ToxSci Advance Access originally published online on February 16, 2008
Toxicological Sciences 2008 103(1):68-76; doi:10.1093/toxsci/kfn034
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Deletion of Yeast CWP Genes Enhances Cell Permeability to Genotoxic Agents
* Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei, 430072 China
Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon SK S7N 5E5, Canada
1 To whom correspondence should be addressed at Institute of Hydrobiology, Chinese Academy of Sciences, 7 Donghu Road South, Wuhan, Hubei, 430072 China. Fax: +86-27-68780123. E-mail: hpdai{at}ihb.ac.cn.
Received December 26, 2007; accepted February 7, 2008
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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We have previously reported the development of a novel genotoxic testing system based on the transcriptional response of the yeast RNR3-lacZ reporter gene to DNA damage. This system appears to be more sensitive than other similar tests in microorganisms, and is comparable with the Ames test. In an effort to further enhance detection sensitivity, we examined the effects of altering major cell wall components on cell permeability and subsequent RNR3-lacZ sensitivity to genotoxic agents. Although inactivation of single CWP genes encoding cell wall mannoproteins had little effect, the simultaneous inactivation of both CWP1 and CWP2 had profound effects on the cell wall structure and permeability. Consequently, the RNR3-lacZ detection sensitivity is markedly enhanced, especially to high molecular weight compounds such as 4-nitroquinoline-N-oxide (> sevenfold) and phleomycin (> 13-fold). In contrast, deletion of genes encoding representative membrane components or membrane transporters had minor effects on cell permeability. We conclude that the yeast cell wall mannoproteins constitute the major barrier to environmental genotoxic agents and that their removal will significantly enhance the sensitivity of RNR-lacZ as well as other yeast-based genotoxic tests.
Key Words: RNR3-lacZ; yeast; cell wall; mannoprotein; genotoxicity test; sensitivity; permeability.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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An RNR3-lacZ genotoxicity testing system has been developed recently based on the induction of a Saccharomyces cerevisiae RNR3-lacZ reporter gene in response to a broad range of DNA-damaging agents (Jia et al., 2002
). This testing system possesses several advantages over existing short-term genotoxicity testing systems. Firstly, the RNR3 promoter is the most sensitive among known yeast genes examined for the genotoxicity test (Afanassiev et al., 2000; Ichikawa and Eki, 2006; Jia et al., 2002). Secondly, use of baker's yeast is safe in the environment, making its use in a field test feasible. Thirdly, as a eukaryotic microorganism, yeast has metabolic systems more resembling those of mammals than bacteria has; indeed, some well know DNA-damaging carcinogens turn out to be positive in the RNR3-lacZ test, but are negative in bacterial testing systems such as the Ames test and SOS chromotest (Jia et al., 2002). Finally, quantitative comparison showed that in many cases, the RNR3-lacZ test is more sensitive than the SOS chromotest, and is comparable to the Ames test (Jia et al., 2002). The Ames test underwent three genetic modifications of the host Salmonella strain to improve its sensitivity, namely, deletion of uvrB to disarm host DNA repair capacity, mutation of rfa to increase permeability, and introduction of the plasmid pKM101 to increase mutability (Ames et al., 1975). In order to further enhance sensitivity of the RNR3-lacZ testing system, we previously examined the effects of inactivation of different DNA repair pathways on the RNR3-lacZ response. It was found that the induction of RNR3-lacZ in some DNA repair-deficient mutants can be greatly enhanced in an agent-specific manner (Jia and Xiao, 2003). A common challenge facing environmental genotoxicity testing is the extreme low dose of testing chemicals in the sample. Dose response studies clearly demonstrated that the detection sensitivity of a biomarker-based testing system is directly related to the accumulation of genotoxic chemicals inside the host cells, which can be regulated at three different levels, namely cell wall, plasma membrane and the cellular detoxification mechanisms. We wish to further enhance the RNR3-lacZ sensitivity by exploring the possibility of increasing cell permeability.
