| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Regular Articles |
1 University of Montreal, Guy-Bernier Research Centre, Montreal, Quebec, Canada and 2 Swiss Federal Institute of Technology Zurich, Institute of Biochemistry, Zurich, Switzerland
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
BLM directly attacks DNA via a free radical-driven process to generate a narrow set of lesions similar to those induced by ionizing radiation (e.g., apurinic/apyrimidinic sites, single-strand breaks containing 3'-blocking groups that inhibit the progression of DNA polymerase) and double-strand breaks (10 , 13 , 14) . Such DNA lesions are known to be highly genotoxic, and, furthermore, it is clear that they account for the potent antitumor effects of the drug (15, 16, 17, 18) . Thus, increased levels of DNA-repair enzymes are likely to contribute to BLM tumor resistance. Indeed, a recent study demonstrated that overproduction of hApe/ref-1, which belongs to a family of enzymes that can directly repair BLM-induced DNA lesions (19, 20, 21, 22) , engenders a 2–3-fold increase in protection against BLM in testicular cancer cells (23) . Whether hApe/ref-1 plays a role in BLM-resistant tumors awaits further investigation.
Previous studies have indicated that processes other than direct removal of BLM-induced DNA damage are required to protect the budding yeast Saccharomyces cerevisiae from the lethal effects of BLM (see review in Ref. 24 ). To better understand such processes, we performed two genome-wide screens using an entire collection of haploid yeast mutants to systematically identify variants that were hypersensitive to BLM. Analysis of these hypersensitive mutants revealed many interesting genes corresponding to various diverse functional groups (see below). However, of particular interest here, the screens were also designed to isolate BLM-resistant strains, a class that had not been sought previously. Only five resistant mutants could be reproducibly isolated by the screens. Among these mutants was one defective in the L-carnitine transporter Agp2, which we demonstrated to regulate the entry of BLM into the cell and, as a consequence, the cytotoxic potential of the drug. Two of the other BLM-resistant mutants were deficient in either of the kinases Ptk2 or Sky1, which are both involved in polyamine transport. Our data clearly indicate a critical role for membrane transport in mediating BLM resistance in yeast.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
High-Throughput BLM Screen and Drug Sensitivity Analysis.
The strains were arrayed in quadruplet, in a clockwise manner to create a dilution in a given square. A total of 96 colonies were arrayed per solid YPD plate containing either no drug or 2.0 or 7.5 µg/ml BLM A5 (BLM, ICN Pharmaceuticals), using a 96-floating pin replicator operated by a Biomek 2000 (Beckman). Plates were incubated for 48 h at 30°C and photographed with a digital camera (Gel Doc 2000, Bio-Rad) to visually compare the growth of every mutant in the presence or absence of BLM (see Fig. 1
). Putative BLM-hypersensitive or -resistant strains were further analyzed by spot-test analysis (26)
. Briefly, exponentially growing cultures in YPD were adjusted to an A600 of 0.6, and 5 µl of a set of serial dilutions, as indicated, were spotted onto YPD supplemented with 1.0 or 4.0 µg/ml BLM to test for BLM hypersensitivity and resistance, respectively.
|
-rays (0 to 40-Krad) in either YDP or selective media. Cells were washed, serially diluted in 20 mM potassium phosphate buffer (pH 7.0), and plated onto solid YPD to score for survivors after 48 h growth at 30°C.
Primer Extension Assay.
This assay was performed as described previously (29)
. Briefly, chromosomal DNA isolated from BLM-untreated and -treated (10 µg/ml for 2 h) cells was assessed for the ability to permit incorporation of [methyl-3H]dTMP by purified E. coli DNA polymerase 1 (Promega). Apn1 used for pretreating the chromosomal DNA was purified in this laboratory (30)
. Specific activity of the labeled [methyl-3H]dTTP (NEN Life Sciences Products) was 1230 cpm/pmol.
Construction of pAGP2-Green Fluorescent Protein and Yeast Transformation.
