Atypical multidrug resistance may be associated with catalytically active mutants of human DNA topoisomerase II α

Gene 272 (2001) 141±148

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Atypical multidrug resistance may be associated with catalytically active mutants of human DNA topoisomerase II a Yoshito Okada a, Aki Tosaka b, Yuji Nimura a, Akihiko Kikuchi c, Shonen Yoshida b, Motoshi Suzuki b,* a The First Department of Surgery, Nagoya University School of Medicine, Nagoya, 466-8550, Japan Laboratory of Cancer Cell Biology, Research Institute for Disease Mechanism and Control, Nagoya University School of Medicine, Nagoya, 466-8550, Japan c Laboratory of Medical Mycology, Research Institute for Disease Mechanism and Control, Nagoya University School of Medicine, Nagoya, 466-8550, Japan b

Received 7 March 2001; received in revised form 16 May 2001; accepted 1 June 2001 Received by T. Sekiya

Abstract In human cells, atypical drug resistance was previously identi®ed with reduced catalytic activity or nuclear localization ef®ciency of DNA topoisomerase IIa (TOP2a). We have shown two etoposide resistant hTOP2a mutants, K798L and K798P confer resistance to etoposide. In this work, we showed these mutants are also resistant against doxorubicin and mAMSA in vivo in the yeast strain ISE2, rad52, top2-4 at the non-permissive temperature. We puri®ed these mutants to characterize the drug resistant mechanism. Puri®ed recombinant proteins were 8to 12-fold more resistant to etoposide and doxorubicin than wild type TOP2a, and 2-fold more resistant to amsacrine, as measured by accumulation of cleavable DNA. These data show that K798L and K798P may be intrinsically resistant against these drugs in vitro and that this character may confer atypical multidrug resistant phenotype in vivo in yeast. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Atypical MDR; DNA replication; Chemotherapy; Cancer; Genetic instability

1. Introduction Type II DNA topoisomerases (TOP2) are essential enzymes for cell division and replicating cells die if they are inactivated. TOP2 catalyzes the breakage and rejoining of double-strand DNA, a reaction that can relax DNA supercoils and catenate or decatenate covalently closed circular DNA. The reaction mechanism involves formation of a transient break in one double-strand helix (a gate), passage of another double-strand helix through the broken DNA, and then resealing of the break. TOP2 plays an important role in chromosome segregation, DNA condensation and regulation of the torsional stress that results from RNA transcription and DNA replication (Wang, 1996). Mammalian cells have a and b isoforms of TOP2. The level of intracellular TOP2a increases during the G2/M phases of the cell cycle, while the level of TOP2b does not change even in a quiescent cell (Tsutsui et al., 1993). Abbreviations: hTOP2a, human type II DNA topoisomerase a; RC, relaxed circular DNA; OC, open circular DNA; CAT, catenated DNA * Corresponding author. Tel.: 181-52-744-2456; fax: 181-52-744-2457. E-mail address: [email protected] (M. Suzuki).

Topoisomerase II isoenzymes showed spotted (a) or reticular (b) nuclear patterns throughout interphase. In mitosis, topoisomerase IIb diffuses into the cytosol, whereas IIa remains chromosome bound (Meyer et al., 1997). It is likely that TOP2a and b play roles in cell division, DNA replication and transcription, although the precise roles of the a and b isoforms of TOP2 are still unknown. The TOP2 enzymes are also important as the speci®c targets of various antibacterial and anticancer chemotherapeutic drugs. However, cancer chemotherapy with antiTOP2 drugs can lose its effectiveness when cells become resistant to the action of the drug. In some cases, drug resistance results from increased expression of MDR1, which changes the permeability of the cell membrane, increasing the rate of drug ef¯ux from the cell and decreasing the intracellular drug concentration (Chen et al., 1986; Hamada and Tsuruo, 1986). However, MDR1 independent pathways of resistance to antiTOP2 drugs have also been reported. In these cases of `atypical MDR', TOP2 is often down-regulated (for example, cf. Lage et al., 2000), decreased in stability (Sabourin et al., 1998) or structurally altered one of three important domains (Vassetzky et al., 1995 and references therein), i.e. the ATP binding domain, the active site/

