Báo cáo khoa học: Phenyl hydroquinone, an Ames test-negative carcinogen, induces Hog1-dependent stress response signaling pptx

Phenyl hydroquinone, an Ames test-negative carcinogen,
induces Hog1-dependent stress response signaling
Implication for aneuploidy development in Saccharomyces
cerevisiae
Ayumi Yamamoto
1
, Tatsuo Nunoshiba
1
, Keiko Umezu
2
, Takemi Enomoto
3
and Kazuo Yamamoto
1
1 Graduate School of Life Sciences, Tohoku University, Sendai, Japan
2 Fukuoka Dental College, Tamura, Japan
3 Pharmaceutical Sciences, Tohoku University, Japan
We have previously observed that phenyl hydroqui-
none (PHQ), a hepatic metabolite of the Ames test-
negative carcinogen ortho-phenylphenol (OPP) [1,2],
arrests the cell cycle at G
1
and G
2
⁄ M and causes aneu-
ploidy [3]. However, potential mechanisms for this
activity have not yet been elucidated. Clarification of
these mechanisms is important in understanding the
mechanisms utilized by the Ames test-negative carcino-
gens OPP and PHQ, as well as the general mechanisms
of aneuploidy development.

A central principle of genetics is that cells
within organisms contain the same complement of
chromosomes. The presence of too many or too few
chromosomes, called aneuploidy, is associated with
diseases including cancer, and accounts for the major-
ity of spontaneous miscarriages [4].
A large number of genes that affect genome stability
in budding yeast have been identified by mitotic defect
mutation screens [5–9]. Many genes identified by other
criteria have also been shown to be necessary for gen-
ome stability, including DNA metabolism enzymes such
as polymerases, recombination enzymes and a ligase
[10,11], as well as components of the chromosome segre-
gation machinery, including tubulins, mitotic spindle
Keywords
aneuploidy; checkpoint; G
2
⁄ M transition;
MAPK; phenyl hydroquinone
Correspondence
K. Yamamoto, Graduate School of Life
Sciences, Tohoku University, Katahira 2-1-1,
Aoba-ku, Sendai 980 8577, Japan
Fax: +81 22 217 5053
Tel: +81 22 217 5054
E-mail:
(Received 21 August 2008, revised
21 September 2008, accepted
22 September 2008)
doi:10.1111/j.1742-4658.2008.06700.x

Recently, we have shown that phenyl hydroquinone, a hepatic metabolite
of the Ames test-negative carcinogen o-phenylphenol, efficiently induced
aneuploidy in Saccharomyces cerevisiae. We further found that phenyl
hydroquinone arrested the cell cycle at G
1
and G
2
⁄ M. In this study, we
demonstrate that phenyl hydroquinone can arrest the cell cycle at the
G
2
⁄ M transition as a result of stabilization of Swe1 (a Wee1 homolog),
probably leading to inactivation of Cdc28 (a Cdk1 ⁄ Cdc2 homolog). Fur-
thermore, Hog1 (a p38 MAPK homolog) was robustly phosphorylated by
phenyl hydroquinone, which can stabilize Swe1. On the other hand, Chk1
and Rad53 were not phosphorylated by phenyl hydroquinone, indicating
that the Mec1 ⁄ Tel1 DNA-damage checkpoint was not functional. Muta-
tions of swe1 and hog1 abolished phenyl hydroquinone-induced arrest at
the G
2
⁄ M transition and the cells became resistant to phenyl hydroquinone
lethality and aneuploidy development. These data suggest that a phenyl
hydroqionone-induced G
2
⁄ M transition checkpoint that is activated by the
Hog1–Swe1 pathway plays a role in the development of aneuploidy.
Abbreviations
ATM, ataxia teleangiectasia mutated; ATR, ATM and Rad3-related; Can, canavanine; CDK, cyclin-dependent kinase; Clb, cyclin B;
DAPI, 4¢6¢-diamidino-2-phenylindole; 5-FOA, 5-fluoro-orotic acid; MAPK, mitogen activated protein kinase; MMS, methylmethane sulfonate;
PHQ, phenyl hydroquinone; OPP, ortho-phenylphenol.

FEBS Journal 275 (2008) 5733–5744 ª 2008 The Authors Journal compilation ª 2008 FEBS 5733
motors, components of the kinetochore, and spindle
pole bodies [12–15]. Defects in spindle checkpoint regu-
latory systems have also been shown to cause a high fre-
quency of aneuploidy [16–18].
We wished to determine how PHQ arrests the cell
cycle at G
2
⁄ M, because aneuploidy that occurs during
mitosis may be linked directly to cell-cycle arrest [3].
Our microscopic observations of PHQ-treated budding
yeast cells revealed that PHQ causes the formation of
elongated buds. In yeast cells whose actin cytoskeleton
has been perturbed, the formation of buds is fre-
quently delayed, and the morphogenesis checkpoint
introduces a compensatory delay in nuclear division at
the G
2
⁄ M boundary until a bud is well formed [19].
The cell-cycle delay is due to inhibitory phosphoryla-
tion of the cyclin-dependent kinase (CDK) Cdc28 [20].
Phosphorylation of Cdc28 is catalyzed by the protein
kinase Swe1 [21]. Stabilization of Swe1 is achieved by
the yeast Hog1 mitogen-activated protein kinase
(MAPK) cascade, which is activated by environmental
stresses such as high osmolarity [22]. We demonstrate
here that PHQ activates and stabilizes the Hog1–Swe1
pathway to arrest the cell cycle at the G
2
⁄ M boundary,

