Abstract
The antiarrhythmic drug amiodarone has fungicidal activity against a broad range of fungi. In Saccharomyces cerevisiae, it elicits an immediate influx of Ca(2+) 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 CaCl(2). 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 CaCl(2), indicating that activation of the starvation response was independent of Ca(2+). 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 G(1), S, and G(2)/M phases, with the Swe1-mediated delay in G(2)/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.
Highlights
Transcriptional Profiling of Amiodarone Response in Yeast revealed that amiodarone appeared to disrupt nutrient sensing and regulatory networks and delay cell cycle progression
Other important cell cycle regulators, including CLB3, CLB4, and CLB5, were repressed, albeit at lower levels (1.5–2-fold). To investigate if this large scale repression of cell cycle genes was mediated by Ca2ϩ, we examined the DNA microarray dataset previously reported for calcium response (10)
We show that acute inhibition of calcineurin with FK506 increased cell death in amiodarone, as evidenced by accumulation of cells with decreased DNA content (Fig. 7A)
Summary
Yeast Strains, Media, and Amiodarone Treatment Conditions—Yeast deletion mutants (swe1⌬, cnb1⌬, crz1⌬), isogenic to BY4742, were from the MAT␣ S. cerevisiae deletion library (Invitrogen). 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. Two independent samples for each treatment (Me2SO control and amiodarone, 10 min and 6 h exposure) were collected and analyzed with DNA microarray. Fluorescence Microscopy—For microscopy with FUN-1 (Invitrogen), BY4742 cells were exposed to amiodarone (15 M) or Me2SO for 10 min or 6 h, collected by centrifugation, and resuspended in 50 l of synthetic complete medium with 4 M FUN-1 dye (diluted from a stock of 200 M in dimethyl sulfoxide). Flow Cytometry and Cell Cycle Manipulations—In experiments with asynchronous cultures, amiodarone (15 M) and FK506 (1 g/ml) were added to 200 ml of BY4742 yeast (OD 0.1) in YPD and samples collected at the specified time intervals. Distribution of cell population in G1, S, and G2/M phases was calculated with FlowJo
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