0378-1119/01/$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0378-111 9(01)00554-6

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CAP domain and the C-terminal NLS-containing domain. Mutations in these regions either lower the catalytic ef®ciency of TOP2 or abolish its transport into the nucleus. Many of the available cell lines that are resistant to antiTOP2 drugs have been obtained from clinical specimens (Andoh et al., 1996) or by culturing drug-sensitive cell lines for long periods in the presence of antiTOP2 drug (for example, see Bugg et al., 1991). These cell lines have been used for isolation of mutations in the TOP2 genes. Alternatively, plasmid-borne TOP2 is also useful to identify mutations that confer drug resistance (Liu et al., 1994; Patel et al., 1997). In our previous study, a plasmid library was created by cassette mutagenesis of the active site region of hTOP2a and the mutants were screened for their ability to confer drug resistance to yeast with a temperature-sensitive allele of the endogenous yeast TOP2. Out of a total of 304 single mutations in the active site region, two etoposide resistant mutants, K798L and K798P, were identi®ed in yeast (Okada et al., 2000). In this study, these mutant proteins were expressed, puri®ed and characterized in vitro. The results indicate that the mutants are catalytically active and are at least resistant to forming cleavable complex in the presence of several antiTOP2 drugs. 2. Materials and methods 2.1. Materials and strains Etoposide, doxorubicin and amsacrine (mAMSA) were purchased from Sigma (St. Louis, MO). The yeast strain JN394t2-4 (MATa, ura3-52, leu2, trp1, his7, ade1-2 ISE2, rad52::LEU2 top2-4) was kindly provided by J. Wang (Harvard University). 2.2. Atypical multidrug resistant phenotype in yeast Identi®cation of etoposide resistant mutants were described previously (Okada et al., 2000). The transformants were incubated on a solid medium, SGAL (6.7 g/l yeast nitrogen base w/o amino acids, 5 g/l casamino acids, 20 g/l galactose, 20 mg/l adenine sulfate, 20 mg/l tryptophan, 15 g/l Bacto agar) that contained various concentration of etoposide, doxorubicin and mAMSA. Drug sensitivity was determined by the ability of colony formation after 7 days incubation at 358C. In experiments carried out, drug concentrations were higher than those required for mammalian cells. We used a yeast cell line in which cell wall increases drug permeability, although it might be possible drug uptake is not equivalently ef®cient in this yeast as in mammalian cell lines. 2.3. Expression of hTOP2a Histidine tagged-wild type-hTOP2a protein and its derivatives, K798L and K798P, were expressed for puri®cation using BAC-TO-BAC HT Baculovirus Expression System (LIFE TECHNOLOGIES, MD). The yeast expression