and therefore may cause aneuploidy.
Results
G
2
/M delay by phenyl hydroquinone
Previously we demonstrated that PHQ efficiently
induced G
2
⁄ M delay and could cause aneuploidy in
budding yeast [3]. To determine the exact point of
arrest, cells were released from G
1
synchrony induced
by a-factor and samples were taken at intervals to
score spindle morphology and for FACScan analysis
of DNA content. As shown in Fig. 1A, 2 mm PHQ
added 70 min after release from G
1
caused accumula-
tion of G
2
–M spindles but not anaphase spindles (spin-
dle elongation). The cell shapes and spindle lengths of
more than 100 cells were observed. Classification of
spindle morphology is based on the images shown in
Fig. S1. FACScan analysis also indicated arrest at
mitosis (Fig. 1B). Thus, we argue that treatment with
PHQ results in substantial lengthening of the G
2
⁄ M

phase and that cells cannot progress to anaphase.
PHQ efficiently induces the formation of
elongated buds
During microscopic observation of PHQ-treated cells,
we found that PHQ caused the formation of elongated
buds (Fig. 2A,B). As controls, 0.1% methylmethane
sulfonate (MMS), which activates a DNA-damage
checkpoint, and 15 lgÆmL
)1
nocodazole, which acti-
vates a spindle checkpoint (metaphase to anaphase tran-
sition), did not cause the formation of elongated buds
(Fig. 2A). In yeast cells whose actin cytoskeleton has
been perturbed, budding is frequently delayed and the
morphogenesis checkpoint introduces a compensatory
delay of nuclear division at the G
2
⁄ M boundary until a
bud is well formed [19]. Thus, PHQ can activate the
morphogenesis checkpoint at the G
2
⁄ M boundary.
180
150
120
90
60
30
0
YPD

100
A
B
80
60
40
20
0
0 50 100 150 200
0 50 100 150 200
100
80
60
40
20
0
2m
M PHQ
% cells
% cells
Minutes after release from G1
Minutes after release from G1
G1-S
G2-meta
Ana
G2-meta
G1-S
Ana
YPD 2 mM PHQ 15 µg·mL
–1

noc
Fig. 1. PHQ-induced G
2
⁄ M delay in wild-
type cells. Haploid YK402 yeast cells at
10
7
cellsÆmL
)1
were synchronized in G
1
using 30 ngÆmL
)1
a-factor for 3 h at 30 °C.
Cells were inoculated into YPD medium and
incubated at 30 °C. PHQ (2 m
M) or noco-
dazole (15 lgÆmL
)1
) was added (arrow)
when 80–90% of cells had very small buds
(approximately 70 min). At the indicated
times, samples were withdrawn and pro-
cessed for spindle morphology observation
(A) and a FACScan analysis of DNA content
(B) as described in Experimental proce-
dures.
PHQ induces stress signals in S. cerevisiae A. Yamamoto et al.
5734 FEBS Journal 275 (2008) 5733–5744 ª 2008 The Authors Journal compilation ª 2008 FEBS
PHQ inhibits degradation of Swe1

The sensors that detect the morphogenesis checkpoint
remain unknown, but the resulting G
2
⁄ M transition
delay is caused by Swe1 (a Wee1 ortholog), which
phosphorylates and negatively regulates the CDK
Cdc28 [21,23]. In the unperturbed cell cycle, Swe1
begins to accumulate in S phase and becomes hyper-
phosphorylated as cells proceed through the cell cycle
[24]. Hyperphosphorylated Swe1 is susceptible to
ubiquitin-mediated degradation [25–27]. We thus
examined whether PHQ affects the accumulation and
degradation of Swe1. G
1
synchronous cells were
inoculated into YPD medium. In the absence of PHQ,
Swe1 accumulated at S phase at 45–60 min and was
then degraded as cells proceeded from S to M phase at
105 min. If the cell cycle moved to next round of
S phase at 150 min (Fig. 1A), Swe1 accumulated again
(data not shown). In the presence of 2 mm PHQ, Swe1
accumulated at S phase but was not degraded at
M phase at 105 min (Fig. 3). We conclude that arrest
at G
2
⁄ M by PHQ is due to stabilization of Swe1.
Stabilization of Swe1 can lead to the formation of
elongated buds and G
2
⁄ M transition arrest due to

inactivation of the cyclin B (Clb)–Cdc28 complex.
G
2
/M arrest by PHQ is abolished by mutation
of swe1
Next, we constructed swe1 deletion mutants and exam-
ined progression of the cell cycle in the presence of
2mm PHQ. The mutants were arrested at G
1
by a-fac-
tor treatment, then inoculated into YPD medium.
PHQ was added 70 min after release. As shown in
Fig. 4A, 2 mm and 0 mm PHQ caused G
2
–M spindles
to accumulate and anaphase spindles to elongate,
YPD 0.1% MMS 15
µ
g·mL
–1
nocodazole 2 mM PHQ
A B
Fig. 2. Elongated bud formation by PHQ. (A) Haploid YK402 cells at the exponential phase were treated with 2 mM PHQ and incubated at
30 °C for 17 h. Samples were observed by light microscopy (upper panel) and fluorescence microscopy (lower panel) after staining with
DAPI. As a control, 0.1% MMS or 15 lgÆmL
)1
nocodazole was used. (B) High-magnification (· 400) images of PHQ-induced elongated buds.
Time (min)
2mM PHQ
YPD

