(Received for publication, June 26, 1996, and in revised form, October 15, 1996)
From the In differentiated tissues, such as muscle and
brain, increased adenosine monophosphate (AMP) levels stimulate
glycolytic flux rates. In the breast cancer cell line MCF-7, which
characteristically has a constantly high glycolytic flux rate, AMP
induces a strong inhibition of glycolysis. The human breast cancer cell
line MDA-MB-453, on the other hand, is characterized by a more
differentiated metabolic phenotype. MDA-MB-453 cells have a lower
glycolytic flux rate and higher pyruvate consumption than MCF-7 cells.
In addition, they have an active glycerol 3-phosphate shuttle. AMP
inhibits cell proliferation as well as NAD and NADH synthesis in both
MCF-7 and MDA-MB-453 cells. However, in MDA-MB-453 cells glycolysis is
slightly activated by AMP. This disparate response of glycolytic flux
rate to AMP treatment is presumably caused by the fact that the reduced
NAD and NADH levels in AMP-treated MDA-MB-453 cells reduce lactate
dehydrogenase but not cytosolic glycerol-3-phosphate dehydrogenase
reaction. Due to the different enzymatic complement in MCF-7 cells,
proliferation is inhibited under glucose starvation, whereas MDA-MB-453
cells grow under these conditions. The inhibition of cell proliferation
correlates with a reduction in glycolytic carbon flow to synthetic
processes and a decrease in phosphotyrosine content of several proteins
in both cell lines.
Both proliferating cells and tumor cells maintain a high
glycolytic rate even under aerobic conditions, a process referred to as
aerobic glycolysis. Observations on aerobic glycolysis in tumor cells
prompted Warburg (1) to postulate an altered respiratory function
leading to an increased glycolytic capacity and a high rate of lactate
formation from glucose in the presence of oxygen. Data from former
reports suggest that there are many factors contributing to the origin
of aerobic glycolysis (2). The altered control of glycolysis by
expression of certain isoenzymes is one important factor (2-12).
Furthermore, the glycerol 3-phosphate shuttle and the malate-aspartate
shuttle are altered in such a way that transport of cytosolic hydrogen
into the mitochondria is reduced, requiring tumor cells to reoxidize
NADH cytosolically by lactate dehydrogenase (13-15). Additionally,
oxidation of pyruvate is reduced in favor of glutamine oxidation
(16-25). Due to the expression of the mitochondrial, NAD-dependent malate decarboxylase, malate is converted to
pyruvate and lactate (22-24). The conversion of glutamine to lactate
is called, in analogy to glycolysis, glutaminolysis (25). In tumor cells the glycolytic capacity can be so great that all of the cell's
energy requirements are derived from glycolysis (2, 26). Therefore,
high glycolytic activity ensures the survival and the migration of
tumor cells in hypoxic areas (2, 26, 27). The main role of the
glutaminolytic pathway is the generation of energy (2, 25). However, a
high glycolytic rate is not always linked to cell proliferation or
tumor formation. There are several cell lines that are able to grow in
a medium with 5 mM galactose or with low glucose supply
(0.5 mM) without producing lactate via glycolysis (19-21,
28-33). Investigations with labeled glucose and galactose have shown
that the carbons of the two carbohydrates can either be used to
synthesize nucleotides, phospholipids, and complex carbohydrates or can
flow through pyruvate kinase to pyruvate and lactate for energy
production (2, 19, 20, 29-33). Under glucose starvation, energy is not
produced by glycolysis but by pyruvate oxidation or by conversion of
glutamine to lactate (18-25). When those cells are replaced in a
medium with a high glucose concentration (5 mM), all
phosphometabolites above pyruvate kinase accumulate until the level of
fructose 1,6-bisphosphate is high enough to activate pyruvate kinase
(34-36). The mass of lactate is then derived from glucose. As a
consequence, all intermediates of glycolysis between hexokinase and
pyruvate kinase increase. By this mechanism the supply of
phosphometabolites for synthetic processes is ensured, although
pyruvate kinase is activated (2, 36). From these observations and the
fact that growth factors and oncogene-dependent
phosphorylation regulate glycolysis and phosphometabolite pools, one
can assume that some phosphometabolites or synthetic products derived
from the phosphometabolites, e.g. sugar phosphates, AMP,
NAD, NADH and serine for sphinganine synthesis, regulate cell
proliferation (2, 4, 6, 36-49). Indeed, by searching for such
metabolic signals we found that extracellular AMP inhibits DNA
synthesis in MCF-7 cells and stops cell proliferation. Extracellular
AMP is split to adenosine by the ecto-5 In order to investigate the mechanisms by which AMP stimulates
glycolysis in differentiated cells and inhibits glycolysis in tumor
cells, we decided to study another human breast cancer cell line
MDA-MB-453, which has a more differentiated metabolic phenotype
(e.g. low aerobic glycolytic flux rate, high pyruvate consumption). In addition, we found that MDA-MB-453 cells grow well in
a medium with a low glucose concentration and with galactose, whereas
MCF-7 cells are unable to grow under these nutrient conditions.
Cell Culture
MCF-7 cells were obtained from Prof. Dr. K. Goerttler, Institute for Experimental Pathology, German Cancer Research
Center, Heidelberg, Germany. MDA-MB-453 cells were from the German
Collection of Microorganisms and Cell Cultures in Braunschweig,
Germany.
For cell culture, the
basic medium used was glucose-free Dulbecco's minimal essential
medium, supplemented with 100 units of penicillin/ml, 100 µg of
streptomycin/ml, 2 mM glutamine, and 4 mM
pyruvate (all from Biochrom, Berlin, Germany). For MCF-7 cells 20%
(v/v) fetal calf serum (FCS)1 from
Biochrom, Berlin, Germany, were added to the basic medium. For
MDA-MB-453 cells the basic medium was supplemented with 10% (v/v) FCS
and 10 mM HEPES, pH 7.0. Glucose and galactose from Sigma
were added to the media of both cell lines as described under
"Results." MCF-7 cells were cultured at 37 °C in a
5% CO2 environment. MDA-MB-453 cells were cultured in a
CO2-free atmosphere. For the proliferation and flux
experiments 4-cm diameter dishes (MCF-7 cells) and
25-cm2 flasks (MDA-MB-453 cells) were used (both from
Nunc, Wiesbaden, Germany). For the intracellular measurements
MCF-7 cells were cultured on 14-cm diameter dishes, and experiments
were started with 1 million cells/dish. MDA-MB-453 cells were cultured
in 83-cm2 flasks, and experiments were started with 2 million cells/flask. MCF-7 cells were passaged every 4 days and
MDA-MB-453 cells every 5-6 days. AMP was derived from Boehringer
Mannheim, Germany, and was added to the media at a final concentration
of 3 mM.
