(CANCER RESEARCH 52, 4248-425.1, August 1. 1992]

DNA Topoisomerase II Immunostaining in Human Leukemia and

Rhabdomyosarcoma Cell Lines and Their Responses toTopoisomerase II Inhibitors1

Judith S. Wolverton, Mary K. Danks, Bernd Granzen, and William T. Beck2

Department of Biochemical and Clinical Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101 [J. S. W., M. K. D., B. G., W. T. B.J,

and Department of Pharmacology, College of Medicine, University of Tennessee, Memphis, Tennessee 38] 63 [W. T. B.]

ABSTRACT

DNA topoisomerase II is an enzyme that affects nuclear structureand function and is the target of a number of anticancer drugs in clinicaluse, including teniposide (VM-26). We have used our polyclonal anti-sera that recognize both the M, 170,000 and 180,000 forms of topoisomerase II to examine the nuclear distribution of topoisomerase II incytospin preparations of drug-sensitive (CEM) and VM-26-resistant(CEM/VM-1 and CEM/VM-1-5) human leukemic lymphoblasts. Wehave also examined the nuclear distribution of topoisomerase II inmonolayer cultures of a human rhabdomyosarcoma (Rh30) cell line. Inthe absence of drug, we observed a focal "patchy" staining of nuclear

topoisomerase II in all cell lines, that was especially notable in thelymphoblastic cells. Treatment of CEM and Rh30 cells with VM-26under conditions that increase the number of covalent topoisomeraseII-DNA complexes increased both the intensity and the homogeneity ofnuclear topoisomerase II staining in a subpopulation of cells; focalstaining was less evident after treatment with drug. These responseswere roughly proportional to the concentration of VM-26 used andrequired only brief (~25-min) incubation with drug. We also found thattreatment of CEM cells with 4'-(9-acridinylamino)methanesulfon-m-

anisidide similarly increased the intensity and homogeneity of nucleartopoisomerase II immunostaining. In contrast, 4'-(9-acridinylamino)-methanesulfon-o-anisidide and 1-0-D-arabinofuranosylcytosine, agentsthat do not inhibit topoisomerase II, did not produce this effect. Finally,the VM-26-mediated alteration in topoisomerase II staining intensityand distribution was attenuated in proportion to the degree of VM-26resistance in the CEM/VM-1 and CEM/VM-1-5 sublines. These results appear to be related to the ability of the drug to stabilize DNAtopoisomerase covalent ("cleavable") complexes in intact cells. Ourfindings indicate that anti-topoisomerase II drugs, such as VM-26, haveprofound effects on the ability to detect topoisomerase II in the nucleusand provide a novel way of examining drug-stabilized DNA topoisomerase II complexes in intact single tumor cells.

INTRODUCTION

DNA topoisomerase II, a component of the nuclear matrix(1), influences nuclear structure and function and, in particular,affects replication, transcription, and chromosome segregation(2). After attachment of topoisomerase II to double-strandedDNA, the enzyme cleaves the DNA, mediates passage of another double-stranded DNA, and then religates the cleaved

Received 3/9/92; accepted 5/13/92.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 in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported in part by Research Grants CA-30103 and CA-40570and Cancer Center Support (CORE) Grant CA-21765, all from the National Cancer Institute, DHHS, and in part by American Lebanese Syrian Associated Charities. B. G. is the recipient of a Fellowship from the Dr. Mildred Scheel Foundation,Federal Republic of Germany.

2 To whom requests for reprints should be addressed, at the Department ofPharmacology, St. Jude Children's Research Hospital, 332 N. Lauderdale, Mem

phis, TN 38101.

strand (3). Anticancer drugs such as VM-26,3 etoposide, and/n-AMSA block this religation (2, 3), resulting in an increase inthe number of drug-stabilized DNA-protein ("cleavable") com

plexes. Increases in the numbers of these complexes are associated with drug cytotoxicity (2). Further, nuclear matrix topoisomerase II is preferentially inhibited by VM-26 and m-AMSAin CEM cells (4). In an attempt to better understand these drugeffects, we have used anti-topoisomerase II antisera that wedeveloped in our laboratory to examine the nuclear distributionof topoisomerase II by immunohistochemical staining, underconditions that increase the number of covalent DNA-proteincomplexes. Our results are the subject of this communication.

