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Review

Oncogene (2003) 22, 72967304. doi:10.1038/sj.onc.1206935

Mechanisms of resistance to topoisomerase I-targeting drugs

Zeshaan A Rasheed1 and Eric H Rubin1
1

The Cancer Institute of New Jersey, Department of Molecular and Cellular Pharmacology, UMDNJ-Robert Wood Johnson Medical School, 195 Little Albany Street, New Brunswick, NJ 08901, USA Correspondence: ZA Rasheed, E-mail: rasheeze@umdnj.edu Topof page

Abstract
DNA topoisomerases are a class of enzymes that alter the topology of DNA and are targets of several anticancer drugs. Camptothecins (CPTs) are a relatively new family of compounds that specifically target topoisomerase I (Top1). These compounds 'poison' Top1 by binding to the Top1DNA complex in a manner that prevents the religation of DNA. Topotecan and irinotecan are two CPTs that are approved for the treatment of a variety of malignancies, including colorectal, ovarian, and small cell lung cancers, as well as myeloid malignancies. Although CPTs have proven to be effective anticancer drugs, resistance is still a critical clinical problem. The mechanisms underlying de novo and acquired clinical resistance to CPTs and the newer classes of Top1 poisons are unclear. However, based on preclinical studies, it is likely that clinical resistance to these drugs is the result of: (1) inadequate accumulation of drug in the tumor, (2) resistance-conferring alterations in Top1, or (3) alterations in the cellular response to the Top1CPT interaction. This review will focus on the current knowledge regarding mechanisms of resistance to CPTs and other Top1-targeting drugs.
Keywords: topoisomerase I, camptothecin, topotecan, irinotecan, drug resistance Topof page

Introduction
DNA topoisomerases are a class of enzymes that alter the topology of DNA (Wang, 1996) and are found in all organisms, including archaebacteria, viruses, yeast, flies, and humans (Wang, 1996). The fundamental need for DNA topoisomerases in all cells is due to the double-helical structure of DNA (Wang, 1985). There are two general classes of topoisomerases, type I and type II. Briefly, type I topoisomerases cleave a single strand of DNA and change the linking number of DNA by one per cycle of activity. In contrast, type II topoisomerases cleave both strands of DNA and change the linking number of DNA by two (Wang, 1996). Both types of topoisomerases are present in most organisms (Wang, 1996). Escherichia coli contain type I (topoisomerases I and III) and type II (gyrase and topoisomerase IV) topoisomerases. Yeast also contain two type I topoisomerases, topoisomerase I (yTop1p) and topoisomerase III (yTop3p). However, yTop1p and yTop3p differ in that yTop1p forms a phosphodiester bond with the 3'phosphate group of the scissile DNA strand and yTop3p with the 5'-phosphate group. Thus, yTop1p is referred to as a type IB enzyme and yTop3p as a type IA enzyme. Yeast also contain a type II topoisomerase, topoisomerase II. Mammalian cells contain one type IB topoisomerase, topoisomerase I (Top1), and two type IA topoisomerases, topoisomerase III (Top3 ) and topoisomerase III (Top3 ). Additionally, mammalian cells contain two type II topoisomerases, topoisomerase II (Top2 ) and topoisomerase II (Top2 ). Mammalian Top1 is especially important in supporting replication fork movement during DNA replication and to relax supercoils generated during transcription (Wang, 1996). Additionally, yeast topoisomerase I yTop1p has been shown to be necessary for chromosome condensation during mitosis (Wang, 1996). Top2 is responsible for unlinking intertwined daughter duplexes during DNA replication and also contributes to DNA relaxation during transcription (Wang, 1996). In mammalian cells Top1, Top2 , and Top2 are essential, as disruption of any of these topoisomerase genes leads to lethality during embryogenesis or at birth (Champoux, 2001). In yeast however, TOP2 is essential, whereas, yeast TOP1knockouts are still viable (Champoux, 2001). Great interest in topoisomerases was generated when they were discovered to be targets for naturally occurring antimicrobial and anticancer drugs (Gellert, 1981;Liu, 1989; Bearden and Danziger, 2001). Among the antimicrobials are the fluoroquinolones, such as ciprofloxacin, which target bacterial DNA gyrase (Gellert, 1981). Human Top2 isozymes are targeted in cancer cells with the use of the anthracyclines, such as doxorubicin, and the epipodophylotoxins, such as etoposide (Liu, 1989). Additionally, a relatively new group of compounds, the camptothecins (CPTs), target Top1 and are promising new anticancer drugs (Liu, 1989). Although CPTs have proven to be effective, drug resistance is still a critical clinical problem. This review will focus on the current knowledge regarding mechanisms of resistance to CPTs and other Top1-targeting agents. Mechanisms of resistance are most easily discussed relative to the mechanism of CPT cytotoxicity. The mechanism of action of Top1 involves an initial noncovalent interaction with DNA. Top1 then cleaves a single strand of DNA and forms a covalent intermediate via a phosphodiester linkage between tyrosine-723 of Top1 and the 3'-phosphate group of the scissile strand of DNA. The intact DNA strand is then passed through the break and then Top1 religates the DNA and releases from the complex (Wang, 1996; Champoux, 2001). Drugs known as Top1 'poisons,' such as CPTs, bind to the covalent complex in a manner that prevents DNA religation. These persistent DNA breaks subsequently induce apoptosis, likely via collisions between these lesions and replication or transcription complexes (Liu, 1989). The parent compound of CPT is a naturally occurring alkaloid found in the Chinese plant Camptotheca accuminata. This compound was first identified in the 1960s in a

