R E S E A R C H A R T I C L E

Metabolic response toMMS-mediatedDNAdamage inSaccharomyces cerevisiae is dependenton theglucoseconcentration in themediumAna Kitanovic1, Thomas Walther2, Marie Odile Loret2, Jinda Holzwarth1, Igor Kitanovic1, FelixBonowski1, Ngoc Van Bui1, Jean Marie Francois2 & Stefan Wolfl1

1Institute for Pharmacy and Molecular Biotechnology, Ruperto-Carola University of Heidelberg, Heidelberg, Germany; and 2Universite de Toulouse,

INSA-UPS-INP, LISBP, Toulouse, France

Correspondence: Stefan Wolfl, Institute for

Pharmacy and Molecular Biotechnology,

Ruperto-Carola University of Heidelberg, Im

Neuenheimer Feld 364, D-69120 Heidelberg,

Germany. Tel.: 149 6221 544 878; fax: 149

6221 544 884; e-mail: wolfl@uni-hd.de

Received 11 September 2008; revised 19

February 2009; accepted 22 February 2009.

First published online 1 April 2009.

DOI:10.1111/j.1567-1364.2009.00505.x

Editor: David Goldfarb

Keywords

DNA damage; energy charge; GAPDH; PYK;

ROS; respiration.

Abstract

Maintenance and adaptation of energy metabolism could play an important role in

the cellular ability to respond to DNA damage. A large number of studies suggest

that the sensitivity of cells to oxidants and oxidative stress depends on the activity

of cellular metabolism and is dependent on the glucose concentration. In fact,

yeast cells that utilize fermentative carbon sources and hence rely mainly on

glycolysis for energy appear to be more sensitive to oxidative stress. Here we show

that treatment of the yeast Saccharomyces cerevisiae growing on a glucose-rich

medium with the DNA alkylating agent methyl methanesulphonate (MMS)

triggers a rapid inhibition of respiration and enhances reactive oxygen species

(ROS) production, which is accompanied by a strong suppression of glycolysis.

Further, diminished activity of pyruvate kinase and glyceraldehyde-3-phosphate

dehydrogenase upon MMS treatment leads to a diversion of glucose carbon to

glycerol, trehalose and glycogen accumulation and an increased flux through the

pentose-phosphate pathway. Such conditions finally result in a significant decline

in the ATP level and energy charge. These effects are dependent on the glucose

concentration in the medium. Our results clearly demonstrate that calorie

restriction reduces MMS toxicity through increased respiration and reduced ROS

accumulation, enhancing the survival and recovery of cells.

Introduction

Modulation in energy metabolism seems to play an impor-

tant role in the cellular response to DNA damage. Gene

expression studies of cells exposed to various stress condi-

tions including DNA damage showed, in addition to other

gene expression changes, significant regulation of genes

involved in glycolysis and gluconeogenesis (DeRisi et al.,

1997; Godon et al., 1998; Dumond et al., 2000; Jelinsky et al.,

2000; Causton et al., 2001; Gasch et al., 2001). A potential

effect of these changes in glucose metabolism could be a shift

from glycolysis to the pentose-phosphate pathway (PPP), as

suggested by Dumond et al. (2000), which could lead to the

generation of reducing equivalents (NADH and NADPH)

required for cellular antioxidant systems. That oxidative

stress response is also crucial in the metabolic response to

DNA damage became evident based on the observation that

DNA damage mediated by methyl methanesulfonate (MMS)

induces the formation of reactive oxygen species (ROS) in

yeast (Salmon et al., 2004; Kitanovic & Wolfl, 2006). More-

over, suppression of ROS formation due to altered (glucose)

metabolism as well as ROS scavenging improves the survival

of MMS-treated cells (Kitanovic & Wolfl, 2006). The diauxic

shift represents a physiological condition that contributes to

increased oxidative-stress tolerance of stationary-phase yeast

cells (Jamieson et al., 1994; Steels et al., 1994). In addition,

transcription of many antioxidant defence genes is repressed

by glucose and their derepression usually follows glucose

exhaustion (Krems et al., 1995).

Glycolytic enzymes were reported to be the target of

oxidative modifications (Albina et al., 1999; Arutyunova

et al., 2003; Osorio et al., 2003, 2004; Shenton & Grant,

2003; Yoo & Regnier, 2004; Almeida et al., 2007). The toxic

effect of oxidants or alkylating agents appeared to be

FEMS Yeast Res 9 (2009) 535–551 c� 2009 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

dependent on the glucose concentration and the cellular

metabolic state (Osorio et al., 2004; Zong et al., 2004; Lopez

et al., 2005). Bearing in mind that cancer cells are strongly

dependent on glycolysis and that they generally exhibit a

poor antioxidant status (reviewed in Verrax et al., 2008),

detailed investigations of the metabolic response and its

alternations upon treatment with cytotoxic and genotoxic

substances could be of considerable interest for future

anticancer therapy.

Despite this interconnection between energy metabolism

and sensitivity to toxic stress in various cell types, no

detailed analysis of the metabolic flux in response to a

DNA-damaging agent has been presented so far. MMS is a

highly toxic DNA-alkylating agent that methylates DNA at

7-deoxyguanine and 3-deoxyadenine, causing base mispair-

ing and replication blocks that activate DNA damage repair

pathways and cell cycle arrest (Evensen & Seeberg, 1982).

There are ample evidences indicating that upon treatment

with alkylating agents such as MMS or N-methyl-N-nitro-

sourea, mtDNA is preferentially alkylated in relation to

nuclear DNA (Wunderlich et al., 1970; LeDoux et al., 1992;

Pirsel & Bohr, 1993). It is also known that DNA alkylation

lesions trigger the mitochondrial apoptotic pathway

(Kaina et al., 1997; Tominaga et al., 1997; Meikrantz et al.,

1998). Because of this ability, DNA-alkylating agents

have been used for many years in cancer research and in the

treatment of several human cancers (Beranek, 1990),

which is, however, hampered by sytemic toxicity and

drug resistance, limiting their clinical efficacy (Ralhan &

Kaur, 2007).

Here we present a detailed analysis of the physiological

and metabolic alterations in yeast cells upon treatment with

MMS. MMS treatment led to a strong suppression of the

enzymatic activity of two major glycolytic enzymes, glycer-

aldehyde-3-phosphate dehydrogenase (GAPDH) and pyru-

vate kinase (PYK), and to an inhibition of oxygen

consumption. The latter effect was strongly dependent on

the glucose concentrations in the medium. Stimulation

of mitochondrial activity before MMS treatment signifi-

cantly reduced oxidative damage in the cells and improved

survival. Altogether, the cellular energy state appeared to

be an important factor in the cellular response to MMS

treatment.