For the proliferation rate and glycolytic and glutaminolytic flux
measurements, AMP was added to the medium at the beginning of each
passage. For the intracellular measurements AMP was added to the media
on the 2nd day of the culture period. After 2 (MCF-7 cells) or 3 days
(MDA-MB-453 cells), the AMP-treated cells arrested at a cell density of
5 million cells/dish, whereas control cells without AMP treatment
continued to proliferate, reaching a density of 10-15 million
cells/dish (MCF-7 cells) or 15-20 million cells/flask (MDA-MB-453
cells).
Proliferation Rate and Glycolytic and Glutaminolytic Flux
Measurements
Every 24 h cell culture supernatants were collected and
immediately frozen in liquid nitrogen. The cells were removed from the
plates with trypsin/EDTA from Biochrom, Berlin, Germany, and counted in
a hemocytometer. The frozen supernatants were heated for 15 min at
80 °C and were subsequently centrifuged at 8000 × g
for 10 min (49). Glucose, lactate, pyruvate, glutamine, and glutamate
concentrations were determined according to Bergmeyer (60). For
galactose measurements a commercially available test kit from
Boehringer Mannheim, Germany, was employed.
Determination of Intracellular Metabolite Concentrations
For the extraction of intracellular lactate, pyruvate, and NAD,
cells were treated at 80 °C in aqua bidest for 15 min as described previously (49). The concentrations of lactate, pyruvate, and NAD were
measured enzymatically (60). The NADH concentration was calculated via
the equation [NADH] = 1.11·10 Determination of Intracellular Enzyme Activities
For the extraction of the intracellular enzymes, a
homogenization buffer, pH 7.4, containing 20 mM
KH2PO4/K2HPO4, 1 mM mercaptoethanol, 1 mM EDTA/Na2,
2 mM Isoelectric Focusing
Cells were extracted with a homogenization buffer containing 10 mM Tris, 1 mM NaF, and 1 mM
mercaptoethanol, pH 7.4. Isoelectric focusing was carried out with a
linear gradient of glycerine (50% to 0% (v/v)) and ampholines (pI
3.5-10.5) as described previously (61).
Immunological Detection of Phosphotyrosine
After separation on a 10% SDS-polyacrylamide gel, the proteins
were transferred onto a nitrocellulose membrane by electroblotting. For
the detection of phosphotyrosine a peroxidase-conjugated monoclonal anti-phosphotyrosine antibody from ICN (Costa Mesa, CA) was used. Immunostaining without anti-phosphotyrosine antibody resulted in no
detectable reaction (61).
Statistical Analysis
For the glycolytic and glutaminolytic flux measurements as well
as for the specific enzyme activities, statistical analysis was
performed by a one- or two-factor (co)variance analysis with the
"statistical program package BMDP," whereby metabolite conversions or enzyme activities were plotted versus cell densities
(62). Possible effects of cell density were taken into consideration. In all other analyses Student's t test was employed.
MCF-7 and MDA-MB-453 cells were cultured in
media with different glucose and galactose concentrations. The basic
medium was glucose-free Dulbecco's minimal essential medium. After
addition of fetal calf serum a final glucose concentration of 0.5 mM was obtained. Galactose was not detectable. In order to
achieve other glucose and galactose concentrations, corresponding
carbohydrates were added to the medium. To obtain a glucose-free medium
fetal calf serum was dialyzed in a dialysis bag three times (for 8 h each) against 40 volumes of phosphate-buffered saline. To ensure that
no vital necessary factors were lost during dialysis, the glucose-free
medium was supplemented with 0.5 mM glucose, and cell
proliferation was checked. There was no difference between the
proliferation rate of MDA-MB-453 cells cultured in the
glucose-supplemented medium (0.5 mM glucose) and that of
the cells held in medium with 0.5 mM glucose from
undialyzed fetal calf serum (data not shown). For cell stock breeding
MCF-7 cells were cultured in the basic DMEM supplemented with 5 mM glucose. MDA-MB-453 cells were cultured in the basic
DMEM supplemented with 5 mM galactose and 0.5 mM glucose from FCS (compare "Experimental
Procedures"). Henceforth, these cells will be referred to as
"proliferating" MCF-7 or MDA-MB-453 cells. For the
described experiments with other glucose and galactose concentrations,
cells were cultured for one passage in the new medium, and the cells of
the second passage were used for the measurements. The effect of AMP on
metabolites and enzymes was always determined in the culture medium in
which the cells grew best. For MCF-7 cells this was basic DMEM
supplemented with 5 mM glucose and for MDA-MB-453 the basic
DMEM supplemented with 5 mM galactose and 0.5 mM glucose (from FCS). The concentration of AMP depends on
the content of adenosine deaminase in the FCS charge (49). Adenosine
deaminase degrades AMP to inosine monophosphate, which has no
inhibitory effect on the proliferation rate of the two cell lines (49).
For the experiments described in this paper an AMP concentration of 3 mM was chosen.
Fig. 1, A and B, shows the effect
of AMP and different glucose and galactose concentrations on the cell
proliferation rate of MCF-7 and MDA-MB-453 cells. In MCF-7 cells the
highest proliferation rate was reached at a glucose concentration of 5 mM in the medium (Fig. 1A). Reduction of the
glucose concentration to 0.5 mM led to an inhibition of
cell proliferation to less than half the maximal rate. In glucose-free
DMEM supplemented with 5 mM galactose, the cells became
adherent but ceased to proliferate. The addition of AMP to the culture
medium (DMEM with 5 mM glucose) totally inhibited cell
proliferation. This inhibition was reversible. After degradation of AMP
in the medium or after reculture in AMP-free medium, cell proliferation
began again and reached normal values (49).
