Functions of yeast helicase Ssl2p that are essential for viability are
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《核酸研究医学期刊》
1 Laboratory of Molecular and Biochemical Toxicology, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai 980-8578, Japan and 2 Laboratory of Analytical Chemistry, School of Pharmaceutical Sciences, Kitasato University, Minato-ku, Tokyo 108-8641, Japan
*To whom correspondence should be addressed. Tel: +81 22 217 6870; Fax: +81 22 217 6869; Email: naganuma@mail.pharm.tohoku.ac.jp
ABSTRACT
We have found that, in the yeast Saccharomyces cerevisiae, overexpression of the DNA helicase Ssl2p confers resistance to adriamycin. Ssl2p is involved, as a subunit of the basic transcription factor TFIIH, in the initiation of transcription and in nucleotide-excision repair (NER), and this helicase is essential for the survival of yeast cells. An examination of the relationship between the known functions of Ssl2p and adriamycin resistance indicated that overexpression of Ssl2p caused little or no increase in the rate of RNA synthesis and in NER. The absence of any involvement of NER in adriamycin resistance was supported by the finding that yeast cells that overexpressed the mutant form of Ssl2p that lacked the carboxy-terminal region, which is necessary for NER, remained resistant to adriamycin. When we examined the effects of overexpression in yeast of other mutant forms of Ssl2p with various deletions, we found that, of the 843 amino acids of Ssl2p, the entire amino acid sequence from position 81 to position 750 was necessary for adriamycin resistance. This region is identical to the region of Ssl2p that is necessary for the survival of yeast cells. Although this region contains helicase motifs, the overexpression of other yeast helicases, such as Rad3 and Sgs1, had little or no effect on adriamycin resistance, indicating that a mere increase in the intracellular level of helicases does not result in adriamycin resistance. Our results suggest that the functions of Ssl2p that are essential for yeast survival are also required for protection against adriamycin toxicity.
INTRODUCTION
Adriamycin is an anthracycline drug that is widely used in the treatment of several types of cancer (1). The proposed mechanisms of the antitumor activity of adriamycin include the inhibition of nucleic acid synthesis via intercalation into DNA, damage to DNA due to promotion of the formation of free radicals, promotion of lipid peroxidation, and promotion of the cleavage of DNA via inhibition of topoisomerase II (2).
The acquisition by neoplastic cells of resistance to adriamycin has emerged as a major obstacle in adriamycin chemotherapy. The best characterized mechanism of acquisition of resistance to adriamycin involves an increase in the concentration of P-glycoprotein, an ATP-binding cassette (ABC) transporter (3,4), which confers resistance to adriamycin by promoting the efflux of this drug from cells. Studies of adriamycin-resistant cell lines have suggested that overexpression of multidrug resistance protein (MRP) (5,6), which is a member of the ABC transporter family, as are P-glycoprotein, anthracycline resistance-associated (ARA) protein/MRP6 (7,8), breast cancer resistance protein (BCRP) (9,10), and lung resistance-related protein (LRP) (11,12), might also be involved in the acquisition of resistance to adriamycin. In addition, the involvement in adriamycin resistance of qualitative and quantitative changes in the expression of topoisomerase II (13,14) and overexpression of glutathione S-transferase (GST) (15) have been reported. However, none of these mechanisms alone explain the development of adriamycin resistance in many types of cancer cell.
To investigate possible novel mechanisms of acquisition of adriamycin resistance, we have been searching for genes that are related to adriamycin resistance using the budding yeast Saccharomyces cerevisiae. Since the functions of many yeast proteins are shared by homologous proteins in higher animals such as humans, yeast provides a useful model for studies of eukaryotes. Two yeast genes have been reported that appear to be involved in adriamycin resistance, namely, the gene for pleiotropic drug resistance 5 (PDR5) (16) and the gene for superoxide dismutase 2 (SOD2) (17).
A subunit of the basic transcription factor TFIIH, Ssl2p, is a protein that is important in nucleotide excision repair (NER) in yeast (18–20). In the present study, we found that overexpression of Ssl2p conferred resistance to adriamycin independently of its involvement in NER and RNA synthesis. In addition, our analysis of functional domain showed that not only the helicase motifs but almost the entire region of Ssl2p that is involved in the maintenance of cell viability are essential for adriamycin resistance. Our findings shed some light on the mechanism of adriamycin resistance and provide new information about the roles of Ssl2p, which have not been fully characterized.
MATERIALS AND METHODS
Yeast strains and media
Saccharomyces cerevisiae W303B (MAT his3 can1-100 ade2 leu2 trp1 ura3) was grown in yeast extract-peptone-dextrose (YPD) medium or in synthetic dextrose (SD) medium. Cells were transformed by the lithium acetate procedure (21–23).
Construction of pRS314-RAD3 and pRS314-SGS1
The RAD3 gene was amplified by the polymerase chain reaction (PCR) with yeast chromosomal DNA as template and the following oligonucleotides as primers: RAD3-F (5'-ATCACCGGAGAACCATTGACCA-3') and RAD3-R (5'-GATAAGCTAGAAAGTTGGTGGTTG-3'). The product of PCR was ligated into the pGEM-T easy vector (Promega, Madison, WI). For construction of pRS314-SGS1, we used pBluescript-SGS1, which was kindly provided by Prof. Takemi Enomoto (Tohoku University). The sequences of interest in these plasmids were subcloned into the TRP1-based single-copy plasmid pRS314.
Construction of Ssl2p-null yeast cells that harbored pRS316-SSL2
The SSL2 gene was disrupted as described elsewhere (24,25). pRS425-SSL2 was digested with BamHI and SalI and the fragment was ligated into pRS316 (a URA3-based plasmid), pRS314 and pBluescript SK(–) to generate pRS316-SSL2, pRS314-SSL2 and pBS-SSL2, respectively. The HIS3 gene was amplified by PCR with primers HIS3/SphI-F (5'-GCATGCCTCTTGGCCTCCTCTAG-3') and HIS3/NcoI-R (5'-CCATGGTCGTTCAGAATGACACG-3') and ligated into the pGEM-T easy vector. To construct pBS-ssl2::HIS3, the SphI-NcoI fragment of the HIS3 gene was ligated into pBS-SSL2 that had been digested with SphI and NcoI. The BamHI-SalI fragment of pBS-ssl2::HIS3 was introduced into the diploid strain of W303B yeast (MATa/ his4/his3 can1-100/can1-100 ade2/ade2 leu2/leu2 trp1/trp1 ura3/ura3) and transformants were selected on agar-solidified SD (–His) medium. Gene disruption was confirmed by PCR with primers SSL2-F (5'-ATGACGGACGTTGAAGGCTA-3') and SSL2-R (5'-TCACTTCTTCAAATTCTTAT-3') and Southern blotting analysis. The yeast strain SSL2/ssl2::HIS3 was transformed with pRS316-SSL2 and transformants were selected on agar-solidified SD (–Ura) medium. Induction of sporulation and tetrad analysis of the yeast strain SSL2/ssl2::HIS3 that harbored pRS316-SSL2 were performed by published methods (26) for isolation of the yeast Ssl2p-null mutant that harbored pRS316-SSL2 (MATa ssl2::HIS3, pRS316-SSL2).
