subcellular redistribution of dna topoisomerase i in ... · isee software was used to perform...

(CANCER RESHARCH 36. I664-I67.V April I. I

Subcellular Redistribution of DNA Topoisomerase I in Anaplastia AstrocytomaCells Treated with Topotecan1

Mary K. Danks,2 Katherine E. Garrett, Raquel C. Marion, and David O. Whipple

Depiintnt'nl af Molecular Phannacohit'y. St. Jude Children's Resenrch Hospital. Memphis. Tennesse

ABSTRACT

DNA topoisomerase I is the cytotoxic target for chemothcrapeuticagents of the camptothecin class. The cytotoxicity of these drugs is thoughtto be mediated by a dose-dependent increase in topoisomerase I molecules

bound to DNA, resulting in DNA damage and cell death. We observed thatin SJ-G5 human anaplastic astrocytoma cells growing in culture, themaximum number of topoisomcrase I-DNA complexes occurred 5-15 minafter the addition of 0.25-25 JIM 9-dimethylaminomethyl-10-hydroxy-camptothecin (topotecan; TPT) or 5 /IM 7-ethyl-10-hydroxycamptothecin(SN-38). We postulated that the decline in number of complexes seen after

15 min might be due to decreases in the amount of topoisomerase I or theredistribution of this enzyme such that it could no longer bind to DNA. Toinvestigate these possibilities, we incubated SJ-G5 cells for 20-60 minwith 0.25-5 (i \i TPT and analyzed the cells for amount and localization of

topoisomerase I by indirect immunofluorescence staining and fluorescence digital imaging microscopy. We verified the results obtained withfluorescence digital imaging microscopy by rapid fractionation of nuclearand cytoplasmic proteins, separation of these proteins by polyacrylamidegel clectrophoresis, and densitometric scanning of immunoblots. Resultsshowed that topoisomerase I dissociated from nucleoli within 60 min aftertreatment with 1-5 /IM TPT. A small (25%) but significant (P < 0.05)

decrease in the amount of nuclear topoisomerase I was also observedduring this time course. Simultaneously, the cytoplasmic content of theM, 67,000 form of topoisomerase 1 increased 50-100%. Preincubation of

cells with 10 /Â¿Mcycloheximidc for 10 min prevented the increase oftopoisomerase I in the cytoplasm, indicating that the increase was due, atleast in part, to de novo protein synthesis. Interestingly, chemotherapeuticagents other than camptothecins were also found to dissociate topoisomerase I from nucleoli. These agents included Â»i-AMSA (4'-(9-acridinylami-

no)methanesulfon-/n-anisidide), mitoxantrone, actinomycin D, and dauno-rubicin. Drugs such as l-/J-l>-arabinofuranosylcytosine or hydroxyurea,which have no elTect on RNA synthesis, did not induce the translocation.The biological significance of the ability of camptothecins to redistributetheir own drug target is under investigation.

INTRODUCTION

Topoisomerase I is a nuclear enzyme localized predominantly tonucleoli ( 1-4). This en/.yme is the cytotoxic target for several camptothecin analogues used clinically, including TPT.3 9-aminocampto-

thecin. and CPT-11 (5-7). Changes in the amount of topoisomerase I

or mutations in this enzyme alter the sensitivity of cells in culture tothe cytotoxicity of camptothecins (8-13).

The cytotoxicity of camptothecins is thought to be mediated by adrug-induced increase in the number of topoisomerase I molecules

Received 8/9/95: accepted 1/31/96.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 withIS U.S.C. Section 1734 solely to indicate this fact.

'This work was supported by NIH Grants CA635I6. CA23099, and CA21765

(CORK) and by the American Lebanese and Syrian Associated Charities.2 To whom requests for reprints should be addressed, at Department of Molecular

Pharmacology. St. Jude Children's Research Hospital. 332 North Lauderdale. Memphis.

TN 38101. Phone: (901 ) 495-3440: Fax: (901) 521-166Â«.'The abbreviations used are: TPT. lopotecan. 9-dimethylaniinomethyl-IO-hydroxy-

camptoihecin: SN-38, 7-ethyl-IO-hydroxycamptolhecin: h'DIM. fluorescence digital im

aging microscopy: MEK2. mitogen-aclivated kinase kinase or MAPK kinase: B23.nucleophosmin: m-AMSA. 4'-(9-acridinylamino)methanesulfon-w-anisidide: DMP S40.(R.R 1-2.2 '-|l. 2-ethanediylbis| Â¡minoli -methy 1-2,1 -ethanediyl)]]-bis|5-nitro-1H-

ben?.(de|isoquinoline-l.3-(2W)-dione|dimethanesulfonale; CPT-ll. 7-ethyl-l()[4-( I-pipendino)- l-piperidino|carbonyl-o\ycamptothecin.

covalently bound to DNA. When these covalent enzyme-DNA complexes occur near replication forks, the single-strand DNA breaksproduced by topoisomerase I are converted to double-strand DNAbreaks. It has been proposed that the double-strand DNA breaks

initiate apoptotic cascades, and cell death follows (14). In cell linesselected for resistance to camptothecins, fewer covalent topoisomerase I-DNA complexes are detectable than in parent cell lines,

which are relatively sensitive to these inhibitors of topoisomerase I(10. 12. 15).

Recently, Beidler and Cheng (16) reported that in HcLa cellsincubated with camptothecin, the number of covalent topoisomeraseI-DNA complexes decreased with time although the drug was present

continuously. In the HeLa cells incubated with camptothecin. levels ofimmunoreactive Mr 1()(),()()()topoisomerase I that was not bound toDNA decreased after exposure to the drug for several hours.

Using a cell line derived from the tumor of a pediatrie patient withanaplastic astrocytoma. we also observed that the level of covalenttopoisomerase I-DNA complexes in intact cells decreased with time,

even in the continuous presence of TPT. We then examined possiblemechanisms that might mediate the observed time-dependent declinein the number of topoisomerase I-DNA complexes in anaplastic

astrocytoma cells in tissue culture.

MATERIALS AND METHODS

Cell Line and Chemicals

The SJ-G5 cell line was derived from a biopsy sample of a tumor atdiagnosis of a 10-year-old female with anaplastic astrocytoma. The tumor

specimen was obtained in accordance with the guidelines of the InstitutionalReview Board of St. Jude Children's Research Hospital. The SJ-CÃŒ5brain

tumor cell line has an aneuploid karyotype with numerous murker chromosomes and chromosomal translocations. By immunohistochemical analysis, thecells express S-100 and neuron-specific enolase. markers of cells of neuronal

or glial lineage. The cell line will he characterized more completely elsewhere.These cells grow as a monolayer in DMEM (BioWhitaker. Walkersville. MD)supplemented with 15% bovine serum (Hyclone, Logan. UT) and O.I mgbovine hypothalamic extract (Upstate Biotechnology Inc., Lake Placid. NY)per 20 ml of tissue culture medium. All experiments were done with cells inlogarithmic growth between passages 20 and 30. Chemicals were purchasedfrom Sigma Chemical Co. (St. Louis. MO) unless otherwise indicated.

