(CANCER RESEARCH 56. 2348-2354. May 15. I996|

Drug-induced Down-Regulation of Topoisomerase I in Human Epidermoid CancerCells Resistant to Saintopin and Camptothecins1

Ken Taniguchi,2 Kimitoshi Kohno, Kiyoshi Kawanami, Morimasa Wada, Takashi Kanematsu, and

Michihiko KuwanoDepartment of Biochemistry, Kyushu University School of Medicine. Maidashi. Hixashi-ku. Fukuoka 812-82 ¡K.T.. K. Ko.. K. Ka„M. W.. M. K. I, and Second Department of

Surgery, Nagasaki University School of Medicine. Sakamoto. Nagasaki 852 ¡T.K.¡,Japan

ABSTRACT

The anticancer agent saintopin induces DNA cleavage mediated by bothtopoisomerase (topo) I and topo II in vitro through stabilization of thereversible enzyme-DNA cleavable complex. We established saintopin-resistant cell lines (KB/STP-1 and KB/STP-2) from human epidermoid

cancer KB cells by stepwise exposure to increasing doses of the drug.KB/STP-1 and KB/STP-2 cells showed 12- and 44-fold increases, re

spectively, in resistance to saintopin relative to that of KB cells. Bothsaintopin-resistant cell lines showed only small reductions in sensitivity tothe topo II inhibitor etoposide but developed marked cross-resistance tothe topo 1-targeting camptothecin derivative CPT-11 |(4s)-4,ll-diethyl-4-hydroxy-9-[(4-piperidinopiperidino)carbonyloxy] dione hydrochloride tri-hydrate] and its active form, SN-38 (7-ethyl-10-hydroxycamptothecin). Incontrast, both KB/STP-1 and KB/STP-2 cells showed increased collateral

sensitivity to cisplatin, a nitrosourea derivative, mitomycin C, and UVlight. The protein concentration, activity, and mRNA abundance of bothtopo I and topo II were similar in KB/STP-1, KB/STP-2, and the parentalKB cells. There were no significant changes in the drug-stabilized topo-DNA cleavable complex formation in KB and KB/STP-2 cells. Two pointmutations were detected in topo I cDNA from KB/STP-2 cells, but these

were also present in KB cells. Topo I mRNA abundance decreased markedly immediately after exposure of KB/STP-2 cells to saintopin; no such

effects were apparent in KB cells. In contrast, topo II mRNA was notmarkedly affected by saintopin in either KB or KB/STP-2 cells. Treatmentwith CPT-11 or SN-38 also induced a markedly greater and more persistent reduction in topo I mRNA abundance in KB/STP-2 cells than in KB

cells. Etoposide had no marked effect on topo I mRNA abundance ineither KB/STP-2 or KB cells. Topo I mRNA was highly unstable inKB/STP-2 cells in comparison to KB cells when incubated with saintopin.This novel regulation of topo I mRNA by topo I-targeting agents could beassociated with acquirement of drug resistance to saintopin or SN-38/CPT-11 in KB/STP-2 cells.

INTRODUCTION

In eukaryotic cells, DNA topo' I and topo II regulate the topolog

ica! conformation of DNA molecules by catalyzing the concertedbreakage of single or double strands and are essential for transcription,recombination, DNA replication and repair, and mitosis (1,2). Inhibitors of these enzymes are widely used in cancer chemotherapy.Camptothecins, such as CPT-11 [(4s)-4,l l-diethyl-4-hydroxy-9-[(4-

piperidinopiperidino)carbonyloxy] dione hydrochloride trihydratel.SN-38 (7-ethyl-l()-hydroxycamptothecin), and topotecan, that inhibittopo I, induce topo I-mediated DNA cleavage and cell death, topo IIinhibitory agents, such as etoposide (VP-16), teniposide (VM-26),4'-(9-acridinylamino) methanesulfon-m-anisidide, doxorubicin, and

Received 12/12/95; accepted 3/19/96.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 with18 U.S.C. Section 1734 solely to indicate this fact.

' This work was supported in part by a Grant-in-Aid for cancer research from the

Ministry of Education. Science, and Culture of Japan, by the Yasuda Memorial MedicalGrant for Cancer Research, and by the Fukuoka Anticancer Research Fund.

•¿�To whom requests for reprints should be addressed.'The abbreviations used are: topo. DNA topoisomerase; VP-16. etoposide; ACNU.

