Myc regulates a transcriptional program that
stimulates mitochondrial glutaminolysis
and leads to glutamine addiction
David R. Wisea, Ralph J. DeBerardinisb, Anthony Mancusoa, Nabil Sayeda, Xiao-Yong Zhangc, Harla K. Pfeifferc,
Ilana Nissimd, Evgueni Daikhind, Marc Yudkoffd, Steven B. McMahonc, and Craig B. Thompsona,1
aDepartment of Cancer Biology, Abramson Cancer Center, University of Pennsylvania, Room 451, Biomedical Research Building II/III, 421 Curie Boulevard,
Philadelphia, PA 19104-6160;bDepartment of Pediatrics and McDermott Center for Human Growth and Development, University of Texas Southwestern
Medical Center, Dallas, TX 75390;cDepartment of Cancer Biology, The Kimmel Cancer Center, Thomas Jefferson Medical College, Philadelphia, PA 19107;
anddDepartment of Pediatrics, Children’s Hospital of Philadelphia, 34th Street and Civic Center Boulevard, Philadelphia, PA 19104
Contributed by Craig B. Thompson, October 10, 2008 (sent for review September 12, 2008)
Mammalian cells fuel their growth and proliferation through the
the remaining metabolites taken up by proliferating cells are not
catabolized, but instead are used as building blocks during ana-
bolic macromolecular synthesis. Investigations of phosphoinositol
3-kinase (PI3K) and its downstream effector AKT have confirmed
that these oncogenes play a direct role in stimulating glucose
glucose for the maintenance of survival. In contrast, less is known
about the regulation of glutamine uptake and metabolism. Here,
we report that the transcriptional regulatory properties of the
oncogene Myc coordinate the expression of genes necessary for
cells to engage in glutamine catabolism that exceeds the cellular
requirement for protein and nucleotide biosynthesis. A conse-
quence of this Myc-dependent glutaminolysis is the reprogram-
ming of mitochondrial metabolism to depend on glutamine catab-
olism to sustain cellular viability and TCA cycle anapleurosis. The
ability of Myc-expressing cells to engage in glutaminolysis does
not depend on concomitant activation of PI3K or AKT. The stimu-
lation of mitochondrial glutamine metabolism resulted in reduced
glucose carbon entering the TCA cycle and a decreased contribu-
tion of glucose to the mitochondrial-dependent synthesis of phos-
pholipids. These data suggest that oncogenic levels of Myc induce
a transcriptional program that promotes glutaminolysis and trig-
gers cellular addiction to glutamine as a bioenergetic substrate.
cancer ? mitochondria
maintained in an oxygen-replete environment (1). This metabolic
phenotype, first observed by Otto Warburg, has been termed
aerobic glycolysis (2). Initially, this high rate of glycolysis was
believed to result from mutations that impair the ability of cancer
cells to carry out oxidative phosphorylation (3). However, such
defects appear to be rare in spontaneously arising tumors (4).
Recent studies have suggested that activating mutations in phos-
phoinositol 3-kinase (PI3K) and its downstream effector AKT
induce the transformed cell to take up glucose in excess of its
bioenergetic needs (5). The resulting high rate of glycolytic metab-
olism leads to the conversion of mitochondria into synthetic or-
ganelles that support glucose-dependent lipid synthesis and non-
essential amino acid production (6, 7). Glycolytic pyruvate that
accumulates in excess of cellular bioenergetic and synthetic needs
is converted to lactate and secreted. A consequence of this meta-
bolic conversion is that cells become addicted to glucose for their
redirected from use as bioenergetic substrates and committed to
use in anabolic synthesis (5). These data suggest that cancer cell
nutrient uptake and metabolism may be under the direct control of
the oncogenic signaling pathways that transform the cell. Strategies
any cancer cell lines depend on a high rate of glucose uptake
and metabolism to maintain their viability despite being
to exploit the glucose addiction of cells transformed by PI3K
mutation for cancer therapy are currently being investigated (4).
