Biomeddtfharmucother (1990)44.41-45

Q Else&r, Pads

JH Hong I, M Okada 3, T Kusano 5, Y Komazawa 3, M Kobayashi 4, A Mi~u~~i 2, N Kamada ‘, A ~ur~rno~~ * ’ Departmeat of ~e~lutu~o~y,Hiroshima University ~ospitff~; 2 Internal Me~~~~e,.$eseurch itmthte for N&ear Medicine and Siol@gy,Himhima liniversity; Division of Blood ~ra~~ion~ Hiroshima Un~~~ersity; ’ ~part~;el~t of Pediatrics, ffiroshi~~ ~nive~~ty Hospice; ~irosh~n~aWomen’s Coifege. ~trosh~ma 734. &pun (Received 3 Oct&er 1988; accepted 6 November 1989)

Summary - VP-16 resistant cells, Fvp?3350 (SOB-3). were isolated from mouse breast cancer cell line FM3A. SOB-3 cells showed 84-fold higher resistance than their parent cells. Reduced drug uptake was not found in resistant cells. Quantitative analysis of drug-stimulated DNA cleavage activity using 313*Pend-labeled pBR322 restriction fragments showed that VP-16 stimulated DNA-topoisomerase II cleavable complex forming activity in crude nuclear extract from SOB-3 cells was app~ximateiy one-fifth as compared with that of FM3A wild-type cells. Dot-blot analysis of RNA extracted from the two ceil lines showed that mR~A levels of t~oisomerase II in 5OB-3 cells dr~t~cal~y decreased and catalytic activity was aiso l/2-1/3 as compared with that of parent ceils. SOB-3 cells showed cross resistance to VM-26, m-AMSA, adriamycin. These findings suggest that reduced topoisomerase II activity (cellular levels) and cleavable complex forming activity may be significant factors in the marked drug resistance, DNA topoisumefase If t VP-16 I drug resistance Resume - R~d~t~~n de t~~~~~rn~~s~ ff-ADN dans nne fig&e ~eiIul~;r~ de cancer mamm~~re r&&&We au VF16. L)es cell&s rksistantes au VP-16 et dksignks sous le nom de Fsp%350 @OBS) ont &t; isolPes B part& de la li_gttPe

cellulaire de caucer mammaire murin FM3A. Les cellules SOB-3 ant montrk une r&istance 84 fois suplrrieure d celie de leurs cellules d’origine. 11 n’a pas PtLitrot& de d&jkit de fixation du produit dans les cellules re’sistantes. Une analyse quantitative de l’activitt! de clivage de I’ADN sous stimulation par le produit avec ~11etechnique utilisant des segments de restrictiei;s BR322 margut% eu terminal au 3”‘P a montrP que I’activitP de formation de complexes ADN-topoisomi%wsc 11 ~livables, a&W stimulee par le VP-I& repr&sentait darts un extrait brut de uoyau provenant de celfules SOB-3 environ flS de cefle des resettles FM3A de type sattva~e. Une a~~u~yse par ~ot~lot de I’ARN extrait de ~~rttne des deux ii~~~es de celfules a montrri que Ie taux 8ARNm portatit le code de la to~o~sum~raseI1 daus les cekles S#B-3 dkroissait dune fagott sprectwultaire et que I’activite?cataiytique repr&entait &lemeut 112 ci 113 de relle des celfrrfes-m&es, Les celluies SOB-3 ont montrt”une r~s~stattcecroise’e au VM26, B la m-A~SA et cl ~adri~my~ine, Ces r~stt~tatssi~~~~rentque la r&duction de l’actl~~it~topoisom~rasc If {au rtiveattcel~ttiaire~et de laacti~Iit~ de ~orrnatioll de complexes ~li~~ablesp~~ttrraie~it jotter un rile ~ntportu~ttdans la prof~ttde r&istance constat&epar rapport au m~~~arnettt.