The yeast cell wall is composed of glucans, which constitute the inner layer of cell wall, mannoproteins, which form an external cell wall layer, and chitin, which is the minor component of the cell wall (Klis et al., 2006; Zlotnik et al., 1984). Yeast cell walls not only determine the shape of the cell, but also provide physical and osmotic protection with their dynamic structure, and the outer protein layer also limits the permeability of the cell wall (Stratford, 1994). Several reports have described mutations that affect the cell wall and increase the sensitivity of a yeast test to genotoxic chemicals (Pesheva et al., 2005; Staleva et al., 1996; Terziyska et al., 2000). In addition, genes involved in the synthesis of the wall or maintaining the wall integrity (Levin, 2005) have been identified and characterized; some null mutations among these genes can increase the cell's permeability to chemicals (Dielbandhoesing et al., 1998; Leduc et al., 2003). In particular, mannoproteins, which comprise 40% of the cell wall mass and are extensively O- and N-glycosylated (Klis et al., 2006), appear to play an important role in the permeability of the cell wall (de Nobel et al., 1990; Zlotnik et al., 1984). CWP1 and CWP2 encode two major mannoproteins of the outer cell wall; their deletion caused increased sensitivities to some chemicals (Dielbandhoesing et al., 1998; van der Vaart et al., 1995).
The yeast cell membrane is composed of sterol, phospholipids, sphingolipids, sterols, and proteins. Ergosterol, as the predominant sterol component, can affect membrane permeability (Hemmi et al., 1995; Karst and Jund, 1976). Eleven genes specifically required for ergosterol synthesis have been identified (Lees et al., 1995). Mutations in these genes can be lethal; however, cells lacking ERG6 exhibit normal vegetative growth. It has been reported that inactivation of ERG6 alters membrane permeability and renders cells sensitive to several chemicals (Emter et al., 2002; Welihinda et al., 1994).
Once a toxic chemical enters cells, yeast cells may sense this stress and turn on a pleiotropic drug resistance (PDR) pathway. PDR5 encodes one of the major ABC transporters that serves as a efflux pump to expel a variety of xenobiotics (Balzi and Goffeau, 1995). Inactivation of PDR5 leads to an increased drug sensitivity (Leonard et al., 1994; Meyers et al., 1992).
In this report, we describe the systematic analysis of inactivation of the above three components, namely cell wall, cell membrane, and the efflux pumps, and their effects on the enhancement of the RNR3-lacZ genotoxicity test with selected chemicals.
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MATERIALS AND METHODS |
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Yeast strains, cell culture, and transformation.
The parental yeast haploid BY4741 (MATa his3
1 leu20 met150 ura30) and its gene deletion derivatives were created by the Saccharomyces Gene Deletion Project and were purchased. The identity of the deletion strains was confirmed by PCR amplification of genomic DNA using primers designed by SGD (http://www-sequence.stanford.edu/group/yeast_deletion_project/deletions3.html).
Yeast cells were grown at 30°C in YPD (Sherman et al., 1983). Plasmid DNA was transformed into yeast cells by a modified lithium acetate protocol (Hill et al., 1991) and selected on minimal SD medium (Sherman et al., 1983). Transformants were streaked on a fresh selective plate before being utilized for further analysis.
Plasmids and mutant strain construction.
Plasmid pZZ2 (Zhou and Elledge, 1992) was obtained from Dr S. Elledge (Harvard University, Boston, MA) and utilized for the RNR3-lacZ test as previously described (Jia and Xiao, 2003; Jia et al., 2002). Plasmid YEp-DDI1-lacZ has been reported previously (Liu and Xiao, 1997).
To obtain the cwp1 cwp2 double mutant, the cwp1::LEU2 disruption cassette was constructed. A 1.4-kb yeast genomic DNA fragment containing the entire CWP1 open reading frame (ORF) plus the 0.6-kb upstream and 0.86-kb downstream sequences was amplified by PCR using CWP1-1 (5'-GTACCGAATCGTAGCTCGAGG') and CWP1-2 (5'–GGGAATGTGAGAGCTGCATGC-3') as primers. The PCR primers were designed to encompass an XhoI site upstream and an SphI site downstream (underlined in the primers) of the CWP1 ORF. The PCR product was cloned into pGEM-T (Promega, Madison, MI). A 0.62-kb StyI fragment within the 0.72-kb CWP1 ORF was then deleted and replaced by a 1.6-kb BamHI fragment from YDp-L (Berben et al., 1991) containing the LEU2 gene. The cwp1::LEU2 cassette was released by XhoI–SphI digestion and then transformed into the cwp2 mutant.
