Global Disruption of Cell Cycle Progression and Nutrient Response by the Antifungal Agent Amiodarone
2007; Elsevier BV; Volume: 282; Issue: 52 Linguagem: Inglês
10.1074/jbc.m707593200
ISSN1083-351X
Autores Tópico(s)Fungal Biology and Applications
ResumoThe antiarrhythmic drug amiodarone has fungicidal activity against a broad range of fungi. In Saccharomyces cerevisiae, it elicits an immediate influx of Ca2+ followed by mitochondrial fragmentation and eventual cell death. To dissect the mechanism of its toxicity, we assessed the transcriptional response of S. cerevisiae to amiodarone by DNA microarray. Consistent with the drug-induced calcium burst, more than half of the differentially transcribed genes were induced by high levels of CaCl2. Amiodarone also caused rapid nuclear accumulation of the calcineurin-regulated Crz1. The majority of genes induced by amiodarone within 10 min were involved in utilization of alternative carbon and nitrogen sources and in mobilizing energy reserves. The similarity to nutrient starvation responses seen in stationary phase cells, rapamycin treatment, and late stages of shift to diauxic conditions and nitrogen depletion suggests that amiodarone may interfere with nutrient sensing and regulatory networks. Transcription of a set of nutrient-responsive genes was affected by amiodarone but not CaCl2, indicating that activation of the starvation response was independent of Ca2+. Genes down-regulated by amiodarone were involved in all stages of cell cycle control. A moderate dose of amiodarone temporarily delayed cell cycle progression at G1, S, and G2/M phases, with the Swe1-mediated delay in G2/M phase being most prominent in a calcineurin-dependent manner. Overall, the transcriptional responses to amiodarone revealed by this study were found to be distinct from other classes of antifungals, including the azole drugs, pointing toward a novel target pathway in combating fungal pathogenesis. The antiarrhythmic drug amiodarone has fungicidal activity against a broad range of fungi. In Saccharomyces cerevisiae, it elicits an immediate influx of Ca2+ followed by mitochondrial fragmentation and eventual cell death. To dissect the mechanism of its toxicity, we assessed the transcriptional response of S. cerevisiae to amiodarone by DNA microarray. Consistent with the drug-induced calcium burst, more than half of the differentially transcribed genes were induced by high levels of CaCl2. Amiodarone also caused rapid nuclear accumulation of the calcineurin-regulated Crz1. The majority of genes induced by amiodarone within 10 min were involved in utilization of alternative carbon and nitrogen sources and in mobilizing energy reserves. The similarity to nutrient starvation responses seen in stationary phase cells, rapamycin treatment, and late stages of shift to diauxic conditions and nitrogen depletion suggests that amiodarone may interfere with nutrient sensing and regulatory networks. Transcription of a set of nutrient-responsive genes was affected by amiodarone but not CaCl2, indicating that activation of the starvation response was independent of Ca2+. Genes down-regulated by amiodarone were involved in all stages of cell cycle control. A moderate dose of amiodarone temporarily delayed cell cycle progression at G1, S, and G2/M phases, with the Swe1-mediated delay in G2/M phase being most prominent in a calcineurin-dependent manner. Overall, the transcriptional responses to amiodarone revealed by this study were found to be distinct from other classes of antifungals, including the azole drugs, pointing toward a novel target pathway in combating fungal pathogenesis. Fungal infections are a persistent problem, especially in immunocompromised patients undergoing treatment for AIDS, cancer, cystic fibrosis, and other diseases. Existing antifungal drugs have limitations in that there are relatively few classes with distinct mode of action; of these, the widely prescribed azole drugs are fungistatic and depend upon a healthy immune system for fungal clearance. The need for new drugs that combat resistance and improve the efficacy of existing antifungals is pressing. Amiodarone has been approved to treat ventricular arrhythmias since 1985. Pharmacological studies have shown its property