INTRODUCTION

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Farnesol (FOH) is an isoprenoid alcohol that may be endogenously generated within the cells by enzymatic dephosphorylation of farnesyl pyrophosphate (FPP), an intermediate of the metabolic pathway yielding sterols and other isoprenoid compounds from mevalonate (4). In addition to geranylgeranyl pyrophosphate, FPP also plays an important role as a precursor of protein prenylation such as in the posttranslational modification of oncogenic RAS proteins and other GTP-binding proteins (11). When exogenously added to the medium, FOH is subjected to either phosphorylation, yielding FPP, or oxidation, to give farnesal, farnesoic acid, and prenyldicarboxylic acid in mammalian cells (4). Recently, FOH has attracted much attention since it causes apoptotic cell death of human acute leukemia CEM-C1 cells (15, 20) and HL-60 cells (23). Interference with a phosphatidylinositol type of signaling has been proposed to be a cause of apoptosis in FOH-treated mammalian cells. In our previous study, FOH was found to exhibit a static growth inhibitory effect on the yeast Saccharomyces cerevisiae in which the intracellular diacylglycerol (DAG) level was significantly decreased and accompanied by downregulation of cell cycle gene expression (16). Mammalian and yeast cells may have a common mechanism in their responses to FOH regardless of whether these organisms are subjected to fatal or static damage from it.

Reactive oxygen species (ROS), including hydrogen peroxide, superoxide anion, and hydroxyl radical, are highly toxic oxidants which are inevitably produced to a certain extent under aerobic conditions. Among them, superoxide anions are generated in a wide variety of enzymatic reactions catalyzed by mitochondrial respiratory chain enzymes, cytochrome P-450 systems, nitric oxide synthase, NADPH oxidase, and lipoxygenase (9, 29, 31, 32). ROS generation is remarkably enhanced in K-ras-transformed murine cells by an inhibitor of protein farnesylation such as (-hydroxyfarnesyl)phosphonic acid and by lovastatin, which should also affect this reaction by inhibiting 3-hydroxy-3-methylglutaryl coenzyme A reductase (27). However, no evidence has been obtained to elucidate the mechanism of ROS generation under the conditions in which protein farnesylation or the corresponding mevalonate biosynthetic reaction is inhibited. It is highly probable that FOH-induced events depend on oxidative stress in both yeast and mammalian cells since FOH can ultimately participate in the reaction of protein farnesylation. In this case, the mitochondrial respiratory chain seems to play an important role since it has been evaluated as the major source of superoxide anion in yeast cells (13).

In this study, the mechanism of FOH-induced growth inhibition of S. cerevisiae was studied in terms of its promotive effect on ROS generation. The mitochondrial electron transport chain was considered a target for inhibition of ROS generation by FOH. We hereby consider the possibility that FOH can indirectly inhibit the mitochondrial function via interference with a phosphatidylinositol type of signal.

	MATERIALS AND METHODS

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Yeast strains and media. The S. cerevisiae strains used in this study were X2180-1A (MATa) (grande) and its isogenic [rho⁰] petite mutants, which had been generated by ethidium bromide treatment (7). The loss of mitochondrial respiratory chain activity in the mutant cells was confirmed by the absence of molecular oxygen consumption, which was measured as described below. As is often the case with [rho⁰] mutants lacking only mitochondrial DNA (5), the cells of our mutants could grow in minimal medium where mitochondrial proteins encoded by nuclear DNA have to be functional. Unless stated otherwise, the growth properties of these yeast cells were examined in YPD medium, which contained 10 g of yeast extract, 20 g of polypeptone, and 20 g of glucose per liter at 30°C. For the preparation of mitochondria as well as the assay of cellular respiratory activity, yeast cells were grown in semisynthetic lactate medium (SSM), which contained 3 g of yeast extract, 0.5 g of glucose, 0.5 g of CaCl₂ · 2H₂O, 0.5 g of NaCl, 0.6 g of MgCl₂ · 2H₂O, 1.0 g of KH₂PO₄, 1.0 g of NH₄Cl, 22 ml of 90% DL-lactate, and 8.0 g of NaOH pellets per liter, where the pH was appropriately adjusted to 5.5 with 6 N NaOH (10).