Materials and methods

Strains, media and growth conditions

The yeast strains used in this study are: FF 18984 (MATa

leu2-3,112 ura3-52, lys2-1, his7-1), Dhap4 (MATa leu2-3,112

ura3-52, lys2-1, his7-1;hap4::KanMX4). Yeast cells were

grown in rich (YPD) medium containing 10 g L�1 yeast

extract, 20 g L�1 Bacto peptone and different glucose con-

centrations (as indicated; 20, 10, 5 or 2.5 g L�1). YPKG

contained 10 g L�1 Bacto yeast extract, 20 g L�1 Bacto pep-

tone, 10 g L�1 potassium acetate and 5 g L�1 glucose. Cell

growth was followed by optical absorbance readings at

600 nm. For treatment with MMS, medium was inoculated

with overnight precultures and grown at 30 1C to the mid-

log phase (OD600 nm 0.6–0.8). Cultures were divided and

either mock-treated or treated with indicated concentra-

tions of MMS. For dry-weight measurements, polyamide

filters (pore size 0.45 mm; Sartorius, UK) were used. After

removal of the medium by filtration, the filter was washed

with demineralized water and dried to a constant weight in a

glass beaker exicator under vacuum.

Metabolic byproduct determination and glucoseconsumption assay

In order to quantify glucose consumption and metabolic

byproduct accumulation, the culture supernatant was col-

lected at the indicated time points. Glucose, glycerol,

ethanol and acetate were determined by HPLC. The cell-free

supernatant was filtered through 0.22-mm pore-size nylon

filters before loading on an HPX-87H Aminex ion exclusion

column. The column was eluted at 48 1C with 5 mM H2SO4

at a flow rate of 0.5 mL min�1 and the concentration of the

compounds was determined using a Waters model 410

refractive index detector.

Quantitative assessment of glycogen andtrehalose content

The procedure was performed as described previously

(Parrou & Francois, 1997). Briefly, the cell pellet (collected

from 20 OD600 nm units of culture) was suspended in 250mL

0.25 M Na2CO3 and heated at 95 1C for 4 h with occasional

stirring. The suspension was adjusted to pH 5.2 with

150 mL 1 M acetic acid and 600 mL 0.2 M sodium acetate

buffer, pH 5.2. Half of this mixture was incubated overnight

at 57 1C with continuous shaking on a rotary shaker

in the presence of 100mg of a-amyloglucosidase from

Aspergillus niger (Sigma). The second half of the mixture

was incubated overnight at 37 1C in the presence of

3 mU trehalase (Sigma). The glucose released was deter-

mined using the glucose oxidase/peroxidase method

(Cramp, 1967).

Metabolite extraction and analysis of nucleotidecontent

The sampling method was performed as described pre-

viously (Loret et al., 2007). Briefly, portions (20 OD600 nm

units) of the cell culture were rapidly collected by filtration,

suspended in 5 mL buffered ethanol (75% ethanol in 10 mM

HEPES, pH 7.1) and incubated for 4 min at 85 1C. The

FEMS Yeast Res 9 (2009) 535–551c� 2009 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

536 A. Kitanovic et al.

solvent was evaporated to dryness at 37 1C under vacuum

and the cell extract was suspended in 0.5 mL deionized

water. The nucleotide content was analysed by high perfor-

mance ionic chromatography (HPIC) carried out on a DX

500 chromatography workstation (Dionex, Sunnyvale, CA)

equipped with a GP50 gradient pump, ED50 electrochemi-

cal and UV detectors as described in Loret et al. (2007).

Energy charge

Energy charge is defined in terms as the sum of hydrolysable

phosphate bonds in adenine nucleotides over the total

concentration of these nucleotides, calculated from the

actual concentrations as ([ATP]10.5[ADP])/([ATP]1[ADP]

1[AMP]) (Atkinson, 1966).

Preparation of cell-free extracts and enzymeassays

All procedures were carried out at 0–4 1C. Crude extracts

were prepared from 20 OD600 nm units of cells with 1-g glass

beads (0.4–0.5 mm diameter) in 0.5 mL 20 mM HEPES, pH

7.1, 100 mM KCl, 5 mM MgCl2, 1 mM EDTA and 1 mM

dithiothreitol. Samples were vortexed (3� 5 min with cool-

ing on ice in between) in Mixer Mill MM 300 (Retsch). After

centrifugation at 16 000 g for 15 min at 4 1C, the super-

natants were immediately used for enzymatic assays. The

protein content was determined using the method of

Bradford (1976). All chemicals and enzymes for enzymatic

assays were purchased from Sigma.

GAPDH (EC 1.2.1.12) activity was measured at 30 1C by

coupling the production of glycerate-1,3-diphosphate from

3-phosphoglyceric acid to the consumption of NADH, using

a spectrophotometric assay in a coupled 3-phosphoglyceric

phosphokinase–GAPDH system (Bergmeyer et al., 1974).

The reaction was performed in 0.15 mL buffer containing

50 mM Tris-HCl, pH 7.1, 5 mM KCl, 2.5 mM MgCl2, 10 mM

ATP/Mg, 0.7 mM NADH, 6.7 mM 3-phosphoglyceric acid

and 10 U of 3-phosphoglyceric phosphokinase (EC 2.7.2.3).

The reaction was initiated by the addition of crude extract

and was followed by a decrease in A340 nm.

PYK (EC 2.7.1.40) activity was measured at 30 1C by

coupling the production of pyruvate from phospho(enol)-

pyruvate and ADP to the consumption of NADH, using a

spectrophotometric assay in a coupled PYK–lactic dehydro-

genase system (Bergmeyer et al., 1974). The reaction was

performed in 0.15 mL buffer containing 50 mM Tris-HCl,

pH 7.1, 5 mM KCl, 2.5 mM MgCl2, 10 mM ADP/Mg,

0.7 mM NADH, 1.7 mM phospho(enol)pyruvate and 10 U

of lactic dehydrogenase (EC 1.1.1.27). The reaction was

initiated by the addition of crude extract, and decrease in

A340 nm was monitored.

Glucose-6-phosphate dehydrogenase (G6PDH; EC

1.1.1.49) activity was measured at 30 1C by measuring

NADPH production, using a spectrophotometric assay

(Noltmann et al., 1961). The reaction was performed in

0.15 mL buffer containing 50 mM imidazole, pH 7.1,

100 mM KCl, 5 mM MgSO4, 5 mM EDTA, 0.7 mM NADP1

and 3.4 mM glucose-6-phosphate. The reaction was initiated

by the addition of crude extract, and increase in A340 nm was

monitored.

Citrate synthase (CS; EC 2.3.3.1) activity was measured at

30 1C by coupling the production of oxalacetate and NADH

from malic acid and NAD1 to the consumption of oxalace-

tate by CS, using a spectrophotometric assay in a coupled

malic dehydrogenase–CS system (Srere, 1969). The reaction

was performed in 0.15 mL buffer containing 50 mM Tris-

HCl, pH 7.1, 5 mM KCl, 2.5 mM MgCl2, 0.16 mM acetyl-

CoA, 1.67 mM NAD1, 6.67 mM malic acid and 3 U of

malate dehydrogenase (EC 1.1.1.37). The reaction was

initiated by the addition of crude extract, and increase in

A340 nm was monitored.

Toxicity assay

Wild-type strain FF18984 was grown in YPD and YPKG

media until the mid-log phase (OD600 nm = 0.6–0.8).

OD600 nm was adjusted to 0.5 and five additional fivefold

serial dilutions were made. Four microlitres of each serial

dilution were spotted onto agar plates containing the

indicated media and incubated at 30 1C for 3 days.