as a cation channel blocker although it has multiple targets and a complex mechanism. Recent in vitro research in unicellular organisms demonstrated its microbicidal activity against a broad range of fungal species (1Courchesne W.E. J. Pharmacol. Exp. Ther. 2002; 300: 195-199Crossref PubMed Scopus (84) Google Scholar), bacteria (2Rosa S.M. Antunes-Madeira M.C. Matos M.J. Jurado A.S. Madeira V.M. Biochim. Biophys. Acta. 2000; 1487: 286-295Crossref PubMed Scopus (20) Google Scholar), and protozoa (3Benaim G. Sanders J.M. Garcia-Marchan Y. Colina C. Lira R. Caldera A.R. Payares G. Sanoja C. Burgos J.M. Leon-Rossell A. Concepcion J.L. Schijman A.G. Levin M. Oldfield E. Urbina J.A. J. Med. Chem. 2006; 49: 892-899Crossref PubMed Scopus (161) Google Scholar). In Saccharomyces cerevisiae, it elicits an immediate Ca2+ burst (4Courchesne W.E. Ozturk S. Mol. Microbiol. 2003; 47: 223-234Crossref PubMed Scopus (76) Google Scholar, 5Gupta S.S. Ton V.K. Beaudry V. Rulli S. Cunningham K. Rao R. J. Biol. Chem. 2003; 278: 28831-28839Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar) and subsequently a mitochondrial-mediated cell death program (6Pozniakovsky A.I. Knorre D.A. Markova O.V. Hyman A.A. Skulachev V.P. Severin F.F. J. Cell Biol. 2005; 168: 257-269Crossref PubMed Scopus (223) Google Scholar). Similarly, 12.5 μm amiodarone elevated cytosolic Ca2+ in Trypansoma cruzi but not in the host Vero cells (3Benaim G. Sanders J.M. Garcia-Marchan Y. Colina C. Lira R. Caldera A.R. Payares G. Sanoja C. Burgos J.M. Leon-Rossell A. Concepcion J.L. Schijman A.G. Levin M. Oldfield E. Urbina J.A. J. Med. Chem. 2006; 49: 892-899Crossref PubMed Scopus (161) Google Scholar). Low levels of amiodarone (1-4 μm), within the therapeutic range achieved in patients, were reported to exhibit synergistic fungicidal effects with azole drugs against pathogenic species of fungi (Candida and Cryptococcus) and protozoa (Trypanosoma), suggesting that the drug may be useful as a sensitizing agent in antimicrobial therapy (5Gupta S.S. Ton V.K. Beaudry V. Rulli S. Cunningham K. Rao R. J. Biol. Chem. 2003; 278: 28831-28839Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). A comprehensive view of the impact of amiodarone on microbial cellular pathways is prerequisite for its potential in vivo use as antifungal adjunct, and to understand mechanisms of drug toxicity and resistance. Phenotypic profiling of the set of S. cerevisiae single gene deletions for amiodarone hypersensitivity revealed the importance of genes involved in membrane trafficking and transport pathways, protein fate, and interaction with the cellular environment (5Gupta S.S. Ton V.K. Beaudry V. Rulli S. Cunningham K. Rao R. J. Biol. Chem. 2003; 278: 28831-28839Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 7Yadav J. Muend S. Zhang Y. Rao R. Mol. Biol. Cell. 2007; 18: 1480-1489Crossref PubMed Google Scholar). Extending the analysis to include additional drugs (tunicamycin, sulfometuron methyl, wortmannin) and ion (Ca2+, Mn2+) stress revealed that the major cellular components responsive to drug toxicity and ion stress localize to the endomembrane system. Notably, disruption of calcium and proton homeostasis by deletion of PMR1 (Golgi Ca2+, Mn2+-ATPase) and VMA (endo-membrane/vacuolar H+-ATPase) genes led to multidrug hypersensitivity. Genes involved in ergosterol biogenesis (ERG6, ERG24), lipid flipping and remodeling (SAC1, LEM3, CDC50, OPI1), and compartmental trafficking (RIC1, COG6, VPS20) were important for growth tolerance to toxic drugs. Together, these represented the first line of defense against diverse forms of toxic stress. Phenotypic profiling of multidrug sensitivity also pointed to a significant transcriptional response: genes involved in chromatin remodeling (HAF4, SNF5, SNF6, SWI3), histone modification (HFI1, GCN5), and transcription activation (SUT2, SRB8, SIN4, HCM1, SIP3, SFP1, UGA3) were important for survival in amiodarone (7Yadav J. Muend S. Zhang Y. Rao R. Mol. Biol. Cell. 2007; 18: 1480-1489Crossref PubMed Google Scholar). To gain a global perspective on the impact of this drug on gene expression networks, we profiled the genome-wide transcriptional response of S. cerevisiae to amiodarone by DNA microarray. Our findings demonstrate a prominent