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Drug Resistance Mechanisms of Topoisomerase I Drugs
Drug Resistance Mechanisms of Topoisomeruse f Drugs Toshiwo Andoh* and Kosuke Okadat * Laboratory of Biochemistry
Aichi Cancer Center Research Institute 1-1 Kanokoden, Chikusa-ku, Nagoya 464, Japan f Department of Blood Transfusion Hiroshima University Hospital 1-2-3 Kasumi, Minami-ku, Hiroshima 734, Japan
1. Introduction DNA topoisomerase I (topo I) has been implicated in various genetic processes, such as replication, transcription, and recombination (CozzareIli and Wang, 1990; Potmesil and Kohn, 1991). CPT-11 (7-ethyl- 10-[4-(1-piperidyl)-1-piperidyl]carbonyloxy-camptothecin], a hydrophilic derivative of camptothecin (CPT), has been developed as an antitumor drug in Japan. The drug was tested in experimental animal systems (Kunimoto et al., 1987; Tsuruo et al., 1987) and in clinical studies of various forms of tumors with a considerably high degree of efficacy (Fukuoka et al., 1992;Andoh et al., 1993). Recently, it was shown that CFT was a specific inhibitor of top0 I by stabilizing an intermediary form of top0 I-DNA complex called the cleavable complex (Hsiang et al., 1985). In order to study the mechanism of resistance to the drug, we have established a CPT-11-resistant cell line, CPT-KS, from a human Tcell-derived acute lymphoblastic leukemia cell line RPMI 8402, by adaptation and selection over a long period in media containing gradually increasAduunces in Pharmacology, Volume 298 Copyright 6 1994 by Academic hess, Inc. All rights of reproduction in any form reserved
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ing concentrations of CPT-11 (Okada et al., 1989; Andoh et al., 1987). The mutant cells possessed an altered form of top0 I which is highly resistant to CPT and which has some differences in enzymatic properties (Andoh et al., 1987; Kjeldsen et al., 1988a; Gromova et al., 1993). Recently, several other cell lines resistant to CPT or derivatives have been established and their mechanism of resistance has been characterized (Gupta et al., 1988; Eng et al., 1990; Kanzawa et al., 1990; Sugimoto et al., 1990a,b; Tanizawa and Pommier, 1992; Takeda et al., 1992; Chang et al., 1992; Madelaine et al., 1993). From these studies at least two different mechanisms for CPT resistance have been seen. Quantitative reduction of top0 I seems to be a common mechanism. Sugimoto et at. (1990a) found a reduction in cellular top0 I content in three of four resistant tumor cell lines. Top0 I purified from one of these cell lines showed specific activity similar to that of the parental enzyme. Similar quantitative reduction of top0 I was also observed in CPT-resistant CPT-KS cells (Andoh et al., 1987), Chinese hamster ovary cells (Gupta et al., 1988), P388 cells (Eng et al., 1990), PC7/CPT cells (Kanzawa et al., 1990), V79' cells (Chang et al., 1992), CPT-resistant human pancreatic tumor cell lines (Takeda et al., 1992), and P388/CPT cells (Madelaine et al., 1993). In P388/CPT+ cells rearrangement and hypermethylation occurred in one allele of the top0 I gene, which may account for the reduction in the enzyme (Eng et al., 1990). As observed in CPT-KS human lymphoblastic leukemia cells, qualitative alterations in top0 I enzyme in specific enzymatic activity (Tanizawa and Pommier, 1992) and in CPT resistance (Gupta et al., 1988; Kanzawa et al., 1990; Tanizawa and Pommier, 1992; Madelaine et al., 1993) have been observed. These findings in various CPT-resistant cell lines point to a mechanism of acquisition of CPT resistance. During early stages of adaptation or selection in media containing the drug, cells may acquire a low degree of resistance by a reduction in top0 I gene expression. Upon further selection in media containing higher concentrations of the drug, cells may be selected that have mutations on the top0 I gene which render cells highly resistant to the drug. These two-step events may take place sequentially in some cases and may overlap in others. This is, in fact, what was observed in the development of a CPT-resistant tumor line during the course of serial passage of cells in uiuo in the presence of the drug (Madelaine et al., 1993). A third mechanism conferring resistance on cells is the reduced accumulation of the drug in the cell. Takeda et al. (1992) and Chang et al. (1992) observed a reduced uptake of CPT-11 and CPT, respectively.
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II. Mutant Cells, CPT-KS, Possess an Altered Form of Top0 I Resistant to CPT Hsiang et al. (1985)have shown that CPT inhibits mammalian top0 I in v i m by stabilizing the intermediate enzyme-linked DNA breaks. However, it was not known whether this enzyme is the sole cellular target of CPT and whether this mode of action is responsible for its cytotoxicity and inhibition of nucleic acid metabolism in uivo. We have approached this problem by isolating and characterizing a cell line, CPT-K5, from a human acute lymphoblastic leukemia cell line, RPMI 8402, resistant to CPT-11 (Andoh et al., 1987). We measured top0 I content in the resistant cells by assaying relaxation activity and by immunoblotting a 1 M NaCl extract of parental (wildtype, or WT) and CPT-K5 cells. About one-third of the WT activity was recovered from the mutant cells. The total amount of the enzyme in the mutant cells seemed to be reduced to less than half that of the WT cells, suggesting that the specific activities of the two enzymes are similar. Since the content of mRNA was the same in WT and CPT-K5 cells, the mutant enzyme may have a lower metabolic stability than the parental enzyme (Tamura ef al., 1991). Top0 I was purified to apparent homogeneity from WT and CPT-K.5 cells according to the method of Ishii et al. (1983), and its sensitivity to CPT was measured. As shown in Fig. 1 , the activity of WT top0 I was inhibited at 1 p M or higher concentrations of CPT, whereas K5 top0 I was not inhibited by the drug at 125 p M , the resistance index being more than 125, indicating that the cellular resistance to the drug is primarily, if not entirely, due to the resistance of a structurally altered top0 I. This in turn establishes top0 I as the primary cellular target of CPT.