Construction of yeast strains that overexpressed various mutant forms of Ssl2p
We constructed a variety of mutant SSL2 genes by creating pairs of BamHI sites in the open reading frame (ORF) of the SSL2 gene and excising the fragments between the respective pairs of BamHI sites. We created the BamHI sites with a Quikchange kit for site-directed mutagenesis (Stratagene, La Jolla, CA) as described by the manufacturer. Mutagenesis by PCR was performed using plasmid pRS314-SSL2 as the template and the various oligonucleotides shown in Table 1 as primers: 1F and 1R to create a BamHI site at position 1, as shown in Figure 5; 2F and 2R to create a BamHI site at position 2; 3F and 3R to create a BamHI site at position 3, etc. After creation of a pair of BamHI sites, the plasmid was cleaved with BamHI and self-ligated. Each resultant plasmid for expression of a mutant form of Ssl2p (pRS314-ssl2 mutants) was introduced into W303B cells and the transformants were selected on agar-solidified SD (–Trp) medium.
Table 1. Oligonucleotide primers used for construction of mutant forms of Ssl2p
Figure 5. Schematic representation of the structural domains of Ssl2p and of mutant proteins. The amino acids of Ssl2p are indicated by a scale below the Arabic numerals. The Roman numerals below the diagram of Ssl2p indicate the numbering of the helicase domains. Small numbers above the diagrams of Ssl2p-deletion mutants indicate the positions at which BamHI sites were created by site-directed mutagenesis. A summary of the results shown in Figures 6 and 7 is given on the right. ‘ADRr’ indicates the effect of overexpression of each mutant form of Ssl2p on the sensitivity of wild-type yeast to adriamycin (+, conferred resistance; –, did not confer resistance). ‘Survival’ indicates the ability of each mutant form of Ssl2p to support the survival of Ssl2p-null cells (ssl2). ‘NERa’ indicates the ability of each mutant form of Ssl2p to restore NER in ssl2 cells (+, exhibited NER activity; –, did not exhibit NER activity; N.D., not determined).
Construction of ssl2 mutant strains
We generated ssl2 mutant strains by replacing pRS316-SSL2, in strain MATa ssl2::HIS3 that harbored pRS316-SSL2 with pRS314-ssl2 mutants. The yeast strain MATa ssl2::HIS3 that harbored pRS316-SSL2 was transformed with each pRS314-ssl2 mutant and transformants were selected on agar-solidified SD (–Trp) medium. Colonies were inoculated into SD (–Trp) medium and cultured for 24 h. Then yeast cells were plated on agar-solidified SD (–Trp) medium that contained 0.5 mg/ml 5-fluoroorotic acid (5-FOA) which suppressed the growth of yeast cells that carried pRS316-SSL2 plasmids.
Quantitation of transcriptional activity
Yeast cells (1 x 107 cells) were cultured in 1 ml of SD (–Leu) medium in the presence of adriamycin for 30 min and then uracil (NENTM Life Science Products, Inc., Boston, MA) was added to a final concentration of 5 μCi/ml. After a 90-min incubation, cells were washed with TE buffer and resuspended in TES buffer . An equal volume of acid phenol was added to the suspension and the mixture was incubated for 60 min at 65°C and for 5 min on ice and then it was centrifuged at 20 000 g. The supernatant was subjected to phenol extraction and nucleic acids were precipitated in ethanol. The precipitate, containing total RNA, was resuspended in elution buffer (10 mM Tris–HCl (pH 7.5), 1 mM EDTA, 0.5% SDS), and the radioactivity of the suspension was measured with a liquid scintillation counter. Poly(A)+ RNA (mRNA) was purified from the total RNA with OligotexTM-dT30 Super (JSR, Tokyo, Japan) and radioactivity was quantitated by liquid scintillation counting.
Construction of plasmid for expression of a fusion protein of Ssl2 or Rad3 with GST
For construction of the GST-Ssl2 or GST-Rad3 plasmid, the SSL2 gene or RAD3 gene was amplified by PCR with the following respective primers: GST-SSL2-F (5'-CGTGGG ATCCCGGATGACGATGACAAAATGACGGACGTTGAAGGCTA-3') and GST-SSL2-R (5'-GATGAATTCACTT CTTCAAATTCTTAT-3'); GST-RAD3-F (5'-CGTGGG ATCCCGGATGACGATGACAAAATGAAGTTTTATGATGA-3') and GST-RAD3-R (5'-CGGCTCTTCGTATATTT CCCGGG-3'). Each product of PCR was digested with appropriate restriction enzymes and inserted into the multicloning site of pGEX-3X (Amersham Pharmacia Biotech, Piscataway, NJ).
Purification of GST fusion protein
Escherichia coli BL21 cells transformed with pGEX-SSL2, pGEX-RAD3 or pGEX-3X plasmids were grown in the medium in the presence of isopropyl-?-D-thiogalactopyranoside (0.1 mM) at 16°C for 4 h. The cells were harvested by centrifugation, and disrupted by sonication. The GST fusion proteins were purified by glutathione-Sepharose 4B (Amersham Pharmacia Biotech).
Quantitation of helicase activity
The helicase substrate was prepared as follows. 17mer universal primer (M13 primer M3) (Takara Bio Inc., Shiga, Japan) was annealed to single-stranded M13 mp18 DNA in 5 mM Tris–HCl buffer (pH 7.9) containing 25 mM NaCl and 2.5 mM MgCl2. The primer was then elongated with BcaBEST. DNA polymerase (2 U) (Takara Bio Inc.) in the presence of 50 μM dGTP, 50 μM dATP and 5 μCi of dCTP (3000 Ci/mmol) (NEN Life Science Products, Inc.). After 20 min at 50°C, the reaction mixture was incubated in the presence of 50 μM dCTP for another 20 min at 50°C. After phenol–chloroform extraction, the unincorporated nucleotides were removed by gel filtration on ProbeQuant. G-50 Micro Columns (Amersham Pharmacia Biotech). The DNA helicase assay was performed by the method of Roy et al. (27).
RESULTS AND DISCUSSION
To characterize the mechanisms of toxicity and of the cellular defenses against adriamycin, we searched for factors that might determine the sensitivity of yeast cells to adriamycin. We screened yeast cells that had been transformed with a yeast genomic DNA library for resistance to adriamycin and isolated clones that grew in the presence of a normally toxic concentration of adriamycin. Analysis of the resistant clones revealed that the SSL2 gene conferred resistance to adriamycin (28). As shown in Figure 2A, yeast cells that overexpressed the product of the SSL2 gene exhibited significant resistance to adriamycin. The SSL2 gene is a homolog of the human XPB gene and mutations in the latter gene are responsible for xeroderma pigmentosum (XP). The SSL2 gene encodes Ssl2p, a DNA helicase that is involved in the initiation of transcription and NER as a subunit of the basic transcription factor TFIIH (29,30). The human XPB protein appears to function similarly to Ssl2p in yeast. However, to our knowledge, there have been no reports of a relationship between the overexpression of Ssl2p and drug resistance.