MDA-MB-453 cells demonstrated a totally different association between
proliferation rate and availability of glucose and galactose in the
medium. MDA-MB-453 cells grew best in a medium containing 5 mM galactose (Fig. 1B). In the absence of
galactose, cell proliferation was inhibited in MDA-MB-453 cells. If
glucose was also removed cell proliferation was totally arrested. The
cells did not become confluent. In the presence of glucose in the
medium, the cell proliferation rate was only about half the rate in
galactose containing medium. An increase of the glucose concentration
from 0.5 to 5 mM had no effect on the proliferation rate.
As in MCF-7 cells, incubation of MDA-MB-453 cells with AMP led to a
total inhibition of cell proliferation. After reculture of the
AMP-treated MDA-MB-453 cells in AMP-free medium, cell proliferation
once again reached normal rates (data not shown).
For flux
measurements, two different forms of calculations were chosen. The
first calculation is in nmol/(h·dish) and describes the direct
correlation between the consumption of a specific carbon source
(glucose, galactose, glutamine, or pyruvate) and lactate or glutamate
production. The second form of calculation in nmol/(h·105
cells) describes the consumption or production of a certain metabolite by each cell.
In MCF-7 cells the measurements of the glycolytic flux in
nmol/(h·dish) showed a close linkage between glucose consumption and
lactate production, independent of the glucose concentration in the
medium (0.5 mM or 5 mM) (Fig.
2A and Table I). The slope of
the regression line with 5 mM glucose was 1.7, with a
correlation coefficient of 0.932. This value approaches the ideal
maximal value of 2 for the ratio of lactate production:glucose
consumption. In glycolysis 1 mol of glucose is converted into 2 mol of
lactate; therefore, a ratio between lactate production and glucose
consumption of nearly 2 indicates that all lactate produced must be
derived from glucose. A slope of 1.7 means that 85% of the glucose
consumed was converted to lactate. Therefore, in MCF-7 cells 37 nmol of glucose consumed were converted to lactate, and 7 nmol were used for
synthetic processes (calculated with data from Table III). The
intercept of the regression line reflects the lactate production without glucose consumption (Fig. 2 and Tables I and II). This lactate
can derive from glutamine and/or pyruvate consumption (Tables I and
II). In MCF-7 cells with 5 mM glucose 91 nmol of lactate/(h·dish) were derived from sources other than glucose (Table
I). If cultured in a medium with 5 mM glucose, there was a
significant correlation between glutamine consumption and lactate production (Table I) but not between pyruvate consumption and lactate
production (data not shown). Therefore, when no glucose was consumed by
MCF-7 cells, the mass of lactate derived from glutamine. Furthermore,
glutamine consumption increased with glucose consumption when the cells
were cultured in 5 mM glucose (slope = 0.08;
intercept = 6.2 nmol/(h·dish); r = 0.777;
n = 33). A reduction of the glucose concentration to
0.5 mM led to an inhibition of cell proliferation (Fig.
1A) and to a reduction of the total glucose conversion
(Table III), but the strong linkage between glucose consumption and
lactate production was not influenced at low glucose concentrations
(Fig. 2A and Table I). The slope of the regression line was
1.6 with a regression coefficient of 0.642. The intercept of the
regression line dropped from 91 nmol/(h·dish) in medium with 5 mM glucose to 25 nmol/(h·dish) in medium with 0.5 mM glucose (Table I). Galactose was not converted in
measurable amounts in MCF-7 cells. The AMP-induced proliferation stop
was correlated with disruption of the strong linkage between glucose
consumption and lactate production in MCF-7 cells (Table I). The slope
of the regression line was
Correlation between glucose consumption or glutamine consumption and
lactate production in MCF-7 cells
Comparison of glycolytic and glutaminolytic flux rates
Correlation between glucose consumption or pyruvate consumption and
lactate production in MDA-MB-453 cells
Institut for Biochemistry and Endocrinology,
-nucleotidase. Adenosine is
transported into the cells via an adenosine translocator and
rephosphorylated by the cytosolic adenosine kinase to AMP (49-53). The
increase in intracellular AMP inhibits P-ribosePP synthetase and
reduces NAD and NADH synthesis (49-54). NADH levels drop so low that
lactate dehydrogenase is no longer able to transfer the hydrogen from
NADH to pyruvate. As a consequence, glycolysis is inhibited at the
level of the NADH producing glyceraldehyde-3-phosphate dehydrogenase
reaction (49). The metabolic behavior of MCF-7 cells is in complete
contrast to differentiated tissues and cells where the increase of AMP
under hypoxic conditions drastically activates 6-phosphofructo-1-kinase
and the glycolytic flux rate (55-59).
4·[NAD]·[lactate]:[pyruvate] (47). The
protein content in the pellet was determined using the commercially
available Bio-Rad test-kit (Bio-Rad, Munich, Germany).
-amino-n-caproic acid, and 0.2 mM phenylmethylsulfonyl fluoride was used. For the
measurements of malate dehydrogenase a homogenization buffer containing
10 mM Tris, 1 mM NaF, and 1 mM
mercaptoethanol, pH 7.4, was used. The extractions and the measurements
of enzyme activities were carried out as described in Ref. 49. Protein
concentrations were measured by the Biuret method using a commercially
available test kit from Boehringer Mannheim, Germany. The pyruvate
kinase subunits (dimer and tetramer) were separated by gel filtration
as described in Ref. 36.
Effect of Glucose, Galactose, and AMP on the Proliferation of MCF-7
and MDA-MB-453 Cells
Fig. 1.
Effect of different culture conditions on
cell proliferation of MCF-7 cells (A) and MDA-MB-453 cells
(B). For MCF-7 cells the cultures were started with
1·105 cells per dish (4 cm diameter), and cells were
counted after 4 days. For MDA-MB-453 cells the cultures were started
with 2.5·105 cells per 25-cm2 flask, and
cells were counted after 6 days. Glucose (gluc) and galactose (gal) concentrations were varied as described
below the bars. AMP was added to the medium, in which the
cells grew best: MCF-7 cells = 5 mM glucose;
MDA-MB-453 cells = 5 mM galactose with 0.5 mM glucose. Cell densities are given as ± S.D., n = 5.