3-((4-amino-2-methyl-5-pyrimidinyl)methyl|-l-(2-chloroethyl)-1 -nitrosourea; GAPDH.glyceraldehyde-3-phosphate dehydrogenase: 1C*,. 90^ inhibitory concentration.

mitoxantrone. induce topo Il-mediated DNA cleavage (3). Inhibitorsof topo I and topo II become covalently linked to the 3'-phosphoryl (4,5) and to the 5'-phosphoryl (6) termini of nicked DNA. They are often

used clinically in combination with DNA-damaging agents and showsynergistic antitumor effects (7-10). However, during long-term treatment with both topo I- and topo Il-targeting antitumor agents, the

appearance of tumor cells that exhibit decreased sensitivity to thesedrugs is often an obstacle to further chemotherapy. It is thereforeimportant to determine the underlying mechanism for the appearanceof drug-resistant tumor cells. Isolation and characterization of tumorcell lines resistant to topo I- and topo II- targeting agents have

provided systems in which to investigate the mechanisms of drugresistance. Studies with such cell lines suggest that qualitative andquantitative changes in the topo enzymes, decreased drug accumulation, and altered double-strand break rejoining during repair of DNA

damage contribute to drug resistance (11, 12). Recent observationshave also shown that overexpression of the MRP gene is oftenapparent in tumor cell lines resistant to topo II targeting epipodophyl-lotoxins or doxorubicin (13-15).

One approach to overcoming drug resistance to either topo I- ortopo Il-targeting agents is to develop anticancer drugs that inhibit both

topo I and topo II. Yamashita et al. (16. 17) have shown that saintopin,a new antitumor antibiotic isolated from Paecilomvces sp., inducesDNA cleavages mediated by both topo I and topo II in vitro as a resultof stabilization of the reversible enzyme-DNA cleavable complex andshows a potency similar to that of 4'-(9-acridinylamino) methanesul-

fon-m-anisidide or VP-16. Leteurtre et al. ( 18) further showed thatsaintopin directly interacts with guanine at the 5' termini of theenzyme-induced DNA breaks (G+l), possibly through hydrogen

bonding. Saintopin may enter the cavity near the catalytic tyrosine oftopo I or topo II, and thereby inhibit ligation of the DNA. Interactionwith the G+ ' may further stabilize saintopin in the catalytic site. To

investigate further the mechanism of action of saintopin, we have nowisolated and characterized human epidermoid cancer KB cell linesresistant to this drug.

MATERIALS AND METHODS

Establishment of Saintopin-resistant Lines of Human Epidermoid Cancer KB Cells. Saintopin-resistant lines were selected from KB cells after

exposure to increasing doses of saintopin in the absence of mutagen by aprocedure described previously (19, 20). KB cells in l(X)-mm plastic dishes

were first exposed to saintopin at 0.5 fig/ml to reduce the surviving fraction to40-50%, and were then cultured in medium containing progressively increas

ing concentrations of saintopin at 1,2, and 5 fig/ml, respectively. The cellswere finally exposed to 10 /xg/ml saintopin for 24 h and then incubated indrug-free medium for 3 weeks. One of the resulting colonies was purified,cloned, and named KB/STP-1. KB/STP-1 cells were further exposed to saintopin at 10 and 20 /ig/ml. each for 1 month, after which the saintopin-resistantcell line KB/STP-2 was isolated. KB/STP-1 and KB/STP-2 cells showed

doubling times of 21 ±3 h (mean ±SD) compared to 20 ±4 h for parentalKB cells. Parental and saintopin-resistant KB cells were grown as monolayer

in MEM (Nissui Seiyaku, Tokyo, Japan) containing 107r newborn calf serum(Microbiological Associates, Bethesda, MD), glutamine (0.292 mg/ml), kana-mycin (100 ¿ig/ml),and penicillin (100 units/ml; Refs. 19-21).

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SAINTOPIN RESISTANCE AND TOPO I EXPRESSION

Chemicals. Saintopin and mitomycin C were donated by Kyowa HakkoKogyo (Tokyo. Japan): VP-16 and cisplatin were obtained from Nihon Kayaku(Tokyo, Japan); CPT-11 and SN-38 were obtained from Yakult (Tokyo,

Japan); and ACNU was obtained from Sankyo (Osaka, Japan). Unless mentioned otherwise, all other drugs and chemicals were obtained from Sigma (St.Louis, MO).

