The Stable Isotope-based Dynamic Metabolic Profile of Butyrate-induced HT29 Cell Differentiation*
Joan Boren
,
Wai-Nang Paul Lee
¶,
Sara Bassilian
,
Josep Joan Centelles
,
Shu Lim
,
Sayed Ahmed
,
László G. Boros
and
Marta Cascante
||
From the
Department of Biochemistry and Molecular
Biology, Centre Recerca en Química Teòrica-Parc Cientific de
Barcelona, Institut d'Investigacions Biomèdiques August Pi i Sunyer,
University of Barcelona, Martí i Franquès 1, 08028 Barcelona,
Catalonia, Spain and
UCLA School of Medicine,
Harbor-UCLA Research and Education Institute, Torrance, California 90502
Received for publication, March 21, 2003
, and in revised form, May 8, 2003.
 |
ABSTRACT
|
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Stable isotope-based dynamic metabolic profiling is applied in this paper
to elucidate the mechanism by which butyrate induces cell differentiation in
HT29 cells. We utilized butyrate-sensitive (HT29) cells incubated with
[1,2-13C2]glucose or
[1,2-13C2]butyrate as single tracers to observe the
changes in metabolic fluxes in these cells. In HT29 cells, increasing
concentrations of butyrate inhibited glucose uptake, glucose oxidation, and
nucleic acid ribose synthesis in a dose-dependent fashion. Glucose carbon
utilization for de novo fatty acid synthesis and tricarboxylic acid
cycle flux was replaced by butyrate. We also demonstrated that these changes
are not present in butyrate-resistant pancreatic adenocarcinoma MIA cells. The
results suggest that the mechanism by which colon carcinoma cells acquire a
differentiated phenotype is through a replacement of glucose for butyrate as
the main carbon source for macromolecule biosynthesis and energy production.
This provides a better understanding of cell differentiation through metabolic
adaptive changes in response to butyrate in HT29 cells, demonstrating that
variations in metabolic pathway substrate flow are powerful regulators of
tumor cell proliferation and differentiation.
 |
INTRODUCTION
|
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Butyrate is a four-carbon short chain fatty acid produced by fermentation
of fiber polysaccharides by the intestinal microflora of the human colon
(1). Butyrate is utilized
primarily by colon epithelial cells as a substrate for energy production
(2). Previous studies
demonstrated that deficiency in the availability or utilization of butyrate
causes colitis and may be involved in colon carcinogenesis
(3).
Butyrate has been shown to induce dose dependent differentiation of various
malignant cell lines
(46).
Studies performed in recent years in colorectal cancer have shown that there
is a cell cycle arrest in G1 phase due to the activation of cyclin
D3 and p21Waf1/Cip1 after incubation with butyrate
(7). These studies implicate
many known cell signaling events in mediating the cell-differentiating effect
of butyrate, including cyclin-dependent kinase inhibitors
(8), mitogen-activated
phosphorylase kinases (9),
down-regulation of c-myc
(10), and the proinflammatory
transcription factor NF-
B
(11,
12). Among the genes studied
using gene array technology, the most significantly affected were those of
transcription factors related to cell growth, apoptosis, and oxidative
metabolism (13,
14). How butyrate induces
these specific molecular changes is mostly unknown. To assess how known
genetic modifications can be translated into metabolic changes characteristic
of differentiated cells, techniques allowing analysis of the levels of low
molecular weight compounds are required. Stable isotope-based dynamic
metabolic profiling using gas chromatography/mass spectrometry
(GC/MS)1 is a new tool
with a largely untapped potential in the field of functional genomics. In this
paper we demonstrate the usefulness of this technique to elucidate the
metabolic mechanism underlying butyrate induced cell differentiation.