QuantitÃ¤ten of Topoisomerase I-DNA Covalent Complexes in

Intact Cells

The assay was modified from that reported by /welling el al. (17) and hasbeen reported previously ( 18). Briefly. SJ-G5 cells were plated at a density of2 X 10s cells/2 ml DMEM in a 35-mm tissue culture dish (Falcon. Oxnard.CA) and allowed to adhere to the tissue culture dish. |'H|Thymidine (0.6/iCi/nil: XI Ci/mmol: Amersham. Arlington Heights. IL) and [ l4C|leucine (0.2

/iCi/ml: 300 mCi/mmol: DuPont NEN. Wilmington. DE) were then added tothe medium, and the DNA and protein in the cells were allowed to incorporateradioactivity overnight. TPT or SN-38 was then added directly to the tissueculture medium and incubated at 37Â°Cfor the indicated times. Proteins weredenatured and precipitated by the addition of K ' -SDS as described. DNA was

sheared by passing the lysed cells and medium through a 22-gauge needle 20

times. Precipitated protein was washed three times, and the amount of DNAcovalently hound to the protein in this precipitate was quantitated by liquidscintillation counting. The results are expressed as |'H|DNA/'4C-laheled

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REDISTRIBUTION OF TOPOISOMERASE I

protein, with the protein cpm us the internal control. Variability betweenduplicate determinations in this assay is less than 8%.

Immunofluorcsccnce Staining for Topoisomerase I

Topoisomerase I was detected by indirect immunofluorescence stainingwith a rabbit polvclonal antiserum generated with a 21-amino acid synthetic

peptidc complexcd to keyhole limpet hemocyanin. as reported previously (1).Cells were grown on plastic chamber well slides (Nunc. Naperville. ID andwere fixed with freshly prepared 1% paraformaldehyde at 4Â°Cfor 20 min. The

remainder of the procedure was as reported previously. After the immunoflu-

orescence staining procedure, each slide was incubated for 2 min in a solutionof Hoechst 33342 (30 jxM) to allow visualization of the DNA by fluorescencemicroscopy. Appropriate filters were used for visualization of topoisomerase Ior DNA; the filters used excluded signal overlap between the two fluorescenceprobes (below, and as has been reported ( 11|.

FDIM

Image Acquisition. Specimens were observed with an Axiovert 135 TVmicroscope (Carl Zeiss, Inc.. Thornwood. NY) through an UV light transmitting 63X oil immersion 1.25 numerical aperture objective (Plan-NEOFLUAR,

no. 44 04 61; Zeiss). Excitation energy was provided by a 75 W xenon lamp.The filter set used to visuali/e and quantitate topoisomerase I allowed detection of fluorescence emission from 520-560 nm. The filter set used to visualize

and quantitate DNA allowed detection of fluorescence emission between4(X)-450 nm. Two images were recorded for each field of cells, an image of

topoisomerase I and an image of DNA. These images were recorded with athermoelectric-ally cooled charge-coupled device camera (Model CE200A with

LC200 liquid cooling unit; Photometries. Tuscon. AZ). The images wererecorded with a Crimson/XS computer (Silicon Graphics. Mountain View.CA) running ISee image processing software (Inovision Corp.. Durham. NC)and printed with an NP-600 Photographic Network Printer (Codonics. Middle-

burg Heights. OH). The above configuration of equipment is capable ofrecording a 1024 x 1024 pixel array. However, only the middle 512 X 512pixels were read and recorded, since the image quality is optimal in the centerof the microscopic field.

ISee software was used to perform background subtraction and shadingcorrection on each image prior to storage on hard drives. Corrections wereperformed using a background image of the camera dark current and a flatfieldimage of a blank, out-of-focus area of the slide containing the cells to be

analyzed (19). In none of the experiments in this study was the fluorescence ofthe image obtained with preimmune serum subtracted from the image obtainedwith antiserum to topoisomerase I. The images are as they appeared in themicroscope, with only light intensity distortions introduced by the charge-

coupled device camera, microscope optics, and light sources subtracted fromthe image seen visually (20).

Image Processing. A binary mask was used to select pixels tor blockingout nuclear areas of SJ-G5 cells. A copy of the DNA image (Hoechst fluores

cence) became the binary mask image after all pixels were changed to a valueof one or zero (binary). Pixels containing fluorescence above threshhold in theDNA Â¡magewere assigned a value of zero; pixels containing fluorescencebelow threshhold in the DNA image were assigned a value of one. The binarymask image was then used in a pixel-by-pixel multiplication of the topoisomer

ase I image for that field of cells. The product of the multiplication reproducedthe original image only for those pixels that had a binary mask value of one.The binary mask pixels with a value of zero result in black areas in thereproduced image, in this case blocking out indirect imniunofluorescenee oftopoisomerase I in nuclei of SJ-G5 cells (Fig. 3).

Quantitation of Topoisomerase I and DNA by FDIM. Quantitation oftopoisomerase I was done by FDIM techniques similar to that reported byBaker et al. ( I ). Briefly, the topoisomerase I image was multiplied by a binarymask created from the DNA image. The product image contained the exactfluorescence intensity for those pixels that represented a nuclear area setagainst a zero background. The ISee software was programmed to: select thepixels making up each nucleus; sum the fluorescence intensities of the pixels;and create a text file containing the quantitation data.

Because of the irregular shape of SJ-G5 cells, the quantitalion of cytoplas-

mic topoisomerase I was much less precise and subjective. Therefore. FDIMquantitation of cytoplasmic fluorescence was not included in this study.

Drugs

TPT was obtained from Peter J. Houghton (St. Jude Children's Research

Hospital). A stock solution of K) ~ MTPT was made in water. Serial dilutions

of this stock solution were made in 10 mM Tris-HCI (pH 7.4). The pH of theresulting stock solutions of TPT from 10~3 to 10 '" M was 7.4. The solutions

were stored at 4Â°Cand maintained potency for at least 4 months under these

storage conditions. DMP 840 was obtained from Janet Dzubow (The DuPontMerck Pharmaceutical Co.. Glenolden. PA). This compound was dissolved inDMSO and stored at -20Â°C as a 10~3 M stock solution. Vincristine (Eli Lilly.

Indianapolis. IN) was obtained from the St. Jude Children's Research Hospital

Pharmacy as the solution used clinically. Dilutions of the drug were made with0.9% NaCl. Hydroxyurea. aphidicolin, actinomycin D. doxorubicin. Hoechst33342. etoposide. 1-ÃŸ-D-arabinofuranosylcytosine. and vincristine were allfrom Sigma Chemical Co. (St. Louis. MO). Mitoxantrone and m-AMSA were

obtained from John L. Nitiss (Department of Molecular Pharmacology.St. Jude Children's Research Hospital).