In addition to glucose, glutamine can be an essential nutrient for
cell growth and viability (8, 9). In vitro addiction to glutamine as a
found to be a universal property of cancer cell lines. In cancer
patients, some tumors have been reported to consume such an
abundance of glutamine that they depress plasma glutamine levels
(10, 11). Despite these observations, the high rates of glutamine
understood. Recently, we reported that glioma cells can exhibit
glutamine uptake and metabolism that exceeds the cell’s use of
the excess glutamine metabolites produced were found to be
was found to be beneficial because it provided the cell a high rate
of NADPH production that was used to fuel lipid and nucleotide
biosynthesis (see supporting information (SI) Fig. S1 for schematic
of glutaminolytic pathway). However, not all tumor cells exhibit
glutaminolysis. This suggested that the use of glutamine as a
bioenergetic substrate is not induced as an indirect consequence of
cell growth, but as a direct consequence of a specific oncogenic
In the present studies, the oncogenes known to contribute to
malignant transformation of glial cells were tested for the ability to
induce glutaminolysis. Here, we report that the glutaminolytic
phenotype exhibited by tumor cells correlates with a cellular
addiction to glutamine metabolism for the maintenance of cell
viability. In contrast to glucose, glutamine uptake was not found to
be under the direct or indirect control of the PI3K/AKT pathway.
Inhibitors of either PI3K or AKT, despite suppressing glucose
metabolism in a dose-dependent fashion, had no effect on the
glutaminolytic phenotype. In contrast, high level expression of Myc
was required to maintain the glutaminolytic phenotype and addic-
tion to glutamine as a bioenergetic substrate. When an inducible
Myc transgene was introduced in mouse embryonic fibroblasts
(MEF), induction of Myc expression resulted in the induction of
glutamine transporters, glutaminase, and lactate dehydrogenase A
(LDH-A). Induction of these key regulatory genes involved in
glutaminolysis correlated with the Myc-induced increases in glu-
tamine uptake and glutaminase flux. This increase in glutamine
Author contributions: D.R.W., R.J.D., A.M., N.S., X.-Y.Z., E.D., M.Y., S.B.M., and C.B.T.
designed research; D.R.W., R.J.D., A.M., N.S., X.-Y.Z., H.K.P., I.N., and E.D. performed
research; D.R.W., R.J.D., A.M., N.S., X.-Y.Z., H.K.P., I.N., E.D., and M.Y. analyzed data; and
D.R.W., R.J.D., A.M., N.S., X.-Y.Z., and C.B.T. wrote the paper.
The authors declare no conflict of interest.
1To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2008 by The National Academy of Sciences of the USA
December 2, 2008 ?
vol. 105 ?
uptake was not a compensatory response to increased glutamine
incorporation into proteins as a result of Myc-induced protein
after Myc induction was secreted as lactate. Myc-induced repro-
gramming of intermediate metabolism resulted in glutamine ad-
diction, despite the abundant availability of glucose. Glutamine
addiction correlated with Myc-induced redirection of glucose car-
bon away from mitochondria as a result of LDH-A activation. As
a result, Myc-transformed cells became dependent on glutamine
anapleurosis for the maintenance of mitochondrial integrity and
the glutamine dependence of Myc-transformed cells. In addition,
Myc-transformed cells were sensitive to inhibitors of glutamate
conversion to ?-ketoglutarate in a Myc-dependent fashion and this
form of the mitochondrial substrate ?-ketoglutarate. Taken to-
consequence of Myc-induced transformation.
The Human Glioma Line SF188 Depends on Glutamine Catabolism to
Maintain Viability. When SF188 cells were cultured in the presence
of14C-labeled glutamine, ?15% of the glutamine the cells took up
from the medium was incorporated into newly synthesized protein
was used for anabolic synthesis, SF188 glioma cells were unable to
survive in glutamine-deficient medium despite the presence of 25
mM glucose in the medium (Fig. 1B). ?-ketoglutarate is the
replacement of glutamine with a cell-penetrant form of ?-ketoglu-
death observed when the cells were cultured in glutamine-deficient
not result from glutamine’s role as an amide donor in nucleotide
biosynthesis or as the source of nitrogen for the maintenance of
nonessential amino acid production because dimethyl ?-ketogluta-
rate is devoid of nitrogen groups and cannot participate in these
PI3K/AKT Signaling Regulates the Consumption of Glucose but Not of
Glutamine in Glioma Cells. Previous work has demonstrated that the
PI3K/AKT pathway can regulate the expression and surface trans-
location of a variety of nutrient transporters (5, 13). We therefore
up-regulate glutamine uptake and metabolism. To determine
the effects of the PI3K inhibitor LY294002 or the AKT inhibitor,
SF188 cells with a Bcl-xL transgene were used in this study to
prevent apoptosis induced by drug treatment. As presented in Fig.
