bind antibodies under in

vivo conditions where the living organism actually metabo-lizes the parent compound benzola]pyrene. The ability ofChironomus thummi larvae to metabolize benzolalpyrenewas studied with the aryl hydrocarbon hydroxylase assay ofNebert and Gelboin (29), which was used to measure Cyto-chrome P-450 mixed function oxidase activity, and withHPLC, which was used to resolve the types of metabolitesproduced. Uninduced larvae exhibited a constitutive arylhydrocarbon hydroxylase activity of <0.1 pmol of 3-hydroxybenzo[a]pyrene formed per min/mg of protein.HPLC analysis of the metabolites of benzo[alpyrene (notshown) revealed that the major products were 1-, 3-, and9-hydroxy derivatives and the 9,10-, 4,5-, and 7,8-dihydro-diols (dihydrodihydroxy derivatives). The 7,8-dihydrodiol isthe precursor of the ultimate carcinogen BPDE-1 (30).To study possible site-specific carcinogen binding in living

Chironomus larvae, the unmetabolized, biologically inert,benzo[a]pyrene was labeled with 3H and added to Chirono-mus larvae. Autoradiograms of squashes prepared from thesalivary glands revealed that the polytene chromosomes wererandomly labeled in vivo, resembling the autoradiographicpatterns observed when the chromosomes were treated invitro with BPDE-1 (Fig. 1). In contrast, immunofluorescencestudies indicated that the binding of the BPDE-1, produced invivo from benzo[a]pyrene, to the DNA in the chromosomeswas highly targeted. Photomicrographs (Fig. 3) clearly dem-onstrate that the antibodies preferentially bound to regionBlc/e of chromosome III. In fact, the targeting of thecarcinogen to that region was significantly more specific invivo than in vitro. Interestingly, we found that prolonged(8-24 hr) exposures of the larvae to benzo[alpyrene, ratherthan yielding highly modified chromosomes, resulted inunlabeled chromosomes, suggesting that Chironomus larvaehave an active DNA repair system.

Preferential Carcinogen Binding to Transcribed Loci. Sinceguanine residues are potential targets for BPDE-1 binding, weinvestigated whether region Blc/e of chromosome III is aG-C satellite which, by virtue of its base composition, servesas a preferential target for carcinogen binding. The relativebase composition of the various regions in the chromosomewas studied by staining the chromosome either with

FIG. 3. Localization of the BPDE-1-DNA adducts in chromosomes isolated from larvae treated in vivo with benzo[a]pyrene. Late fourthinstar Chironomus thummi larvae were immersed in 1% dimethyl sulfoxide containing 200 ,uM benzo[a]pyrene (Sigma; recrystallized inmethanol). After a 30-min incubation, the larvae were washed with H20, salivary glands were isolated, chromosome squashes were prepared,and the squashes were processed for immunofluorescence. (A and B) Squashes treated in vitro with BPDE-1 as described in Fig. 2. (C-F)Squashes prepared from larvae incubated with benzo[a]pyrene. Note that the specific staining of region Bic/e of chromosome III (o) is morepronounced in the chromosomes modified in vivo than in vitro.

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quinacrine [a DNA-binding dye whose fluorescence is en-hanced when bound to A-T and quenched when bound to G-C(31)] or mithramycin, which is a dye whose fluorescence isenhanced when bound to G-C (32). The data presented in Fig.4 indicate that region Blc/e of chromosome III is notdisproportionately enriched in G-C sequences. The phase-contrast micrograph (Fig. 4A) shows that this region (openarrow) is puffed, as indicated by its wide, light, diffuseappearance. As noted by others (33, 34), the centerofthe puffcollapsed into a thin dark line. The immunofluorescence data(Fig. 4B) indicate that, when stained with anti-BPDE-1, thisregion fluoresced more brightly than the surrounding regions.Compared to other regions, the fluorescent band was notenriched in either A-T or C-G sequences (Fig. 4 C and D).The presence of a puff in region Bic/e of chromosome III

suggests a possible correlation between the intensity offluorescence with anti-BPDE-1 and chromosomal loci activein transcription. Loci active in transcription can be visualizedin these chromosomes by autoradiography after incubatingeither the isolated salivary glands or isolated chromosomeswith [3H]uridine (23, 25). Photomicrographs (Fig. 5) indicatethat the anti-BPDE-1 binding region Bic/e ofchromsome IIIis also one of the most active in transcription. Generally,there was a correlation between the staining intensity and thedensity of autoradiographic grains. Interestingly, region A2aofchromosome III (hollow arrow) was active in transcriptionyet did not display prominent antibody binding. Examination

