Mechanisms of Resistance to Topoisomerase I-Targeting Drugs

Mechanisms of resistance to topoisomerase I-targeting drugs

Zeshaan A Rasheed1 and Eric H Rubin*,1

1The 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

DNA topoisomerases are a class of enzymes that alter the 1996). Escherichia coli contain type I (topoisomerases I topology of DNA and are targets of several anticancer and III) and type II (gyrase and topoisomerase IV) drugs. Camptothecins (CPTs)are a relatively new family topoisomerases. Yeast also contain two type I topoi- of compounds that specifically target topoisomerase I somerases, topoisomerase I (yTop1p) and topoisome- (Top1). These compounds ‘poison’ Top1 by binding to the rase III (yTop3p). However, yTop1p and yTop3p differ Top1–DNA complex in a manner that prevents the in that yTop1p forms a phosphodiester bond with the 30- religation of DNA. Topotecan and irinotecan are two phosphate group of the scissile DNA strand and yTop3p CPTs that are approved for the treatment of a variety of with the 50-phosphate group. Thus, yTop1p is referred malignancies, including colorectal, ovarian, and small cell to as a type IB enzyme and yTop3p as a type IA enzyme. lung cancers, as well as myeloid malignancies. Although Yeast also contain a type II topoisomerase, topoisome- CPTs have proven to be effective anticancer drugs, rase II. Mammalian cells contain one type IB topoi- resistance is still a critical clinical problem. The mechan- somerase, topoisomerase I (Top1), and two type IA isms underlying de novo and acquired clinical resistance to topoisomerases, topoisomerase IIIa (Top3a) and topoi- CPTs and the newer classes of Top1 poisons are unclear. somerase IIIb (Top3b). Additionally, mammalian cells However, based on preclinical studies, it is likely that contain two type II topoisomerases, topoisomerase IIa clinical resistance to these drugs is the result of: (1) (Top2a) and topoisomerase IIb (Top2b). inadequate accumulation of drug in the tumor, (2) Mammalian Top1 is especially important in support- resistance-conferring alterations in Top1, or (3)altera- ing replication fork movement during DNA replication tions in the cellular response to the Top1–CPT interac- and to relax supercoils generated during transcription tion. This review will focus on the current knowledge (Wang, 1996). Additionally, yeast topoisomerase I regarding mechanisms of resistance to CPTs and other yTop1p has been shown to be necessary for chromo- Top1-targeting drugs. some condensation during mitosis (Wang, 1996). Top2a Oncogene (2003) 22, 7296–7304. doi:10.1038/sj.onc.1206935 is responsible for unlinking intertwined daughter du- plexes during DNA replication and also contributes to Keywords: topoisomerase I; camptothecin; topotecan; DNA relaxation during transcription (Wang, 1996). In irinotecan; drug resistance mammalian cells Top1, Top2a, and Top2b 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 TOP1 knockouts are still viable Introduction (Champoux, 2001). Great interest in topoisomerases was generated when DNA topoisomerases are a class of enzymes that alter they were discovered to be targets for naturally occurring the topology of DNA (Wang, 1996) and are found in all antimicrobial and anticancer drugs (Gellert, 1981; Liu, organisms, including archaebacteria, viruses, yeast, flies, 1989; Bearden and Danziger, 2001). Among the anti- and humans (Wang, 1996). The fundamental need for microbials are the fluoroquinolones, such as ciproflox- DNA topoisomerases in all cells is due to the double- acin, which target bacterial DNA gyrase (Gellert, helical structure of DNA (Wang, 1985). There are two 1981). Human Top2 isozymes are targeted in cancer general classes of topoisomerases, type I and type II. cells with the use of the anthracyclines, such as Briefly, type I topoisomerases cleave a single strand of doxorubicin, and the epipodophylotoxins, such as DNA and change the linking number of DNA by one etoposide (Liu, 1989). Additionally, a relatively new per cycle of activity. In contrast, type II topoisomerases group of compounds, the camptothecins (CPTs), target cleave both strands of DNA and change the linking Top1 and are promising new anticancer drugs (Liu, number of DNA by two (Wang, 1996). Both types of 1989). Although CPTs have proven to be effective, drug topoisomerases are present in most organisms (Wang, resistance is still a critical clinical problem. This review will focus on the current knowledge regarding mechanisms of resistance to CPTs and other Top1- *Correspondence:ZA Rasheed; E-mail:[email protected] targeting agents. Resistance to topoisomerase I-targeting drugs ZA Rasheed and EH Rubin 7297 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 30-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 colli- sions 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 synthe- sized, 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 B30% 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, 9-nitrocamptothecin, exatecan mesylate (DX- 8951f), ST1481, and karenitecin (BNP1350)], as well as Figure 1 Structures of the CPTs structurally unique compounds including the indolocarbazole NB-506, protoberberines, intoplicine (a dual Top1 and Top2 inhibitor), idenoisoquinolones, and form of CPT is the active form of the drug (Hertzberg benzo-phenazines. The mechanisms underlying de novo et al., 1989). Achieving high enough intracellular and acquired clinical resistance to CPTs and the newer concentrations of the active form of CPT is dependent classes of Top1 poisons are unclear. However, based on on cellular uptake, metabolism, and efflux mechanisms preclinical studies, it is likely that clinical resistance to (Figure 2). these drugs might be the result of (1) inadequate Few studies have addressed mechanisms of cellular accumulation of drug in the tumor, (2) alterations in CPT uptake. Both active and passive transport mechan- the target (Top1), or (3) alterations in the cellular isms are implicated in intestinal cell uptake of CPT response to the Top1–CPT interaction. (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 Cellular accumulation and transport of CPTs addition to uptake, cellular metabolism may be parti- cularly important for the prodrug CPT-11, which is Cell culture data indicate that only brief exposures to converted to its active form, SN-38, by cellular submicromolar concentrations of CPT are required to carboxylesterases (Danks et al., 1998; Ahmed et al., target Top1 and to kill proliferating cancer cells 1999; Humerickhouse et al., 2000; Khanna et al., 2000). (Goldwasser et al., 1995). Additionally, the lactone Increased levels of cellular carboxylesterases correlate