The primers AGP2-F1: 5'-CCCTGCCTCTGTGAGCTCTGCATTGTATACTATATAC-3' and AGP2-GFPR1: 5'-CATTATGCTTTGGTACCATATTGAAATTTTTCGAAGGAGC-3' (restriction sites SacI and KpnI underlined) were used to amplify the entire AGP2 gene (i.e., coding region and 430-bp of the putative promoter region) from yeast genomic DNA using PCR. The amplified product, digested with SacI and KpnI, was cloned into the multi-copy plasmid pYEp-GFP lacking a promoter (31)
to produce the expression plasmid pAGP2-GFP. In a similar manner, the AGP2 gene with 200 nucleotides downstream was cloned into the single and multi-copy vectors YEplac33 and YEplac195 to generate the plasmids pAGP2-1 and pAGP2-2, respectively. pYEp-GFP, pAGP2-GFP, pAGP2-1, and pAGP2-2 were separately introduced into the indicated yeast strains by the lithium acetate method (32)
.
Immunoblot Analysis.
Total protein extracts were prepared from cells carrying either pYEp-GFP or pAGP2-GFP and probe by Western analysis using anti-GFP monoclonal antibody (Clontech) as described previously (31)
.
Coupling of FITC to BLM and Uptake Analysis.
A 100-µl aliquot of 2.1 mM of the fluorescent molecule 5-(and-6)-carboxyl fluorescein [5(6)
-FAM] succinimidyl ester (SE; succinimidyl-FITC, Molecular Probes) in 0.2 M NaHCO3 (pH 9.0) was added to 300 µl of 0.6 mM BLM (prepared in 0.2 M NaHCO3, pH 8.3), and the mixture was incubated for 2 h at room temperature. The reaction was stopped by the addition of 10 µl of 1.5 M hydroxylamine (pH 8.5). A control reaction was done in parallel, except for the addition of BLM. The reaction products were resolved on 1% agarose gel for 2 h at 100 V using 40 mM 2-(N-morpholino) ethanesulfonic acid monohydrate (USB) buffer (pH 6.0). The active product (slower migrating band toward the cathode) containing FITC coupled to BLM was visualized by a hand-held long wavelength UV lamp, excised, placed at -80°C until frozen, centrifuged at 9,000 x g/5 min, lyophilized, and resuspended in 300 µl of sterile water. A fluorescently labeled for of BLM (F-BLM) was aliquoted and stored at -20°C. The concentration of F-BLM was assessed against natural BLM by absorbance at 292 nm and by DNA fragmentation of the purified plasmid pBluescript (data not shown). The genotoxicity measurement was determined by clonogenic assays of a parent strain YW465 and the DNA repair deficient mutant YW778 (Ref. 30
; data not shown). For F-BLM uptake studies, exponentially growing cultures were washed twice in water and resuspended in 50 mM citrate acetate buffer (pH 5.5) containing 2% glucose and 0.05% Tween 20 at a density of 2 x 108 cells/ml. F-BLM (0.1 µg) was added to 100 µl of the cells, incubated at 30°C in the dark with mild shaking for the indicated times, and uptake was stopped by adding 1 ml of stop buffer [50 mM citrate acetate buffer (pH 5.5), 2 mM sodium azide]. Cells were washed three times with PBS buffer, resuspended in 500 µl of PBS, sonicated at 30% for 15 s, and the extent of F-BLM uptake was measured using a fluorescent spectrophotometer at 495/525 nm (excitation/emission).
Fluorescence Microscopy.
Cells were grown to a density of 2 x 108 cells/ml, washed two times in water, and resuspended in uptake buffer. An aliquot (100 µl) of cells was incubated with either F-BLM (0.72 µg/ml) in the absence or presence of L-carnitine (at the concentrations indicated in the text) at 30°C in the dark with mild shaking for 1 h. Cells were washed three times with 1 ml of PBS, resuspended in 100 µl of PBS, and 3 µl were mounted on microscope slides for fluorescent microscopy. For the negative and positive controls, cells were incubated with FITC (0.72 µg/ml for 1 h) and FM4–64 (30 µM for 15 min, Molecular Probes), respectively. Cells were photographed at x100 magnification by imaging camera (Retiga GX 32–002TB-303) attached to a Leica DMRE immunofluorescent microscope, and images were processed by the Macintosh OpenLab program.