The task will be performed using metabolomic and fluxomic analysis to
investigate whether butyrate-induced differentiation in HT29
(butyrate-sensitive) colon and MIA (butyrate-resistant) pancreatic
adenocarcinoma cells involves the reversion of metabolic reactions
characteristic of undifferentiated cells. Such results would strongly support
the importance of gene-nutrient interactions on the platform of cellular
metabolic pathways and the level of their intermediates as key metabolic
signals/ligands to the transcriptional regulation of mammalian cell growth and
differentiation.
 |
MATERIALS AND METHODS
|
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Cell Lines and CultureHT29 human colon adenocarcinoma cells
and MIA pancreatic adenocarcinoma cells (obtained from the American Type
Culture Collection) were grown in minimum essential medium in the presence of
10% fetal bovine serum at 37 °C in 95% air, 5% CO2 for 3 days
before the start of the experiments. Just prior to butyrate treatment and
stable isotope labeling, the medium was removed and fresh Dulbecco's modified
Eagle's medium (with L-glutamine, without glucose and without
sodium pyruvate; Invitrogen) was added with 10% fetal bovine serum, 10
mM of glucose and increasing doses of butyrate (0, 0.1, 1, and 5 or
10 mM), for 72 h. Two separate experiments were performed in each
cell line: 1) to study the effect of butyrate on glucose metabolism, cells
were incubated with [1,2-13C2]glucose (50% isotope
enrichment) and increasing doses of butyrate; and 2) when the metabolism of
butyrate itself was studied, the cultured cells were incubated with unlabeled
glucose but increasing doses of [1,2-13C2]butyrate (50%
isotope enrichment). Stable
[1,2-13C2]D-glucose and
[1,2-13C2]butyrate isotopes were purchased with >99%
purity and 99% isotope enrichment for each position (Isotec Inc., Miamisburg,
OH).
Cell cultures were started with the same cell number (2 x
105), which was achieved using standard cell counting techniques.
At the end of the experiment, the final cell numbers were also measured with
these standard procedures. HT29 and MIA cells were selected for the study
because HT29 differentiate with butyrate, whereas MIA cells do not. The
metabolic differences of these cell lines are reported in order to reveal
differentiation-specific carbon flow and substrate redistribution changes in
tumor cells.
Alkaline Phosphatase ActivityThis enzyme activity was
measured as a marker of the degree of cell differentiation after butyrate
treatment according to the published procedures
(15). Alkaline phosphatase was
expressed as nmol of substrate converted per min and per mg of protein.
Protein determination was done using the BCA protein assay (Pierce).
Glucose and LactateThe levels of glucose and lactate in
media were measured using a Cobas Mira chemistry analyzer (Roche Applied
Science). Glucose and butyrate oxidation by the cells was determined by media
13C/12C ratios in released CO2 by a Finnegan
Delta-S isotope ratio mass spectrometer (IRMS)
(16). To obtain the
CO2 from the medium, 200 µl of the cell medium were added to a
hermetically closed vial, and then 50 µlof0.1 M
NaHCO3 and 50 µl of 0.12 N HCl were added to react
with the medium and liberate the CO2, which was injected directly
into the IRMS. The value obtained from the spectrometer is
13/12, which is equal to the ratio between 13C
and 12C from the sample minus the 13C/12C
from the reference (air) divided from the ratio of the reference. Release of
13CO2 was measured to estimate glucose or butyrate
carbon utilization through oxidation by the cell lines and was expressed as
the difference between the amount released by each treatment and the amount
released by the control.
Lactate from the cell culture medium was extracted by ethyl acetate after
acidification with HCl. Lactate was derivatized to its
propylamide-heptafluorobutyric form and the m/z 328 (carbons
13 of lactate, chemical ionization) was monitored for the detection of
m1 (recycled lactate through the pentose cycle) and m2 (lactate produced by
glycolysis) for the estimation of pentose cycle activity
(17). The unlabeled species
(m0) represents corrected lactate mass isotopomer distribution without
13C label, m1 with one 13C label and m2 with two
13C labels. m2 originates from glucose that is converted to lactate
directly by glycolysis, whereas m1 originates from glucose metabolized by
direct oxidation via the oxidative steps of the pentose phosphate pathways and
then recycled to glycolysis via the non-oxidative pentose cycle. From these
results, we can predict pentose cycle flux relative to the glycolytic flux,
and it is calculated by the m1/m2 ratio in lactate
(17).
RNA RiboseRNA ribose was isolated by acid hydrolysis of
cellular RNA after Trizol purification of cell extracts. Ribose isolated from
RNA was derivatized to its aldonitrile acetate form using hydroxyl-amine in
pyridine and acetic anhydride. We monitored the ion cluster around the
m/z 256 (carbons 15 of ribose, chemical ionization)
to find the molar enrichment and positional distribution of 13C
labels in ribose (17).