Figure 2. Sensitivity of yeast cells that overexpressed Ssl2p to adriamycin, cisplatin and to UV-B. (A and B) Yeast cells that harbored pRS425 (control) or pRS425-SSL2 were suspended in liquid-SD (–Leu) medium at 1 x 104, 1 x 103, 1 x 102 and 1 x 10 cells/μl. Five microliters of each suspension of cells were spotted on agar-solidified SD (–Leu) medium prepared with and without adriamycin (80 μM) or cisplatin (150 μM). Plates were photographed after incubation for 48 h at 30°C. (C) Yeast cells that harbored pRS425 (control) or pRS425-SSL2 were spread on agar-solidified SD (–Leu) medium as indicated above and irradiated with UV-B (302 nm) for the indicated times. After incubation for 48 h at 30°C, each plate was photographed. Three separate experiments were performed and the results were reproducible.
The intercalation of adriamycin into DNA inhibits transcription of the DNA by RNA polymerase II (2). The Ss12p protein is one of the subunits of the basic transcription factor TFIIH and, thus, the possibility cannot be excluded that the transcriptional activity of TFIIH is enhanced by overexpression of Ss12p. However, overexpression of Ssl2p had no statistically significant effect on the synthesis of total RNA and of mRNA in yeast, as shown in Figure 1. Moreover, overexpression of Ssl2p had no significant effect on the inhibitory effect of adriamycin on RNA synthesis (Fig. 1). These observations suggest that adriamycin might cause some injury to cells at concentrations lower than those that are sufficient to inhibit transcription and that the overexpression of Ssl2p allows yeast somehow to avoid such injury, thereby exhibiting resistance to adriamycin. It seems plausible that adriamycin might exert its cytotoxic effects through some mechanism other than the inhibition of RNA synthesis. Moreover, a specific effect of Ssl2p on transcription of particular genes that are involved in the resistance to adriamycin could not be excluded.
Figure 1. Effects of overexpression of Ssl2p on the adriamycin-induced inhibition of transcription in yeast. Yeast cells (1 x 107 cells) were incubated in the presence of uracil for 90 min. Incorporation of uracil into total RNA (A) and into mRNA (B) in yeast cells was measured by liquid scintillation counting. Each column and bar represent the mean value and S.D. of results from three cultures. Differences between respective levels of uracil in total RNA and in mRNA were not statistically significant (Student’s t-test). See text for details.
The Ssl2p protein has NER activity and yeast cells that produce Ssl2p with deletions of the carboxy-terminal amino acids from position 751 and beyond are very sensitive to ultraviolet B (UV-B) irradiation and to cisplatin, an antitumor drug, because of their very low NER activity (31,32). This observation suggests that the adriamycin resistance of Ssl2p-overexpressing yeast cells might be a result of increased intracellular NER activity. However, we found that the sensitivity of Ssl2p-overexpressing yeast cells to cisplatin (Fig. 2B) and to UV-B (Fig. 2C) was similar to that of wild-type yeast cells. This finding indicates that overexpression of Ssl2p does not induce a marked increase in NER activity. Damia et al. (33) measured levels of XPB mRNA and NER activity in eight types of human cancer cell, and they reported that there was no correlation between levels of XPB mRNA and NER activity. Our results indicate that overexpression of Ssl2p had little or no effect on transcriptional and NER activity. An increase in the level of Ssl2p or of XPB might not result in an increase in the level of the TFIIH complex because the amount of Ssl2p (or of XPB) that is constitutively expressed in cells might be stoichiometrically equivalent to that of other components of TFIIH and might, thus, be sufficient for production of adequate TFIIH.
However, it appears that Ssl2p by itself has helicase activity in the absence of formation of the TFIIH complex (Fig. 4) and it is possible that this activity of Ssl2p might be involved in the acquisition of resistance to adriamycin. Adriamycin has been reported to inhibit both the helicase activity of SV40 T antigen and the helicase activity in extracts of human HeLa cells and of mouse FM3A cells (34,35). We cannot exclude the possibility that Ssl2p might be the target molecule of adriamycin, which directly inhibits the helicase activity of Ssl2p to suppress cell proliferation. Such suppression of cell proliferation would lead to apparent resistance to adriamycin because Ssl2p-overexpressing yeast would require higher concentrations of adriamycin for inhibition of the helicase activity of Ssl2p than would normal yeast cells. To examine the possible involvement of increased helicase activity in adriamycin resistance, we investigated the effects on the sensitivity of yeast cells to adriamycin of overexpression of helicases other than Ssl2p, namely, Sgs1p (36,37), which is involved in DNA recombination, and Rad3p (38), a subunit of the basic transcription factor TFIIH that is similar to Ss12p. Ssl2p, Sgs1p and Rad3p are members of helicase superfamily II. These proteins all have seven helicase motifs, whose amino acid sequences are strongly conserved (18) (Fig. 5). Yeast cells that overexpressed Ssl2p were significantly more resistant to adriamycin than control yeast cells that had been transformed with the empty vector (pRS314). In contrast, the sensitivity of Sgs1p- or Rad3p-overexpressing yeast cells to adriamycin was similar to that of the control yeast cells (Fig. 3). These results suggest that the acquisition of resistance to adriamycin as a result of overexpression of the Ssl2p helicase does not translate to all helicases, but is specific to Ssl2p.
Figure 4. Effects of adriamycin on the helicase activity of GST-Ssl2 or GST-Rad3. Reaction mixtures containing GST-Ssl2 or GST-Rad3 protein (300 μg), a partial duplex of circular single-stranded M13mp18 DNA as helicase substrate and a 32P-labeled 24-base complementary fragment were incubated in the presence of adriamycin at 37°C for 45 min. The displacement of the 24-base fragment from the single-stranded circle was determined by nondenaturing polyacrylamide gel electrophoresis and autoradiography.
Figure 3. Sensitivity to adriamycin of yeast cells that harbored plasmids that encoded Rad3, Ssl2p or Sgs1. Yeast cells (1 x 104 cells/200 μl) that harbored pRS314, pRS314-RAD3, pRS314-SSL2 or pRS314-SGS1 were grown in SD (–Trp) medium supplemented with the indicated concentrations of adriamycin. After a 48-h incubation, absorbance of cultures at 620 nm (A620) was measured spectrophotometrically. Each point represents the mean value and S.D. of results from three cultures. The absence of a bar indicates that the S.D. falls within the symbol.
We next examined the effect of adriamycin on the helicase activity in vitro of Ssl2p fused with GST. We found that adriamycin inhibited the helicase activity of both GST-Ssl2p and GST-Rad3 under identical conditions, with no difference in the extent of inhibition (Fig. 4). Similarly, aclarubicin, a derivative of adriamycin, inhibited the helicase activity of both GST-Ssl2p and GST-Rad3 (data not shown). When we consider these results together with the findings that Rad3-overexpressing yeast cells were not resistant to adriamycin (Fig. 3) and that Ssl2p-overexpressing yeast cells were not resistant to aclarubicin (28), it seems unlikely that adriamycin specifically inhibits the helicase activity of Ssl2p.