[View Larger Version of this Image (13K GIF file)]
0.4 with a correlation coefficient of
0.136. The intercept of the regression line with 121 nmol/(h·dish) indicates that more lactate was derived from carbon sources other than
glucose (Table I).
Fig. 2.
Relationship between glucose consumption and
lactate production in MCF-7 (A) and MDA-MB-453 cells
(B) dependent on the glucose concentration in the
medium. Compare Tables I and II.
[View Larger Version of this Image (17K GIF file)]
MCF-7 cells
Culture
conditions
Slope
Intercept
n
Regression
coefficient
nmol/(h·dish)
Lactate production versus
glucose consumption
5 mM
glucose
1.7
91
33
0.932
0.5 mM
glucose
1.6
25
24
0.642
AMP
0.4
121
29
0.136
Lactate production
versus glutamine consumption
5 mM
glucose
10.7
150
34
0.607
0.5 mM
glucose
0.1
56
16
0.050
AMP
1.5
91
33
0.209
g · DF±1 is
the delogarithmic form of the
± SD. p
values are given under "Results." In brackets: number of dates.
Metabolites
Proliferating, 5 mM
glucose
Inhibited, 0.5 mM glucose
Arrested, AMP
± S.E.,
nmol/(h·105 cells)
MCF-7 cells
Glucose
consumption
43.8 ± 0.5 (33)
13.0 ± 3.3 (17)
10.9 ± 0.5 (36)
Lactate
production
109.9 ± 0.9 (37)
20.4 ± 6.4 (17)
95.6 ± 1.5 (37)
g
· DF±1, nmol/(h·105 cells)
Pyruvate
consumption
2.9 · 2.8 (40)
2.2 · 3.7 (17)
4.3 · 3.1 (38)
Glutamine
consumption
4.5 · 3.0 (39)
0.1 · 3.6 (17)
6.0 · 5.0 (38)
Glutamate
production
0.7 · 2.6 (27)
1.4 · 2.3 (17)
1.2 · 2.7 (38)
Inhibited
Proliferating
Arrested,
AMP
± S.E.,
nmol/(h·105 cells)
MDA-MB-453 cells
Glucose
consumption
10.6 ± 0.9 (42)
4.7 ± 0.1 (27)
5.3 ± 0.2 (23)
Lactate
production
19.5 ± 1.8 (42)
27.4 ± 0.6 (26)
32.3 ± 0.5 (27)
g
· DF±1, nmol/(h·105 cells)
Pyruvate
consumption
33.6 · 1.7 (42)
35.8 · 3.1 (24)
5.1 · 2.9 (26)
Glutamine
consumption
0.5 · 4.4 (41)
4.3 · 3.8 (27)
8.8 · 4.3 (27)
Glutamate
production
0.9 · 2.5 (41)
0.2 · 3.0 (39)
0.6 · 3.8 (27)
MDA-MB-453 cells
Culture
conditions
Slope
Intercept
n
Regression
coefficient
nmol/(h·dish)
Lactate production versus
glucose consumption
5 mM
glucose
0.7
60
41
0.573
0.5 mM
glucose
0.7
108
25
0.153
AMP
1.3
60
24
0.362
Lactate production
versus pyruvate consumption
5 mM
glucose
0.4
51
40
0.594
0.5 mM
glucose
0.2
83
30
0.189
AMP
0.0
67
33
0.001
In contrast to MCF-7 cells, in MDA-MB-453 cells glucose consumption and lactate production were not closely linked (Fig. 2B and Table II). The slope of the regression line was 0.7 for both glucose concentrations tested (0.5 and 5 mM). Therefore, 35% of the glucose consumed was converted to lactate. This reveals that in a medium with 5 mM glucose, about 4 nmol of glucose consumed were converted to lactate, whereas 7 nmol of glucose were used for synthetic processes (calculated with data from Table III). In contrast to MCF-7 cells there existed a correlation between pyruvate consumption and lactate production in MDA-MB-453 cells when cultured in a medium with 5 mM glucose (Table II). When all pyruvate is converted to lactate the slope of the regression line reaches the ideal maximal value of 1. For MDA-MB-453 cells, the estimated value of the regression line was 0.4; therefore, 40% of the pyruvate consumed was converted to lactate. No correlation was observed between glutamine consumption and lactate production or between glucose consumption and glutamine consumption in MDA-MB-453 cells (data not shown). MDA-MB-453 cells need galactose for an optimal proliferation rate (Fig. 1B). Nevertheless, glucose was converted first when 5 mM galactose and 0.5 mM glucose were available in the medium. Galactose consumption was only measurable when no glucose was present in the medium. Under these conditions there existed a correlation between galactose consumption and lactate production with a regression line slope of 0.25 (r = 0.679, n = 17). Therefore 13% of the galactose consumed was converted to lactate, and 87% was used for synthetic processes. In AMP-arrested MDA-MB-453 cells, the slope of the regression line (lactate production plotted versus glucose consumption) is enhanced from 0.7 (= 35%) to 1.3 (= 65%) with a correlation coefficient of 0.362 (Table II). The increased slope and the reduced intercept (108 nmol/(h·dish) in proliferating cells and 60 nmol/(h·dish) in AMP-arrested cells) might indicate that AMP-arrested MDA-MB-453 cells are more glycolytic than the untreated proliferating cells (Table II).
The calculation in nmol/(h·105cells) revealed a highly
significant correlation between pyruvate consumption and cell density for both cell lines under optimal proliferation conditions (5 mM glucose for MCF-7 cells and 0.5 mM glucose
with 5 mM galactose for MDA-MB-453 cells). In MCF-7 cells
the curve follows the equation y = 9.88·e(0.26x) (49). In
MDA-MB-453 cells the curve follows the equation y = 24.81·e(
0.42x). In both
cell lines pyruvate consumption declined exponentially with cell
density. At high cell densities pyruvate consumption approached zero
values. The dependence of cell density on glutamine consumption and
glutamate production was not significant in either cell line. Glucose
consumption, galactose consumption, and lactate production all declined
with cell density in MDA-MB-453 cells (slope for glucose
consumption =
0.6, slope for galactose consumption =
0.17, and slope for lactate production =
0.14). In MCF-7 cells no correlation to cell density was found for these metabolite conversions.