overlap between amiodarone and calcium stress responses, consistent with the drug-induced Ca2+ burst reported earlier. Unexpectedly, transcriptional profiling revealed that amiodarone appeared to disrupt nutrient sensing and regulatory networks and delay cell cycle progression. In addition to Ca2+ stress, both cell cycle block and nutrient starvation may contribute to the antifungal mechanism of amiodarone. Yeast Strains, Media, and Amiodarone Treatment Conditions—Yeast deletion mutants (swe1Δ, cnb1Δ, crz1Δ), isogenic to BY4742, were from the MATα S. cerevisiae deletion library (Invitrogen). Yeast strains were grown in standard synthetic complete medium or YPD medium at 30 °C. Liquid cultures were incubated at 30 °C with shaking (250 rpm) and growth was determined by absorbance at 600 nm. Amiodarone (Sigma) was added from a stock solution of 50 mm in dimethyl sulfoxide to 100-ml log phase culture (OD 0.1, synthetic complete medium) to concentrations specified in the figure legends. Me2SO was added to 0.03% (v/v) in the control culture. These conditions were used for determination of doubling time, viability, DNA microarray, and fluorescent microscopy. To determine cell viability, samples were collected 10 min and 6 h after addition of amiodarone (15 μm) or Me2SO and equal numbers of cells for each treatment (determined after reading A600) were spread on YPD plates, incubated at 30 °C for 36 h before counting 500-1500 colonies to calculate cell viability. Microarray Hybridization and Bioinformatic Analysis—S. cerevisiae BY4742 strain was used for DNA microarray experiment. Culture and amiodarone treatment conditions are as described above. The Me2SO control samples were collected 10 min after Me2SO addition. The amiodarone-treated samples were collected 10 min and 6 h after amiodarone addition. Cells were spun down at 4000 rpm and flash-frozen after removing the supernatant. Two independent samples for each treatment (Me2SO control and amiodarone, 10 min and 6 h exposure) were collected and analyzed with DNA microarray. Total RNA was isolated by the hot acidic phenol extraction method as previously described (8Schmitt M.E. Brown T.A. Trumpower B.L. Nucleic Acids Res. 1990; 18: 3091-3092Crossref PubMed Scopus (1152) Google Scholar). RNA concentration and purity were determined spectrophotometrically by measuring absorbance at 260 and 280 nm. The integrity of the RNA samples was confirmed by polyacrylamide gel electrophoresis. cDNA synthesis, labeling and hybridization, image scanning, and processing were conducted at the Johns Hopkins Microarray Core Facility. Briefly, first and second strand cDNA was synthesized with SuperScript II (Invitrogen) and DNA polymerase I (Invitrogen). Biotin-labeled cRNA was synthesized with T7 RNA Polymerase (ENZO Life Sciences, Inc.) and fragmented. Sample mixture was hybridized to Yeast Genome 2.0 Arrays (Affymetrix). The arrays were stained and washed using the Affymetrix GeneChip Fluidics Station 450 and Mini_euk2V3_450 fluidics script. All arrays were scanned in the Affymetrix GeneChip Scanner 3000 and raw analysis performed with Affymetrix GeneChip Operating System (GCOS) 1.4. Subsequently microarray data were imported to GeneSpring 7.0 (Agilent Technologies) for normalization and analysis. Data for genes showing 2-fold or greater response to amiodarone were imported to Gene Cluster 3.0 (9de Hoon M.J. Imoto S. Nolan J. Miyano S. Bioinformatics. 2004; 20: 1453-1454Crossref PubMed Scopus (2336) Google Scholar) for hierarchical and k-means clustering analyses, along with previously published DNA microarray data for these genes in response to CaCl2 (10Yoshimoto H. Saltsman K. Gasch A.P. Li H.X. Ogawa N. Botstein D. Brown P.O. Cyert M.S. J. Biol. Chem. 2002; 277: 31079-31088Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar), rapamycin (11Hardwick J.S. Kuruvilla F.G. Tong J.K. Shamji A.F. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14866-14870Crossref PubMed Scopus (466) Google Scholar), amino acid or nitrogen depletion, growth in YPD (12Gasch A.P. Spellman P.T. Kao C.M. Carmel-Harel O. Eisen M.B. Storz G. Botstein D. Brown P.O. Mol. Biol. Cell. 2000; 11: 4241-4257Crossref PubMed Scopus (3720) Google Scholar), diauxic shift (13DeRisi J.L. Iyer V.R. Brown P.O. Science. 