MATERIALS AND METHODS

Chemicals and Supplies. The following chemicals were purchased:BrdUrd, BSA, goat serum, fluorescein-conjugated goat anti-rabbit IgG,diaminobenzidine, hydrogen peroxide, and DABCO from Sigma (St.Louis, MO); paraformaldehyde, EM grade, from Polysciences (War-rington, PA); Triton X-100 (10% solution) from Pierce (Rockford, IL);peroxidase-conjugated goat anti-rabbit IgG from Zymed (San Francisco, CA); anti-BrdUrd monoclonal antibody from Becton-Dickson(San Jose, CA); and rhodamine-conjugated goat anti-mouse IgG fromTago (Burlingame, CA). [3H]Thymidine (specific activity, 6.7 Ci/mmol)and [14C]leucine (specific activity, 54 mCi/mmol) were purchased from

NEN/DuPont (Boston, MA). SMEM was from Whittaker Bioproduces(Walkersville, MD), and fetal bovine serum was from Hyclone (Logan,UT). VM-26 was a generous gift of Bristol-Myers Squibb (Wallingford,CT), m-AMSA and o-AMSA were gifts of Dr. Bruce C. Baguley (University of Auckland, New Zealand), and l-/3-D-arabinofuranosylcy-tosine in the clinical formulation was from Upjohn (Kalamazoo, MI).

Antibodies. Anti-topoisomerase II antisera were raised by conventional methods. Rabbits were immunized with a M, 72,000 humantopoisomerase II carboxyl-terminal peptide, expressed in Escherìchiacoli from the plasmid generously provided by Dr. Leroy Liu (JohnsHopkins University) (5). Preimmune sera were collected prior to immunization. These antisera, collectively termed MAC, recognize boththe M, 170,000 and 180,000 topoisomerase II forms on Western blotsand will be described in detail elsewhere.4 Animals were housed andtreated according to the Public Health Service "Policy on Humane Careand Use of Laboratory Animals."

Cell Lines. The human leukemic cell line CEM and its sublinesCEM/VM-1 and CEM/VM-1-5, which we selected for resistance toVM-26, have been described (6). The human rhabdomyosarcoma cellline Rh30 was provided by Dr. P. J. Houghton (St. Jude Children's

Research Hospital) and has also been described (7). Cells for experiments were in logarithmic growth and had previously tested negativefor Mycoplasma. For staining of S-phase cells, cells were grown for30 min with 10 /iM BrdUrd.

3 The abbreviations used are: VM-26, teniposide; m-AMSA, 4'-(9-acridinylami-no)methanesulfon-/n-anisidide; o-AMSA, 4'-(9-acridinylamino)methanesulfon-o-anisidide; BrdUrd, bromodeoxyuridine; BSA, bovine serum albumin; PBS,phosphate-buffered saline: CEM, CCRF-CEM; DABCO, 1,4-diazobicyclo-(2,2,2)-octane; SMEM, Spinner's minimal essential medium (Eagle).

4 M. K. Danks, C. V. Catapano, D. J. Fernandes, C. A. Schmidt, and W. T.

Beck, manuscript in preparation.

