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Departments of Medicine (K.H., B.T.), Biology (G.L., A.-M.S.), Biochemistry (B.T.), and Microbiology and Immunology (B.T.), McGill University, Montréal, Québec, Canada
Abstract
Tirapazamine (TPZ) is an anticancer drug that targets topoisomerase II. TPZ is preferentially active under hypoxic conditions. The drug itself is not harmful to cells; rather, it is reduced to a toxic radical species by an NADPH cytochrome P450 oxidoreductase. Under aerobic conditions, the toxic compound reacts with oxygen to revert back to TPZ and a much less toxic radical species. We have used yeast (Saccharomyces cerevisiae) as a model to better understand the mechanism of action of TPZ. Overexpression of NCP1, encoding the yeast ortholog of the human P450 oxidoreductase, results in greatly increased sensitivity to TPZ. Likewise, overexpression of TOP2 (encoding topoisomerase II) leads to hypersensitivity to TPZ, suggesting that topoisomerase II is also a target of TPZ in yeast. Thus, our data show that yeast mimics human cells in terms of TPZ sensitivity. We have performed robot-aided screens for altered sensitivity to TPZ using a collection of approximately 4600 haploid yeast deletion strains. We have identified 117 and 73 genes whose deletion results in increased or decreased resistance to TPZ, respectively. For example, cells lacking various DNA repair genes are hypersensitive to TPZ. In contrast, deletion of genes encoding some amino acid permeases results in cells that are resistant to TPZ. This suggests that permeases may be involved in intracellular uptake of TPZ. Our discoveries in yeast may lead to a better understanding of TPZ biology in humans.
Nonsurgical treatment of cancer includes radiotherapy and chemotherapy. A major drawback of these treatments is that they do not specifically target cancer cells. Approaches under current study include the use of hypoxic-selective drugs (Brown and Giaccia, 1998; Rooseboom et al., 2004; Seddon et al., 2004). The approach is derived from the fact that oxygen levels are generally lower in the center of a tumor because of poor vascularization (Brown and William, 2004). Because these hypoxic cells are generally more resistant to radiation and conventional chemotherapy (Gatenby et al., 1988; Hockel et al., 1993; Okunieff et al., 1993; Teicher, 1994; Nordsmark et al., 1996), drugs specifically active under low oxygen levels are of great interest for cancer treatment (Brown, 1999). The best prototype is probably 3-amino-1,2,4-benzotriazine-1,4-dioxide (also called tirapazamine or SR4233, and hereafter referred to as TPZ). Phase II and III clinical trials have shown the efficacy of TPZ when used in combination with radiotherapy or chemotherapy (Bedikian et al., 1999; Craighead et al., 2000; von Pawel et al., 2000; Rischin et al., 2001).
The hypoxic toxicity of TPZ is believed to be caused by the addition of one electron to TPZ by enzymatic reductases, yielding a radical species that causes single- and double-strand DNA breaks, leading to chromosome aberration and cell death (Patterson et al., 1998). The radical species is unstable and, under normal oxygen levels, reacts with oxygen to revert back to TPZ and a much less toxic radical species (Lloyd et al., 1991). The exact mechanism of TPZ's action is not known. Under hypoxic conditions, a protonated neutral form of a TPZ nitroxide radical is formed, but there is no formal proof that this compound is responsible for the toxicity (Patterson et al., 1998). The TPZ nitroxide is unstable and reacts with biomolecules such as DNA to form a nontoxic two-electron product called SR4317 (Lloyd et al., 1991). It is interesting that only a fraction (30–70%) of TPZ is converted to SR4317. This may explain why the rate of formation of SR4317 does not always correlate with toxicity (Siim et al., 1996). It has been shown recently that TPZ inhibits DNA replication (Peters et al., 2001) and that it mediates its effect through topoisomerase II (Peters and Brown, 2002). Topoisomerase II unwinds DNA by introducing transient double-stranded breaks. Therefore, TPZ treatment probably leads to covalent binding of the topoisomerase II subunit to DNA, stabilizing topoisomerase II-induced double-strand breaks and resulting in cell toxicity (Peters and Brown, 2002).