111. The Mutant Enzyme Possesses Higher Affinity for Recognition Sequences
Several types of cleavage sites have been identified, using CPT in mapping studies (Kjeldsen et al., 1988b; Gromova et al., 1990). These sites were classified into three categories according to their response to drug treatment. Class A sites include strong cleavage sites only slightly affected by CPT treatment. Class B sites are greatly enhanced in the presence of CPT. Thus, CPT altered the cleavage pattern of the WT, but not the mutant, enzyme. At class A sites the mutant enzyme cleaves the recognition
Toshiwo Andoh ond Kosuke Okoda
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RPM18402-Top0 I
CPT-K5 ToPo I
Fig. 1 Effect of CFT on the activity of top0 I from WT (lanes 1-5) and CPT-K5 cells (lanes 6-10). Enzymatic activity was assayed in the absence (lanes 1 and 6) and presence of CPT at 1.0 (lanes 2 and 7), 5.0 (lanes 3 and 8). 25.0 (lanes 4 and 9), and 125.0 pM (lanes 5 and lo), respectively. Lane 1 1 , Substrate colEl DNA only. Form I and form Ir represent supercoiled form I and relaxed form I DNA, respectively.
sequence with twofold higher efficiency than the WT enzyme and forms more stable complexes. The mutant enzyme does not seem to recognize and cleave class B sites. However, by the use of oligonucleotides with a class B recognition site, Gromova et al. (1993) demonstrated that K5 top0 I strongly interfered with the WT top0 I-mediated cleavage of class B site sequence which is otherwise strongly stimulated in the presence of CPT. Furthermore, the mutant enzyme catalyzed the strand transfer reaction via the class B site with higher efficiency regardless of the presence of CPT, and the equilibrium between cleavage and religation appears to be shifted and a higher rate of catalysis at class B recognition sites, explaining the apparent inability of the mutant enzyme to recognize the class B sites. These unique properties of the mutant top0 I may well account for the resistance to CPT.
IV. Determination of Mutation Sites of K5 Top0 I Responsible for CPT Resistance In order to determine the mutation(s) responsible for CPT resistance of K5-top0 I, we have attempted to determine the whole amino acid sequence of top0 I from WT and mutant cells by nucleotide sequencing of its cDNAs (Tamura et al., 1991). Amino acid sequence of human top0 I has been
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Drug Resistance Mechanisms
determined by D’Arpa et al. (1988). cDNA libraries of WT and CPT-K5 cells were constructed and screened with a 3.1-kb fragment of a human top0 I cDNA that was originally isolated from human brain stem cells. Several clones were isolated and their nucleotide sequences were determined. Comparison of the sequences revealed two nucleotide substitutions of A to G, changing Asp-533 (GAC) and Asp-583 (GAC) of the WT top0 I to Gly (GGC) in K5 top0 I (numbered according toD’Arpa et al., 1988) (see Fig. 2), Thus, unexpectedly, we have found mutations at two sites in the K5 top0 I. The next question is whether both mutations are necessary for the high degree of resistance or whether either one of these is sufficient for it. Comparison of the amino acid sequences of yeast and human top0 I shows that the regions where the mutations occurred are highly conserved. As shown in Fig. 3, it is of particular relevance to refer to the finding that the amino acid residues corresponding to position 533 of the WT top0 I are Asp in all species compared, including mouse top0 I, which shares 96% of amino acids in common with the human enzyme (Koiwai et al., 1993), with the exception of Gly in K5 top0 I, whereas residues corresponding to position 583 are variable (i.e., Asp in WT and human brain stem cell top0 I, and Gly in all other species compared). Taking into account that yeast, mouse, and some of human enzymes are of the Gly type at position 583 and are sensitive to CPT, one could argue that the amino acid variation at residue 583 among human enzymes is the result of a polymorphism of the human genome unrelated to CPT resistance, and the mutation from Asp to Gly at 533 is responsible for the resistance. To address this point, the enzyme with a single mutation was created by sitedirected mutagenesis and expression in Escherichia coli. AGCT
AGCT
WT
K5
Asp533
Fig. 2 Sequence difference between the WT and K5 top0 I cDNAs. Portions of an autoradiogram around the mutation site at amino acid residue 533 are shown, where GAC was changed to GGC. -
98
, NH2
S. pombe mouse
Toshiwo Andoh and Kosuke Okada Hydrophilic domain
Central conserved domain
A
765
tt
K5 Mutation
COOH
CPT Sensitivity
533 D-
583
G
S
G(GSC)
G (GPC)
R
G-
D
R
placenta human
in vitro
DG -G-
S
G
s
Fig. 3 Relationship between amino acid substitutions and CPT sensitivity of topo I from various eukaryotes: S. cereuisiae, Schizosaccharomyces pombe, mouse, and humans. CPT sensitivities of the products of in uitro rnutagenesis were also compared. Only amino acids at positions 533 and 583 were shown with sensitivity (S) or resistance (R) to CPT.