The comparison of proliferating MCF-7 cells (5 mM glucose) and proliferating MDA-MB-453 cells (5 mM galactose and 0.5 mM glucose) revealed that MCF-7 cells are much more glycolytic than MDA-MB-453 cells (Table III). Glucose consumption was 9-fold (p < 0.001), and lactate production was 4-fold higher (p < 0.001) in proliferating MCF-7 cells than in proliferating MDA-MB-453 cells. In contrast, MDA-MB-453 cells consumed 12-fold more pyruvate than MCF-7 cells (p < 0.001). Glutamine consumption was the same in the two cell lines. Glutamate production was slightly higher in MCF-7 cells (p < 0.001).
An enhancement of the glucose concentration in the medium of MDA-MB-453 cells from 0.5 to 5 mM led to a 2-fold enhancement of glucose consumption (p < 0.001), whereas lactate production was reduced under these conditions (p < 0.026). Glutamine consumption was reduced (p < 0.001) and glutamate production was enhanced (p < 0.001); pyruvate consumption was not influenced (Table III).
The inhibition of cell proliferation in MCF-7 cells by glucose starvation (medium with 0.5 mM glucose) was characterized by a drastic reduction of glucose consumption (p < 0.001), glutamine consumption (p < 0.001), and lactate production (p < 0.001). Glutamate production was doubled under glucose starvation (p < 0.004), whereas pyruvate consumption was not significantly influenced (Table III).
In MCF-7 cells the total inhibition of cell proliferation by AMP was correlated with a drastic reduction of glucose consumption (p < 0.001, Table III), whereas lactate production was not significantly affected. Pyruvate and glutamine consumption as well as glutamate production were enhanced in AMP-arrested MCF-7 cells (p < 0.05). In contrast to MCF-7 cells there was a strong reduction of pyruvate consumption in AMP-arrested MDA-MB-453 cells compared with proliferating cells (p < 0.001, Table III), whereas glucose consumption and lactate production were not affected by AMP treatment in MDA-MB-453 cells. Glutamine consumption (p < 0.05) and glutamate production (p < 0.001) were enhanced in AMP-arrested MDA-MB-453 cells.
Effect of Glucose, Galactose, and AMP on Intracellular Metabolite Levels in MCF-7 and MDA-MB-453 CellsMeasurements of the
intracellular metabolite concentrations revealed a strong correlation
between lactate, NAD concentrations, and cell density. For the lactate
concentration in MCF-7 cells, the slope of the regression line was
30.3 with an intercept of 53.7 nmol/mg protein (n = 17, r =
0.876). For the NAD concentration a slope of
0.7 was calculated with an intercept of 3.2 nmol/mg protein
(n = 41, r =
0.754). This dependence
on cell density has been taken into account for the following
comparison of the metabolite concentrations between the different cell
groups. For intracellular pyruvate concentration no such correlation
could be demonstrated. All measurements were done at the same cell
density of about 5 million cells/dish.
Proliferating MDA-MB-453 cells cultured in medium with 5 mM
galactose and 0.5 mM glucose had a higher lactate and
pyruvate content than proliferating MCF-7 cells cultured in medium with 5 mM glucose (Fig. 3A). The
absolute NAD levels did not significantly differ between the two cell
lines (Fig. 3B). However, there was a great difference in
the ratio of free NADH:NAD between the two cell lines. In proliferating
MCF-7 cells the NADH:NAD ratio was 1:160, whereas in proliferating
MDA-MB-453 cells the ratio was 1:920. MCF-7 cells had a 5-fold higher
NADH content than MDA-MB-453 cells (Fig. 3B). In MCF-7 cells
inhibition of cell proliferation by glucose starvation (0.5 mM glucose) or total inhibition of cell proliferation by
AMP led to a drastic reduction in the intracellular lactate and NADH
concentrations, whereas the pyruvate concentration increased (Fig.
3, A and B). Furthermore, in AMP-arrested MCF-7 cells the NAD content was reduced (Fig. 3B), and the
NADH:NAD ratio was 1:1000. Glucose starvation had no significant effect on the NAD level and the ratio between NADH and NAD was 1:2400. In
MDA-MB-453 cells lactate and pyruvate levels were not significantly different between AMP-arrested and uninhibited proliferating cells (Fig. 3A). NAD as well as NADH levels decreased under AMP
treatment (Fig. 3B). The NADH:NAD ratio was 1:1400 in
AMP-arrested MDA-MB-453 cells compared with 1:920 in proliferating
cells.
Comparison of Glycolytic Enzymes between MCF-7 and MDA-MB-453 Cells
A correlation between specific glycolytic enzyme activities
and cell density was found in both cell lines. In Fig. 4
this relationship is shown for glyceraldehyde-3-phosphate dehydrogenase in both cell lines. Glyceraldehyde-3-phosphate dehydrogenase activity increased with cell density. Significant correlations between the
specific enzyme activity and cell density were also found for the
mitochondrial hexokinase (slope = 2.2 in both cell lines), the
cytosolic hexokinase (slope = 3.1 in both cell lines),
6-phosphofructo-1-kinase (slope in MCF-7 cells = 90.0; in
MDA-MB-453 cells = 52.8), glucose-6-phosphate dehydrogenase (slope
in MCF-7 cells = 0.3; in MDA-MB-453 cells =
0.4),
6-phosphogluconate dehydrogenase (slope = 21.4 in both cell
lines), enolase (slope = 105.6 in both cell lines), and pyruvate kinase (slope in MCF-7 cells =
0.1; in MDA-MB-453 cells = 0.35).
The differences in the glycolytic flux rates between proliferating MCF-7 and proliferating MDA-MB-453 cells correlate with a striking difference in the specific glycolytic enzyme activities (Table IV). MCF-7 cells, which had the higher glycolytic capacity, had much higher specific 6-phosphofructo-1-kinase, glyceraldehyde-3-phosphate dehydrogenase, and pyruvate kinase activities than MDA-MB-453 cells. On the other hand, MDA-MB-453 cells contain cytosolic glycerol-3-phosphate dehydrogenase, which was not detectable in MCF-7 cells. Furthermore, MDA-MB-453 cells had a higher specific cytosolic hexokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and enolase activity than MCF-7 cells (Table IV).