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Drug Treatment, Doubling Times, and Cytospin Preparation. Cellswere treated with VM-26 as described previously (6). Doubling timeswere determined by counting exponentially growing cells with a Coultercounter (model ZM), after seeding of the wells with about 3 x IO5cells/ml. Samples (1 ml, 4 x IO5 cells) used for cytospin preparationwere washed once with ice-cold PBS (pH 7.4), resuspended in 3 ml PBScontaining 1% BSA, and kept on ice no longer than ~1 h beforecytospin preparations of 200-f¿laliquots were made. Cytospin preparations were air dried overnight, foil wrapped, and stored at —¿�20°Cuntil

immunostaining.Immunostaining. ( I \1 cytospins were Fixed and permeabilized for

10 min at 4°C,by the method of Clevenger et al. (8), using freshly

prepared 0.5% paraformaldehyde in PBS, followed by 3 min in 0.1%Triton X-100 in PBS at 4°C.Rh30 cells grow as monolayer cultures; for

immunostaining experiments, they were grown on Permonox 4-welltissue culture chamber slides (LabTek no. 177437; Nunc, Inc., Napier-ville, IL) and were fixed and permeabilized in the same way as CEMcells. Slides were first incubated with 10% goat serum in PBS and thenincubated with MAC anti-topoisomerase II antiserum, diluted 1:500 in

PBS containing 1% BSA. Slides were then rinsed three times in PBS/0.1% Triton X-100, incubated with a 1:80 dilution in PBS of eitherperoxidase-conjugated or fluorescein-conjugated goat anti-rabbit IgG,and rinsed three times in PBS/0.1% Triton X-100. Incubations were atroom temperature, for 40-90 min. Controls were prepared similarly,substituting preimmune serum for antiserum, and routinely gave virtually no nuclear stain. For development of the peroxidase stain, slideswere incubated for 10-20 min at room temperature with freshly prepared diaminobenzidine (0.3 mg/ml) and 0.015% hydrogen peroxide, in0.05 MTris (pH 7.6), and were rinsed three times with deionized water.Slides were mounted in 90% glycerol/10% PBS, pH 8.6, containingDABCO (23 mg/ml). Appropriate dimethylsulfoxide controls were routinely compared with the VM-26-treated sample. For dual staining withanti-BrdUrd and anti-topoisomerase II antibodies, the parafor-maldehyde/Triton X-100 method was used, with a 2-min incubation in0.07 N NaOH after Triton permeabilization.

Formation of Covalent Topoisomerase II-DNA Complexes in IntactCEM, CEM/VM-1, and CEM/VM-1-5 Cells. This assay is a modification of previously published methods (9-11). Briefly, cellular DNAand protein were metabolically labeled by incubating ~4 x IO5 CEMcells/ml at 37'C for 18 h with both [14C]leucine (0.2 ¿iCi/ml)and ('H]-

thymidine (0.6 ¿iCi/ml),in SMEM supplemented with 10% fetal bovineserum. The cells were then centrifuged at 400 x g for 5 min andresuspended to ~8 x IO5 cells/ml in sterile PBS. The indicated con

centrations of VM-26 were added to cell suspensions, and the cells wereincubated for an additional 30 min at 37°C.After incubation with drug,

each sample was divided into 0.5-mI aliquots, and reactions werestopped by adding 0.5 ml of a solution containing 2.5% sodium dodecylsulfate, 10 min EDTA (pH 8.0), and 0.8 mg/ml salmon sperm DNA(37°C).Cell lysates were passed 20 times through a 22-gauge needle andthen heated to 65°Cfor 15 min. KC1 (100 mM, final concentration) was

then added to each tube. The tubes were vortexed for 10 s, put on ice for5 min, and then centrifuged at 10,000 x g for 10 min at 4°C.Each pellet

was washed three times with 1.0 ml of a solution containing 10 mMTris-HCl (pH 8.0), 100 imi KC1, 1 m\i EDTA, pH 8.0, and 0.1 mg/mlsalmon sperm DNA. The pellets were then dissolved in 0.5 ml water at65°Cfor 15 min and centrifuged for 10 s at 10,000 x g at room

temperature, and 0.5 ml was transferred to a vial for scintillation counting. Results are expressed as the ratio of 3H-DNA/l4C-protein, with the

cpm of protein precipitated as the internal control for all samples.