Rapid Separation of Nuclear and Cytoplasmic Fractions of SJ-G5 Cells

SJ-G5 cells were grown in 75-cnr flasks (Costar, Cambridge, MA). The

cells were washed twice with PBS and then scraped into cold isotonic homog-enization buffer 110 mM Tris-HCI (pH 7.4), 140 mM KC1, and 1.5 mM MgCK|

containing the following protease inhibitors: 2 mM phenylmethylsulfonyl fluoride. 50 /ig aprotinin/ml, and 6 |ig leupeptin/ml. Cytoplasmic membranes

were disrupted by a total of 10 strokes of a glass homogenizer. A drop of buffercontaining homogenized cells was then examined microscopically for trypanblue exclusion to confirm permeabilization of >909c of cells. The homogenatewas then centrifuged for 10 min at 1000 x g to produce a crude nuclear

fraction as the pellet and a cytoplasmic supernatant. This procedure takes amaximum of 4 min before centrifugation is begun. The resultant "nuclear" and"cytoplasmic" fractions are described more accurately as nucleus-enriched and

cytoplasm-enriched fractions (21). Using this procedure, cellular organdÃes

such as mitochondria and lysozomes. as well as fragments of the cell membrane, are components of the "nuclear" fraction. The efficiency of fractionation

was determined by immunoblolting of each fraction with an antibody toMEK2, a cytoplasmic marker (22), and by quantitation of the DNA content ofeach fraction. Immunoblolting procedures are detailed below. The quantitationof DNA with Hoechst 33258 was done by standard methods using a Hoefer

TKO 100 fluorometer (San Francisco. CA). according the the directions of themanufacturer.

Immunoblotting for Topoisomerase I and MEK2

SDS-PAGE and immunoblotting were carried out as detailed previously(23). Protein samples were prepared for SDS-PAGE by heating to 95Â°Cfor 2

min in Laemmli sample buffer. The antiserum to topoisomerase I was generated with a 21-amino acid peptide and is the same antiserum used for immu-

nohislochemical analyses in this study (1). Antiserum to MEK2 cytoplasmicmarker protein (Santa Cruz Biotechnology, Inc.. Santa Cruz, CA) was usedaccording to the directions of the manufacturer. Antibody binding was detectedby a secondary antibody conjugated to alkaline phosphatase and incubatedwith nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate, as

reported previously (23). Where indicated, protein samples were incubatedwith DNase (Boehringer Manneheim. Indianapolis. IN) prior to suspension andheating in sample buffer (23, 24). Equal loading of protein in each lane onpolyacrylamide gels was verified by densitometric scanning. A film positive ofthe gel stained with Coomassie blue R250 was scanned onto a disk with aScanJet He (Hewlett Packard), and a nonspecific high molecular weight handwas quantitated by standard methods using ImageQuant program on an IBM

compatible personal computer. Bands on immunoblots were quantitated in asimilar manner. Comparisons of amounts immunodetectable protein (Figs.6-8) were corrected, if necessary, to account for any slight differences in

protein loading on the gels. Prints for publication of immunoblots wereprepared using the Adobe Photoshop Program for Macintosh and were printedon the NP-n(X) Photographic Network Printer. Contrast and brightness values

within the Photoshop Program were chosen to reflect the original blot asclosely as possible.

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HI-DISTRIBUTION OF TOPOISOMERASK 1

RESULTS

Covalent Topoisomera.se I-DNA Complexes in SJ-G5 Cells

SJ-G5 cells were incubated with 0.25, 2.5, or 25 JU.MTPT for 3 h as

shown in Fig. 1A. TPT was present continuously and was not removed from the medium prior to denaturation of topoisomerase I bySDS. Culture medium and TPT stock solutions were each equilibratedto a pH of 7.4 before the drug was added to each tissue culture dishcontaining cells. Compared to no-drug controls, cells incubated for 15min with 0.25, 2.5 or 25 JU.MTPT contained 3.5-, 10.1- and 15.6-foldmore covalent protein-DNA complexes than did the no-drug controls.

Irrespective of the concentration of TPT added to the cultures, themaximum number of complexes was detected 5-15 min after the

addition of TPT to the medium. The number of detectable complexesdecreased thereafter. After 3 h incubation with TPT, the number ofcomplexes detected ranged from 60% (at 25 /J.M)to less than 5(Wc(for0.25 and 2.5 /XM)of the level seen 15 min after addition of the drug.

A similar experiment was then done with SN-38, another drug ofthe camptothecin class and the active form of CPT-11. Results obtained after 2.5 min to 7 h incubation of SJ-G5 cells with 5 /J.MSN-38

were similar to those seen with TPT (Fig. IÃŸ).The maximum numberof topoisomerase I-DNA complexes (10-fold increase compared tono-drug controls) was detected 5-15 min after the addition of SN-38.This level decreased to 7.6-fold at 3 h and 4.0-fold (40%) at 7 h.

Topotecan

60 120

Minutes

180

B /.S-,

-OII 5.0-

2.5HO o.!=â€¢<J

0.0

SN-38

O 60 120 180 240 300 360 420

MinutesFig. 1. A, quantitation of covalent lopoisomerase I-DNA complexes in SJ-G5 cells

incubated wiih 0.25. 2.5. or 25 /IM TPT. The protein and DNA of SJ-G5 cells were labeledmetabolically by an overnight incubation with |'H|thymidine and |IJC]leucine and were

then incubated with the indicated concentrations of TPT for limes ranging from 5-180min. Covalent protein-DNA complexes were precipitated by the K ' -SDS method, and

radioactivity in the precipitate was quantitaled by liquid scintillation counting. Details ofthe method are in "Materials and Methods." B, quanlitation of covalent topoisomerase

I-DNA complexes in SJ-G5 cells treated with 5 fiM SN-38 for times ranging from 2.5 minto 7 h. Details of the method arc in "Materials and Methods."

If the number of covalent topoisomerase I-DNA complexes were

dependent on a simple equilibrium between enzyme, DNA, and drug,then we would predict that covalent topoisomerase 1-DNA complexesshould increase in a dose- and time-dependent manner and then

plateau. Although alterations in one or more of these componentscould be responsible for the decrease seen in topoisomerase I-DNA

complexes seen in Fig. 1, we focused on possible changes in theenzyme itself. Our working hypothesis was that after the addition ofTPT or SN-38, topoisomerase I was either degraded rapidly by cel

lular proteases or, as has been shown for other nucleolar proteins, thesubcellular distribution of the enzyme was altered (25, 26) such thatit could no longer bind to DNA.

Subcellular Localization of Topoisomerase I by FDIM

DNA topoisomerase I has been reported to have a predominantlynucleolar distribution ( 1-4) and to be involved in rRNA synthesis (3,27). Fig. 2 shows SJ-G5 anaplastic astrocytoma cells stained by

indirect immunofluorescence for topoisomerase I. No fluorescencewas visible when the reaction was carried out with control preimmunerabbit serum with or without incubation with TPT (Fig. 2. left panel).Nucleolar staining was clearly seen when SJ-G5 cells were incubated

with antiserum that recognized topoisomerase I (Fig. 2. right panel),as has been reported for other cell types. Using the same antiserum,we verified in a previous study that nuclear localization of topoisomerase I immunofluorescence was indistinguishable from that ofB23, a nucleolar marker (25, 26).