2, AKT inhibitor VIII suppressed glucose metabolism and lactate
production in a dose-dependent fashion. In contrast, neither glu-
of glutamine metabolism in response to increasing doses of the
inhibitor. Similar results were observed using the PI3K inhibitor
LY294002 (data not shown).
a poor prognosis in glial tumors is Myc (14). SF188 cells were
originally isolated from a patient whose tumor displayed amplifi-
cation of Myc (15). Western blot analysis of the SF188 cells used in
these studies revealed Myc protein expression in excess of that
observed in proliferating fibroblasts or a tumor cell line lacking
the maintenance of oxidative glutamine metabolism in SF188 cells,
cells were transduced with a lentivirus containing shRNA against
MYC (shMYC) or a lentivirus containing a control shRNA (shC-
TRL) and the rate of glutamine consumption and ammonia
production was examined. shMYC cells had an ?80% reduction in
their Myc level (Fig. 3A). This level of Myc reduction lead to a
statistically significant reduction in glutamine consumption (P ?
0.01) and ammonia production (P ? 0.05) (Fig. 3B).
Myc Activates the Transcription of Genes Required for Glutamine
Uptake and Metabolism. Myc’s transforming properties depend on
its ability to bind to DNA and modify gene transcription (16). By
expressed significantly lower levels of the high affinity glutamine
supplemented with 0.01% [14C-U5]glutamine relative to unenriched glutamine
for 4 h. [14C-U5]glutamine in SSA precipitated protein (striped bar) and total
glutamine consumed from the medium (gray bar) are presented as the mean ?
standard deviation (SD) of four independent experiments. (B) The requirement
for glutamine can be satisfied by alpha-ketoglutarate. SF188 cells were allowed
to plate in complete medium and then cultured in either glutamine-depleted
medium supplemented with 7 mM dimethyl ?-ketoglutarate (? glutamine ?
?-ketoglutarate). Cell viability was determined at the time points shown by
trypan blue dye exclusion. The data presented are the mean ? SD of triplicate
samples. Representative data from one of three independent experiments are
Glutamine catabolism in the human glioma line SF188. (A) Protein
glutamine, and ammonia. The rates shown were calculated from the difference
in metabolite concentration between the medium at the time point shown and
PI3K/Akt signaling regulates the consumption of glucose but not of
Wise et al.PNAS ?
December 2, 2008 ?
vol. 105 ?
no. 48 ?
importers ASCT2 and SN2 (P ? 0.01) without expressing signifi-
cantly lower levels of the control transcript EIF1A (Fig. 3C).
Furthermore, when Myc antibodies were used to perform chroma-
to the promoter regions of both ASCT2 and SN2 (Fig. 3D). This
selectivity was comparable to that of the established Myc target
CYCLIN D2 (Fig. 3D). Thus, Myc appears to bind to the promoter
elements of glutamine transporters and this binding is associated
with enhanced levels of glutamine transporter mRNA.
Myc Activates Glutaminolysis in MEF. The above data demonstrate
exhibited by SF188 glioma cells. We next wanted to determine
whether the induction of Myc transcription was sufficient to induce
increased glutamine metabolism. To address this question, immor-
talized MEF that stably express a 4-hydroxy tamoxifen-inducible
MycER construct (17) were analyzed to study the effects of Myc
activation on glutamine metabolism. Treatment of cells with 4-hy-
droxy tamoxifen for 24 h resulted in increased levels of the
transcripts for not only the glutamine transporter ASCT2, but also
4A). These increases in mRNA levels correlated with enhanced
statistically significant increases in glutamine uptake (P ? 0.05)
(Fig. 4B), glutaminase flux (P ? 0.005) (Fig. 4C), and production
of glutamine-derived lactate (P ? 0.05) (Fig. 4D) in the MEF
expressing MycER. As a result, the rate at which glutamine was
consumed from the medium was significantly greater (Fig. 4E).
reach an asymptote by 6 h, whereas the glutamine metabolized by
the 4-hydroxy tamoxifen treated cells increased linearly over the
culture period (Fig. 4E). Thus, the ability of Myc-induced cells to
metabolize glutamine does not appear to be saturatable over this
period as would be predicted for glutaminolysis, which ends not in
the cellular accumulation of glutamine-derived metabolites over
time, but in the secretion of glutamine-derived lactate into the
medium. Despite increasing glutamine consumption, Myc induc-
tion did not increase the proliferative expansion of normal MEF
under the same conditions. After 24 h of 4-hydroxy tamoxifen or
vehicle treatment of cells that were initially plated at 1 ? 105cells
per well the day before induction, the control cells increased to
5.2 ? 0.5 ? 105cells per well (mean ? SD) whereas the Myc-
induced cells had only increased to 3.5 ? 0.5 ? 105cells per well
(mean ? SD).
Myc Diverts Glucose Away from Mitochondrial Metabolism. Previous
studies have suggested that proliferating nontransformed cells
maintain de novo phospholipid biosynthesis from glucose (7).
