(CANCER RESEARCH 51, 3857-3861. August 1. 1991]

Interaction between Ionizing Radiation and Supercoiled DNA withinHuman Tumor Cells'

Andrew T. M. Vaughan2, Anne M. Milner, David G. Gordon, and Jeffery L. Schwartz

Department of Immunology, University of Birmingham, Birmingham A/5 2TT, United Kingdom [A. T. M. K, A. M. M., D. G. G.], andBiological and Medical Division, Argonne National Laboratory, Argonne, Illinois 60439-4833 fJ. L. S.J

ABSTRACT

We have analyzed DNA supercoiling within histone-free nuclei (nu-cleoids) using four human squamous cell carcinoma cell lines that expressvarying degrees of radiosensitivity. The entire DNA, arranged as negativesupercoiled loops attached to the nuclear matrix, was extracted fromsingle cells, stained with ethidium bromide, and passed through a flowcytometer recording both light scatter and red (DNA) fluorescence.Supercoiled loops of DNA from all cells were unwound with a lowconcentration of ethidium bromide, as seen by increased light scatter.Nucleoids from radiosensitive but not radioresistant cells resisted thetransition from zero to positive supercoiling at higher concentrations ofethidium bromide. The profile of red DNA fluorescence from ethidiumbromide-stained nucleoids showed that the radiosensitive cells expresseda greater variation in the total amount of ethidium bromide bound. After12 Gy of ->-radiation, radiosensitive cell lines produced nucleoids that

contained a greater proportion of relaxed supercoiled DNA, making themlarger than those from radioresistant cell lines. We suggest these observations are secondary effects resulting from an altered affinity betweensupercoiled looped DNA and the nuclear matrix. Combined with radiationdamage, these structural alterations may lead to a more complex type ofdamage to repair within the radiosensitive cell lines.

INTRODUCTION

The reasons why radiotherapy may fail to achieve local tumorcontrol are still a matter of speculation. Theoretically at least,the presence of hypoxic regions within a tumor may provide asource of radioresistant cells. While such resistant cells may bea factor in some tumors, combinations of either hyperbaricoxygen or hypoxic cell sensitizers with radiotherapy have thusfar met with only limited clinical success (1, 2). More recently,there has been increased interest in the biological diversity ofindividual tumors as a factor which may determine tumorpersistence after therapy. Variations in cell proliferation rate,DNA damage repair, and intrinsic radioresistance have all beenproposed as explanations for local disease recurrence (3-5).The clinical interest in these factors has been stimulated by thepotential application of such knowledge to individual patients.This information, gained prior to therapy, would permit construction of treatment schedules tailored to the specific biological parameters of a patient's tumor. For example, rapidly

dividing tumors may best be treated with a short course offractionated radiation (6).

A number of workers have shown a correlation between thekilling of cells with radiation and either the rate of repair orabsolute numbers of DNA double-strand breaks (4, 7-9). Thisfinding is supported by the description of radiosensitive rodentcell lines that are also deficient in the rejoining of DNA double-strand breaks (10, 11). No such correlation has been found

Received 3/1/91; accepted 5/23/91.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.

' Supported in part by the Cancer Research Campaign, United Kingdom.2To whom requests for reprints should be addressed, at Loyola-Hines Depart

ment of Radiotherapy, 114B, Bldg. 1. HiñesVA Hospital. General Delivery,Hiñes,IL 60141-9999.

between radiosensitivity and other DNA lesions, such as DNAsingle-strand breaks or DNA-protein cross-links (9). The mechanism^) whereby DNA double-strand breaks lead to cell deathare still not fully understood. While it is clear that the majorityof DNA double-strand breaks are repaired, breaks that remainmay produce a disruption of normal cellular activity. In particular, the process of DNA replication, especially mitosis, isknown to be acutely sensitive to the effects of ionizing radiation(12). In this study we have examined the effect of radiation onthe repetitive loops of supercoiled DNA that are implicated inthe process of DNA replication (13).