Test chemicals.
Genotoxic chemicals used in this study include methyl methanesulfonate (MMS), hydroxyurea (HU), 4-nitroquinoline-N-oxide (4-NQO), and phleomycin. All the above chemicals were purchased from Sigma-Aldrich (St Louis, MO). The 4-NQO stock solution was made in acetone at a concentration of 10 mg/ml, aliquoted, and stored at –20°C. After treatment, yeast cells were precipitated by centrifugation, washed twice with sterile distilled water, and resuspended in Z buffer (60mM Na2HPO4·7H2O, 40mM NaH2PO4·H2O, 10mM KCl, 1mM MgSO4·7H2O, 40mM β-mercaptoethanol, pH 7. 0) for the β-galactosidase (β-gal) assay. MMS and HU represent low molecular weight mutagens, 4-NQO represents a medium molecular weight mutagen, whereas phleomycin is considered to be bulky molecule. In addition, tetracycline (Tet) was used as a nongenotoxic agent and the stock solution was made by dissolving Tet in 95% ethanol. Congo red was purchased from British Drug Houses, whereas Calcofluor white (fluorescent brightener) and 4,6-diamidino-2-phenylindole (DAPI) were from Sigma-Aldrich.
DNA-damage treatment and β-gal assay.
The β-gal assay was performed as described previously (Xiao et al., 1993). Briefly, 0.5 ml of overnight yeast culture was used to inoculate 2.5 ml of fresh SD selective medium and incubation was continued for another 2 h. At this point, chemicals were added at the concentration indicated and cells were incubated for another 4 h. Conditions for ultraviolet (UV) and 
-irradiation treatments have been previously described (Jia et al., 2002). After the incubation, 1 ml of the above unsynchronized log-phase cell suspension was used to determine cell titer by measuring OD600 nm, and the remaining cells were used for the β-gal assay. The β-gal activity is expressed in Miller units (Guarente, 1983).
Toxicity test.
Cell survival rates were determined as previously described (Jia et al., 2002). At the end of incubation and prior to the β-gal assay, untreated and treated cells were collected by centrifugation, diluted, and plated on YPD in duplicate. The plates were incubated at 30°C for 3 days, and the number of colonies was counted. The toxic effect is expressed as a percentage of colonies from treated samples versus untreated samples.
Cell survival was also assessed by a plate-based serial dilution assay. An overnight culture was used to inoculate fresh YPD and allowed to grow until mid-log phase. The cell density was adjusted to 2 x 106 cells/ml and further diluted serially 10-fold with sterile ddH2O. Five µl aliquots of each dilution were spotted onto YPD and YPD plus test chemicals as indicated. For radiation treatments, the spotted YPD plates were exposed to different doses of the radiation as indicated followed by incubation at 30°C. UV-treated plates were incubated in the dark.
Microscopic methods.
To visualize cellular staining with fluorochromes such as Congo red, Calcofluor white and DAPI, wild-type cells and the cwp1 cwp2 double mutant were treated with various concentrations of test chemicals side-by-side for variable time at 30°C. After cells were harvested by centrifugation at 16,110 RCF for 1 min, they were rinsed with phosphate buffered saline (PBS) five times over 30 min and proceed for fluorescent microscopic analysis.
Fluorescence microscopy was performed with an inverted microscope (Olympus model IX70) equipped with an UPlanFLN 60x/1.25 oil immersion objective at room temperature. All micrographs were taking by a camera (SPOT RT Slider; Diagnostic Instruments, Sterling Heights). Photographic images were obtained by Image-Pro Plus version 4.1 and cropped using Adobe Photoshop version 6. All phase-contrast images were adjusted to acquire similar brightness and contrast.
For electron microscopy, log-phase cells were harvested, washed with PBS and fixed with 2% glutaraldehyde for 2 h at room temperature. The fixed cells were mixed with 2% agarose at 40°C and cut into 1-mm2 blocks. The blocks were then dehydrated by several rounds of 70% and then 90% ethanol treatments, followed by increasing proportions of the LR white embedding medium (Sigma 71K9812) in 90% ethanol. After incubation of blocks with pure LR white overnight, the blocks were embedded into LR white-filled gelatine capsules, which were polymerized in a 50°C over for 24 h. Thin sections were produced and floated on 300 mesh copper grids ready for imaging using a JEOL JEM 1230 electron microscope.
