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 Yamamoto1, Tatsuo Nunoshiba1, Keiko Umezu2, Takemi Enomoto3 and Kazuo Yamamoto1 Graduate School of Life Sciences, Tohoku University, Sendai, Japan Fukuoka Dental College, Tamura, Japan Pharmaceutical Sciences, Tohoku University, Japan Keywords aneuploidy; checkpoint; G2 ⁄ 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: yamamot@m.tains.tohoku.ac.jp (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 G1 and G2 ⁄ M In this study, we demonstrate that phenyl hydroquinone can arrest the cell cycle at the G2 ⁄ M transition as a result of stabilization of Swe1 (a Wee1 homolog), probably leading to inactivation of Cdc28 (a Cdk1 ⁄ Cdc2 homolog) Furthermore, 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 Mutations of swe1 and hog1 abolished phenyl hydroquinone-induced arrest at the G2 ⁄ M transition and the cells became resistant to phenyl hydroquinone lethality and aneuploidy development These data suggest that a phenyl hydroqionone-induced G2 ⁄ M transition checkpoint that is activated by the Hog1–Swe1 pathway plays a role in the development of aneuploidy We have previously observed that phenyl hydroquinone (PHQ), a hepatic metabolite of the Ames testnegative carcinogen ortho-phenylphenol (OPP) [1,2], arrests the cell cycle at G1 and G2 ⁄ M and causes aneuploidy [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 carcinogens 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 majority 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 genome stability, including DNA metabolism enzymes such as polymerases, recombination enzymes and a ligase [10,11], as well as components of the chromosome segregation machinery, including tubulins, mitotic spindle 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 PHQ induces stress signals in S cerevisiae A Yamamoto et al motors, components of the kinetochore, and spindle pole bodies [12–15] Defects in spindle checkpoint regulatory systems have also been shown to cause a high frequency of aneuploidy [16–18] We wished to determine how PHQ arrests the cell cycle at G2 ⁄ 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 frequently delayed, and the morphogenesis checkpoint introduces a compensatory delay in nuclear division at the G2 ⁄ M boundary until a bud is well formed [19] The cell-cycle delay is due to inhibitory phosphorylation 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 G2 ⁄ M boundary, and therefore may cause aneuploidy Results G2/M delay by phenyl hydroquinone Previously we demonstrated that PHQ efficiently induced G2 ⁄ M delay and could cause aneuploidy in budding yeast [3] To determine the exact point of arrest, cells were released from G1 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, mm PHQ added 70 after release from G1 caused accumulation of G2–M spindles but not anaphase spindles (spindle 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 G2 ⁄ 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 lgỈmL)1 nocodazole, which activates a spindle checkpoint (metaphase to anaphase transition), 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 G2 ⁄ M boundary until a bud is well formed [19] Thus, PHQ can activate the morphogenesis checkpoint at the G2 ⁄ M boundary A YPD 100 G1-S 60 B 40 G2-meta 150 Ana 0 50 100 150 200 mM PHQ 100 80 G2-meta G1-S 60 40 120 90 60 30 20 Ana 0 50 100 150 200 Minutes after release from G1 5734 mM PHQ 15 µg·mL–1 noc 180 20 % cells YPD Minutes after release from G1 % cells 80 Fig PHQ-induced G2 ⁄ M delay in wildtype cells Haploid YK402 yeast cells at 107 cellsỈmL)1 were synchronized in G1 using 30 ngỈmL)1 a-factor for h at 30 °C Cells were inoculated into YPD medium and incubated at 30 °C PHQ (2 mM) or nocodazole (15 lgỈmL)1) was added (arrow) when 80–90% of cells had very small buds (approximately 70 min) At the indicated times, samples were withdrawn and processed for spindle morphology observation (A) and a FACScan analysis of DNA content (B) as described in Experimental procedures FEBS Journal 275 (2008) 5733–5744 ª 2008 The Authors Journal compilation ª 2008 FEBS A Yamamoto et al PHQ induces stress signals in S cerevisiae A B YPD 0.1% MMS 15 µg·mL–1 nocodazole mM PHQ Fig Elongated bud formation by PHQ (A) Haploid YK402 cells at the exponential