1997; 278: 680-686Crossref PubMed Scopus (3695) Google Scholar), and four classes of antifungals (caspofungin, ketoconazole, 5-fluorocytosine, and amphotericin B14), downloaded from the publisher's website or requested from the authors. Results were displayed with Java Tree View software and edited in Adobe Photoshop (Adobe Systems Inc.). Geneset enrichment analyses were performed on the server at the Munich Information center for Protein Sequences (MIPS) data base (mips.gsf.de/proj/funcatDB/search_main_frame.html). Quantitative RT-PCR—Aliquots of the same RNA samples used for DNA microarray were saved for quantitative RT-PCR. 2The abbreviation used is: RT-PCRreverse transcriptase-PCR The RNA samples were treated with DNase I (Roche Diagnostics) to remove residual genomic DNA. First strand cDNA was synthesized from 1 μg of total RNA with SuperScript III Reverse Transcriptase (Invitrogen) according to the manufacturer's instructions. Quantitative RT-PCR was conducted with SYBR Green PCR Master Mix in a 7500 Real Time PCR system (Applied Biosystems, Foster City, CA). A dissociation curve was generated at the end of each PCR to verify that a single DNA species was amplified. ACT1 was amplified as the reference gene to calculate -fold change for genes of interest. All quantitative RT-PCR experiments were conducted in triplicate. Data were analyzed with Sequence Detection Software (Applied Biosystems). -Fold changes and standard deviations were calculated with the standard curve method according to the manufacturer's instructions. reverse transcriptase-PCR Fluorescence Microscopy—For microscopy with FUN-1 (Invitrogen), BY4742 cells were exposed to amiodarone (15 μm) or Me2SO for 10 min or 6 h, collected by centrifugation, and resuspended in 50 μl of synthetic complete medium with 4 μm FUN-1 dye (diluted from a stock of 200 μm in dimethyl sulfoxide). Following incubation at 30 °C for 1 h, cells were examined under a Zeiss Axiophot fluorescence microscope equipped with a Photometrics Coolsnap fx camera. The fluorescent dye was excited by UV light. Conversion of FUN-1 into cylindrical intravacuolar structures was monitored by recording fluorescent micrographs at emission wavelengths of 645 nm (metabolically active and inactive cells) or 525 nm (metabolically inactive cells only). To monitor Crz1p translocation, crz1Δ yeast cells expressing GFP-Crz1 from plasmid pKK249 (15Kafadar K.A. Cyert M.S. Eukaryot. Cell. 2004; 3: 1147-1153Crossref PubMed Scopus (66) Google Scholar) were grown to OD 0.1 and treated with CaCl2 (50 mm) or amiodarone (15 μm) for specified time. Cells were collected by rapid centrifugation and visualized with the Zeiss Axiophot fluorescence microscope at emission wavelength of 525 nm. A total of 300-500 cells were counted for each condition to calculate percentage of cells showing nuclear translocation of Crz1p. Nuclei were stained with 1 μg/ml 4′,6-diamidino-2-phenylindole (Roche Diagnostics). Pseudo-colorization was done with Adobe Photoshop. Flow Cytometry and Cell Cycle Manipulations—In experiments with asynchronous cultures, amiodarone (15 μm) and FK506 (1 μg/ml) were added to 200 ml of BY4742 yeast (OD 0.1) in YPD and samples collected at the specified time intervals. For cell cycle synchronization, 0.2 m hydroxyurea was used to arrest the yeast strains at S phase. The cells were then released into YPD medium for 15 min or 1 h before addition of amiodarone (15 μm). For flow cytometry, cultures were spun down, resuspended in 0.3 ml of 0.2 m Tris-HCl (pH 7.5) buffer, and fixed by addition of 0.7 ml of ethanol (95%) at 4 °C for 2 h. The cells were washed with the above buffer once before treatment with RNase A solution (1 mg/ml in 0.2 m Tris-HCl buffer, pH 7.5) for 2 h at 37 °C. The cells were washed once and stained with 1 μm SYTOX Green (Invitrogen) in 50 mm Tris-HCl (pH 7.5). Samples (20,000 cells) were analyzed with a FACScan instrument (BD Biosciences). Results were visualized with FlowJo software (Tree Star, Inc., Ashland, OR) and edited with Adobe Photoshop. Distribution of cell population in G1,S, and G2/M phases was calculated with FlowJo. Dose-dependent Growth Inhibition and Metabolic Arrest by Amiodarone—To reveal a substantive transcriptional response to amiodarone