Measurement of yeast cell growth. The yeast cells were grown overnight in YPD medium with vigorous shaking and were inoculated into freshly prepared medium to give an initial cell density of approximately 10⁷ cells/ml. Cells were then grown with or without each effector or inhibitor with vigorous shaking, and portions were withdrawn at various time intervals to measure the cell density at A₆₁₀. The cell suspension (10⁷ cells/ml) gave an A₆₁₀ value of approximately 1.0.

Measurement of ROS production. Cellular ROS production was examined by a method dependent on intracellular deacylation and oxidation of 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) to the fluorescent compound 2',7'-dichlorofluorescein (DCF). This probe was highly reactive with hydrogen peroxide and has been used in evaluating ROS generation in mammalian (14, 25) and yeast (3, 34) cells. After preincubation of the yeast cells (10⁷ cells/ml) in YPD medium with 40 µM DCFH-DA at 30°C for 60 min, the cell suspensions (1.0 ml) were withdrawn and further treated with each chemical for the indicated time and then washed and resuspended in 100 µl of phosphate-buffered saline. Fluorescence intensity of the cell suspension (100 µl) containing 10⁷ cells was read with a Cytoflow 2300 fluorescence spectrophotometer (Millipore Co.) with excitation at 480 nm and emission at 530 nm. The arbitrary units were based directly on fluorescence intensity.

Preparation of mitochondria and the cytosol fraction. Cells of strain X2180-1A were aerobically grown in 10 liters of SSM at 30°C for 15 h. Mitochondria were isolated from the cell lysate, which had been prepared by enzymatic digestion with Zymolyase 20 T (Seikagaku Kogyo Co.) according to the method of Glick and Pon (10). Cells from an overnight culture were further incubated in 100 ml of YPD medium (10⁷ cells/ml) containing 200 µM FOH for 15 min, collected, suspended in 1.0 ml of cold lysis buffer (0.3 M D-sorbitol, 0.1 M NaCl, 5 mM MgCl₂, 10 mM Tris-HCl, 1 mM phenylmethylsulfonyl fluoride [PMSF; pH 7.4]) at the cell density of 10⁹/ml, and disrupted by repeated vortexing with glass beads (6). The supernatant obtained after removing the beads and cell debris by centrifugation at 1,500 × g for 15 min was used as the cytosol fraction. One milliliter of the fraction was thus expected to contain FOH and its metabolites, if any, from approximately 10⁹ cells. Protein was measured by the method of Bradford (2) by using bovine serum albumin as a standard.

Assay of cellular and mitochondrial respiratory chain activity. Cells of strain X2180-1A were grown in SSM with vigorous shaking at 30°C for 15 h, collected, and suspended in HEPES buffer (pH 7.4) containing 50 mM glucose at a cell density of 10⁷ cells/ml. After preincubating the cell suspension with shaking at 30°C for 10 min, the respiratory activity of yeast cells was measured polarographically with an oxygen electrode (model 100; Rank Brothers, Ltd., Cambridge, United Kingdom). We also measured cellular oxygen consumption under the condition in which the mitochondrial respiration was fully stimulated with 100 µM 2,4-dinitrophenol (DNP) prior to the addition of FOH.

Chemicals. The following chemicals were purchased from Sigma: geraniol (GOH), FOH, farnesylacetate, FPP, geranyl geraniol (GGOH), antimycin A, myxothiazol, thenoyltrifluoroacetone (TTFA), rotenone, -tocopherolacetate, probucol, N-acetylcysteine (NAC), TMPD, and 1-oleoyl,2-acetyl-sn-glycerol (OAG) as a membrane-permeable DAG analog. DCFH-DA was a product of Molecular Probe. A stock solution of each chemical was routinely prepared in either phosphate-buffered saline, ethanol, acetone, or dimethyl formamide due to its solubility. Farnesoic acid and farnesal were kindly provided by G. Asanuma (Kurare, Co., Osaka, Japan). The other chemicals were of analytical reagent grade.