V. Site-Directed Mutagenesis of Top0 I and Expression in Escherichia coli In a previous communication we have shown that the C-terminal twothirds of top0 I (amino acid residues 163-765), when expressed as a fusion protein with protein A’, exhibited a relaxation activity, and that the activity of the fusion protein from K5 top0 I was resistant to CPT, indicating that the CPT-K5 cDNA for top0 I in fact encodes a functional mutant-type enzyme (Tamura et af., 1991). To confirm and verify the assumption above we have constructed a series of plasmids containing inserts of top0 I cDNA (amino acid residues 163-765) with a single mutation at either residue 533 or 583 by site-directed mutagenesis as a fusion protein with glutathione S-transferase. Fusion proteins of mutant top0 I were expressed in E. coli and were purified from the bacterial lysates as described earlier (Ishii et al., 1983).
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As depicted in the bottom of Fig. 3, the relaxation activity of the fusion proteins of the parental type (-D-D-) and that with a single mutation at 583 (-D-G-, M2) was as sensitive to CPT as the native WT top0 I, whereas that of the fusion proteins with a single mutation at 533 (-G-D-, MI) and with double mutations at 533 and 583 (-G-G-, M3) was as resistant to CPT as the native K5 top0 I. These results verity the assumption above and establish that the single mutation changing Asp to Gly at 533 is responsible for CPT resistance of K5 top0 I and CPT-KS cells. One model for inhibition of top0 I by CPT is that the drug binds avidly to a specific site(s) or pocket(s) of the enzyme on formation of the cleavable complex with the recognition sequence of DNA, thereby stabilizing the complex and blocking the strand passage and/or religation step of the catalytic cycle of the enzyme (Hsiang et id.,1985; Liu, 1989) (Fig. 4). It is reasonable to assume that the amino acid residue at 533 is contained
5‘
3 DNA
3
5’
- It
Cleavable
~7) ‘ 7 Complex v
$3 a v WT - top0 I
z+
Stabilization
J
T -
K5 toPo I
Fig. 4 A model of catalysis by WT and K5 top0 I. As proposed by Hsiang et al. (1985). top0 I catalyzes topological changes of DNA through several steps: binding, cleavable complex formation, and religation. CPT interferes with the reaction by interacting with and stabilizing the cleavable complex. The mutation changing Asp-533 to Gly confers CPT resistance on the enzyme with a higher affinity for the recognition sequences.
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Toshiwo Andoh and Kosuke Okodo
in the pocket(s) or domain(s) involved in or affecting the interaction with CPT, and that the mutation changing Asp to Gly at this site confers CPT resistance on the enzyme by altering this drug binding domain(s) of the complex. This hypothesis is supported by the finding that K5 top0 I binds to the recognition sequences with higher affinity and with a higher rate of catalysis, as described above. Furthermore, the predictions of the secondary structure of the enzyme, that the region containing the mutation is protruding toward the outer surface of the protein and that the change of Asp to Gly makes the region retract inwardly (Tamura et al., 1991), and the finding that the region is within the most conserved region along the sequence of the enzyme (Lynn ef al., 1989), strongly suggest that this region plays an important role in the catalysis, especially in the latter half of the catalytic cycle (i.e., the strand passage and/or religation step). Thus, this constitutes the second functional site detected in addition to the hitherto described active-site Tyr-723. It is ofgreat relevance to refer to some other papers describing a mutation in the top0 I gene conferring CPT resistance (Kubota et al., 1992; Caron and Wang, 1993; Pommier et al., 1993; Levin et ai., 1993). All of the mutations were found in the central conserved region except the one located close to the active-site Tyr-723 in the conserved C-terminal domain of the enzyme, which showed, however, a smaller degree of resistance. Of great interest is the mutation topl-203 of Saccharomyces cereuisiae top0 I, which stimulates mitotic recombination due to an elevated intrinsic stability of the enzyme-mediated cleavable complex, mimicking the effect of CPT on WT top0 I (Levin et al., 1993). These results imply that the amino acid residues identified in the mutant enzymes are involved in the domain(s) interacting with CPT, playing an important role in the enzymatic catalysis. Further study of the structure-drug sensitivity relationship should define more precisely the presumptive domains(s) interacting with the drug, which we may term the “CPT pocket,” and participating in the catalysis of the enzyme.
VI. Conclusion We have described the establishment and characterization of CPTresistant cell lines in our group. We have obtained a definitive answer to the problem of how mammalian cells acquire CPT resistance (i.e., by mutation of the top0 I gene producing a CPT-resistant form of the enzyme in resistant cells). This is, in fact, one way of acquiring resistance to the drug. However, cells could become resistant by some other ways (e.g., by lowering the permeability of the drug or by lowering the content of
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top0 I within the cells). Some reports have described lowered top0 I content in CPT-resistant cells (Sugimoto et al., 1990a; Eng et al., 1990; Tanizawa and Pommier, 1992; Chang et al., 1992; Madelaine et al., 1993). Some others have described cell lines with reduced cellular accumulation of CPT or a CPT derivative (Chang et at., 1992; Takeda et al., 1992). The CPT-resistant cell lines characterized so far has been obtained by adaptation in in uitro culture or in serial passage in experimental animals. Further efforts obviously are needed in the characterization of CPTresistant cells which might prevail and develop in patients treated with CPT derivatives.