phase were treated with 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 lgỈmL)1 nocodazole was used (B) High-magnification (· 400) images of PHQ-induced elongated buds (data not shown) In the presence of mm PHQ, Swe1 accumulated at S phase but was not degraded at M phase at 105 (Fig 3) We conclude that arrest at G2 ⁄ M by PHQ is due to stabilization of Swe1 Stabilization of Swe1 can lead to the formation of elongated buds and G2 ⁄ M transition arrest due to inactivation of the cyclin B (Clb)–Cdc28 complex PHQ inhibits degradation of Swe1 The sensors that detect the morphogenesis checkpoint remain unknown, but the resulting G2 ⁄ 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 hyperphosphorylated 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 G1 synchronous cells were inoculated into YPD medium In the absence of PHQ, Swe1 accumulated at S phase at 45–60 and was then degraded as cells proceeded from S to M phase at 105 If the cell cycle moved to next round of S phase at 150 (Fig 1A), Swe1 accumulated again Time (min) Fig Stabilization of Swe1 by PHQ G1 synchronous AYU901 cells (SWE1myc:TRP1) were inoculated in YPD medium, mM PHQ was added at 70 (arrow), and cells were sampled at intervals and processed for western blotting experiments Proteins extracted were resolved by 10% SDS–PAGE and subjected to immunoblotting using an antibody that recognizes Myc proteins G2/M arrest by PHQ is abolished by mutation of swe1 Next, we constructed swe1 deletion mutants and examined progression of the cell cycle in the presence of mm PHQ The mutants were arrested at G1 by a-factor treatment, then inoculated into YPD medium PHQ was added 70 after release As shown in Fig 4A, mm and mm PHQ caused G2–M spindles to accumulate and anaphase spindles to elongate, 30 45 60 75 90 105 Swe1 YPD Arp3 30 45 60 75 90 105 Swe1 mM PHQ FEBS Journal 275 (2008) 5733–5744 ª 2008 The Authors Journal compilation ª 2008 FEBS Arp3 5735 PHQ induces stress signals in S cerevisiae A Yamamoto et al A YPD 100 G1-S 60 B mM PHQ 15 µg·mL–1 noc 180 20 G2-meta Ana 0 50 100 150 150 200 mM PHQ 100 G1-S 80 % cells YPD 40 60 40 G2-meta 20 Minutes after release from G1 % cells 80 120 90 60 30 Fig PHQ-induced G2 ⁄ M delay in swe1D cells Experiments were performed as in Fig 1, but using haploid AYU902 (swe1D) yeast cells instead of YK402 (SWE1) Ana 0 50 100 150 200 Minutes after release from G1 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 mm PHQ FACScan analysis also indicated that mm PHQ did not stop the cell cycle at G2 ⁄ 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 G1-synchronized cells were plated on microscope slides with YPD agar containing mm PHQ, and the growth of individual cells was monitored over the next h Figure shows that SWE1 cells divided slowly and continuously in the presence of mm PHQ, with approximately four cells formed after h of incubation The swe1 mutant cells divided more rapidly than the SWE1 cells in the presence of mm PHQ, with approximately nine cells formed after h However, division of swe1 cells on PHQ-containing plates was slower than that of SWE1 or swe1 cells on plates without PHQ (Fig 5) This effect of PHQ was probably due to G1 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 incu5736 bation (data not shown) Our results suggest that, in wild-type cells, mm PHQ is able to induce cell-cycle arrest but not cell death During microscopic observation, we observed elongated buds in SWE1 cells treated 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 phosphorylation of Hog1 by mm PHQ We quantified activation of Hog1 by measuring the amount of the phosphorylated form (Thr174) of Hog1 using a selective antibody After 30 of incubation of asynchronous wild-type cells with mm PHQ, it was possible to detect the phosphorylated form of Hog1 (Fig 6A) The level of phosphorylated Hog1 decreased by 60 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 lgỈmL)1 nocodazole resulted in weak phosphorylation of Hog1 at 60 As a loading control, the same blot membrane was FEBS Journal 275 (2008) 5733–5744 ª 2008 The Authors Journal compilation ª 2008 FEBS A Yamamoto et al PHQ induces stress signals in S cerevisiae A 9h B Cell number per microcolony WT (YPD) WT (PHQ) swe1 (PHQ) 10 7.5 5.0 2.5 0 2.5 7.5 10 Incubation time (h) Fig Microcolony assay of PHQ-treated SWE1 and swe1D cells (A) G1-synchronized cells of YK402 (SWE1) and AYU902 (swe1D) were plated on microscope slides with YPD agar containing mM PHQ at 30 °C, and the growth of individual cells was monitored over the next 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 mM PHQ; closed circle, swe1D without PHQ; open circle, swe1D with mM PHQ A NaCl 30 60 PHQ 30 DMSO 60 30 60 MMS 30 nocodazole 60 30 60 (min) p-Hog1 Hog1 Arp3 Fig Phosphorylation of Hog1, Chk1 and Rad53 by PHQ An exponentially growing asynchronous culture of YK402 was treated with mM PHQ or with 0.8 M NaCl, 0.2% dimethylsulfoxide (DMSO), 0.1% MMS or 15 lgỈmL)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 immunoblotting using antibodies against phospho-p38 MAPK (T180 ⁄ Y182) (A), against Myc proteins for Chk1 (B), and against Myc proteins for Rad53 (C) B PHQ 15 30 MMS 60 15 30 60 (min) p-Chk1 Chk1 Arp3 PHQ C 15 reprobed against total Hog1 using a monoclonal Hog1 antibody (Fig 6A) Next, we examined the phosphorylation of Chk1 (Fig 6B) and Rad53 (Fig 6C), which are phosphorylated if upstream Mec1 ⁄ Tel1 is activated by DNA damage After 60 of incubation of asynchronous wild-type cells with 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 Thus, we conclude that PHQ treatment causes 30 MMS 60 15 30 60 (min) p-Rad53 Rad53 Arp3 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 G2 ⁄ 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 FEBS Journal 275 (2008) 5733–5744 ª 2008 The Authors Journal compilation ª 2008 FEBS 5737 PHQ induces stress signals in S cerevisiae A Yamamoto et al mutation Fig 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 CanR 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 CanR mutants that can be classified by phenotype CanR 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 CanR 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 mM of PHQ, as shown in (B), were estimated to be gene conversion, crossover and chromosome loss, as shown in (A) The CanR frequencies in mM PHQ were · 10)3 for wild-type, · 10)4 for swe1D and 1.05 · 10)4 for hog1D (D) The surviving fraction (upper panel) and 5-FOAR 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-FOAR mutants identified in the middle panel At least 250 5-FOAR colonies were isolated for each treatment condition, and 5-FOAR Leu) Ade) in RD301 (wild-type; filled column) and AYU401 (swe1D; open column) is classified as a chromosome loss (lower panel) 5738 FEBS Journal 275 (2008) 5733–5744 ª 2008 The Authors Journal compilation ª 2008 FEBS A Yamamoto et al homologous recombination [3] In this study, we demonstrate that PHQ induced arrest at the G2 ⁄ 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 carrying CAN1 ⁄ can1D::LEU2 heterozygous alleles (Fig 7A) were exposed to various concentrations of PHQ for 17 h Cell survival and the frequency of the CanR mutation were measured (Fig 7B upper and lower panels, respectively) PHQ did not induce cell death or CanR 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 · 106 and 1.90 · 106 per mL, respectively, meaning that growth was not strongly retarded when compared to wild-type cells (3.95 · 106 cellsỈmL)1) As a reference, swe1 and hog1 mutants were sensitive to NaCl treatment (data not shown), consistent with previous results [22] We then classified CanR mutants using V11::LYS2 and V565::ADE2 markers (Fig 7A) and found that PHQ generates CanR 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 CanR mutation were increased by PHQ as efficiently as in the YAS3001 strain (wild-type), and CanR mutants were induced through loss of chromosome V but not gene conversion or crossover (Fig S2), indicating no involvement of the DNA-damage pathway in PHQ-induced aneuploidy 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-FOAR mutations did not increase (Fig 7D upper and middle panels, respectively) Again, PHQ exclusively induced aneuploidy 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 cellstatic effect rather than a cell-killing effect We argue PHQ induces stress signals in S cerevisiae that PHQ-induced activation of the Hog1–Swe1 pathway 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 G2 ⁄ 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 G2 ⁄ M boundary