screen of plant extracts for antineoplastic drugs (Wall and Wani, 1995). Subsequently, many derivatives of the parent compound have been synthesized, and two have been approved by the US FDA for clinical use (topotecan and irinotecan) (Figure 1). Topotecan, a 10-hydroxyl modification of CPT, is approved for treatment of metastatic ovarian and small cell lung cancer, as well as myeloid malignancies. Irinotecan (CPT-11) is a prodrug, which is converted to the active compound SN-38 by plasma and cellular carboxylesterases, and is approved for use in the treatment of metastatic colon and rectal carcinomas. Approval for clinical use of both of these drugs was based on 30% response rates (Rothenberg et al., 1996; ten Bokkel Huinink et al., 1997). However, based on studies in mouse xenograft models, these response rates are disappointing (Giovanella et al., 1989; Houghton et al., 1995; Zamboni et al., 1998) and clinical responses to CPTs are typically transient. Additionally, other Top1 poisons (Figure 1) are being tested in clinical trials, including other CPT analogues [9-aminocamptothecin, 9nitrocamptothecin, exatecan mesylate (DX-8951f), ST1481, and karenitecin (BNP1350)], as well as structurally unique compounds including the indolocarbazole NB-506, protoberberines, intoplicine (a dual Top1 and Top2 inhibitor), idenoisoquinolones, and benzo-phenazines. The mechanisms underlying de novo and acquired clinical resistance to CPTs and the newer classes of Top1 poisons are unclear. However, based on preclinical studies, it is likely that clinical resistance to these drugs might be the result of (1) inadequate accumulation of drug in the tumor, (2) alterations in the target (Top1), or (3) alterations in the cellular response to the Top1CPT interaction. Figure 1.

Structures of the CPTs Full figure and legend (20K)

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Cellular accumulation and transport of CPTs

Cell culture data indicate that only brief exposures to submicromolar concentrations of CPT are required to target Top1 and to kill proliferating cancer cells (Goldwasser et al., 1995). Additionally, the lactone form of CPT is the active form of the drug (Hertzberg et al., 1989). Achieving high enough intracellular concentrations of the active form of CPT is dependent on cellular uptake, metabolism, and efflux mechanisms (Figure 2). Figure 2.

Processes involved in cellular uptake and efflux of CPT. Efflux is greater than uptake in this example, which is representative of a CPT-resistant cell Full figure and legend (59K)