Detection of ROS level, cardiolipin content (CL),mitochondrial activity and mitochondrialmembrane potential (W) by flow cytometryanalysis

Control and MMS-treated cells were grown until the mid-

log or the stationary phase. An aliquot of cells was taken at

the indicated time and cells were dissolved in phosphate-

buffered saline (PBS). For the ROS level, samples were

incubated for 10 min with dihydroethidium (Molecular

Probes) at a final concentration (f.c.) of 5 mg mL�1. Cells

were washed and suspended in PBS. ROS production was

quantified using FACSs Calibur (Becton Dickinson) and

CELLQUEST PRO ANALYSIS software. Excitation and emission

settings were 488 and 564–606 nm (FL2 filter). The CL was

determined by 15-min staining with 10 nonyl acridine

orange (f.c. 0.5 mM) (NAO; Molecular Probes). Cells were

washed and fluorescence was quantified using FACSs

Calibur (Becton Dickinson) and CELLQUEST PRO ANALYSIS soft-

ware. Excitation and emission settings were 488 and

525–550 nm (FL1 filter). The mitochondrial membrane

potential was analysed after 15 min of incubation with

rhodamine 123 (f.c. 10 mM) (Molecular Probes; Chen et al.,

1982). The excitation and emission settings were 488 and

564–606 nm (FL2 filter). Active mitochondria were quanti-

fied by 15 min Mito Tracker Green FM labelling (f.c.

FEMS Yeast Res 9 (2009) 535–551 c� 2009 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

537Metabolic control upon DNA damage

100 nM) (Molecular Probes). Excitation and emission set-

tings were 488 and 564–606 nm (FL2 filter). Dead cells were

excluded from the statistical analysis of CL and Mito Tracker

Green intensity by parallel staining with propidium iodide

(PI).

Monitoring of oxygen consumption

Oxygen consumption was monitored in OxoPlates (Pre-

Sens, Germany) covered with a breathable membrane (Di-

versified Biotech) in 150mL volume containing the indicated

medium and substances and 0.1 OD600 nm units of cells.

The signal of the oxygen fluorescence sensor and the OD

of the culture at 600 nm were measured continuously

during the indicated time at kinetic intervals of 30 min.

The calibration of the fluorescence reader was performed

using a two-point calibration curve with oxygen-free

water (80 mM Na2SO3) and air-saturated water. The partial

pressure of oxygen was calculated from the calibration

curve.

Determination of cofactors

The cellular levels of NADP1 and NADPH were determined

using the method of Klingenberg (1989). Cells from 24 h

MMS-treated cells were divided into two parts, and acid

(0.5 M perchloric acid) or alkaline extracts (0.5 M alcoholic

potassium hydroxide solution) were prepared to measure

NADP1 and NADPH levels, respectively. The level of

NADPH produced in an enzymatic reaction with G6PDH

was analysed spectrophotometrically at 340 nm.

The cellular levels of NAD1 and NADH were determined

using the cycling method of Matsumura & Miyachi (1983).

Cells from 24 h MMS-treated cells were divided into two

parts, and acid (0.1 M hydrochloric acid) or alkaline extracts

(0.1 M sodium hydroxide solution) were prepared to mea-

sure NAD1 and NADH levels, respectively. The method

involves coupling of a NAD1-dependent (alcohol) dehydro-

genase reaction with the reduction of tetrazolium salt. The

reaction was performed in 0.15 mL buffer containing 87 mM

triethanolamine hydrochloride, pH 7.4, 9.6% ethanol, 2 mM

phenazine ethosulphate, 2 mM 3-(4,5-dimethylthiazolyl-2)-

2,5-diphenyltetrazolium bromide, 4 mM EDTA and 1.5 U of

alcohol dehydrogenase. The reaction was initiated by the

addition of acid or alkaline extracts, and increase in A570 nm

was monitored.

Mitochondria isolation and oxygenconsumption of isolated mitochondria

Mitochondria were isolated from the stationary culture of

cells grown in full (YPD) medium. Cells were spheroplasted

with zymolase and subsequently broken by glass beads in an

ice-cold sorbitol buffer (0.6 M sorbitol, 20 mM HEPES,

1 mM EDTA and 0.2% bovine serum albumin, pH 7.4).

Lysate was purified from unbroken cells and cell debris by

centrifugation at 500 g for 5 min and mitochondria were

pelleted by centrifugation at 10 000 g for 10 min. Mitochon-

dria were suspended in ice-cold sucrose buffer (250 mM

sucrose, 20 mM HEPES and 1 mM EDTA, pH 7.4). Mito-

chondrial activity was stimulated with 12.5/12.5 mM gluta-

mate/malate and 1.65 mM ADP and oxygen consumption

was measured in sorbitol buffer containing MMS (0.01%,

0.02% and 0.04%) or 3 mM H2O2 in OxoPlates.

Results

Bioenergetic conditions in yeast cells upon MMStreatment-growth rate and glucoseconsumption

We discovered earlier that changes in the expression of the

gluconeogenic protein, fructose-1,6-bisphosphatase, signifi-

cantly modulate cellular sensitivity to the DNA-damaging

agent MMS (Kitanovic & Wolfl, 2006). To understand better

the role of glucose metabolism and bioenergetic conditions

in the cellular sensitivity to treatment with this alkylating

agent, we analysed whether treatment with MMS alters

carbon flux through the main metabolic pathways, glycoly-

sis, tricarboxylic acid (TCA) and PPP. Wild-type strain

FF18984 was cultivated in full medium (YPD; with 2%

glucose), mid-log phase cultures were split and either mock-

or MMS-treated. Filtrated medium from several time points

during a 24-h time course was collected and the concentra-

tions of extracellular metabolites, glucose, ethanol, glycerol

and acetate were analysed by HPLC. Upon treatment with

0.03% MMS cellular growth is immediately affected, result-

ing in a reduced OD at all time points (Fig. 1a), which is

consistent with a rapid cell cycle arrest in the S phase

reported earlier (Gasch et al., 2001). After a 24-h treatment

with 0.03% MMS, OD is reduced 4 50%. The analysis of

glucose consumption from the medium revealed that con-

trol cells grown in full medium consumed the entire glucose

within 6 h. In contrast, in MMS-treated cultures, glucose

consumption started to reduce after 4 h of treatment, but

still the entire glucose was consumed after 24 h. This

consumption occurred in the absence of growth as MMS

strongly inhibited biomass production during this period

(Fig. 1a).

Measurement of ethanol, glycerol and acetate production

revealed that the maximal byproduct accumulation in con-

trol cultures is reached within 6 h of cultivation in full

medium corresponding to the complete exhaustion of

glucose (Fig. 1b–d). After this time point, the concentra-

tions of ethanol, glycerol and acetate started to decrease over

time, indicating a switch from fermentative to respiratory

metabolism and utilization of nonfermentative carbon

FEMS Yeast Res 9 (2009) 535–551c� 2009 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

538 A. Kitanovic et al.

sources. In cultures treated with MMS, ethanol, glycerol and

acetate were also produced along with glucose consumption.

However, no decrease in the extracellular concentration of

these byproducts was observed upon glucose exhaustion,

indicating that MMS-treated cells were not able to switch

from fermentative to respiratory metabolism. In contrast,

MMS-treated cells produced more glycerol than nontreated

ones (Fig. 1c).