The Ssl2p protein contains a sequence that resembles a nuclear localization signal (NLS), a DNA-binding domain, and, in the central part of the amino acid sequence, seven helicase motifs that are conserved in helicase superfamily II (18). In addition, a region necessary for NER is present at the carboxyl terminus (31) (Fig. 5). To identify the functional domain(s) in Ssl2p that is related to adriamycin resistance, we prepared plasmids for the expression of mutant forms of Ssl2p with various deletions, as shown in Figure 5, and then we examined the adriamycin resistance of yeast cells that had been transformed with the individual plasmids. We found that deletion of the amino-terminal region (amino acids 14–80), which contains the NLS-like sequences, did not affect resistance to adriamycin (Fig. 6, N2), but the additional deletion of 93 amino acids (amino acids 81–173) towards the carboxyl terminus abolished the resistance to adriamycin (Fig. 6, N4). Although the NLS-like sequence in this region is also conserved in the human XPB gene, which is homologous to the gene for Ssl2p, it has been reported that this sequence in the product of XPB does not function as a NLS (39). Therefore, the nuclear localization of Ssl2p might not be necessary for the acquisition of Ssl2p-induced resistance to adriamycin. Alternatively, this region in the yeast protein might not function as a NLS. The mutant forms of Ssl2p that lacked the sequence that resembled a DNA-binding domain (N5), the acidic domain (N6), or the seven helicase motifs (H1–H5) also failed to confer resistance to adriamycin (Fig. 6, N4–N6, H1–H5). In contrast, the mutant that lacked the carboxy-terminal region (amino acids 751–833) that is necessary for NER did confer resistance to adriamycin (Fig. 6, C2), supporting the hypothesis that the mechanism of acquisition of adriamycin resistance does not involve the NER activity of Ssl2p. However, deletion of an additional 50 amino acids in the direction of the amino terminus abolished the resistance to adriamycin (Fig. 6, C1). These results suggest that the intervening region between the region that contains the amino-terminal NLS-like sequence and the carboxy-terminal NER-related region is necessary for the adriamycin resistance that is associated with overexpression of Ssl2p.
Figure 6. Effects of overexpression of mutant forms of Ssl2p on the sensitivity of wild-type yeast to adriamycin. Yeast cells (1 x 104 cells/200 μl) harboring pRS314, pRS314-SSL2, pRS314-N1, pRS314-N2, pRS314-N3, pRS314-N4, pRS314-N5, pRS314-N6 (A), pRS314-H1, pRS314-H2, pRS314-H3, pRS314-H4, pRS314-H5, pRS314-C1 or pRS314-C2 (B) were grown in SD (–Trp) medium that contained the indicated concentrations of adriamycin. After a 48-h incubation, absorbance of cultures at 620 nm (A620) was measured spectrophotometrically. For other details, see legend to Figure 3. For structures of mutant proteins, see Figure 5.
Since Ssl2p is essential for the survival of yeast cells (18), we analyzed the requirements for various regions within Ssl2p by a plasmid-shuffling method. In this procedure, we prepared yeast cells (MAT a ssl2::HIS3, pRS316-SSL2) that lacked the chromosomal SSL2 gene but had been transformed with a full-length SSL2-containing plasmid (pRS316-SSL2) and, in addition, with plasmids that encoded various partially deleted mutant forms of Ss12p. Then we eliminated the pRS316-SSL2 plasmids by selection with 5-FOA (40). This procedure allowed the growth exclusively of yeast cells that had been transformed with plasmids that encoded mutant forms of Ssl2p that were able to support cell survival. We found that deletion of the amino-terminal amino acids at positions 14–86 or of the carboxy-terminal amino acids at positions 751–833 was compatible with survival. In contrast, almost the entire intervening region was essential for the survival of yeast cells (Fig. 7A). This essential region within Ssl2p was identical to the intra-Ssl2p region that was essential for the acquisition of resistance to adriamycin (Figs 6 and 7A).
Figure 7. Effects of expression of mutant forms of Ssl2p on the viability and sensitivity of Ssl2p-null yeast cells. (A) An overnight culture of Ssl2p-null yeast cells (ssl2) harboring pRS316-SSL2 plus pRS314, pRS314-SSL2, pRS314-N1, pRS314-N2, pRS314-N3, pRS314-N4, pRS314-N5, pRS314-N6, pRS314-H1, pRS314-H2, pRS314-H3, pRS314-H4, pRS314-H5, pRS314-C1 or pRS314-C2 was diluted in SD (–Trp) medium (1 x 105 cells/μl), and 5 μl of each suspension of cells were spotted onto agar-solidified SD (–Trp) medium that contained 5-FOA, to eliminate pRS316-SSL2, and cultured for 72 h. (B) Ssl2p-null yeast cells (ssl2) harboring pRS314-SSL2, pRS314-N1, pRS314-N2, or pRS314-C2 were examined. An overnight culture of each yeast strain was diluted in SD (–Trp) medium at 1 x 104, 1 x 103, 1 x 102 and 1 x 10 cells/μl. Five microliters of each suspension of cells were spotted on agar-solidified SD (–Trp) medium and exposed to UV-B for the indicated times. To examine the sensitivity to cisplatin and adriamycin, the yeast cells were also grown on plates of agar-solidified SD (–Trp) medium that contained cisplatin (100 μM) or adriamycin (80 μM). After incubation for 48 h at 30°C, each plate was photographed. Three separate experiments were performed and the results were reproducible.
We examined the NER activity of the yeast cells that survived in the above experiment (Fig. 7A), determining the sensitivity of these cells to UV-B and to cisplatin as indicators of this activity. We found that the sensitivity to UV-B, to cisplatin and to adriamycin of cells that express the mutant form of Ssl2p that lacked the region that included amino-terminal NLS-like sequence (N2; 14–80 amino acids) was similar to that of yeast cells that expressed full-length Ssl2p (Fig. 7B). Compared with the latter cells, cells that expressed the mutant form of Ssl2p that lacked the carboxy-terminal region necessary for NER (C2; 751–833 amino acids) had markedly enhanced sensitivity to UV-B and to cisplatin (Fig. 7B), as reported elsewhere (31,32).
In this study, we found that overexpression of Ssl2p conferred resistance to adriamycin independently of other functions of this protein, for example, in NER and RNA synthesis, and that the central region of Ssl2p, which is involved in the maintenance of cell viability, is also essential for the acquisition of adriamycin resistance. However, the essential role played by Ssl2 in cell viability remains to be clarified. Lee et al. (41) reported that an Ssl2p mutant yeast strain (ssl2-rtt, Glu556Lys) undergoes frequent translocations of the translocation factor retrotransposon Ty1 to other genetic loci. The authors suggested that wild-type Ssl2p might inhibit the translocation of Ty1 via some activity different from the known activities of Ssl2 (41). It seems plausible that Ssl2p might have an as yet unidentified activity that is related to the maintenance of cell survival and that also confers resistance to adriamycin. Our findings not only shed some light on the mechanism of adriamycin resistance but also provide information about the roles and activities of Ssl2p.