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In tumor cells, the pyruvate kinase type M2 isoenzyme exists in two different forms, an active tetrameric form and an inactive dimeric form (36, 49). In MCF-7 cells the ratio between the inactive dimeric and the active tetrameric form of pyruvate kinase was 6:1. AMP had no effect on this relationship in MCF-7 cells. MDA-MB-453 cells had the same ratio between the two forms of pyruvate kinase when cultured in a medium with 5 mM glucose. Culture of MDA-MB-453 cells in a medium with 5 mM galactose and 0.5 mM glucose led to a shift to the inactive dimeric form. Under these conditions the ratio between the dimeric and tetrameric form of pyruvate kinase was 10:1 in MDA-MB-453 cells.
Comparison of the Glutaminolytic Enzyme Activities between MCF-7 and MDA-MB-453 CellsThe measurement of the glutaminolytic enzyme
activities revealed a strong dependence of glutamate dehydrogenase
activity as well as malate dehydrogenase activity (measured in NADH NAD direction) on cell density in both cell lines. The slope of
glutamate dehydrogenase is 66 in MCF-7 cells and 28 in MDA-MB-453
cells. The slope of malate dehydrogenase is 0.5 in MCF-7 cells and
0.1 in MDA-MB-453 cells.
Concerning the glutaminolytic enzymes, the greatest difference between
the two cell lines was found in the case of malate dehydrogenase. MCF-7
cells showed a 7-fold higher malate dehydrogenase activity when
measured in NADH NAD direction (MDH Ox.) than MDA-MB-453 cells
(Table V). Measured in NAD
NADH direction (MDH Ma.)
MDA-MB-453 cells had a slightly higher activity.
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The ratio between malate dehydrogenase activity measured in the NADH
NAD direction and the NAD
NADH direction was 83:1 in MCF-7
cells, whereas MDA-MB-453 cells had a ratio of 7:1. This difference is
a result of the different malate dehydrogenase isoenzyme features and
kinetic properties of the isoenzymes in the two cell lines. Both cell
lines basically have two malate dehydrogenase isoenzymes that can be
distinguished by their different isoelectric points. The cytosolic
isoenzyme has an isoelectric point of 5.0, the mitochondrial isoenzyme
of 9.5 (Fig. 5, A and B). In MCF-7 cells a third form of malate dehydrogenase can be detected. This form
has an isoelectric point of 7.8 and represents a precursor of the
mitochondrial isoenzyme located in the cytosol (Fig. 5A). This form of malate dehydrogenase could not be found in MDA-MB-453 cells. In contrast to MCF-7 cells, MDA-MB-453 cells showed a broad mitochondrial peak, which ranged from pI 8.5 up to pI 10.5 (Fig. 5, A and B). Furthermore in MCF-7 cells the pI
7.8 form of malate dehydrogenase was associated with p36, whereas the
pI 5.0 and the pI 9.5 forms were not (61). In MDA-MB-453 cells p36 was found in considerably higher amounts than in MCF-7 cells, but these
were spread over all malate dehydrogenase fractions (data not
shown).
The cytosolic and mitochondrial malate dehydrogenase isoenzymes had
different capacities when measured in the NADH NAD direction or in
the NAD
NADH direction in MCF-7 and MDA-MB-453 cells (Table VI). In MCF-7 cells the cytosolic isoenzyme (pI 5.0)
preferred the NAD
NADH conversion, whereas the mitochondrial forms
(pI 7.8 and pI 9.5) had the greatest capacity when measured in the NADH
NAD direction (Table VI). Measured in the NADH
NAD direction only 26% of the total malate dehydrogenase activity focused at pI 5.0, and 72% of the malate dehydrogenase activity was found in the
fractions corresponding to the mitochondrial forms. Measured in the NAD
NADH direction this ratio was just the other way around. In
MDA-MB-453 cells the mitochondrial isoenzyme showed almost no capacity
for the NAD
NADH direction (8.5%). Ninety percent of the activity
was found in the cytosolic fractions. Measured in the NADH
NAD
direction, the ratio between the cytosolic and the mitochondrial
isoenzymes was nearly 50:50% in MDA-MB-453 cells.
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Glutamate dehydrogenase and cytosolic glutamate oxaloacetate transaminase activities were slightly higher in MDA-MB-453 cells than in MCF-7, whereas MCF-7 cells had higher mitochondrial glutamate oxaloacetate transaminase and cytosolic malic enzyme activities (Table V).
Effect of AMP on Glycolytic and Glutaminolytic Enzymes in MCF-7 and MDA-MB-453 CellsAddition of AMP led to an enhancement of most of
the specific glycolytic enzyme activities in both cell lines (Table
IV). Glucose-6-phosphate dehydrogenase, 6-phosphogluconate
dehydrogenase, and pyruvate kinase activities were enhanced after AMP
treatment in both cell lines. Differences between the two cell lines
were seen in the case of 6-phosphofructo-1-kinase,
glyceraldehyde-3-phosphate dehydrogenase, glycerol-3-phosphate
dehydrogenase, enolase, and lactate dehydrogenase (interaction
p 0.001). In AMP-arrested MCF-7 cells
6-phosphofructo-1-kinase and glyceraldehyde-3-phosphate dehydrogenase
activity were enhanced when compared with untreated proliferating
cells. In contrast, in MDA-MB-453 cells AMP treatment had no effect on
these enzyme activities. Glycerol-3-phosphate dehydrogenase activity
increased in AMP-arrested MDA-MB-453 cells, whereas MCF-7 cells
contained no cytosolic glycerol-3-phosphate dehydrogenase. Enolase and
lactate dehydrogenase activities were enhanced in AMP-treated
MDA-MB-453 cells but were not involved in AMP-treated MCF-7 cells. The
only glycolytic enzyme that was reduced under AMP treatment in both
cell lines was mitochondrial hexokinase. Cytosolic hexokinase was not
affected by AMP treatment in either of the cell lines.
In glutaminolysis, inhibition of cell proliferation by AMP was coupled with an increase of glutamate dehydrogenase and mitochondrial glutamate oxaloacetate transaminase activities in both cell lines (Table V). The mitochondrial and cytosolic isoenzymes of malic enzyme were reduced in AMP-treated MDA-MB-453 cells, whereas in MCF-7 cells AMP treatment had no effect on the two isoenzymes. The cytosolic glutamate oxaloacetate transaminase activity was not affected by AMP treatment in either cell line.