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
70% growth of the parent and <20% for hypersensitive mutants. In contrast, the higher concentration of BLM significantly reduced growth of the parent and completely prevented growth of all hypersensitive mutants, but permitted normal proliferation of resistant mutants. Although this type of screen has the limitation of potentially overlooking mutants with unstable or weak phenotypes and/or possessing a redundant function, we have nonetheless successfully identified over 200 mutants.
The two screens reproducibly identified 260 BLM-hypersensitive and, remarkably, only 5 BLM-resistant mutants (Fig. 1
, representative plate depicting two BLM-hypersensitive and one-resistant mutant). A total of 67 BLM-hypersensitive mutants could not be reproduced between screens and were, therefore, eliminated from further analysis. To confirm the results of the initial screen, the 260 BLM-hypersensitive and 5 BLM-resistant mutants were subjected to spot-test analysis on solid YPD plates containing BLM (Fig. 2A
shows randomly tested strains). The data revealed 29 strains that were not actually hypersensitive to BLM, leaving a total of 231 displaying 4–20-fold higher levels of sensitivity to the drug relative to the parental counterpart (Table 1)
. Thus, the two independent high-throughput screens generated a relatively low fraction (12%) of false positives, analogous to other screens performed with growth assessment on plates containing drugs, such as hydroxyurea, to select for DNA-replication defective mutants (33)
.3
The spot-test analysis also clearly confirmed that all five putative BLM-resistant mutants (Fig. 2B)
indeed manifested extremely high resistance to BLM (see below).
|
|
|
with those reported previously to protect yeast against other DNA damaging agents, i.e., either the alkylating agent methyl methane sulfonate (MMS), or -rays (34
, 35)
. We observed that 27 BLM-hypersensitive genes were also involved in sensitivity to MMS (Table 1
, denoted by superscripted a). There were also 38 genes in common that protected cells from -ray exposure (Table 1
, superscripted b), and 14 of which overlapped with those identified in the MMS screen (Table 1
, superscripted a + b). In summary, among BLM-hypersensitive genes, 52 exhibited cross-sensitivity to other DNA-damaging agents; thus, the remaining 179 could have been expected to play various roles in protecting yeast against BLM-induced lethality. To test this notion further, we randomly checked 84 of the 179 mutants mediating BLM-specific protection for cross-sensitivity to MMS, as well as to 4-nitroquinoline-1-oxide (4-NQO), which induces bulky DNA adducts. None of the 84 mutants showed any significant sensitivity to either MMS or 4-NQO, as assessed by gradient plate assay (data not shown).
Functional Assignment of the Defective Genes Causing BLM Hypersensitivity.
Searches performed with the Saccharomyces Genome Database (SGD) and YPD databases revealed a diverse collection of BLM-hypersensitive genes (Table 1
and Supplementary Table 2, showing a brief summary of the function encoded by each gene). Loci implicated in vacuolar function represented the largest number of BLM-hypersensitive mutants (40 genes), followed by 34, 24, 23, 16, and 15 genes involved in mitochondrial function, transcription machinery, the DNA damage response, protein synthesis, and cell wall biosynthesis, respectively. At least 22 genes were relegated to "miscellaneous" pathways, and a further 15 had no previously assigned biological function. The database search also revealed that 79 of the BLM hypersensitivity genes encode proteins that share between 22 and 68% homology with potential human functional counterparts (Table 1
, conserved genes are indicated by an asterisk). Thus, it is apparent that human cells may use some similar cellular processes as yeast to mitigate BLM-induced lethality.
BLM-Resistant Mutants.