References Andoh, T., Ishii, K., Suzuki, Y., Ikegami, Y., Kusunoki, Y., Takemoto, T., and Okada, K. (1987). Characterization of a mammalian mutant with a camptothecin-resistant DNA topoisomerase I. Proc. Natl. Acad. Sci. USA 84, 5565-5569. Andoh, T., Ikeda, H., and Oguro, M., eds. (1993). “Molecular Biology of DNA Topoisomerases and Its Application to Chemotherapy.” CRC Press, Boca Raton, Florida. Caron, P. R., and Wang, J. C. (1993). DNA topoisomerases as targets of therapeutics: A structural overview. in “Molecular Biology of DNA Topoisomerases and Its Application to Chemotherapy” (T. Andoh, H. Ikeda, and M. Oguro, eds.), pp. 1-18. CRC Press, Boca Raton, Florida. Chang, J.-Y., Dethlefsen, L. A,, Barley, L. B., Zhou, B.-S., and Cheng, Y.-C. (1992). Characterization of camptothecin-resistant Chinese hamster lung cells. Biochem. PharmaC O ~ 43, . 2443-2452. Cozzarelli, N. R., and Wang, J. C., eds. (1990).“DNA Topology and Its Biological Effects.” Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. D’Arpa, P., Machlin, P. S., Ratrie, H., 111, Rothfield, N. F., Cleaveland, D. W., and Earnshaw, W. C. (1988). cDNA cloningof human DNA topoisomerase I: Catalytic activity of a 67.7-kDa carboxy-terminal fragment. Proc. Natl. Acad. Sci. USA 85, 2543-2547. Eng, W.-K., McCabe, F. L., Tan, K. B., Mattern, M. R., Hofman, G. A., Woessner R. D., Hertzberg, R. P., and Johnson, R. K. (1990). Development ofastable camptothecinresistant subline of P388 leukemia with reduced topoisomerase I content. Mol.Pharmacol. 38,471-480. Fukuoka, M., Niitani, H., Suzuki, A., Motomiya, M., Hasegawa, K., Nishiwaki, Y., Kuriyama, T., Ariyoshi, Y., Negoro, S., Masuda, N., Nakajima, S., and Taguchi, T. (1992). A phase I1 study of CPT-11, a new derivative of camptothecin, for previously untreated non-small cell lung cancer. J . Clin. Oncol. 10, 16-20. Gromova, I. I., Buchman, V. L., Abagyan, R. A., Ulyanov, A. V., and Bronstein I. B. (1990). Sequence dependent modulating effect of camptothecin on the DNA-cleaving activity of the calf thymus type I topoisomerase. Nucleic Acids Res. 18, 637-645. Gromova, I. I., Kjeldsen, E., Svejstrup, J. Q . , Alsner, J . , Chritiansen, K.. and Westergaard, 0. (1993). Characterization of a n altered DNA catalysis of acamptothecin-resistant eukaryotic topoisomerase I. Nucleic Acids Res. 21, 593-600. Gupta, R. S., Gupta, R., Eng, B., Lock, R. B., Ross, W. E., Hertzberg, R. P., Caranfa, M. J., and Johnson, R. K. (1988). Camptothecin-resistant mutants of Chinese hamster ovary cells containing a resistant form of topoisomerase. I. Cancer Res. 48,6404-6410. Hsiang, Y.-H., Hertzberg, R., Hecht, S., and Liu, L. F. (1985). Camptothecin induces
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protein-linked DNA breaks mediated via mammalian DNA topoisomerase I. J. Biol. Chem. 260, 14873-14878. Ishii, K., Hasegawa, T., Fujisawa, K., and Andoh, T. (1983). Rapid purification and characterization of DNA topoisomerase I from cultured mouse mammary carcinoma FM3A cells. J. Biol. Chem. 258, 12728-12732. Kanzawa, F., Sugimoto, Y., Minato, K., Kasahara, K., Bungo, M., Nakagawa, K., Fujiwara, Y., Liu, L. F., and Saijo, N . (1990). Establishment o f a camptothecin analogue (CPT-11)resistant cell line of human non-small cell lung cancer: Characterization and mechanism of resistance. Cancer Res. 50,5919-5924. Kjeldsen, E., Bonven, B. J., Andoh, T., Ishii, K., Okada, K., Bolund, L., and Westergaard, 0. (1988a). Characterization of a camptothecin-resistant human DNA topoisomerase I. J . Biol. Chem. 263, 3912-3916. Kjeldsen, E., Mollerup, S., Thomsen, B., Bonven, B. J., Bolund, L., and Westergaard, 