ACKNOWLEDGEMENTS
The authors thank Kyowa Hakko Kogyo Co., Ltd (Tokyo, Japan) and Nippon Kayaku Co., Ltd (Tokyo, Japan) for providing the adriamycin and cisplatin, respectively, used in this study.
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*To whom correspondence should be addressed. Tel: +81 22 217 6870; Fax: +81 22 217 6869; Email: naganuma@mail.pharm.tohoku.ac.jp
ABSTRACT
We have found that, in the yeast Saccharomyces cerevisiae, overexpression of the DNA helicase Ssl2p confers resistance to adriamycin. Ssl2p is involved, as a subunit of the basic transcription factor TFIIH, in the initiation of transcription and in nucleotide-excision repair (NER), and this helicase is essential for the survival of yeast cells. An examination of the relationship between the known functions of Ssl2p and adriamycin resistance indicated that overexpression of Ssl2p caused little or no increase in the rate of RNA synthesis and in NER. The absence of any involvement of NER in adriamycin resistance was supported by the finding that yeast cells that overexpressed the mutant form of Ssl2p that lacked the carboxy-terminal region, which is necessary for NER, remained resistant to adriamycin. When we examined the effects of overexpression in yeast of other mutant forms of Ssl2p with various deletions, we found that, of the 843 amino acids of Ssl2p, the entire amino acid sequence from position 81 to position 750 was necessary for adriamycin resistance. This region is identical to the region of Ssl2p that is necessary for the survival of yeast cells. Although this region contains helicase motifs, the overexpression of other yeast helicases, such as Rad3 and Sgs1, had little or no effect on adriamycin resistance, indicating that a mere increase in the intracellular level of helicases does not result in adriamycin resistance. Our results suggest that the functions of Ssl2p that are essential for yeast survival are also required for protection against adriamycin toxicity.
INTRODUCTION
Adriamycin is an anthracycline drug that is widely used in the treatment of several types of cancer (1). The proposed mechanisms of the antitumor activity of adriamycin include the inhibition of nucleic acid synthesis via intercalation into DNA, damage to DNA due to promotion of the formation of free radicals, promotion of lipid peroxidation, and promotion of the cleavage of DNA via inhibition of topoisomerase II (2).
The acquisition by neoplastic cells of resistance to adriamycin has emerged as a major obstacle in adriamycin chemotherapy. The best characterized mechanism of acquisition of resistance to adriamycin involves an increase in the concentration of P-glycoprotein, an ATP-binding cassette (ABC) transporter (3,4), which confers resistance to adriamycin by promoting the efflux of this drug from cells. Studies of adriamycin-resistant cell lines have suggested that overexpression of multidrug resistance protein (MRP) (5,6), which is a member of the ABC transporter family, as are P-glycoprotein, anthracycline resistance-associated (ARA) protein/MRP6 (7,8), breast cancer resistance protein (BCRP) (9,10), and lung resistance-related protein (LRP) (11,12), might also be involved in the acquisition of resistance to adriamycin. In addition, the involvement in adriamycin resistance of qualitative and quantitative changes in the expression of topoisomerase II (13,14) and overexpression of glutathione S-transferase (GST) (15) have been reported. However, none of these mechanisms alone explain the development of adriamycin resistance in many types of cancer cell.
To investigate possible novel mechanisms of acquisition of adriamycin resistance, we have been searching for genes that are related to adriamycin resistance using the budding yeast Saccharomyces cerevisiae. Since the functions of many yeast proteins are shared by homologous proteins in higher animals such as humans, yeast provides a useful model for studies of eukaryotes. Two yeast genes have been reported that appear to be involved in adriamycin resistance, namely, the gene for pleiotropic drug resistance 5 (PDR5) (16) and the gene for superoxide dismutase 2 (SOD2) (17).
A subunit of the basic transcription factor TFIIH, Ssl2p, is a protein that is important in nucleotide excision repair (NER) in yeast (18–20). In the present study, we found that overexpression of Ssl2p conferred resistance to adriamycin independently of its involvement in NER and RNA synthesis. In addition, our analysis of functional domain showed that not only the helicase motifs but almost the entire region of Ssl2p that is involved in the maintenance of cell viability are essential for adriamycin resistance. Our findings shed some light on the mechanism of adriamycin resistance and provide new information about the roles of Ssl2p, which have not been fully characterized.
MATERIALS AND METHODS
Yeast strains and media
Saccharomyces cerevisiae W303B (MAT his3 can1-100 ade2 leu2 trp1 ura3) was grown in yeast extract-peptone-dextrose (YPD) medium or in synthetic dextrose (SD) medium. Cells were transformed by the lithium acetate procedure (21–23).
Construction of pRS314-RAD3 and pRS314-SGS1
The RAD3 gene was amplified by the polymerase chain reaction (PCR) with yeast chromosomal DNA as template and the following oligonucleotides as primers: RAD3-F (5'-ATCACCGGAGAACCATTGACCA-3') and RAD3-R (5'-GATAAGCTAGAAAGTTGGTGGTTG-3'). The product of PCR was ligated into the pGEM-T easy vector (Promega, Madison, WI). For construction of pRS314-SGS1, we used pBluescript-SGS1, which was kindly provided by Prof. Takemi Enomoto (Tohoku University). The sequences of interest in these plasmids were subcloned into the TRP1-based single-copy plasmid pRS314.
Construction of Ssl2p-null yeast cells that harbored pRS316-SSL2
The SSL2 gene was disrupted as described elsewhere (24,25). pRS425-SSL2 was digested with BamHI and SalI and the fragment was ligated into pRS316 (a URA3-based plasmid), pRS314 and pBluescript SK(–) to generate pRS316-SSL2, pRS314-SSL2 and pBS-SSL2, respectively. The HIS3 gene was amplified by PCR with primers HIS3/SphI-F (5'-GCATGCCTCTTGGCCTCCTCTAG-3') and HIS3/NcoI-R (5'-CCATGGTCGTTCAGAATGACACG-3') and ligated into the pGEM-T easy vector. To construct pBS-ssl2::HIS3, the SphI-NcoI fragment of the HIS3 gene was ligated into pBS-SSL2 that had been digested with SphI and NcoI. The BamHI-SalI fragment of pBS-ssl2::HIS3 was introduced into the diploid strain of W303B yeast (MATa/ his4/his3 can1-100/can1-100 ade2/ade2 leu2/leu2 trp1/trp1 ura3/ura3) and transformants were selected on agar-solidified SD (–His) medium. Gene disruption was confirmed by PCR with primers SSL2-F (5'-ATGACGGACGTTGAAGGCTA-3') and SSL2-R (5'-TCACTTCTTCAAATTCTTAT-3') and Southern blotting analysis. The yeast strain SSL2/ssl2::HIS3 was transformed with pRS316-SSL2 and transformants were selected on agar-solidified SD (–Ura) medium. Induction of sporulation and tetrad analysis of the yeast strain SSL2/ssl2::HIS3 that harbored pRS316-SSL2 were performed by published methods (26) for isolation of the yeast Ssl2p-null mutant that harbored pRS316-SSL2 (MATa ssl2::HIS3, pRS316-SSL2).