Colony Formation. To assay colony formation, we seeded 300 parental or400 resistant KB cells into 35-mm dishes and incubated the cells in the absenceof drugs at 37°Cfor 24 h. Cells were then incubated for 6 days with various

concentrations of anticancer drugs without a change of medium. Alternatively,cells were irradiated with UV light 24 h after seeding and then incubated for6 days. Colonies were counted after Giemsa staining. Results were expressed

as a percentage of the control number of colonies formed in the absence ofdrug or irradiation.

Preparation of Crude Nuclear Extracts. Crude nuclear extracts wereprepared as described (22) from I X 10s cells in the early log phase of growth

under the indicated conditions. Briefly, cells were washed three times withice-cold PBS, harvested with a rubber scraper, and separated by centrifugation.Cell pellets were lysed in a solution containing K) mM Tris-HCI (pH 7.5). 1.5

mM MgCI,, 10 mM NaCl, and 1% NP40. Nuclei were separated by centrifugation at 600 x g for 10 min, suspended in 2 ml 50 min Tris-HCI (pH 7.5)-25mM KCI-2 mM CaCU-5 mM MgCI, (TKM buffer), and layered over a 0.6-ml

cushion of the same buffer containing 0.6 M sucrose. After centrifugation at2000 X g for 10 min. the nuclear pellets were washed once with 2 ml TKM

buffer containing 0.25 M sucrose and resuspended in 0.2 ml of the same buffer.Twenty ¿¿1of 0.2 M EDTA (pH 8.0) followed by two volumes of 80 mMTris-HCI (pH 7.5), 1 mM DTT, 2 mM EDTA, 0.53 M NaCl. and 20% (v/v)

glycerol were added to the nuclear suspension, and the mixture was incubatedon ice for 60 min and then centrifuged at 17.000 X g for 15 min. The protein

concentration of the supernatant (crude nuclear extract) was immediatelydetermined according to the method of Bradford (23). Small portions of theextract were adjusted to 40% (v/v) glycerol and. after addition of BSA to afinal concentration of 1 mg/ml, stored at —¿�80°C.

Topo I and Topo II Activity Assays. The standard reaction mixture forassay of topo I contained 20% (v/v) glycerol, 100 mM Tris-HCI (pH 7.5), 200

mM KC1, 20 mM MgCI,, 2 mM DTT, and 0.2 mM EDTA. The relaxation ofsupercoiled pRYG plasmid DNA (l /ig) was assayed with serial dilutions ofnuclear extract in a final volume of 20 ¿tlat 37°Cfor 15 min. The standard

reaction mixture for assay of topo II activity contained 50 mM Tris-HCI (pH

7.5), 85 HIMKC1. 10 mM MgCl2,5 mM DTT, 0.5 mM EDTA, 0.03 mg/ml BSA.and 1 mM ATP. The decatenation reaction of catenated DNA (0.25 /xg

kinetoplast DNA from Crithidia fasciculata; Ref. 19) was assayed with serialdilutions of nuclear extract in a final volume of 20 fil at 30°Cfor 30 min. Both

reactions were terminated with 4 p.1 0.25% bromophenol blue, 0.25% xylenecyanol, and 15% (v/v) Ficoll (Pharmacia). Samples were then subjected toelectrophoresis through a 1% agarose gel, which was subsequently stained withethidium bromide and photographed under UV illumination.

Cleavable Complex Formation Assay. The cleavage assay was done withnuclear extracts of KB and KB/STP-2 cells as described previously (20). Fortopo I assay, pUCIS DNA was digested with Hindlll and Seal, then the 3' endwas labeled with |a-'2P]dCTP. For topo II assay, pUC18 was digested with

Hind\ll and was dephosphorylated with bacterial alkaline phosphatase, thenthe 5' end was labeled with T4 polynucleotide kinase and [•y-32P|ATP.Finally,

the fragment was digested with Aat\\ and subjected to assay. Unincorporatednucleotides were removed by three sequential precipitations with ethanol-

ammonium acetate.Immunoblot Analysis. Equivalent amounts of crude nuclear extracts were

subjected to SDS-PAGE on 6% gels, and separated proteins were electro-

phoretically transferred to a nitrocellulose membrane containing 25 mM Tris(pH 8.3), 92 mM glycine, and 20% (v/v) methanol. Nitrocellulose filters wereincubated with human antibodies to human topo I ( 1:200 dilution; Topogen,

Columbus, OH) or mouse antibodies to human topo II (1:5000 dilution;Topogen) for l h at room temperature and then with antibodies to humanimmunoglobulin for topo I or antibodies to mouse immunoglobulin for topo II.Immune complexes were detected with the ECL system (Amersham). Filterswere exposed to Hyperfilm-ECL X-ray film (Amersham) for 2 h at room

temperature (24. 25), and the amounts of topo I and topo II were quantified bydensitometry and NIH image version 1.44. software.