Oncogene Resistance to topoisomerase I-targeting drugs ZA Rasheed and EH Rubin 7298 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 9- aminocamptothecin 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 Figure 2 Processes involved in cellular uptake and efflux of CPT. efflux. To date, there are relatively few published studies Efflux is greater than uptake in this example, which is representa- of BCRP gene expression in clinical samples. BCRP tive of a CPT-resistant cell 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 with increased cellular sensitivity to CPT-11 (Danks carcinoma cells does not seem to correlate with response et al., 1998; Wierdl et al., 2001). to doxorubicin-based chemotherapy (Kanzaki et al., SN-38 is also conjugated and detoxified by UDP- 2001), nor is it upregulated in patients with breast cancer glucuronosyltransferase (UGT) to yield an SN-38- that were previously treated with anthracyclines versus glucuronide (Ciotti et al., 1999). SN-38 glucuronidation those patients that were not treated with these drugs is specifically catalysed by human liver UGT1A1, (Faneyte et al., 2002). Another recent study found that UGT1A3, UGT1A6, and UGT1A9 isoforms (Hanioka BCRP protein expression was increased in leukemia et al., 2001). Furthermore, glucuronidation of SN-38 is cells from three out of four patients following an associated with increased efflux the drug from colon infusion of topotecan and arabinoside-C (Gounder cancer cells (Cummings et al., 2002), and glucuronida- et al., 2003). However, more clinical studies are needed tion of CPTs has been associated with altered chemo- in order to determine the role of BCRP overexpression sensitivity of breast cancer and lung cancer cells and mutations in resistance to CPTs. (Takahashi et al., 1997; Brangi et al., 1999). Interest- ingly, indolocarbazole analogs are also glucuronidated in cells; however, it is still unclear whether this process Alterations in Top1 that confer resistance to CPTs plays a role in indolocarbazole-mediated cytotoxicity (Takenaga et al., 2002). CPT causes DNA damage by stabilizing a normally Several groups have shown that ATP-binding cassette transient covalent complex between Top1 and DNA (ABC) proteins are involved in efflux and cellular (Hsiang et al., 1985). Furthermore, genetic studies in resistance to CPTs in yeast and mammalian cells. In yeast identified Top1 as a unique cellular target for the yeast, mutations in the ABC protein, Snq2, result CPTs (Eng et al., 1988; Nitiss and Wang, 1988; Bjornsti in CPT resistance (Reid et al., 1997). Furthermore, in et al., 1989). With this in mind, it is not surprising that mammalian cells, MDR1 (P-glycoprotein, P-gp) over- Top1 mutations conferring resistance to CPT have been expression confers resistance to CPT derivatives, albeit identified in various mammalian and yeast cells, and, to a lesser degree than to other substrates of P-gp, such more recently, in tumor tissue from a patient treated as the anthracyclines (Chen et al., 1991). Also, antisense with irinotecan (Tsurutani et al., 2002). Most of the oligonucleotides directed against the MRP2 gene can point mutations can be found clustered in three regions increase cellular sensitivity to CPT-11 and SN-38 (Koike of the protein, one of which is near the catalytic tyrosine et al., 1997). at position 723 (Tamura et al., 1991; Kubota et al., 1992; Recently, several groups have shown that cells Benedetti et al., 1993; Tanizawa et al., 1993; Rubin et al., selected for resistance to doxorubicin, mitoxantrone, 1994; Fujimori et al., 1995; Takatani et al., 1997; Wang or topotecan are crossresistant to SN-38 and 9- et al., 1997; Urasaki et al., 2001b; Chang et al., 2002; aminocamptothecin, but not CPT (Allen et al., 1999; Tsurutani et al., 2002) (Figure 3). Furthermore, the Brangi et al., 1999; Maliepaard et al., 1999; Yang et al., elucidation of the crystal structure of the Top1–DNA 2000). The mechanism for multidrug resistance in these covalent complex (Redinbo et al., 1998) as well as cells involves overexpression of the BCRP gene (also models of the Top1–DNA–drug ternary complex (Fan known as MXR or ABCP), an ATP-binding cassette et al., 1998; Redinbo et al., 1998; Kerrigan and Pilch, half-transporter (Honjo et al., 2001; Kawabata et al., 2001; Stewart et al., 2001) has enabled structural 2001). Additionally, a mutant form of BCRP (R482G/ mapping of these mutations (Figure 4). Models of the T) was preferentially selected for in human cells treated ternary complex implicate CPT in an intercalated with anthracyclines (Allen et al., 2002). Interestingly, the position at the À1 and þ 1 base pairs of the cleavage mutant form of BCRP was also preferentially selected site, with hydrogen bonding between the drug and both for in cells treated with the indolocarbazole NB-506 Top1 and DNA, stabilizing the ternary complex. (Komatani et al., 2001). In both the studies, the mutant Mutations in the regions between amino acids 361– form of BCRP exhibits greater resistance to anthracy- 364, 533, and 722 explain resistance to CPT since these clines and relative lower resistance to topotecan regions of Top1 are associated with the minor and major

