(CANCER RESEARCH 48, 5805-5809, October 15, 1988]

Bromine-80m Radiotoxicity and the Potential for Estrogen Receptor-directedTherapy with Auger Electrons1

Eugene R. DeSombre,2 Paul V. Harper, Alun Hughes, Ronnie C. Mease,3 S. John Gatley, Onofre T. DeJesus,4 and

Jeffrey L. SchwartzBen May Institute [E. R. D., A. H.J, Franklin McLean Institute [P. V. H., S. J. G., O. T. D.J, and Department of Radiation Oncology ¡J.L. S.J, University of Chicago,Chicago, Illinois 60637; and Argonne National Laboratory [R. C. M., O. T. D.], Argonne, Illinois 60439

ABSTRACT

While theoretically feasible, estrogen receptor (ER)-directed radiotherapy of hormone-dependent cancers has not been realized because noER-seeking ligand with an appropriate radiotoxic potential has beenidentified. Since an appropriate nuclide is a key component we studiedthe 4.4-h half-life, Auger electron-emitting nuclide bromine-SOm. Whenincorporated into DNA this nuclide was radiotoxic to cells in culture andcaused substantial chromosomal damage, while similar concentrations ofbromine-SOm as bromide, or bromoantipyrine were without effect. Themean lethal dose for bromine-80m was 45 atoms per nucleus which isconsistent with use in receptor-positive cancers with limited numbers ofER.

INTRODUCTION

The concept of ER5-directed radiotherapy was suggested 6

years ago by Bronzert, Hochberg, and Lippman who treatedER+ MCF-7 cells in culture with [I25l]16a-iodoestradiol and

showed reduced cloning efficiency of the cells stored frozen toaccumulate sufficient disintegrations (1). However, the longhalf-life (60 days) makes iodine-125 a poor nuclide for such usein patients. There is good evidence that electrons emitted in thenucleus as a result of nonradiative Auger and Coster-Kronigprocesses can be highly effective in causing double strandedDNA breaks (2-4) with minimum radiation hazard outside theaffected cells (5, 6). Since estrogen, when complexed with ER,is tightly associated with nuclear DNA and chromatin (7-10),such a ligand is an attractive vehicle to carry an Auger electron-emitting isotope to the nuclei of ER+ cancer cells. An appropriate estrogen receptor-directed ligand must have a sufficientlyshort half-life to decay while associated with the ER, known toitself have a biological half-life of only around 4 h (11). Wehave recently reported the synthesis of several estrogens labeledwith bromine-80m (12, 13), a nuclide with a half-life of 4.4 hwhich, on the basis of its Auger electron-emission spectrum(14, 15), would be expected to be highly radiotoxic whenassociated with cellular DNA. However to establish the feasibility of such an approach using a bromine-80m-labeled estrogen it is necessary to demonstrate the radiotoxicity of thisnuclide, and especially to determine that the number of decaysper cell needed for cell killing is compatible with the numberof ER molecules found in ER-positive cancers.

Received 3/16/88; revised 7/6/88; accepted 7/15/88.The costs of publication of this article were defrayed in pan 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.

1These studies were supported by the NIH (CA27476 and HD1S5I3), DOE(contract W-31-109-Eng-38), and by the Julius J. Reingold Fellowship Fund.

1To whom requests for reprints should be addressed, at the Ben May Institute,

University of Chicago, 5841 South Maryland Avenue, Chicago, IL 60637.' Current address: Medical Division, Brookhaven National Laboratories, As

sociated Universities, Inc., Upton, Long Island, NY 11973.4Current address: Department of Medical Physics, University of Wisconsin

Medical Center, 1300 University Avenue, Madison, WI 53706.5The abbreviations used are: ER, estrogen receptor; CHO, Chinese hamster

ovary; BrdUrd, 5-bromo-2'-deoxyuridine; TCA, trichloroacetic acid; MEM, min

imal essential medium; pen/strep, penicillin plus streptomycin; PBS, phosphatebuffered saline.