Northern Blot Analysis. Total RNA was extracted from exponentiallygrowing cells by the acid guanidinium thiocyanate-phenol-chloroform method

(24, 25). RNA (15 jig) was subjected to electrophoresis in a 1% agarose gelcontaining 2.2 M formaldehyde and then transferred to a nylon membrane(Hybond N +; Amersham). The filters were hybridized for 24 h at 42°Cwith a'-P-labeled topo I or topo Ila cDNA probe in a solution comprising 50%

deionized formamide, lOx Denhardfs buffer, 5x SSC[lx SSC: 0.15 M NaCl

and 0.015 M sodium citrate (pH 7.0)], and 0.1% SDS, and then washed twiceat 42°Cin 2|tomes| SSC containing 0.1% SDS. The abundance of mRNA

encoding topo I or topo II was quantified with a Fujix BAS 2(XX) imageanalyzer (Fuji. Tokyo, Japan). Sources and properties of topo I, topo Ila, andGAPDH cDNA were described previously (26).

Stability of Topo I mRNA. To determine the stability of topo I mRNA,parental and resistant cells were treated with I(X)ng/ml actinomycin D with orwithout 20 fig/ml saintopin. and total RNA was prepared at O min. 15 min. 30min, I h, 3 h, 6 h, and 12 h after addition of actinomycin D (26). Total RNAwas analyzed by hybridization with "P-labeled cDNA probes. The abundance

of mRNA encoding topo I was quantified with a Fujix BAS 2000 ¡mageanalyzer (Fuji).

Sequencing of Topo I cDNA. Topo I cDNA was synthesized by reversetranscription from 4 /j.g total RNA from KB and KB/STP-2 cells. PCR primers

were synthesized with a DNA synthesizer (model 381 A; Applied Biosystems,Tokyo. Japan) and purified with a DNASTEC-1000 DNA Refinement system

(ASTEC. Fukuoka. Japan). Primer sequences were as described by Kubota etal. (27). Ten regions of topo I cDNA from KB or KB/STP-2 cells were

amplified by PCR with the synthesized primers. The amplified fragments wereligated into plasmids with a pMOSBlue T-vector kit (Amersham) and purifiedwith QIAGEN-tip 20 (QIAGEN. Chatsworth, CA). Chain elongation and

termination were performed with a DyeDeoxy Terminator Cycle Sequencingkit. and nucleotide sequencing was performed with a DNA sequencing system(model 373A; Applied Biosystems). Six to 10 independent clones were se-

quenced. and data were aligned and analyzed with GeneWorks software(IntelliGenetics. Mountain View, CA).

RESULTS

Sensitivities of Saintopin-resistant Cell Lines to Various Anti-cancer Agents. Two saintopin-resistant lines. KB/STP-1 and KB/STP-2, were isolated from human epidermoid cancer KB cells. The

sensitivities of these two cell lines to various anticancer drugs and UVlight were examined using colony formation assays. The resistance ofKB/STP-1 and KB/STP-2 cells to saintopin was 12- and 44-fold thatof KB cells, respectively (Table 1). KB/STP-1 and KB/STP-2 cellsshowed cross-resistance to the topo I-targeting camptothecin derivative CPT-11 and its active form SN-38, whereas their sensitivities tothe topo Il-targeting VP-16 were increased only slightly in comparison to KB cells. The resistance of KB/STP-2 cells to CPT-11 andSN-38 was 5- and 58-fold relative to that of KB cells, respectively(Table 1). In contrast, the saintopin-resistant cell lines showed collat-

Table 1 Sensitivi^ of taintapin-reiiltatt cell lines to various anlicancer agents

AnticanceragentSaintopinCPT-11SN-38VP-

16CisplatinACNUMitomycin

CUVlightKB

cell IC«*,"

(Hg/ml)0.451.81.7e0.110.24260.02VfRelative

resistance''KB/STP-

112.03.04.01.40.30.40.60.7KB/STP-244.05.058.01.80.20.20.40.3

"Concentration required to reduce Ihe initial surviving cell fraction to 109! as deter

mined by the dose-response curves for the colony formation assays.'' The relative resistance of KB/STP-1 and KB/STP-2 cells was determined by dividing

the ICC«value for each cell line by that of the parental KB cells. Mean values for each cellline were derived from triplicate trials.