Few studies have addressed mechanisms of cellular CPT uptake. Both active and passive transport mechanisms are implicated in intestinal cell uptake of CPT (Gupta et al., 2000). Additionally, ovarian cancer cells contain active transporters that are required for the influx of topotecan and SN-38 (Ma et al., 1998). In addition to uptake, cellular metabolism may be particularly important for the prodrug CPT-11, which is converted to its active form, SN-38, by cellular carboxylesterases (Danks et al., 1998; Ahmed et al., 1999; Humerickhouse et al., 2000; Khanna et al., 2000). Increased levels of cellular carboxylesterases correlate with increased cellular sensitivity to CPT-11 (Danks et al., 1998; Wierdlet al., 2001). SN-38 is also conjugated and detoxified by UDP-glucuronosyltransferase (UGT) to yield an SN-38-glucuronide (Ciotti et al., 1999). SN-38 glucuronidation is specifically catalysed by human liver UGT1A1, UGT1A3, UGT1A6, and UGT1A9 isoforms (Hanioka et al., 2001). Furthermore, glucuronidation of SN-38 is associated with increased efflux the drug from colon cancer cells (Cummings et al., 2002), and glucuronidation of CPTs has been associated with altered chemosensitivity of breast cancer and lung cancer cells (Takahashi et al., 1997;Brangi et al., 1999). Interestingly, indolocarbazole analogs are also glucuronidated in cells; however, it is still unclear whether this process plays a role in indolocarbazole-mediated cytotoxicity (Takenaga et al., 2002). Several groups have shown that ATP-binding cassette (ABC) proteins are involved in efflux and cellular resistance to CPTs in yeast and mammalian cells. In yeast, mutations in the ABC protein, Snq2, result in CPT resistance (Reid et al., 1997). Furthermore, in mammalian cells, MDR1 (P-glycoprotein, P-gp) overexpression confers resistance to CPT derivatives, albeit to a lesser degree than to other substrates of P-gp, such as the anthracyclines (Chen et al., 1991). Also, antisense oligonucleotides directed against the MRP2 gene can increase cellular sensitivity to CPT-11 and SN-38 (Koike et al., 1997). Recently, several groups have shown that cells selected for resistance to doxorubicin, mitoxantrone, or topotecan are crossresistant to SN-38 and 9-aminocamptothecin, but not CPT (Allen et al., 1999; Brangi et al., 1999;Maliepaard et al., 1999; Yang et al., 2000). The mechanism for multidrug resistance in these cells involves overexpression of the BCRP gene (also known as MXR or ABCP), an ATP-binding cassette half-transporter (Honjo et al., 2001;Kawabata et al., 2001). Additionally, a mutant form of BCRP (R482G/T) was preferentially selected for in human cells treated with anthracyclines (Allen et al., 2002). Interestingly, the mutant form of BCRP was also preferentially selected for in cells treated with the indolocarbazole NB-506 (Komatani et al., 2001). In both the studies, the mutant form of BCRP exhibits greater resistance to anthracyclines and relative lower resistance to topotecan compared to the wild-type protein (Komatani et al., 2001; Allen et al., 2002). Furthermore, cells that overexpress both the native and mutant R482T form of BCRP are more resistant to and accumulate less 9aminocamptothecin compared to a close analog, 9-nitrocamptothecin (Rajendra et al., 2002). Similarly, another study showed that a lipophilic 7-modified CPT analogue (ST1481) is not a substrate for BCRP (Perego et al., 2001). These results suggest that certain CPT analogs may be less susceptible to BCRP-mediated efflux. To date, there are relatively few published studies of BCRP gene expression in clinical samples. BCRP seems to be expressed at low levels in breast cancer cells as well as leukemic cells (Ross et al., 2000;Kanzaki et al., 2001). Furthermore, the expression of BCRP in breast carcinoma cells does not seem to correlate with response to doxorubicin-based chemotherapy (Kanzaki et al., 2001), nor is it upregulated in patients with breast cancer that were previously treated with anthracyclines versus those patients that were not treated with these drugs (Faneyte et al., 2002). Another recent study found that BCRP protein expression was increased in leukemia cells from three out of four patients following an infusion of topotecan and arabinoside-C (Gounder et al., 2003). However, more clinical

studies are needed in order to determine the role of BCRP overexpression and mutations in resistance to CPTs.
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Alterations in Top1 that confer resistance to CPTs