RESULTS

Immunostaining of Topoisomerase II in CEM, CEM/VM-1,and CEM/VM-1-5 Cells. In the absence of drug, visualizationof cellular topoisomerase II with our antiserum revealed thatthe enzyme was localized to the nucleus. The nuclear staining,however, was focal and heterogeneous, as seen in Fig. 1. Topoisomerase II staining appeared to be similar among theCEM, CEM/VM-1, and CEM/VM-1 -5 cells but was somewhatless intense in the CEM/VM-1-5 cells than in the other two

CEM cell lines. Staining intensity was heterogeneous amongthe cells lines, but almost 100% of the cells in the three cell linesshowed some specific staining for topoisomerase II. Dual immunostaining of CEM cells for both topoisomerase II and Br-dUrd indicated that all S-phase cells stained for topoisomeraseII, but many cells not in S-phase also stained for this enzyme(data not shown). Also, cells appearing to have just completedmitosis were observed occasionally, and these showed virtuallyno staining, suggesting that early GÃŒcells had little topoisomerase II. Visualization of topoisomerase II staining withfluorescein gave patterns similar to those seen with peroxidase(Fig. 2, Control). Preimmune serum gave a barely detectablesignal in the peroxidase-stained cells and appeared as a faint

CEM CEM / VM-1 CEM / VM-1-5

•¿�

No Drug •¿�.

+VM-26 f 1

Fig. 1. Topoisomerase II immunoperoxidase staining in CEM, CEM/VM-1, and CEM/VM-1-5 cells and effect of VM-26. Cells were treated with 2 UMVM-26or dimethylsulfoxide control for 135 min, and cytospin preparations were made and stained for topoisomerase II with peroxidase, as detailed in "Materials andMethods." Slides were routinely viewed with oil immersion, using a Zeiss microscope with Planapo 40/1.0 or 63/1.4 objectives. Photomicrographs were taken witha Nikon UFX-11 microscope equipped for photography, with oil immersion and a xlOO objective.

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Control 2\M VM-26 VM-26Fig. 2. Effect of VM-26 on ¡mmunofluorescence staining of topoisomerase II and its distribution in CEM cells. CEM cells were treated with the indicated

concentrations of VM-26 for 70 min, and cytospin preparations were made and stained for topoisomerase II immunofluorescence, as described in "Materials andMethods." Photomicrographs were taken with oil immersion, using a Zeiss microscope and Planapo 63/1.4 objective. Arrows, bright homogeneous staining.

aVl

.

t

f'

No drug VM-26 10/iM VM-26Fig. 3. Effect of VM-26 on topoisomerase II immunostaining in Rh30 cells. Human rhabdomyosarcoma Rh30 cells were grown on Permonox chamber slides. Cells

in mid-logarithmic phase growth were treated with VM-26 for 60 min, after which they were washed and stained with anti-topoisomerase II antiserum by the peroxidasemethod, as described in "Materials and Methods." Preimmune serum gave virtually no staining. Shown are representative panels.

cytoplasmic stain surrounding an essentially unstained nucleusin the fluorescein-stained cells (data not shown). We also examined topoisomerase II distribution by immunofluorescencein unfixed CEM cells that were permeabilized with TritonX-100, using a method that allows retention of the native chro-

matin structure (12). This topoisomerase II immunofluorescence staining revealed the same type of focal "patchy" heter

ogeneous staining pattern as seen with fixed cells in Figs. 1 and2 (results not shown), suggesting that the distribution of topoisomerase II in fixed cells reflects that existing in the nuclei ofintact cells.