When nutrients become exhausted, yeast cells accumulate

the storage carbohydrates, trehalose and glycogen. The

intracellular concentration of trehalose and glycogen

was also linked to the survival of yeast cells under various

stress conditions (Parrou et al., 1997; Hounsa et al., 1998).

Analysing cellular glycogen stores, a high accumulation

of glycogen upon MMS treatment in glucose-rich

medium could be detected (Fig. 2a). Consistent with the

inability of MMS-treated cells to utilize nonfermentative

carbon products from glucose fermentation, these cells

mobilized their glycogen stores when glucose was exhausted

(Fig. 2a). A similar profile of rapid glycogen mobilization

was reported by Enjalbert et al. (2000) in respiratory-

deficient mutants. Nontreated cells showed a very low

glycogen content when grown in full (YPD) medium.

Concerning trehalose synthesis, nontreated cells started to

accumulate high levels of trehalose after glucose was con-

sumed. The trehalose level in these cells remained high

over 48 h, reaching 12% of cell dry weight at 24 h of

cultivation (Fig. 2b). In MMS-treated cells trehalose accu-

mulation occurred more rapidly but to a much lower level.

At later time points, MMS-treated cells mobilized this

carbohydrate storage to continue growth. Therefore, our

results clearly suggest that MMS treatment reduces glucose

utilization and glycolytic flux while increasing glycerol,

trehalose and glycogen production at an early point of

cultivation in a glucose-rich medium. The rapid mobiliza-

tion of stored glucose from glycogen and trehalose as

MMS-treated cells ran out of an exogenous carbon source

is consistent with the inability of MMS-treated cells

to utilize nonfermentative carbon sources and hence

must thrive on their endogenous carbon stores for

energy maintenance (Enjalbert et al., 2000; Jules et al.,

2008).

Fig. 1. (a) Effect of MMS treatment on cell growth and glucose consumption. Wild-type FF18984 cells were cultured to the mid-log phase

(OD600 nm = 0.6–1.0) in full (YPD) medium. Cultures were divided and either mock- (control) or MMS-treated with a concentration of 0.03% (MMS).

The OD of the culture (OD600 nm) was monitored at several time points in a 24-h time course (a). At the same time points, medium was filtrated and the

concentration of glucose (a), ethanol (b), glycerol (c) and acetate (d) in the medium (given in g L�1) was determined by HPLC. Graphs present

representative experiments of at least three similar repetitions.

FEMS Yeast Res 9 (2009) 535–551 c� 2009 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

539Metabolic control upon DNA damage

MMS effects on the activity of glycolysisenzymes and the PPP

The above results clearly show that MMS treatment changes

the function of the main energetic pathways and the carbon

flux into various end products. One explanation could be a

direct effect of MMS on the activity of metabolic enzymes.

To test this hypothesis, we analysed the enzymatic activity of

some key enzymes of glycolysis, TCA and PPP: PYK;

GAPDH(yeast TDH); CS; and G6PDH (Fig. 3). Activities

of PYK and GAPDH, two important enzymes of the lower

part of glycolysis, were strongly reduced already after 4 h of

MMS treatment, regardless of the medium in which cells

were cultivated. In cells grown in low-glucose/acetate

(YPKG) medium, PYK and GAPDH activities were signifi-

cantly lower even in the untreated control (Fig. 3a and b).

These lower activities might be due to lower expression

under gluconeogenic conditions, as reported earlier (Gasch

et al., 2000), which is in agreement with an increased

requirement for oxidative phosphorylation under low-

glucose conditions (Pronk et al., 1996).

Considering that MMS, similar to peroxide, triggers the

formation of ROS even at lower concentrations (Salmon

et al., 2004; Kitanovic & Wolfl, 2006), redirection of the

metabolic flux may also reflect an adjustment of antioxida-

tive defence in response to MMS treatment. It was reported

earlier that treatment with 1.5 mM H2O2 results in GAPDH

inhibition under both low- and high-glucose conditions

(Osorio et al., 2004). We observed that peroxide treatment

not only inhibited‘ GAPDH but also reduced PYK activity

in vivo treating cells with 3 mM H2O2 (data not shown).

Inhibition of the lower glycolytic pathway, namely the

processes controlled by GAPDH and PYK, could signifi-

cantly influence NADH regeneration and by this, increase

the NAD1/NADH ratio and redirect the carbon flux to

glycerol. Our measurements of these important redox

cofactors show a significant increase of NAD1 upon MMS

treatment (Fig. 4a). The increase in glycerol production

shown above (Fig. 1c) is also consistent with the GAPDH

and PYK inhibition observed.

Adjustment of the cellular redox potential is also obser-

vable in the PPP. While the activity of G6PDH was increased

by about 30–50% when cells were treated for 4 and 24 h with

MMS (Fig. 3c), we did not observe an increase of the

NADPH/NADP1 ratio (Fig. 4b). Although G6PDH is a

major supplier of the reducing equivalent NADPH, fast

recycling of NADPH in antioxidant defence reactions may

explain the lack of a concomitant increase in the NADPH/

NADP1 ratio. This assumption is reflected in the reduced

half-cell reduction potential upon MMS treatment. Interest-

ingly, in cells grown in low-glucose/acetate medium

(YPKG), we observed a significantly higher reduction po-

tential due to an elevated level of NADPH (Fig. 4b). In

YPKG medium, the reducing power is utilized upon MMS

treatment, changing the NADPH/NADP1 ratio despite the

enhanced activity of G6PDH. This enhanced activity of

G6PDH is probably an integral part of the antioxidant

defence and is in accordance with early observations that

enzymes of the PPP play an important role in protection

from oxidative stress in both yeast and mammals (Pandolfi

et al., 1995; Slekar et al., 1996).

Analysis of mitochondrial function through determina-

tion of CS activity revealed a slight increase of its activity at

the beginning of treatment, and significantly decreased

activity upon 24 h of incubation with MMS in full medium

(Fig. 3d), indicating persistent glucose repression in MMS-

treated cells. As expected, CS activity was elevated in

respiratory active cells, for example cells from the YPD

stationary phase (control culture upon 24 h cultivation) or

those grown in YPKG medium, confirming glucose dere-

pression and a switch from fermentative to oxidative

Fig. 2. (a) Effect of MMS treatment on accumulation of glycogen and

trehalose. Wild-type FF18984 cells were cultured in 0.6 L full (YPD), well-

aerated medium. Cultures were splitted and either mock- (con) or MMS-

treated with a concentration of 0.03% (MMS). Kinetic of glycogen (a)

and trehalose (b) accumulation was monitored during 48 h of cultivation.

The arrow indicates the time of glucose exhaustion in the control culture,

and the star in the MMS-treated culture. Graphs present representative

experiments of at least three repetitions.

FEMS Yeast Res 9 (2009) 535–551c� 2009 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

540 A. Kitanovic et al.

metabolism. It is important to note that we did not detect a

significant influence of MMS on CS activity in YPKG

medium. This suggests that enhanced mitochondrial meta-

bolism, reflected in enhanced CS activity, could serve

as a protective mechanism against MMS, enabling more

cells to survive and continue proliferation. Indeed, cells

cultivated in YPKG survive MMS treatment much better,

which is clearly observable in the plate sensitivity assay

(Fig. 4c).