The five BLM-resistant mutants identified by our genome-wide screens are deleted for the following genes: AGP2, SKY1, PTK2, FES1, and YGL007W (Table 1)
. Remarkably, three among the 5 BLM-resistant genes, i.e., AGP2, PTK2, and SKY1, encode proteins that execute a role in plasma membrane transport. Agp2 has been shown previously to transport L-carnitine, whereas Ptk2 and Sky1 were shown to be members of two different kinase families that act independently to positively influence polyamine transport (36, 37, 38)
. The two other BLM-resistant genes, FES1 and YGL007W, have no documented role in transport function. The protein encoded by FES1 is implicated in a complex that regulates protein translation (39)
. In the case of YGL007W, there are currently no clues regarding its function.
Clonogenic survival analysis revealed that all five mutants (agp2
, ptk2, sky1, fes1, and ygl007w) were extremely resistant to BLM, as compared with the parent (see Fig. 3D
and below). The most resistant to BLM was agp2, followed by ptk2, sky1, fes1, and ygl007w, all of which showed nearly equal levels of resistance to the drug (see Fig. 3D
and below). A similar result was obtained when each of these five genes was independently deleted in a different parental background (SEY6210), suggesting that the BLM-resistant phenotype is not strain specific (data not shown). On the basis of the above-mentioned information, it is reasonable to postulate that Agp2 may act as a transporter of BLM.
|
, F-BLM uptake was significantly reduced in the agp2 mutant, as compared with the parent. The fractional uptake of F-BLM into the agp2 mutant was attributable to fluid endocytosis into vacuoles (data not shown). It is noteworthy that the other 4 BLM-resistant mutants also showed similar reduction in F-BLM uptake as the agp2 mutant (data not shown).
We next tested the effect of Agp2 overexpression on F-BLM uptake. Introduction of a multi-copy plasmid pAGP2-GFP into either the parent or the agp2 mutant expressed the expected Mr 93,000 Agp2-GFP fusion protein, which appeared as a broad band perhaps attributable to posttranslational modifications, e.g., glycosylation or phosphorylation (Fig. 3B)
. Agp2-GFP overproduction greatly enhanced the uptake of F-BLM into the cells (Fig. 3A
; for simplicity, shown only for the parent). Because the only documented function for the Agp2 protein is to transport L-carnitine into the cell, we tested whether this natural substrate of the transporter could compete with and prevent F-BLM entry into the parent strain overexpressing Agp2-GFP. As shown in Fig. 3A
, L-carnitine clearly diminished F-BLM uptake into the cells to the residual level observed in the agp2 mutant. These data strongly indicate that the L-carnitine transporter Agp2 is also required to efficiently transport BLM into the cell. To further assess this, the intracellular distribution of F-BLM was examined in the parent and agp2 mutant using fluorescent microscopy. F-BLM intensely stained the vacuoles of the parent strain, but only weakly those of the agp2 mutant (Fig. 3C)
. In control experiments, fluorescein (FITC) showed no detectable staining of the vacuoles (Fig. 3C)
, whereas the vacuolar marker FM4–64 was localized to this organelle of both strains (data not shown). Introduction of a single-copy plasmid pAGP2-1, bearing an untagged form of the entire AGP2 gene, into the agp2 mutant restored parental level of F-BLM in the vacuoles (Fig. 3C)
. Likewise, a multi-copy plasmid pAGP2-2 carrying the AGP2 gene caused accumulation of a higher level of F-BLM in the vacuoles of the agp2 mutant, although this was difficult to document because of the increased brightness (Fig. 3C)
. As expected from the uptake studies (Fig. 3A)
, the addition of L-carnitine (1 mM) blocked the accumulation of F-BLM in the vacuoles of either the parent or agp2 mutant carrying pAGP2-2 (Fig. 3C
, shown for the parent). Taken together, these findings confirm that the agp2 mutant is indeed defective for the AGP2 gene and further strengthen the notion that the encoded protein Agp2 can act as a transporter of BLM. The data also indicate that after F-BLM uptake, the drug is channeled to the vacuoles for detoxification. In fact, mutants (e.g., end3) that are defective in the endocytic pathway to the vacuoles accumulated F-BLM in the cytoplasm and displayed hypersensitivity to the drug (Table 1
, Fig. 2A
, and data not shown).