Effects of VM-26 on the Topoisomerase II ImmunostainingPattern in CEM, CEM/VM-1, and CEM/VM-1-5 Cells. Aftertreatment of CEM cells with VM-26 for 135 min (conditionsthat permit formation of DNA-topoisomerase II complexes),the topoisomerase II stain was more intense and more homogeneous in a subpopulation of the CEM cells (Fig. 1). Similardrug-induced alterations were seen by immunofluorescence detection in several experiments. Fig. 2 shows a representativeimmunofluorescence experiment. Treatment of CEM cells with2 MMVM-26 increased the intensity of staining of a subpopulation of ~16% of the cells (Fig. 2, arrows), and 10 MMdrugcaused more of the cells (~35%) to be homogeneously andintensely stained. Appropriate dimethylsulfoxide controls confirmed that this response was drug specific. Results similarto those in Fig. 2 were obtained when the time of exposure toVM-26 was increased, between 75 and 245 min (data notshown). In other experiments (data not shown), we found thatthe homogeneous staining response of topoisomerase II toVM-26 reached a maximum of ~48% and then decreased.

In contrast to the results with drug-sensitive CEM cells, theresponse of drug-resistant cells was attenuated or absent. Thus,while ~23% of the CEM cells showed homogeneous and intense topoisomerase II staining in response to treatment with10 MMVM-26, only about 15% and 6%, respectively, of theCEM/VM-1 and CEM/VM-1-5 cells exhibited this response toVM-26 (Fig. 1). We observed similar responses using immuno

fluorescence (data not shown).Topoisomerase II Immunostaining in Human Rhabdomyo

sarcoma Cells and Effects of VM-26. We asked whether theimmunostaining response of topoisomerase II could be seen inanother cell type. Accordingly, we treated Rh30 rhabdomyosarcoma cells growing in monolayer culture with 3 and 10 MMVM-26 for 60 min (these are conditions that stabilize DNA-topoisomerase II complexes in these cells5), washed them free

of drug, and then stained them for topoisomerase II. It can beseen in Fig. 3 (left) that untreated Rh30 cells displayed a lowlevel of topoisomerase H-specific staining, although the nucleidid not express the same type of patchy focal staining seen withthe CEM cells. After treatment with VM-26, however, it is clearthat the topoisomerase H-specific staining was considerablymore intense. It can also be seen that, with increasing concentrations of VM-26, this intense staining affected a subpopula-tion of cells (Fig. 3, middle and right), being ~43% and ~59%at 2 MMand 10 MMVM-26, respectively. Thus, we conclude thatthe increase in topoisomerase II staining in response to treatment with an epipodophyllotoxin is not cell type specific;

' B. Granzen, M. K. Danks, and W. T. Beck, manuscript in preparation.

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Table 1 Effect of FA/-26 on cellular doubling lime and DNA-protein complex formation in intact cells

Cell line

Doubling time (hr)° DNA-prolein complexes*

+VM-26 VM-26

No drug l /IM 10 MM No drug I >IM 10 >IM

CEMCEM/VM-1CEM/VM-1-5212728226r3228No growthNo

growth310.26

±0.020.25

±0.030.23

±0.020.63

±0.05(2.40)rf0.34

+0.03(1.36)0.24

±0.02(1.04)2.20

±0.17(8.46)0.80

±0.17(3.20)0.38

+0.14(1.65)

" Determined as described in "Materials and Methods."''Determined as described in "Materials and Methods," after 30-min incubation with VM-26. Results are ratios of 'H-DNA cpm:'4C-protein cpm and are the

means ±SD of three separate experiments.' Projected.d Numbers in parentheses represent fold increases in DNA-protein complexes in drug-treated cells, compared to no-drug controls.

o-AMSA m-AMSA

Fig. 4. Effects of m-AMSA or o-AMSA ontopoisomerase II immunostaining in CEMcells. CEM cells were treated for 100 min witheither 10 UMm-AMSA or 10 UMo-AMSA andwere then prepared for topoisomerase II immunostaining by the peroxidase method, as described in "Materials and Methods." Shown

are representative panels.

rather, it appears to be a general response related to the stabilization of DNA-topoisomerase II complexes formed by inhibitors of this enzyme.