The swe1 mutation abolished PHQ-induced G2 ⁄ 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 G2 ⁄ 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 G1 and G2 [31–33] Entry from G2 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 hyperphosphorylated [24,35], and undergoes ubiquitin-mediated degradation [25,26,36] Defects in septin filament assembly at the bud neck result in the hypophosphorylation 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 G2 ⁄ 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, phosphorylation 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 G2 ⁄ 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 G2 ⁄ M boundary through the Hog1–Swe1 pathway is a prerequisite for aneuploidy Obviously, arrest at the G2 ⁄ M boundary is not sufficient for aneuploidy FEBS Journal 275 (2008) 5733–5744 ª 2008 The Authors Journal compilation ª 2008 FEBS 5739 PHQ induces stress signals in S cerevisiae A Yamamoto et al because high osmolarity, such as that caused by NaCl treatment, also activates the Hog1–Swe1 pathway (Fig 6) but does not strongly induce aneuploidy (data not shown) PHQ causes G2 ⁄ 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 G2 ⁄ M transition and increase the rate of mini-chromosome loss in fission yeast Thus, stress-induced G2 ⁄ M transition arrest is involved in aneuploidy development Cell-cycle progression from the G2 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 G2 ⁄ 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 G2 ⁄ 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 aneuploidy in the chk1D strain (Fig S2) Thus, the DNAdamage response did not occur in response to PHQ treatment In the swe1 strain, we observed that PHQ does not cause cell arrest at G2 ⁄ M (Fig 4) or cell death (Fig 7B) As the Hog1–Swe1 pathway is a key pathway 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 G2 ⁄ M is required for cell survival 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 G2 ⁄ M arrest Recently, it was shown that Neurospora crassa and Candida albicans 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 5740 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 G2 ⁄ 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 G2 ⁄ M arrest but also G1 arrest [3] As mentioned above, Hog1 activation also results in G1 arrest [31,33] It has been reported that Hog1 controls the G1 transition via a dual mechanism that involves downregulation of cyclin expression and direct phosphorylation of the CDK inhibitor protein Sic1 Phosphorylation of Sic1 in combination with cyclin downregulation results in Sic1 stabilization and the inhibition of cell-cycle progression to prevent premature entry into S phase without proper cell adaptation [47] Thus, PHQ may activate the Hog1–Sic1 pathway, causing G1 ⁄ S arrest Our results raise a question regarding the mechanism of PHQ-induced aneuploidy, especially the difference between the presence and absence of Hog1–Swe1 Activation of the Hog1 MAPK is physiologically significant for cell adaptation to environmental 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], oxidative stress [49], bacterial endotoxins [50] and citric acid [51] The yeast high-osmolarity sensor has been determined to consist of two independent branch sensors: 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 urinary bladder hyperplasia and tumors [2], but not produce gene mutations or genetic recombination They are therefore classified as typical Ames test-negative carcinogens [1] Many of the chemicals considered to be carcinogenic are categorized as Ames test-negative [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 FEBS Journal 275 (2008) 5733–5744 ª 2008 The Authors Journal compilation ª 2008 FEBS A Yamamoto et al PHQ induces stress signals in S cerevisiae Experimental procedures Yeast strains and plasmids The yeast strains used in this study are listed in Table 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, RAD53Myc13 and CHK1-Myc13 under endogenous promoter control, 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 indicated, 10 lgỈmL)1 of adenine sulfate, 20 lgỈmL)1 of uracil and 20 lgỈmL)1 of amino acids were added to the SC medium 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 Biotechnology 