0. (1988b). Sequence-dependent effect of camptothecin on human topoisomerase I DNA cleavage. J. Mol. Biol. 202, 333-342. Koiwai, O., Yasui, Y., Sakai, Y., Watanabe, T., Ishii, K., Yanagihara, S., and Andoh, T. (1993). Cloning of the mouse cDNA encoding DNA topoisomerase I and chromosomal location of the gene. Gene US,21 1-216. Kubota, N., Kanzawa, F., Nishio, K., Takeda, Y ., Ohmori, T., Fujiwara, Y., Terashima, Y., and Saijo, N. (1992). Detection of the topoisomerase 1 gene point mutation in CPT11 resistant lung cancer cell line. Biochem. Biophys. Res. Commun. 188, 571-577. Kunimoto, T., Nitta, K., Tanaka, T., Uchida, N., Baba, H., Takeuchi, M., Yokokura, T., Sawada, S., Miyasaka, T., and Mutai, M. (1987). Antitumor activity of 7-ethyl-10-[4-(1piperidin0)-1-piperidino]carbonyloxy-camptothecin, a novel water-soluble derivative of camptothecin, against murine tumors. Cancer Res. 47, 5944-5947. Levin, N. A., Bjornsti, M.-A,, and Fink, G. R. (1993). A novel mutation in DNA topoisomerase I of yeast causes DNA damage and RAD9-dependent cell cycle arrest. Genetics 133, 799-8 14. Liu, L . F. (1989). DNA topoisomerase poisons as antitumor drugs. Annu. Rev. Biochem. 58, 351-375. Lynn, R. M., Bjornsti, M.-A., Caron, P. R., and Wang, J. C. (1989). Peptide sequencing and site-directed mutagenesis identify tyrosine-727 as the active site tyrosine of Saccharomyces cereuisiae DNA topoisomerase I. Proc. Nail. Acad. Sci. USA 86, 3559-3563. Madelaine, I., Prost, S., Naudin, A., Riou, G . , Lavelle, F., and Riou, J.-F. (1993). Sequential modifications of topoisomerase I activity in a camptothecin-resistant cell line established by progressive adaptation. Biochem. Pharmacol. 45, 339-348. Okada, K., Mizutani, A., Kusunoki, Y., Takemoto, Y., and Kuramoto, A. (1989). Antileukemic effects of CPT-I 1 (a new derivative of camptothecin) on rat leukemias and the isolation of resistant human leukemia cells. in “Cancer Chemotherapy: Challenges for the Future’’ (K. Kimura, K. Ota, S. K . Carter, and H. M. Pinedo, eds.), Vol. 4, pp. 313-316. Pommier, Y., Tanizawa, A., Okada, K., and Andoh, T. (1994). Cellular determinants of sensitivity and resistance to camptothecins in “Camptothecin: A New Class of Anticancer Agents” (M. Potmesil and H. M. Pinedo, eds.). CRC Press, Boca Raton, Florida. In press. Potmesil, M., and Kohn, K. W., eds. (1991). “DNA Topoisomerases in Cancer.” Oxford University Press, New York. Sugimoto, Y., Tsukahara, S., Oh-hara, T., Isoe, T., and Tsuruo, T. (1990a). Decreased expression of DNA topoisomerase I in camptothecin-resistant tumor cell lines as determined by a monoclonal antibody. Cancer Res. 50, 6925-6930.
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Sugimoto, Y., Tsukahara, S., Oh-hara, T., Liu, L. F., and Tsuruo, T. (1990b). Elevated expression of DNA topoisomerase I1 in camptothecin-resistant human tumor cell lines, Cancer Res. 50, 7962-7965. Takeda, S ., Shimazoe, T., Sato, K . , Sugimoto, Y., Tsuruo, T., and Kono, A. (1992). Differential expression of DNA topoisomerase I gene between CFT-I 1 acquired- and native-resistant human pancreatic tumor cell lines: Detected by RNA/PCR-based quantitation assay. Biochem. Biophys. Res. Commun. 184, 618-625. Tanizawa, A., and Pommier, Y. (1992). Topoisomerase I alteration in a camptothecinresistant cell line derived from Chinese hamster DC3F cells in culture. Cancer Res. 52, 1849- 1854. Tsuruo, T., Matsuzaki, T., Matsushita, M., Sato, H., and Yokokura, T. (1988). Antitumor effect of CPT-I 1, a new derivative of camptothecin, against pleiotropic drug-resistant tumors in vitro and in vivo. Cancer Chemorher. Pharmacol. 21, 11-74.