Effects of VM-26 on DNA-Topoisomerase II Complex Formation and Cell Doubling Times. The effects of VM-26 ontopoisomerase II immunostaining may reflect its action to increase the number of covalent DNA-topoisomerase II complexes and to inhibit cell growth. Approximately 43 h afterseeding, all three cell lines achieved more than one doubling inthe absence of drug and were still in exponential growth. It isseen in Table 1 that 1 MMVM-26 increased the projected CEMdoubling time by ~ 10-fold and 10 MMdrug prevented growth ofthese cells. While this higher concentration of VM-26 alsoprevented the growth of CEM/VM-1 cells, 1 MMdrug was without effect, and the doubling time of CEM/VM-1-5 cells wasunaffected by these concentrations of VM-26. It is also seen inTable 1 that increasing concentrations of VM-26 stabilized increasing numbers of DNA-topoisomerase II complexes in CEMcells. Thus, 1 and 10 MMVM-26 caused a 2.4- and 8.5-foldincrease, respectively, in DNA-protein complexes. This increase, however, was progressively attenuated in drug-resistantcells, so that 10 MMVM-26 produced only a 3.2-fold and 1.65-

fold increase in DNA-protein complexes in CEM/VM-1 andCEM/VM-1-5 cells, respectively. These dose-response relationships are consistent with the immunostaining observations.

Effects of/n-AMSA, o-AMSA, and 1-0-D-Arabinofuranosyl-cytosine on Topoisomerase II Immunostaining in CEM Cells.Our data suggest that the effects of VM-26 on nuclear topoisomerase II immunostaining is related to its effect on DNA-protein complex formation. An indirect test of this hypothesisis to ask whether other cytotoxic drugs do the same. It can beseen in Fig. 4 that w-AMSA, another drug that stabilizes DNA-protein complexes, also produced intense and more homogeneous topoisomerase II staining in about one third of the cells.In contrast, the isomer o-AMSA, which does not stabilizeDNA-protein complex formation in our cells,6 did not producethese changes; only control staining was seen. Finally, a 2-hexposure to 1-0-D-arabinofuranosylcytosine, at a concentration5 times higher than its 50% inhibitory concentration, producedlittle alteration in topoisomerase II immunostaining, comparedto controls (data not shown).

6 M. K. Danks and B. Granzen, unpublished observations.

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DISCUSSION

We have used polyclonal antisera to human DNA topoi-somerase II to study nuclear distribution of this enzyme inCEM cells and their altered topoisomerase H-associated nuil-tidrug-resistant (at-MDR) derivatives. Most cells not exposedto drug showed specific topoisomerase II staining of varyingintensities and considerable focal heterogeneous structure. Thispattern of topoisomerase II staining is consistent with thosereported for a paraformaldehyde-fixed, detergent-extracted,chicken cell line (13), for cryosections of Drosophila larvae (1),and for MELC cells of murine origin (14). In the Drosophilastudy, topoisomerase II appeared to be excluded from the nucleoli (1). It should be noted that our antisera recognize bothforms of topoisomerase II, Mr 170,000 (a) and 180,000 (0), asindicated above (15, 16), whereas the other studies (1, 13) usedantisera that recognized only topoisomerase II a. Accordingly,we cannot yet determine whether the nuclear distribution anddrug-induced changes in topoisomerase II represent effects onprimarily a, ß,or both. We are presently attempting to developa-specific and /3-specificantibodies that should resolve this issue. Finally, our preliminary studies using another method (12)also suggest that the staining pattern in fixed cells is similar tothat in nonfixed permeabilized cells and therefore probably reflects the distribution of the enzyme in intact cells.