We next checked whether the level of Agp2 expression correlated directly with cell survival after BLM exposure. As shown in Fig. 3D
, the agp2 mutant was at least 1000-fold more resistant than the parent. Overproduction of the Agp2-GFP fusion caused parental, or agp2 mutant, cells to become exquisitely sensitive to BLM (Fig. 3D
, shown for the parent). No difference in cell survival was observed between the parent and the agp2 mutant (either with or without overexpression of Agp2-GFP) after exposure to other DNA-damaging agents including MMS, cisplatin, -rays, and 4-NQO (Fig. 3E
, shown for 4-NQO). Thus, the data clearly indicate that Agp2 levels mediate cellular susceptibility to BLM-induced killing.
Agp2 Overproducing Cells Accumulate High Levels of BLM-Induced DNA Lesions.
Using a primer extension assay (29
, 40)
, we next examined whether the BLM sensitization of cells overproducing Agp2-GFP was a result of increased damage to chromosomal DNA. This assay detects a major class of BLM-induced oxidative DNA lesions that are processed into 3'-hydroxyl groups in vivo. These 3'-hydroxyl groups can then be used as substrates to monitor the in vitro incorporation of [methyl-3H]dTMP by purified E. coli DNA polymerase I (29
, 40)
. Briefly, exponentially growing cultures of the parent strain (BY4741) carrying either the plasmid pYEp-GFP or pAGP2-GFP was treated with BLM and the isolated chromosomal DNA analyzed for the ability to incorporate [methyl-3H]dTMP. BLM-damaged DNA isolated from strain BY4741/pAGP2-GFP showed a higher level of [methyl-3H]dTMP incorporation, as compared with the damaged DNA derived from strain BY4741/pYEp-GFP (Fig. 4)
. Preincubation of the BLM-damaged DNA obtained from strain BY4741/pAGP2-GFP with the purified yeast DNA repair enzyme Apn1, which can remove BLM-induced DNA lesions (29)
, caused a stimulation of [methyl-3H]dTMP incorporation (Fig. 4)
. In contrast, no Apn1-stimulated [methyl-3H]dTMP incorporation was observed into the BLM-damaged DNA isolated from strain BY4741/pYEp-GFP (Fig. 4)
. In control experiments, DNA isolated from the drug-untreated strain BY4741/pAGP2-GFP showed a very weak level of [methyl-3H]dTMP incorporation in the absence or presence of Apn1-preincubation (Fig. 4)
. These data indicate that cells overexpressing Agp2-GFP are subject to increase levels of BLM-induced DNA lesions, which are not efficiently removed.
|
mutant challenged with BLM. As shown in Fig. 4
, DNA derived from the BLM-exposed agp2 cells showed no detectable [methyl-3H]dTMP incorporation, as compared with the treated parent cells (Fig. 4)
. This finding distinctly indicates that the Agp2 transporter plays a critical role in allowing BLM to eventually attack cellular DNA. |
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
, which underscores the validity of our experimental approach. It is noteworthy that the remaining genes RAD50, RAD51, RAD52, and RAD55 of this DNA repair pathway were not recovered by the screens and may be a reflection of the genetic instability of the corresponding mutants causing an unstable phenotype (33)
. However, it should be noted that unlike genes of the DNA double-strand break repair pathway, the known genes (FKS1, SLG1, and GAS1) of the cell wall biosynthetic pathway involved in BLM-defense were recovered (26)
. Thus, of the 15 previously reported nonessential genes that protect yeast cells from BLM lethality, 4 were not isolated, suggesting a false-negative rate of 26%, although this could be an overestimate (24)
.