CPT causes DNA damage by stabilizing a normally transient covalent complex between Top1 and DNA (Hsiang et al., 1985). Furthermore, genetic studies in yeast identified Top1 as a unique cellular target for the CPTs (Eng et al., 1988;Nitiss and Wang, 1988; Bjornsti et al., 1989). With this in mind, it is not surprising that Top1 mutations conferring resistance to CPT have been identified in various mammalian and yeast cells, and, more recently, in tumor tissue from a patient treated with irinotecan (Tsurutani et al., 2002). Most of the point mutations can be found clustered in three regions of the protein, one of which is near the catalytic tyrosine at position 723 (Tamura et al., 1991; Kubota et al., 1992;Benedetti et al., 1993; Tanizawa et al., 1993; Rubin et al., 1994; Fujimori et al., 1995; Takatani et al., 1997; Wang et al., 1997; Urasaki et al., 2001b; Chang et al., 2002; Tsurutani et al., 2002) (Figure 3). Furthermore, the elucidation of the crystal structure of the Top1DNA covalent complex (Redinbo et al., 1998) as well as models of the Top1DNAdrug ternary complex (Fan et al., 1998; Redinbo et al., 1998; Kerrigan and Pilch, 2001; Stewart et al., 2001) has enabled structural mapping of these mutations (Figure 4). Models of the ternary complex implicate CPT in an intercalated position at the -1 and +1 base pairs of the cleavage site, with hydrogen bonding between the drug and both Top1 and DNA, stabilizing the ternary complex. Mutations in the regions between amino acids 361364, 533, and 722 explain resistance to CPT since these regions of Top1 are associated with the minor and major grooves of DNA and are in close proximity to the intercalated drug. Figure 3.

Schematic of resistance-conferring mutations of Top1. The catalytic Y723 residue is indicated as well as the three main clusters of mutations. All mutations confer CPT resistance. Boxed mutations confer indolocarbazole resistance as well as CPT resistance Full figure and legend (50K)

Figure 4.

Model of the Top1CPTDNA ternary complex. (Top) A portion of the refined structural model for the ternary 20(S)-CPTDNATop1 cleavable complex (view is looking down the axis of the DNA helix). Note that the drug is intercalated between the -1 and +1 base pairs of the DNA. The color-coding scheme for the DNA nucleotides is as follows: -1 thymine, orange; adenine complement to -1 thymine, green; +1 guanine, magenta; +2 guanine, cyan. The Top1 amino-acid residues are depicted in dark blue, while the drug molecule is depicted in standard CPK colors (carbon in gray, nitrogen in blue,

oxygen in red, and hydrogen in white). (Bottom) A schematic representation of the potential hydrogen bonding contacts between the bound 20(S)-CPT molecule and both the DNA and Top1. In this representation, the drug is depicted in cyan, the DNA functionalities are depicted in blue, and the Top1 amino-acid residues are depicted in red. The distance cutoff used in the determination of the hydrogen bonding contacts was 3.1 . Note that the dynamics of the 20-OH moiety are such that it is capable of forming hydrogen bonds with either the N3 atom of the +1 guanine or the furanose ring oxygen atom of the +2 guanine nucleotide. The figure was taken fromKerrigan and Pilch (2001) (reprinted with permission) Full figure and legend (144K)

Some Top1 point mutations, including Y723F and Y727F, which confer CPT resistance are also implicated in resistance to the indolocarbazole, rebeccamycin (Woo et al., 2002). Other CPT-resistant cell lines that express mutant Top1, including R364H, G503S, and N722S, are crossresistant to rebeccamycin, albeit to a lesser degree (Urasaki et al., 2001a). Additionally, the F361S Top1 mutant is catalytically crossresistant to a rebeccamycin analogue (Bailly et al., 1999). These results indicate that CPT and the indolocarbazoles may share binding sites in the Top1DNA complex. In contrast, some Top1 mutants that confer resistance to CPT retain sensitivity to rebeccamycin, including N726S/A (Woo et al., 2002). In addition to point mutations, a mutant Top1 containing an internal duplication of residues 20609 has been described in a murine cell line resistant to another Top1targeting drug, the indolocarbazole NB-506 (Komatani et al., 1999). Analyses of the recombinant form of this protein indicate that this alteration is sufficient to confer enzymatic resistance to NB-506 and CPT (Komatani et al., 1999), although the mechanistic basis for this alteration is unclear. Recent studies also indicate that interactions between Top1 and other proteins may affect cellular sensitivity to CPTs. The Top1-binding protein nucleolin may recruit Top1 to the nucleolus as a result of the high rate of transcription in this region (Bharti et al., 1996). Studies of a nucleolin orthologue in yeast, Nsr1p, indicate that the absence of this protein is associated with relocalization of Top1 from a predominantly nucleolar localization to a diffuse nuclear localization (Edwards et al., 2000). Furthermore, loss of Nsr1p results in resistance to CPT (Edwards et al., 2000). These data suggest that loss of the nucleolar localization of Top1 may result in a decrease in the overall amount of Top1 associated with DNA, and thus reduce the effects of CPT (Figure 5). Additionally, Top1 is known to move rapidly from the nucleolus to the nucleus or even cytoplasm after cellular exposure to CPT (Buckwalter et al., 1996; Danks et al., 1996). This relocalization has been associated with SUMO (small ubiquitin-like modifier) modification of Top1 (Mo et al., 2001) and may decrease Top1DNA interactions, and thus minimize Top1mediated DNA damage induced by CPT (see further discussion below). Notably, altered localization of topoisomerase II (Top2 ) (as a result of loss of nuclear localization sequences) was identified in mammalian cell lines resistant to the Top2-targeting drugs etoposide and mitoxantrone (Harker et al., 1995; Mirski and Cole, 1995; Wessel et al., 1997). In these cases, the mutant Top2 localizes to the cytoplasm, which presumably results in resistance to Top2-targeting drugs by decreasing interactions between the enzyme and DNA. Altered localization of Top2 has also been implicated in clinical resistance to Top2-targeting drugs (Valkov et al., 2000). Figure 5.