Introduction DNA non-intercalating agent VP-16 is one of the important cancer chemotherapeutic drugs. The drug has been known to inhibit mammalian DNA topoisomerase II (Top0 II) [t 1. The mechanism of

and cytatoxicity of VP-16 has been investigated by several other ~~yestigator~ [2, 9, 11, 12j. Resistance to the anti-Topo II drugs such as nt-AMSA, adriamycin, VM-26 owe to a variety of causes. For example, reduced intracellular Topo II activity, decreased drug uptake accomaction

f H Hong et nl

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panyiog Topo II reduction, unknown factor{s) may be r&at& to the interaction between lbpo ff, i.xw and drug p-81. It has also been shown that intrac~~~~~Topo II level (Top0 II activity) had a relationship with protein-DNA cleavable complex formation [13, 141. We have previously shown an altered cellular level but similar activity of Topo II in a VP-16 resistant human lymphoblastic cell line. To clarify the mechanism of resistance in the present study, we isolated resistant cells which demonstrated marked resistance to VP-16 from mouse breast cancer cell line FM3A. A relationship between the reduced intracellular Topo II levels, cleavable complex forming activity and drng resistance was found in the resistant cells.

containing 0.3 M KCI. The active fractions were eiutcd at about 0.6 M (KC&

DNA cleavage a&tjv~~ ~s~~g~~strand br~aka~~~ The quantitative analysis of DNA cleavage activity of Topo II (cleavable complex formation) was assayed using 3’32P-labeled pBR322 restriction fragments and the sodium dodecyl sulfate(SDS)-KC1 precipitation of DNA topoisomerase II-DNA complex as originally described by Liu [9].

The qu~ti~tive analysis of Topo IT mRNA was carried out as described by Hong et al 161. pCl5 DNA containing 1.8 kb of human Topo II cDNA 1101 fragment was labeled with radioisotope and used in the hybridization. Other methods

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VP-16 (Nippon Kayak@, CPT-1 I (Yak& Co), 4’-(9acridinyl-amino) methanesutfon-~-anisidide (nt-AMSA: Warner Lambert Co) and teniposide (VM-26; Bristol Myers) were used in the experiment. [3H]-VP-i6 (388 m~nmol~ was obtained from Chemicine Sci Lab through the courtesy of Nippon Kayaku.

FM3A and their resistant cells were maintained in RPM1 1640 medium (Gibco) supplemented with 10% fetal calf serum, streptomycin and penicillin. 5OB-3 c&s were isolated by treatment with ~-methyl-~‘-nitrous-nitrosoguanidine ~MNNG~.

Wild-type and resistant cells were injected ip into 6week old ddY mice irradiated with 400 rad of 6JCo at S X 16 cellsfmouse and the cefls were harvested while in exponential growth after g-9 post-transplantation days. Frozen cells were thawed at 0’ C and suspeuded in buffer A(20 mM KH$O4, pH 7.5, 0.1 mM EDTA, 1 mM phenylmethanesulfonylfluoride, 0.1% Triton X100 and 0.2s mM 2-mercaptoethanol. 20% ethylene glycol), and homogenized. The extraction was carried out for 30 min at 0” C in buffer A containing 0.3 M KCl. The extracts were then centrifuged twice at 21000 8 for 20 min. The supematant was applied on celfulose phosphate column (bed volume: 20 ml). Topo II activity was eluted by Iinear gradient from 0.3-1.0 M KC1 in buffer A. The active fractions were then applied and fractionated by hydroxy~apatite HT column (bed volume: S ml) ~uiiib~ated with buffer A containing 0.3 M KCI and then active frac$ions were efured in linear gradient from 20 to SO0 mM phosphate buffer

The drug uptake and efflux, the colony forming ability assay were estimated as described by Hong et al [6]. The preparation of crude nuclear extract and the Topo II assay were carried out as described by Hong et al [6],

results Isajat~an af resjstant teifs 503-3 cells were about 84-fold more resistant to VP-16 than wild-type (WT) cells and expressed cross resistance to VM-26, m-AMSA and adriamycin. SOB-3 cells, however, did not show reduced sensitivity to CPT-11 (table I).

Table I, ICtio values of various t~oj~rnem~s

inhibitor for WT and SOB-3 ce&.. f I&J values are ~flcentrations causing SO 8 inhibition of cofony forming ability relative to that of untreated cells. Values are shown in $tg!rnl (VP16, CPT-11) or ng/mt (VM-26, no-AMSA and adriamycin). 2 Ratio of ICSOvalues for SOB-3 cells to that for FM3A cells, 3 Mean It SD for 2-3 separate experiments.