Oncogene Resistance to topoisomerase I-targeting drugs ZA Rasheed and EH Rubin 7299

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 grooves of DNA and are in close proximity to the intercalated drug. 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 Top1–DNA 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 20–609 has been described in a murine cell line resistant to another Top1-targeting drug, the indolocarbazole NB- Figure 4 Model of the Top1–CPT–DNA ternary complex. (Top) 506 (Komatani et al., 1999). Analyses of the recombi- A portion of the refined structural model for the ternary 20(S)- nant form of this protein indicate that this alteration is CPT–DNA–Top1 cleavable complex (view is looking down the axis sufficient to confer enzymatic resistance to NB-506 and of the DNA helix). Note that the drug is intercalated between the et al À1 and þ 1 base pairs of the DNA. The color-coding scheme for CPT (Komatani ., 1999), although the mechanistic the DNA nucleotides is as follows: À1 thymine, orange; adenine basis for this alteration is unclear. complement to À1 thymine, green; þ 1 guanine, magenta; þ 2 Recent studies also indicate that interactions between guanine, cyan. The Top1 amino-acid residues are depicted in dark Top1 and other proteins may affect cellular sensitivity to blue, while the drug molecule is depicted in standard CPK colors CPTs. The Top1-binding protein nucleolin may recruit (carbon in gray, nitrogen in blue, oxygen in red, and hydrogen in white). (Bottom) A schematic representation of the potential Top1 to the nucleolus as a result of the high rate of hydrogen bonding contacts between the bound 20(S)-CPT transcription in this region (Bharti et al., 1996). Studies molecule and both the DNA and Top1. In this representation, of a nucleolin orthologue in yeast, Nsr1p, indicate that the drug is depicted in cyan, the DNA functionalities are depicted the absence of this protein is associated with relocaliza- in blue, and the Top1 amino-acid residues are depicted in red. The distance cutoff used in the determination of the hydrogen bonding tion of Top1 from a predominantly nucleolar localiza- contacts was 3.1 A˚ . Note that the dynamics of the 20-OH moiety tion to a diffuse nuclear localization (Edwards et al., are such that it is capable of forming hydrogen bonds with either 2000). Furthermore, loss of Nsr1p results in resistance the N3 atom of the þ 1 guanine or the furanose ring oxygen atom to CPT (Edwards et al., 2000). These data suggest that of the þ 2 guanine nucleotide. The figure was taken from Kerrigan loss of the nucleolar localization of Top1 may result in a and Pilch (2001) (reprinted with permission) decrease in the overall amount of Top1 associated with DNA, and thus reduce the effects of CPT (Figure 5). Notably, altered localization of topoisomerase IIa Additionally, Top1 is known to move rapidly from the (Top2a) (as a result of loss of nuclear localization nucleolus to the nucleus or even cytoplasm after cellular sequences) was identified in mammalian cell lines exposure to CPT (Buckwalter et al., 1996; Danks et al., resistant to the Top2-targeting drugs etoposide and 1996). This relocalization has been associated with mitoxantrone (Harker et al., 1995; Mirski and Cole, SUMO (small ubiquitin-like modifier) modification of 1995; Wessel et al., 1997). In these cases, the mutant Top1 (Mo et al., 2001) and may decrease Top1–DNA Top2a localizes to the cytoplasm, which presumably interactions, and thus minimize Top1-mediated DNA results in resistance to Top2-targeting drugs by decreas- damage induced by CPT (see further discussion below). ing interactions between the enzyme and DNA. Altered