Model of the regulation of Top1 localization and its role in resistance to CPT. (a) Nucleolin (or Nsr1p in yeast) may normally serve to shuttle Top1 into the nucleolus. Top1 localization is predominantly nucleolar, therefore, the Nsr1p-mediated (or nucleolin-mediated) cytotoxicity of CPT may be largely due to 'poisoning' of nucleolar Top1, rather than nucleoplasmic Top1. (b). CPT treatment induces relocalization of Top1 and topors to a diffuse nuclear pattern. The Top1topors complex may induce ubiquitination and/or sumoylation of Top1 in response to Top1-mediated DNA damage, thereby affecting CPT cytotoxicity Full figure and legend (100K)

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Alterations in the cellular response to ternary complex formation

It is well established that Top1 is the specific target of CPT, and that CPT induces the formation of CPTTop1DNA ternary complexes (Hsiang et al., 1989). However, CPTinduced complexes are reversible and formation of these complexes is insufficient to explain the cytotoxic effects of CPT (Hsiang et al., 1989). CPT cytotoxicity is S-phase selective and can be ameliorated in cell culture by treatment with the DNA polymerase inhibitor, aphidocolin (D'Arpa et al., 1990). Furthermore, collision of replication forks with the ternary complex leads to double-stranded DNA breaks and is necessary for induction of cell death (Hsianget al., 1989). However, relatively little is known about the pathways downstream to CPTTop1DNA ternary complex formation that ultimately lead to repair of DNA damage or cell death. Studies in yeast and cell culture models have implicated several DNA replication, DNA damage checkpoint, and DNA repair proteins in response to cleavable complex formation (Table 1). Cellular exposure to CPT results in the activation of S-checkpoint proteins, such as Chk1 (Wan et al., 1999), ATR (Cliby et al., 2002), ATM (Wang et al., 2002), as well as the DNA-dependent protein kinase (DNA-PK) multimer (Shao et al., 1999; Wang et al., 2002). Furthermore, loss of function of Chk1 or ATR is associated with increased cellular sensitivity to CPT (Wan et al., 1999; Cliby et al., 2002). Additionally, loss of RAD9, a checkpoint protein that is activated by DNA damage and induces G2 arrest, was shown to enhance Top1-induced cell death (Megonigal et al., 1997; Simon et al., 2000). In addition to checkpoint proteins, proteins involved in DNA replication are also involved in CPT cytotoxicity. A yeast screen for conditional mutants with enhanced sensitivity to Top1-mediated DNA damage led to the identification of the yeast replication proteins, Dpb11p and Cdc45p, as important determinants of CPT sensitivity (Reidet al., 1999). Dpb11p and Cdc45p are implicated in DNA polymerase switching from priming to processive replication (Reid et al., 1999). Also, studies in murine cells implicate the loss of the Werner syndrome protein in CPT hypersensitivity (Lebel and Leder, 1998). The Werner protein is a helicase that interacts with Top1 and copurifies with the DNA replication complex (Lebel et al., 1999; Lebel, 2001). Table 1 - Genes implicated in the cellular response to CPT-induced DNA damage.