However, when MEF stably expressing MycER were treated with
4-hydroxy tamoxifen, the use of glucose as a precursor for phos-
pholipid synthesis was suppressed (Fig. 5A). Concomitantly, an
increased amount of the glucose-derived carbon was secreted from
the cell as lactate (Fig. 5B). In contrast, the contribution of
glutamine to phospholipid synthesis in Myc-induced cells was
maintained and even increased despite increased secretion of
glutamine-derived lactate (Figs. 4D and 5C). Together, these data
demonstrate that Myc-induction leads to the diversion of glucose-
and secretion from the cell. As a result, Myc induction enhances
cellular dependence on glutamine to maintain phospholipid syn-
thesis and TCA cycle anapleurosis.
The Glutamine Addiction Exhibited by SF188 Glioma Cells Is Myc-
Dependent. The above data suggest that Myc is both necessary and
SF188 cells. To confirm that Myc is also involved in the glutamine
addiction observed by these cells, SF188 cells were transduced with
shRNA (shCTRL). The resulting cells were incubated in glu-
tamine-depleted or complete medium. Cells transduced with
MYC-shRNA had a statistically significant increase (P ? 0.01) in
their resistance to glutamine starvation relative to cells transduced
with a control shRNA (Fig. 6A).
As a further confirmation that this glutamine addiction is Myc-
dependent, the cells were treated with aminooxyacetate (AOA), a
vert glutamate into ?-ketoglutarate in the glutaminolytic pathway
control shRNA without affecting the viability of cells transduced
with MYC-shRNA (P ? 0.01). Although AOA is a well-
characterized inhibitor of the transaminases (19), a chemical inhib-
the specificity of AOA’s effects, the ability of dimethyl ?-ketoglu-
tarate to reverse the AOA-induced toxicity to SF188 cells was
examined. Addition of 7 mM dimethyl ?-ketoglutarate completely
and control transduced cells (P ? 0.01) (Fig. 6B and data not
The factors that regulate glutamine uptake and metabolism during
cell growth and transformation have remained poorly understood.
In this manuscript, we provide evidence that oncogenic levels of
Myc reprogram intermediate metabolism, leading to glutamine
addiction for the maintenance of mitochondrial TCA cycle integ-
rity. Previous work has demonstrated that LDH-A induction by
and metabolism. (A) Myc protein is over-expressed in SF188 cells. Western blot
and another glioblastoma cell line, LN229. (B) Myc is required for glutamine
Knockdown of Myc protein is depicted in (A). (C) Myc is required for the expres-
and shCTRL cells and quantified using quantitative RT-PCR (qPCR). The bars
shown are normalized to a ?-actin control and represent the mean ? SD of
triplicate samples. Representative data from one of two independent experi-
ments are shown. EIF1A is included as a negative control. (D) Myc is enriched at
the regulatory binding sites of genes involved in glutamine uptake. Sheared
chromatin from fixed and lysed SF188 cells was immunoprecipitated using the
Myc activates the transcription of genes involved in glutamine uptake
www.pnas.org?cgi?doi?10.1073?pnas.0810199105Wise et al.
Myc is required for Myc-transformation (20). This results in diver-
sion of glucose-derived pyruvate into lactate. Despite this, Myc-
transformed cells display an increased mitochondrial mass and
(22) have reported that Myc-over-expressing cells are exquisitely
To explain this apparent paradox, they suggested that mitochon-
drial respiration might be maintained by catabolizing alternative
substrate is glutamine. Myc-transformation leads to conversion
from glucose to glutamine as the oxidizable substrate used to
maintain TCA cycle activity and cell viability. Myc binds to the
promoters and induces the expression of several key regulatory
genes involved in glutaminolytic metabolism. Our studies suggest
that supraphysiological levels of Myc associated with oncogenic
transformation are both necessary and sufficient for the induction
of glutaminolysis to levels that result in glutamine addiction.
Yuneva et al. (23) have reported that some, but not all, Myc
transformants were dependent on glutamine. They also demon-
MEF. (A) Oncogenic levels of Myc induce
the expression of genes involved in glu-
treated with 200 nM 4-hydroxytamox-
ifen (4-OHT) or vehicle (EtOH) for 24 h.
The bars shown are normalized to an
mean ? SD of triplicate samples. Repre-
sentative data from one of three inde-
pendent experiments are shown. (B) On-
cogenic levels of Myc induce glutamine
cultured for 1 min with medium supple-
mented with [U-14C5]glutamine. Uptake
of the label was quantified by scintilla-
tion counting of the cellular lysate. The
data presented are the mean ? SD of
Myc induce flux through glutaminase.