vector containing hTOP2a was digested with Not l and Xho l. The coding fragment was used for replacement with the Not l-Xho l fragment of pFastBac HTb (pFastBac-WThTOP2a). An E. coli strain DH10bac (Gibco BRL Co. MD) was transformed by pFastBac-WThTOP2a. A single colony containing the recombinant bacmid (bacmid-hTOP2a) was picked for 2 ml culture in LB medium (10 g/l Bacto-trypton, 5 g/l Bacto-yeast extract, 10 g/l NaCl) at 378C overnight. The bacmid-hTOP2a was isolated by alkaline minipreparation and used for transfection into Sf9 cells (Gibco BRL Co., MD). The virus was ampli®ed to a titer of 10 8 plaque forming units/ml, and inoculated in a fresh culture of con¯uent Sf9 cells in 75 cm 2 ¯asks at a M.O.I. of 2.0. 2.4. Puri®cation of recombinant phTOP2a carrying a histidine-tag At 72 h postinfection cells (2.5 £ 10 7) were harvested by centrifugation at 500 £ g, lysed in an ice cold-lysis buffer [20 mM Tris±HCl (pH 7.9), 500 mM NaCl, 5 mM imidasole, 10 mM 2-mercaptoethanol, 1 mM PMSF, 1% Nonidet P-40, 1 mM DTT] at 48C, and sonicated three times on ice, each for 15 s at 50 W (Micro Ultrasonic Cell Disrupter, KONTES, Vineland, NJ). The sample was kept on ice for 1 h, centrifuged at 25,000 £ g for 10 min, and the supernatant was loaded onto In-resin column (Nonagon, His-Bind Resin WI) equilibrated with buffer A [20 mM Tris±HCl (pH 8.5), 500 mM KCl, 20 mM imidazole, 10 mM 2-mercaptoethanol, 10% glycerol]. The column was washed with 10 ml of buffer A, followed by 2 ml of buffer B [20 mM Tris± HCl (pH 8.5), 1 M KCl, 10 mM 2-mercaptoethanol, 10% glycerol] and 2 ml of buffer A again. Each 1 ml of enzyme preparation was eluted and collected in 10 ml of elution buffer [20 mM Tris±HCl (pH 8.5), 100 mM KCl, 1 M imidazole, 10 mM 2-mercaptoethanol, 10% glycerol]. Peak fractions were concentrated by dialysis against 1 l of a buffer [25% glycerol, 50 mM potassium phosphate (pH 7.1), 0.5 mM DTT, 1 mM EDTA, 1 mM EGTA, 260 mM NaCl] for 4 h with a change dialysis buffer at 2 h, and stored at - 808C for several months without loss of activity. The expression, puri®cation and dialysis of hTOP2a mutants were carried out using the same procedures as wild type. Protein concentration of each stock was determined using the Bradford reagent (BioRad, Richmond, CA). 2.5. Kinetoplast DNA decatenation assay An assay for the decatenation of kinetoplast DNA was carried out using TOPOISOMERASE II ASSAY KIT (TopoGEN, Columbus, Ohio). Each 20 ml of reaction mixture contains 100 ng of kinetoplast DNA from Crithidia fasiculata and 5 ml dialyzed wild type or mutant TOP2a in 50 mM Tris±HCl (pH 8.0), 120 mM KCl, 10 mM MgCl2, 0.5 mM dithiothreitol, 30 mg/ml BSA, and 1 mM ATP. After an incubation for 30 min at 378C, 4 ml stop buffer (5% Sarkosyl, 0.0025% bromophenol blue, and 25% glycerol) was