Inactivation of GAPDH and PYK activity by MMS as well

as H2O2 (data not shown) raised the question of whether

these enzymes were directly affected by MMS treatment or

rather through subsequent ROS production and oxidative

stress. Purified GAPDH and PYK or cell lysates were treated

with different MMS and H2O2 concentrations for 2 h. While

MMS did not significantly inhibit the activity of both

enzymes, neither purified, nor in cellular lysate (Fig. 5),

3 mM H2O2 strongly inhibited GAPDH activity of the

purified enzyme as well as in cell extracts by more than

80%. PYK activity was not influenced by H2O2. These results

indicate that GAPDH inhibition upon MMS treatment is

the result of high ROS production, while the effect of MMS

or H2O2 on PYK activity in vivo is not a direct effect of ROS

generated by these agents.

Bioenergetic conditions in yeast cells upon MMStreatment -- nucleotide levels and energy charge

Our results showed that upon MMS treatment, all glucose is

catabolized into byproducts, without concomitant growth.

On the other hand, the activity of enzymes involved in

dephosphorylation of C3 units is significantly decreased,

suggesting reduced flux into pyruvate production. Changes

in the glycolytic flux could significantly influence cellular

energy homeostasis, which should affect the cellular antiox-

idative and DNA damage response, both high energy-

demanding processes. To address this question, we measured

the levels of nucleotide metabolites and estimated the energy

status of MMS-treated cells. MMS treatment significantly

reduced ATP levels already after 5 h of treatment in cells

growing on high glucose, while ADP and AMP levels were

hardly modified (Fig. 6a). In contrast, in low-glucose

medium (YPKG), incubation with MMS did not result

in a significant ATP reduction (Fig. 6b). The calculated

energy charge (Fig. 6c), a measure of energy stored in adenine

nucleotide phosphates [(ATP11/2ADP)/(ATP1ADP1

AMP)], clearly shows a progressive reduction of the cellular

energetic pool upon MMS treatment under high-glucose

conditions to a value o 0.5. This reduction was mostly

Fig. 3. (a) Effect of 0.03% MMS treatment on the activity of metabolic enzymes. A culture of yeast cells growing exponentially in full (YPD) or low

glucose/acetate (YPKG) medium was divided into two batches, one of which was mock-treated (con), and the other challenged was with 0.03% MMS.

At the indicated times of incubation, aliquots were withdrawn and (a) PYK, (b) GAPDH, (c) G6PDH and (d) CS activity were measured from the cellular

protein extract. The values are given in mU mg�1 protein� SE (U =mmol min�1).

FEMS Yeast Res 9 (2009) 535–551 c� 2009 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

541Metabolic control upon DNA damage

caused by depletion of cellular ATP and only a slight decrease

of the ADP pool. When grown in low-glucose/acetate med-

ium (YPKG), the overall energy charge was significantly

lower. MMS treatment did not cause a further reduction

(Fig. 6c).

The significant decline of the total intracellular pool of

adenine nucleotides (AXP) in control samples cultured in

both YPD and YPKG media (Fig. 6a and b) during the first

hours of incubation is probably a result of adenine nucleo-

tide consumption in DNA and RNA synthesis. At this time

point, exponentially growing cells require and rapidly con-

sume large numbers of building blocks. When cells are grown

in the low-glucose medium, YPKG, the total intracellular

ATP content is slightly lower (accompanied by a higher AMP

content), the growth rate is reduced and the reversible

decline of the AXP level in the exponentially growing culture

is shorter. In treated cells, MMS triggers growth arrest and,

therefore, ATP is not consumed in DNA and RNA synthesis,

resulting in a constant level of the cellular ATP pool.

Prolonged MMS treatment leads to a persistent inhibition

of respiratory activity and a diversion of the glycolytic flux,

which finally results in an inability of the cells to maintain

their energy level and a strong decline of the ATP pool. Our

results strongly suggest that the increased resistance of yeast

cells to MMS treatment could be related to a better ability of

the cells to maintain their energy balance, when grown under

calorie restriction conditions.

ROS production and mitochondrial activity inresponse to MMS treatment

mtDNA undergoes 3–10-fold more damage than chDNA

following oxidative stress in numerous cell types from yeast,

mouse, rats and humans (reviewed in Van Houten et al.,

2006) and it was reported earlier that mtDNA is a preferred

target for MMS-mediated DNA damage (Pirsel & Bohr,

1993). mtDNA damage can lead to loss of expression of

mitochondrial polypeptides, a subsequent decrease in elec-

tron transport and an increase in ROS generation, loss of

mitochondrial membrane potential and release of signals for

cell death, such as CytC and AIF. By releasing several

Fig. 4. (a) Effect of MMS treatment on the half-cell redox potential and

survival of Saccharomyces cerevisiae cells incubated in different media. A

culture of yeast cells growing exponentially in full (YPD) or low-glucose/

acetate (YPKG) medium was divided into two batches; one batch was

challenged with 0.03% MMS. Aliquots were withdrawn at 24 h and the

levels of (a) NAD1 and NADH or (b) NADP1 and NADPH were analysed.

The levels are expressed as nanomoles per milligram of dry weight (DW)

(n4 = 3). The half-cell redox potential for NADPH/NADP1 redox couple

was calculated as Ehc = –315–59.1/2 log [NADPH]/[NADP1] taking the

standard electromotive force from these redox pair to be –315. (c) MMS

sensitivity of yeast cells precultivated in different media. Precultures were

grown overnight in full (YPD) or low-glucose/acetate (YPKG) medium.

Cultures were prepared in the same medium by inoculation with

precultures, and grown until the mid-log phase. Serial fivefold dilutions

were prepared and spotted onto agar plates containing YPD,

YPD10.0225% MMS or YPD10.025% MMS and incubated for 48 h at

30 1C.

Fig. 5. Direct effect of MMS and H2O2 on the enzymatic activity of

purified or cell-extracted PYK and GAPDH. 0.1 U of purified enzymes or

10 mg of cellular protein extract (cell lysate) were incubated in Tris/Mg/K

buffer (see Materials and methods) with MMS or H2O2 2 h before

measurements of enzymatic activity. The percentage of enzymatic

activity was calculated in comparison with the activity of mock-treated

enzymes.

FEMS Yeast Res 9 (2009) 535–551c� 2009 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

542 A. Kitanovic et al.

proteins that trigger programmed cell death, mitochondria

act as the ‘executioners’ in apoptosis (review in Garrido &

Kroemer, 2004). We showed earlier that ROS formation

upon MMS treatment could be reduced by alterations in

glucose metabolism (Kitanovic & Wolfl, 2006). The results

presented above indicated that the diauxic shift induced by

lower glucose concentrations could prevent inhibition of

mitochondrial function upon MMS treatment (preserved

CS activity, Fig. 3d) and protect cells from energetic collapse.

As mitochondria are major suppliers of ATP and also the

major source of ROS, we further investigated mitochondrial

features in response to MMS treatment: mitochondrial

membrane potential (C), mitochondrial activity and CL.

Cells cultivated in full (YPD) or low-glucose/acetate med-

ium (YPKG) were treated with MMS for 16 h and stained

with dihydroethidium (ROS formation) and rhodamine 123

(mitochondrial membrane potential C) (Fig. 7).