Under hypoxia, there is good evidence that NADPH cytochrome P450 oxidoreductase (EC 1.6.2.4 [EC] ) is involved in the metabolism of TPZ to a toxic compound (Patterson et al., 1997; Chinje et al., 1999; Saunders et al., 2000). Hypoxic sensitivity of human breast cancer cell lines to TPZ correlates with the expression of P450 oxidoreductase (Patterson et al., 1995). Furthermore, stable transfection of an expression vector encoding P450 oxidoreductase results in increased sensitivity to TPZ in human breast and lung cancer cell lines (Patterson et al., 1997; Saunders et al., 2000). In addition to P450 oxidoreductase, a nuclear enzyme is probably involved in the conversion of TPZ to a toxic molecule (Evans et al., 1998). Using a human lung cancer cell line, nuclei were found to be responsible for only 20% of the TPZ metabolism, but DNA damage was similar to what was observed for whole cells. These results suggest that an enzyme(s), other than the P450 oxidoreductase, is responsible for the conversion of TPZ to a toxic compound. Thus, the relevant enzyme(s) seem to be nuclear, unlike the oxidoreductase, which is located at the membrane of the endoplasmic reticulum. In addition, other enzymes such as cytochrome P450 and DT-diaphorase can also metabolize TPZ (Brown and Giaccia, 1998; Patterson et al., 1998).
Saccharomyces cerevisiae (referred to as yeast hereafter) has been a useful model organism to study various drugs (Barret and Hill, 1998). In keeping with these results, our study shows that TPZ targets topoisomerase II and that overexpression of the NCP1 gene (encoding an ortholog of the human P450 oxidoreductase) results in increased TPZ sensitivity in yeast cells. Screening of a panel of yeast deletion strains has allowed the identification of many genes that confer resistance or sensitivity to TPZ, including genes involved in DNA repair and amino acid transport.
Materials and Methods
Yeast Strains. Wild-type strains used were BY4741 (MATa his31 leu20 met150 ura30) (Brachmann et al., 1998) and a derivative of BY4741, R1158 (Hughes et al., 2000) (MATa his31 leu20 met150 URA3::CMV-tTA). Strains yTH-NCP1 and yTH-TOP2 were obtained from Open Biosystems (Huntsville, AL). Haploid deletion strains were derived from BY4741 (Winzeler et al., 1999) and were arrayed on 16 768-format plates (Tong et al., 2001).
Media and Drug Assays. Media were prepared according to the methods described by Adams et al. (1997). Yeast extract peptone dextrose (YEPD) contained 1% yeast extract, 2% peptone, and 2% glucose. TPZ was obtained from Sigma Chemical (St. Louis, MO) or Sanofi-Synthélabo (Malvern, PA) and dissolved in 50% methanol or 50% ethanol. Anaerobic conditions were obtained using an anaerobic jar (BD Biosciences, San Jose, CA) and gas pack (BBL GasPak Plus; BD Biosciences). Anaerobic conditions were verified by using an anaerobic indicator (BBL; BD Biosciences) and monitoring growth of the strict anaerobe Clostridium tetanomorphum (Supplementary Fig. S3). Growth assays were all performed at 30°C.
Ncp1 and Top2 Overexpression. Haploid wild-type strain R1158 and strains carrying a doxycycline-repressible promoter integrated at the NCP1 or the TOP2 loci were grown overnight in YEPD in the absence or the presence of doxycycline (20 μg/ml; Sigma Chemical). Cells were serially diluted and spotted on YEPD plates containing various concentrations of TPZ and 20 μg/ml doxycycline for cells grown overnight in the presence of the antibiotic.
Western Blot Analysis of Top2. Extracts were prepared as described previously (Akache et al., 2004), and proteins were run on a 7.5% polyacrylamide gel. Western blot analysis was performed with a polyclonal antibody against S. cerevisiae Top2 (TopoGEN, Port Orange, FL).