0 30 45 60 75 90 105
0 30 45 60 75 90 105
Swe1
Swe1
Arp3
Arp3
Fig. 3. Stabilization of Swe1 by PHQ.
G
1
synchronous AYU901 cells (SWE1-
myc:TRP1) were inoculated in YPD medium,
2m
M PHQ was added at 70 min (arrow),
and cells were sampled at intervals and pro-
cessed for western blotting experiments.
Proteins extracted were resolved by 10%
SDS–PAGE and subjected to immunoblot-
ting using an antibody that recognizes Myc
proteins.
A. Yamamoto et al. PHQ induces stress signals in S. cerevisiae
FEBS Journal 275 (2008) 5733–5744 ª 2008 The Authors Journal compilation ª 2008 FEBS 5735
which was different from the response to PHQ
observed in the wild-type strain (Fig. 1A) in which no
accumulation of anaphase spindles was observed with
2mm PHQ. FACScan analysis also indicated that
2mm PHQ did not stop the cell cycle at G
2
⁄ M, but
nocodazole arrested swe1 cells at M phase (Fig. 4B).
Thus, we argue that the PHQ-induced cell-cycle arrest

in budding yeast is mediated by the swe1 gene.
Microcolony analysis in swe1 cells
To observe the growth of mutant yeast cells in the
presence of PHQ, we performed microcolony assays.
G
1
-synchronized cells were plated on microscope slides
with YPD agar containing 2 mm PHQ, and the growth
of individual cells was monitored over the next 9 h.
Figure 5 shows that SWE1 cells divided slowly and
continuously in the presence of 2 mm PHQ, with
approximately four cells formed after 9 h of incuba-
tion. The swe1 mutant cells divided more rapidly than
the SWE1 cells in the presence of 2 mm PHQ, with
approximately nine cells formed after 9 h. However,
division of swe1 cells on PHQ-containing plates was
slower than that of SWE1 or swe1 cells on plates with-
out PHQ (Fig. 5). This effect of PHQ was probably
due to G
1
arrest [3] (see Discussion). The microcolony
assay also indicated that incubation of swe1 and
SWE1 cells on plates containing PHQ did not stop cell
division but rather slowed it, and that similar sizes of
microcolonies as those for SWE1 or swe1 cells on
plates without PHQ were obtained after 24 h of incu-
bation (data not shown). Our results suggest that, in
wild-type cells, 2 mm PHQ is able to induce cell-cycle
arrest but not cell death. During microscopic observa-
tion, we observed elongated buds in SWE1 cells trea-

ted with PHQ (Fig. 5A, arrow) but not in swe1 cells
(data not shown).
PHQ causes phosphorylation of Hog1 but not
of Chk1 or Rad53
Accumulation and stabilization of Swe1 are known to
occur via phosphorylation of Hog1 (a mammalian p38
MAPK homolog), activated by environmental stresses
including osmotic stress [22,28], or of Mec1 ⁄ Tel1
[a mammalian ataxia teleangiectasia mutated (ATM) ⁄
ATM and Rad3-related (ATR) homolog], activated by
DNA damage [28,29]. First we examined the phos-
phorylation of Hog1 by 2 mm PHQ. We quantified
activation of Hog1 by measuring the amount of the
phosphorylated form (Thr174) of Hog1 using a selec-
tive antibody. After 30 min of incubation of asynchro-
nous wild-type cells with 2 mm PHQ, it was possible
to detect the phosphorylated form of Hog1 (Fig. 6A).
The level of phosphorylated Hog1 decreased by
60 min. As controls, treatment with 0.8 m NaCl
resulted in phosphorylation of Hog1 at 30 min, after
which there was a decline by 60 min, treatment with
0.1% MMS did not result in phosphorylation of
Hog1, and treatment with 15 lgÆmL
)1
nocodazole
resulted in weak phosphorylation of Hog1 at 60 min.
As a loading control, the same blot membrane was
A
B
100

80
60
40
20
0
0 50 100 150 200
100
80
60
40
20
0
% cells
% cells
Minutes after release from G1
180
150
120
90
60
30
0
Minutes after release from G1
0 50 100 150 200
G1-S
G2-meta
Ana
G1-S
G2-meta
Ana

YPD
2m
M PHQ
YPD 2 m
M PHQ 15 µg·mL
–1
noc
Fig. 4. PHQ-induced G
2
⁄ M delay in swe1D
cells. Experiments were performed as in
Fig. 1, but using haploid AYU902 (swe1D)
yeast cells instead of YK402 (SWE1).
PHQ induces stress signals in S. cerevisiae A. Yamamoto et al.
5736 FEBS Journal 275 (2008) 5733–5744 ª 2008 The Authors Journal compilation ª 2008 FEBS
reprobed against total Hog1 using a monoclonal Hog1
antibody (Fig. 6A). Next, we examined the phosphory-
lation of Chk1 (Fig. 6B) and Rad53 (Fig. 6C), which
are phosphorylated if upstream Mec1 ⁄ Tel1 is activated
by DNA damage. After 60 min of incubation of asyn-
chronous wild-type cells with 2 mm PHQ, we did not
detect broad phosphorylated bands for Chk1 and
Rad53. As a control, 0.1% MMS treatment resulted in
efficient phosphorylation of both Chk1 and Rad53 at
60 min. Thus, we conclude that PHQ treatment causes
phosphorylation of Hog1 but not Rad53 nor Chk1,
followed by accumulation and stabilization of Swe1,
which in turn leads to inhibition of mitosis with arrest
at the G
2