MEF MycER treated as in (A) were cul-
tivity was determined by measuring the
culture medium by GC-MS. The bars
shown represent the mean ? SD of trip-
licate cultures. (D) Oncogenic levels of
was subsequently removed and analyzed with13C NMR spectroscopy. [2,3-13C]lactate is metabolically derived from [U-13C5]glutamine, while [3-13C]lactate is
of Myc induce the consumption of glutamine from the medium. The glutamine concentration in medium from MEF MycER treated as in (A) was analyzed at the time
points shown by the Nova Flex. The data points shown represent the mean ? SD of triplicate samples.
Myc activates glutaminolysis in
Oncogenic levels of Myc suppress the contribution of glucose to phospholipid
synthesis. MEF MycER treated as in Fig. 4A were cultured with medium supple-
mented with D-[U-14C]-glucose for 8 h. After the culture period, lipids were
harvested and14C enrichment in phospholipids (PL) was determined by scintilla-
tion counting. The bars shown represent the mean ? SD of triplicate samples.
Representative data from one of three experiments are shown. (B) Oncogenic
Nova Flex Metabolite Analyzer. Each time point is the mean ? SD of triplicate
samples. Representative data from one of three experiments are shown. (C)
of oncogenic levels of Myc. MEF MycER treated as in Fig. 5A were cultured with
lipids were harvested and14C enrichment in PL was determined by scintillation
counting. The bars shown represent the mean ? SD of triplicate samples. Rep-
resentative data from one of three independent experiments are shown.
Myc diverts glucose away from mitochondrial metabolism in MEF. (A)
dependent. (A) Myc-suppressed SF188 cells are resistant to glutamine starva-
in the presence of glutamine and then cultured in the absence of glutamine.
Cell viability was determined at the time points shown by trypan blue dye
exclusion. The data points shown represent the mean ? SD of triplicate
samples. (B) Myc-suppressed SF188 cells are resistant to an inhibitor of glu-
taminolysis. shMYC and shCTRL SF188 cells, described in Fig. 3B, were allowed
to plate in the presence of glutamine and then were treated with 500 ?M
aminooxyacetate (AOA). Cell viability was determined at the time points
shown by trypan blue dye exclusion. AOA- treated shCTRL cells were also
treated with 7 mM dimethyl ?-ketoglutarate (AOA ? ?-ketoglutarate). The
data points shown represent the mean ? SD of triplicate samples.
The glutamine addiction exhibited by SF188 glioma cells is Myc-
Wise et al. PNAS ?
December 2, 2008 ?
vol. 105 ?
no. 48 ?
strated that over-expression of Bcl-2 suppressed the death of
Myc-transformants deprived of glutamine. Cell types and cell lines
this may account for the differences observed between cell lines.
they underwent cell cycle arrest but did not die when deprived of
glutamine (data not shown). Like Yuneva et al. (23), we also found
that cell-penetrant TCA cycle intermediates could suppress Myc-
induced apoptosis. Together, these results suggest that glutamine
addiction does not result from the use of glutamine as an amine
donor, but rather because glutamine metabolism is essential to
maintain mitochondrial integrity and function in Myc-transformed
Whether levels of Myc expression induced in response to mito-
genic stimulation also play a critical role in glutamine uptake and
metabolism in the growth of nontransformed cells remains to be
protein synthesis and cell growth will also stimulate the incorpo-
ration of glutamine into newly synthesized proteins (24, 25). There
are undoubtedly additional signaling pathways that contribute to
the regulation of glutamine uptake. Cells lacking oncogenic Myc
levels, while not glutamine dependent, still take up sufficient
glutamine to fuel both nucleotide and protein biosynthesis for cell
growth and proliferation. Perhaps that is what is most surprising
about the current results. Little of the glutamine uptake stimulated
by Myc is used for macromolecular synthesis. Previous13C-NMR
studies found that during glutaminolysis, ?60% of glutamine-
derived carbon is released from the cell as either lactate or CO2
(12). Although the TCA cycle was also replenished by glutamine,
only 5% of glutamine fluxing through the TCA cycle was incor-
porated into fatty acids. Here, we show that only 15% of the
glutamine carbon taken up by the cell is incorporated in protein.
Nevertheless, the additional stimulation of glutaminolysis by onco-
genic levels of Myc results in cellular addiction to glutamine.