We further verified the nuclear characteristic of the fluorescencesignal for topoisomerase I in the SJ-G5 cells by using digital imaging

software (Fig. 3). After indirect immunofluorescence staining hadbeen done for topoisomerase I in SJ-G5 cells (Fig. 3, lower left panel ).

cells were incubated in Hoechst 33342 to allow visualization of DNAofthat same field of cells (Fig. 3, upper left panel). We then used thearea of DNA fluorescence to define the area of the nucleus in each celland created a binary mask for each cell (Fig. 3, upper right-hand

panel). We then blocked out the area of the binary mask in the imageshowing topoisomerase I immunofluorescence. The lower right-hand

panel of Fig. 3. therefore, shows indirect Â¡mmunofluorescence staining of only the cytoplasm of SJ-G5 cells. The majority of the fluo

rescence seen in the intact cells was no longer visible. TopoisomeraseI had a predominantly nuclear distribution in SJ-G5 cells. Nucleolartopoisomerase I was evident in approximately 60% of SJ-G5 cells inexponential growth (Figs. 2, 3, and 5), with some apparent nucleo-

plasmic or cytoplasmic staining also evident.An experiment similar to that shown in Fig. 3 was then repeated

with control SJ-G5 cells (Fig. 4A) and was also done with SJ-G5 cells

that had been incubated for 1 h with 2.5 JU.MTPT (Fig. 4B). In the twoleft panels of Fig. 4A are fields of control SJ-G5 cells. Nucleolar

staining of topoisomerase I was evident, and the nuclear localizationof the fluorescence was confirmed by the images in the two right-

hand panela in which areas occupied by nuclei have been blocked out.In contrast to the results in Fig. 4A are those shown in Fig. 4B forSJ-G5 cells that had been incubated with 2.5 /*MTPT for 60 min. Indrug-treated cells, nucleolar staining of topoisomerase I was absent,

and fluorescence appeared to be predominantly cytoplasmic. Whenthe nuclei of these two fields of cells were blocked out (Fig. 4B,right-hand panels), specific fluorescence was still visible in the cy

toplasm of the cells treated with TPT. When cells were analyzed byelectron microscopy, no change in character or number of nucleoliwas detected between drug-treated and untreated cells (data notshown). From the data in Fig. 4, we concluded that in SJ-G5 cells

treated for I h with 2.5 p.M TPT. topoisomerase I dissociated fromnucleoli, and the amount of topoisomerase I in the cytoplasm in-

1666



R[-;niSTRIBl'TION OF TOPOISOMERASE I

Fig. 2. Indirect immunofluorescence staining ofSJ-G5 anaplastic astrocytoma cells for DNA topoi-somerase I. SJ-G5 cells were grown on chamberwell slides and were fixed with paraformaldehyde.Cells were then incubated with either preimmunerabbit serum or a polyclonal antiserum that recognizes topoisomerasc I. The experiment was done 25times. At least 12 fields of cells were analyzed ineach experiment. A representative field of cells isshown. Details of the procedures are in "Materialsand Methods."

Preimmune Immune

DNA Binary mask

Fig. 3. Immunofluorescence staining for topoi-

somerase I in whole cells and in the cytoplasm ofSJ-G5 cells. Cells were stained for topoisomerase Iand then incubated with Hoechst 33342 to visualizethe DNA for each field of cells. DNA and topoisomerase I images were recorded with separate narrow band filters, and a binary mask was created fromthe DNA image. The areas of the binary mask representing the nuclear area of the cells was then blockedout of the topoisomerase I image to visualize indirectimmunofluorescence of topoisomerase I in the cytoplasm of SJ-G5 cells. The experiment was done 10

times. An average of 20 fields of cells were recordedfor each experiment. A representative field of cells isshown. Details of the procedures are in "Materialsand Methods."

Topoisomerase I,Intact cell

Topoisomerase I,Cytoplasm

1667




Fig. 4. A, comparison of indirect immunofluo-

rescence staining for topoisomerase I in intactSJ-G5 cells and in the cytoplasm of the same cells.See the legend to Fig. 3 for details of the methods.B, comparison of indirect immunofluorescencestaining for topoisomerase I in SJ-G5 cells or cy

toplasm after treatment of the cells with 2.5 Â¿IMTPT. Cells were incubated for l h with TPT, fixed,and stained for topoisomerase I. See the legend toFig. 3 for details of the methods.

Intact Cells Cytoplasm

B

Intact Cells Cytoplasm

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No Drug Control

REDISTRIBUTION OF TOPO1SOMERASE

0.25 Â¡M 1.

2. 5.0pÂ¿M Preimmune SerumControl

Fig. 5. Dose-dependent dissociation of topoisomerase I from nucleoli of TPT-treated SJ-G5 cells. Cells were incubated for I h with the indicated concentrations of TPT and thenwere stained for topoisomerase I by indirect Â¡mmunofluorescence. The topoisonierase I amount and subcellular locali/alion were analyzed by FDIM procedures. The experiment wasdone eight times, with an average of three fields of cells (an image for DNA and an image for topoisomeruse I ) recorded at each concentration of TPT for each experiment. Resultsfrom a representative experiment are shown. See the legend to Fig. 2 and "Materials and Methods" for details of the procedures.

creased. We repeated the experiments shown in Figs. 3 and 4 withGC, colon carcinoma cells and with SJ-G2 gliohlastoma multiforme

cells: similar results were obtained with all three cell lines (data notshown).

Dose Dependence of Drug-induced Redistribution ofTopoisomerase I

SJ-G5 cells were incubated with 0.25, 1.0. 2.5. and 5 /AMTPT for

1 h. Cells treated with 0.25 JUMTPT had a normal nucleolar distribution oftopoisomerase I (Fig. 5). In cells treated with 1, 2.5, or 5 /MMTPT. a dose-dependent decrease in nucleolar topoisomerase I was

seen. In cells incubated for 1 h with 5 Â¡i\iTPT, no nucleolar topoisomerase I could be detected. Since nucleoli were still visible byelectron microscopy after SJ-G5 cells had been treated with 5 /MMTPT

(data not shown), these results suggest that topoisomerase I haddissociated from nucleoli in SJ-G5 cells treated with TPT. It also

appeared that in cells incubated with 5 Â¿Â¿MTPT, total nuclearMr 67.000 topoisomerase I immunofluorescence had decreased. Thisdecrease in nuclear fluorescence of cells treated with 5 /MMTPT was

verified by FDIM quantitation of the total nuclear fluorescence foreach field of cells in Fig. 5. Control cells contained 1.93 X l(ffluorescence units of topoisomerase I. Cells treated with 0.25. 1.0, 2.5,and 5 /MMTPT contained 2.20, 2.44, 1.41, and 1.41 X IO6 fluores

cence units, respectively. Statistically, the decreases in amounts ofnuclear topoisomerase I seen after incubation with 2.5 and 5 /IM TPTwere significantly different from control (P < 0.05, two-tailed I test).

We also attempted to quantitate cytoplasmic topoisomerase I in thecells in Fig. 5. However, because of the irregular shape of SJ-G5 cells,

this quantitation would necessarily be biased by the person operatingthe digital imaging software. These analyses were, therefore, notincluded in this report.