Fig. 6. (a) Changes in the level of ATP, ADP and AMP upon MMS

treatment. Yeast cells were grown in (a) full medium (YPD) and (b) low-

glucose/acetate (YPKG) medium until the mid-log phase

(OD600 nm = 0.6–1.0). Cells were treated with 0.03% MMS (MMS) or

mock-treated (con). At the indicated time points, aliquots were with-

drawn, cells were quickly collected by filtration, quenched in boiling

buffered ethanol and the intracellular nucleotide content was analysed

by HPIC. The values are presented in mmol g�1 dry weight (DW).

AXP =ATP1ADP1AMP. (c) The energy charge was calculated at indi-

cated time points as (ATP11/2ADP)/(ATP1ADP1AMP). Graphs present

representative experiments of at least three repetitions.

Fig. 7. (a) MMS treatment induced ROS production and mytochondria

hyperpolarization. Wild-type cells were grown until the mid-log phase in

full (YPD) and low-glucose/acetate (YPKG) medium, cultures were

divided and either mock-treated or treated with 0.03% of MMS (a) or

0.1% (b; only in YPD medium). After 16 h, aliquots were taken and

stained with dihydroethidium (detection of ROS production) or rhoda-

mine 123 to measure the mitochondrial membrane potential (c).

Fluorescence intensity was quantified by flow cytometry and results are

given as the mean fluorescence value. Graphs present representative

experiments of at least three repetitions.

FEMS Yeast Res 9 (2009) 535–551 c� 2009 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

543Metabolic control upon DNA damage

Fluorescence intensities were quantified by flow cytometry.

Treatment with 0.03% MMS resulted in a strong induction

of ROS and mitochondrial hyperpolarization (Fig. 7a).

Under low-glucose conditions, induction of ROS by MMS

treatment was significantly lower (Fig. 7a). Treatment with

0.1% MMS over the same time period had a rather opposite

effect (Fig. 7b). At this high MMS concentration, neither

ROS formation nor significant mitochondrial membrane

potential (C) could be detected, most likely reflecting the

severe damage that occurred with this highly cytotoxic

concentration (Fig. 7b).

It had been shown earlier that a decrease in the metabolic

rate could cause specific perturbations of mitochondrial

ATP/ADP exchange that results in ATP synthase stagnation

and mitochondrial hyperpolarization (Vander Heiden et al.,

1999). The inability of F1F0ATPase to utilize the H1 ion

gradient facilitates further ROS production and mtDNA

damage (Esposito et al., 1999; Vander Heiden et al., 1999).

To further analyse mitochondrial function, mitochondria

were stained with NAO and Mito Tracker Green FM

(MTGreen) (Fig. 8a and b). While NAO specifically stains

caridolipin in the inner mitochondrial membranes, fluores-

cence staining with Mito Tracker requires mitochondrial

activity and is specific for active mitochondria. Dead cells,

which could be in particular critical for cardiolipin staining,

were excluded from statistical analysis of both NAO and MT

Green fluorescence, by costaining with PI.

Long-term treatment of cells with 0.03% MMS (24 h)

under high-glucose conditions (Fig. 8a) shows a reduced

amount of functional mitochondria. Cultivation under low-

glucose conditions prevented the loss of functional mito-

chondria, which agrees well with the observation that MMS

treatment did not cause a significant decline of the ATP level

in low glucose media level (Fig. 6b). In both media, addition

of MMS led to elevated staining of cardiolipin already after

2 h of treatment, which was even more pronounced in 24-h

treated cells (Fig. 8b).

Our results indicate that enhanced mitochondrial capa-

city may be critical for better tolerance of MMS treatment

under low-glucose conditions. Thus, while controlling in-

duction of apoptosis in severely damaged cells, mitochon-

dria are also crucial to maintain the energy supply required

for DNA-damage repair and antioxidant defence, enabling

cellular survival.

Oxygen consumption upon MMS treatment

The mitochondrial function in energy metabolism is directly

dependent on oxygen consumption. Therefore, we mea-

sured oxygen consumption of MMS-treated cells using

oxygen sensor 96-well microtitre plates (PreSens). To test

the correlation between glucose availability in the medium

and the influence of MMS on mitochondrial respiration, we

analysed oxygen consumption of cells treated with 0.01%

MMS cultivated at different glucose concentrations (YPD

medium with 2%, 1%, 0.5% and 0.25% glucose). The results

are summarized in Fig. 9. As expected, biomass production

strongly depended on the amount of glucose in the medium,

visible as a progressive reduction of biomass yield at lower

glucose concentrations (Fig. 9a). MMS treatment caused

similar growth inhibition in all cultures. Upon addition of

MMS, oxygen consumption of cells was significantly re-

duced, but resumed at different time points depending on

the glucose concentration in the medium (Fig. 9b). At lower

glucose levels, the recovery of oxygen consumption was

faster, and correlated with glucose depletion from the

medium, starting when the glucose concentration declined

o 0.1%. Under control conditions, the respiration rate was

only slightly influenced by glucose availability, although

biomass production correlated directly with the glucose

concentrations (Fig. 9).

To test whether MMS directly blocks mitochondrial

respiratory activity, we isolated mitochondria from station-

ary cultures of cells grown in full (YPD) medium. For

this, cells were spheroplasted and mitochondria were

isolated in ice-cold sorbitol buffer. Mitochondria were

resuspended in ice-cold sucrose buffer and mitochondrial

activity was stimulated with glutamate/malate and ADP.

Oxygen consumption was measured in sorbitol buffer

containing MMS (0.01%, 0.02% and 0.04%) or 3 mM

H2O2 with oxoplates. The results showed that both MMS

and H2O2 directly block oxygen consumption of isolated

mitochondria (Fig. 10). Therefore, we concluded that

MMS treatment can directly block mitochondrial respira-

tion, which consequently leads to increased ROS produc-

tion, induced oxidative stress and inhibition of GAPDH

activity.

The effect of MMS treatment directly dependson the cellular content of respiratory chaincompounds and the respiration rate

Our results suggest that enhanced mitochondrial activity

and cellular respiration is beneficial for cellular survival by

enabling a fast and better recovery upon MMS treatment. To

test this hypothesis, we included treatment with a mito-

chondrial uncoupling agent, CCCP (carbonyl cyanide m-

chloro phenyl hydrazone), and performed experiments in

hap4 deletion mutants, in which the synthesis of proteins

involved in the TCA cycle, the electron transport chain, ATP

generation and mitochondrial biogenesis is impaired (re-

viewed in Schuller, 2003). To ensure highly energized and

functional mitochondria in the cells, the experiments were

performed in low-glucose/acetate medium. Addition of

CCCP increased oxygen consumption of control cells

and promoted faster recovery of mitochondrial activity in

FEMS Yeast Res 9 (2009) 535–551c� 2009 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

544 A. Kitanovic et al.

MMS-treated cells (Fig. 11a). Moreover, the initial block of

oxygen consumption was significantly lower in CCCP-con-

taining cultures. In contrast, disruption of the heme-acti-

vated protein complex Hap2/3/4/5 through the deletion of

HAP4 reduced the speed of oxygen utilization and strongly

enhanced the MMS-mediated respiratory block (Fig. 11b).