Construction of yeast strains that overexpressed various mutant forms of Ssl2p
We constructed a variety of mutant SSL2 genes by creating pairs of BamHI sites in the open reading frame (ORF) of the SSL2 gene and excising the fragments between the respective pairs of BamHI sites. We created the BamHI sites with a Quikchange kit for site-directed mutagenesis (Stratagene, La Jolla, CA) as described by the manufacturer. Mutagenesis by PCR was performed using plasmid pRS314-SSL2 as the template and the various oligonucleotides shown in Table 1 as primers: 1F and 1R to create a BamHI site at position 1, as shown in Figure 5; 2F and 2R to create a BamHI site at position 2; 3F and 3R to create a BamHI site at position 3, etc. After creation of a pair of BamHI sites, the plasmid was cleaved with BamHI and self-ligated. Each resultant plasmid for expression of a mutant form of Ssl2p (pRS314-ssl2 mutants) was introduced into W303B cells and the transformants were selected on agar-solidified SD (–Trp) medium.
Table 1. Oligonucleotide primers used for construction of mutant forms of Ssl2p
Figure 5. Schematic representation of the structural domains of Ssl2p and of mutant proteins. The amino acids of Ssl2p are indicated by a scale below the Arabic numerals. The Roman numerals below the diagram of Ssl2p indicate the numbering of the helicase domains. Small numbers above the diagrams of Ssl2p-deletion mutants indicate the positions at which BamHI sites were created by site-directed mutagenesis. A summary of the results shown in Figures 6 and 7 is given on the right. ‘ADRr’ indicates the effect of overexpression of each mutant form of Ssl2p on the sensitivity of wild-type yeast to adriamycin (+, conferred resistance; –, did not confer resistance). ‘Survival’ indicates the ability of each mutant form of Ssl2p to support the survival of Ssl2p-null cells (ssl2). ‘NERa’ indicates the ability of each mutant form of Ssl2p to restore NER in ssl2 cells (+, exhibited NER activity; –, did not exhibit NER activity; N.D., not determined).
Construction of ssl2 mutant strains
We generated ssl2 mutant strains by replacing pRS316-SSL2, in strain MATa ssl2::HIS3 that harbored pRS316-SSL2 with pRS314-ssl2 mutants. The yeast strain MATa ssl2::HIS3 that harbored pRS316-SSL2 was transformed with each pRS314-ssl2 mutant and transformants were selected on agar-solidified SD (–Trp) medium. Colonies were inoculated into SD (–Trp) medium and cultured for 24 h. Then yeast cells were plated on agar-solidified SD (–Trp) medium that contained 0.5 mg/ml 5-fluoroorotic acid (5-FOA) which suppressed the growth of yeast cells that carried pRS316-SSL2 plasmids.
Quantitation of transcriptional activity
Yeast cells (1 x 107 cells) were cultured in 1 ml of SD (–Leu) medium in the presence of adriamycin for 30 min and then uracil (NENTM Life Science Products, Inc., Boston, MA) was added to a final concentration of 5 μCi/ml. After a 90-min incubation, cells were washed with TE buffer and resuspended in TES buffer . An equal volume of acid phenol was added to the suspension and the mixture was incubated for 60 min at 65°C and for 5 min on ice and then it was centrifuged at 20 000 g. The supernatant was subjected to phenol extraction and nucleic acids were precipitated in ethanol. The precipitate, containing total RNA, was resuspended in elution buffer (10 mM Tris–HCl (pH 7.5), 1 mM EDTA, 0.5% SDS), and the radioactivity of the suspension was measured with a liquid scintillation counter. Poly(A)+ RNA (mRNA) was purified from the total RNA with OligotexTM-dT30 Super (JSR, Tokyo, Japan) and radioactivity was quantitated by liquid scintillation counting.
Construction of plasmid for expression of a fusion protein of Ssl2 or Rad3 with GST
For construction of the GST-Ssl2 or GST-Rad3 plasmid, the SSL2 gene or RAD3 gene was amplified by PCR with the following respective primers: GST-SSL2-F (5'-CGTGGG ATCCCGGATGACGATGACAAAATGACGGACGTTGAAGGCTA-3') and GST-SSL2-R (5'-GATGAATTCACTT CTTCAAATTCTTAT-3'); GST-RAD3-F (5'-CGTGGG ATCCCGGATGACGATGACAAAATGAAGTTTTATGATGA-3') and GST-RAD3-R (5'-CGGCTCTTCGTATATTT CCCGGG-3'). Each product of PCR was digested with appropriate restriction enzymes and inserted into the multicloning site of pGEX-3X (Amersham Pharmacia Biotech, Piscataway, NJ).
Purification of GST fusion protein
Escherichia coli BL21 cells transformed with pGEX-SSL2, pGEX-RAD3 or pGEX-3X plasmids were grown in the medium in the presence of isopropyl-?-D-thiogalactopyranoside (0.1 mM) at 16°C for 4 h. The cells were harvested by centrifugation, and disrupted by sonication. The GST fusion proteins were purified by glutathione-Sepharose 4B (Amersham Pharmacia Biotech).
Quantitation of helicase activity
The helicase substrate was prepared as follows. 17mer universal primer (M13 primer M3) (Takara Bio Inc., Shiga, Japan) was annealed to single-stranded M13 mp18 DNA in 5 mM Tris–HCl buffer (pH 7.9) containing 25 mM NaCl and 2.5 mM MgCl2. The primer was then elongated with BcaBEST. DNA polymerase (2 U) (Takara Bio Inc.) in the presence of 50 μM dGTP, 50 μM dATP and 5 μCi of dCTP (3000 Ci/mmol) (NEN Life Science Products, Inc.). After 20 min at 50°C, the reaction mixture was incubated in the presence of 50 μM dCTP for another 20 min at 50°C. After phenol–chloroform extraction, the unincorporated nucleotides were removed by gel filtration on ProbeQuant. G-50 Micro Columns (Amersham Pharmacia Biotech). The DNA helicase assay was performed by the method of Roy et al. (27).
RESULTS AND DISCUSSION
To characterize the mechanisms of toxicity and of the cellular defenses against adriamycin, we searched for factors that might determine the sensitivity of yeast cells to adriamycin. We screened yeast cells that had been transformed with a yeast genomic DNA library for resistance to adriamycin and isolated clones that grew in the presence of a normally toxic concentration of adriamycin. Analysis of the resistant clones revealed that the SSL2 gene conferred resistance to adriamycin (28). As shown in Figure 2A, yeast cells that overexpressed the product of the SSL2 gene exhibited significant resistance to adriamycin. The SSL2 gene is a homolog of the human XPB gene and mutations in the latter gene are responsible for xeroderma pigmentosum (XP). The SSL2 gene encodes Ssl2p, a DNA helicase that is involved in the initiation of transcription and NER as a subunit of the basic transcription factor TFIIH (29,30). The human XPB protein appears to function similarly to Ssl2p in yeast. However, to our knowledge, there have been no reports of a relationship between the overexpression of Ssl2p and drug resistance.
