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: [email protected]
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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551Metabolic control upon DNA damage
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