Comparison of the Phosphotyrosine Content in MCF-7 and MDA-MB-453 CellsStaining with anti-phosphotyrosine antibodies revealed that
in both cell lines many proteins were phosphorylated in tyrosine under
optimal proliferation conditions (5 mM glucose for MCF-7 cells and 5 mM galactose and 0.5 mM glucose for
MDA-MB-453 cells). Total phosphotyrosine content was inversely
correlated to the proliferation rate in both cell lines (Fig.
6). Inhibition of cell proliferation by glucose
starvation (0.5 mM glucose) in MCF-7 cells and galactose
starvation (0 mM galactose) in MDA-MB-453 cells led to a
weak reduction of the total phosphotyrosine content. In AMP-arrested
MCF-7 cells as well as in AMP-arrested MDA-MB-453 cells,
phosphotyrosine staining was drastically reduced.
MCF-7 cells have a much higher glycolytic flux rate than MDA-MB-453 cells (Tables I, II, III, Fig. 2, A and B). The different glycolytic flux rates in MCF-7 and MDA-MB-453 cells can be correlated with different enzyme expressions and regulation mechanisms in the two cell lines. MCF-7 cells have much higher 6-phosphofructo-1-kinase, glyceraldehyde-3-phosphate dehydrogenase, and pyruvate kinase activities than MDA-MB-453 cells (Table IV). It is generally assumed that the mitochondrial bound hexokinase has a strong influence on the glycolytic flux rate (3). However, the mitochondrial hexokinase activity did not differ between MCF-7 and MDA-MB-453 cells, whereas the cytosolic hexokinase was higher in MDA-MB-453 cells, which had the lower glycolytic flux rate (Table III and IV). In both cell lines 6-phosphofructo-1-kinase, glyceraldehyde-3-phosphate dehydrogenase, and enolase activities strongly increased with cell density, whereas the glycolytic flux rate decreased at high cell densities in MDA-MB-453 cells or remained unchanged in MCF-7 cells. This type of increase in enzyme activity related to cell density has been reported for several other cell lines and seems to be a general phenomenon (63-65). Only the cytosolic hexokinase activity decrease correlates with the reduced glycolytic flow rates at high cell densities. These data confirm several of those in other studies reporting that the glycolytic flux rate depends upon the availability of nutrients and is not regulated by a single enzyme but by the interaction of all enzymes involved (66-69).
The complex of the glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, pyruvate kinase, and enolase is not involved in the regulation of the glycolytic flux rates because under extreme variations of the glycolytic flux rates no alterations in the complex formation could be demonstrated. This complex exists in MCF-7 cells with a high glycolytic flux rate (61) as well as in MDA-MB-453 cells with a low glycolytic flux rate (data not shown). Furthermore, the complex was not affected by AMP treatment of MCF-7 cells, which correlates with a drastic inhibition of glycolysis (61). The complex contains a specific AU-rich RNA. Therefore, it seems that the main function of this complex is the regulation of RNA stability (61, 70).
Glutaminolysis and Malate-Aspartate ShuttleActive
proliferating MCF-7 and MDA-MB-453 cells consumed glutamine in equal
amounts (Table III). Accordingly, there is no great difference in the
glutaminolytic enzyme equipment between the two cell lines. An
exception is malate dehydrogenase, measured in the NADH NAD
direction (Table V). Cytosolic and mitochondrial malate dehydrogenase
isoenzymes are part of the malate-aspartate shuttle (Fig.
7A). MCF-7 cells had a 7-fold higher total
malate dehydrogenase activity than MDA-MB-453 cells when measured in the NADH
NAD direction (=oxaloacetate
malate direction) due to
an increase in the mitochondrial malate dehydrogenase activity (Tables
V and VI, Fig. 5, A and B) (61). In MCF-7 cells
the amount of the mitochondrial malate dehydrogenase is so high that the precursor of the mitochondrial isoenzyme is retained in the cytosol
presumably by the interaction with a protein referred to as p36
(annexin II). In MCF-7 cells the p36-associated form of malate
dehydrogenase, which has an isoelectric point of 7.8, might be
responsible for the increase of cytosolic malate and for the flow of
glycolytic hydrogen derived from the glyceraldehyde-3-phosphate dehydrogenase reaction from the cytosol into the mitochondria (Fig.
7A) (61). The availability of cytosolic malate determines whether glutamine is converted to lactate or excreted as glutamate (2,
21, 23, 61, 71). In MCF-7 cells, which contain the pI 7.8 form of
malate dehydrogenase, the glycolytic flux rate increases the flow of
glutamine to lactate via glutaminolysis (Table I) (2, 61). Therefore,
under glucose starvation (0.5 mM glucose) glutamine
consumption was reduced, whereas glutamate production increased (Table
III). MDA-MB-453 cells had a low mitochondrial malate dehydrogenase
activity and no p36-associated precursor of the mitochondrial malate
dehydrogenase in the cytosol. In complete contrast to MCF-7 cells, in
MDA-MB-453 cells the flow of glutamine to lactate was reduced, and
glutamate production was enhanced at high glucose concentrations (Table
III). Such a difference in the interaction between cell proliferation
and glutamine and glucose consumption has also been reported for other
cell lines, and it has been demonstrated that this interaction can be
overcome by inhibitors of the malate-aspartate shuttle (19-23, 28-30,
71-74). Therefore, under glucose starvation, energy production from
glutaminolysis is drastically reduced in MCF-7 cells but increases in
MDA-MB-453 cells (Table III). In MDA-MB-453 cells but not in MCF-7
cells the increased glutamine consumption can compensate for the
reduced glycolytic energy production and represents a commitment for
cell proliferation under glucose starvation.
Influence of Metabolism on Cell Proliferation, Carbohydrate Consumption for Glycolysis and Synthetic Processes
The different metabolic behavior of the two cell lines correlates with different nutrient requirements. MCF-7 cells are unable to grow in a galactose medium, whereas MDA-MB-453 cells need galactose for optimal growth (Fig. 1, A and B). The inability to grow in media, which contain galactose, fructose, or other carbohydrates instead of glucose, is commonly linked with the inability to grow under glucose starvation (75-78). Those cells are characterized by high NADH levels (75, 77-80). The high NADH levels are caused by a disruption of the glycerol 3-phosphate shuttle and a reduction of glutaminolysis as found in MCF-7 cells (77).