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RESULTS |
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Inactivation of CWP Genes Enhances Sensitivity of the RNR3-lacZ Genotoxicity Testing
It has been previously reported that mannoproteins form an external cell wall layer, which determines wall porosity (Klis et al., 2002, 2006
). CWP1 and CWP2 are two main genes encoding the cell wall mannoproteins (van der Vaart et al., 1995). We created a cwp1 cwp2 double deletion yeast mutant and compared it with the isogenic wild-type and cwp single mutants for DNA damage-induced RNR3-lacZ expression. MMS is a direct acting, monofunctional alkylating agent with a low molecular weight (110 Da). The cwp1 cwp2 double mutant, but not the single cwp mutants, is more sensitive to killing by MMS compared with wild-type cells (Fig. 1A). In the cwp1 or cwp2 single mutant, the induction profile of RNR3-lacZ after MMS treatment was indistinguishable from that of wild-type cells over a wide range of MMS concentrations (Fig. 1B). However, in the cwp1 cwp2 double mutant, MMS-induced expression of RNR3-lacZ was observed at a significantly lower dose than in the wild-type or single cwp mutants. For example, after 0.005% MMS treatment, the double mutant displayed over 20-fold induction, compared with less than 10-fold induction in the wild-type or single cwp mutants (Fig. 1B). More importantly, the maximum induction was achieved by 0.02% MMS in the double mutant compared with 0.04% in other strains. Further examination using low MMS doses shows that significant enhancement of RNR3-lacZ expression by cwp mutations can be observed at the MMS concentration of 25 ppm, but not at 12.5 ppm (Fig. 1C). Hence, deletion of both CWP genes only slightly enhances induction but has little (< twofold) effect on MMS detection limit by the RNR3-lacZ assay. Similar results were obtained with the replication-blocking small molecule (76 Da) HU (Figs. 1D–F). In this case, however, the RNR3-lacZ induction by HU peaks at 6 mg/ml in the double mutant instead of 4 mg/ml in other strains and differences in kinetics with that of MMS treatment are noticed (Fig. 1E).
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4-NQO (190 Da) has been referred to as a "UV-mimetic" agent as it produces bulky adducts that are mainly repaired by the nucleotide excision repair pathway (Friedberg et al., 2006
). A slight but consistent increase of sensitivity to killing by 4-NQO in the cwp2 single and cwp1 cwp2 double mutants was observed (Fig. 2A). The differential response to 4-NQO between cwp1 and cwp2 mutants is consistent with previous reports that the cwp2 null mutant was more susceptible than the cwp1 null mutant to chemicals such as Calcofluor white or Congo red (van der Vaart et al., 1995), as well as proteins such as zymolyase (van der Vaart et al., 1995) or nisin (Dielbandhoesing et al., 1998). Correspondingly, both cwp2 single and cwp1 cwp2 double mutations, but not cwp1, cause an enhanced RNR3-lacZ expression induced by 4-NQO at high doses (Fig. 2B), which is more apparent than with MMS or HU treatment. Interestingly, at very low doses (up to 1 µg/ml), only the cwp double mutation enhances the induction of RNR3-lacZ (Fig. 2C). It is noted that a twofold enhancement can be observed at the lowest 4-NQO dose tested (0.125 µg/ml), whereas the RNR3-lacZ induction cannot be detected in wild-type and single mutants by 0.5 µg/ml 4-NQO (Fig. 2C). A linear regression analysis using low dose data indicates that the cwp double mutation (y = 6.12x + 1.21) enhances the RNR3-lacZ sensitivity by 7.65 fold compared with wild-type (y = 0.8x + 1.16).