Time Dependence of TPT-induced Redistribution ofTopoisonierase I

Rapid Fractionation of Cells and Immunoblots of Nuclear andCytoplasmic Proteins for Topoisomerase I. We next wanted toverify by a second method the decrease in nuclear immunofluorescence and the increase in cytoplasmic immunofluorescence of topoi-

1669



REDISTRIBUTION OF TOPO1SOMERASE

-DNase +DNase

Topotecan 0 20 40 60 0 20 40 60 min.

â€”¿�lOOkDa

â€”¿�67 kDa

BTopotecan 0 20 40 60 min.

â€”¿�100kDa

â€”¿�67 kDa

Fig. 6. A, immunoblot for topoisomerase I of preparations of nuclear proteins of SJ-G5cells treated with 5 JUMTPT for 0, 20, 40, or 60 min. Cells were incubated for the indicatedtimes with TPT, after which nuclear and cytoplasmic proteins were separated by rapidfractionation of the cells, followed by SDS-PAGE. Proteins were transferred to nitrocellulose paper, and immunoblots were done to detect topoisomerase I by standard methods(see "Materials and Methods"). The experiment was done eight times: each set of samples

was run on at least two SDS gels. A representative blot is shown. B. immunoblot fortopoisomerase I of preparations of cytoplasmic proteins of SJ-G5 cells treated with 5 Â¡at

TPT for 0, 20. 40, or 60 min. See the legend to Fig. M for details of the methods.

somerase I in TPT-treated cells. Therefore, we incubated SJ-G5 cells

for 0, 20, 40, or 60 min with 5 /MMTPT; separated nuclear fromcytoplasmic proteins by rapid fractionation methods; and analyzedeach fraction for topoisomerase I by immunoblot.

Nuclear Topoisomerase I. Fig. 6/4 shows an immunoblot of thenuclear fractions of SJ-G5 cells treated with TPT for the indicated

times. The four lanes on the left contain samples that have not beenincubated with DNase; therefore, only topoisomerase I not bound toDNA migrated into the gel. Nuclear preparations from cells incubatedfor 60 min with TPT contained 25% less free A/r 100,000 topoisomerase I than control cells; however, this decrease was not statisticallysignificant in the eight experiments done. The four lanes on the rightof this figure contain aliquots of the same samples incubated withDNase for 20 min before boiling in sample buffer. After DNasetreatment, no change in the amount of immunoreactive nuclear topoisomerase I was observed in samples incubated from 20-60 min with

TPT.The most obvious difference between the samples treated and not

treated with DNase was the presence of a prominent Mr 67,000immunoreactive band in the samples treated with DNase. This Mr67,000 band has been reported to be a proteolytic breakdown productof the MT 100.000 enzyme (28). but the catalytic activity of M, 67,000topoisomerase I is equivalent to that of the Mr 100,000 enzyme. Theobservation that no Mr 67.000 immunoreactive band was seen in

samples not treated with DNase suggests several possibilities. Thesmaller molecule may be bound to DNA in intact cells and maysimply not enter the gel until the DNA has been digested. Alternatively, the form of the enzyme bound to DNA may be the Mr 100,000protein, but this protein may be more readily degraded after DNAdigestion, even in the presence of protease inhibitors. Quantitation bydensitometric scanning verified that the amount of immunoreactiveMr 67.000 protein did not change over the time course of the experiment.

The results obtained by immunoblot showed no change in theamount of topoisomerase I in the nuclei of TPT-treated SJ-G5 cells.

Quantitation by FDIM indicated a significant decrease in nucleartopoisomerase I content from 1.93 X IO6 fluorescence units in untreated cells to 1.44 X IO6 fluorescence units in cells treated for 60

min with 5 JUMTPT (P < 0.05). The FDIM quantitation was done ona small population of cells and is a very sensitive assay, whereasimmunoblots reflect results from large populations of cells and are aless sensitive technique. It is likely the lack of a significant decreasein topoisomerase I after drug treatment in the immunoblots can beattributed to these two factors. Alternatively, it is also possible thatupon the addition of drug, topoisomerase I assumes a different molecular conformation, and the epitope is no longer accessible to theantiserum. The antiserum may not recognize all conformations oftopoisomerase I in situ, and what appears to be dissociation oftopoisomerase I from nucleoli might be, instead, a conformationalchange of enzyme still associated with nucleoli. We consider this aless likely interpretation of the data since: (a) we used a polyclonalantiserum: and (b) at the 1.0 /UMdose of TPT (Fig. 5), a change intopoisomerase I subnuclear localization was evident, but no decreasein the amount of nuclear topoisomerase I was seen. We conclude thata small (25%) but significant decrease in topoisomerase I occurred inthe nuclei of SJ-G5 cells treated for 60 min with 2.5 or 5 /MMTPT.

Cytoplasmic Topoisomerase I. Although analysis of the topoisomerase I content in nuclei of SJ-G5 cells treated with 5 /IM TPT

showed only a slight decrease after a 60 min incubation with TPT, thecytoplasmic content of topoisomerase I in these cells obviously increased during this time course. Fig. 6ÃŸshows immunoblots fortopoisomerase I of the cytoplasmic fractions of SJ-G5 cells treated for

0, 20, 40, or 60 min with 5 /MMTPT. Immunoreactive topoisomeraseI in the cytoplasm first decreased at 20 min and then increased at 40and 60 min. The cytoplasmic topoisomerase I content was 51% lowerin cells incubated for 20 min with TPT than in control cells. Conversely, the cytoplasmic content of topoisomerase I was 98% higherin cells incubated for 60 min with TPT than in untreated cells. Theonly immunoreactive band detected in cytoplasmic preparations hadan apparent molecular weight of Mr 67,000. DNase treatment ofcytoplasmic fractions had no effect on immunodetectable bands (datanot shown).

Verification of Separation of Nuclear and Cytoplasmic Proteins

Before investigating further the increase in cytoplasmic topoisomerase I after incubation of SJ-G5 cells with TPT, it was necessary

to determine the efficiency of separation of nuclear and cytoplasmicproteins. To estimate the purity of the cytoplasmic fractions (21), weseparated "nuclear" and "cytoplasmic" proteins by SDS-PAGE and

then did immunoblot analysis with a polyclonal antiserum to MEK2,a cytoplasmic marker (22). Quantitation of immunoblots for MEK2verified that the preparation we designated as "cytoplasmic" con

tained â€”¿�93%of immunodetectable MEK2 protein (data not shown).

To estimate the purity of the nuclear fractions, we quantitated byHoechst 33342 intercalation and fluorometry the amount of DNApresent in "nuclear" and "cytoplasmic" fractions. Nuclear fractions

1670



REDISTRIBUTION OF TOPOISOMERASE 1

/

â€”¿�100kDa

â€”¿�67 kDa

Fig. 7. Immunoblot for topoisomerase I of preparations of cytoplasmic proteins ofSJ-G5 cells treated with either 10 /J.Mcycloheximide. 5 JIMTPT. or 10 JIMcycloheximideplus 5 JIM TPT. The methods are as in the legend for Fig. 6A.

contained â€”¿�95%of the total DMA content of both fractions. We

concluded that the separation of nuclear and cytoplasmic proteins inthe above experiments was adequate.