As anticipated, mutants of the nucleotide excision-repair pathway were not isolated, because this pathway is ostensibly not required to repair BLM-induced DNA lesions. Interestingly, several unexpected gene products, possibly involved in DNA repair, were also recovered. Of particular interest are chromatin remodeling factors such as Snf6 (a subunit of SWI/SNF ATP-dependent chromatin assembly factor), Spt10 (a putative histone acetylase), and Spt20 (a component of the SAGA coactivator complex; Refs. 41, 42, 43
). The involvement of such factors suggests a requirement for remodeling BLM-damaged DNA to promote repair. This possibility is consistent with previous studies showing that mutations in the INO80 ATPase chromatin remodeling complex of yeast, and in the TIP60 histone acetylase of mammalian cells, cause sensitivity to MMS and -rays, respectively (44, 45, 46)
.
The isolation of several BLM-hypersensitive mutants deleted for genes (e.g., IMP2, RPB7, and SLG1) previously identified by different methods further emphasizes the utility of the current experimental approach (26
, 29
, 47)
. Although we previously showed that IMP2 encodes a transcriptional co-activator, neither the gene(s) that it controls nor its mechanism of action is known (29)
. Thus, the current list of BLM-hypersensitive genes (Table 1)
should facilitate the identification of Imp2 transcriptional target(s). Whether the Imp2-target gene(s) encodes a protein that either repairs BLM-induced DNA lesions, and/or detoxifies the drug will require additional investigation. In the current study, we have identified several transcriptional activators including Ccr4, Swi4, Pop2, and Spt7 (Table 1)
, which Imp2 could co-activate in response to BLM exposure.
In addition to the relatively large collection of 231 BLM hypersensitive genes, it is remarkable that our screen revealed only 5 that cause resistance to BLM when deleted, i.e., AGP2, PTK2, SKY1, FES1, and YGL007W. Among these latter genes, AGP2 was previously shown to encode a plasma membrane transporter of L-carnitine (36)
. Herein, we have demonstrated a novel function for Agp2 by clearly showing that yeast mutants deleted for this gene are deficient in transport of BLM. Furthermore, the overproduction of Agp2 increases drug influx, resulting in severe damage to the genome and leading to rapid cell death. It is worthy to note that although Agp2 overproduction caused only a 2-fold increase in F-BLM uptake, the cytotoxic effect was much more dramatic (Fig. 3)
. This apparent discrepancy could be explained if F-BLM transport is impeded as Agp2-overproducing cells are undergoing cell death from the drug influx. Alternatively, the 2-fold increase in F-BLM uptake may lead to more toxic DNA lesions. In fact, it is known that distinct DNA lesions are generated in yeast cells in a manner that depends on the BLM intracellular concentration (48)
. Nonetheless, the consequences of altering Agp2 levels appear to be specific for BLM, because either Agp2-overproducing or -null strains show parental resistance to several other diverse genotoxic agents including MMS, 4-NQO, cisplatin, and camptothecin (Fig. 3
and data not shown). In short, Agp2 exhibits substrate specificity toward BLM, and modulation of its activity impacts BLM resistance. On the other hand, the precise mechanism by which the other four BLM-resistance genes regulate protection awaits further experimentation. However, although the potential roles of Fes1 and Ygl007w in BLM resistance remain completely unknown, the Ptk2 and Sky1 kinases have been shown to control transporter activity (49
, 50)
. Indeed, Ptk2 has been shown to be a regulator of the proton pump Pma1 in yeast (50)
. Moreover, Ptk2 and Sky1 are independently involved in polyamine transport in yeast (37
, 38)
. As such it is distinctly possible that Ptk2 and/or Sky1 may also directly regulate Agp2 activity.