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Phleomycin is a bleomycin-family glycopeptide antibiotic and induces DNA strand breaks in vitro (Sleigh, 1976
). Phleomycin is a bulky DNA-damaging agent with a molecular weight of 1526 Da. It was observed that both cwp2 single and cwp1 cwp2 double mutants, but not the cwp1 single mutant, display enhanced sensitivity to killing by phleomycin; nonetheless the double mutant is much more sensitive than the cwp2 single mutant (Fig. 2D). Consistent with the killing experiment, the cwp1 cwp2 double mutant displayed extreme sensitivity to phleomycin-induced RNR3-lacZ expression, whereas the cwp2 single mutation slightly enhanced the detection sensitivity (Fig. 2E). At a phleomycin concentration of 2 µg/ml, the double mutant displayed a fivefold induction, whereas the induction in the wild-type and cwp1 single mutant was barely detectable. Furthermore, the maximum induction was also elevated also elevated from four to fivefold in the wild-type and single cwp mutants to 13-fold in the double mutant (Fig. 2E). A linear regression analysis at even lower doses (Fig. 2F) showed that the detection sensitivity was increased by 13.3-fold in the cwp double mutant (y = 2.045x + 1.27) and by 3.16-fold in the cwp2 single mutant (y = 0.486x + 1.056) compared with the wild-type cells (y = 0.154x + 1.09). The above results collectively suggest that the yeast cell wall mannoproteins indeed provide primary protection against environmental carcinogens, especially compounds with high molecular weights.
Enhanced RNR3-lacZ Expression Is Due to an Increased Cell Permeability
Although the above results are most consistent with Cwp proteins serving as cell wall barrier to the tested chemicals, one cannot rule out the possibility that mannoproteins specifically interfere with the RNR3 expression. We noted that the basal-level β-gal activity in the cwp1 cwp2 double mutant carrying the RNR3-lacZ reporter is comparable to that of wild-type and cwp single mutants (Figs. 1 and 2), indicating that inactivation of CWP genes does not increase endogenous RNR3 expression. To determine whether the observed effects in the cwp mutants are specifically attributed to RNR gene expression, we measured DDI1-lacZ expression in response to MMS and phleomycin treatment in the wild-type and cwp double mutant cells. DDI1 is also inducible by a variety of DNA-damaging agents but does not share promoter sequences with the RNR genes (Liu and Xiao, 1997); indeed, it is controlled by a different set of transcriptional regulators than that of RNR genes (Zhu and Xiao, 2004). As shown in Figure 3, the DDI1-lacZ induction can also be enhanced by deletion of both CWP genes, and the enhancement to phleomycin treatment (Fig. 3A) is more pronounced than to MMS (Fig. 3B). Hence, enhancement of DNA damage by removal of cell wall mannoproteins appears to be a general phenomenon instead of being specific to the RNR3 gene.
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We further argue that if the cell wall permeability is solely responsible for the observed phenotypes in the cwp mutants, RNR3-lacZ induction by agents that do not require cell permeability will not be affected, and that nongenotoxic chemicals that differentially inhibit cwp mutant growth still cannot induce RNR3-lacZ expression. Indeed we found that UV (Fig. 4A) and
-irradiation (Fig. 4B) induced RNR3-lacZ expression was not significantly affected by inactivation of both CWP genes and that the double mutant is no more sensitive to UV and -irradiation than the wild-type cells (Fig. 4D). To address the second prediction, we used the antibiotic Tet, which kills yeast cells without damaging DNA or inducing RNR3-lacZ (Jia et al., 2002). Under our experimental conditions, Tet was unable to induce RNR3-lacZ expression in wild-type or the cwp mutants (Fig. 4C), although it killed more double mutant cells than wild-type cells (Fig. 4D).