DNA replication is initiated at the base of supercoiled loopsof DNA, replicons, attached to the nuclear matrix. Synthesisproceeds as coordinated waves of DNA replication throughoutthe nucleus (13, 14). It is possible to examine this level oforganization by extracting from cells DNA still attached to thenuclear matrix, and unwinding the negatively supercoiled loopswith ethidium bromide (15, 16). At a concentration of approximately 5-10 fig/ml ethidium bromide, the loops lose super-coiling and are completely unwound. Higher concentrations ofthe intercalator continue to impose a winding torque, introducing positive supercoiling and contracting the size of intact loops,but not those which contain broken DNA. Therefore at highethidium bromide concentrations nucleoids from irradiatedcells will be larger than controls due to the presence of damagedDNA loops (17). These variations in nucleoid size can bemonitored by centrifugation, microscopy, or, in our case, flowcytometry (17-19). Using the latter technique, the amount oflaser light scattered from individual nucleoids is recorded asthey pass through a focused laser beam.

In a previous study we analyzed the organization of super-coiled DNA within the Chinese hamster V79.379A cell line(20). These cells are more radiosensitive when grown in mono-layer culture than as mult ¡cellularspheroids (21). Nucleoidsextracted from radiosensitive cells were associated with both agreater increase in size after irradiation and a larger variationin the amount of ethidium bromide bound per nucleoid. Weconcluded from this data that the organization of supercoiledlooped DNA had changed in association with the change ingrowth form and radiosensitivity. In the study reported here wehave examined the organization of supercoiled DNA using fourhuman squamous cell carcinoma cell lines of different intrinsicradiosensitivities. To achieve this, we have measured the effectof both radiation and ethidium bromide intercalation on DNAsupercoiling within nucleoid structures.

MATERIALS AND METHODS

Cell Lines. The squamous cell carcinoma lines SCC-25, SQ-9G, andSQ-20B were derived from patients with tumors of the head and neckwho had failed conventional radiotherapy treatment (5). The cell lineSCC-12B.2 was derived from a patient with squamous cell carcinomaof the skin, isolated after immunosuppressive treatment following renaltransplantation (22). Each cell line was routinely grown as a subcon-fluent monolayer in 70% Dulbecco's modification of Eagle's mediumand 20% Ham's F12, supplemented with 10% fetal calf serum, 50

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SUPERCOILED DNA AND RADIOSENSITIVITY

units/ml penicillin (Sigma, St. Louis, MO), 50 Mg/ml streptomycin(Sigma), and 0.5 Mg/ml hydrocortisone (Calbiochemi, San Diego, CA).For each experiment, a flask of the cell line was rinsed once in salinesolution and trypsinized for approximately 10 min in 0.25% trypsinand 0.03% EDTA in Hanks' balanced salt solution (Sigma). Prior to

use, each sample was washed once in complete medium and resus-pended in fresh medium.

Cytometry Analysis. Approximately 1 x IO6 cells were placed into

small plastic test tubes, pelleted by centrifugation, and irradiated on icewith a 60Co-7 source. Immediately prior to analysis the supernatant

was aspirated, and the cells were mixed with 1 ml of lysis buffercontaining 2 M NaCl, IO mividisodium EDTA, IO mM Tris buffer, and0.1% Triton X-100. Triplicate samples of irradiated and control cellswere stained with 0-50 Mg/ml ethidium bromide immediately beforeanalysis. Data from 10,000 cells were accumulated using a BectonDickinson FACS 440 flow cytometer, set to collect low-angle forwardlight scatter. Data acquisition was triggered by either red (DNA)fluorescence or, when not stained with ethidium bromide, forwardscatter. Forward scatter and red (DNA) fluorescence histograms wererecorded for each sample. To analyze the light scatter response themean of the forward scatter distributions was used. Some cell sampleswere also suspended in a 0.6% Nonidet 40:0.2% bovine serum albuminin phosphate-buffered saline solution and stained with 20 Mg/ml ethidium bromide, and a conventional cell cycle was recorded.

supercoils, contracting their size below that of the native unstained material and therefore scattering less light.