Increase of Cytoplasmic Topoisomerase I Content after TPTTreatment Is Due to Increased Synthesis of Topoisomerase I

Having determined that nuclear and cytoplasmic preparations werereflective of the localization of proteins in intact cells, we next lookedat a possible cellular mechanism to account for the increase in topoisomerase I content in the cytoplasm of cells incubated with TPT. Anincrease in total cellular protein could be due to either increasedsynthesis or decreased breakdown of the protein of interest. Weinvestigated the first possibility, that the increase of topoisomerase Iin the cytoplasm was due to de novo protein synthesis, by attemptingto prevent the increase by incubating SJ-G5 cells with cycloheximideprior to the addition of TPT. SJ-G5 cells were incubated under the

following conditions: 10 /UMcycloheximide for 70 min, 5 JJ.MTPT for60 min, or 10 /MMcycloheximide for 10 min, followed by the additionof 5 /MMTPT for an additional 60 min. Preparations of the cytoplasmicproteins of these cells and of untreated control cells were separated bySDS-PAGE and topoisomerase I detected by immunoblotting. Fig. 7

shows that in cytoplasmic preparations of cells treated with TPT for60 min, the anticipated increase in topoisomerase I was evident(compare first and third lanes). However, in cells preincubated for 10min with cycloheximide and then TPT for an additional 60 min (Fig.7, compare third and fourth lanes), the magnitude of the increasediminished by 50%. A similar effect was seen on topoisomerase Icontent in cells not treated with TPT (Fig. 7, compare first and secondlanes). The experiment was then repeated but with a 30-min incubation instead of a 60-min incubation with TPT. Similar to results shown

in Fig. 7, the cytoplasmic fraction of cells incubated with cycloheximide contained ~71% less Â¡mmunoreactive A/r 67,000 topoisomerase

I than control cells incubated with TPT alone (data not shown). Weconclude that the increase in cytoplasmic topoisomerase I after theaddition of TPT to SJ-G5 cells can be accounted for, at least in part.

by de novo protein synthesis. Additional studies on the rates ofsynthesis of topoisomerase I in the presence and absence of TPT arein progress.

Effect of Other Drugs on Subcellular Distribution of DNATopoisomerase I

It was also of interest to determine whether the dissociation oftopoisomerase I by TPT from nucleoli of SJ-G5 cells could be inducedby compounds other than TPT. Therefore, we incubated SJ-G5 cells

with compounds known to inhibit cellular proliferation by differentmechanisms, including: SN-38 (a camptothecin analogue: Ref. 29),DMP 840 (a bis-naphthalimide compound that inhibits DNA, RNA,

and protein synthesis; Ref. 30), vincristine (a Vinca alkaloid that bindsto tubulin), hydroxyurea (an inhibitor of ribonucleotide reducÃase),aphidicolin (an inhibitor of DNA polymerase), actinomycin D (aninhibitor and topoisomerases I and II), mitoxantrone and etoposide(inhibitors of topoisomerase II). 1-ÃŸ-o-arabinofuranosylcytosine (a

nucleoside analogue: Ref. 31), and Hoechst 33342 (a minor groovebinder and inhibitor of topoisomerase I; Ref. 32). SJ-G5 cells were

incubated for 60 min at the indicated concentrations of each drug. Thefilters used for detecting immunofluorescence of topoisomerase I didnot permit visualization of any of the above compounds; therefore,fluorescence seen in photomicrographs of SJ-G5 cells treated with

each compound can be attributed to immunoreactive topoisomerase I.Fig. 8 shows indirect immunofluorescence of topoisomerase I in

SJ-G5 cells treated with either vincristine (l /UM),aphidicolin (5 /Â¿M),wi-AMSA (3 /UM),or actinomycin D (25 JU.M).Neither vincristine nor

aphidicolin altered the anticipated fluorescence pattern of topoisomerase I; topoisomerase I was localized to nucleoli after incubation witheach drug. In contrast, both Â«Â¡-AMSAand actinomycin D altered the

fluorescence pattern of topoisomerase I such that topoisomerase I nolonger had a nucleolar distribution. Although the pattern of immunofluorescence produced by m-AMSA and actinomycin D did not ex

actly mimic that produced by TPT, the result common to all threedrugs was that topoisomerase I no longer appeared to be associatedwith nucleoli. In addition, in Table 1 are listed the compounds shownin Fig. 8, as well as others that were tested for the effect on subnucleardistribution of topoisomerase I. With each of the above compounds,nuclear membranes remained intact throughout the 60 min of theexperiment, as determined by DNA fluorescence.

Results tabulated here suggest that the compounds that alter thesubnuclear distribution of topoisomerase I are those that inhibit RNAsynthesis. Compounds that do not affect RNA synthesis have no effecton the subnuclear distribution of DNA topoisomerase I. As mentionedabove, an alternative but less likely interpretation of the data would bethat each of the above compounds that inhibits RNA synthesis altersthe conformation of topoisomerase I indirectly to limit antibody-

epitope binding and, therefore, limit detection of topoisomerase I. Weconclude that compounds that inhibit RNA synthesis induce dissociation of topoisomerase I from nucleoli of SJ-G5 cells.

DISCUSSION

One of the novel findings reported in this study is that in anaplasticastrocytoma cells incubated with TPT, topoisomerase I rapidly dissociated from nucleoli. Simultaneously, the cytoplasmic concentrationof topoisomerase I in these cells increased. [Similar results wereobtained in SJ-G5 cells in an immunofluorescence assay with the IgMmonoclonal antibody used in the study reported in the accompanyingpaper by Buckwalter et al. (33) in this issue of Cancer Research; datanot shown]. The increase of topoisomerase I in the cytoplasm wasdue, at least in part, to new protein synthesis. Interestingly, the

1671




Vincristine Actinomycin D

Fig. 8. Effect of different types of inhibitors ofcell growth on the subcellular localisation of topoisomerase I. SJ-G5 cells were treated with 1 Â¿IMvincristine. 25 Â¿IMactinomycin D, 5 Â¿IMaphidico-lin, or 3 /UMm-AMSA for I h. Cells were then fixed

with parafornialdehyde and unuly/ed by indirectimmunofluorescence for subcellular locali/ation oftopoisomerasc I. The experiment was done threetimes. An average of six fields of cells were recorded tor each experiment. Representative fieldsof cells are shown. Details of the metruxJs are in thelegend to Fig. 2 and in "Materials anil Methods."

Aphidicolin m-AMSA

dissociation of topoisomerase I from nucleoli was also induced bydrugs other than TPT that inhibit RNA synthesis (33). This observation is similar to observations of Yung el al. (25) and Chan et al. (26)that actinomycin D alters the subnuclear distribution of nucleophos-

min or B23. a nucleolar protein involved in the assembly of ribosomalproteins into ribosomes. The dissociation of B23 from nucleoli ofHeLa cells occurs within the same time frame (less than I h) asreported in this study for TPT-induced dissociation of topoisomeraseI from nucleoli of SJ-G5 cells. Chan et al. (26) also observed that in

P388 cells selected for resistance to doxorubicin. higher concentrations of doxorubicin are required to induce nucleolar to nucleoplasmic

Table I Kffecl of inhihitors of wll growth on the snhnnt'lt'nr ilislribnlion of DNAlopoistitni'rusÃ¯ 1

SJ-G5 cells were incubated for W) min with the indicated concentrations of the abovecompounds. The cells were then fixed with parafornialdehyde and stained by immuno-fluorescence methods for DNA topoisomerase I. Control cultures were treated with water.0.9% NaCI. or DMSO. Immunolluorescence patterns were recorded with an NP-MXIPhotographic Network Printer and assessed visually.