The fact that our genome-wide screen revealed altered transport as a critical mechanism of BLM resistance in yeast raises the possibility that mammalian cells may use similar genes to regulate protection against BLM. Thus far, two high-affinity L-carnitine transporters, CT2 and OCTN2, have been identified in humans (51 , 52) . It is, therefore, possible that CT2 and/or OCTN2 may also act as BLM transporters. Interestingly, CT2 is expressed exclusively in human testis, whereas OCTN2 is expressed strongly in kidney, skeletal muscle, heart, and prostate (51 , 52) . The fact that CT2 is expressed exclusively in testis and that testicular cancers have a high cure rate with BLM therapy is striking, offering strong support for the notion that CT2 could be the human transporter of BLM. Because transporter-dependent drug resistance is a frequent problem in clinical therapy, it is possible that patients who develop resistance to BLM could have defects in CT2. We note that further compelling evidence for a conserved BLM-resistance, transport-based mechanism in human cells is bolstered by a recent report showing that a dominant negative form of the mammalian Sky1 homologue (SRPK1) confers BLM-resistance in Chinese hamster lung fibroblast and HeLa cells, but not to various other DNA damaging agents (53) . Thus, elucidation of homologous BLM resistance genes in human would be expected to reveal multiple novel molecular targets toward enhancing the antitumor properties of the drug.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Notes: M. Aouida and N. Pagé contributed equally to this work. Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org).
Requests for reprints: Dindial Ramotar, University of Montreal, Guy-Bernier Research Centre, 5415 de l’Assomption, Montreal, Quebec, H1T 2M4 Canada. Phone: (514) 252-3400, extension 4684; Fax: (514) 252-3430, E-mail: dramotar{at}hmr.qc.ca
Received 8/29/03. Revised 11/24/03. Accepted 11/26/03.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
phage. Mutat. Res., 240: 93-100, 1990.[CrossRef][Medline]
This article has been cited by other articles:
|
|
 |
|
  J. Chen, M. K. Ghorai, G. Kenney, and J. Stubbe Mechanistic studies on bleomycin-mediated DNA damage: multiple binding modes can result in double-stranded DNA cleavage Nucleic Acids Res., June 1, 2008; 36(11): 3781 - 3790. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Dawson, W. M. Toone, N. Jones, and C. R. M. Wilkinson Loss of Regulators of Vacuolar ATPase Function and Ceramide Synthesis Results in Multidrug Sensitivity in Schizosaccharomyces pombe Eukaryot. Cell, June 1, 2008; 7(6): 926 - 937. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. D. Strome, X. Wu, M. Kimmel, and S. E. Plon Heterozygous Screen in Saccharomyces cerevisiae Identifies Dosage-Sensitive Genes That Affect Chromosome Stability Genetics, March 1, 2008; 178(3): 1193 - 1207. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. L. Pike and J. Heierhorst Mdt1 Facilitates Efficient Repair of Blocked DNA Double-Strand Breaks and Recombinational Maintenance of Telomeres Mol. Cell. Biol., September 15, 2007; 27(18): 6532 - 6545. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Cai, S. Kauffman, F. Naider, and J. M. Becker Genomewide Screen Reveals a Wide Regulatory Network for Di/Tripeptide Utilization in Saccharomyces cerevisiae Genetics, March 1, 2006; 172(3): 1459 - 1476. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Hellauer, G. Lesage, A.-M. Sdicu, and B. Turcotte Large-Scale Analysis of Genes that Alter Sensitivity to the Anticancer Drug Tirapazamine in Saccharomyces cerevisiae Mol. Pharmacol., November 1, 2005; 68(5): 1365 - 1375. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Thornton, C. R. M. Wilkinson, W. M. Toone, and N. Jones A novel pathway determining multidrug sensitivity in Schizosaccharomyces pombe Genes Cells, October 1, 2005; 10(10): 941 - 951. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R.-Y. Huang, M. Eddy, M. Vujcic, and D. Kowalski Genome-Wide Screen Identifies Genes Whose Inactivation Confer Resistance to Cisplatin in Saccharomyces cerevisiae Cancer Res., July 1, 2005; 65(13): 5890 - 5897. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Aouida, A. Leduc, R. Poulin, and D. Ramotar AGP2 Encodes the Major Permease for High Affinity Polyamine Import in Saccharomyces cerevisiae J. Biol. Chem., June 24, 2005; 280(25): 24267 - 24276. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. G. Perrone, C. M. Grant, and I. W. Dawes Genetic and Environmental Factors Influencing Glutathione Homeostasis in Saccharomyces cerevisiae Mol. Biol. Cell, January 1, 2005; 16(1): 218 - 230. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