The ability of Myc to induce glutaminolysis does have a poten-
tially beneficial effect for the transformed cell. Glutaminolysis
results in the robust production of NADPH, thus providing an
energy source for a wide variety of synthetic reactions required for
cell growth (12). It has long been believed that the major source of
NADPH production during cell growth occurs through the oxida-
tive arm of the pentose phosphate shunt (26). However, recent
evidence suggests that transformed cells exhibiting aerobic glycol-
ysis derive the majority of their ribose biosynthesis through the
conditions, G6PD cannot be used to produce a supply of NADPH
to support macromolecular synthesis of fatty acids or nucleotides.
nucleotide synthesis using ribose produced in the nonoxidative arm
of the pentose phosphate shunt would rapidly lead to intracellular
depletion of NADPH. The ability of Myc to stimulate NADPH
transformed cell with a mechanism to produce the quantities of
NADPH needed to meet the demands of cell proliferation.
is controlled independently of glucose uptake. Although the PI3K/
AKT pathway plays a major role in regulating glucose uptake, it
does not appear to be required for the uptake and catabolism of
glutamine in Myc-transformed cells. Thus, the two main bioener-
getic substrates used by proliferating cells appear to be under
able ability of Myc and Akt to cooperate in transforming cells may
result in part from their ability to complement each other in
stimulating the uptake of these two critical nutrients.
In conclusion, the results presented here provide evidence that
Myc transformation is associated with induction of a level of
glutamine metabolism that results in glutamine addiction. Such
addiction may ultimately be exploited through the use of inhibitors
of the enzymes involved in the glutaminolytic pathway. The ability
of the transaminase inhibitor AOA to induce the death of Myc-
transformed cells but not isogenic cells in which Myc is suppressed
by a MYC-shRNA provides evidence that such an intervention
would have a selectively toxic effect on Myc-transformed cells.
events observed in a wide variety of cancers and is known to drive
and small cell lung cancer (31). Despite this, therapeutics that
discovery efforts. The identification of Myc’s role in glutaminolysis
may provide a number of enzymatic targets through which to
selectively impair the growth and survival of Myc-transformed
tumor cells. Finally, the present results demonstrate that nutrient
uptake in mammalian cells is under distinct and specific regulation
as a result of the properties of known oncogenes. Previous results
have suggested that an activating mutation in PI3K or AKT
facilitate the uptake and metabolism of glucose in an mTOR-
to hypoxia or mitochondrial ROS can also reprogram the intracel-
lular fate of glucose (6). The present studies suggest that oncogenic
Myc activation selectively induces addiction to glutamine, the other
major catabolic substrate used by mammalian cells to maintain
bioenergetics during cell growth and proliferation. Whether other
essential nutrients are under similar control by these or other
oncogenic signaling pathways remains to be determined.
Cell Culture and Media. SF188cells(UCBrainTumorResearchCenter,SF,CA)and
SV40-immortalized MEF stably transfected with MycER (a gift from Drs. AT
were cultured in DMEM (Invitrogen), 10% FBS (Gemini Biosystems), 100 units/ml
in a 5% CO2incubator. To avoid depleting oxygen, all experiments were carried
out under subconfluent conditions. For glutamine starvation experiments,
(Gemini Biosystems). For metabolic tracing experiments, DMEM without glu-
tamine and with 10% dialyzed FBS was supplemented with either L-glutamine
that was unenriched or with L-[U-13C5]glutamine (Isotec), [U-14C5]glutamine (GE
Amersham), and [?-15N]glutamine (Cambridge Isotope Laboratories). DMEM
without glucose (Sigma) was supplemented with [U-14C6]glucose (Sigma). To
activate MycER, cells were incubated with 200 nM 4-hydroxytamoxifen for 24 h.
Lentiviral transduction, qPCR, immunoblotting, and metabolite analysis were
performed as previously described (6, 12, 33, 34).
Glutamine Uptake. Cells were incubated with 4 mM glutamine and 1 ?M
[U-14C5]glutamine in 2 ml of medium in a 6-well plate for 1 min at 37°C in a 5%
CO2incubator. After incubation, the medium was aspirated, and cells were
2N HCl, and analyzed with a beta scintillation counter (PerkinElmer Life
NMR Analysis. Cells were grown to 80% confluency, after which the culture
medium was removed and replaced with medium that contained 4 mM
MHz. A 90° excitation pulse was applied every 20 seconds (fully relaxed), with
ment). Spectra were acquired with 16384 points, a bandwidth of 25,000 Hz and
3,000 excitations. Free induction decays were apodized with exponential multi-
plication (3 Hz line broadening). Peak intensities in Fourier transformed spectra
were determined with Nuts NMR (Acorn NMR, Livermore, CA). Carbon-3 of
lactate derived from glutamine produced a doublet (21.5 and 20.3 ppm) due to
splitting from13C at carbon-2, whereas lactate derived from the natural abun-
from each other.