Figure 2. Sensitivity of yeast cells that overexpressed Ssl2p to adriamycin, cisplatin and to UV-B. (A and B) Yeast cells that harbored pRS425 (control) or pRS425-SSL2 were suspended in liquid-SD (–Leu) medium at 1 x 104, 1 x 103, 1 x 102 and 1 x 10 cells/μl. Five microliters of each suspension of cells were spotted on agar-solidified SD (–Leu) medium prepared with and without adriamycin (80 μM) or cisplatin (150 μM). Plates were photographed after incubation for 48 h at 30°C. (C) Yeast cells that harbored pRS425 (control) or pRS425-SSL2 were spread on agar-solidified SD (–Leu) medium as indicated above and irradiated with UV-B (302 nm) for the indicated times. After incubation for 48 h at 30°C, each plate was photographed. Three separate experiments were performed and the results were reproducible.
The intercalation of adriamycin into DNA inhibits transcription of the DNA by RNA polymerase II (2). The Ss12p protein is one of the subunits of the basic transcription factor TFIIH and, thus, the possibility cannot be excluded that the transcriptional activity of TFIIH is enhanced by overexpression of Ss12p. However, overexpression of Ssl2p had no statistically significant effect on the synthesis of total RNA and of mRNA in yeast, as shown in Figure 1. Moreover, overexpression of Ssl2p had no significant effect on the inhibitory effect of adriamycin on RNA synthesis (Fig. 1). These observations suggest that adriamycin might cause some injury to cells at concentrations lower than those that are sufficient to inhibit transcription and that the overexpression of Ssl2p allows yeast somehow to avoid such injury, thereby exhibiting resistance to adriamycin. It seems plausible that adriamycin might exert its cytotoxic effects through some mechanism other than the inhibition of RNA synthesis. Moreover, a specific effect of Ssl2p on transcription of particular genes that are involved in the resistance to adriamycin could not be excluded.
Figure 1. Effects of overexpression of Ssl2p on the adriamycin-induced inhibition of transcription in yeast. Yeast cells (1 x 107 cells) were incubated in the presence of uracil for 90 min. Incorporation of uracil into total RNA (A) and into mRNA (B) in yeast cells was measured by liquid scintillation counting. Each column and bar represent the mean value and S.D. of results from three cultures. Differences between respective levels of uracil in total RNA and in mRNA were not statistically significant (Student’s t-test). See text for details.
The Ssl2p protein has NER activity and yeast cells that produce Ssl2p with deletions of the carboxy-terminal amino acids from position 751 and beyond are very sensitive to ultraviolet B (UV-B) irradiation and to cisplatin, an antitumor drug, because of their very low NER activity (31,32). This observation suggests that the adriamycin resistance of Ssl2p-overexpressing yeast cells might be a result of increased intracellular NER activity. However, we found that the sensitivity of Ssl2p-overexpressing yeast cells to cisplatin (Fig. 2B) and to UV-B (Fig. 2C) was similar to that of wild-type yeast cells. This finding indicates that overexpression of Ssl2p does not induce a marked increase in NER activity. Damia et al. (33) measured levels of XPB mRNA and NER activity in eight types of human cancer cell, and they reported that there was no correlation between levels of XPB mRNA and NER activity. Our results indicate that overexpression of Ssl2p had little or no effect on transcriptional and NER activity. An increase in the level of Ssl2p or of XPB might not result in an increase in the level of the TFIIH complex because the amount of Ssl2p (or of XPB) that is constitutively expressed in cells might be stoichiometrically equivalent to that of other components of TFIIH and might, thus, be sufficient for production of adequate TFIIH.
However, it appears that Ssl2p by itself has helicase activity in the absence of formation of the TFIIH complex (Fig. 4) and it is possible that this activity of Ssl2p might be involved in the acquisition of resistance to adriamycin. Adriamycin has been reported to inhibit both the helicase activity of SV40 T antigen and the helicase activity in extracts of human HeLa cells and of mouse FM3A cells (34,35). We cannot exclude the possibility that Ssl2p might be the target molecule of adriamycin, which directly inhibits the helicase activity of Ssl2p to suppress cell proliferation. Such suppression of cell proliferation would lead to apparent resistance to adriamycin because Ssl2p-overexpressing yeast would require higher concentrations of adriamycin for inhibition of the helicase activity of Ssl2p than would normal yeast cells. To examine the possible involvement of increased helicase activity in adriamycin resistance, we investigated the effects on the sensitivity of yeast cells to adriamycin of overexpression of helicases other than Ssl2p, namely, Sgs1p (36,37), which is involved in DNA recombination, and Rad3p (38), a subunit of the basic transcription factor TFIIH that is similar to Ss12p. Ssl2p, Sgs1p and Rad3p are members of helicase superfamily II. These proteins all have seven helicase motifs, whose amino acid sequences are strongly conserved (18) (Fig. 5). Yeast cells that overexpressed Ssl2p were significantly more resistant to adriamycin than control yeast cells that had been transformed with the empty vector (pRS314). In contrast, the sensitivity of Sgs1p- or Rad3p-overexpressing yeast cells to adriamycin was similar to that of the control yeast cells (Fig. 3). These results suggest that the acquisition of resistance to adriamycin as a result of overexpression of the Ssl2p helicase does not translate to all helicases, but is specific to Ssl2p.
Figure 4. Effects of adriamycin on the helicase activity of GST-Ssl2 or GST-Rad3. Reaction mixtures containing GST-Ssl2 or GST-Rad3 protein (300 μg), a partial duplex of circular single-stranded M13mp18 DNA as helicase substrate and a 32P-labeled 24-base complementary fragment were incubated in the presence of adriamycin at 37°C for 45 min. The displacement of the 24-base fragment from the single-stranded circle was determined by nondenaturing polyacrylamide gel electrophoresis and autoradiography.
Figure 3. Sensitivity to adriamycin of yeast cells that harbored plasmids that encoded Rad3, Ssl2p or Sgs1. Yeast cells (1 x 104 cells/200 μl) that harbored pRS314, pRS314-RAD3, pRS314-SSL2 or pRS314-SGS1 were grown in SD (–Trp) medium supplemented with the indicated concentrations of adriamycin. After a 48-h incubation, absorbance of cultures at 620 nm (A620) was measured spectrophotometrically. Each point represents the mean value and S.D. of results from three cultures. The absence of a bar indicates that the S.D. falls within the symbol.
We next examined the effect of adriamycin on the helicase activity in vitro of Ssl2p fused with GST. We found that adriamycin inhibited the helicase activity of both GST-Ssl2p and GST-Rad3 under identical conditions, with no difference in the extent of inhibition (Fig. 4). Similarly, aclarubicin, a derivative of adriamycin, inhibited the helicase activity of both GST-Ssl2p and GST-Rad3 (data not shown). When we consider these results together with the findings that Rad3-overexpressing yeast cells were not resistant to adriamycin (Fig. 3) and that Ssl2p-overexpressing yeast cells were not resistant to aclarubicin (28), it seems unlikely that adriamycin specifically inhibits the helicase activity of Ssl2p.