MATERIALS AND METHODS

Materials. Chemicals were reagent grade or better. UnlabeledBrdUrd, thymidine, aphidicolin, colcemid, hematoxylin, and crystalviolet were from Sigma Chemical Co., St. Louis, MO. The 100-mmplastic culture plates were from Corning, 6-well plates were from LinbroDivision of Flow Laboratories, McLean, VA, and slide culture plateswere from Lab-Tek Division of Miles Laboratories, Napierville, IL.MEM, McCoys F12, insulin, fetal calf serum, penicillin, streptomycin,and glutamine were from GIBCO, Grand Island, NY. Emulsion NTB3was from Kodak. TCA, Giemsa, and Gurr buffer were obtained fromFisher Scientific, Chicago, IL.

Bromine-SOm Labeled Compounds. Bromine-SOm was prepared byeither the 32Kr(d, n, a) 80mBrreaction (12) or the »°Se(p,n) 80mBrreaction (13). [80mBr]-4-Bromo-1,2-dihydro-1,5-dimethyl-2-phenylpyr-azol-3-one (bromoantipyrine) and [80mBr]-5-Bromo-2'-deoxyuridine([80raBr]BrdUrd)were prepared from antipyrine and 2'-deoxyuridine as

reported elsewhere (16) in detail. Briefly they were prepared by oxidation of bromine-SOm bromide, in the presence of the antipyrine or 2'-

deoxyuridine with yV-chlorosuccinimide in either l M sulfuric acid for10 min at SO'C (BrdUrd) or at room temperature with 2 N HC1 in

acetonitrile (bromoantipyrine), followed by separation of the labeledproducts on a Cm reversed-phase high-performance liquid chromatog-raphy column, eluting with mixtures of acetonitrile and water. Theradioactive peaks were collected and concentrated by evaporating thesolvents at 50"C under a stream of nitrogen. Specific activities were

determined by relating the measured radioactivity in the eluted radioactive product to the normalized UV absorption of the respectiveproduct on the high-performance liquid chromatography tracing. Ra-diochemical yields varied from 50 to 90% for these compounds invarious preparations.

MCF-7 Studies. MCF-7 cells were plated at 1000 cells per 100-mmdish, 24 h prior to exposure to [80nlBr]BrdUrd,specific activity, 465 Ci/mmol; [80l"Br]bromoantipyrine, specific activity, 2000 Ci/mmol; sodium [80l"Br]bromide or control medium [Dulbecco's MEM with 5%

fetal calf serum, 100 I¡/nilpenicillin, 100 iig/rnl streptomycin (pen/strep) and 2 /ug/nil insulin] each in triplicate. Additional controlsincluded dishes incubated with 0.5 /uniol unlabeled BrdUrd (10 timesthe highest concentration of the radioactive BrdUrd) and the inclusionof 1 HIM unlabeled thymidine with the 3 ¿tCi/mlconcentration of[80mBr]BrdUrd, the former to assess possible light-induced damage due

to BrdUrd itself under the actual experimental conditions used, thelatter to determine whether incorporation of bromine-SOm into DNAwas essential for radiotoxicity. The dishes were handled in a darkenedtissue culture hood and kept in an incubator with aluminum foil overthe glass door to minimize exposure to light. After 16 h at 37°Call the

media were replaced with fresh control medium and the dishes returnedto the CO2 incubator for colony growth. After 14 days the cells wererinsed, fixed with methanohacetic acid (3:1), and stained with crystalviolet. Colonies with more than -50 cells were counted and the resultscalculated as the ratio of mean number of colonies in treated culturesto the number in control cultures.