Remarkably, hap4 mutant cells were not able to regain

mitochondrial activity and oxygen consumption even

upon treatment with lower MMS concentrations (0.01%

and 0.02%), conditions under which wild-type cells

can overcome respiratory inhibition and continue prolifera-

tion. Thus, the results obtained support our hypothesis

that high activity of the respiratory chain, which must be

even higher in the presence of an uncoupling agent, reduces

the development of oxidative stress and subsequent cell

death.

Fig. 8. (a) Modulation of mitochondrial bioenergetic conditions of yeast cells in response to MMS treatment. Exponentially growing wild-type cells

were treated with 0.03% MMS in full (YPD) or low-glucose (YP-0.5% Glu) media. Samples were withdrawn after 2 h and 24 h of treatment and double-

stained with Myto Tracker Green/PI (a) or NAO/PI (b). Histogram plots present the fluorescence intensity of Myto Tracker (a) and NAO staining (b). PI-

positive cells are excluded from gates. The mean values of viable cells (non-PI positive) are presented for each measurement. The number of dead cells

(excluded from analysis) in each sample is given as % of PI-positive cells.

FEMS Yeast Res 9 (2009) 535–551 c� 2009 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

545Metabolic control upon DNA damage

Discussion

Our study shows that MMS treatment results in a strong and

rapid inhibition of oxygen consumption, indicating a clear

decrease of respiratory activity, as well as a drastic increase in

ROS production. The observed effects on mitochondrial

function and ROS production could be based on mtDNA

damage, which triggers ROS production (Salmon et al.,

2004; Kitanovic & Wolfl, 2006) and further impairs the

activity of mitochondrial proteins. This could be shown for

CS, whose activity is reduced after longer exposure to MMS.

While oxygen consumption is decreased, other markers of

mitochondrial activity are less significantly changed or even

higher. Measuring oxygen consumption in isolated mito-

chondria, we could show that inhibition of mitochondrial

respiration by MMS is intrinsic to mitochondria, support-

ing our hypothesis that it could be the primary mode of

action for MMS.

A major effect triggered by the alkylating agent MMS on

yeast cells was to induce a dramatic metabolic modification

indicated by the inability of yeast cultures to shift the

metabolism from fermentation to oxidative phosphoryla-

tion. As a consequence, the glucose flow is diverted into the

glycerol, PPP and glucose stores, glycogen and trehalose,

which were consumed later as the sole endogenous carbon

source remaining for cell survival. This rapid mobilization

of stored glucose from glycogen and trehalose as MMS-

treated cells ran out of an exogenous carbon source is

consistent with the notion that these cells cannot resume

growth on a nonfermentative carbon source and hence must

thrive on their endogenous carbon source for energy main-

tenance (Enjalbert et al., 2000; Jules et al., 2008). As a

consequence, the energy charge was strongly reduced with a

net depletion of cellular ATP.

The inhibitory effect of MMS treatment on cellular

proliferation and energy content was concomitant with

persistent glucose consumption and increased glycerol and

Fig. 10. Influence of MMS and H2O2 on the respiratory activity of

isolated mitochondria. Mitochondria were isolated from 500 OD units

of a stationary culture of wild-type cells grown in full (YPD) medium.

Mitochondrial activity was stimulated with 12.5/12.5 mM glutamate/

malate and 1.65 mM ADP, and oxygen consumption was measured in a

96-well OxoPlate in sorbitol buffer containing MMS (0.01%, 0.02% and

0.04%) or 3 mM H2O2. Sorbitol buffer containing 3 mM H2O2 and no

mitochondria was used as a control (marked as medium). Kinetic of

oxygen consumption was followed during 25 h of incubation at 30 1C.

Measurements and calculations were performed as described in Fig. 9.

Fig. 9. (a) Inhibition of respiratory activity upon MMS treatment. Wild-

type (wt) yeast cells were incubated in a full medium (YP-) containing

increasing concentrations of glucose (0.1%, 0.5%, 1% and 2% Glu)

until the mid-log phase (OD600 nm = 0.6–1.0). Equal amounts of cells

were plated in each well of 96-well OxoPlates with a round bottom. Cells

were either mock-treated (con) or treated with 0.01% MMS in triplicate.

The kinetic of fluorescence signal (650 nm for the oxygen sensor and

590 nm for the reference sensor) together with the OD of culture

(OD600 nm) was monitored in a plate reader. The percentage of oxygen

saturation of the medium was calculated based on a two-point calibra-

tion curve that was made with oxygen-saturated water (representing

100% saturation) and sodium sulphite-treated water (oxygen-free

water). The SE values of the measurements were all o 5% of the

calculated values and are therefore not presented here. (a) Growth

curves of cultures. (b) Oxygen consumption of cells treated with MMS

at different glucose concentrations was monitored during 18 h of

cultivation. The legend for both figures is explained in (b). The stars

indicate the time when the glucose concentration in the medium

declines o 1 g L�1; the arrows indicate the time when the total glucose

is consumed.

FEMS Yeast Res 9 (2009) 535–551c� 2009 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

546 A. Kitanovic et al.

ethanol production. This implies that the oxidation of three-

carbon fragments in the glycolytic pathway and their

subsequent utilization in mitochondria for TCA cycle and

respiration should be impaired by MMS. Indeed, MMS

strongly inhibited GAPDH and PYK activity independent

of the glucose concentration in the medium. In contrast, the

activity of G6PDH was strongly enhanced by MMS, which

could reflect a high requirement for NADPH in antioxidant

defence. MMS has no direct inhibitory effect on these

enzymes. Instead, inhibition of GAPDH upon MMS chal-

lenge seemed to be the result of high ROS accumulation by

repressed mitochondria. The mechanism of inhibition of

GAPDH in this specific condition has not been investigated,

but results from the literature indicate that these enzymes

may be subjected to post-translational modification such as

S-nitrosation or oxidation in response to H2O2 treatment,

which results in inhibition of its dehydrogenase activity

(Shenton & Grant, 2003; Osorio et al., 2004; Almeida et al.,

2007). How PYK is repressed remains unclear, because its

activity was not affected directly by peroxide in vitro.

Interestingly, genetic analysis of yeast PYK showed that it is

involved in the cell division cycle, and is also named CDC19.

Temperature-sensitive cdc19 mutants arrest growth in G1 at

the restrictive temperature of 36 1C (Aon et al., 1995) as a

result of carbon metabolism and energy uncoupling. Thus,

cell cycle arrest and reduced proliferation by MMS-induced

DNA damage is probably enhanced upon PYK inhibition,

redirecting carbon flow to glycerol, glycogen and trehalose

accumulation.

The concentration of glucose in the medium seems to be

a key factor in the response to MMS. Preincubation under

low-glucose conditions enables yeast cells to survive better

upon MMS treatment. Inhibition of cellular respiration

upon MMS treatment is also directly correlated with the

glucose concentration in the medium. At low glucose, the

glycolytic rate is reduced, resulting in lower repression of

respiration and TCA enzymes, which is reflected in de-

creased PYK and GAPDH activities and increased CS

activity. As a consequence, pyruvate is utilized more effec-

tively in mitochondria, enabling NADH production in the

TCA cycle and NAD1 regeneration in the respiratory chain.