In addition to the light scatter response the red (DNA)fluorescence profiles from nucleoids were also recorded (Fig.2). This distribution is collected in a manner identical to thatfor a conventional cell cycle. In each case, nucleoids extractedfrom the radiosensitive cell lines gave rise to a more dispersedfluorescence distribution. To compare the individual fluorescence profiles the dispersion of the Gìpeak is defined as theratio of peak height divided by the width of the Gìdistributionat half-maximum peak height. Nucleoids from both radiosensitive cell lines give rise to a decreased value for this parameter,

Table 1 Biological parameters of the cell lines usedThe radiosensitivity D0 is recorded as the average of 3-5 observations. The

DNA index is the DNA content of each cell line referenced to normal fibroblasts;% S phase was determined by flow cytometry. All data have been reportedpreviously (27).

CelllineSCC-25

SQ-9GSQ-20BSCC-12B.2D0

(Gy)1.42

1.462.392.66DNA

index1.93

1.531.731.88<Z

SPhase38.8

±2.311.1 ±1.237.3 ±1.942.1 ±3.1

RESULTS

The radioresistant classification used here is a relative termbased on normal fibroblast sensitivity; for these cells a range ofDo values between 0.89 and 1.75 Gy has been found (23). Thetotal DNA content, referenced to normal DNA fibroblasts, theproportion of cells in S phase, and the amount of DNA presentall show no radiosensitivity-related differences (Table 1). Thislatter finding is supported here by the DNA fluorescence histograms shown in Figs. 2 and 3; radiosensitive and resistantlines overlap in the amount of DNA fluorescence expressed.

Nucleoids analyzed by flow cytometry generate more lightscatter per particle than either cells or nuclei/' This finding is

consistent with relaxation of the supercoiled DNA after removalof both cell and nuclear membranes, generating a larger targetarea for the laser interaction. Inspection of the mean lightscatter response from nucleoids of each line shows that theSCC12-B.2 line scattered less light than the other three (Table2). As the total DNA content of SSC-12B.2 cells is not substantially different from that of the other cell lines, SCC12-B.2nucleoids may contain a more compact arrangement of DNA.

The response of nucleoids from each cell line to titration withethidium bromide is shown in Fig. 1. The biphasic expansionand contraction of nucleoids in response to ethidium bromideexposure is due to the relaxation of negative DNA supercoilsat low concentrations and the subsequent imposition of positiverewinding at higher concentrations. This was one of the firstobservations which indicated that DNA was organized intorepetitive loops anchored to an immobile base (13, 15, 16).Here, at high ethidium bromide concentrations (>30 /ug/ml),nucleoids from the radiosensitive lines fail to be contracted tothe same extent as those derived from radioresistant cells. Atthe highest concentration of ethidium bromide used, nucleoidsfrom radiosensitive cells scattered the same amount of light asthose from unstained material. Thus in these nucleoids, afterthe negative supercoils have been unwound, excess ethidiumbromide is able to reimpose only the same amount of positivesupercoiling. Nucleoids from the radioresistant cells are notsimilarly constrained, and these continue to accumulate positive

3A. T. M. Vaughan, unpublished observations.

Table 2 Nucleoid light scatter and fluorescenceThe coefficient G, peak height/full width half-maximum is a measure of the

dispersion of the G, distribution shown in Fig. 2 and is derived by dividing theG, peak height by its width at half-maximum peak height. Low values representa greater dispersion. The light scatter recorded is the average value (±1SE) ofthree separate experiments »herethe mean light scatter distribution was measuredfrom unstained nucleoids.