⁄ M transition.
Disruption of swe1 and hog1 genes and
aneuploidy
We have previously shown that PHQ treatment induced
aneuploidy, but not base substitution mutations or
0 30 60 30 60 30 60 30 60 30 60 (min)
NaCl PHQ DMSO MMS nocodazole
p-Hog1
A
0 15 30 60 15 30 60 (min)
PHQ MMS
p-Chk1
Chk1
p-Rad53
Rad53
B
C
PHQ MMS
0 15 30 60 15 30 60 (min)
Hog1
Arp3
Arp3
Arp3
Fig. 6. Phosphorylation of Hog1, Chk1 and
Rad53 by PHQ. An exponentially growing
asynchronous culture of YK402 was treated
with 2 m
M PHQ or with 0.8 M NaCl, 0.2%
dimethylsulfoxide (DMSO), 0.1% MMS or
15 lgÆmL

)1
of nocodazole as controls.
Cultures were sampled at 0, 30 and 60 min,
and proteins were resolved by 8%
SDS–PAGE and subjected to immunoblot-
ting using antibodies against phospho-p38
MAPK (T180 ⁄ Y182) (A), against Myc
proteins for Chk1 (B), and against Myc
proteins for Rad53 (C).
WT
AB
(YPD)
WT
(PHQ)
swe1
(PHQ)
0 3 6 9 h
10
7.5
5.0
2.5
0
Cell number per microcolony
0 2.5 5 7.5 10
Incubation time (h)
Fig. 5. Microcolony assay of PHQ-treated SWE1 and swe1D cells. (A) G
1
-synchronized cells of YK402 (SWE1) and AYU902 (swe1D) were
plated on microscope slides with YPD agar containing 2 m
M PHQ at 30 °C, and the growth of individual cells was monitored over the next

9 h. The arrow indicates an elongated bud formed as a result of PHQ treatment. (B) The number of cells in each of 20 microcolonies was
counted under a light microscope at the indicated time, and the mean number of cells per microcolony was calculated. Each visible object
was scored as a cell, so that a cell plus a bud (but not a-factor activated shmoo) was scored as two cells. Closed diamond, SWE1 without
PHQ; open diamond, SWE1 with 2 m
M PHQ; closed circle, swe1D without PHQ; open circle, swe1D with 2 mM PHQ.
A. Yamamoto et al. PHQ induces stress signals in S. cerevisiae
FEBS Journal 275 (2008) 5733–5744 ª 2008 The Authors Journal compilation ª 2008 FEBS 5737
mutation
Fig. 7. Lethality and induction of canavanine or 5-FOA resistance by PHQ in diploid strains carrying can1D::LUE2 ⁄ CAN1 heterozygous allele
of YAS3001 (WT), AYU9100 (swe1D ⁄ swe1D) and AYU9200 (hog1D ⁄ hog1D) (A–C), and in strains carrying the URA3
+
heterozygous allele of
RD301 (WT) and AYU402 (swe1D ⁄ swe1D) (D). (A) Analysis of chromosomal structures generated during the development of Can
R
mutants.
The chromosome V pair in diploid cells (white with can1D and black with V-11::LYS2
+
, V-565::ADE2
+
and CAN1) makes three possible Can
R
mutants that can be classified by phenotype. Can
R
Ade
)
Lys
+
clones were not expected to occur at high levels as they are associated with
a non-disjunction. This classification is in accordance with that described by Hiraoka et al. [30] and Ohnishi et al. [58]. The open circle shows
the centromere. (B) The surviving fraction (upper panel) and Can

R
mutation frequency (lower panel) of diploid cells treated with various
concentrations of PHQ. Open triangle, wild-type; open circle, swe1D; open diamond, hog1D. (C) Possible chromosome events causing
canavanine resistance after treatment with 2 m
M of PHQ, as shown in (B), were estimated to be gene conversion, crossover and chromo-
some loss, as shown in (A). The Can
R
frequencies in 2 mM PHQ were 6 · 10
)3
for wild-type, 2 · 10
)4
for swe1D and 1.05 · 10
)4
for hog1D.
(D) The surviving fraction (upper panel) and 5-FOA
R
mutation frequency (middle panel) for RD301 (filled triangle) and AYU401 (swe1D) (open
circle) treated with various concentrations of PHQ. Chromosome loss percentages were estimated by replica plating of the 5-FOA
R
mutants
identified in the middle panel. At least 250 5-FOA
R
colonies were isolated for each treatment condition, and 5-FOA
R
Leu
)
Ade
)
in RD301
(wild-type; filled column) and AYU401 (swe1D; open column) is classified as a chromosome loss (lower panel).

PHQ induces stress signals in S. cerevisiae A. Yamamoto et al.
5738 FEBS Journal 275 (2008) 5733–5744 ª 2008 The Authors Journal compilation ª 2008 FEBS
homologous recombination [3]. In this study, we
demonstrate that PHQ induced arrest at the G
2
⁄ M
transition that was Hog1–Swe1-dependent but not
DNA damage-dependent. We then determined how
swe1 or hog1 mutants respond to PHQ. Diploid
AYU9100 (swe1D) or AYU9200 (hog1D) strains carry-
ing CAN1 ⁄ can1D::LEU2 heterozygous alleles (Fig. 7A)
were exposed to various concentrations of PHQ for
17 h. Cell survival and the frequency of the Can
R
mutation were measured (Fig. 7B upper and lower
panels, respectively). PHQ did not induce cell death or
Can
R
mutations in swe1 and hog1 cells (Fig. 7B). The
numbers of surviving swe1 and hog1 diploid cells in
SC medium in the stationary phase were 2.26 · 10
6
and 1.90 · 10
6
per mL, respectively, meaning that
growth was not strongly retarded when compared to
wild-type cells (3.95 · 10
6
cellsÆmL
)1