Lipid Biochemistry. To determine the rate of lipid synthesis from glucose or
supplemented with either 2.2 ?M D-[U-14C6] glucose (GE Amersham) or 0.54 ?M
L-[U-14C5]glutamine (GE Amersham), both supplemented to 0.01% of their re-
spective unenriched nutrients. After 8 h, cells were trypsinized, washed in PBS,
www.pnas.org?cgi?doi?10.1073?pnas.0810199105 Wise et al.
and lysed in 0.4 ml of 0.5% Triton X-100. Total lipids were extracted and phos-
ChIP Analysis. SF188 cells were plated on 15-cm dishes and were fixed in 1%
formaldehyde. Chromatin was sheared to an average size of 500–1,000 bp by
sonication (30 times with 30-s pulses, on a Diagenode Bioruptor). Lysates corre-
IgG. Precipitated DNA fragments were quantified using PCR.
Akt Inhibitor. SF188 cells stably expressing Bcl-xLwere plated at a density of 4 ?
before treatment with AKT inhibitor VIII (Calbiochem) at 0, 0.1, 2, 5, and 10 ?M
concentrations in triplicate. After 8 h, medium samples were collected for me-
tabolite analysis, and viable cells were counted using trypan blue exclusion.
Contribution of Glutamine to Protein Synthesis. SF188 cells were cultured in
medium supplemented with 0.01% [U-14C5]glutamine relative to unenriched
Triton-X, acidified using 10% (vol/vol) of 35% (wt/v) sulfosalicylic acid, and then
spun down at 13,000 rpm for 15 min at 4°C. The pellet was then resolubilized in
NaOH at 37°C.14C incorporated into the protein product were quantified using
a scintillation counter (PerkinElmer Life Sciences). Efficiency of protein recovery
Recovery of intracellular [U-14C5]glutamine in its free form was controlled for by
calculating the recovery of [U-14C5]glutamine added just before SSA precipita-
tion. The recovered counts were sensitive to 5 ?g/ml cycloheximide. The glu-
(Sigma). The data presented are the mean ? SD from four independent
dish. At 80% confluency, they were fed with 1.5 ml of DMEM containing 4 mM
l-[?-15N]glutamine (Cambridge Isotope Laboratories). Every 2 h, medium was
Analyzer. An aliquot was used to determine isotopic enrichment in NH3with
published methods (35).
AOA Inhibitor Experiments. SF188 cells were treated with AOA (Sigma) at doses
ranging from 500 nM – 500 ?M and viability was assessed 24 h post-treatment
cells. SF188 cells with Myc or control shRNA were replated 3 days post viral
transduction. After allowing to plate overnight, cells were treated with 500 ?M
for 24 h, and then viability was assessed using trypan blue dye exclusion.
ACKNOWLEDGMENTS. We thank members of the Thompson laboratory for
thoughtful discussions and Tullia Lindsten and Tamar Schwartz for their
review of the manuscript. This work was supported by grants from the
National Cancer Institute; the National Institutes of Health, including K08
DK072565 (to R.J.D.) and HD26979 and NS054900 (to M.Y.); the Abramson
Family Cancer Research Institute (C.B.T.); a Medical Scientist Training grant
and a Cancer Research Institute grant (to D.R.W.). S.B.M. was supported by
grants from the National Institutes of Health and Commonwealth Universal
Research Enhancement Program, Pennsylvania Department of Health.
1. Warburg O (1956) On the origin of cancer cells. Science 123:309–314.
2. Warburg O (1925) u ¨ber den Stoffwechsel der Carcinomzelle. J Mol Med 4:534.
3. Warburg O (1956) On respiratory impairment in cancer cells. Science 124:269–270.
5. Elstrom RL, et al. (2004) Akt stimulates aerobic glycolysis in cancer cells. Cancer Res
6. Lum JJ, et al. (2007) The transcription factor HIF-1alpha plays a critical role in the
growth factor-dependent regulation of both aerobic and anaerobic glycolysis. Genes
7. Bauer DE, Hatzivassiliou G, Zhao F, Andreadis C, Thompson CB (2005) ATP citrate lyase is
an important component of cell growth and transformation. Oncogene 24:6314–6322.
8. Coles NW, Johnstone RM (1962) Glutamine metabolism in Ehrlich ascites-carcinoma
cells. Biochem J 83:284–291.