Aichi Cancer Center Research Institute 1-1 Kanokoden, Chikusa-ku, Nagoya 464, Japan f Department of Blood Transfusion Hiroshima University Hospital 1-2-3 Kasumi, Minami-ku, Hiroshima 734, Japan
1. Introduction DNA topoisomerase I (topo I) has been implicated in various genetic processes, such as replication, transcription, and recombination (CozzareIli and Wang, 1990; Potmesil and Kohn, 1991). CPT-11 (7-ethyl- 10-[4-(1-piperidyl)-1-piperidyl]carbonyloxy-camptothecin], a hydrophilic derivative of camptothecin (CPT), has been developed as an antitumor drug in Japan. The drug was tested in experimental animal systems (Kunimoto et al., 1987; Tsuruo et al., 1987) and in clinical studies of various forms of tumors with a considerably high degree of efficacy (Fukuoka et al., 1992;Andoh et al., 1993). Recently, it was shown that CFT was a specific inhibitor of top0 I by stabilizing an intermediary form of top0 I-DNA complex called the cleavable complex (Hsiang et al., 1985). In order to study the mechanism of resistance to the drug, we have established a CPT-11-resistant cell line, CPT-KS, from a human Tcell-derived acute lymphoblastic leukemia cell line RPMI 8402, by adaptation and selection over a long period in media containing gradually increasAduunces in Pharmacology, Volume 298 Copyright 6 1994 by Academic hess, Inc. All rights of reproduction in any form reserved
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ing concentrations of CPT-11 (Okada et al., 1989; Andoh et al., 1987). The mutant cells possessed an altered form of top0 I which is highly resistant to CPT and which has some differences in enzymatic properties (Andoh et al., 1987; Kjeldsen et al., 1988a; Gromova et al., 1993). Recently, several other cell lines resistant to CPT or derivatives have been established and their mechanism of resistance has been characterized (Gupta et al., 1988; Eng et al., 1990; Kanzawa et al., 1990; Sugimoto et al., 1990a,b; Tanizawa and Pommier, 1992; Takeda et al., 1992; Chang et al., 1992; Madelaine et al., 1993). From these studies at least two different mechanisms for CPT resistance have been seen. Quantitative reduction of top0 I seems to be a common mechanism. Sugimoto et at. (1990a) found a reduction in cellular top0 I content in three of four resistant tumor cell lines. Top0 I purified from one of these cell lines showed specific activity similar to that of the parental enzyme. Similar quantitative reduction of top0 I was also observed in CPT-resistant CPT-KS cells (Andoh et al., 1987), Chinese hamster ovary cells (Gupta et al., 1988), P388 cells (Eng et al., 1990), PC7/CPT cells (Kanzawa et al., 1990), V79' cells (Chang et al., 1992), CPT-resistant human pancreatic tumor cell lines (Takeda et al., 1992), and P388/CPT cells (Madelaine et al., 1993). In P388/CPT+ cells rearrangement and hypermethylation occurred in one allele of the top0 I gene, which may account for the reduction in the enzyme (Eng et al., 1990). As observed in CPT-KS human lymphoblastic leukemia cells, qualitative alterations in top0 I enzyme in specific enzymatic activity (Tanizawa and Pommier, 1992) and in CPT resistance (Gupta et al., 1988; Kanzawa et al., 1990; Tanizawa and Pommier, 1992; Madelaine et al., 1993) have been observed. These findings in various CPT-resistant cell lines point to a mechanism of acquisition of CPT resistance. During early stages of adaptation or selection in media containing the drug, cells may acquire a low degree of resistance by a reduction in top0 I gene expression. Upon further selection in media containing higher concentrations of the drug, cells may be selected that have mutations on the top0 I gene which render cells highly resistant to the drug. These two-step events may take place sequentially in some cases and may overlap in others. This is, in fact, what was observed in the development of a CPT-resistant tumor line during the course of serial passage of cells in uiuo in the presence of the drug (Madelaine et al., 1993). A third mechanism conferring resistance on cells is the reduced accumulation of the drug in the cell. Takeda et al. (1992) and Chang et al. (1992) observed a reduced uptake of CPT-11 and CPT, respectively.
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II. Mutant Cells, CPT-KS, Possess an Altered Form of Top0 I Resistant to CPT Hsiang et al. (1985)have shown that CPT inhibits mammalian top0 I in v i m by stabilizing the intermediate enzyme-linked DNA breaks. However, it was not known whether this enzyme is the sole cellular target of CPT and whether this mode of action is responsible for its cytotoxicity and inhibition of nucleic acid metabolism in uivo. We have approached this problem by isolating and characterizing a cell line, CPT-K5, from a human acute lymphoblastic leukemia cell line, RPMI 8402, resistant to CPT-11 (Andoh et al., 1987). We measured top0 I content in the resistant cells by assaying relaxation activity and by immunoblotting a 1 M NaCl extract of parental (wildtype, or WT) and CPT-K5 cells. About one-third of the WT activity was recovered from the mutant cells. The total amount of the enzyme in the mutant cells seemed to be reduced to less than half that of the WT cells, suggesting that the specific activities of the two enzymes are similar. Since the content of mRNA was the same in WT and CPT-K5 cells, the mutant enzyme may have a lower metabolic stability than the parental enzyme (Tamura ef al., 1991). Top0 I was purified to apparent homogeneity from WT and CPT-K.5 cells according to the method of Ishii et al. (1983), and its sensitivity to CPT was measured. As shown in Fig. 1 , the activity of WT top0 I was inhibited at 1 p M or higher concentrations of CPT, whereas K5 top0 I was not inhibited by the drug at 125 p M , the resistance index being more than 125, indicating that the cellular resistance to the drug is primarily, if not entirely, due to the resistance of a structurally altered top0 I. This in turn establishes top0 I as the primary cellular target of CPT.