Full table

With regard to repair of CPT-induced DNA damage, both mismatch repair and base excision repair systems are implicated (Table 1). Cells lacking the mismatch repair protein, MSH2, are hypersensitive to CPT (Pichierri et al., 2001). Recently, Meijeret al. (2002) showed that a eucaryotic polynucleotide kinase, Pnk1, also plays a role in CPT-induced DNA damage repair, and cells lacking this gene are hypersensitive to CPT. Additionally, Nash and colleagues identified a tyrosine-DNA phosphodiesterase (TDP) that specifically cleaves Top1 that is covalently linked to DNA (Pouliot et al., 1999). Studies in yeast indicate that loss of TDP in the presence of mutant RAD9 confers hypersensitivity to CPT (Pouliot et al., 1999). Importantly, while most of these studies indicate that loss of function of a DNA repair protein enhances cellular sensitivity, to date only a single study demonstrated that overexpression of a DNA repair protein confers CPT hypersensitivity. Park et al. (2002) have shown that overexpression of a protein involved in base excision repair, X-ray repair cross-complementing gene I protein (XRCC), leads to CPT resistance in cells. Cellular processes downstream from induction and repair of DNA damage may also be important in the resistance to CPT. For example, the cytotoxicity of CPT may be related to the induction of apoptotic pathways in cells as well as other cell death pathways (Nieves-Neira and Pommier, 1999). Studies have shown that proapoptotic proteins, such as p53 and Bax, are upregulated following CPT treatment, while bcl-2 expression is decreased (Zhang and Xu, 2000). Additionally, proteasome inhibition-mediated stabilization of NF- B is associated with enhanced CPT cytotoxicity (Cusack et al., 2001). Furthermore, CPT treatment of cells leads to the activation of caspases and cleavage of Top1, a substrate for caspase-3 (Samejima et al., 1999; Zhang and Xu, 2000). Overexpression of bcl-2 has been associated with relative resistance to CPT (Walton et al., 1993; Nieves-Neira and Pommier, 1999). Furthermore, a multidrug-resistant lung cancer cell line that was selected for resistance to CPT was found to overexpress bcl-2 and p21Waf1/Cip1 (Zhang et al., 1999). Taken together, these data indicate that proapoptotic and antiapoptotic proteins may regulate the cellular response to CPT. Recently, post-translational modifications of Top1 were reported after CPT treatment of cells and may be involved in resistance. Top1 is ubiquitinated and degraded after cells are treated with CPT, which appears to occur in the context of the ternary complex rather than free Top1, since nuclease treatment of cell lysates is necessary to visualize Top1ubiquitin conjugates by Western blotting (Desai et al., 1997). Recently, tumor cells deficient in CPT-induced Top1 downregulation were found to be more sensitive to CPT, implicating ubiquitination of Top1 as an important determinant of cellular sensitivity (Desai et al., 2001). Additionally, CPT-induced Top1DNA covalent complex formation results in transcriptional arrest and 26S proteasome-mediated degradation of Top1 and the large subunit of RNA polymerase II (Desai et al., 2002). Degradation of the transcriptional machinery then initiates transcription-coupled repair (Desai et al., 2002). Furthermore, recovery from transcriptional arrest depends on degradation of Top1 and functional transcription-coupled repair, affecting cellular sensitivity to CPT (Desai et al., 2002). In another study, Cusack et al. (2001) showed that pretreatment of cells with PS341 (a dipeptide proteasome inhibitor) enhanced SN-38-mediated cellular cytotoxicity. In yeast, two proteins related to the ubiquitination pathway were discovered using genetic screens for mutants that alter CPT sensitivity. Overexpression of a ubiquitin-