The unit for SN-38 is ng/ml.The unit for UV light is J/nT.

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SAINTOPIN RESISTANCE AND TOPO I EXPRESSION

18SGAPDH"

Topo I

•¿�ft

TopoIIa

B

Topo I TopoIIa

Nuclear extract(Hg/ml)

KB

0 llZ.sl 25 I 50 llOO

KB/STP-2

0 llZ.sl 25 I 50 llOO

Relaxed form »•Supercoiled form »-

»-t «-»« ^ •¿�•^ M M

Fig. 1. Cellular levels and activities of topo I and topo Ila from KB. KB/STP-1, andKB/STP-2 cells. A, Northern blot analysis of topo I and topo Ila mRNA. Samples of totalRNA (15 jig) extracted from KB. KB/STP-I. and KB/STP-2 cells were all subjected toelectrophoresis in a 1% agarose gel containing 2.2 Mformaldehyde, transferred to a nylonmembrane, and hybridized with each '12P-labeled cDNA probe. The equivalent loading of

total RNA was shown by hybridization of the GAPDH probe. B. immunoblot analysis oftopo I and topo Ila. Nuclear extracts from KB. KB/STP-1. and KB/STP-2 cells were runin 6% SDS-PAGE. and then were transferred to nitrocellulose filters. The filters werestained with antibodies for topo I or topo Ila. C, comparison of relaxation activities bynuclear extracts from KB and KB/STP-2 cells. Relaxation assays were carried out asdescribed in "Materials and Methods." Reaction mixtures were incubated in the absence

or presence of various concentrations of nuclear extracts.

eral sensitivity to various DNA-interacting or -alkylating agents,

including cisplatin, ACNU, mitomycin C, and UV light (Table 1).Concentration, Activity, and mRNA Abundance for Topo I and

Topo II. Northern blot and immunoblot analyses of crude nuclearextracts demonstrated similar amounts of mRNA (Fig. \A) and protein

(Fig. \B) for topo I and topo II in the saintopin-resistant cell lines andKB cells, topo I activity, assayed by relaxation of pRYG-supercoiledplasmid DNA, was similar in nuclear extracts of saintopin-resistantcells and parental KB cells under drug-free conditions (Fig. 1C), topoII activity, assessed by decatenation of kinetoplast DNA to mi-nicircles, also did not differ among KB/STP-1, KB/STP-2, and KB

cells (data not shown).Effect of Saintopin on DNA Cleavages by Topo I or Topo II. An

alteration in the drug-stabilized topo I or topo II-DNA cleavable

complex is the obvious mechanism of drug resistance (11). To evaluate the effect of saintopin on the ability of topo I or topo II from KBand KB/STP-2 cells to cleave DNA, we quantified the protein-DNAcomplexes stabilized by saintopin. 3'-Labeled DNA was used toestimate topo I (Fig. 2A), and 5'-labeled DNA was used to estimate

topo II (Fig. 2B). There appeared to be no significant changes indrug-mediated cleavage formation with nuclear extracts from KB/STP-2 cells compared with parental KB cells, consistent with cellular

topo I and topo II levels in both cell lines.Comparison of Topo I cDNA Sequences between Saintopin-

resistant Cells and KB Cells. Acquired drug resistance to CPT-11 orother topo I-targeting agents is often associated with mutations of the

topo I gene (11, 12). We thus sequenced topo I cDNA from KB andKB/STP-2 cells. We cloned PCR products corresponding to the topoI cDNA into the pMOSBlue T-vector and determined their sequence.

Sequence data were aligned with the previously determined sequenceof human topo I cDNA (28). Two point mutations at nucleotidepositions 645 (T to C) and 1984 (G to A) were detected in the topo IcDNA from KB/STP-2 cells (Table 2). These two mutations, how

ever, were also observed in the topo I cDNA of the parental KB cells.Effects of Anticancer Agents on the Expression of Topo I and

Topo II in Saintopin-resistant Cells, topo I enzymatic activity is

decreased after exposure cells to ionizing radiation (29). We thereforeinvestigated the effects of topo inhibitors on topo I expression, topo ImRNA abundance decreased markedly immediately after exposure ofKB/STP-2 cells to saintopin, but was suppressed to a much smaller

Table 2 Nucleotide changes in lopo I cDNA from KB anil KB/STP-2 cells

Nucleotideno."645

1984Nucleotide

changeT-»C

G -»AAmino

acidchangeVal145-* Ala

No change (Thr591)

"Based on the human placentul topo I cDNA sequence (HUMTOPII determined byD'Arpa el al. (281. The first base of the published HUMTOPI sequence is numbered I, so

that the A of the AUG start codon correspond to nucleotide 212. Both mutations existedon the coding region of the cDNA and were present in both KB and KB/STP-2 cells.