Dissociated topoisomerase IfromnucleoliTopotecan

(1-5 Â¿IM)Daunorubiein (5 Â¿IM)Hoechst 33342 (30 /AM)Mitoxantrone (20 /AM)DMP 840 (1 /AM)Â»f-AMSA (3 /AM)VP-l6a(5 /AM)

Actinomycin D (25 /AM)No

effect on localizationof topoisomerase1Cytosine

arabinoside (3 /AM)Vincristine ( 1 Â¿IM)Aphidicolin (5 Â¿IM)Hydroxyurea (5 /AM)Cycloheximide (7Â¿IM)"

VP-16, etoposide.

translocation of nucleophosmin compared to drug-sensitive cells.

Other nucleolar proteins, such as nucleolin, move freely among nucleoli, nucleoplasm. and cytoplasm (21). but the effect of chemother-

apetitic agents on the subcellular localization of nucleolar proteinsother than nucleophosmin has not been reported.

The second observation reported here, that TPT treatment increasesthe cytoplasmic content of topoisomerase I. raises the question ofwhether the rate of synthesis of topoisomerase I is increased followingtreatment with TPT or whether the rate of catalysis of the protein isdecreased. Alternatively, the rate of nuclear import of newly synthesized enzyme might somehow be slowed, either directly or indirectly.by TPT. The simplest explanation for the observed increase of cytoplasmic immtinoreactive topoisomerase I is that the rate of nuclearimport of newly synthesized protein is slower than the rate ofsynthesis, as has been shown in other systems (34. 35). In fact,photomicrographs showing indirect immunofluorescence of topoisomerase 1 in SJ-G5 cells treated with TPT (Fig. 40) look remarkablysimilar to photomicrographs of ATP-deprived Vero cells, with an

inhibited rate of nuclear import of nucleoplasmin reported by Richardson et al. (34). In each case, the fluorescent-labeled protein appears

to be sequestered just outside of the nuclear membrane, possiblybound to extranuclcar fibrils, "waiting" to be transported into the

nucleus.A third observation in this report relates to different immunoreac-

tive forms of topoisomerase I detected in nuclear versux cytoplasmicfractions of the cells. Free topoisomerase I in the nucleus of SJ-G5

cells has an apparent molecular weight of Mt KK).(MK).with another

1672



RlÃDlSTRIBlTION ()l: TOPOISOMERASE I

prominent Mr 67,000 form evident only after treatment of nuclearprotein preparations with DNase. In contrast, the only form detectable in the cytoplasm was the Mr 67,000 form of the enzyme, apostulated proteolytic product of topoisomerase I (28). Because theMr 67,000 form of topoisomerase I is catalytically active in vitro,it is possible that the two forms of the enzyme exist in the cell andhave unique functions in intact cells. A simpler explanation wouldbe that even in the presence of protease inhibitors, the Mr 100,000enzyme in the cytoplasm is unstable and is simply a proteolyticbreakdown product. It is intriguing that the Mr 67,000 form of theenzyme was detectable only in nuclear fractions that had beentreated with DNase.

In summary, the biological significance of the ability of TPT to alterthe subcellular distribution of its own cytotoxic target is unknown. Alsounknown is the clinical relevance of the observation that classes ofchemotherapeutic agents other than inhibitors of topoisomerase I alter thesubcellular distribution of topoisomerase I. Studies to address the effectof TPT on the nuclear import of topoisomerase I and the effect ofredistribution of topoisomerase I on the cytotoxicity of camptothecins andof combinations of chemotherapeutic agents are in progress.

ACKNOWLEDGMENTS

We thank Frank Harwood, Chris Morton, and Phil Potter for their invaluableassistance in leading me (M. K. D.) through the St. Jude computer network and"helping" me print the immunoblot figures. We also thank Donna Davis for her

beautiful electron micrographs of TPT-treated SJ-G5 cells.

REFERENCES

1. Baker. S. D.. Wadkins. R. M.. Stewart. C. F., Beck. W. T.. and Danks. M. K. Cellcycle analysis of amount of distribution of nuclear DNA topoisomerase 1 as determined by fluorescence digital imaging microscopy. Cytometry. 19: 134-145. 1995.

2. Fleischmann, G.. Pflugfelder, G., Steiner, E. K.. Javaherian, K.. Howard, G. C.,Wang. J. C.. and Elgin. S. C. Drosophila DNA topoisomerase I is associated withtranscriptionally active regions of the genome. Proc. Nati. Acad. Sci. USA. 81:6958-6962. 1984.

3. Muller, M. T., Pfund. W. P.. Mehta. V. B., and Trask. D. K. Eukaryotic type Itopoisomerase is enriched in the nucleolus and cataiytically active on ribosomalDNA. EMBO. J.. 4: 1237-1243, 1985.

4. Negri. C.. Chiesa, R.. Cerino, A.. Bestagno. M.. Sala. C.. Zini. N., Maraldi, N. M., andAstaldi-Ricotti, G. C. B. Monoclonal antibodies to human DNA topoisomerase I andthe two isoforms of DNA topoisomerase II: 170- and 180-kDa isozymes. Exp. CellRes.. 200: 452-459, 1992.

5. Hsiang. Y-H.. and Liu. L. F. Identification of mammalian DNA topoisomerase I as anintracellular target of the anticancer drug camptothecin. Cancer Res., 48: 1722-1726,1988.

6. Slichenmyer. W. J.. Rowinsky. E. K.. Donehower, R. C., and Kaufmann. S. H. Thecurrent status of camptothecin analogues as antilumor agents. J Nati. Cancer Inst., 85:271-291, 1993.

7. Tanizawa, A., Fujimori. A., Fujimori, Y.. and Pommier, Y. Comparison of topoisomerase I inhibition. DNA damage, and cytotoxicity of camptothecin derivativespresently in clinical trials. J. Nail. Cancer Inst.. 86: 836-842, 1994.

8. Sugimoto. Y.. Tsukahara, S., Oh-hara, T., Isoe, T., and Tsuruo, T. Decreasedexpression of DNA topoisomerase I in camptolhecin-resistant tumor cell lines asdetermined by a monoclonal antibody. Cancer Res., 50: 6925-6930. 1990.

9. Kubota. N.. Kanzawa. F.. Nishio. K.. Takeda. Y.. Ohmori. T.. Fujiwara. Y.. Terashima.Y.. and Saijo. N. Detection of topoisomerase I gene point mutation in CPT-II resistantlung cancer cell line. Biochem. Biophys. Res. Commun.. 188: 571-577.1992.

10. Benedetti. P., Fiorani. P., Capuani, L., and Wang. J. C. Camptothecin resistance froma single mutation changing glycine 363 of human DNA topoisomerase I to cysteine.Cancer Res., 53: 4343-4348, 1993.