	RESULTS AND DISCUSSION

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Correlation between FOH-induced growth inhibition and ROS generation. We first examined whether the yeast cells were subjected to oxidative stress due to ROS generation as reflected by DCFH-DA oxidation when the cells were grown or incubated with FOH. DCFH-DA was not directly oxidized even with 200 µM FOH in the absence of the yeast cells (data not shown), indicating that DCFH-DA oxidation depended only on the cellular response to FOH treatment. As summarized in Table 1, FOH significantly promoted cellular ROS generation in a dose-dependent manner, demonstrating its clear relation to the growth inhibitory effect. ROS generation was kept almost at the control level in medium containing other isoprenoid compounds, with growth inhibitory effect demonstrated even at a quite high concentration (200 µM). Surprisingly, GGOH was as effective as FOH in inducing apoptosis (23), suggesting that these isoprenoid alcohols are commonly involved in the mechanism of causing oxidative stress in mammalian cells. The susceptibility of mammalian cells to the cytotoxic effect of farnesylacetate (19) may depend on the ability to hydrolyze it to FOH as an active form. The level of ROS generation was linearly increased up to fivefold of the control level during the initial 30 min of incubation in the medium with 25 µM FOH, as shown in Fig. 1. Such a time-dependent increase in the fluorescence intensity indicated that ROS production occurred at a constant rate in FOH-treated cells.

View larger version (17K): [in this window] [in a new window]   FIG. 1.   Time course of FOH-induced ROS generation. ROS generation was assayed by using at the indicated times 100 µl of yeast cell suspension (equivalent to 107 cells) which had been incubated in YPD medium with 0 () and 25 () µM FOH after pretreatment with DCFH-DA for 60 min. Values are means ± standard deviations (n = 4).

Resistance of respiration-deficient petite mutant against FOH-induced events. To assess the role of mitochondrial function in FOH-induced ROS generation, the FOH sensitivity of a [rho⁰] petite mutant was compared with that of the wild-type grande strain. As shown in Fig. 2B, the mutant cells could grow in YPD medium depending on fermentation equally well with or without 200 µM FOH. In accordance with this fact, cellular ROS generation stayed at the control level even after 30 min of incubation of the mutant cells with FOH (Fig. 2A). The same results were obtained with another four petite mutants which had been independently isolated by ethidium bromide treatment of the parent strain. These mutant strains were all characterized by the rate of cellular uptake of FOH in comparison to that of the parent strain (data not shown), which strongly supported the dependence of FOH-induced ROS generation on mitochondrial function alone. The electron transport chain could be a target of FOH since the [rho⁰] petite mutant generally lacks cytochrome b of complex III and various subunits of cytochrome c oxidase in addition to F₁F₀-ATPase (5).

View larger version (32K): [in this window] [in a new window]   FIG. 2.   Effects of respiration competence on ROS production (A) and FOH-induced growth inhibition (B). (A) ROS generation was assayed as described in the legend to Fig. 1 except that FOH was added at 25 µM for cells of the wild-type strain and at 200 µM for cells of the mutant strain. Values are means ± standard deviations (n = 4). (B) Cells of parental grande strain ( and ) and [rho0] petite mutant ( and ) were grown in YPD medium with ( and ) or without ( and ) FOH at 30°C for 6 h.

Effect of blocking electron flow in complexes I, II, III, and IV of the respiratory chain on FOH-induced ROS generation. Wild-type cells were pretreated with a respiratory chain inhibitor(s) for 10 min to identify the critical site of ROS generation during the subsequent incubation with 25 µM FOH. Each respiratory chain inhibitor was used at the concentration that entirely inhibited cellular oxygen consumption but that did not influence cell growth upon fermentation in YPD medium. As shown in Fig. 3, ROS generation was reduced by almost 50% when yeast cells were pretreated with rotenone, which specifically inhibits complex I, and TTFA, a specific inhibitor of complex II (24). Protection against ROS generation and growth inhibition was not provided by treatment with either antimycin A, which inhibits cytochrome reductase of complex III, or KCN, a typical inhibitor of complex IV (28). Of the various respiratory chain inhibitors tested, myxothiazol, which inhibits the oxidation of ubiquinol to the ubisemiquinone radical via the Rieske iron-sulfur center of complex III (29), eliminated cellular ROS generation most effectively. With myxothiazol pretreatment, the yeast cells treated with FOH (25 µM) could grow normally upon fermentation in YPD medium, in which the relative cell growth (A₆₁₀ = 3.9) was around 50% of the control level at 6 h. This strongly supported that the protection against ROS generation provided by mxyothiazol depended solely on its role as a respiratory chain inhibitor.