Y. Okada et al. / Gene 272 (2001) 141±148

added to terminate the reaction. 20 ml of the reaction mixture was used for analysis. Reaction products were separated by 1% agarose gel electrophoresis in the presence of 1.0 mg/ml ethidium bromide. Electrophoresis was carried out at 100 V for 45 min in TAE buffer (0.04 M Tris acetate, 0.001 M EDTA) containing 1.0 mg/ml ethidium bromide. After electrophoresis, the gel was destained in water for 2 h and reaction products were visualized under UV light. 2.6. Quanti®cation of catalytic activity To determine enzyme units, we carried out a relaxation assay. A 20 ml reaction mixture contained various amounts of enzyme up to 20 ng, 200 ng of supercoiled pBluescript, 50 mM Tris±HCl (pH 8.0), 120 mM KCl, 10 mM MgCl2, 0.5 mM dithiothreitol, 30 mg/ml BSA, and 1 mM ATP (relaxation reaction mixture). Incubation was carried out for 30 min at 378C. Reaction was terminated by 0.5% SDS. Samples were further incubated with 100 mg/ml proteinase K for 15 min at 378C. Reaction products were separated by 1% agarose gel electrophoresis at 100 V for 45 min in TAE buffer. After electrophoresis, the gel was stained in TAE buffer containing 1.0 mg/ml ethidium bromide and reaction products were visualized under UV light. The amount of each reaction product was quanti®ed and photographed using Densitograph and Lane analyzer (ATTO, Tokyo, Japan). 2.7. Quanti®cation of inhibition by anticancer drugs A ®nal concentration of 50, 100, 500 or 1000 mM etoposide was used in a DNA relaxation reaction with 100 ng of wild type hTOP2a for 30 min at 378C. Reactions were terminated with 0.5% SDS. After samples were further incubated with 100 mg/ml proteinase K for 15 min at 378C, 20 ml of Phenol/Chloroform/Isoamyl alcohol (25:24:1) was added and mixed vigorously. The aqueous phase was recovered by centrifugation and electrophoresis was carried out. Reaction products were separated by 1% agarose gel electrophoresis. Electrophoresis was carried out at 100 V for 45 min in TAE buffer. After electrophoresis, the gel was stained in the presence of 1.0 mg/ml ethidium bromide for 30 min. It was further destained in water for 1 h and reaction products were visualized under UV light. The amount of each reaction product was quanti®ed as described above. In some experiments, reactions were carried out in the presence of either 500 mM VP-16, 10 mM ADM, or 100 mM mAMSA, with 10 to 100 ng of wild type or a mutant hTOP2a. Other conditions were the same as described above. 3. Results 3.1. Atypical multidrug resistance phenotype of Lys798 mutants of hTOP2a in yeast We have used a yeast system to isolate and characterize

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drug resistant mutants in human DNA topoisomerase IIa (hTOP2a). Transformants expressing hTOP2a K798L or K798P were viable and formed colonies at concentrations of 42±340 mM etoposide, but transformants expressing wild type hTOP2a were not (Okada et al., 2000 and Fig. 1). hTOP2a mutants K798C and P803S showed marginal resistance at the low drug concentrations. Besides etoposide resistance, hTOP2a mutants K798L, K798P and K798C also formed colonies on plates with 0.9±3.4 mM doxorubicin or 2.3±46 mM mAMSA (Fig. 1F±H,I±K). These data indicate that both K798L and K798P are resistant to three anticancer drugs. 3.2. Expression and puri®cation of wild-type and mutant hTOP2a Histidine-tagged wild type human DNA topoisomerase IIa (hTOP2a) and the mutants K798L and K798P were expressed and puri®ed using a baculovirus expression system and nickel af®nity column chromatography. Each puri®ed fraction contained one major polypeptide of the expected molecular mass (170 kDa) when analyzed by SDS polyacrylamide gel electrophoresis (Fig. 2A), and was active in decatenation of kinetoplast DNA (Fig. 2B). As a control, a mock puri®cation was carried out from Sf9 cells lacking a hTOP2a bacmid; the control fraction did not have detectable protein at 170 kDa or TOP2 activity (data not shown). In the following experiments, the activity of wild type and mutant hTOP2a was measured using a relaxation assay with supercoiled pBluescript plasmid DNA as a substrate. The relaxation assay is more sensitive and quantitative than the decatenation assay. All reactions were carried out in the presence and absence of ATP to determine if type I topoisomerase activity (ATP-independent relaxation activity) was present in the enzyme preparation. These control reactions demonstrate that no TOP1 activity contaminates the puri®ed hTOP2a enzyme preparations (Fig. 2C). In this study of drug resistant TOP2 mutants, the catalytic ef®ciency of each mutant enzyme was carefully compared to wild type hTOP2a. The relative catalytic ef®ciency of each enzyme was considered when determining appropriate conditions for measuring formation of the drug-induced cleavable complex. This is necessary in order to correctly measure how much cleavable complex is stabilized, because both the catalytic activity and the level of drug resistance in¯uences formation of the cleavable complex. Relaxation activity was measured in the presence of each enzyme at a concentration of 0 to 20 ng enzyme per reaction (Fig. 3). The amount of the remaining supercoiled DNA substrate was quanti®ed (data not shown) and the speci®c activity of the wild type and mutant enzymes was calculated. One unit of enzyme activity is de®ned as the amount which relaxes 50% of the supercoiled substrate DNA under standard conditions (i.e. 200 ng substrate, buffer and assay conditions as described in Section 2). As shown in Fig. 3,