without eliciting secondary effects, we sought a drug concentration that would prolong the doubling time of the culture by 2-fold. Fig. 1A shows that addition of amiodarone to an exponentially growing yeast culture in synthetic complete medium caused a dose-dependent inhibition of growth, with a doubling time of 110 min in the absence of drug increasing progressively to 150 (9 μm), 170 (12 μm), 230 (15 μm), and 380 min (18 μm). Additional amiodarone completely inhibited growth. At 15 μm, amiodarone was moderately fungicidal, with a19 ± 2% decrease in colony forming units after a 10-min exposure to drug. Longer exposure times increased the fungicidal effect, with a loss in colony forming units of 34 ± 8% after 6 h. Similar effects were seen in cells loaded with the FUN-1 dye (16Millard P.J. Roth B.L. Thi H.P. Yue S.T. Haugland R.P. Appl. Environ. Microbiol. 1997; 63: 2897-2905Crossref PubMed Google Scholar), which monitors loss of metabolic activity by a change in fluorescence from red cylindrical structures to diffuse green-yellow stain (Fig. 1B). Based on these results, we chose to monitor both early (10 min) and late (6 h) transcriptional response to a moderate dose of amiodarone (15 μm). Overview of Differentially Transcribed Genes—Within 10 min of exposure to 15 μm amiodarone, we observed differential transcription of 352 genes, using a 2-fold margin as cut-off. Of these, 218 gene transcripts increased, whereas 134 decreased (supplemental data Table 1S). At 6 h, the response was considerably muted, with 106 genes maintaining a ≥2-fold increase and only 9 genes remaining at ≥2-fold decrease. These data suggest that the transcriptional response reached its peak shortly after drug exposure and diminished over time, consistent with the fungicidal efficacy of the drug. Because this was confirmed using additional time intervals (not shown), we focused our analysis on the transcriptional response at 10 min of drug exposure. Relative to the yeast proteome, the differentially up-regulated gene products were significantly enriched in functional categories of metabolism (p value, 5.66 × 10-6) and energy production (p value, 4.56 × 10-9), with protein localization predominantly in membranes (plasma membrane and endo-membranes) and in peroxisomes, as shown in Fig. 2. In contrast, down-regulated genes were highly enriched in functional categories of cell cycle and DNA processing (p value, 1.19 × 10-7), cell type differentiation (p value, 1.60 × 10-5), and cell communication (p value, 2.49 × 10-5). Protein localization of down-regulated gene products showed significant enrichment in the yeast bud and cell periphery, whereas other cell compartments were represented at levels similar to the yeast proteome (Fig. 2). To validate our DNA microarray data, we performed real time quantitative PCR on 26 genes representing a variety of enriched functional categories. Results from real time PCR were in good agreement with DNA microarray results (supplemental data Table 2S), with -fold change in transcript levels being similar to, or greater than results from microarray. This indicated that the DNA microarray results faithfully represented the transcriptional response to amiodarone. Amiodarone Triggers a Large Transcriptional Response to Ca2+ Burst—Among the genes showing differential transcription in response to amiodarone, many are also known to respond to elevation of cytosolic Ca2+ (RCN1, ENA1-5, CMK1, CMK2, and GYP7), consistent with the drug-induced calcium burst reported previously (4Courchesne W.E. Ozturk S. Mol. Microbiol. 2003; 47: 223-234Crossref PubMed Scopus (76) Google Scholar, 5Gupta S.S. Ton V.K. Beaudry V. Rulli S. Cunningham K. Rao R. J. Biol. Chem. 2003; 278: 28831-28839Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). Fig. 3A shows that a large percentage of the amiodarone responsive gene set was also induced (137 of 218 genes; 63%) or repressed (77 of 134 genes; 57%) upon exposure to 200 mm CaCl2 (10Yoshimoto H. Saltsman K. Gasch A.P. Li H.X. Ogawa N. Botstein D. Brown P.O. Cyert M.S. J. Biol. Chem. 2002; 277: 31079-31088Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar). A hierarchical clustering analysis confirmed that the transcriptional response to amiodarone most closely resembled the