111. The Mutant Enzyme Possesses Higher Affinity for Recognition Sequences
Several types of cleavage sites have been identified, using CPT in mapping studies (Kjeldsen et al., 1988b; Gromova et al., 1990). These sites were classified into three categories according to their response to drug treatment. Class A sites include strong cleavage sites only slightly affected by CPT treatment. Class B sites are greatly enhanced in the presence of CPT. Thus, CPT altered the cleavage pattern of the WT, but not the mutant, enzyme. At class A sites the mutant enzyme cleaves the recognition
Toshiwo Andoh ond Kosuke Okoda
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RPM18402-Top0 I
CPT-K5 ToPo I
Fig. 1 Effect of CFT on the activity of top0 I from WT (lanes 1-5) and CPT-K5 cells (lanes 6-10). Enzymatic activity was assayed in the absence (lanes 1 and 6) and presence of CPT at 1.0 (lanes 2 and 7), 5.0 (lanes 3 and 8). 25.0 (lanes 4 and 9), and 125.0 pM (lanes 5 and lo), respectively. Lane 1 1 , Substrate colEl DNA only. Form I and form Ir represent supercoiled form I and relaxed form I DNA, respectively.
sequence with twofold higher efficiency than the WT enzyme and forms more stable complexes. The mutant enzyme does not seem to recognize and cleave class B sites. However, by the use of oligonucleotides with a class B recognition site, Gromova et al. (1993) demonstrated that K5 top0 I strongly interfered with the WT top0 I-mediated cleavage of class B site sequence which is otherwise strongly stimulated in the presence of CPT. Furthermore, the mutant enzyme catalyzed the strand transfer reaction via the class B site with higher efficiency regardless of the presence of CPT, and the equilibrium between cleavage and religation appears to be shifted and a higher rate of catalysis at class B recognition sites, explaining the apparent inability of the mutant enzyme to recognize the class B sites. These unique properties of the mutant top0 I may well account for the resistance to CPT.
IV. Determination of Mutation Sites of K5 Top0 I Responsible for CPT Resistance In order to determine the mutation(s) responsible for CPT resistance of K5-top0 I, we have attempted to determine the whole amino acid sequence of top0 I from WT and mutant cells by nucleotide sequencing of its cDNAs (Tamura et al., 1991). Amino acid sequence of human top0 I has been
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Drug Resistance Mechanisms
determined by D’Arpa et al. (1988). cDNA libraries of WT and CPT-K5 cells were constructed and screened with a 3.1-kb fragment of a human top0 I cDNA that was originally isolated from human brain stem cells. Several clones were isolated and their nucleotide sequences were determined. Comparison of the sequences revealed two nucleotide substitutions of A to G, changing Asp-533 (GAC) and Asp-583 (GAC) of the WT top0 I to Gly (GGC) in K5 top0 I (numbered according toD’Arpa et al., 1988) (see Fig. 2), Thus, unexpectedly, we have found mutations at two sites in the K5 top0 I. The next question is whether both mutations are necessary for the high degree of resistance or whether either one of these is sufficient for it. Comparison of the amino acid sequences of yeast and human top0 I shows that the regions where the mutations occurred are highly conserved. As shown in Fig. 3, it is of particular relevance to refer to the finding that the amino acid residues corresponding to position 533 of the WT top0 I are Asp in all species compared, including mouse top0 I, which shares 96% of amino acids in common with the human enzyme (Koiwai et al., 1993), with the exception of Gly in K5 top0 I, whereas residues corresponding to position 583 are variable (i.e., Asp in WT and human brain stem cell top0 I, and Gly in all other species compared). Taking into account that yeast, mouse, and some of human enzymes are of the Gly type at position 583 and are sensitive to CPT, one could argue that the amino acid variation at residue 583 among human enzymes is the result of a polymorphism of the human genome unrelated to CPT resistance, and the mutation from Asp to Gly at 533 is responsible for the resistance. To address this point, the enzyme with a single mutation was created by sitedirected mutagenesis and expression in Escherichia coli. AGCT
AGCT
WT
K5
Asp533
Fig. 2 Sequence difference between the WT and K5 top0 I cDNAs. Portions of an autoradiogram around the mutation site at amino acid residue 533 are shown, where GAC was changed to GGC. -
98
, NH2
S. pombe mouse
Toshiwo Andoh and Kosuke Okada Hydrophilic domain
Central conserved domain
A
765
tt
K5 Mutation
COOH
CPT Sensitivity
533 D-
583
G
S
G(GSC)
G (GPC)
R
G-
D
R
placenta human
in vitro
DG -G-
S
G
s
Fig. 3 Relationship between amino acid substitutions and CPT sensitivity of topo I from various eukaryotes: S. cereuisiae, Schizosaccharomyces pombe, mouse, and humans. CPT sensitivities of the products of in uitro rnutagenesis were also compared. Only amino acids at positions 533 and 583 were shown with sensitivity (S) or resistance (R) to CPT.
V. Site-Directed Mutagenesis of Top0 I and Expression in Escherichia coli In a previous communication we have shown that the C-terminal twothirds of top0 I (amino acid residues 163-765), when expressed as a fusion protein with protein A’, exhibited a relaxation activity, and that the activity of the fusion protein from K5 top0 I was resistant to CPT, indicating that the CPT-K5 cDNA for top0 I in fact encodes a functional mutant-type enzyme (Tamura et af., 1991). To confirm and verify the assumption above we have constructed a series of plasmids containing inserts of top0 I cDNA (amino acid residues 163-765) with a single mutation at either residue 533 or 583 by site-directed mutagenesis as a fusion protein with glutathione S-transferase. Fusion proteins of mutant top0 I were expressed in E. coli and were purified from the bacterial lysates as described earlier (Ishii et al., 1983).