Screen for Altered Sensitivity to TPZ. Deletion strains were propagated on standard YEPD or YEPD supplemented with 200 μg/ml G418 (Invitrogen, Carlsbad, CA) using a colony picker (Bio-Rad, Hercules, CA). Hypersensitive mutants were screened by pinning the deletion collection on YEPD supplemented with and without 300 μM TPZ and then scoring the colony size after a 3.5-day incubation. Resistant mutants were screened by pinning the deletion collection on YEPD and then on YEPD supplemented with 750 mM TPZ. After 48 h, plates were replicated on fresh YEPD containing 750 μM TPZ, and growth was scored after a 48-h incubation. Of two screens for hypersensitive and a single screen for resistant mutants, 256 and 263 mutants were identified, respectively.
The sensitivity of these mutants was confirmed by the following spotting procedure: cells were grown in liquid YEPD to log phase, diluted to an optical density at 600 nm of 0.5, serially diluted 10-fold four times, and 5 μl was spotted on YEPD plates supplemented with and without 200 and 500 μM TPZ, respectively. After 2 days of incubation, growth of mutants in the presence or absence of TPZ was scored and compared with that of the wild-type BY4741 strain. Mutants showing significant growth defect or absence of growth in the presence of 200 μM TPZ were scored as "- -" or "- - -", respectively. Mutants showing similar or more vigorous growth than the fre1 mutant in the presence of 500 μM TPZ were scored as "++" or "+++", respectively. Finally, 73 and 117 mutants exhibited hypersensitivity and resistance to TPZ, respectively.
Search for Human Proteins with Yeast Homologs Involved in Modulating TPZ Sensitivity. A list of approximately 34,000 human protein sequences was obtained from Ensembl database (http://www.ensembl.org) and was used as query in a search for homologs against the yeast proteome (approximately 6000 protein sequences; http://www.yeastgenome.org). We found approximately 26,000 human proteins matching a yeast protein sequence (E value 0.001). Of this set, 614 human peptides showed significant homology to yeast product of genes involved in sensitivity or resistance to TPZ (data not shown). A partial list of these genes can be found in Tables 3 and 4.
Results
To determine whether yeast can be used as a model for studying the mode of action of the anticancer drug TPZ, wild-type yeast cells were grown overnight under aerobic conditions, serially diluted, and spotted on plates containing increasing concentrations of TPZ. Cells were then grown under anaerobic or aerobic conditions for approximately 24 (aerobia) or 48 h (anaerobia) (Fig. 1). It is interesting to note that TPZ was somewhat more toxic to cells grown under anaerobic conditions. For example, with 200 μM TPZ, growth was almost completely abolished under, anaerobia whereas only a moderate effect was observed in the presence of oxygen (Fig. 1E). Similar growth was observed in the absence of TPZ (Fig. 1A). However, the difference in TPZ toxicity of cells grown under aerobic and hypoxic conditions is much more pronounced in human tumor cell lines. For example, equal cell killing for human tumor cells grown under aerobic conditions requires approximately 300-times higher TPZ concentration compared with hypoxic cells (Brown, 1993). The basis for this species difference is unknown, but it may be related to the fact that yeast is a facultative aerobe (see below).
There is good evidence that the human NADPH oxidoreductase (EC 1.6.2.4 [EC] ) is responsible for metabolizing TPZ to a toxic compound (Patterson et al., 1997; Chinje et al., 1999; Saunders et al., 2000). We were interested in determining whether a related enzyme would perform a similar function in yeast. The essential gene NCP1 encodes the yeast ortholog of human P450 oxidoreductase. To study the involvement of NCP1 in TPZ toxicity in yeast, the NCP1 promoter of a haploid strain was replaced with a doxycycline-repressible promoter (Mnaimneh et al., 2004). Use of this promoter results in the overexpression of the targeted gene in the absence of doxycycline and reduced expression in the presence of the antibiotic.
Overexpression of NCP1 did not affect growth under aerobic or anaerobic conditions compared with a wild-type strain (Fig. 1A), whereas reduced expression of NCP1 impaired growth only under anaerobic conditions (Fig. 1B). The nearly normal aerobic growth under repressible conditions is probably caused by leaky expression of NCP1, as observed for some other genes (Mnaimneh et al., 2004). Overexpression of NCP1 was highly toxic to cells grown in the presence of TPZ (Fig. 1, C–F). This suggests that, as observed in human cells, high levels of P450 oxidoreductase result in increased production of a toxic metabolite (Patterson et al., 1997; Saunders et al., 2000). This provides further evidence that yeast NADPH oxidoreductase, as its human counterpart, is responsible, at least in part, for the conversion of TPZ to a toxic compound. Thus, yeast mimics human cells with regard to TPZ toxicity.