). As a reference,
swe1 and hog1 mutants were sensitive to NaCl treat-
ment (data not shown), consistent with previous results
[22]. We then classified Can
R
mutants using
V11::LYS2 and V565::ADE2 markers (Fig. 7A) and
found that PHQ generates Can
R
mutants through loss
of chromosome V carrying the CAN1
+
allele in
wild-type cells (Fig. 7C, upper panel), but rates of gene
conversion, crossover and chromosome loss were not
increased over spontaneous levels in swe1 and hog1
cells (Fig. 7C, middle and lower panels, respectively).
Furthermore, the spontaneous level of chromosome
loss was lower in the swe1 and hog1 strains than the
wild-type strain. In the AYU9300 (chk1D) strain, cell
survival and the frequency of the Can
R
mutation were
increased by PHQ as efficiently as in the YAS3001
strain (wild-type), and Can
R
mutants were induced
through loss of chromosome V but not gene conver-
sion or crossover (Fig. S2), indicating no involvement
of the DNA-damage pathway in PHQ-induced aneu-

ploidy development. For reproducibility, we examined
the effect of PHQ on the swe1D strain derived from
RD301 [30] in which the heterozygous target
URA3 ⁄ ura3D resides on chromosome III. The swe1D
strain in this background was resistant to PHQ when
compared to the SWE1 strain, and 5-FOA
R
mutations
did not increase (Fig. 7D upper and middle panels,
respectively). Again, PHQ exclusively induced aneu-
ploidy in RD301 cells but not in the swe1D strain
(Fig. 7D, lower panel). Thus, the Hog1–Swe1 pathway,
but not the Mec1 ⁄ Tel1 DNA-damage pathway, is
involved in PHQ-induced aneuploidy. As mentioned
above (Fig. 5), growth of the swe1 strain in the
presence of PHQ was not strongly inhibited when
compared to the wild-type strain. Thus, the lethality
observed in this study may have been due to a cell-
static effect rather than a cell-killing effect. We argue
that PHQ-induced activation of the Hog1–Swe1 path-
way causes a lethal cell-static effect and chromosome
loss (aneuploidy).
Discussion
In this study, we demonstrate that PHQ treatment
arrests the cell cycle at the G
2
⁄ M transition as a result
of stabilization of Swe1, resulting in phosphorylation of
the mitosis-promoting factor Clb-bound Cdc28 (a
Cdk1 ⁄ Cdc2 homolog) and subsequent arrest of the cell

cycle at the G
2
⁄ M boundary. The swe1 mutation abo-
lished PHQ-induced G
2
⁄ M arrest. Furthermore, PHQ
activated the phosphorylation of Hog1, which stabilizes
Swe1. When either the swe1 or hog1 gene was disrupted,
the mutant became resistant to PHQ and aneuploidy
was not likely to occur. Previously, we observed that
PHQ is lethal to cells and is prone to cause aneuploidy
in wild-type budding yeast (Fig. 7). We thus conclude
that PHQ activates the Hog1–Swe1 pathway to arrest
the cell cycle at G
2
⁄ M transition, leading to aneuploidy.
It has recently been shown that activation of Hog1
MAPK by high osmolarity results in arrest of the cell
cycle at G
1
and G
2
[31–33]. Entry from G
2
into mitosis
is controlled by activation of the Clb–Cdc28 complex,
which is controlled by the protein kinase Swe1 in
S. cerevisiae [34]. During a normal cell cycle, Swe1
accumulates in S phase, becomes sequentially hyper-
phosphorylated [24,35], and undergoes ubiquitin-medi-

ated degradation [25,26,36]. Defects in septin filament
assembly at the bud neck result in the hypophosphory-
lation and stabilization of Swe1 and lead to
Swe1-dependent inhibition of Clb–Cdc28 [24,37,38].
Activation of Hog1 also facilitates the accumulation
and stabilization of Swe1 [22], keeping Clb–Cdc28
inactive and thus arresting the cell cycle at the G
2
⁄ M
boundary. This Swe1-imposed delay leads to elongated
buds because the cells fail to switch from polarized to
isotropic growth during budding [39]. Thus, phosphor-
ylation and degradation of Swe1 appear to be critical
for efficient activation of Clb–Cdc28 and timely
mitotic entry.
Here we showed that PHQ could efficiently induce
the phosphorylation of Hog1 (Fig. 6) as well as the
hypophosphorylation and stabilization of Swe1
(Fig. 3), leading to elongation of buds (Fig. 2) and
G
2
⁄ M transition arrest (Fig. 1). This effect is directly
linked to genetic instability, especially aneuploidy,
because strains lacking swe1 and hog1 did not show
aneuploidy (Fig. 7). Thus, arrest at the G
2
⁄ M
boundary through the Hog1–Swe1 pathway is a pre-
requisite for aneuploidy. Obviously, arrest at the
G