GlutamateGlutamate was separated from the cell medium using
ion-exchange chromatography
(18). Glutamate was converted
to its n-trifluoroacetyl-n-butyl derivative and the ion
clusters m/z 198 (carbons 25 of glutamate, electron
impact ionization) and m/z 152 (carbons 24 of glutamate,
electron impact ionization) were monitored. The different isotopomers of
glutamate allowed us to determine the parameter Y, which is the
anaplerotic flux (pyruvate carboxylase) related to the tricarboxylic acid
cycle flux (expressed as the fraction of oxaloacetate entering and completing
a full turn of the tricarboxylic acid cycle)
(19).
Fatty AcidsFatty acids were extracted by saponification of
the Trizol cell extract after removal of the RNA-containing supernatant. Cell
debris was treated with 30% KOH and 100% ethanol overnight, and the extraction
was performed using petroleum ether. Fatty acids were converted to their
methylated derivative using 0.5 N methanolic HCl. Palmitate was
monitored at m/z 270 and stearate at m/z 298. The enrichment
of acetyl units in HT29 and MIA cells in response to butyrate treatment was
determined using the mass isotopomer distribution analysis approach of
different isotopomers of palmitate, an abundant cell membrane lipid readily
recovered by biological mass spectrometry from cell pellets
(20). Lipid synthesis is also
dependent on glucose carbons, as they are the primary source of acetyl-CoA,
which is then incorporated into fatty acids through de novo synthesis
(C16, palmitate). Acetyl-CoA enrichment was calculated from the m4/m2 ratio
using the formula m4/m2 = (n
1)/2·(p/q), where n is the number of acetyl
units, p is the labeled fraction, and q is the unlabeled
fraction (p + q = 1).
Gas Chromatography/Mass SpectrometryMass spectral
data were obtained on the HP5973 mass selective detector connected to an
HP6890 gas chromatograph. The settings are as follows: GC inlet 230 °C,
transfer line 280 °C, MS source 230 °C, MS quad 150 °C. An HP-5
capillary column (30-m length, 250-µm diameter, 0.25-µm film thickness)
was used for analysis of glucose, ribose, glutamate, and lactate. A Bpx70
column (25-m length, 220-µm diameter, 0.25-µm film thickness; SGE
Incorporated, Austin, TX) was used for fatty acid analysis with specific
temperature programming for each compound studied.
Data Analysis and Statistical MethodsIn vitro experiments
were carried out using three cultures each time for each treatment regimen and
then repeated twice. Mass spectral analyses were carried out by three
independent automatic injections of 1 µl of each sample by the automatic
sampler and were accepted only if the standard sample deviation was less than
1% of the normalized peak intensity. Statistical analyses were performed using
the parametric unpaired, two-tailed independent sample t test with
99% confidence intervals. p < 0.01 was considered to indicate
significant differences in glucose carbon metabolism in HT29 and MIA cell
cultures treated with increasing doses of butyrate.
 |
RESULTS
|
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Effect of Butyrate on Cell DifferentiationThe
anti-proliferative/cell differentiating effect of sodium butyrate was evident
in HT29 cells by an increase in cell differentiation, as assessed by the
increase in the activity of the differentiation marker enzyme alkaline
phosphatase (Fig. 1) as well as
by a visible change in cell morphology. Glucose consumption decreased in a
dose-dependent manner in HT29 cells after 0.1, 1 and 5 mM of
butyrate, whereas MIA cells did not show any decrease in glucose consumption
or cell differentiation (Fig.
2). Lactate production was also decreased in a dose-dependent
manner in HT29 cells after 0.1, 1, 5, and 10 mM butyrate treatment,
whereas MIA cells continued high lactate production (see
Fig. 2). These results indicate
that glycolysis substrate flow readily responds to butyrate only in
differentiating HT29, but not in MIA cells.

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FIG. 1. Effect of butyrate on cell differentiation after 72 h of incubation.
Alkaline phosphatase activity, measured as a marker of cell differentiation,
is expressed as
nmol·min1·mg1
of protein in the cell extract. *, p < 0.05; **, p <
0.01.
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FIG. 2. Effect of butyrate on glucose consumption and on lactate production.