response to high CaCl2 (Fig. 4).FIGURE 4Amiodarone elicits a starvation response by a mechanism independent of Ca2+. A, hierarchical clustering of 343 genes that were up- or down-regulated by at least 2-fold when exposed to 15 μm amiodarone for 10 min. Other transcriptionally regulated genes included in this analysis were downloaded from the publisher's websites or requested from the authors: exposure to CaCl2 (5, 15, and 30 min), rapamycin (30 min), time course series for diauxic shift (successive samples 1-7 were collected between 9 and 20.5 h growth), amino acid starvation (0.5-6 h), nitrogen depletion (1 h and 5 days), and growth in YPD (2 h and 5 days). -Fold change values under each condition were log2 transformed and clustered with Gene Cluster 3.0 (9de Hoon M.J. Imoto S. Nolan J. Miyano S. Bioinformatics. 2004; 20: 1453-1454Crossref PubMed Scopus (2336) Google Scholar). Distances between genes and arrays were computed based on correlation (centered). Both genes and arrays were clustered with average linkage method. The data were visualized with Java Tree View (36Saldanha A.J. Bioinformatics. 2004; 20: 3246-3248Crossref PubMed Scopus (2334) Google Scholar). B, K-means clustering of 343 genes that were differentially regulated by at least 2-fold when exposed to 15 μm amiodarone for 10 min. Euclidean distance method was used to calculate similarity matrix. The set of 52 genes induced by amiodarone but not by Ca2+ is listed.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The transcription factor Crz1 is the major effector of calcineurin, a highly conserved Ca2+/calmodulin-activated protein phosphatase that couples Ca2+ signals to downstream responses. Analogous to the NFAT family of transcription factors in mammalian cells, yeast Crz1 is dephosphorylated by calcineurin and migrates to the nucleus where it induces expression of target genes in response to a Ca2+ signal. We monitored the intracellular localization of Crz1-GFP upon exposure to amiodarone (15 μm) and high CaCl2 (50 mm). There was an immediate relocalization of Crz1 to the nucleus upon addition of amiodarone, similar to but more transient than the effect of 50 mm extracellular Ca2+ (Fig. 3, B and C). Combined with the microarray data, this result demonstrates that amiodarone-induced Ca2+ influx is capable of mounting a robust downstream transcriptional response. Amiodarone Induces a Stress Response—Previous studies have identified a number of genes whose expression is altered under stress. In one study, expression of 216 genes was induced in response to seven stress conditions, namely heat, high salt, acid, alkali, H2O2, hyperosmolarity, and diauxic shift (17Causton H.C. Ren B. Koh S.S. Harbison C.T. Kanin E. Jennings E.G. Lee T.I. True H.L. Lander E.S. Young R.A. Mol. Biol. Cell. 2001; 12: 323-337Crossref PubMed Scopus (1068) Google Scholar). Of these, 54 gene transcripts (Fig. 3A and supplemental data Table 3S) were also induced by amiodarone, mostly as a response to Ca2+ stress; fewer (11 genes) were repressed in common with general stress response. Transcription of most (∼60%) of these 54 genes is under the control of the MSN2/MSN4 stress-regulated factors. This indicates that the cell initiates its defense mechanism to cope with amiodarone toxicity. Amiodarone Induces a Unique Nutrient Starvation Response—The majority of amiodarone-induced genes were involved in utilization of alternative carbon and nitrogen sources and mobilizing energy reserves (Table 1). This included genes for metabolizing the storage carbohydrates trehalose and glycogen (TPS2, GAC1, GLC3, GPH1, GSY1, GSY2, and others), fermenting non-glucose carbohydrates (FDH1, ACS1, ALD4, CYB2, and others), and metabolizing fatty acids (FOX2, POX1, POT1, ECI1, FAA2, CTA1, PXA1, PXA2, IDP3, TES1, and YPL156C). Concomitantly, we observed an induction of transporters for galactose (GAL2), maltose (MAL31), and high-affinity and moderate-affinity scavengers of glucose (HXT4, HXT5, and HXT6). Numerous genes involved in regulating glucose metabolism were up-regulated (MTH1, CAT8, REG2, NRG1, and MIG2) or repressed (STD1, CYR1, SRB8, MIG1, and GAL11) upon exposure to amiodarone. Similarly, genes under Nitrogen Catabolite Repression, including GAP1, MEP2, DAL80, DAL4, DAL7, PUT1, PUT4, UGA4, and