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Fig. 1. Drug resistance in yeast. JN394t2-4 (ISE2, top2-4) was transformed by wild-type or a mutant in hTOP2a (K798P, K798L, K798C, or P803S). Each clone was cultured and streaked on an SGAL-URA plate with or without anticancer drugs. Drug sensitivity was determined after incubation at 358C for 1 week. (A) Positions of the clones are illustrated. (B) No drug. (C±E) 2542, 100170, 200 340 mg/mMl etoposide. (F±H) 0.59, 1.7, 2 3.4 mg/mlM doxorubicin. (I±K) 12.3, 125, 20 46 mg/mlM of mAMSA.

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145

drug concentration. Similar experiments were carried with doxorubicin and mAMSA, and suitable drug concentrations were determined for further analyses (data not shown). 3.4. Multidrug inhibition of wild type and mutant hTOP2a in vitro

Fig. 2. Characterization of recombinant wild type and mutant hTOP2a. (A) SDS-polyacrylamide gel electrophoresis. Ten ml puri®ed hTOP2 were loaded onto a 6% SDS-PAGE gel. After electrophoresis, the gel was stained with Coomassie Brilliant Blue. Lane M, molecular weight marker (Kaleidoscope Prestained Standards, Bio-Rad, CA): lane 1, wild type: lane 2, K798L; lane 3, K798P. Positions of molecular sizes (208, 127, 90 and 50 kDa) are indicated by arrowheads. An open arrowhead indicates the position of TOP2 (170 kDa). (B) Kinetoplast DNA decatenation assay. Catalytic activity of the wild type hTOP2a and the mutants (K798L, K798P) was assayed by decatenation of kinetoplast DNA. Lane M, molecular weight marker (1 kb DNA ladder, NEB, MA): lanes 1 to 4; wild type, K798L, K798P, and no enzyme, respectively. Arrowheads indicate the positions of catenated DNA (CAT), and decatenated DNA [OC (open circular) and RC, (relaxed circular)] (C) TOP1 activity assay. TOP1 activity was determined by relaxation of supercoiled DNA in the absence (lanes 1, 3 and 5) of ATP. Control reactions were in the presence of ATP (lanes 2, 4 and 6). Lane 1, no enzyme (N); lanes 2 and 3, wild type; lanes 4 and 5, K798L; lanes 6 and 7, L798P. Positions of the relaxed circular DNA (RC) and supercoiled circular DNA (SC) are indicated by arrowheads.

As mentioned above, the relative speci®c activity of wild type and mutant TOP2a may affect the amount of cleavable complex formed by that enzyme. That is, an enzyme with low speci®c activity is likely to produce less linear product than an enzyme with high speci®c activity. Therefore, enzyme concentration was varied (other reaction conditions constant) and the amount of linear product was determined in the presence of 500 mM etoposide, 10 mM doxorubicin or 100 mM mAMSA (Fig. 5A±C,D±F). Wild type TOP2a produced several-fold more linear product than K798L or K798P at any enzyme concentration for each of the three drugs tested. Under our reaction conditions, extent of drug sensitivity may be assessed by comparing the enzyme units between wild type and a mutant TOP2 that produce the same amount of the linear DNA, i.e. to produce 2 ng of the linear DNA, for example, K798L and K798P requires 8- and 10-fold more enzyme unit and are resistant to etoposide than wild type, 8- and 12-fold more resistant to doxorubicin, and 1.5- and 2-fold more resistant to mAMSA, respectively. Results of the in vitro and in vivo experiments were summarized in Table 1.