CHO Studies. Clone AA8 of CHO cells were plated in pentuplicateat 350 cells per dish in McCoy's F12 medium supplemented with 10%

fetal calf serum, 2 HIMglutamine and pen/strep and incubated with theindicated concentrations of [""""BrjBrdUrd, specific activity 540 Ci/

mmol, in the dark for 2 or 18 h. In parallel CHO cells, plated at 10,000cells per chambered slide or 100,000 cells per well, were also incubatedfor 2 h with ["'""HrjBrdUrd to assess the labeling index of the cells inthe chambered slides and to assay TCA-precipitable incorporation of

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BROMINE-80m RADIOTOXICITY

bromine-80m with the multiwell plates, respectively. After the incubations the experimental media were removed by aspiration and controlmedium added. The 100-mm dishes for colony assays were returned tothe darkened incubator to grow for 7 days and the colonies were fixed,stained, and counted as described for the MCF-7 cells. The slide cultureswere immediately rinsed in PBS, fixed, washed in PBS, and dried. Theywere then dipped in Kodak NTB3 emulsion at 38'C in the dark, allowed

to dry and exposed overnight in a light-tight box. The emulsion wasthen developed, and the cells stained with hematoxylin. At least 100cells were counted from each of several slides to calculate the proportionof cells with silver grains over nuclei (labeling index). Parallel slidecultures using ['Hjthymidine to assess the labeling index gave similar

results after 1week of exposure. To assess the incorporation of bromine-80m into DNA, parallel incubations were carried out with 100.000 cellsper well in triplicate in 6-well plates. After the 2-h incubations the cellswere washed, exposed for 15 min to trypsin, scraped, and rinsed withPBS containing 1 mM unlabeled BrdUrd into 1.5-ml Eppendorf tubesand centrifuged in the cold. After a second PBS wash the cells wereresuspended in PBS, sonicated, and precipitated by adding an equalvolume of cold 20% TCA. After 20 min in ice to allow precipitation,the tubes were centrifuged in the cold, decanted, washed with cold 10%TCA, decanted, and the TCA-precipitated pellet counted in a gammacounter.

The studies with synchronized cells were carried out in a similarfashion except that the cells were synchronized by mitotic shake off,held at the Gl/S border by incubating with 2 ¿ig/mlaphidicolin, andreleased from the cell cycle block by replating in fresh medium about 4h prior to incubation with [80mBr]BrdUrd, specific activity 1180 Ci/

mmol. Colonies were counted after 8 days.Chromosomal Analyses. Exponentially growing CHO cells (subline

AA8) were seeded at 5 x IO6 cells in 20 ml McCoy's F12 medium

supplemented with 10% fetal calf serum. 2 HIMglutamine. 100 units/ml penicillin, and 100 Mg/ml streptomycin for 24 h prior to use. Thecontrol media were replaced with similar media containing the indicatedconcentration of [80mBr]BrdUrd(specific activity, 540 Ci/mmol) aloneor with l IHMunlabeled thymidine. After 2 h at 37°Cthe cells were

washed and resuspended in 20 ml of control medium with 0.2 fiMcolcemid. At 2-h intervals for 10 h mitotic cells were collected byshaking and treated with 0.075 M KC1 for 15 min to spread thechromosomes before fixing in methanol:acetic acid (3:1). The chromosomes were stained in a 2% solution of Giemsa in Gurr buffer. Onehundred cells per treatment were analyzed for chromatid-type aberrations, classified as chromatid or isochromatid deletions (distinguishedfrom gaps by displacement of the chromatids) and chromatid exchanges. Inter- and intra arni and chromatid-isochromatid exchangeswere scored individually, but combined for data analysis.

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0.3 0.4

Fig. 1. Cytotoxicity of bromine-80m in MCF-7 cells. MCF-7 cells wereexposed to """BrdUrd (solid line), [80"Br]bromoantipyrine (dotted line), or Na"""'Hr (dashed line) at the concentrations indicated, for 16 h as described in"Materials and Methods." After replacing the media the cells were grown for 14

days in the dark, and the resultant colonies stained and counted. Results presentedas the ratio of the number of colonies in the treated (S) and control (50) cultures.