CelllinesSCC-25

SQ-9GSQ-20BSCC-12B.2G,

peak heightfull width

half-maximum11.5

16.531.043.8Mean

forwardlight scatter(channels)240.7

±9.9228.3 ±8.8256.6 ±7.3172.8 ±8.1

125

100

75

50

25

-25

-5010 20 30 40 50 60

Ethidium bromide (

Fig. I. Nucleoids were obtained as described in the text and stained withethidium bromide. Columns, data from one of three experiments. The means ofeach forward scatter distribution are plotted after the subtraction of the meanvalue from unstained nucleoids. Bars, SE from triplicate samples. Cell lines: SCC-12B.2 (0): SQ-20B: (H): SQ-9G (Q); and SCC-25 (@).

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SUPERCOILED DNA AND RADIOSENSITIVITY

-Q

E

TJ'o

600 r

500 -

400 -

300 -

200 -

100 -

160

Ethidium bromide fluorescence (channels)

Fig. 2. Fluorescence histograms from nucleoids stained with 50 fig/ml ethidiumbromide. , nucleoids derived from cells of radioresistant origin; left, SQ-20B;right, SCC-12B.2. , nucleoids from radiosensitive cells; left, SCC-25; right,SQ-9G. Comparable traces were seen in all nucleoid experiments.

1000

800

600JD

E

= 400

200

40 80 120 160

Ethidium bromide fluorescence

Fig. 3. Cell cycles obtained from either SQ20B ( ) or SQ-9G ( ) drawnon the same scale as the nucleoid profiles in Fig. 2. The cell cycles from the othercell lines gave similar profiles, omitted here for clarity.

indicating a higher dispersion of the G\ distribution (Table 2).No comparable differences were seen between the cell cyclesrecorded from ethidium bromide-stained cells (Fig. 3).

After a single dose of 12 Gy 7-radiation to viable cells, allcell lines produced ethidium bromide-stained nucleoids thatscattered more light and were assumed to be larger than controls. We have previously shown in human and rodent cells thatthere is an approximately linear dose-response relationshipbetween the irradiation dose and nucleoid light scatter (19, 20).This expansion was seen at all ethidium bromide concentrations; however, the differential between control and irradiated

cell lines is greatest at the higher concentrations, when all intactloops are contracted to their maximum extent (19). Nucleoidsfrom irradiated radiosensitive cells fail to be condensed byethidium bromide as effectively as those of radioresistant origin,leading to a larger increase in light scatter (Fig. 4). Inspectionof the original histogram data indicates that for the cells studied, the increase in mean light scatter is produced by an effectin all nucleoids, rather than a large effect on a smallsubpopulation.

DISCUSSION

Ethidium bromide is a DNA-specific intercalating agentwhich exerts a concentration-dependent strain on the DNAdouble helix, producing a straightening of the molecule. Thisforce unwinds the repetitive loops of negatively supercoiledDNA attached to the nuclear matrix, replacing them at higherconcentrations with positive supercoils (13, 14). Using time-delayed autoradiography of radiolabeled DNA precursors, Vo-gelstein et al. (13) have shown that new DNA is synthesized atthe base of these loops which then gradually moves to the loopperiphery as DNA synthesis proceeds. The site of DNA synthesis initiation on the nuclear matrix has been documented as thelocation of a multienzyme complex, the reputase, containingamong other enzymes DNA polymerase «and topoisomeraseII, all associated with the replication of DNA (24). By definition, the enzyme-rich reputase location must also be closelyassociated with the site for DNA attachment to the nuclearmatrix and represents the fixed location for each DNAsupercoil.