7000

Fig. 2. Effect of saintopin on DNA cleavage bytopo I (/t) and topo II {B} in nuclear preparationsfrom KB and KB/STP-2 cells. }' End-labeled (A)or 5' end-labeled (B) 1:P-pUC18 DNA was incu

bated with nuclear extracts in the presence of various doses of saintopin. Each poini. average ofthree to four samples, with variability of <10%.Radioactivity of *2P-labeled DNA precipitate frac

tion from KB and KB/STP-2 cells in the presenceof saintopin did not differ significantly at each doseof saintopin. Cellular radioactivities at 0.01 p-Msaintopin in both topo I and topo II assays showedalmost the same values as the control in the absenceof the drug. ».KB: O, KB/STP-2.

- 1000

2000Q.O!

saintopin (

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SAINTOPIN RESISTANCE AND TOPO I EXPRESSION

Time

KB

KB/STP-2

KB

KB/STP-2

Saintopin (20 ng/ml)0 5' 15' 30' 2 h 4h 8h 12 h

~ -

~

Time

KB

Topo I*+ KB/STP-2

«»•»»ittiTopo IKB

KB/STP-2

SN-38(0.1 M.g/ml)5f 15' 30' 1h 2h 4h 8h 12h

<**«»*«****

Topo I

Topo II

BTime (h)

Nuclear extract

M)

KB

KB/STP-2

KB

KB/STP-2

Saintopin (20 i»g/ml)

1 12D

Time

TOPO 1

KB

KB/STP-2

KB

Topo II^ KB/STP-2

VP-16 (0.4|»g/ml)

0 | 5' 15' 30' 1 h 2 h 4h 8h 12h

---- ---

«.»«*•»«*

Topo I

Topo II

£_£*<5

4)

oíE

-100

-10

saintopin (hr) SN-38 (hr)

Fig. 3. Cellular levels of topo I and topo Ila in KB and KB/STP-2 cells during incubation with saintopin, SN-38, and VP-16. In Northern blot analysis, cells were treated with 20/ig/ml saintopin (A}, 0.1 p.g/ml SN-38 (C), or 0.4 ^g/rnl VP-16 (D) for the indicated time periods, and total RNA was transferred to a nylon membrane. In immunoblol analysis (B)of topo I and topo Ila in KB and KB/STP-2 cells, cells were treated with 20 /xg/ml saintopin for the indicated time periods. The concentration of each drug roughly correspondingto ICy,, for KB/STP-2 cells was used for treatment: ICyo was determined from colony formation assays as seen in Table 1. Both KB and KB/STP-2 cell lines at subconfluent condition

were not detached from dishes when treated with these drugs for 24 h. In E and F. cellular mRNA abundance of topo I (•.O) and topo II (A. A) was determined when mRNA abundanceat the indicated periods was normalized by the mRNA abundance at lime O min in KB (•,A) and KB/STP-2 (O, A) cells treated with 20 fig/ml saintopin (see A', E) or 0.1 /'•.•mlSN-38 (see C; F).

extent in KB cells for up to 12 h in the presence of the drug (Fig. 3,A and E). In contrast, topo II mRNA increased 1.6-1.8-fold higher in

both cell lines after exposure to saintopin. Immunoblot analysis alsorevealed that the amount of topo I in KB/STP-2 cells, but not that in

KB cells, was decreased after exposure to saintopin for &3 h (Fig.

3ß).Saintopin had only a slight effect on the amount of topo II in KBor KB/STP-2 cells. Treatment with SN-38, the active form of CPT-11,

induced an immediate marked decrease in the abundance of topo ImRNA in KB/STP-2 cells; the amount of topo I mRNA had not

returned to the initial level after 12 h (Fig. 3, C and F) or even after

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iTim«Actinomycin D 100ng/mlKB015'30'1

h3h6h12hKB/STP-2015'30'1 h3h6 h12 h

SAINTOPIN RESISTANCE AND TOPO I EXPRESSION

100

Topo I"

28S-

18S-

iTim«ActinomycinD lOOng/ml + Saintopin20ng/mlKB015'30'1

h3 h6h12hKB/STP-2015'30'1 h3 h6h12 h

re

ce.