5806

Fig. 2. Survival curve of CHO cells treated with ("""BrlBrdUrd. Top, cellswere treated with ["""BrlBrdUrd for 2 h (x), for 18 h (x in circle) alone, or in the

presence of 1 HIMunlabeled thymidine (A, TdR). Dashed line, approximates theextension of the curve derived from the low concentrations (< S ¿iCi/ml).Labelingindex was found to be 29%. Bottom, synchronized CHO cells as described in"Materials and Methods." Labeling index was found to be 75%.

RESULTS AND DISCUSSION

Fig. 1 shows the survival curve of MCF-7 cells, treated for16 h with [80mBr]BrdUrd. The toxicity is clearly due to the

incorporation of the brominated nucleoside into DNA since thepresence of excess thymidine, which inhibited the incorporationof the labeled precursor into the TCA-precipitable fraction, alsoinhibited its effect on survival. Labeled bromide, which is excluded from viable cells, and bromoantipyrine, which distributesuniformly in cells, were without substantial effect. Furthermore,while nonradioactive BrdUrd incorporated into DNA is knownto destabilize DNA subject to light-inducible DNA breaks (17),unlabeled bromodeoxyuridine, at 10 times the highest concentration of [80inBr]BrdUrd used, did not reduce the survival of

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BROMINE-SOm RAD1OTOXICITY

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Time After Treatment (hours)

14 6 8 IO 13 14 16 18 20 22 24 26 28

Dose (MCi/ml)

Fig. 3. (80mBr]BrdUrd-induced chromosome damage in CHO cells. Left, time course of damage. Mitotic cells were obtained by shaking at the times indicated,following a 2-h exposure to the concentrations (as ^Ci/ml) indicated (to the right of the curves) of ["""BrlBrdUrd. The dotted line gives the percentage of maximumcell labeling with [3H]thymidine to illustrate the timing of the S phase. Right, chromosome breaks related to the dose of ["""BrlBrdUrd at 10 h. O, data from

incubations including 1 HIMcold thymidine.

Table 1 Induction ofchromatid-type aberrations in CHO cellsCHO cells were exposed to different concentrations of [*0mBr]BrdUrd for 2 h

and then washed and cultured for 10 h before harvest and analysis of aberrationfrequency. The results presented are the number of chromatid breaks (Breaks),isochromatid breaks (ISO), and chromatid-type exchanges (Exchanges) per cell.Included are also the fraction of cells with more than 10 aberrations per cell (>/0ABS/cell) and the per cent of cells with aberrations (% with AUS). 100 cells perexperiment were counted.

Dose % withBreaks ISO Exchanges >10 ABS/Cell ABS

02.33.56.726.72.331.2+

dThd°+dThd"0.032.092.674.204.350.120.080.020.190.300.190.410.000.020.000.090.180.460.350.000.020.000.010.130.150.210.000.003.576.075.584.093.212.08.0

°dThd, 1 mM unlabeled thymidine.

the cells (data not shown). Therefore it is clear that bromine-80m, like other Auger electron emitters, is radiotoxic whenincorporated into cellular DNA.

Because the 36-h cell cycle of the MCF-7 cells made dosim-etry studies difficult with the short-lived [80mBr]BrdUrd, furtherstudies used 2-h incorporation with the AA8 Chinese hamsterovary cell line which has a 16-h cell cycle (Fig. 2, top). Thesurvival curve was biphasic with an asymptotic initial curve atabout 70% survival showing no shoulder, consistent with Augerelectron effects. It is not entirely clear what the reasons are forthe biphasic nature of the survival curve. Autoradiography ofcells in slide culture, also treated for 2 h established that thelabeling index was 29%, in good agreement with the apparent