In the data presented here, nucleoids respond to increasingethidium bromide concentrations by first scattering more light,then less (Fig. 1). This result is characteristic of the changefrom negative to positive supercoiling. For both the radiosensitive cell lines, the contraction in nucleoid size as measured by

50

40

30

20

10

SCC-12B.2 SQ-20B SQ-9G SCC-25

Fig. 4. Each cell line was irradiated with 12 Gy; both these and an unirradiatedsample were analyzed by flow cytometry. The percentage increase in the mean ofthe forward light scatter distribution is compared to control samples. Bars, SE offour independent experiments carried out in triplicate. Each radiosensitive cellline (SCC-25; SQ-9G) gives rise to nucleoid scatter data that is significantlydifferent from each radioresistant line (SQ-20B; SCC-12B.2) as shown by theMann-Whitney U test: P = 0.05.

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SUPERCOILED DNA AND RADIOSENSITIVITY

light scatter does not proceed beyond the size of the freshlyextracted, unstained material. One conclusion from this data isthat the imposition of positive supercoiling does not continuebeyond a mirror image of the original amount of negativesupercoiling. No similar constraint is seen in the radioresistantlines, which continue to contract in response to higher concentrations of ethidium bromide, similar to our previous findingswith human lymphocytes (19). It is unlikely that the size of theindividual DNA loops is the cause of this difference. A difference in loop size would affect the maximum expansion of thenucleoids, because there would be different amounts of DNAin each loop to unwind. No consistent difference in absoluteexpansion is seen here. A more likely explanation is that thenucleoids from the radiosensitive cell lines contain DNA thathas a greater freedom of movement at the site of its insertioninto the nuclear matrix. At high concentrations of ethidiumbromide a progressively greater coiling torque will be generatedwithin each loop, applied across the loop/matrix anchoragesite. Any relative movement between the DNA and the matrixwill limit the torsional energy in each loop, and the loop willbe restricted in the degree of supercoiling possible (15, 16). Ifthe insertion of the DNA into the nuclear matrix is physicallyassociated with the reputase complex, as seems likely, such anincrease in relational movement between the DNA and thereplication enzymes may affect the process of DNA replicationitself (25).

Inspection of the nucleoid fluorescence profiles from unirra-diated cells also shows a difference between radiosensitive andradioresistant cells (Fig. 2 and Table 2). Both radiosensitivelines exhibit nucleoid DNA histograms that are more dispersedthan those from resistant cells. A similar observation was notedwithin nucleoids derived from the radiosensitive growth formof V79.379A cells (20). In each case, conventionally recordedcell cycles showed no such radiosensitivity-related differences.In addition, no evidence of disproportionate loss of DNA fromthese nucleoids was detected from either cell system. Becausethese histograms are produced from the sum of individualnucleoid events, it would seem that each nucleoid from radiosensitive cells exhibits a greater variability in binding ethidiumbromide than that from radioresistant cells. We have suggestedpreviously that the alteration in fluorescence distributions maybe linked to both differences in chromatin structure, limitingthe accessibility of ethidium bromide to the DNA and/or theability to repair DNA double-strand breaks (26). It is not clearat this time time what are the limiting factors which determinethe binding of ethidium bromide to nucleoid DNA.

Considering the effect of radiation damage in these cells, bothradiosensitive cell lines release nucleoids after irradiation thatare less well condensed by ethidium bromide than those of theradioresistant lines (Fig. 4). Although it has been found thatthese relatively radioresistant cell lines repair double-strandbreaks faster than the radiosensitive lines, it is unlikely thatcellular repair is involved here, since the nucleoid assay iscarried out entirely at ice temperature (4, 5, 22). Nevertheless,with the neutral elution technique, the two radiosensitive celllines do accumulate more DNA double-strand breaks at low(5-25 Gy) induction doses, although the difference is small. Inaddition, during neutral elution experiments, the radiosensitivelines are also more prone to DNA fragmentation without anyprior irradiation (27). It is unlikely that the double-strandbreaks are directly responsible for the differential compactionof irradiated nucleoids. Alterations in supercoil compactionafter irradiation are thought to be due primarily to the insertion

of single-strand breaks in the DNA (17). DNA double-strandbreaks are relatively rare events and are unlikely to be thepredominant species affecting supercoiling in a background ofnumerically greater numbers of single-strand breaks.