specific protease, Ubp11, confers resistance to Top1-mediated DNA damage (Byrne and Bjornsti, 2001) and loss of DOA4, a 26S proteasome-associated C-terminal ubiquitin hydrolase, sensitizes cells to Top1-mediated DNA damage (Fiorani et al., 2001). Together, these studies provide compelling evidence that ubiquitin/proteasome pathways are important in cellular sensitivity to CPTs. Top1 is also modified by SUMO following CPT treatment (Mao et al., 2000). Although SUMO is similar to ubiquitin in structure, SUMO does not target proteins for degradation, but is implicated in control of cellular localization (Muller et al., 2001). Indeed, sumoylation of Top1 is associated with relocalization of the protein from the nucleolus to a more diffuse nuclear pattern following CPT treatment (Moet al., 2001), whereas, Top1 mutants that cannot be sumoylated remain more concentrated in nucleoli of cells even after CPT treatment (Rallabhandi et al., 2002). Together these studies strongly suggest that sumoylation regulates Top1 localization in the nucleus, and that sumoylation of Top1 may function to decrease Top1DNA interactions and thus minimize Top1-mediated DNA damage induced by CPT. Although the mechanisms related to CPT-induced Top1 ubiquitination and sumoylation are currently unknown, recent studies of a Top1-binding protein, named topors, suggests that this protein may be involved (Haluska et al., 1999). Topors is a RING protein that functions in vitro as an E3 ubiquitin ligase (Rasheed et al., 2000) and as an E3-type SUMO ligase (Rasheed et al., 2002a). Moreover, topors sumoylates Top1 in vitro (Rasheed et al., 2002a). In cells, topors is associated with PML nuclear bodies and rapidly disperses to a diffuse nuclear pattern upon cellular treatment with CPT, similar to Top1 (Rasheedet al., 2002b). It is possible that topors play a role in the cellular response to CPT, and regulates the function of Top1 following CPT-induced DNA damage (Figure 5).
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Resistance to CPT in the clinical setting

Studies of clinical specimens are needed to determine whether the resistance mechanisms detected in yeast and cell culture models are clinically relevant. Cellular metabolism, via carboxylesterases and UGTs, plays an important role in the cytotoxicity of CPT-11 in cell culture models. Little is known regarding the clinical relevance of this finding, although varied carboxylesterase activity has been reported in clinical specimens (Ahmed et al., 1999; Khanna et al., 2000). BCRP seems to be expressed at low levels in breast cancer cells as well as leukemic cells (Ross et al., 2000; Kanzaki et al., 2001). Recently, BCRP protein expression was found to increase in leukemia cells following infusion of topotecan and arabinoside-C in patients (Gounder et al., 2003). To date, altered expression of BCRP in clinical samples has not been correlated with altered CPT sensitivity or treatment. There are a limited number of clinical studies that have analysed clinical specimens (tumor tissue or surrogates) for mutations in Top1 and most have yielded negative results (Ohashi et al., 1996; Takatani et al., 1997). Recently,Tsurutani et al. (2002) reported a Top1 mutation in a tumor specimen from a patient with large cell carcinoma of the lung. The mutation results in two changes, a stop codon at position 736 and a glycine to serine missense mutation at codon 737. Interestingly, the patient with this mutation did not respond to a chemotherapy regimen consisting of cisplatin and irinotecan (Tsurutani et al., 2002). However, it remains to be determined if these mutations result in enzymatic resistance to CPT. Other studies utilizing clinical specimens found alterations in Top1 and Top2 levels following treatment with CPT. Analyses in clinical study of 11 patients with nonhematologic malignancies treated with oral CPT for 14 days showed decreases in Top1 protein levels in nonmalignant peripheral blood mononuclear cells (PBMNCs) that were not due to cleavable complex formation, suggesting that Top1 is degraded following CPT exposure in nonmalignant cells (Gupta et al., 1998). Analyses in a similar

clinical study of nonmalignant PBMNCs in patients treated with a 72 h infusion of 9aminocamptothecin (9AC) also indicated decreases in Top1 protein levels at 48 or 72 h in two out of three patients (Rubin et al., 1995). In contrast, two out of four patients with leukemia who were treated with a 72 h infusion of 9AC showed no change in Top1 protein levels in their malignant blast cells during the infusion (Saleem et al., 2000), suggesting that if Top1 degradation occurs in these cells, the timing is distinct from that of nonmalignant PBMNCs. It is possible that the apparent difference in 9AC-induced Top1 degradation may relate to alterations in ubiquitinproteasome pathways in malignant versus nonmalignant cells. These findings are consistent with the observation that malignant and nonmalignant cultured cells differ in their capacity to degrade Top1 (Desai et al., 2001). Interestingly, topors protein and mRNA expression are decreased in tumor tissues versus matched normal tissues, suggesting that topors may be involved in the apparent differences in CPT-induced Top1 degradation in these tissues (Saleem et al., 2002). Other mechanisms of CPT resistance that have been identified in yeast and cell culture models need to be evaluated clinically, including the role of Top1 localization and specific repair processes. Pharmacogenetic and biochemical understanding of clinical CPT resistance will improve the use of CPTs in the treatment of malignancy.
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We thank Drs Leroy F Liu, Daniel Pilch, and Ahamed Saleem for helpful discussions in writing this review.

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