Topo I•¿�•10 -

Time after actinomycin D (h)

Fig. 4. Stability of topo I mRNA in KB and KB/STP-2 cells. Each cell line was treated with actinomycin D ( 100 ng/ml) and with or without 20 /¿g/mlsaintopin. After the indicatedtime periods, tolal RNAs were prepared and hybridized with the topo I 12P-labeled cDNA probe (A and ß).The amount of RNA in each lane was monitored using ribosome RNA.

In C. decay rates of topo I mRNA were determined when mRNA abundance al the indicated periods was normalized by the mRNA abundance at time 0. •¿�.KB; O, KB/STP-2.

24 h (data not shown), topo I mRNA abundance also showed a rapiddecrease in KB/STP-2 cells after treatment with CPT-11, but theeffect was found to be less than that of SN-38 (data not shown), topoII mRNA abundance in KB/STP-2 or KB cells was increased to asmall extent (1.3-1.7-fold) by SN-38 (Fig. 3, C and D or by CPT-11(data not shown). The topo II-targeting agent VP-16 induced a slightincrease in the abundance of topo I mRNA (Fig. 3D), topo II mRNAabundance also increased 1.5-2-fold by exposure of KB or KB/STP-2cells to VP-16 (Fig. 3D).

Stability of Topo I mRNA in the Presence of Saintopin. Toexamine whether the decreased topo I mRNA abundance in thepresence of saintopin was due to altered stability of mRNA, weperformed Northern blots in the presence of actinomycin D with orwithout saintopin. The degradation kinetics of topo I mRNA in thepresence of actinomycin D demonstrated that topo I mRNA hadhalf-lives of about 7-8 h in both KB and KB/STP-2 cells (Fig. 4, Aand C). In contrast, topo I mRNA degraded much faster in KB/STP-2cells than in KB cells when treated with actinomycin D and saintopin(Fig. 4, B and C), topo I mRNA from saintopin-treated KB/STP-2cells had half-lives of 2-4 h while that of saintopin-treated KB cellshad half-lives of 10-11 h. Treatment with saintopin thus appeared tomarkedly destabilize topo I mRNA in KB/STP-2 cells.

DISCUSSION

We have isolated and characterized human cancer cell lines resistant to the anticancer antibiotic saintopin. This agent was originallyshown to induce both topo I- and topo II-mediated DNA cleavagethrough stabilization of the reversible enzyme-DNA cleavable complex (16, 17), suggesting that this drug is a dual inhibitor of both topoI and II. Leteurtre et al. (18) further showed that the topo I-cleavablecomplex is preferentially stabilized by saintopin at DNA sites with aguanine at the 5' terminus of the enzyme-induced breaks, suggesting

that the topo Il-cleavable complex may also be stabilized by saintopinin a similar manner. Both saintopin-resistant KB/STP-1 and KB/STP-2 cell lines showed a markedly higher cross-resistance to SN-38and CPT-11 than KB cells, but they exhibited only weak cross-resistance to VP-16. Development of resistance to saintopin in KB

cells thus appeared to be associated with preferential changes in thetopo I-mediated pathway rather than in that mediated by topo II.

KB/STP-2 cells had 5- and 58-fold increases in resistance toCPT-11 and SN-38, respectively. CPT-11, a prodrug, is converted intoits active form, SN-38, by carboxylesterase, with SN-38 being ~ 1000times more potent than CPT-11 with regard to cytotoxicity (30, 31).Either the conversion of CPT-11 to SN-38 or degradation of CPT-11may be altered in KB/STP-2 cells, resulting in the observed differential sensitivity to the two drugs. We compared conversion activitiesfrom CPT-11 to SN-38 in KB and KB/STP-2 cells and found thatthe conversion rates were almost identical in both cell lines.4

Other unknown mechanisms should be considered in the differentialsensitivities.

