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[CANCER RESEARCH 64, 1102–1109, February 1, 2004]
A Genome-Wide Screen in Saccharomyces cerevisiae Reveals Altered Transport As
a Mechanism of Resistance to the Anticancer Drug Bleomycin
Mustapha Aouida,
1
Nicolas Page´,
2
Anick Leduc,
1
Matthias Peter,
2
and Dindial Ramotar
1
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
The potent DNA damaging agent bleomycin (BLM) is highly effective
for treating various cancers, although, in certain individuals, the devel-
opment of cellular resistance to the drug can severely diminish its antin-
eoplastic properties. We performed two independent genome-wide screens
using a Saccharomyces cerevisiae mutant collection to isolate variants
exhibiting either sensitivity or resistance to BLM. This procedure repro-
ducibly identified a relatively large collection of 231 BLM-hypersensitive
mutants, representing genes belonging to diverse functional groups. In
contrast, only five BLM-resistant mutants could be recovered by our
screens. Among these latter mutants, three were deleted for genes involved
in plasma membrane transport, including the L-carnitine transporter
Agp2, as well as the kinases Ptk2 and Sky1, which are involved in
regulating polyamine transport. We further showed that Agp2 acts as a
transporter of BLM and that overexpression of this transporter signifi-
cantly enhances BLM-induced cell killing. Our data strongly implicate
membrane transport as a key determinant in BLM resistance in yeast.
This finding is critical, given that very little is known about BLM trans-
port in human cells. Indeed, characterization of analogous mechanisms in
humans may ultimately lead to enhancement of the antitumor properties
of BLM.
INTRODUCTION
Bleomycin (BLM) is used to treat various cancers, including lym-
phoma, squamous cell carcinoma of the cervix, head, and neck, and
Hodgkin’s disease (1–4). In the case of testicular cancer, BLM in
combination with cisplatin and etoposide is particularly effective,
yielding a striking cure rate of 70–80% (4, 5). However, the remain-
der of patients eventually develop resistance to BLM therapy and
relapse (4, 6). Furthermore, there is clear evidence that individuals
afflicted with various other human cancers (e.g., Daudi lymphoma and
colon carcinoma) are highly resistant to BLM therapy at the outset (7,
8). Although the mechanism of BLM-induced cytotoxicity has been
well studied (see below and Refs. 3, 9–12), much less attention has
been devoted to establishing the determinants that regulate tumor
resistance to the drug.
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 con-
taining 3⬘-blocking groups that inhibit the progression of DNA po-
lymerase) 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 (15–18).
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 (19–22), engen-
ders 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
Yeast Strains and Media. The wild-type strains used were BY4741 and
SEY6210 (25, 26). The collection of nonessential haploid MATa deletion
strains, derived from the parent BY4741, was obtained from EUROSCARF
(Frankfurt, Germany; Ref. 27). Standard YPD (yeast-peptone-dextrose) and
selective growth media were used as described previously (28).
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
g/ml BLM A5 (BLM, ICN Pharma-
ceuticals), 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 A
600
of 0.6, and 5
l of a set of serial dilutions, as indicated, were spotted
onto YPD supplemented with 1.0 or 4.0
g/ml BLM to test for BLM hyper-
sensitivity and resistance, respectively.
Gradient Plate and Survival Curve Assays. These assays were per-
formed as described previously (26). In the case of gradient plate assays, the
bottom layer contained either 0.5
g/ml 4-nitroquinoline-1-oxide (4-NQO) or
0.03% methyl methane sulfonate (MMS). For the survival curves, exponen-
tially growing cultures were treated at an A
600
of 1.0 for 1 h with 4-NQO (0 to
6
g/ml) or MMS (0 to 0.3%) or cisplatin (0 to 10
g/ml) or camptothecin (0
to 10
g/ml), and or
␥
-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.
Received 8/29/03; revised 11/24/03; accepted 11/26/03.
Grant support: This research was supported by funds from the National Cancer
Institute of Canada (with funds from the Canadian Cancer Society) to D. Ramotar and to
M. Peter by the Swiss National Science Foundation and the ETH/Zurich. D. Ramotar is a
senior fellow of the Fonds de la Recherche en Sante´ du Que´bec. N. Page´ holds a
fellowship from Roche Research Foundation.
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. Page´ 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 Re-
search Centre, 5415 de l’Assomption, Montreal, Quebec, H1T 2M4 Canada. Phone:
(514) 252-3400, extension 4684; Fax: (514) 252-3430, E-mail: dramotar@hmr.qc.ca.
1102
Research.
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