2
⁄ M boundary is not sufficient for aneuploidy
A. Yamamoto et al. PHQ induces stress signals in S. cerevisiae
FEBS Journal 275 (2008) 5733–5744 ª 2008 The Authors Journal compilation ª 2008 FEBS 5739
because high osmolarity, such as that caused by
NaCl treatment, also activates the Hog1–Swe1 path-
way (Fig. 6) but does not strongly induce aneuploidy
(data not shown). PHQ causes G
2
⁄ M boundary
arrest (this study) and damage to tubulin [3]. On the
other hand, high osmolarity does not cause tubulin
damage. This difference may be one of the reasons
why NaCl does not induce aneuploidy. Recently, on
the other hand, Kawasaki et al. [40] observed that
stresses such as 1.2 m sorbitol in the medium cause
arrest at the G
2
⁄ M transition and increase the rate
of mini-chromosome loss in fission yeast. Thus,
stress-induced G
2
⁄ M transition arrest is involved in
aneuploidy development.
Cell-cycle progression from the G
2
to M phase is
controlled by not only the Hog1 MAPK pathway but
also the ATM ⁄ ATR (Tel1 ⁄ Mec1) pathway [28,41]. In
the latter case, the protein kinases ATM ⁄ ATR are

activated in response to DNA damage, resulting in
phosphorylation and activation of Chk1 and Chk2
(yeast Rad53 homologs). Chk1 and Chk2 stimulate
inhibitory phosphorylation of CDK1, causing G
2
⁄ M
transition arrest. Previously, it was reported that PHQ
neither binds to nor cleaves DNA in vitro or in vivo
[1,42]. On the other hand, some reports suggested that
PHQ causes oxidative DNA damage [43,44]. Thus, the
observed G
2
⁄ M transition arrest and aneuploidy may
have been caused by PHQ-induced DNA damage but
not MAPK activation. We demonstrate here that PHQ
cannot phosphorylate Chk1 (Fig. 6B) or Rad53
(Fig. 6C). Moreover, PHQ efficiently induced aneu-
ploidy in the chk1D strain (Fig. S2). Thus, the DNA-
damage response did not occur in response to PHQ
treatment.
In the swe1 strain, we observed that PHQ does
not cause cell arrest at G
2
⁄ M (Fig. 4) or cell death
(Fig. 7B). As the Hog1–Swe1 pathway is a key path-
way of PHQ-induced arrest as mentioned above, it is
logical that PHQ does not cause cell-cycle arrest in
swe1. With respect to cytotoxicity, swe1 and hog1
cells are quite sensitive to NaCl treatment [22].
Therefore, arrest at G

2
⁄ M is required for cell sur-
vival under osmotic stress. On the other hand, this
is not the case for PHQ stress, as budding yeast
showed increased survival and decreased aneuploidy
in the absence of G
2
⁄ M arrest. Recently, it was
shown that Neurospora crassa and Candida albi-
cans hog1 mutants are resistant to dicarboximide and
phenylpyrrole fungicides [45,46]. Although the precise
lethal mechanisms of these drugs have not yet been
elucidated, the Hog1 MAPK pathway of N. crassa,
C. albicans and S. cerevisiae is the target of several
fungicides including PHQ. Here, we reported that
both hog1 and swe1 cells are resistant to PHQ, thus
PHQ may not be toxic to budding yeast but may
simply cause Hog1–Swe1-dependent arrest. In the
presence of PHQ, wild-type cells can stay at G
2
⁄ M
or grow very slowly until the PHQ is washed off
(Fig. 5). We therefore argue that the lethality in this
study may be due to a cell-static effect rather than a
killing effect.
It is worth noting that PHQ causes not only
G
2
⁄ M arrest but also G
1

arrest [3]. As mentioned
above, Hog1 activation also results in G
1
arrest
[31,33]. It has been reported that Hog1 controls the
G
1
transition via a dual mechanism that involves
downregulation of cyclin expression and direct phos-
phorylation of the CDK inhibitor protein Sic1. Phos-
phorylation of Sic1 in combination with cyclin
downregulation results in Sic1 stabilization and the
inhibition of cell-cycle progression to prevent prema-
ture entry into S phase without proper cell adapta-
tion [47]. Thus, PHQ may activate the Hog1–Sic1
pathway, causing G
1
⁄ S arrest.
Our results raise a question regarding the mecha-
nism of PHQ-induced aneuploidy, especially the
difference between the presence and absence of
Hog1–Swe1. Activation of the Hog1 MAPK is phy-
siologically significant for cell adaptation to environ-
mental stresses such as osmotic stress. On the other
hand, we demonstrated here that activation of Hog1
is required for the development of aneuploidy, which
inflicts stress on cells. This paradox needs to be
resolved.
A second question raised by our results is that of
the sensing mechanism for PHQ stress in yeast. Hog1

is activated by osmotic stress [33], heat stress [48], oxi-
dative stress [49], bacterial endotoxins [50] and citric
acid [51]. The yeast high-osmolarity sensor has been
determined to consist of two independent branch sen-
sors: the ‘two-component’ osmo-sensing complex Sln1–
Ypd1–Ssk1 [52] and the membrane-bound protein
Sho1 [53]. In contrast, the pathways by which yeast
signals the presence of PHQ are unknown as this
report provides the first demonstration that yeast can
respond to PHQ.
OPP and its hepatic metabolite PHQ can cause uri-
nary bladder hyperplasia and tumors [2], but do not
produce gene mutations or genetic recombination.
They are therefore classified as typical Ames test-nega-
tive carcinogens [1]. Many of the chemicals considered
to be carcinogenic are categorized as Ames test-nega-
tive [54]. Thus, how these carcinogens exert their
effects and whether all Ames test-negative carcinogens
produce aneuploidy through the Hog1–Swe1 pathway
requires further investigation.
PHQ induces stress signals in S. cerevisiae A. Yamamoto et al.
5740 FEBS Journal 275 (2008) 5733–5744 ª 2008 The Authors Journal compilation ª 2008 FEBS
Experimental procedures
Yeast strains and plasmids
The yeast strains used in this study are listed in Table 1.
For cell-cycle analysis and western blotting, strains derived
from YK402 were used. Complete deletions of ORFs
(swe1D::kanMX4, hog1D::kanMX4 and chk1D::kanMX4)
were generated by PCR-based one-step gene disruption
[55]. To construct strains expressing SWE1-Myc13, RAD53-