asymptote of the first curve at about 70% survival. Althoughthe bromine-80 daughter decays by emission of two hard betas(approximately 2.0 and 1.38 MeV), the radiation dose calculated for such low linear energy transfer radiation from thehighest concentration during the 2-h exposure is only 60 rads(assuming uniform distribution of the ßparticles and a 1-cmmaximum range), insufficient by itself to explain the > 90%cell kill. However it is also likely that nuclide incorporated intocellular nucleotide pools, not washed out by media changes,could explain, in part, the additional cytotoxicity at higherdoses. Since cells for the labeling index were washed and fixedat 2 h and only cells with nuclear labeling counted, they wouldonly reflect the proportion of cells which had incorporated thelabeled precursor into DNA by the end of the 2-h exposure.The cells for colony assay, no longer incubated with the labeledprecursor, would continue to incorporate nuclide remaining inthe nucleotide pool, although at decreased efficiency due todecay and dilution of labeled nucleotide triphosphate from thecomplete medium. In the presence of excess thymidine onewould expect inhibition of killing due to both the nuclideincorporated into DNA and incorporation of the nuclide intothe nucleotide triphosphate pool but not due to the generaleffect of the energetic ßemissions of the daughter bromine-80.As seen in Fig. 2, top, even at > 30 MCi/m! [80mBr]BrdUrd, 1

HIM unlabeled thymidine increased the survival to > 90%.Indeed, the general cell labeling background was seen to bereduced substantially in the autoradiograms of cells incubatedwith [80mBr]BrdUrd in the presence of unlabeled thymidine.Furthermore, in the experiment in which MCF-7 cells were

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BR()MINE-80m RADIOTOXICITY

*v, A •

,Fig. 4. Effect of ("""BrJBrdUrd on chromosome breakage. Left, normal CHO metaphase; right, metaphase from CHO cells exposed to 26.7 »iCi/ml[*°™Br]-

BrdUrd for 2 h followed by culture for an additional 10 h before, harvest and chromosome analysis.

exposed for 16 h (Fig. 1), by the end of which time > 90% of age was induced after DNA replication. While over half of thethe nuclide would have decayed, such a secondary effect wasnot seen. On the other hand, when the CHO cells were exposedto (""""BrJBrdUrd alone for 18 h, more than one cell cycle, even

at a lower concentration, 3.5 jiCi/ml, survival was less than10~3 (Fig. 2, top), as would be expected when all cells would

have incorporated the nuclide into DNA.To increase the proportion of cells incorporating the nuclide

in a 2-h pulse, AA8 cells were synchronized prior to treatment,Fig. 2, bottom. Again the plateau of the survival curve wasconsistent with the labeling index (75%). The greater toxicityin this experiment allowed direct estimation of the mean lethaldose (i.e., the 37% survival dose) at about 40 femtocuries percell, corresponding to about 45 molecules per cell after correction for labeling index. This compares favorably with the reported D37 for bromine-77 of 130 fCi/cell (6). Similar estimation of the D37 for the first curve by extrapolation of theinitial linear portion of the curve for the experiment shown inFig. 2, top, was about 50 molecules per cell.

Effects of the short [80l"Br]BrdUrd exposure at different times

in the cell cycle were examined. The frequency of chromosomalaberrations dramatically increased with time, and the peakeffects for each of the four doses of [80mBr]BrdUrd used was 10h after treatment, which corresponds to mid-S phase as shownby the [3H]thymidine incorporation curve, Fig. 3, top. Thatinduction of aberrations by [80mBr]BrdUrd mirrored [3H]thy-midine uptake suggests that [80mBr]BrdUrd acts after incorpo

ration into the DNA. This is supported by the observation thatthymidine inhibited the induction of aberrations (Fig. 3, bottom). Furthermore, as shown in Table 1, most of the aberrationswere of the chromatid type, suggesting that chromosome dam-

cells exposed to 26.7 ¿/Ci/ml[80mBr]BrdUrd had aberrations 2

h after treatment, it is likely that this was not an Auger electroneffect. These cells were not in S phase during exposure andcocultivation with cold thymidine failed to significantly reducethe aberration frequency (Fig. 3, top) unlike the cells in S phaseas shown in Table 1.

While [80mBr]BrdUrd exposure increased chromatid-type

breaks, isochromatid breaks and exchanges, the most prevalentform of damage was chromatid-type breaks, Table 1. Thenumber of breaks per cell increased linearly as the dose increased from 0 to 6.7 ¿¿Ci/ml[80mBr]BrdUrd (Fig. 3, bottom).