Because a recent study in animal cells shows that TPZ targets topoisomerase II (Peters and Brown, 2002), we tested whether this enzyme also mediates TPZ toxicity in yeast cells. Overexpression of topoisomerase II results in hypersensitivity of yeast to some anticancer drugs (Nitiss et al., 1992) when tested in a DNA repair-deficient rad52 background. Because yeast topoisomerase II is encoded by the essential gene TOP2 (Wang, 1996), we altered TOP2 expression by using a doxycycline-repressible promoter as described above for NCP1. Using this system, greatly increased expression of TOP2 was observed with cells grown aerobically in the absence of doxycycline compared with cells treated with doxycycline or a wild-type strain (Fig. 2A, left). It is surprising that the expression of TOP2 in wild-type cells was greatly increased under anaerobic conditions, whereas the overexpression system gave only a modest increase in TOP2 levels compared with wild-type cells (Fig. 2A, right). Growth of these strains was similar when assayed under aerobic and anaerobic conditions in the presence or absence of doxycycline (Fig. 2B, panels A and B). The addition of doxycycline is likely not to lead to full repression of the promoter driving TOP2 expression because TOP2 is an essential gene (as suggested by the Western blot analysis). Under aerobic conditions, overexpression of TOP2 in a wild-type background resulted in increased cell sensitivity to TPZ, whereas cells with reduced expression of TOP2 behaved as wild-type cells (Fig. 2B, compare panels C and F). This effect was less apparent under anaerobic conditions, which is in agreement with the Western blot analysis of TOP2 expression. These results suggest that topoisomerase II is a target of TPZ in yeast cells. Thus, our data show that yeast mimics human cells in terms of TPZ sensitivity.
Genome-Wide Screen for Altered Sensitivity to TPZ. The identification of yeast mutants (other than NCP1 and TOP2) showing an altered sensitivity to TPZ should give insights into the mode of TPZ action and tools to design more effective drug treatments. As stated above, the difference of TPZ toxicity with regard to oxygen levels is much less pronounced in yeast than in human cells. It is well-established that growth of yeast under anaerobic conditions results in global changes in gene expression (Becerra et al., 2002). For example, anaerobia results in cell wall and membrane remodeling (Aguilar-Uscanga and Francois, 2003). Altered TPZ entry into the cells may explain the relatively weak sensitivity of yeast cells grown under anaerobic conditions. Anaerobicity also results in more rapid response to osmotic shock (Krantz et al., 2004) and in altered expression of genes encoding NCP1 and cytochrome P450. Because oxygen levels have only a minor effect on TPZ sensitivity of yeast and for easier manipulation of a large number of strains, we decided to perform a large-scale screen under aerobic conditions.
We performed robot-aided screens for altered sensitivity to TPZ using a collection of 4600 haploid deletion mutants corresponding to most nonessential yeast genes. Phenotypes were confirmed by individually spotting serial dilutions of deletion strains on TPZ and control plates (Supplementary Fig. S1). Figure 3 shows examples of strains that are resistant or sensitive to TPZ. In all, 73 strains were sensitive to the drug (Table 1), whereas 117 strains showed increased resistance to TPZ (Table 2 shows a list of the strongest resistance phenotypes, and Supplementary Table S1 shows a list of weaker resistance phenotypes). Genes were grouped in categories according to their known or inferred function and are discussed accordingly. It should be stressed that we do not know what mechanism of TPZ action renders some deletion mutants sensitive to the drug. For example, the effect could be mediated by topoisomerase II or by DNA damage produced by a TPZ metabolite.