Myc13 and CHK1-Myc13 under endogenous promoter con-
trol, a Myc13::TRP1 fragment obtained from PCR using
pFA6a-13Myc-TRP1 [56] as a template was integrated into
YK402. For loss-of-heterozygosity analysis, diploid strains
carrying the can1D ⁄ CAN1 heterozygote allele (YAS3001
and derivatives) [57])and the ura3D ⁄ URA3 heterozygote
allele (RD301 and its derivative) [30] were used.
Reagents and media
Rich medium (YPD) and synthetic complete medium (SC)
were prepared as described previously [57]. Where indi-
cated, 10 lg Æ mL
)1
of adenine sulfate, 20 lgÆmL
)1
of uracil
and 20 lgÆmL
)1
of amino acids were added to the SC med-
ium. Canavanine (Can) and 5-fluoro-orotic acid (5-FOA)
were purchased from Sigma-Aldrich (St Louis, MO, USA).
PHQ (CAS 1079-21-6) was obtained from Tokyo Kasei Co.
(Tokyo, Japan) 4¢6¢-Diamidino-2-phenylindole (DAPI) was
purchased from Sigma-Aldrich. Monoclonal antibody
against Myc proteins, anti-Hog1 IgG, anti-Arp3 IgG and
anti-actin IgG were purchased from Santa Cruz Biotechnol-
ogy Inc (Santa Cruz, CA, USA). The monoclonal antibody
against tubulin (YOL1 ⁄ 34) was obtained from Novus
Biological (Littleton, CO, USA), and the Cy2-conjugated
secondary antibody was purchased from Jackson Immuno-
Research Laboratory Inc. (West Grove, PA, USA).

Antibody against phospho-p38 MAPK (T180 ⁄ Y182) was
obtained from R&D Systems Inc. (Minneapolis, MN,
USA). HRP-conjugated anti-mouse and anti-rabbit serum
were purchased from GE Healthcare UK Ltd. (London,
UK). HRP-conjugated anti-goat serum was purchased from
Santa Cruz Biotechnology Inc. a-factor was obtained from
Sigma-Aldrich. Enhanced Chemiluminescent (ECL) mem-
brane was purchased from GE Healthcare UK Ltd.
Canavanine resistance and 5-FOA resistance
assays in yeast diploid cells
Diploid cells (approximately 10
6
cellsÆmL
)1
) were treated
with various concentrations of PHQ in SC medium at
30 °C for 17 h. After two washes with sterile double-dis-
tilled water, appropriate dilutions were plated on SC plates
with or without 25 lgÆmL
)1
of Can. Colonies were scored
after 2–3 days of incubation at 30 °C. The frequency of the
Can
R
mutation was calculated by dividing the number of
Can
R
colonies by the total number of viable colonies. The
colonies on the Can plate were then replica-plated onto
SC-Ade (adenine-deficient SC), SC-Lys or SC-Ade-Lys

plates to classify Can
R
colonies as Ade
+
Lys
+
(gene con-
version or any form of point mutation in the CAN1 allele),
Ade
+
Lys
)
(crossover or any form of mutation in the
CAN1–LYS region such as CAN1–LYS deletion), or Ade
)
Lys
)
(chromosome loss) (see Fig. 7A) [58] after incubation
at 30 °C for 2–4 days. In the case of RD301 strains, a
5-FOA resistance (5-FOA
R
) assay was performed as
described previously [30].
Cell-cycle experiments
Yeast haploid YK402 and its derivatives were grown in
YPD medium to approximately 10
7
cellsÆmL
)1
and arrested

Table 1. Sacchromyces cerevisiae strains used in this study.
Strain Genotype Reference
YK402 MATa ade2-1 can1-100 his3-11,-15 leu2-3,112 trp1-1 ura3-1 bar1D::HISG [59]
AYU901 As YK402 except SWE1myc:TRP1 This study
AYU902 As YK402 except swe1D::kanMX This study
AYU903 As YK402 except RAD53myc:TRP1 This study
AYU904 As YK402 except CHK1myc:TRP1 This study
YAS3001 MATa ⁄ a can1D::LUE2 ⁄ CAN1 ade2-1 ⁄ ade2-1 lys2-1 ⁄ lys2-1 ILV ⁄ ilv2 ura3-52 ⁄ ura3-52 leu2-3,
112 ⁄ leu2-3,112 his3-200D ⁄ HIS trp1-901 ⁄ TRP V-11::LYS2
a
V-565::ADE2
a
[58]
AYU9100 As YAS3001 except swe1D::kanMX ⁄ swe1D::kanMX This study
AYU9200 As YAS3001 except hog1D::kanMX ⁄ hog1D::kanMX This study
AYU9300 As YAS3001 except chk1D::kanMX ⁄ chk1D::kanMX This study
RD301
b
MATa ⁄ MATa lys2D202 ⁄ lys2D202 trp1D63 ⁄ TRP1 hisD200 ⁄ HIS3 ade2D::hisG ⁄ ade2D::hisG
III-205::URA3 ⁄ III-205 ura3-52 ⁄ ura3-52 III-314::ADE2 LEU2 ⁄ leu2-1
[30]
AYU402 As RD301 except swe1D::kanMX ⁄ swe1D::kanMX This study
a
V-11::LYS2 signifies that the LYS2 fragment is inserted at the 11 kb position of chromosome V; V-565::ADE2 signifies that the ADE2
fragment is inserted at the 565 kb position of chromosome V [13].
b
Three heterozygous markers reside on chromosome III, which are
III-205::URA3, III-314::ADE2 and LEU2.
A. Yamamoto et al. PHQ induces stress signals in S. cerevisiae
FEBS Journal 275 (2008) 5733–5744 ª 2008 The Authors Journal compilation ª 2008 FEBS 5741