Instead, it is suggested that the reduced ability of ethidiumbromide to compact nucleoid supercoils in both control andirradiated DNA is related to a physical weakness at the site ofthe DNA insertion into the nuclear matrix. This possibility issupported by the observation that a radiation-induced breakwithin one DNA loop affects the ethidium bromide windingand physiological behavior of more than just that loop (17, 28,29). To have such an effect, the damage must be propagatedthrough the locations of DNA attachment to sites distant fromthe original lesion. The reduced compaction of nucleoids fromradiosensitive cells after irradiation may therefore be due to anincrease in the number of affected supercoiled loops, leavingmore loops unable to be rewound by the ethidium bromide.Thus the increased number of DNA double-strand breaks detected within the radiosensitive cell lines after irradiation andtheir reduced rate of repair may be a consequence, not a cause,of the alteration in chromatin stability.

Results very similar to the nucleoid scatter data presentedhere have been obtained by other workers using a range ofhuman and rodent cell systems. Kapiszewska (18) has studiedradiosensitive and radioresistant mutants of murine lymphomacells. Here, the radiosensitive cell line LY5178S produces nucleoids that fail to be compacted as effectively as those fromthe resistant, LY5178R line. Also, in a comparison between theinherently radiosensitive human ataxic telangiectasia fibro-blasts with cells derived from normal individuals, the ataxiatelangiectasia cells demonstrated a larger nucleoid expansionafter irradiation (30). Finally, in studies with lymphocyte systems using a centrifugation technique to quantify loop domainsize, a correlation has been made between radiosensitivity anda larger effective DNA domain size (31, 32).

Using a different technique, Cramp (33) has studied theassociation of newly synthesized DNA with its template strand.Nascent DNA from radiosensitive cells was found to be preferentially separated from its partner DNA in comparison toradioresistant cell lines, suggesting a physically weaker association between new DNA and its template within radiosensitivecells. These data indicate differences related to radiosensitivityinvolving the structure or process surrounding DNA synthesisinitiation at the nuclear matrix site, the same location that isimplicated in radiosensitivity changes in this study.

As discussed previously, the site of DNA insertion into thenuclear matrix is associated with the reputase complex containing the DNA unwinding enzyme topoisomerase II. Both theLY5178S line and the double-strand break repair-deficient mutant, xrs-5, are cross-sensitive to the topoisomerase II inhibitor,VP 16 (34, 35), as is the radiosensitive human line SCC-25studied here.' This correlation may simply highlight a defect inDNA double-strand break repair, because VP 16 stabilizes anintermediate form of the enzyme which surrounds a DNAdouble-strand break. Alternatively, it is possible that the structural weakness we propose at this location may involve alterations to this enzyme or the reputase enzyme complex of whichit is a part (24).

The consistency of the data reported here with that of otherworkers indicates that there may be a common theme underlying the differences in nucleoid behavior. Cell lines for which anenhanced nucleoid response to radiation has been seen includeboth those containing a defect in DNA double-strand break

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SUPERCOILED DNA AND RADIOSENSITIVITY

repair and those where no such defect is as yet recognized. Thedata we have generated strongly implicate chromatin structureas a common factor associated with radiosensitivity. A structural weakness at any DNA location, but particularly at thecritical site of DNA synthesis initiation, may increase thepossibility of errors in repair. This would apply equally to cellswith documented genetic repair defects and to those, such astumor cells from different patients, carrying out normal DNAdouble-strand break repair. If we are correct in this interpretation of the effect of chromatin structure, it would imply thatthe measurements we document here may be a useful generalindicator of the ability any cell may have at repairing potentiallylethal double-strand breaks.

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1991;51:3857-3861. Cancer Res   Andrew T. M. Vaughan, Anne M. Milner, David G. Gordon, et al.   within Human Tumor CellsInteraction between Ionizing Radiation and Supercoiled DNA

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