The results of each isotope experiment were combined to show the changes in
medium glucose or lactate concentration after a 72-h incubation. Triplicates
were performed for each butyrate concentration with a total n = 12.
**, p < 0.01.
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Butyrate Metabolism in HT29 and MIA Cells
-Oxidation
of butyrate produces acetyl-CoA, which can either be oxidized in the
mitochondria via the tricarboxylic acid cycle or reutilized for de
novo lipogenesis. Thus, oxidation of butyrate generates acetyl-CoA and
malonyl-CoA, both being potent metabolic regulators of glucose and fatty acid
oxidation.
-Oxidation of [1,2-13C2]butyrate
generates [1,2-13C2]acetyl-CoA, which can be
incorporated into carbon 4 and 5 of glutamate, which is in equilibrium with
-ketoglutarate. The measurement of enrichment in C-4C-5 of
glutamate reflects the utilization of butyrate in energy production. In
experiments with [1,2-13C2]glucose and unlabeled
butyrate, the unlabeled acetyl-CoA from butyrate dilutes the enrichment of
C-4C-5 of glutamate. These results are shown in
Fig. 3. When
[1,2-13C2]butyrate was used as the tracer, the
enrichment of m2 C-4C-5 of glutamate steadily increased in both cell
lines, reflecting the increasing butyrate
-oxidation. The HT29 cells
were able to metabolize butyrate better than the MIA cells, as demonstrated by
the higher m2 enrichment in glutamate. The high acetyl-CoA enrichment in HT29
cells resulted in the combination of 13C in carbons 3 and 4
generating m2 C-2C-3 in
-ketoglutarate, as the first labeled
molecule continues through the tricarboxylic acid cycle as oxaloacetate. When
[1,2-13C2]glucose was used as the tracer, increasing
butyrate
-oxidation and suppression of pyruvate dehydrogenase activity
contributed to the decreasing enrichment in m2 C-4C-5 of glutamate in
both cell lines. [1,2-13C2]Glucose can also label
carbons 2 and 3 of glutamate via pyruvate carboxylase. The decrease in the
label of the upper part of the glutamate molecule (m2 C-2C-3) indicates
a decrease in the pyruvate carboxylase activity. Because anaplerosis relative
to the tricarboxylic acid cycle (Y) remained relatively unchanged or
was increased, unlabeled substrates must have entered the tricarboxylic acid
cycle to make up for the decrease in pyruvate carboxylase activity.
-Oxidation of butyrate also contributes to the precursor pool of
malonyl-CoA for de novo lipogenesis. Incubation with labeled butyrate
showed a dose response increase in the incorporation of labeled acetyl-CoA in
palmitate synthesis, indicating a switch between the utilization of glucose
and butyrate for fatty acid synthesis. This increase was higher in the
differentiated HT29 cells than in MIA cells
(Fig. 4). For the same reason,
the decreased label incorporation into the fatty acids of MIA cells from
[1,2-13C2]glucose due to dilution by unlabeled
acetyl-CoA from butyrate was less prominent than in HT29 cells.

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FIG. 4. Utilization of glucose and butyrate for lipid synthesis as determined
for palmitate isotopomers. Acetyl-CoA enrichment is calculated from the
mass isotopomer ratio of palmitate using the formula p = (m4/m2)/[3.5
+ (m4/m2)]. "% contribution" is the Acetyl-CoA enrichment
divided by the theoretical enrichment derived from glucose or butyrate. *,
p < 0.05; **, p < 0.01.
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Effect of Butyrate on
[1,2-13C2]Butyrate and
[1,2-13C2]-Glucose
Oxidation13CO2 release in the cell media was
determined by IRMS as the enrichment of CO2 after the addition of
an excess amount of NaHCO3 as described under "Materials and
Methods." The difference in enrichment from that of preincubation medium
is expressed as variation in
(
) representing the
released of 13CO2 from oxidation of labeled substrate.
When [1,2-13C2]butyrate was used as a tracer in HT29
cells, there was a dose-dependent increase in 13CO2
release indicating the intense oxidation of butyrate by these cells
(Fig. 5). However, the intense
oxidation of butyrate was not observed in MIA cells. The regulation of
pyruvate dehydrogenase by acetyl-CoA results in decreased glucose oxidation.