View larger version (45K): [in this window] [in a new window]   FIG. 3.   Protective effects provided by blocking the electron transport chain in FOH-induced ROS generation. ROS generation was assayed by using the cell suspension with or without the following treatment, as described in the legend to Fig. 1. Prior to the addition of 25 µM FOH, cells were pretreated with a mixture of 50 µM rotenone (Rot) and 1 mM TTFA, 20 µM antimycin A (AA), 30 µM myxothiazol (Myxo), or 2.5 mM KCN for 10 min. Bars are means ± standard deviations (n = 4).

Effects of FOH on cellular oxygen consumption and mitochondrial oxidase activities. As deduced from the dependence of FOH-induced ROS generation on mitochondrial function, FOH restricted cellular oxygen consumption immediately upon its addition to the cell suspension (Fig. 4). Similar results were obtained under conditions in which mitochondrial respiration was fully stimulated with DNP. This means that FOH inhibited mitochondrial electron transport that was normally coupled with oxygen consumption at complex IV. FOH-induced ROS generation (Fig. 1) should proceed mainly by utilizing the mitochondrial electron flow from the ubiquinol pool which was still remaining since the overall mitochondrial electron transport had been mostly shut off within 5 min of incubation. In fact, FOH could accelerate ROS generation even under conditions in which mitochondrial electron transport was fully repressed with antimycin A (Fig. 3).

View larger version (22K): [in this window] [in a new window]   FIG. 4.   Effects of FOH on the respiratory activity of a whole-cell suspension. Cells were suspended in HEPES buffer (pH 7.4) containing 50 mM glucose at a density of 107 cells/ml and incubated with shaking at 30°C. The rate of oxygen consumption was measured with 0 (, ), 12.5 (), and 25 ( and ) µM FOH. Square symbols indicate the cellular oxygen consumption when it was fully stimulated with 100 µM DNP.

Involvement of a phosphatidylinositol type of signal in FOH-induced ROS generation. In the experiment with a human acute leukemia cell line, the inhibitory effect of FOH on cell proliferation could be restored with the extracellular addition of OAG, a membrane-permeable analog of DAG which can activate PKC from mammalian (15, 20) and yeast (22) cells. As shown in Fig. 5, FOH-induced ROS generation and growth inhibition were apparently diminished to the control level in the presence of 10 µM OAG. This agreed with our previous finding that the PKC activator restored FOH-induced growth inhibition of S. cerevisiae cells in which the endogenous DAG level had been drastically decreased immediately upon incubation with FOH (16). These findings support the idea that FOH inhibits mitochondrial electron transport via interference with a phosphatidylinositol type of signal without having a direct inhibitory effect on the mitochondrion itself. It seems possible that a PKC-dependent mechanism normally functions in yeast cells to regulate mitochondrial electron transport, especially at the Q cycle of complex III, not to promote ROS generation.

View larger version (34K): [in this window] [in a new window]   FIG. 5.   Protective effects of OAG against ROS production (A) and FOH-induced growth inhibition (B). (A) ROS generation with or without various concentrations of OAG was assayed as described in the legend to Fig. 1. A control assay was run with YPD medium without any other ingredient. Values are means ± standard deviations (n = 4). (B) Cells (107 cells/ml) were grown in YPD medium with () or without () 25 µM FOH. FOH was further added at 25 µM to YPD medium containing OAG at either 5 (), 10 (), or 20 () µM.