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 ImmunoResearch 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) membrane was purchased from GE Healthcare UK Ltd Canavanine resistance and 5-FOA resistance assays in yeast diploid cells Diploid cells (approximately 106 cellsỈmL)1) were treated with various concentrations of PHQ in SC medium at 30 °C for 17 h After two washes with sterile double-distilled water, appropriate dilutions were plated on SC plates with or without 25 lgỈmL)1 of Can Colonies were scored after 2–3 days of incubation at 30 °C The frequency of the CanR mutation was calculated by dividing the number of CanR 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 CanR colonies as Ade+ Lys+ (gene conversion 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-FOAR) assay was performed as described previously [30] Cell-cycle experiments Yeast haploid YK402 and its derivatives were grown in YPD medium to approximately 107 cellsỈmL)1 and arrested Table Sacchromyces cerevisiae strains used in this study Strain Genotype Reference YK402 AYU901 AYU902 AYU903 AYU904 YAS3001 MATa ade2-1 can1-100 his3-11,-15 leu2-3,112 trp1-1 ura3-1 bar1D::HISG As YK402 except SWE1myc:TRP1 As YK402 except swe1D::kanMX As YK402 except RAD53myc:TRP1 As YK402 except CHK1myc:TRP1 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::LYS2a V-565::ADE2a As YAS3001 except swe1D::kanMX ⁄ swe1D::kanMX As YAS3001 except hog1D::kanMX ⁄ hog1D::kanMX As YAS3001 except chk1D::kanMX ⁄ chk1D::kanMX 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 As RD301 except swe1D::kanMX ⁄ swe1D::kanMX [59] This This This This [58] AYU9100 AYU9200 AYU9300 RD301b AYU402 study study study study This study This study This study [30] 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 FEBS Journal 275 (2008) 5733–5744 ª 2008 The Authors Journal compilation ª 2008 FEBS 5741 PHQ induces stress signals in S cerevisiae A Yamamoto et al in G1 by adding 30 ngỈmL)1 a-factor at 30 °C for h Cells were monitored microscopically and experiments were performed if G1 synchrony of 85–100% was reached a-factor was washed away with YPD medium and cells were inoculated 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 after release) At each time point and for each strain, more than 100 cells were scored for cytology (budding and spindle 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 mgỈmL)1 of RNase A Following incubation at 37 °C for h, the cells were treated again with 0.25 mgỈmL)1 of proteinase K at 37 °C for h After brief sonication, 0.4 mL of propidium iodide (16 lgỈmL)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 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 References To observe the nuclear position, DNA of ethanol-fixed cells was visualized by staining with DAPI To observe microtubules, formamide-fixed cells were incubated with monoclonal antibody against rat tubulin followed by the Cy2-conjugated secondary antibody Fluorescence microscopy was performed using an Olympus microscope equipped with an Olympus digital camera To observe microcolonies, G1 synchronous cells with pheromone were plated on microscope slides with YPD agar containing mm of PHQ at 30 °C, and the growth of individual cells was monitored over the next h An Olympus 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8, 183– 191 Supporting information The following supplementary material is available: Fig S1 Classification of spindle morphology Fig S2 Lethality and induction of canavanine resistance 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 corresponding author for the article FEBS Journal 275 (2008) 5733–5744 ª 2008 The Authors Journal compilation ª 2008 FEBS ... survival and decreased aneuploidy in the absence of G2 ⁄ M arrest Recently, it was shown that Neurospora crassa and Candida albicans hog1 mutants are resistant to dicarboximide and phenylpyrrole... pair in diploid cells (white with can1D and black with V-11::LYS2+, V-565::ADE2+ and CAN1) makes three possible CanR mutants that can be classified by phenotype CanR Ade) Lys+ clones were not expected... Sigma-Aldrich Monoclonal antibody against Myc proteins, anti-Hog1 IgG, anti-Arp3 IgG and anti-actin IgG were purchased from Santa Cruz Biotechnology Inc (Santa Cruz, CA, USA) The monoclonal antibody against