Although there was little further change as the dose rose above6.7 ííCi/ml,the number of cells with more than 10 breaks percell did increase from 15 to 21% as the [80raBr]BrdUrd concen

tration was changed from 6.7 to 26.7 pCi/ml. In fact, nearly12% of the cells examined in the 26.7 ¿¿Ci/mltreated cultureshad too many aberrations to score accurately (Fig. 4). Therefore, the failure to see an increase in the frequency of breaksinduced at the high dose may simply reflect an inability to scorethe more heavily damaged cells. It is also likely that cells withgreater numbers of aberrations might not be able to progressfrom S phase to mitosis or alternatively might be delayed intheir entry into mitosis.

We conclude from these studies that bromine-80m, whenincorporated into DNA, is clearly radiotoxic. This is consistentwith the considerable body of evidence for the effectiveness ofAuger electron emissions from iodine-125 (17-23) and thereport on bromide-77 (6) incorporated into DNA. Furthermore,our studies show that about 50 atoms of bromine-80m aresufficient to kill cells. This radiotoxicity may thus be compatible

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BROMINE-80m RADIOTOXICITY

with estrogen receptor-directed therapy, since ER+ cancerscontain from 500 to 20,000 free receptor molecules per cell.

Realistically, however, one must also deal with the fact thatthere is heterogeneity in the expression of ER in hormone-

dependent cancers like breast cancer. Nonetheless, results fromimmunohistochemical assays for ER suggest that improvedprognosis is seen in breast cancer patients whose cancers contain at least 40% ER+ cells (24). This implies that even somecancers with fewer than half of their cells expressing ER actlike hormone-responsive cancers. In this regard, it is becomingmore evident that estrogen may at least in part control growthof cancer cells by autocrine or paracrine mechanisms (25), sothat ER+ cells, producing estrogen-dependent growth factors,may regulate the growth of other cancer cells which could beER negative but depend on the growth factors produced by theER+ cells. Such a mechanism of growth control is likely to beoperating with current endocrine therapies as well. However,while current endocrine therapies are basically cytostatic, receptor-directed therapy with Auger electron-emitting nuclidesshould be cytotoxic if the effects are similar to those shownhere for the nuclide incorporated into DNA. Furthermore,unlike cell cycle-dependent therapeutic agents, ER-directedtherapy should work equally well in all ER+ cells, even thosein Go since all that should be needed is for ER to bind thenuclide-bearing estrogen and form an intimate association withnuclear DNA.

An additional advantage to ER-directed therapy with Augerelectron-emitting estrogens is that those cells lacking substantial concentrations of nuclear ER, in which any estrogen takenup would be expected to be largely cytoplasmic (such as liver inwhich cytoplasmic enzymes metabolize estrogens), should notbe adversely affected. This was seen with bromine-80m assodium bromide and bromoantipyrine (Fig. 1) which were notconcentrated in the nucleus, and has been well-documentedpreviously (6, 18, 19) for a number of Auger-emitting nuclides.Clearly the details of the route of administration, tumor uptakeand clearance, potential hazards of the ßdecay of the bromine-daughter, etc. must be worked out prior to successful clinicaluse. However, as shown by studies in our laboratory (12, 13,26, 27) and elsewhere (28-32) there are a number of bromine-containing steroidal and nonsteroidal estrogens with good affinity for the estrogen receptor, likely to be concentrated inER+ tissues and tumors, that can now be investigated for thispurpose. Demonstration of the radiotoxicity of bromine-80mlabeled estrogens in vivo awaits higher specific activities ofbromine-80m-labeled estrogens, but it now appears that thisattractive approach to the treatment of estrogen-receptor-positive cancers in humans is feasible.

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1988;48:5805-5809. Cancer Res   Eugene R. DeSombre, Paul V. Harper, Alun Hughes, et al.   Receptor-directed Therapy with Auger ElectronsBromine-80m Radiotoxicity and the Potential for Estrogen

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