VP-16 VM-26 ADR nt-AMSA CPT-1 I

0.19 25.5 76 I.60 3.0

fO.04 f4.30 f13 fO.18 f0.2

15.9 950 224 16.0 3.8

+ I.203 a230 529 22.40 +0.2

84 37 3 IO 1

VP-16 resistant cells from mouse breast cancer cell line Drug aptake and e&%#x ~~tra~e~lu~~r~~~urnulet~~~ of drug were examined in two cell fines. Figure 1 showed that drug uptakes by two cell lines increased linearly dependent on drug concentration. However, there was no significant difference in WT and 5OB-3 cells (fig 1A). In parallel, drug release was also tested in WT and 5OB-3 cells. The drug efflux occurred mostly within 3 min in both cell lines. However, no difference was found between WT and 50B-3 cells (fig 1B).

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Three units of partially purified Topo II were used in the assay (fig 2), The results showed that the sensitivities of partially purified Topo II against VP-16 were similar in WT and 5OB-3.

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Fig 1. A, Uptakesof @]-VP-l6 into WT ( ) and 508-3 (a} ceils, E, Retease of [‘Elf-VP-16 from WT ( ) and 508-3 ) ceils. Cells were ln~ubated with 10 $@rnl of VP-i& for 30 min and washed 3 times with PBS(-) and allowed to retease the drug in the drug-free medium for the indicated time at 370 C.

Fig 2. Inhibition of strand passing activity of topoisomemse IL Three units of partially purified topoisomerase II were used for assay= AAer i&u&ati&~f& 30 mi&at 30” C, reactions were stopped by addition of SDS and pmteinase K was added Lane l P4 lcaotted DNA or&, lanes 2-6 WT. lanes 7-11 SOB-3 cells. Lanes 2 and 7 VP-IS free, fanes 3 and 8 25 @g/ml of VP-16, lanes 4 and 9 50 pgLgfm1 of VP-I% lane S and IO 100 pgfml of VP-t6, lane 6 and I I 400 pglmt of VP-M. C circular P4 DNA, K knotted P4 DNA.

VP-16 stimulated DNA cleavage activity (single strand breakage) The quantitative analysis of cleavable complex formation showed that Topo II in crude nuclear extract from 5OB-3 cells was approximately Sfold more resistant in drug-stimulate DNA-protein cleavable complex forming activity than that of WT cells. The 3’%-end labeled DNA precipitates sedimented by the treatment of SDS-KC1 solution dramatically increased dependent on drug concentration up to 50 yg/ml in WT Topo II, whereas that of SOB-3 Topo II rose slowly up to 50 pg/ml and reached a plateau at above 50 pg/ml (fig 3). analysis Topo II mRNA were reduced drastically in resistant cells as compared with that of WT cells (fig 4).

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Hong et al

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Fig 3. Quantitative precipitation assay for the formation of the cleavable complex in the presence of VP-16. The reactions were stopped by the addition of SDS-KC1 solution to detect DNA single strand breakage as described in Materials and Delhi. WT. (A), and 503-3 cells, { ). Points, mean of 2 separate experiments. One on the ordinate line, VP-16 free. 12

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5

compared with the WT cells. The result suggested a correlation between drug sensitivity and enzyme activity, in agreement with the findings Deffre et al [3], who found a considemble correlation between Topo II catalytic activity and DNA cleavactivity in adriamycin-resistant P388 age analysis also leukemia cell lines. Dot-blot showed a remarkably reduced Topo II mRNA level in SOB-3 cells compared to WT cells. Reduced enzyme levels may lead to a decreased cleavable complex formation and catalytic activity in SOB-3 cells. The result were consistent with those of Ferguson et al [4], who reported that Topo II protein determined by immunoblotting analysis was reduced in accordance with the relative resistance in VP-16 resistant KB cell lines. Partially purified Topo II was tested to verify whether mutant Tops II was produced in 5OB-3 cells. However, there was no difference in the enzymatic sensitivity to VP-16. Southern blot analysis showed the identical restriction patterns of DNA in WT and resistant cell lines (data not shown). The results may exclude the possibili~ of mutation on Topo II gene itself. 5OB-3 cells exhibited cross resistance to VM-26, m-AMSA, adriamycin. These results shows that the resistant mechanisms may be different between 5OB-3 cells and previously isolated VP-16 resistant K6-1 cells 161, The present study suggests that the drug resistance may be due to the reduced enzyme levels and DNA cleavage activity in our resistant cell line.