the speci®c activity of both hTOP2a mutants (K798L, 94 units/mg and K798P, 340 units/mg) is similar to wild type (140 units/mg). 3.3. Determination of the drug concentration for study of drug inhibition In order to assess if mutant hTOP2a is resistant to etoposide in vitro, 100 ng of enzyme was assayed for relaxation activity and cleavage activity with 200 ng of supercoiled DNA and several concentrations of etoposide (50, 100, 500, and 1000 mM) in the reaction mixture. Linear DNA accumulated proportionally to increasing etoposide concentration (Fig. 4). The amount of the relaxed circular (RC) and open circular (OC) DNA products decreased slightly (Fig. 4A) with high drug concentration. The amount of linear product, which results from the stabilized cleavable complex, was quanti®ed and the results are shown graphically in Fig. 4B. Extent of inhibition is measured quantitatively as an accumulation of linear DNA as a function of

Fig. 3. Determination of TOP2 speci®c activity. To evaluate the activity of recombinant enzymes, supercoiled DNA relaxation assay was carried out using wild type (A), K798L (B) and K798P (C). Reaction products were electrophoresed. Lane 1, 20 ng; lanes 2 to 10, 18 ng and decreasing amounts by intervals of 2 ng; lane 11, 0 ng. Positions of relaxed circular DNA (RC) and supercoiled circular DNA (SC) are indicated by arrows. The enzyme amount required for 50% relaxation was determined (indicated by arrowhead). The speci®c activities of TOP2a preparations were 140 units/mg, 94 units/mg and 340 units/mg for wild type, K798L and K798P, respectively.

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Fig. 4. Quanti®cation of inhibition by etoposide. (A) TOP2 inhibition assay was carried out using 100 ng of wild type enzyme and 0, 50, 100, 500, and 1000 mM etoposide (lanes 1 to 5, respectively). Positions of the relaxed circular DNA (RC), and open circular DNA (OC) are indicated by ®lled arrows, and linear DNA (Linear) by an open arrow. (B) The intensity of each DNA band was quanti®ed. Amount of the linear DNA (vertical axis) was plotted vs. etoposide concentration (horizontal axis).

Fig. 5. TOP2 inhibition assay of wild type hTOP2a, K798L and K798P. To determine the inhibition of each enzyme by etoposide, doxorubicin and mAMSA, assays were carried out using 100 (lanes 1, 4, 7), 50 (lanes 2, 5, 8), 10 ng (lanes 3, 6, 9) of wild type (lanes 1±3), K798L (lanes 4±6) and K798P (lanes 7±9). Reactions were performed with 500 mM etoposide (A), 10 mM doxorubicin (B), or 100 mM mAMSA (C). Positions of the relaxed circular DNA and supercoiled circular DNA are indicated by ®lled arrows, and linear DNA by an open arrow. In panels D, E, and F, amounts of linear DNA in A, B, and C, respectively, were quanti®ed and plotted vs. enzyme units. Rectangle, square and triangle represent wild type, K798L and K798P, respectively.

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Table 1 Relative drug resistance of TOP2a mutants

WT K798P K798L a b

Speci®c activity (units/mg protein)

Drug resistance a in yeast (fold)

Drug resistance b in vitro (fold)

Etoposide

Doxorubicin

mAMSA

Etoposide

Doxorubicin

mAMSA

140 340 94

± 8 8

± 4 4

± 4 4

± 8±12 8±12

± 8±12 8±12

± 2 2

Relative drug resistance was indicated as wild type by comparing the colony formation ability in yeast (Fig. 1). Relative drug resistance was indicated as wild type by comparing the formation of linear DNA (Fig. 5).