The cellular concentration and mutations of topo I often correlatewith the development of resistance to camptothecins (11, 12, 32-35).KB/STP-2 cells showed no changes of topo I and II activity, proteinconcentration, mRNA abundance, topo-mediated cleavable complex,and primary sequence of topo I cDNA. However, in contrast to theireffects in KB cells, saintopin and SN-38 induced a marked andpersistent decrease in the abundance of topo I mRNA in KB/STP-2cells. These drugs induce a slight up-regulation of topo II mRNA inKB/STP-2 cells. This specific drug-induced down-regulation of topoI appears to be correlated with development of resistance to saintopinand SN-38. By contrast, exposure to VP-16 slightly increased cellularlevels of both topo I and topo II mRNA in KB and KB/STP-2 cells.Matsuo et al. (21) have previously demonstrated up-regulation of topoII in response to heat shock in KB cells, but topo I abundance is notaltered under this condition. A novel mechanism might be involved inthe up-regulation of topo I and topo II by the topo II-targeting agent,but any precise mechanism is not known. These data indicate thatregulation of topo I gene expression in response to exogenous stimulidiffers from that of topo II gene expression.

Concerning the underlying mechanism for down-regulation oftopo I by saintopin or SN-38, we observed rapid degradation oftopo I mRNA in KB/STP-2 cells by saintopin (Fig. 4) or SN-38.5

4 H. Nagata, K. Taniguchi. and M. Kuwano. unpublished data.5 K. Taniguchi and M. Kuwano, unpublished data.

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Topo I mRNA degraded at 3-4-fold faster in KB/STP-2 cells thanin KB cells when saintopin was present (Fig. 4C). Nuclear run-onassay with nuclei demonstrated similar transcription activities ofthe topo I gene in KB and KB/STP-2 cells treated with 20 ju.g/mlsaintopin for 3 h.6 These data suggest that down-regulation of topoI mRNA in saintopin- or SN-38-treated KB/STP-2 cells is due tothe drug-induced mRNA destabilization rather than transcriptionalcontrol. A relevant study by Beidler and Cheng (36) have reportedthat time- and concentration-dependent decrease of topo I proteinis induced in camptothecin-treated KB cells. However, they observed no change in topo I mRNA abundance during incubation for2, 8, and 19 h with camptothecin and aphidicolin. Consistent withthis study, we also could not observe any apparent change of topoI mRNA in CPT-11-treated KB cells (data not shown). Amara et al.(37) have reported that stabilization of ribonucleotide reducíasemRNA results in development of resistance to hydroxyurea, andalso that this mRNA stabilization is mediated through specificcis-elements in the 3' untranslated region of the gene. It remains to

be further studied what molecular mechanism is involved in thedestabilization of topo I mRNA by topo I-targeting agents.

Sensitivity to camptothecin is often altered in radiation-sensitivePanconi anemia, Cockayne's syndrome, and ataxia telangiectasia

cells (38-41) as well as in radiation-sensitive mutants of culturedmammalian cells (32, 39). Human ovarian cancer cells selected byresistance to cisplatin also showed altered sensitivity to CPT-11(42), and cisplatin-resistant human bladder cancer cells that exhibited increased topo I expression showed collateral sensitivity toCPT-11 (43). Thus, topo I appears to be important in the repair ofDNA damage induced by radiation or DNA-interacting anticanceragents. In our saintopin-resistant cell lines, cellular topo I abundance was modulated in response to certain topo I-targeting agents.Exposure to cisplatin or UV light also induced down-regulation oftopo I in KB/STP-2 cells, but the effects were much less than thatof saintopin or SN-38.7 Either topo I or other DNA repair enzymes

might be highly vulnerable to exogenous stimuli in KB/STP-2 cellsthrough unknown mechanisms, resulting in altered sensitivity toDNA-damaging agents.

Saintopin-resistant cell lines derived from human epidermoid cancer KB cells showed cross-resistance to topo I-targeting agents(CPT-11 and SN-38) and collateral sensitivity to cisplatin, ACNU,mitomycin C, and UV light. Saintopin or SN-38, but not VP-16,inhibited topo I gene expression to a markedly greater extent inresistant lines than in parental KB cells. The effects of alkylatinganticancer agents are modulated by a combination of topo I-targetingdrugs (44), and consistent with this observation, our present studysuggests that the combination of topo I-targeting agents and DNA-damaging agents may improve therapeutic efficacy.

ACKNOWLEDGMENTS

We thank Takanori Nakamura, Kyoko Yamada. and Yukiko Mine (KyushuUniversity) for experimental help and preparing the manuscript.

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1996;56:2348-2354. Cancer Res   Ken Taniguchi, Kimitoshi Kohno, Kiyoshi Kawanami, et al.   CamptothecinsEpidermoid Cancer Cells Resistant to Saintopin and Drug-induced Down-Regulation of Topoisomerase I in Human

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