PRB1, were derepressed. Induction of these genes is triggered by depletion of preferred nitrogen sources such as ammonium and glutamine. Collectively, the pattern of global transcriptional change induced by amiodarone is reminiscent of a starvation response. Indeed, hierarchical clustering analysis demonstrated that the transcriptional response within 10 min of exposure to amiodarone was similar to glucose and nitrogen starvation observed during stationary phase, rapamycin treatment, and late stages of diauxic shift and nitrogen depletion (Fig. 4A). Thus, the rapid and extensive re-programming of yeast metabolic networks to adapt to nutrient-limiting conditions, despite the availability of glucose and ammonium in the medium, suggests that amiodarone may disrupt nutrient sensing and regulatory circuitry.TABLE 1Enrichment of functional categories for genes up-regulated by amiodaroneFunctional categoryFrequency in amiodarone datasetGenome frequencyp value%Metabolism (83)38.024.65.66E-06C-compound & carbohydrate metabolism (44)20.18.231.24E-08Fatty acid metabolism (6)2.750.390.000148Energy (37)16.95.984.56E-09Fermentation (non-glucose) (9)4.120.763.23E-05Energy reserve metabolism (9)4.120.910.000136Oxidation of fatty acids (5)2.290.146.09E-06Cellular transport (42)19.216.90.197659Anion transport (5)2.290.420.001937Sugar transport (6)2.750.500.000659Amine/polyamine transport (3)1.370.220.012073Lipid/fatty acid transport (5)2.290.710.019083Drug/toxin transport (5)2.290.630.011668Cell rescue, defense, and virulence (28)12.89.030.034936Oxidative stress response (5)2.290.890.044778pH stress response (2)0.910.130.030590Heat shock response (3)1.370.320.032343Catalase reaction (2)0.910.030.001259 Open table in a new tab Comparison of the transcriptional profiles in response to amiodarone and CaCl2 by K-means clustering revealed a signature response of amiodarone; a cluster of 52 genes was induced at least 2-fold by amiodarone but not by CaCl2 (Fig. 4B). Of these, 25 genes are known to be activated upon carbon or nitrogen starvation and include key regulators of glucose metabolism (such as CAT8, MIG2, GAC1, MTH1, and REG2), high affinity glucose transporters (HXT4 and HXT6), and genes for alternative carbon metabolism (MAL31, MAL33, POT1, POX1, and others). Additionally, none of the glucose metabolism regulators (STD1, CYR1, SRB8, MIG1, and GAL11) repressed by amiodarone were repressed by CaCl2 (10Yoshimoto H. Saltsman K. Gasch A.P. Li H.X. Ogawa N. Botstein D. Brown P.O. Cyert M.S. J. Biol. Chem. 2002; 277: 31079-31088Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar). We investigated, by quantitative RT-PCR, the expression of a subset of these genes upon exposure to 200 mm CaCl2: high levels of Ca2+ did not affect these nutrient-responsive genes, whereas expression of the calcium-responsive CMK2 was induced by 24-fold (supplemental data Table 2S). Together, these data indicate that amiodarone activates a starvation response by a mechanism independent of Ca2+ signaling. Amiodarone Blocks Cell Cycle Progression—Genes down-regulated by amiodarone were highly enriched in the category of cell cycle regulation (Table 2). Key cell cycle regulators included CLB6 (promoting DNA replication), CLB1 and CLB2 (promoting nuclear division), FKH2 (promoting G2/M transition), and SWI5 and ACE2 (promoting M/G1 transition). In addition, genes involved in the processes of DNA synthesis and replication, and cytokinesis were repressed. Other important cell cycle regulators, including CLB3, CLB4, and CLB5, were also repressed, albeit at lower levels (1.5-2-fold). To investigate if this large scale repression of cell cycle genes was mediated by Ca2+, we examined the DNA microarray dataset previously reported for calcium response (10Yoshimoto H. Saltsman K. Gasch A.P. Li H.X. Ogawa N. Botstein D. Brown P.O. Cyert M.S. J. Biol. Chem. 2002; 277: 31079-31088Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar). This dataset includes genes repressed by Ca2+ (200 mm) that have not been analyzed, to date. Among the 712 genes repressed by Ca2+ within 15 min, 114 genes are in the functional category of cell cycle and DNA processing. Of the 43 cell cycle genes represse
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