4. Discussion Despite the impact of drug resistance on cancer chemotherapy, very few biochemical experiments have been carried out on human TOP2. Most studies of hTOP2 mutants have examined phenotype, transcription level, and activity in crude cell extracts. According to the current observations most hTOP2 mutants exhibit either decreased activity or decreased level of expression. The hTOP2a mutants reported here, K798L and K798P, have an intact nuclear localization signal, and a nearly wild type (or higher) level of catalytic activity (67% for K798L and 240% for K798P). Nevertheless, amount of the linear DNA produced by these mutants was several-fold lower than wild type (Fig. 5). At present it is not of conclusive that the reduction in an amount the linear DNA is the sole mechanism for the drug resistance because we have not measured inhibition extent in supercoiling relaxation and other assays. However these data suggest that the enzymes are intrinsically drug resistant in vitro. Among human mutants, a double mutant R450Q:P803S that was originally isolated from a human leukemia cell line showed a phenotype known as `atypical MDR' (Bugg et al., 1991; Danks et al., 1993). Recently, the single hTOP2a mutants R450Q and P803S were expressed in yeast, puri®ed and characterized (Mao et al., 1999). R450Q is impaired in ATP utilization, and P803S has no effect on drug induced DNA cleavage in the presence of ATP but showed reduced cleavage in the absence of ATP. Therefore, at least R450Q may belong to a category of mutations that decrease activity. Drug-resistant and catalytically pro®cient mutants of hTOP2a have not been reported previously, although mutations with similar characteristics have been reported in yeast TOP2. Yeast H1012Y, which was selected in vivo against the ¯uoroquinolone CP-115,953, has an activity level similar to wild type in vitro (Elsea et al., 1995). A few-fold resistance against both CP-115,953 and etoposide is associated with yH1012Y. Other mutants, yG748E and yA642S also show a 2- to 3- fold increase in resistance to amsacrine and 10-fold resistance to etoposide (Patel et al., 1997). Yeast S740W also confers resistance to CP-115,953 but is hypersensitivie to etoposide both in vivo and in vitro (Hsiung et al., 1995). Some yeast mutants are associated with changes in their cleavage pattern that may result from altered nucleo-

tide sequence preference when interacting with drug (Strumberg et al., 1999). It is not clear what mechanism caused the drug resistance, in term of reduction in the production of linear DNA by K798L and K798P. The mutants described in this work have a single amino acid substitution of Lys798 in hTOP2a. Another mutant altered at the same site, K798C, also showed reduced sensitivity to antiTOP2 drugs (Fig. 1). In addition, the TOP2 mutant K798N has been reported to be drug resistant in a leukemia cell line (Patel and Fisher, 1993). These results suggest that Lys798 is a critical residue for the drug sensitivity of hTOP2a. In the crystal structure of yeast TOP2 (Berger et al., 1996), the corresponding amino acid sits in the functionally important loop between A 0 b2 and A 0 b3 and faces toward a helix-turn-helix region in the CAP domain (Okada et al., 2000). The yeast residues yGly748 and ySer740 (see above) also lie in the helix-turnhelix region and yAla642 might lie near to this region (Hsiung et al., 1995; Patel et al., 1997). Studies suggest that drugs may interact with TOP2 at or near the helix-turn-helix region. For example, the S83W mutation in the gyrA protein (corresponding to the ySer740) reduces ¯uoroquinolon binding (Reece and Maxwell, 1991; Willmott and Maxwell, 1993). The interaction may not be limited to ¯uoroquinolon because ¯uoroquinolone and etoposide can compete for binding to TOP2 (Corbett et al., 1993). This structural and biochemical information suggests that mutations at Lys798 could also interfere with speci®c contact between these drugs and hTOP2a. So far reduced level of TOP2 activity and/or TOP2 mRNA is often observed in drug resistant cell lines with no mutation in the TOP2 gene. In these cases, reduced number of TOP2 molecules in the nuclear compartment may decrease the probability of trapping TOP2 in the cleavable complex and thus limit the extent to which cell death pathways are triggered. From the viewpoint of clinical chemotherapy, our mutations would cause a problematic case if it ever happens in patients. In malignant tumors, cells that express TOP2 at a high level proliferate aggressively (Yabuki et al., 1996), and higher expression of TOP2a is correlated with poor prognosis (Dingemans et al., 1999; Taniguchi et al., 1999). If those cells acquire a drug resistant form of TOP2a such like K798P and K798L, cells may maintain their ability to proliferate and are resis-