Oncogene Resistance to topoisomerase I-targeting drugs ZA Rasheed and EH Rubin 7300 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 (Mego- nigal 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 (Reid et 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 Figure 5 Model of the regulation of Top1 localization and its role copurifies with the DNA replication complex (Lebel in resistance to CPT. (a) Nucleolin (or Nsr1p in yeast) may et al., 1999; Lebel, 2001). normally serve to shuttle Top1 into the nucleolus. Top1 localiza- With regard to repair of CPT-induced DNA damage, tion is predominantly nucleolar, therefore, the Nsr1p-mediated (or both mismatch repair and base excision repair systems nucleolin-mediated) cytotoxicity of CPT may be largely due to are implicated (Table 1). Cells lacking the mismatch ‘poisoning’ of nucleolar Top1, rather than nucleoplasmic Top1. (b). CPT treatment induces relocalization of Top1 and topors to a repair protein, MSH2, are hypersensitive to CPT diffuse nuclear pattern. The Top1–topors complex may induce (Pichierri et al., 2001). Recently, Meijer et al. (2002) ubiquitination and/or sumoylation of Top1 in response to Top1- showed that a eucaryotic polynucleotide kinase, Pnk1, mediated DNA damage, thereby affecting CPT cytotoxicity also plays a role in CPT-induced DNA damage repair, and cells lacking this gene are hypersensitive to CPT. localization of Top2a has also been implicated in clinical Additionally, Nash and colleagues identified a tyrosine- resistance to Top2-targeting drugs (Valkov et al., 2000). 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 Alterations in the cellular response to ternary complex CPT (Pouliot et al., 1999). Importantly, while most of formation these studies indicate that loss of function of a DNA repair protein enhances cellular sensitivity, to date only It is well established that Top1 is the specific target of a single study demonstrated that overexpression of a CPT, and that CPT induces the formation of CPT– DNA repair protein confers CPT hypersensitivity. Park Top1–DNA ternary complexes (Hsiang et al., 1989). et al. (2002) have shown that overexpression of a protein However, CPT-induced complexes are reversible and involved in base excision repair, X-ray repair cross- formation of these complexes is insufficient to explain complementing gene I protein (XRCC), leads to CPT the cytotoxic effects of CPT (Hsiang et al., 1989). CPT resistance in cells. cytotoxicity is S-phase selective and can be ameliorated Cellular processes downstream from induction and in cell culture by treatment with the DNA polymerase repair of DNA damage may also be important in the inhibitor, aphidocolin (D’Arpa et al., 1990). Further- resistance to CPT. For example, the cytotoxicity of CPT more, collision of replication forks with the ternary may be related to the induction of apoptotic pathways in complex leads to double-stranded DNA breaks and is cells as well as other cell death pathways (Nieves-Neira necessary for induction of cell death (Hsiang et al., and Pommier, 1999). Studies have shown that proa- 1989). However, relatively little is known about the poptotic proteins, such as p53 and Bax, are upregulated pathways downstream to CPT–Top1–DNA ternary following CPT treatment, while bcl-2 expression is complex formation that ultimately lead to repair of decreased (Zhang and Xu, 2000). Additionally, protea- DNA damage or cell death. some inhibition-mediated stabilization of NF-kBis Studies in yeast and cell culture models have associated with enhanced CPT cytotoxicity (Cusack implicated several DNA replication, DNA damage et al., 2001). Furthermore, CPT treatment of cells leads checkpoint, and DNA repair proteins in response to to the activation of caspases and cleavage of Top1, a cleavable complex formation (Table 1). Cellular expo- substrate for caspase-3 (Samejima et al., 1999; Zhang sure to CPT results in the activation of S-checkpoint and Xu, 2000). Overexpression of bcl-2 has been