As expected, increasing doses of butyrate reduced glucose oxidation in both
HT29 and MIA cells (Fig. 5). However, the inhibitory effect of butyrate on glucose oxidation in MIA cells
was less prominent than that in HT29 cells. The data in HT29 and MIA cells
together suggest a diminished mitochondrial oxidation of substrate in butyrate
treatment.
Effect of Butyrate on Flux Distribution between Glycolysis and the
Pentose CycleLactate labeling from
[1,2-13C2]glucose and
[1,2-13C2]butyrate was measured using GC/MS in separate
experiments. Lactate 13C labeling was observed only when glucose
tracer was used and was absent when the butyrate tracer was used. These
findings indicate that 13C in butyrate is not incorporated into
pyruvate through gluconeogenesis in HT29 or MIA cells. The results of lactate
isotopomer production from [1,2-13C2]glucose are shown
in Fig. 6. In addition to m2
lactate ([2,3-13C2]lactate), a significant fraction of
singly labeled lactate was detected. Because recycling of label via the
tricarboxylic acid cycle has been ruled out by the lack of labeling from
butyrate, the slight increase in m1 in HT29 cells indicates an increase of the
oxidative pentose phosphate pathway. The relative amount of glucose that is
converted indirectly to lactate through the pentose cycle as a percentage of
the glycolytic flux can be calculated from the m1/m2 ratio. The pentose cycle
in HT29 cells was about twice that of the MIA cells. A dose-dependent increase
in the pentose cycle was observed in HT29 cells treated with butyrate. No such
effect was observed in MIA cells.

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FIG. 6. Lactate isotopomers from [1,2-13C2]glucose.
This is a graphical representation of the distribution of the different
isotopomers of lactate obtained from the glucose label. The pentose cycle
activity is defined as a percentage of the glycolytic flux calculated from
m1/m2. There are significant differences between the pentose cycle flux in MIA
and HT29 cells. *, p < 0.05; **, p < 0.01.
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Butyrate treatment resulted in a decrease in 13C incorporation
into ribose of nucleic acid from glucose in a dose-dependent manner in HT29
cells (see Fig. 7). The average
number of 13C atoms/molecule was reduced by 40% after 5
mM butyrate treatment. The reduction in ribose synthesis was much
more modest (a reduction of about 15%) in MIA cells. Because butyrate
treatment stimulates the oxidative pentose phosphate pathway (lactate
isotopomer data in Fig. 6), the
reduction of ribose synthesis in HT29 cells was caused by reduced substrate
flux through the non-oxidative steps of the pentose cycle. The reduced
transketolase activity is evident in the decreased m2 ribose fraction in the
HT29 cells. In contrast, the effect of butyrate treatment on MIA cells ribose
synthesis was much less than that observed in HT29 cells.

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FIG. 7. Effect of butyrate on RNA ribose synthesis. Ribose isotopomers
obtained from the experiment with glucose label are shown. The m2 isotopomers
of ribose are indicative of the non-oxidative pentose phosphate pathway flux,
whereas the m1 isotopomers indicate the oxidative combined with the
non-oxidative pentose phosphate pathway flux producing ribose.
mn represents the molar enrichment of 13C in ribose
for each condition and each cell line and is indicative of the ribose that is
synthesized. *, p < 0.05; **, p < 0.01.
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 |
DISCUSSION
|
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The results of this study demonstrate that HT29 colon adenocarcinoma cells
exhibit profound metabolic adaptive changes in connection with phenotype
modification and differentiation in response to butyrate. Colon HT29 cells
responded to increasing doses of butyrate by expressing alkaline phosphatase,
a potent cell differentiation marker. This is in good agreement with the
increasing expression of several differentiation markers in HT29 cells
reported in the literature
(21). As determined from the
metabolic profile, increasing concentrations of butyrate inhibited glucose
uptake, glucose oxidation, and nucleic acid ribose synthesis in HT29 cells. In
contradiction to these changes, there was a dose-dependent increase in de
novo fatty acid synthesis utilizing butyrate carbons, whereas the
utilization of glucose carbons for this purpose was diminished. There was also
a significant increase in pentose cycle activity, affecting primarily the
oxidative branch, which indicates an increase of NADPH production that is
necessary for the increased fatty acid synthesis profile reported herein
(Fig. 8). MIA cells, on the
other hand, did not differentiate in response to butyrate, and the metabolic
profile of MIA cells remained essentially unaffected. In contrast to HT29
cells, butyrate did not reduce glucose utilization, lactate production, or
ribose synthesis in MIA cells.