DNA Repair and Genome Stability. Given that exposure to TPZ results in DNA damage, it was not unexpected that cells lacking various DNA repair genes would be hypersensitive to the drug. These genes encode members of the RAD52 epistasis group (RAD51, RAD52, RAD54, RAD55, RAD57, and RAD59), subunits of the MRX complex (MRE11, RAD50, and XRS2), topoisomerase III (TOP3), factors involved in the repair of replication-dependent DNA damage (ASF1, MMS1, MMS4, MMS22, MUS81, RAD5, RTT101, RTT107, and UBC13) and subunits of the nucleotide excision repairosome (RAD10 and RAD16). In addition, four poorly characterized genes (NCE4, RTT109, WSS1, and YBR094W), whose deletion leads to TPZ hypersensitivity, were included in this category because they show synthetic lethality with genes involved in DNA repair or genome stability. For example, a double deletion of NCE4 and TOP1 (encoding topoisomerase I) is lethal, whereas WSS1 and YBR094W show synthetic lethality with SGS1, a gene encoding a nucleolar DNA helicase involved in the maintenance of genome integrity (Tong et al., 2004). In contrast, deletion of the DNA repair genes RAD18 or DNL4 resulted in increased resistance to TPZ (Table 2). We do not know the reason for these observed resistance phenotypes.
Transporters. A number of resistant strains lack amino acid permeases such as Agp3 (Schreve and Garrett, 2004), Alp1 (Regenberg et al., 1999), or the choline permease Hnm1. These results suggest that uptake of TPZ within the cell could be mediated by permeases (see Discussion). In keeping with these results, genetic interactions suggest that Asi3 is a regulator of permease gene expression (Forsberg et al., 2001). Expression of putative permeases involved in TPZ uptake would be reduced in cells lacking Asi3, resulting in increased resistance to the drug.
Reductases and Related Proteins. As stated above, Ncp1 is very likely to be responsible for metabolizing TPZ to a toxic compound in yeast, as observed in mammalian cells. It is interesting that deletion of other reductase genes leads to resistance to TPZ. For example, cells lacking Fre1 are resistant to the drug. FRE1 encodes a ferric and cupric reductase necessary for the uptake of environmental Cu2+ and Fe3+ (Eide, 1998). Reduced copper is a substrate for the highaffinity transporter Ctr1 and related transporters. Although Fre1 and Ctr1 are functionally linked, deletion of CTR1 does not result in resistance to TPZ, in contrast to what was observed for the anticancer drug cisplatin (Ishida et al., 2002; Lin et al., 2002; Nitiss, 2002). In addition, a strain lacking Utr1 shows increased resistance to TPZ. UTR1 encodes a NAD kinase that enhances the activity of Fre1 (Lesuisse et al., 1996), an observation that may explain the phenotype of an utr1 strain. The other reductase identified in our screen is His4, a multifunctional enzyme bearing dehydrogenase activity and involved in histidine biosynthesis (Alifano et al., 1996).
Cell Stress Signaling and Signal Transduction. Deletion of genes required for resistance to oxidative stress such as LYS7, SOD1, and SOD2 leads to hypersensitivity. SOD1 and SOD2 encode superoxide dismutases, and LYS7 encodes a copper chaperone required for Sod1 activity. Hypersensitivity of strains lacking these stress genes is likely to be explained by the fact that metabolism of TPZ leads to the formation of a superoxide radical toxic to cells (Lloyd et al., 1991). In addition, mutants defective in the protein kinase C MAP-kinase pathway (bck1 and slt2) or affected in signaling through multiple MAP-kinase pathways (sit4) show an increased TPZ sensitivity. In contrast, deletion of HSP104 or WSC2 leads to TPZ resistance. Wsc2 is a putative integral membrane protein and a stress-response component required for cell-wall integrity (Verna et al., 1997).
Vesicular Transport. Deletion of genes involved in protein recycling to the endosomal compartment increases TPZ sensitivity. Included here are members of the ESCRT-I (VPS26), ESCRT-II (SNF8 and VPS25), and ECSRT-III (SNF7) complexes, which are involved in ubiquitin-mediated protein sorting to the vacuole, factors involved in protein sorting from the late-Golgi to the vacuole through adaptor protein (AP)-3 transport vesicles (VAM3 and VPS41), and components of the endosome-to-Golgi recycling pathway (RIC1 and WHI6). In contrast, removal of three genes involved in ER-to-Golgi transport (ERV41, SPO20, and YOS9) conferred resistance to TPZ.