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Deletion of CWP Genes Removes Outer Cell Wall and Increases Cell Permeability
It has been previously reported that deletion of CWP2 significantly reduced the thickness of the electron-dense outer layer of the cell wall (van der Vaart et al., 1995
). During this study, we routinely observed the reduced reflection of cwp mutant cells under the microscope, indicating that the outer layer of cell wall in these cells is indeed altered. To further investigate the morphological difference between wild-type and the cwp double mutant, we examined electron microscopic images and found that wild-type cells contain a much thicker outer layer than the cwp2 single and cwp1 cwp2 double mutants, and this layer is clearly electron dense. Furthermore, the density of outer cell wall also appears to be reduced in the cwp mutants (Fig. 5A). Cell growth of cwp2 single and cwp1 cwp2 double mutants were reported to be inhibited by chemicals such as Calcofluor white and Congo red (van der Vaart et al., 1995), and this phenotype has been utilized to screen and isolate cell wall mutants (Ram and Klis, 2006). Indeed, we found that inactivation of both CWP genes enhances cell staining by Calcofluor white or Congo red (Fig. 5B), confirming that after removal of outer layer mannoproteins, cells are more accessible to the above chemicals. However, Calcofluor white and Congo red primarily bind various β-linked glucans that form the inner layer of cell wall and interact with nascent chitin chains (Herth, 1980), which is a cell wall component and often enriched at the septum of dividing cells (Bowers et al., 1974) (Fig. 5B). In order to address whether removal of cell wall mannoproteins enhances cell permeability to genotoxic chemicals that have to enter the nucleus to be effective, we chose DAPI (molecular weight of 350 Da), a well-known fluorescent groove-binding probe for DNA (Jeppesen and Nielsen, 1989) that is commonly used to stain the nucleus. In our experience, wild-type yeast cells are refractory to DAPI staining and the staining can be enhanced by fixing cells (Hasek, 2006), which is a procedure to increase cell permeability. In this experiment, we found that compared with wild-type cells, the cwp double mutant cells are more readily stained by DAPI (Fig. 5B), indicating that indeed the cwp double mutation increases cell permeability. The observed general increase in the permeability of the cwp1 cwp2 double mutant cells is consistent with its increased sensitivity to chemical carcinogens examined in this study, with respect to both killing and the reporter-based toxicity assays.
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RNR3-lacZ Induction in erg6 or pdr5 Deletion Mutant
In order to examine the roles of the yeast membrane in preventing DNA-damaging chemicals from entering cells, we chose the ERG6 gene because this gene is required for a major biosynthesis pathway of ergosterol (Gaber et al., 1989
), and its inactivation has been reported to enhance cellular permeability (Emter et al., 2002; Welihinda et al., 1994). In this study, deletion of ERG6 did not alter RNR3-lacZ inducibility by MMS (Fig. 6A), HU (Fig. 6B), or 4-NQO (Fig. 6C), but increased the induction by phleomycin (Fig. 6D). However, compared with the cwp1 cwp2 double mutations, the effect of erg6 mutation on phleomycin-induced RNR3-lacZ expression is much less (cf. Figs. 2E,F and Fig. 6D). These results suggest that Erg6 plays a minor role in the RNR3-lacZ sensitivity to large molecular weight compounds.
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Pdr5 is a member of the ABC transporter superfamily involved in multidrug resistance (Balzi et al., 1994
) and its inactivation results in an enhanced sensitivity to a large number of xenobiotics including anticancer drugs (Hirata et al., 1994). Our results show that there is no significant difference in the expression of RNR3-lacZ between wild-type and the pdr5 deletion strain in the presence of all four testing compounds (Figs. 6A–D), indicating that Pdr5, and possibly other individual membrane transporters, do not affect the RNR3-lacZ based genotoxicity test of DNA-damaging carcinogens. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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To date, several genotoxicity testing systems have been developed based on the DNA damage induction of yeast genes (Afanassiev et al., 2000
; Jia et al., 2002), among which the RNR3-lacZ is the most sensitive reporter. In order to further enhance the sensitivity of this testing system, we previously reported the comprehensive characterization of the effects of inactivating various DNA repair pathways on the enhancement of RNR3-lacZ sensitivity (Jia and Xiao, 2003). In this report, we focused our attention on the issue of cell permeability and targeted three potential protection mechanisms, namely, cell wall, cell membrane, and the efflux pump.
The molecular size and type of chemicals appear to play a major role in their penetration through the cell wall and the molecular weight limit for penetration through the intact yeast cell wall has been reported to be around 700 Da (Scherrer et al., 1974). Despite its pivotal importance as a permeability barrier, research into the effects of cell wall defects on cell permeability to chemical carcinogens is surprisingly scarce, and none of the investigations has analyzed the cwp mutations. The introduction of a temperature-sensitive mutation has been reported to enhance cell wall permeability and sensitivity to chemical mutagens, but in a nonspecific manner (Pesheva et al., 2005; Staleva et al., 1996). Slg1 is a sensor protein for cell wall integrity and its inactivation results in hypersensitivity to the antitumor drug bleomycin (de Bettignies et al., 1999; Levin, 2005), which causes DNA strand breaks and is structurally related to phleomycin (Leduc et al., 2003). However, another report failed to show slg1 hypersensitivity to other mutagens such as ethyl methanesulfonate, MMS, and camptothecin (Ichikawa and Eki, 2006). In this study, deletion of both CWP1 and CWP2 genes encoding cell wall mannoproteins markedly increased cell wall permeability, and the effects are apparently synergistic. The enhancement is augmented with respect to large and complex compounds like phleomycin, although sensitivities to even small mutagens such as MMS and HU are also altered. The effect appears to be relatively independent of the nature of DNA damage, as the four selected chemicals cause rather different DNA lesions that are repaired by different mechanisms (Friedberg et al., 2006). These observations collectively indicate that cell wall permeability to different chemicals is a dynamic process and hence may not be as simple as originally thought (Scherrer et al., 1974). This universal effect of cwp mutations on the enhancement of the RNR3-lacZ sensitivity is apparently an asset to the improvement of the current testing systems and should also be applicable to other yeast-based toxicity tests, including perhaps nongenotoxicity tests.