Oncogene Resistance to topoisomerase I-targeting drugs ZA Rasheed and EH Rubin 7301 Table 1 Genes implicated in the cellular response to CPT-induced DNA damage Process Mutated gene Reference

Checkpoint Wang et al. (2002) ATR Cliby et al. (2002) CHK1 Wan et al. (1999) RAD9, RAD17 Megonigal et al. (1997), Simon et al. (2000)

DNA repair CSA, CSB Squires et al. (1993) RAD6 Simon et al. (2000) TDP Pouliot et al. (1999) TRF4 Walowsky et al. (1999) MSM2. MSM3 Pandit et al. (2001), Pichierri et al. (2001) XRCC Park et al. (2002) PNK1 Meijer et al. (2002) DNA replication CDC45, DPB11 WRN Reid et al. (1999) Lebel and Leder (1998) Ubiquitination/sumoylation UBP11 Byrne and Bjornsti (2001) DOA4 Fiorani et al. (2001) 26S proteasome, ubiquitin Desai et al. (2001) UBC9, SUMO Mao et al. (2000), Van Wardenburg et al. (2002) associated with relative resistance to CPT (Walton et al., et al., 2001). Together, these studies provide compelling 1993; Nieves-Neira and Pommier, 1999). Furthermore, a evidence that ubiquitin/proteasome pathways are im- multidrug-resistant lung cancer cell line that was portant in cellular sensitivity to CPTs. selected for resistance to CPT was found to overexpress Top1 is also modified by SUMO following CPT bcl-2 and p21Waf1/Cip1 (Zhang et al., 1999). Taken treatment (Mao et al., 2000). Although SUMO is similar together, these data indicate that proapoptotic and to ubiquitin in structure, SUMO does not target antiapoptotic proteins may regulate the cellular re- proteins for degradation, but is implicated in control sponse to CPT. of cellular localization (Muller et al., 2001). Indeed, Recently, post-translational modifications of Top1 sumoylation of Top1 is associated with relocalization of were reported after CPT treatment of cells and may be the protein from the nucleolus to a more diffuse nuclear involved in resistance. Top1 is ubiquitinated and pattern following CPT treatment (Mo et al., 2001), degraded after cells are treated with CPT, which appears whereas, Top1 mutants that cannot be sumoylated to occur in the context of the ternary complex rather remain more concentrated in nucleoli of cells even after than free Top1, since nuclease treatment of cell lysates is CPT treatment (Rallabhandi et al., 2002). Together necessary to visualize Top1–ubiquitin conjugates by these studies strongly suggest that sumoylation regulates Western blotting (Desai et al., 1997). Recently, tumor Top1 localization in the nucleus, and that sumoylation cells deficient in CPT-induced Top1 downregulation of Top1 may function to decrease Top1–DNA interac- were found to be more sensitive to CPT, implicating tions and thus minimize Top1-mediated DNA damage ubiquitination of Top1 as an important determinant of induced by CPT. Although the mechanisms related to cellular sensitivity (Desai et al., 2001). Additionally, CPT-induced Top1 ubiquitination and sumoylation are CPT-induced Top1–DNA covalent complex formation currently unknown, recent studies of a Top1-binding results in transcriptional arrest and 26S proteasome- protein, named topors, suggests that this protein may be mediated degradation of Top1 and the large subunit of involved (Haluska et al., 1999). Topors is a RING RNA polymerase II (Desai et al., 2002). Degradation of protein that functions in vitro as an E3 ubiquitin ligase the transcriptional machinery then initiates transcrip- (Rasheed et al., 2000) and as an E3-type SUMO ligase tion-coupled repair (Desai et al., 2002). Furthermore, (Rasheed et al., 2002a). Moreover, topors sumoylates recovery from transcriptional arrest depends on degra- Top1 in vitro (Rasheed et al., 2002a). In cells, topors is dation of Top1 and functional transcription-coupled associated with PML nuclear bodies and rapidly repair, affecting cellular sensitivity to CPT (Desai et al., disperses to a diffuse nuclear pattern upon cellular 2002). In another study, Cusack et al. (2001) showed treatment with CPT, similar to Top1 (Rasheed et al., that pretreatment of cells with PS-341 (a dipeptide 2002b). It is possible that topors play a role in the proteasome inhibitor) enhanced SN-38-mediated cellu- cellular response to CPT, and regulates the function of lar cytotoxicity. In yeast, two proteins related to the Top1 following CPT-induced DNA damage (Figure 5). ubiquitination pathway were discovered using genetic screens for mutants that alter CPT sensitivity. Over- expression of a ubiquitin-specific protease, Ubp11, Resistance to CPT in the clinical setting confers resistance to Top1-mediated DNA damage (Byrne and Bjornsti, 2001) and loss of DOA4, a 26S Studies of clinical specimens are needed to determine proteasome-associated C-terminal ubiquitin hydrolase, whether the resistance mechanisms detected in yeast and sensitizes cells to Top1-mediated DNA damage (Fiorani cell culture models are clinically relevant. Cellular