Other Categories. A set of deletion strains hypersensitive to a range of inhibitory compounds has been identified (Parsons et al., 2004). A number of these mutants also show hypersensitivity to TPZ. A first group is involved in the function of the vacuolar H+-ATPase (PPA1, TFP3, VMA4, VMA7, and VMA10). A second group of genes is involved in ergosterol biosynthesis (ERG2, ERG3, and ERG4). The increased sensitivity to TPZ of the second group of deleted genes is probably caused by altered plasma membrane fluidity. Likewise, other genes involved in lipid, fatty acid, or sterol metabolism (DPL1, EKI1, FAA3, and PDR17) resulted in resistance to TPZ when deleted. Removal of these genes may also alter plasma membrane fluidity and integrity, thereby restricting the entry of TPZ into the cells. Other genes that modulate TPZ sensitivity are associated with transcription or RNA processing. For example, the deletion of the RNA polymerase II subunit RPB9, components of the RNA-polymerase II mediator complex (GAL11, PGD1, ROX3, and SRB2), the subunit of the CCR4-NOT1 complex POP2,or transcriptional regulators (DBF2, SPT10, SPT20, and SWI4) confers hypersensitivity to TPZ. These genes may be required for the transcription of TPZ resistance gene(s).
Relevance to Human TPZ Biology. We were interested in determining whether the genes identified in our screen have human counterparts. A selected set of 30 human proteins showing significant homology to products of yeast genes whose deletion leads to resistance to TPZ is shown in Table 3. Some of these human proteins have a role in cell proliferation (e.g., CLK1), cell morphogenesis (DAAM1 and -2), and signal transduction (e.g., HRAS). Others have been reported to exhibit altered expression in cancer cells. For example, OS-9 is amplified in sarcomas (Su et al., 1996). OS-9 is involved in oxygen-dependent degradation of the hypoxia-inducible factor (Baek et al., 2005) and is associated with the ER membrane (Litovchick et al., 2002). Other human proteins, such as the KIST kinase or the XPR1 (Battini et al., 1999) may be involved in signaling and could play a role in TPZ sensing. Table 4 lists a selection of 40 human proteins sharing significant homology with products of yeast genes whose deletion confers TPZ hypersensitivity. These proteins may be important for resistance to TPZ in human cells. For example, the removal of the manganese superoxide dismutase leads to TPZ hypersensitivity in both human (Wouters et al., 2001) and yeast cells (Table 1). Inhibition of processes such as microtubule cytoskeleton assembly, nuclear transport, protein synthesis, transport to the endosome, and proton transport through V-type ATPase is likely to be synergistic with TPZ treatment in human cells as observed in yeast.
Discussion
Yeast has been a useful model organism in better understanding the mode of action of various drugs (Barret and Hill, 1998). In this report, we show that yeast can also be used to study the anticancer drug TPZ. Yeast mimics animal cells with regard to TPZ toxicity. First, overexpression of the NCP1 gene (encoding a P450 oxidoreductase) results in a marked increased sensitivity to TPZ (Fig. 1), in analogy to human cells in which overexpression of the P450 oxidoreductase results in increased TPZ toxicity (Patterson et al., 1997; Chinje et al., 1999; Saunders et al., 2000). Second, we provide good evidence that topoisomerase II is a target of TPZ in yeast (Fig. 2), as observed in animal cells (Peters and Brown, 2002). Thus, these observations reinforce the view that yeast can be used as a model to gain insights into the mechanism of action of TPZ.
We took advantage of the yeast system to perform a large-scale screen of nonessential genes that modulate sensitivity to TPZ (Tables 1 and 2, and Supplementary Table S1). As other similar studies, our screen was not totally comprehensive; for example, essential genes were not tested, and some nonessential genes modulating TPZ sensitivity may not have been identified in this study. However, our work led to the identification of 190 deletion strains that showed an altered growth in the presence of TPZ. For example, a major class of mutants is related to DNA repair or genome stability, in agreement with the model of TPZ action. Similar results were obtained for screens with other anticancer agents such as cisplatin, oxaliplatin, mitomycin, and bleomycin (Aouida et al., 2004; Wu et al., 2004). RAD1 and RAD10 gene products form a complex, and deletion of either gene results in similar phenotypes (Prakash and Prakash, 2000). We were surprised that a rad1 deletion strain was not recovered in our screen, whereas a rad10 strain showed sensitivity to the drug. We manually spotted the rad1 mutant and found it to be similar to that of a wild-type strain. Thus, the phenotype of a rad10 mutant does not always seem to match that of a rad1 mutant.