The Ssl2p protein contains a sequence that resembles a nuclear localization signal (NLS), a DNA-binding domain, and, in the central part of the amino acid sequence, seven helicase motifs that are conserved in helicase superfamily II (18). In addition, a region necessary for NER is present at the carboxyl terminus (31) (Fig. 5). To identify the functional domain(s) in Ssl2p that is related to adriamycin resistance, we prepared plasmids for the expression of mutant forms of Ssl2p with various deletions, as shown in Figure 5, and then we examined the adriamycin resistance of yeast cells that had been transformed with the individual plasmids. We found that deletion of the amino-terminal region (amino acids 14–80), which contains the NLS-like sequences, did not affect resistance to adriamycin (Fig. 6, N2), but the additional deletion of 93 amino acids (amino acids 81–173) towards the carboxyl terminus abolished the resistance to adriamycin (Fig. 6, N4). Although the NLS-like sequence in this region is also conserved in the human XPB gene, which is homologous to the gene for Ssl2p, it has been reported that this sequence in the product of XPB does not function as a NLS (39). Therefore, the nuclear localization of Ssl2p might not be necessary for the acquisition of Ssl2p-induced resistance to adriamycin. Alternatively, this region in the yeast protein might not function as a NLS. The mutant forms of Ssl2p that lacked the sequence that resembled a DNA-binding domain (N5), the acidic domain (N6), or the seven helicase motifs (H1–H5) also failed to confer resistance to adriamycin (Fig. 6, N4–N6, H1–H5). In contrast, the mutant that lacked the carboxy-terminal region (amino acids 751–833) that is necessary for NER did confer resistance to adriamycin (Fig. 6, C2), supporting the hypothesis that the mechanism of acquisition of adriamycin resistance does not involve the NER activity of Ssl2p. However, deletion of an additional 50 amino acids in the direction of the amino terminus abolished the resistance to adriamycin (Fig. 6, C1). These results suggest that the intervening region between the region that contains the amino-terminal NLS-like sequence and the carboxy-terminal NER-related region is necessary for the adriamycin resistance that is associated with overexpression of Ssl2p.
Figure 6. Effects of overexpression of mutant forms of Ssl2p on the sensitivity of wild-type yeast to adriamycin. Yeast cells (1 x 104 cells/200 μl) harboring pRS314, pRS314-SSL2, pRS314-N1, pRS314-N2, pRS314-N3, pRS314-N4, pRS314-N5, pRS314-N6 (A), pRS314-H1, pRS314-H2, pRS314-H3, pRS314-H4, pRS314-H5, pRS314-C1 or pRS314-C2 (B) were grown in SD (–Trp) medium that contained the indicated concentrations of adriamycin. After a 48-h incubation, absorbance of cultures at 620 nm (A620) was measured spectrophotometrically. For other details, see legend to Figure 3. For structures of mutant proteins, see Figure 5.
Since Ssl2p is essential for the survival of yeast cells (18), we analyzed the requirements for various regions within Ssl2p by a plasmid-shuffling method. In this procedure, we prepared yeast cells (MAT a ssl2::HIS3, pRS316-SSL2) that lacked the chromosomal SSL2 gene but had been transformed with a full-length SSL2-containing plasmid (pRS316-SSL2) and, in addition, with plasmids that encoded various partially deleted mutant forms of Ss12p. Then we eliminated the pRS316-SSL2 plasmids by selection with 5-FOA (40). This procedure allowed the growth exclusively of yeast cells that had been transformed with plasmids that encoded mutant forms of Ssl2p that were able to support cell survival. We found that deletion of the amino-terminal amino acids at positions 14–86 or of the carboxy-terminal amino acids at positions 751–833 was compatible with survival. In contrast, almost the entire intervening region was essential for the survival of yeast cells (Fig. 7A). This essential region within Ssl2p was identical to the intra-Ssl2p region that was essential for the acquisition of resistance to adriamycin (Figs 6 and 7A).
Figure 7. Effects of expression of mutant forms of Ssl2p on the viability and sensitivity of Ssl2p-null yeast cells. (A) An overnight culture of Ssl2p-null yeast cells (ssl2) harboring pRS316-SSL2 plus pRS314, pRS314-SSL2, pRS314-N1, pRS314-N2, pRS314-N3, pRS314-N4, pRS314-N5, pRS314-N6, pRS314-H1, pRS314-H2, pRS314-H3, pRS314-H4, pRS314-H5, pRS314-C1 or pRS314-C2 was diluted in SD (–Trp) medium (1 x 105 cells/μl), and 5 μl of each suspension of cells were spotted onto agar-solidified SD (–Trp) medium that contained 5-FOA, to eliminate pRS316-SSL2, and cultured for 72 h. (B) Ssl2p-null yeast cells (ssl2) harboring pRS314-SSL2, pRS314-N1, pRS314-N2, or pRS314-C2 were examined. An overnight culture of each yeast strain was diluted in SD (–Trp) medium at 1 x 104, 1 x 103, 1 x 102 and 1 x 10 cells/μl. Five microliters of each suspension of cells were spotted on agar-solidified SD (–Trp) medium and exposed to UV-B for the indicated times. To examine the sensitivity to cisplatin and adriamycin, the yeast cells were also grown on plates of agar-solidified SD (–Trp) medium that contained cisplatin (100 μM) or adriamycin (80 μM). After incubation for 48 h at 30°C, each plate was photographed. Three separate experiments were performed and the results were reproducible.
We examined the NER activity of the yeast cells that survived in the above experiment (Fig. 7A), determining the sensitivity of these cells to UV-B and to cisplatin as indicators of this activity. We found that the sensitivity to UV-B, to cisplatin and to adriamycin of cells that express the mutant form of Ssl2p that lacked the region that included amino-terminal NLS-like sequence (N2; 14–80 amino acids) was similar to that of yeast cells that expressed full-length Ssl2p (Fig. 7B). Compared with the latter cells, cells that expressed the mutant form of Ssl2p that lacked the carboxy-terminal region necessary for NER (C2; 751–833 amino acids) had markedly enhanced sensitivity to UV-B and to cisplatin (Fig. 7B), as reported elsewhere (31,32).
In this study, we found that overexpression of Ssl2p conferred resistance to adriamycin independently of other functions of this protein, for example, in NER and RNA synthesis, and that the central region of Ssl2p, which is involved in the maintenance of cell viability, is also essential for the acquisition of adriamycin resistance. However, the essential role played by Ssl2 in cell viability remains to be clarified. Lee et al. (41) reported that an Ssl2p mutant yeast strain (ssl2-rtt, Glu556Lys) undergoes frequent translocations of the translocation factor retrotransposon Ty1 to other genetic loci. The authors suggested that wild-type Ssl2p might inhibit the translocation of Ty1 via some activity different from the known activities of Ssl2 (41). It seems plausible that Ssl2p might have an as yet unidentified activity that is related to the maintenance of cell survival and that also confers resistance to adriamycin. Our findings not only shed some light on the mechanism of adriamycin resistance but also provide information about the roles and activities of Ssl2p.
ACKNOWLEDGEMENTS
The authors thank Kyowa Hakko Kogyo Co., Ltd (Tokyo, Japan) and Nippon Kayaku Co., Ltd (Tokyo, Japan) for providing the adriamycin and cisplatin, respectively, used in this study.
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