The changes produced specifically in topoisomerase II patterns in a subpopulation of CEM and Rh30 cells by inhibitorsof topoisomerase II are striking and consist of increases in bothintensity and apparent homogeneity of stain. This response iscomplete within 25 min after exposure to VM-26, and the sizeof the responding subpopulation is dependent on the drug concentration. The short time required for complete response isconsistent with the rapid attainment of steady state levels ofDNA-topoisomerase II complexes in intact cells treated withtopoisomerase H-directed drugs (16). Our results with o-AMSAand 1-/3-D-arabinofuranosylcytosinesuggest that the immuno-staining response most likely is not due to general DNA damage, and they support the idea that the response is specific forinhibitors of topoisomerase II. In a recent study, intense homogeneous staining of topoisomerase II was shown in subpopula-tions of both confluent (i.e., "resistant") and nonconfluent("sensitive") HT-69 cells (17); however, drug effects on topo

isomerase II staining were not described in that report.The alteration in topoisomerase II staining induced by

VM-26 in the two drug-resistant CEM cell lines was less thanthat seen in the drug-sensitive CEM line and was inverselyrelated to the resistance of the cells to VM-26. Thus, while10MMVM-26 caused the intense and homogeneous staining oftopoisomerase II in ~23% of CEM cells, only about 15% and6%, respectively, of the CEM/VM-1 and CEM/VM-1-5 cellsshowed this response. These relative staining responses of thecells are consistent with their relative responses to VM-26 ingrowth inhibition and DNA-protein complex formation assays(Table 1). Although the progressive decrease in staining of topoisomerase II in the resistant cells in response to VM-26 mostlikely reflects their decreased drug sensitivity, it must be notedthat these cells are ~40- and 150-fold resistant to VM-26 ingrowth inhibition assays. The finding that there is not a directrelation between topoisomerase II staining or DNA-proteincomplex formation and the degree of resistance of the cellsmost likely reflects the fact that these assays measure differentfunctions of topoisomerase II activity and do not distinguishbetween the a- and /3-isozymesof topoisomerase II.

The pronounced VM-26- and m-AMSA-mediated shift intopoisomerase II staining in CEM and Rh30 cells, from heterogeneous to a more intense, homogeneous pattern, is limited toa subpopulation of the cells, approximately 30-35% for CEM.We do not yet understand the molecular mechanism for thischange in topoisomerase II staining pattern. The brief timerequired for its induction suggests that an increase in de novosynthesis of topoisomerase II does not account for the response.Some possibilities to account for this effect include: decreasedproteolytic degradation of topoisomerase II in the drug-stabilized enzyme-DNA complex; a shift in localization of the enzyme from nucleoplasm to nuclear matrix; drug-inducedchanges in spatial interactions of topoisomerase II with othernuclear components; or drug-mediated exposure of newepitopes. While this list is not meant to be exhaustive, it doesoffer several readily testable hypotheses.

In conclusion, we have demonstrated a change in nucleartopoisomerase II staining by topoisomerase Il-active drugs thatparallels their ability to stabilize DNA-protein complexes. Thiseffect does not appear to be restricted to a particular cell type,because it is seen in human leukemic cells that grow in suspension and also in human rhabdomyosarcoma cells that grow inmonolayer. The observations that the phenomenon is apparently specific for inhibitors of topoisomerase II and is progressivelyattenuated in mutant cell lines of increasing resistance tothese agents suggest that the drug-induced stabilization of topoisomerase II-DNA cleavable complexes is accompanied bymorphological and structural changes in the cell. This studyprovides a new way of looking at drug-stabilized DNA-topoisomerase II complexes and drug resistance in intact single tumor cells and may have application in evaluating the drug-responsiveness of tumor specimens from patients.

ACKNOWLEDGMENTS

We thank Dani Harris and John Zacker of the BiomédicalCommunications Department for excellent preparation of the artwork andguidance with photomicrography, and we thank Fabrienne Hollowayfor her skilled typing of the manuscript.

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1992;52:4248-4253. Cancer Res   Judith S. Wolverton, Mary K. Danks, Bernd Granzen, et al.   Topoisomerase II InhibitorsRhabdomyosarcoma Cell Lines and Their Responses to DNA Topoisomerase II Immunostaining in Human Leukemia and

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