Besides Ncp1, two other reductases were found to confer resistance to TPZ when removed: Fre1 and His4. Both enzymes may metabolize TPZ to a toxic compound in analogy to Ncp1. To our knowledge, there is no human homolog of His4, ruling out the possibility that a human His4-like protein would be responsible for TPZ metabolism. However, various human proteins have domains that show similarity to Fre1 (Lambeth et al., 2000) and may modulate sensitivity to the drug.
Our screen identified three transporters encoding genes whose deletion enhances TPZ resistance: the choline permease gene HNM1, and the amino acid permease genes AGP3 and ALP1. This suggests a role for these genes in TPZ uptake within the cells. Such membrane permeases have been shown previously to mediate the uptake and toxicity of other compounds. For example, Hnm1 is involved in the uptake of the alkylating agent nitrogen mustard, and an hnm1 mutant is resistant to this drug (Li and Brendel, 1994). Bleomycin action was found to be modulated by the level of the L-carnitine transporter Agp2. Drug uptake and toxicity were decreased and increased upon deletion and overexpression of AGP2, respectively (Aouida et al., 2004). Likewise, the copper transporter Ctr1 mediates cisplatin uptake in yeast and human cells (Ishida et al., 2002; Lin et al., 2002; Nitiss, 2002). Thus, it seems that TPZ, as other anticancer drugs, uses membrane transporters to enter the cells. Because related amino acid transporters are found in humans (e.g., SLC7A2, Table 3), it will be interesting to determine whether these transporters are involved in mediating uptake of TPZ in human cells.
This hypothesis is reinforced by our findings that deletion of a number of genes involved in ubiquitin-regulated protein trafficking alters the resistance to TPZ. Indeed, ubiquitination is known to regulate the transport of the general amino acid permease Gap1 (Soetens et al., 2001) and may regulate the transport of other amino acid permeases as well. According to this hypothesis, mutations affecting this ubiquitin-regulated endocytosis pathway (such as snf7, snf8, vps25, or vps26) would perturb the turnover of permeases, resulting in their accumulation at the plasma membrane. The TPZ hypersensitivity of these mutants may be explained by the resulting increased TPZ uptake into the cells. On the other hand, a defect in forward permease trafficking (for example, erv41 or yos9) would result in a decreased efficiency of TPZ entry into cells and, as a result, in an increased resistance to TPZ. In summary, we have shown that yeast can be used as a model to study the anticancer drug TPZ. This allowed the identification of many yeast genes that modulate sensitivity to the drug. These observations will be invaluable to further increase our understanding of the mode of action of TPZ in human cells. Moreover, our results suggest that yeast could be used to design derivatives of TPZ and related bioreductive drugs.
Acknowledgements
We thank Sanofi-Synthelabo for providing TPZ. We are extremely grateful to Dr. Howard Bussey (Department of Biology, McGill University) for providing access to robotic facilities. We thank Dr. Ed Chan (McGill University) for advice and providing access to his anaerobic chamber for preliminary experiments. We thank MarcAndré Sylvain, Edith Sirard, and Stella Drury for input in this project. We are grateful to Sarah MacPherson for critical review of the manuscript. We also thank Drs. Fred Sherman, Simon Labbé, John White, Dindial Ramotar, and Stéphane Laporte for useful comments.
Footnotes
This work was supported by a grant from the Cancer Research Society (Montréal) (to B.T.) and the Canadian Institutes of Health Research (to G.L.) (principal investigator: Dr. Howard Bussey). B.T. was supported by a scholarship from the Fonds de la Recherche en Santé du Québec.
K.H. and G.L. contributed equally to this work.
ABBREVIATIONS: TPZ, tirapazamine; ORF, open reading frame; YEPD, yeast extract peptone dextrose; SR4317, 3-amino-1,2,4-benzotriazine-1-N-oxide; MAP, mitogen-activated protein; ER, endoplasmic reticulum; P450, cytochrome P450.
The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
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