9. Eagle H (1955) Nutrition needs of mammalian cells in tissue culture. Science 122:501–
10. Klimberg V, McClellan JL (1996) Glutamine, cancer, and its therapy. Am J Surgery
11. Chen MK, Espat NJ, Bland KI, Copeland EM, III, Souba WW (1993) Influence of pro-
gressive tumor growth on glutamine metabolism in skeletal muscle and kidney. Ann
Surg 217:655–666, discussion 666–667.
12. DeBerardinis RJ, et al. (2007) Beyond aerobic glycolysis: Transformed cells can engage
in glutamine metabolism that exceeds the requirement for protein and nucleotide
synthesis. Proc Natl Acad Sci USA 104:19345–19350.
13. Edinger AL, Thompson CB (2002) Akt maintains cell size and survival by increasing
mTOR-dependent nutrient uptake. Mol Biol Cell 13:2276–2288.
14. Ben-Porath I, et al. (2008) An embryonic stem cell-like gene expression signature in
poorly differentiated aggressive human tumors. Nat Genet 40:499–507.
15. Trent J, et al. (1986) Evidence for rearrangement, amplification, and expression of
c-myc in a human glioblastoma. Proc Natl Acad Sci USA 83:470–473.
Mol Cell Biol 19:1–11.
17. Thomas-Tikhonenko A, et al. (2004) Myc-transformed epithelial cells down-regulate
clusterin, which inhibits their growth in vitro and carcinogenesis in vivo. Cancer Res
18. Moreadith RW, Lehninger AL (1984) The pathways of glutamate and glutamine
oxidation by tumor cell mitochondria. Role of mitochondrial NAD(P)?-dependent
malic enzyme. J Biol Chem 259:6215–6221.
19. Rej R (1977) Aminooxyacetate is not an adequate differential inhibitor of aspartate
aminotransferase isoenzymes. Clin Chem 23:1508–1509.
20. Lewis BC, et al. (2000) Tumor induction by the c-myc Target Genes rcl and lactate
dehydrogenase A. Cancer Res 60:6178–6183.
21. Li F, et al. (2005) Myc stimulates nuclearly encoded mitochondrial genes and mito-
chondrial biogenesis. Mol Cell Biol 25:6225–6234.
22. Morrish F, Neretti N, Sedivy JM, Hockenbery DM (2008) The oncogene c-Myc coordi-
nates regulation of metabolic networks to enable rapid cell cycle entry. Cell Cycle
23. Yuneva M, Zamboni N, Oefner P, Sachidanandam R, Lazebnik Y (2007) Deficiency in
24. Johnston LA, Prober DA, Edgar BA, Eisenman RN, Gallant P (1999) Drosophila myc
regulates cellular growth during development. Cell 98:779–790.
25. Iritani BM, Eisenman RN (1999) c-Myc enhances protein synthesis and cell size during
B lymphocyte development. Proc Natl Acad Sci USA 96:13180–13185.
of DHEA, GSH depletion and phenylarsine oxide. Biochem Biophys Res Commun
27. Serkova N, Boros LG (2005) Detection of resistance to imatinib by metabolic profiling:
Clinical and drug development implications. Am J Pharmacogenomics 5:293–302.
28. Dalla-Favera R, et al. (1982) Human c-myc onc gene is located on the region of
chromosome 8 that is translocated in Burkitt lymphoma cells. Proc Natl Acad Sci USA
29. Taub R, et al. (1982) Translocation of the c-myc gene into the immunoglobulin heavy
chain locus in human Burkitt lymphoma and murine plasmacytoma cells. Proc Natl
Acad Sci USA 79:7837–7841.
30. Schwab M, et al. (1983) Amplified DNA with limited homology to myc cellular onco-
gene is shared by human neuroblastoma cell lines and a neuroblastoma tumour.
31. Nau MM, et al. (1985) L-myc, a new myc-related gene amplified and expressed in
human small cell lung cancer. Nature 318:69–73.
32. Bode BP, Souba WW (1994) Modulation of cellular proliferation alters glutamine
transport and metabolism in human hepatoma cells. Ann Surg 220:411–424.
modulation of carnitine palmitoyltransferase 1A expression regulates lipid metabo-
lism during hematopoietic cell growth. J Biol Chem 281:37372–37380.
34. Patel JH, McMahon SB (2007) BCL2 is a downstream effector of MIZ-1 essential for
blocking c-myc-induced apoptosis. J Biol Chem 282:5–13.
J Biol Chem 276:31876–31882.
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vol. 105 ?
no. 48 ?