11. Fujimori. A.. Harker. W. G.. Kohlhagen, G.. Hoki. Y., and Pommier. Y. Mutation at

the catalytic site of topoisomerase lin CEM/C2. a human leukemia cell line resistantto camptothecin. Cancer Res., 55: 1339-1346. 1995.

12. Gupta, R. S., Gupta. R.. Eng, B., Lock. R. B.. Ross, W. E., Hertzberg. R. P.. Caranfa,M. J., and Johnson. R. K. Camptothecin-resistant mutants of Chinese hamster ovary cellscontaining a resistant form of topoisomerase I. Cancer Res., 48: 6404-6410, 1988.

13. Rubin. E.. Pantazis. P., Bharti, A.. Toppmeyer. D.. Giovanella, B., and Kufe. D.Identification of a mutant human topoisomerase I with intact catalytic activity andresistance to 9-nitro-camptothecin. J. Biol. Chem., 269: 2433-2439, 1994.

14. D'Arpa, P., and Liu. L. F. Topoisomerase-targeting antitumor drugs. Biochim.

Biophys. Acta, 989: 163-177, 1989.

15. Harker. W. G., Kapoor. R., Slade, D. L., Fujimori, A., and Pommier, Y. AltÃ©rationsin DNA topoisomerase I content and sensitivity in camptothecin-resistant human

CEM leukemia cell lines. Proc. Am. Assoc. Cancer Res., 35: 456. 1994.16. Beidler. D. R.. and Cheng, Y-C. Camptothecin induction of a time- and concentration-

dependent decrease of topoisomerase I and its implication in camptothecin activity.Mol. Pharmacol., 47: 907-914. 1995.

17. Zwelling, L. A.. Hinds. M.. Chan, D., Mayes, J., Sie, K. L., Parker, E., Silberman, L.,Radcliffe, A., Beran, M.. and Blick. M. Characterization of an amsacrine-resistant lineof human leukemia cells: evidence for a drug-resistant form of topoisomerase II. J.Biol. Chem., 264: 16411-16420, 1989.

18. Wolverton. J. S., Danks, M. K.. Granzen, B., and Beck, W. T. DNA topoisomeraseII immunostaining in human leukemia and rhabdomyosarcoma cell lines and theirresponses to topoisomerase II inhibitors. Cancer Res., 52: 4248-4253, 1992.

19. Masters, B. R.. and Kino. G. S. Charge-coupled devices for quantitative Nipkow diskreal-time scanning confocal microscopy. In: D. Shotton (ed.). Electronic LightMicroscopy, Chap. 14, pp. 315-327. New York: Wiley-Liss, 1993.

20. Castleman. K. R. Digital Image Processing. Englewood Cliffs, NJ: Prentice-Hall,

1979.21. Borer, R. A., Lahner. C. F.. Eppenberger. H. M., and Nigg. E. A. Major nucleolar

proteins shuttle between nucleus and cytoplasm. Cell, 56: 379-390. 1989.22. Zheng, C-F., and Guan, K-L. Cytoplasmic localization of the mitogen-activated

protein kinase activator MEK. J. Biol. Chem.. 269: 19947-19952, 1994.23. Femandes. D. F.. Danks, M. K., and Beck, W. T. Resistance to VM-26 is associated

with selective decrease in nuclear matrix DNA topoisomerase II. Biochemistry, 29:4235-4241. 1990.

24. Danks, M. K., Qiu, J., Catapano, C. V.. Schmidt, C. A.. Beck, W. T.. and Femandes,D. J. Subcellular distribution of the a and ÃŸtopoisomerase II-DNA complexesstabilized by VM-26. Biochem. Pharmacol., 48: 1785-1796, 1994.

25. Yung, BY-M., Busch. H., and Chan. P-K. Effects of luzopeplins on protein B23translocation and ribosomal RNA synthesis in HeLa cells. Cancer Res.. 46: 922-925,

1986.26. Chan, P-K.. Aldrich, M. B., and Yung, BY-M. Nucleolar protein B23 translocation

after doxorubicin treatment in murine tumor cells. Cancer Res., 47: 3798-3801.1987.

27. Zhang, H.. Wang, J. C.. and Liu, L. F. Involvement of DNA topoisomerase I intranscription of human ribosomal RNA genes. Proc. Nati. Acad. Sci. USA. 85:1060-1064. 1988.

28. Liu. L. F.. and Miller. K. G. Eukaryotic DNA topoisomerases: two forms of type IDNA topoisomerases from HeLa cell nuclei. Proc. Nati. Acad. Sci. USA, 78:3487-3491.1981.

29. Araki. E.. Ishikawa. M.. ligo. M.. Koide, T., Ilabashi. M., and Hoshi. A. Relationshipbetween development of diarrhea and the concentration of SN-38. an active metabolite of CPT-11, in the intestine and the blood plasma of athymic mice followingÂ¡ntraperitoneal administration of CPT-11. Jpn. J. Cancer Res., 84: 697-702, 1993.

30. Kirshenbaum, M. R., Chen, S. F., Behrens, C. H., Papp, L. M.. Stafford, M. M., Sun,J. H., Behrens, D. L.. Fredericks, J. R., Polkus, S. T., Sipple, P.. Patten. A. D., Dexter,D., Seitz. S. P., and Gross, J. L. (Ã„,/?)-2,2'-[l,2-ethanediylbis|imino(l-methyl-2.1-

ethanediyl)]]-bis[5-nitro-l//-benz[de]isoquinoline-l,3-(2W)-dione]dimethanesulfon-ate (DMP 840), a novel bis-naphthalimide with potent nonselective tumoricidalactivity in vitro. Cancer Res.. 54: 2199-2206. 1994.

31. Chabner. B. A., and Collins, J. M. Cancer Chemotherapy. Principles and Practice.New York: J. B. Lippincott Company. 1990.

32. Chen. A. Y.. Chiang, Y., Bodley, A., Peng, L. F., and Liu, L. F. A new mammalianDNA topoisomerase I poison Hoechst 33342: cytotoxicity and drug resistance inhuman cell cultures. Cancer Res., 53: 1332-1337, 1993.

33. Buckwalter, C. A., Lin, A. H., Tanizawa, A., Pommier, Y. G., Cheng, Y-C., andKaufmann. S. H. RNA synthesis inhibitors alter the subnuclear distribution of DNAtopoisomerase I. Cancer Res., 56: 1674-1681. 1996.

34. Richardson, W. D., Mills, A. D., Dilworth, S. M., Laskey, R. A., and Dingwall, C.Nuclear protein migration involves two steps: rapid binding at the nuclear envelopefollowed by slower translocation through nuclear pores. Cell, 52: 655-664. 1988.

35. Newmeyer, D. D.. and Forbes, D. J. Nuclear import can be separated into distinctsteps in vitro: nuclear pore binding and translocation. Cell, 52: 641-653, 1988.

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1996;56:1664-1673. Cancer Res Mary K. Danks, Katherine E. Garrett, Raquel C. Marion, et al. Anaplastic Astrocytoma Cells Treated with TopotecanSubcellular Redistribution of DNA Topoisomerase I in

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