1 Fig 4. Dot-blot analysis. Serial dilutions of 15, 5 and 1 pg of total RNA were applied to each well. Hyb~dization of bIot with beta-actin probe demonstrated comparable amount of RNA loaded in all wells (Pig 4, columns 3, 4). Columns I and 3 WT, columns 2 and 4. SOB-3.

iscussim Intracellular radioactivity were measured to determine whether reduced drug uptake was related to resistance. However, no evidence of low drug uptake was found. Quantitative analysis of DNA cleavage activity showed that Topo II-mediated DNA cleavage activity was reduced by about 5fold in 50%3 cells and catalytic activity was also decreased by about 2-3-fold in 5OB-3 cells as

We thank Drs T Andoh (Aichi Cancer Center, Japan) and F Hanaoka (Tokyo University, Japan) for the generous gifts of pC 15 DNA and P4 phage seed, respectively.

aferences Chen GL, Yang L, Rowe TC, Halligan BD (1984) Non~tercalative antitumor drugs interfere with the breakage-reunion reaction of mammalian DNA topoisomerase Ii. J Biol Chem 259, 13560 Drilica K, Franc0 RJ (1988) Inhibitors of DNA topoisomerase. Biochemistry 27, 2253 Deffie AM, Batra JK, Goldenberg GJ (1989) Direct correlation between DNA topoisomerase H activity and cytotoxici~ in adriamy~in-sensitive and -re-

VP-16 resistant cells from mouse breast cancer cell line sistant P388 leukemia cell lines. Cancer Res 49,5862 4 Ferguson PJ, Fisher MH, Stephenson J. Li DH, Zhou BS, Cheng YC (1988) Combined modalities of resistance in etoposide-resistant human KB cell lines. Cancer Res 48, 5956 5 Hwang J, Shyy S, Chen AY, Juan CC, gang-Peng J (1989) Studies of topoisomerase specific antitumor drugs in human lymphocytes using rabbit antisera against recombinant human topoisomerase II polypeptide. Cancer Res 49, 958 6 Hong JH, Okada K. Kusunoki Y, Komazawa Y, Kobay~hi M, Mizu~ni A, Mizuno T, Kuramoto A, Kamada N (1990) Isolation and cha~cterization of VP-16 resistant human leukemia cell line. Biomed & Pharmacather 44, 7 Ishida R, Nishizawa M, Nishimoto T, Takahashi T

(1988) Cross-resistance of novobiocin-resistant BHK ceil line to topoisomerase II inhibitors. Som CeR Mel Genet 14, 489 8 Ken&an D. Pommier Y, Kohn KW (1987) Proteinlinked DNA strand breaks produced by etoposide and teniposide in mouse L1210 and human VA-13 and HT-29 cell lines: relationship to cytotoxicity. NC1 Monogr 4, 117 9 Liu LF, Rowe TC, Yang L, Tewey KM, Chen GL (1983) Cleavage of DNA by mammalian DNA topoisomerase II. J Biol Chem 258, 15365

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IO Monika TP, Liu LF, Liu AA, Tewey KM, Jacqueline WI? Knutsen T; Huebner K, Croce CM, Wang JC (1988) Cloning and sequencing of cDNA encoding human DNA topoisomerase II and location of the gene to chromosome region 17q21-22. Proc Nat1 Acad Sci LISA 857177

11 Levin M, Silver R, Israel M, Gol~tein A, Khetarpai VK, Potmesii M (1981) Protein-associated DNA breaks and DNA-protein cross-links caused by DNA nonbinding derivatives of adriamycin in L1210 cells. Cancer Res 41, 1006 12 Nelson EM, Tewey KM, Liu LF (1984) Mechanism of antit~or drug action: poisoning of mammalian DNA to~isomerase II on DNA by 4’-(9-acridinylamino)-methanesulfon-m-anisidide. Proc Nat1 Acad Sci USA 81, 1361 13 Potmesil M, Hsiang YH, Liu LF, Bank B. Grossberg H, Kirschenbaum S, Kanganis D, Knowles D, Traganos F, Silber R (1988) Resistance of human leukemic and normal lymph~~s to dug-index DNA cleavage and low levels of DNA topoisomerase Il. Cancer Res 48, 3537 14 Sinha BK, Haim N, Dusre L, Kerrigan D, Pommier Y (1988) DNA strand breaks produced by etoposide (VP-16,213) in sensitive and resistant human breast tumor cells: implication for the mechanism of action. Cancer Res 48, 5096

Reduced DNA topoisomerase II in VP-16 resistant mouse breast cancer cell line.

VP-16 resistant cells, FvprB350 (50B-3), were isolated from mouse breast cancer cell line FM3A. 50B-3 cells showed 84-fold higher resistance than thei...
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