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Drug Resisiance Mechanisms
As depicted in the bottom of Fig. 3, the relaxation activity of the fusion proteins of the parental type (-D-D-) and that with a single mutation at 583 (-D-G-, M2) was as sensitive to CPT as the native WT top0 I, whereas that of the fusion proteins with a single mutation at 533 (-G-D-, MI) and with double mutations at 533 and 583 (-G-G-, M3) was as resistant to CPT as the native K5 top0 I. These results verity the assumption above and establish that the single mutation changing Asp to Gly at 533 is responsible for CPT resistance of K5 top0 I and CPT-KS cells. One model for inhibition of top0 I by CPT is that the drug binds avidly to a specific site(s) or pocket(s) of the enzyme on formation of the cleavable complex with the recognition sequence of DNA, thereby stabilizing the complex and blocking the strand passage and/or religation step of the catalytic cycle of the enzyme (Hsiang et id.,1985; Liu, 1989) (Fig. 4). It is reasonable to assume that the amino acid residue at 533 is contained
5‘
3 DNA
3
5’
- It
Cleavable
~7) ‘ 7 Complex v
$3 a v WT - top0 I
z+
Stabilization
J
T -
K5 toPo I
Fig. 4 A model of catalysis by WT and K5 top0 I. As proposed by Hsiang et al. (1985). top0 I catalyzes topological changes of DNA through several steps: binding, cleavable complex formation, and religation. CPT interferes with the reaction by interacting with and stabilizing the cleavable complex. The mutation changing Asp-533 to Gly confers CPT resistance on the enzyme with a higher affinity for the recognition sequences.
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Toshiwo Andoh and Kosuke Okodo
in the pocket(s) or domain(s) involved in or affecting the interaction with CPT, and that the mutation changing Asp to Gly at this site confers CPT resistance on the enzyme by altering this drug binding domain(s) of the complex. This hypothesis is supported by the finding that K5 top0 I binds to the recognition sequences with higher affinity and with a higher rate of catalysis, as described above. Furthermore, the predictions of the secondary structure of the enzyme, that the region containing the mutation is protruding toward the outer surface of the protein and that the change of Asp to Gly makes the region retract inwardly (Tamura et al., 1991), and the finding that the region is within the most conserved region along the sequence of the enzyme (Lynn ef al., 1989), strongly suggest that this region plays an important role in the catalysis, especially in the latter half of the catalytic cycle (i.e., the strand passage and/or religation step). Thus, this constitutes the second functional site detected in addition to the hitherto described active-site Tyr-723. It is ofgreat relevance to refer to some other papers describing a mutation in the top0 I gene conferring CPT resistance (Kubota et al., 1992; Caron and Wang, 1993; Pommier et al., 1993; Levin et ai., 1993). All of the mutations were found in the central conserved region except the one located close to the active-site Tyr-723 in the conserved C-terminal domain of the enzyme, which showed, however, a smaller degree of resistance. Of great interest is the mutation topl-203 of Saccharomyces cereuisiae top0 I, which stimulates mitotic recombination due to an elevated intrinsic stability of the enzyme-mediated cleavable complex, mimicking the effect of CPT on WT top0 I (Levin et al., 1993). These results imply that the amino acid residues identified in the mutant enzymes are involved in the domain(s) interacting with CPT, playing an important role in the enzymatic catalysis. Further study of the structure-drug sensitivity relationship should define more precisely the presumptive domains(s) interacting with the drug, which we may term the “CPT pocket,” and participating in the catalysis of the enzyme.
VI. Conclusion We have described the establishment and characterization of CPTresistant cell lines in our group. We have obtained a definitive answer to the problem of how mammalian cells acquire CPT resistance (i.e., by mutation of the top0 I gene producing a CPT-resistant form of the enzyme in resistant cells). This is, in fact, one way of acquiring resistance to the drug. However, cells could become resistant by some other ways (e.g., by lowering the permeability of the drug or by lowering the content of
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top0 I within the cells). Some reports have described lowered top0 I content in CPT-resistant cells (Sugimoto et al., 1990a; Eng et al., 1990; Tanizawa and Pommier, 1992; Chang et al., 1992; Madelaine et al., 1993). Some others have described cell lines with reduced cellular accumulation of CPT or a CPT derivative (Chang et at., 1992; Takeda et al., 1992). The CPT-resistant cell lines characterized so far has been obtained by adaptation in in uitro culture or in serial passage in experimental animals. Further efforts obviously are needed in the characterization of CPTresistant cells which might prevail and develop in patients treated with CPT derivatives.
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Sugimoto, Y., Tsukahara, S., Oh-hara, T., Liu, L. F., and Tsuruo, T. (1990b). Elevated expression of DNA topoisomerase I1 in camptothecin-resistant human tumor cell lines, Cancer Res. 50, 7962-7965. Takeda, S ., Shimazoe, T., Sato, K . , Sugimoto, Y., Tsuruo, T., and Kono, A. (1992). Differential expression of DNA topoisomerase I gene between CFT-I 1 acquired- and native-resistant human pancreatic tumor cell lines: Detected by RNA/PCR-based quantitation assay. Biochem. Biophys. Res. Commun. 184, 618-625. Tanizawa, A., and Pommier, Y. (1992). Topoisomerase I alteration in a camptothecinresistant cell line derived from Chinese hamster DC3F cells in culture. Cancer Res. 52, 1849- 1854. Tsuruo, T., Matsuzaki, T., Matsushita, M., Sato, H., and Yokokura, T. (1988). Antitumor effect of CPT-I 1, a new derivative of camptothecin, against pleiotropic drug-resistant tumors in vitro and in vivo. Cancer Chemorher. Pharmacol. 21, 11-74.