The yeast cell wall plays an important role in maintaining cell integrity and morphogenesis, as well as in protecting against environmental stresses. A study of four single mutants (not including cwp), each affecting a cell wall component, revealed a global transcriptional regulation affecting about 300 genes (Lagorce et al., 2003), or 5% of the yeast genome. Genes affected by these cell wall mutations do not include RNR3, which is consistent with our observation that the basal-level RNR3-lacZ expression is not affected in cell wall mutants. We also demonstrated that the increased RNR3-lacZ sensitivity in the cwp double mutant is not unique for the RNR3-lacZ reporter, but is specific for genotoxic chemicals, which makes our cwp1 cwp2 cell wall mutant strain suitable for improving the RNR3-lacZ genotoxicity testing system.
Compared with the pronounced and synergistic effects of cwp mutations, the effects of erg6 or pdr5 mutation on the RNR3-lacZ genotoxicity testing system is much less dramatic. One can argue that Erg6 and Pdr5 may not represent the best choice of membrane protection and efflux pump to the genotoxic compounds, that these mutations may specifically affect certain chemicals, and that in each case, functional redundant proteins may exist that undermine the effect of single gene deletion. Deletion of ERG6 has been reported to alter the membrane fluidity and permeability and increase sensitivity to many chemicals (Emter et al., 2002; Gaber et al., 1989). In this study, the erg6 null mutant showed an increased but moderate sensitivity to crystal violet (data not shown) and phleomycin, but not to other genotoxic chemicals examined. A previous report (Aouida et al., 2003) indicated that the cell wall and plasma membrane partly account for bleomycin resistance by acting as two independent barriers. However, this study did not address which protein(s) in each component creates such barriers. Nevertheless, it would be of great interest to see whether the erg6 cwp triple mutations further enhance phleomycin and bleomycin sensitivity.
Although yeast ABC transporter systems have a broad range of chemical substrates with very different chemical structures and cellular targets, the presence of several transporters with overlapping specificities often prevents the observation of phenotypes associated with their single deletion mutants (Kolaczkowski and Goffeau, 1997), which is probably the case in this study using the pdr5 single mutant. Although it remains interesting to understand such functional redundancy, we feel that given the genetic complexity of yeast membrane transporters and their substrate specificity, inactivation of ABC transporter genes does not appear to be an attractive approach to enhancing general genotoxicity for the RNR3-lacZ test.
Previous attempts to disrupt yeast cell wall often required the culturing of cells under osmotic pressure; however, our cwp1 cwp2 mutant cells do not have such a problem. Deletion of both CWP1 and CWP2 genes has dramatically altered the outer cell wall structure and cell permeability has been significantly increased, especially with regard to large compounds. We have thus removed a major barrier to a broad range of genotoxic compounds and likely other uncharacterized chemicals. Hence, our results presented in this report provide a very useful approach to improving toxicity tests in yeasts.
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FUNDING |
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Natural Science Foundation of China grant (30440420603) to H.D. and W.X.; Natural Sciences and Engineering Research Council of Canada Discovery grant (OPG0138338) to W.X.; and Chinese Academy of Sciences creative direction program (KSCX2-SW-128) and a Chinese 863 program (2006AA06Z424) to H.D.
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ACKNOWLEDGMENTS |
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We wish to thank Dr S. Elledge for the RNR3-lacZ reporter plasmid, Michelle Hanna for proofreading the manuscript, and members from both laboratories for helpful discussion.
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