in G
1
by adding 30 ngÆmL
)1
a-factor at 30 °C for 3 h. Cells
were monitored microscopically and experiments were per-
formed if G
1
synchrony of 85–100% was reached. a-factor
was washed away with YPD medium and cells were inocu-
lated under experimental conditions. PHQ (2 mm) was
added if the cell cycle progressed to early S phase (about
70–80% of cells form small buds approximately 70 min
after release). At each time point and for each strain, more
than 100 cells were scored for cytology (budding and spin-
dle morphology). For FACScan analysis, cell samples were
fixed with 70% ethanol overnight, washed twice with
50 mm sodium citrate, and resuspended in 50 mm sodium
citrate containing 0.25 mgÆmL
)1
of RNase A. Following
incubation at 37 °C for 1 h, the cells were treated again
with 0.25 mgÆmL
)1
of proteinase K at 37 °C for 1 h. After
brief sonication, 0.4 mL of propidium iodide (16 lgÆmL
)1
)
was added to the samples. A Becton-Dickinson (Franklin
Lakes, NJ, USA) flow cytometer (FACS Calibar) was used

to analyze the samples. More than 20 000 cells were
assayed for each time point.
Fluorescence staining and microscopy
To observe the nuclear position, DNA of ethanol-fixed cells
was visualized by staining with DAPI. To observe micro-
tubules, formamide-fixed cells were incubated with mono-
clonal antibody against rat tubulin followed by the
Cy2-conjugated secondary antibody. Fluorescence micros-
copy was performed using an Olympus microscope equipped
with an Olympus digital camera. To observe microcolonies,
G
1
synchronous cells with pheromone were plated on micro-
scope slides with YPD agar containing 2 mm of PHQ at
30 °C, and the growth of individual cells was monitored
over the next 9 h. An Olympus microscope equipped with a
digital camera was used to count the number of cells (buds).
Protein techniques
Log-phase yeast cells were used to prepare protein samples
for time-course experiments. Yeast cells were harvested by
centrifugation (780 g, 5 min at room temperature), and then
pellets were stored frozen at )80 °C. Frozen pellets were
thawed in 0.1 n NaOH and harvested by centrifugation
(3120 g, 5 min at room temperature). Yeast lysates were pre-
pared by resuspending pellets in protein loading buffer
(0.125 m Tris ⁄ HCl, 10% 2-mercaptoethanol, 4% SDS, 10%
sucrose and 0.004% bromophenol blue), incubated at 95 °C
for 10 min, and centrifuged (3120 g, 5 min at room
temperature) to obtain supernatants for application to poly-
acrylamide gels. Protein samples were resolved on 8 or 10%

SDS–PAGE and transferred to ECL membranes. To detect
phospho-Rad53 or phospho-Chk1, membranes were probed
with mouse monoclonal antibody against Myc proteins as
the primary antibody and HRP-conjugated anti-mouse IgG
as the secondary antibody. To detect phospho-Hog1,
membranes were probed with antibody against phospho-p38
MAPK (T180⁄ Y182) as the primary antibody and HRP-
conjugated anti-mouse IgG as the secondary antibody. To
detect Hog1, membranes were probed with goat monoclonal
anti-Hog1 as the primary antibody and HRP-conjugated
anti-goat IgG as the secondary antibody. To detect Arp3p as
a loading control, membranes were probed with goat
monoclonal anti-Arp3 as the primary antibody and
HRP-conjugated anti-goat IgG as the secondary antibody.
The antibody-treated membranes were incubated with
ECL Plus western blotting detection reagent (GE Healthcare
UK) and visualized using an image analyzer.
Acknowledgements
This work was supported by a grant-in-aid from the
Japanese Ministry of Education, Science, Culture,
Sport and Technology, and by a Research Proposal
for Long-Range Research Initiatives from the Japan
Chemical Industry Association.
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Supporting information
The following supplementary material is available:
Fig. S1. Classification of spindle morphology.
Fig. S2. Lethality and induction of canavanine resis-
tance by PHQ in the chk1D strain, AYU9300
(chk1D ⁄ chk1D).
This supplementary material can be found in the
online version of this article.
Please note: Wiley-Blackwell is not responsible for
the content or functionality of any supplementary
materials supplied by the authors. Any queries (other

than missing material) should be directed to the corre-
sponding author for the article.
PHQ induces stress signals in S. cerevisiae A. Yamamoto et al.
5744 FEBS Journal 275 (2008) 5733–5744 ª 2008 The Authors Journal compilation ª 2008 FEBS