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tant against multidrugs. On the other hand, if the function of TOP2 is impaired or insuf®cient, as is the case of known drug resistant mutants, it could be potential disadvantage for the cells to proliferate (Matsuo et al., 1990; Jannatipour et al., 1993; Kawanami et al., 1996). Acknowledgements We are grateful to the people in Laboratory of Cancer Cell Biology for helpful discussion. This study was supported by Grants-in Aid from the Ministry of Education, Science, Sports and Culture of Japan to AK, SY and YN, and by Yokoyama and Aichi Cancer Research Foundations to MS. References Andoh, T., Nishizawa, M., Hida, T., Ariyoshi, Y., Takahashi, T., Ueda, R., 1996. Reduced expression of DNA topoisomerase II confers resistance to etoposide (VP-16) in small cell lung cancer cell lines established from a refractory tumor of a patient and by in vitro selection. Oncol. Res. 8, 229±238. Berger, J.M., Gamblin, S.J., Harrison, S.C., Wang, J.C., 1996. Structure and mechanism of DNA topoisomerase II. Nature 379, 225±232. Bugg, B.Y., Danks, M.K., Beck, W.T., Suttle, D.P., 1991. Expression of a mutant DNA topoisomerase II in CCRF-CEM human leukemic cells selected for resistance to teniposide. Proc. Natl. Acad. Sci. USA 88, 7654±7658. Chen, C.J., Chin, J.E., Ueda, K., Clark, D.P., Pastan, I., Gottesman, M.M., Roninson, I.B., 1986. Internal duplication and homology with bacterial transport proteins in the mdr1 (P-glycoprotein) gene from multidrugresistant human cells. Cell 47, 381±389. Corbett, A.H., Hong, D., Osheroff, N., 1993. Exploiting mechanistic differences between drug classes to de®ne functional drug interaction domains on topoisomerase II. Evidence that several diverse DNA cleavage-enhancing agents share a common site of action on the enzyme. J. Biol. Chem. 268, 14394±14398. Danks, M.K., Warmoth, M.R., Friche, E., Granzen, B., Bugg, B.Y., Harker, W.G., Zwelling, L.A., Futscher, B.W., Suttle, D.P., Beck, W.T., 1993. Single-strand conformational polymorphism analysis of the M(r) 170,000 isozyme of DNA topoisomerase II in human tumor cells. Cancer Res. 53, 1373±1379. Dingemans, A.M., Witlox, M.A., Stallaert, R.A., van der Valk, P., Postmus, P.E., Giaccone, G., 1999. Expression of DNA topoisomerase IIalpha and topoisomerase IIbeta genes predicts survival and response to chemotherapy in patients with small cell lung cancer. Clin. Cancer Res. 5, 2048±2058. Elsea, S.H., Hsiung, Y., Nitiss, J.L., Osheroff, N., 1995. A yeast type II topoisomerase selected for resistance to quinolones. Mutation of histidine 1012 to tyrosine confers resistance to nonintercalative drugs but hypersensitivity to ellipticine. J. Biol. Chem. 270, 1913±1920. Hamada, H., Tsuruo, T., 1986. Functional role for the 170- to 180-kDa glycoprotein speci®c to drug-resistant tumor cells as revealed by monoclonal antibodies. Proc. Natl. Acad. Sci. USA 83, 7785±7789. Hsiung, Y., Elsea, S.H., Osheroff, N., Nitiss, J.L., 1995. A mutation in yeast TOP2 homologous to a quinolone-resistant mutation in bacteria. Mutation of the amino acid homologous to Ser83 of Escherichia coli gyrA alters sensitivity to eukaryotic topoisomerase inhibitors. J. Biol. Chem. 270, 20359±20364. Jannatipour, M., Liu, Y.X., Nitiss, J.L., 1993. The top2-5 mutant of yeast

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