FIG. 4. Base composition ofchromosome III. (A) Phase-contrastmicrograph of part of chromosome III containing region Blc/e. (B)Immunofluorescence of the corresponding region. (C) Fluorescencemicrograph ofa chromosome treated with 0.001% quinacrine (Sigma)in 50mM sodium acetate (pH 7.5) for 30 sec at 24°C. The fluorescencevisualizes the relative content of A-T pairs. (D) Fluorescencemicrograph of a chromosome treated with mitramycin (Sigma) in 50mM Tris4HCl, pH 7.5/15 mM MgCl2/33% ethanol. The fluorescencevisualizes the relative content of G-C pairs. The arrow points toregion Bic/e of chromosome III.

FIG. 5. Immunofluorescence with anti-BPDE-1-DNA correlateswith transcription. (A) Phase contrast micrograph of a region ofchromosome III treated in vitro with BPDE-1. (B) Correspondingfluorescence micrograph after treatment with anti-BPDE-1-DNA. (Cand D) Two examples ofcorresponding segments ofthe chromosomein which the loci active in transcription are visualized by autoradi-ography using [3H]uridine as described (24). The filled arrow pointsto region Blc/e; the open arrow points to region A2a, both ofchromosome III.

of the phase-contrast micrographs shows that this region wasrelatively condensed, as evidenced by the diameter of thechromosome. Therefore, the binding ofthe carcinogen seemsto be dependent on the chromatin decondensation associatedwith transcriptional processes rather than on the rate oftranscription.

DISCUSSIONThe results presented indicate that the binding in vivo of theultimate carcinogen to the DNA in chromosomes is notrandom. Immunofluorescent data reveals variability in thelevel ofmodification among various loci in the chromosomes.Region Bic/e of chromosome III, which is the most prefer-entially modified under in vitro conditions, is also the mosthighly BPDE-1-modified DNA region under in vivo condi-tions. The chromosomes were isolated from larvae that hadbeen continuously exposed to benzo[a]pyrene. The presenceofthe adduct in a particular chromosomal region reflects boththe tendency of a locus to be modified and the ability of therepair system to remove the adduct from that locus. Becauseregion Bic/e of chromosome III is a preferentially modifiedlocus under in vitro conditions, it would seem that theconcentration of the adducts in this locus reflects its prefer-ential modification rather than its resistance to repair.Our data support previous experiments with cloned DNA

fragments that indicated that carcinogens such as BPDE-1 orAFB1 can attack the DNA in a gene in a site-specific manner(14, 21). Furthermore, the binding of the carcinogen to theDNA in chromatin is not random; the internucleosomal linkerDNA is 3-5 times more accessible to modification than in thecore-particle DNA (17-20, 23). The DNA in transcribingribosomal regions seems to constitute a preferential target forcarcinogen binding, suggesting that perturbation in chroma-tin structure associated with gene activity or compartmen-

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talization of active genes in the nucleus around nuclearmatrixes (35) may significantly influence the manner in whichthe carcinogen binds to the genome. It may be significant thatregion Bic/e of chromosome III has a relatively high contentof Z-DNA (36). Thus, the data accumulated so far suggestthat the combined effects of DNA sequence, chromatinstructure, and gene localization in the nucleus may lead toselective targeting of carcinogens to specific genes. At thelevel of a single chromatid, these combined targeting effectsmay lead to a 4- to 10-fold preferential binding to a specificlocus over random binding to the rest of the chromatid. Sincein each chromatid the same locus is targeted over randombinding to the rest of the genome, the targeting effect isamplified by the level of polyteny of the chromosome. Thus,in a polytene chromosome, the ratio of specific binding overrandom binding could be amplified as much as 1000-fold,thereby making the polytene chromosome a uniquely suitablesystem for analyzing and studying site-specific interactions ofcarcinogens with the genome.

We thank Drs. M. Poirier and H. Slor for antibodies; Dr. M.Seidman for helpful suggestions; Drs. M. Seidman, L. Einck, and K.Kraemer for critical reading of the manuscript; and Ms. D. West andMr. D. Robinson for help with aryl hydrocarbon hydroxylase andHPLC assays.

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