At high glucose (fermentative growth), pyruvate is mostly

converted to ethanol, due to strong repression of mitochon-

dria. This is in accordance with earlier observations that

calorie restriction (CR)-mediated metabolic shift results in a

more efficient electron transport in the mitochondrial

respiratory chain (Weindruch et al., 1986; Sohal & Wein-

druch, 1996). Faster and more efficient electron transport

will enhance oxygen consumption and therefore reduce

oxygen concentrations in the mitochondrial microenviron-

ment. Thus, it is quite conceivable that the final outcome of

enhanced respiration of calorie-restricted cells when chal-

lenged with MMS is reduced leakage of electrons from the

respiratory chain and a lower production of ROS. This

should improve cellular survival upon MMS treatment,

which is the case in our experiments, showing better survival

of cells on MMS plates when cells were precultured under

CR conditions.

Scavenging of ROS, which increased the survival of MMS-

treated cells (Kitanovic & Wolfl, 2006), has a similar effect on

survival as caloric restriction. This is in good agreement with

the observation that CR decreases the ratio of ROS produced

over O2 consumed in isolated mammalian mitochondria

(Gredilla et al., 2001). It is also known that CR enhances the

expression of mitochondrial uncoupling proteins (Merry,

2002; Bevilacqua et al., 2004), which could be an explanation

for the reduced ROS produced/O2 consumed ratio.

These observations suggest that mitochondrial activity

and its roles in maintaining cellular energy levels as well as

Fig. 11. (a) Influence of mitochondria respiratory conditions on MMS-

induced inhibition of oxygen consumption. Yeast cells were incubated in

a full (YPD) or a low-glucose/acetate (YPKG) medium until the mid-log

phase (OD600 nm = 0.6–1.0). Equal amounts of cells were plated in each

well of 96-well OxoPlates with a round bottom. Cells were either mock-

treated (con) or treated with different MMS concentrations in triplicate.

Experiments and calculations were performed as detailed in Fig. 9. (a)

Kinetic of oxygen consumption of wild-type cells treated with MMS with

or without addition of 5mM CCCP was monitored during 18 h of

cultivation in YPKG medium. (b) Oxygen consumption of wild-type (wt)

and Dhap4 cells treated with MMS was monitored during 17 h of

cultivation in YPD medium.

FEMS Yeast Res 9 (2009) 535–551 c� 2009 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

547Metabolic control upon DNA damage

ROS formation are the critical link between CR and

decreased sensitivity to MMS. In fact, analysing mitochon-

drial parameters, we found that treatment with MMS not

only leads to ROS formation but also induces changes in

mitochondrial activity. Blocking the last step, in the respira-

tory chain, oxygen consumption, electrons must be trans-

ferred to other targets, leading to ROS formation. In this

condition, the membrane potential is increased, despite

reduced activity, which may result in mitochondrial reorga-

nization reflected in enhanced cardiolipin staining. This fits

very well with a dual role of ROS signalling in response to

mitochondrial stress. At low levels, ROS would mediate

retrograde signalling inducing synthesis of mitochondrial

genes for mitochondrial biogenesis, while in response to

severe damage, cell death is induced by high levels of ROS

(Piantadosi & Suliman, 2006; Passos et al., 2007). Under CR

conditions, the membrane potential and respiratory activity

is significantly enhanced to ensure energy and redox home-

ostasis. Modulation of membrane uncoupling and the need

for mitochondrial ATP production ensures constant activity

of the respiratory chain and suppresses ROS formation.

Thus, when the flux in the respiratory chain is artificially

increased by an uncoupling agent, cells should more effi-

ciently tolerate toxic challenges, while suppression of re-

spiratory activity should inhibit the efficient response to the

toxic challenge. Indeed, treatment with the uncoupling

agent CCCP leads to an improved recovery of respiration,

indicating a more efficient flux in the respiratory chain.

Reduction of the respiratory capacity, which is the case in

the hap4 deletion mutant, in which synthesis of electron

transport chain components is repressed (high glucose leads

to similar repression) (Lin et al., 2002, 2004; Schuller, 2003),

prevents the recovery from MMS treatment. As a result,

although the activity of the respiratory chain is decreased,

the block of respiration by MMS in this mutant leads to an

accumulation of electrons at intermediate levels of the

respiratory chain, favouring electron leakage and very high

ROS formation (data not shown).

In summary, the above data demonstrate that the cellular

metabolic status determines the cellular fate in response to

treatment with DNA-damaging agents. High respiratory

activity significantly reduces ROS production and thus

serves as a protective mechanism against treatment with

agents that damage mtDNA. In addition, we could show

Fig. 12. Schematic representation of the proposed effects of MMS on yeast metabolism. MMS treatment directly inhibits the mitochondrial respiratory

chain (probably by damaging mtDNA), leading to a strong decrease of oxygen consumption and high accumulation of electrons (e-) at intermediate

levels of the respiratory chain, thereby increasing ROS production and induction of oxidative stress. ROS directly inhibit the activity of GAPDH and

decrease the flux towards lower parts of the glycolytic pathway, which further suppresses the activity of PYK, aiding the cells to initiate cell-cycle

arrest. As a consequence, ethanol and acetate production, as well as ATP synthesis, are considerably decreased, while the flux of glucose carbon is

redirected towards glycerol, trehalose and glycogen accumulation. In addition, the activity of G6PDH also increases, resulting in a higher flux into the

penthose-phosphate pathway (PPP) and enhanced production of NADPH and nucleotides important for antioxidative defence and DNA repair. Glu6P,

glucose-6-phosphate; Fru1,6P2, fructose-1,6-bisphosphate; GA3P, glyceraldehyde 3-phosphate; DHAP, dihydroxyacetone phosphate; 1,3BPG,

1,3-bisphosphoglycerate; 3PG, 3-phosphoglycerate; and PEP, phosphoenolpyruvate.

FEMS Yeast Res 9 (2009) 535–551c� 2009 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

548 A. Kitanovic et al.

that the mechanism by which alkylating agents, such as

MMS, induce apoptosis is closely related to inhibition of the

lower parts of glycolysis as well as of the mitochondrial

respiratory activity, thereby increasing ROS accumulation

and induction of oxidative stress (Fig. 12). Promoting the

efficient generation of free radicals by respiratory inhibition

could enhance the cytotoxic activity of DNA-damaging

agents, which could be exploited to increase the efficiency

of anticancer therapy. In a recent publication, Raffaghello

et al. (2008) showed that starvation-induced stress resistance

protected yeast and mammalian cells from oxidative or

alkylating agents. Interestingly, they showed that the same

conditions did not improve survival of cancer cells. This

effect was connected to Ras, Akt and mTor in the nutrient

response pathway, which is deregulated in cancer cells. Our

results are in direct agreement with this observation. In fact,

tumour cells are often defective in antioxidative stress

response (reviewed in Verrax et al., 2008) and are highly

dependent on glycolysis. Taken together, our results strongly

support the idea that preservation of mitochondrial activity

is crucial for cellular defence to DNA damage and oxidative

stress. Therefore, it should be expected that starvation could

selectively increase the resistance of normal cells. Our results

also support the possibility of using yeast cells to screen for a

drug combination that can sensitize cancer cells to antic-

ancer agents.

Acknowledgements

We thank Catharina Scholl and Elke Lederer for many

stimulating discussions and technical support. This work

was supported by the SysMO Project Network (EU-BMBF)

on Systems Biology of Microorganisms (MOSES, WP 4.3,

S.W. and A.K.).

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