Oncogene Resistance to topoisomerase I-targeting drugs ZA Rasheed and EH Rubin 7302 metabolism, via carboxylesterases and UGTs, plays an formation, suggesting that Top1 is degraded following important role in the cytotoxicity of CPT-11 in cell CPT exposure in nonmalignant cells (Gupta et al., culture models. Little is known regarding the clinical 1998). Analyses in a similar clinical study of nonmalig- relevance of this finding, although varied carboxylester- nant PBMNCs in patients treated with a 72 h infusion of ase activity has been reported in clinical specimens 9-aminocamptothecin (9AC) also indicated decreases in (Ahmed et al., 1999; Khanna et al., 2000). BCRP seems Top1 protein levels at 48 or 72 h in two out of three to be expressed at low levels in breast cancer cells as well patients (Rubin et al., 1995). In contrast, two out of four as leukemic cells (Ross et al., 2000; Kanzaki et al., 2001). patients with leukemia who were treated with a 72 h Recently, BCRP protein expression was found to infusion of 9AC showed no change in Top1 protein increase in leukemia cells following infusion of topote- levels in their malignant blast cells during the infusion can and arabinoside-C in patients (Gounder et al., (Saleem et al., 2000), suggesting that if Top1 degrada- 2003). To date, altered expression of BCRP in clinical tion occurs in these cells, the timing is distinct from that samples has not been correlated with altered CPT of nonmalignant PBMNCs. It is possible that the sensitivity or treatment. apparent difference in 9AC-induced Top1 degradation There are a limited number of clinical studies that may relate to alterations in ubiquitin–proteasome path- have analysed clinical specimens (tumor tissue or ways in malignant versus nonmalignant cells. These surrogates) for mutations in Top1 and most have findings are consistent with the observation that yielded negative results (Ohashi et al., 1996; Takatani malignant and nonmalignant cultured cells differ in et al., 1997). Recently, Tsurutani et al. (2002) reported a their capacity to degrade Top1 (Desai et al., 2001). Top1 mutation in a tumor specimen from a patient with Interestingly, topors protein and mRNA expression are large cell carcinoma of the lung. The mutation results in decreased in tumor tissues versus matched normal two changes, a stop codon at position 736 and a glycine tissues, suggesting that topors may be involved in the to serine missense mutation at codon 737. Interestingly, apparent differences in CPT-induced Top1 degradation the patient with this mutation did not respond to a in these tissues (Saleem et al., 2002). chemotherapy regimen consisting of cisplatin and Other mechanisms of CPT resistance that have been irinotecan (Tsurutani et al., 2002). However, it remains identified in yeast and cell culture models need to be to be determined if these mutations result in enzymatic evaluated clinically, including the role of Top1 localiza- resistance to CPT. tion and specific repair processes. Pharmacogenetic and Other studies utilizing clinical specimens found biochemical understanding of clinical CPT resistance alterations in Top1 and Top2a levels following treat- will improve the use of CPTs in the treatment of ment with CPT. Analyses in clinical study of 11 patients malignancy. with nonhematologic malignancies treated with oral CPT for 14 days showed decreases in Top1 protein levels Acknowledgements in nonmalignant peripheral blood mononuclear cells We thank Drs Leroy F Liu, Daniel Pilch, and Ahamed Saleem (PBMNCs) that were not due to cleavable complex for helpful discussions in writing this review.

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