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FIG. 8. Metabolic profile changes associated with butyrate-induced
differentiation. Colon cells normally utilize butyrate as the major fuel
substrate. In malignant transformation, these cells acquire the ability to
utilize glucose as the major substrate. When HT29 cells, a colon cancer cell
line, are exposed to high concentration of butyrate, butyrate induces HT29
cells to differentiate back to their butyrate-utilizing phenotype. These
metabolic profile changes in HT29 cells between these two metabolic phenotypes
are shown. In the figure, the intensity of the arrows represents the magnitude
of substrate flux. In the gray box, we have listed some of the major
genetic changes accompanying butyrate utilization by these cells
(13,
14). Metabolic profiling
provides a roadmap by which regulation of cell cycle as well as gene
expression by metabolic intermediates may be investigated.
|
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The ability of HT29 cells and the inability of MIA cells to respond to
butyrate present an interesting paradigm of nutrient gene interactions. HT29
cells originate from colon epithelial cells, which have the physiological role
of metabolizing butyrate to prevent it from reaching toxic levels in the
circulation. The transformation of colon epithelia into malignant HT29 cells
requires prompt adaptation to a different nutrient environment in the absence
of butyrate. The metabolic profile under such nutrient condition is similar to
that of cultured MIA cells. However, when HT29 cells are exposed to butyrate,
these cells re-adapt to a high butyrate nutrient condition, altering the
expression of many genes of the glucose and lipid metabolic pathways. As a
result, HT29 cells exhibit a different metabolic profile when treated with
butyrate, whereas MIA cells are incapable of adapting to the high butyrate
nutrient environment. This is demonstrated by the fact that labeled butyrate
was poorly utilized by MIA cells for mitochondrial oxidation and
lipogenesis.
The present study also offers an example of how cells sense changes in the
nutrient environment and how they respond to these changes. The metabolism of
butyrate significantly alters glucose metabolic pathways and the rate of
energy production in responsive cells. The inhibition of glucose metabolic
pathways and excess butyrate
-oxidation potentially changes the
intracellular concentrations of many glucose and fatty acid metabolic
intermediates, which serve as signals for transcriptional, translational, and
post-translational events that alter the metabolic phenotype of responsive
cells.
In summary, HT29 cells assume two distinct metabolic phenotypes in response
to changes in the nutrient environment. In the presence of butyrate, HT29
cells decrease the rate of glucose utilization and readily substitute glucose
with butyrate. This change in metabolic phenotype is accompanied by molecular
and morphological changes. The metabolic intermediates of butyrate may play a
role not only as substrates for macromolecule synthesis and energy production
but also as nuclear receptor-signaling ligands. Depending on the genetic
background, changes in substrate availability result in the transformation of
cells that exhibit a glucose-metabolizing phenotype, which is highly
implicated in the proliferative cellular phenotype in cancer
(22). This study demonstrates
that metabolic enzymes and their substrates may serve as high efficiency,
non-genetic novel targets and agents, respectively, for future cancer
therapies (23).
 |
FOOTNOTES
|
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* This work was supported by a grant from the Spanish government Science and
Technology Ministry (SAF 2002-02785) and a grant from the Gaspar de
Portolà Program of collaboration between Catalonia and UCLA. The GC/MS
facility is supported by Public Health Services Grants P01-CA42710 (to the
UCLA Clinical Nutrition Research Unit, Stable Isotope Core) and M01-RR00425
(to the General Clinical Research Center). The costs of publication of this
article were defrayed in part by the payment of page charges. This article
must therefore be hereby marked "advertisement" in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 
¶ Supported by Grant DK56090-A1 from the National Institutes of Health. 
||
To whom correspondence should be addressed: Dept. of Biochemistry and
Molecular Biology, C/Martí i Franquès 1, E-08028, Barcelona,
Spain. Tel.: 34-93-4021593; Fax: 34-93-4021219; E-mail:
marta{at}bq.ub.es.
1 The abbreviations used are: GC/MS, gas chromatography/mass spectrometry;
IRMS, isotope ratio mass spectrometer. 
 |
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