High NADH levels inhibit the enzyme UDP-galactose epimerase, which regulates the flow of glucose 6-phosphate to galactose or the inverse reaction (75). MDA-MB-453 cells have an active glycerol 3-phosphate shuttle and have much lower NADH levels than MCF-7 cells (Table IV and Fig. 3B). In MDA-MB-453 cells the UDP-galactose epimerase favors the flow of galactose to glucose 6-phosphate and into glycolysis (75). Galactose is preferentially used for synthetic processes and is not converted to pyruvate and lactate for energy production (2, 19, 20, 31, 81). Another factor that regulates the ability to grow under glucose starvation is pyruvate kinase. For this purpose, proliferating cells express a particular pyruvate kinase isoenzyme, termed type M2. Under glucose starvation this enzyme stays primarily in the inactive dimeric form to guarantee that sufficient glycolytic carbons are channeled into synthetic processes (34-36). In the case of an over-supply of glycolytic carbons, high fructose 1,6-bisphosphate levels induce the association of the enzyme to the active tetrameric form and lead to an increased flow of the glycolytic carbons to pyruvate (34-36). In accordance, MDA-MB-453 cells, which are able to grow under glucose starvation, had a much lower pyruvate kinase activity and a lower amount of the active tetrameric form than MCF-7 cells. Indeed, in MDA-MB-453 cells only 35% of glucose was converted to lactate, whereas in MCF-7 cells 85% of glucose consumed was channeled to lactate independent of the glucose concentration in the medium (Tables I and II, Fig. 2, A and B). HT-29 cells, which like MCF-7 cells need high glucose concentrations to grow, are also characterized by a high pyruvate kinase activity and an 85% conversion of glucose to lactate. Accordingly, specific selected HT-29 cells, which like MDA-MB-453 cells are able to grow under glucose starvation, have a much lower pyruvate kinase activity than the wild type, and only 65% of the glucose consumed is converted to lactate (76, 82).
Effect of AMP on the Metabolism of MCF-7 and MDA-MB-453 CellsThe addition of AMP into the culture medium totally inhibits proliferation of both cell lines (Fig. 1, A and B). However, the effect of AMP on the metabolism of the two cell lines is completely different. In MDA-MB-453 cells AMP treatment had no effect on glucose consumption or lactate production but led to an increase in the amount of glucose converted to pyruvate and lactate from 35 to 65%, whereas the amount of glucose used for synthetic processes decreased (Tables II and III). The availability of glycolytic pyruvate led to a reduction in the consumption of extracellular pyruvate in AMP-arrested MDA-MB-453 cells. In contrast, in MCF-7 cells AMP treatment led to a drastic reduction of glucose consumption and glucose to pyruvate conversion (Tables I and III). On the other hand, lactate production was not influenced in AMP-arrested MCF-7 cells. Therefore, AMP-arrested MCF-7 cells need more extracellular pyruvate and glutamine for energy production than proliferating MCF-7 cells. The mass of the lactate produced in AMP-arrested MCF-7 cells is derived from the increased degradation of the amino acid glutamine (Table III).
Measurements of glycolytic enzyme activities in AMP-arrested MCF-7 cells revealed that, in contrast to the inhibition of the glycolytic flux rate, most glycolytic enzymes were up-regulated under AMP treatment (Table IV). An increase of some glycolytic enzyme activities was also found in AMP-arrested MDA-MB-453 cells, in which the glycolytic flux rate was not affected by AMP (Table IV). Therefore, the alteration in the glycolytic flux rate by AMP is not induced by an increase of the glycolytic enzyme activities. Furthermore, the glycolytic enzyme complex and the ratio between the dimeric and tetrameric forms of pyruvate kinase were not affected by AMP. Therefore, the main difference in the response of these two cell lines to AMP must be caused by the different shuttle systems (Fig. 7, A and B). MDA-MB-453 cells contain the cytosolic glycerol 3-phosphate shuttle, whereas MCF-7 cells do not. In proliferating MCF-7 cells the mass of hydrogen produced in the cytosolic glyceraldehyde-3-phosphate dehydrogenase reaction must be excreted as lactate. The reduction of NAD and free NADH levels under AMP treatment reduce lactate dehydrogenase activity. Thus the generation of NAD in the cytosol is limited, and the glyceraldehyde-3-phosphate dehydrogenase reaction is inhibited. As a consequence, total glycolysis is inhibited in AMP-arrested MCF-7 cells (Fig. 7A) (49). In contrast to MCF-7 cells, MDA-MB-453 cells contain cytosolic glycerol-3-phosphate dehydrogenase, which was further activated under AMP treatment (Table IV). In MDA-MB-453 cells the hydrogen produced in the glyceraldehyde-3-phosphate dehydrogenase reaction is transported into the mitochondria by the glycerol 3-phosphate shuttle and is not excreted as lactate (Fig. 7B). The slope of the regression line between lactate production and pyruvate consumption dropped to zero under AMP treatment (Table II). Therefore, the drop in NAD levels reduces the flow of extracellular pyruvate to lactate by the lactate dehydrogenase reaction but does not affect the glyceraldehyde-3-phosphate dehydrogenase reaction and glucose consumption in MDA-MB-453 cells.
Metabolism and PhosphotyrosineIn MCF-7 and MDA-MB-453 cells, inhibition of cell proliferation either by AMP treatment or by glucose starvation (MCF-7 cells) or galactose starvation (MDA-MB-453 cells) correlates with a decrease in the phosphotyrosine content in several cytosolic proteins (Fig. 6). One of these proteins has been characterized as glyceraldehyde-3-phosphate dehydrogenase (61). The decrease in phosphotyrosine is either induced by an inhibition of tyrosine kinases or an activation of tyrosine phosphatases. The complex interaction between protein kinases and phosphatases makes it difficult to define exactly which protein kinase or phosphatase is altered. However, there are previous reports that tyrosine kinases are modified by phosphometabolites such as fructose 1,6-bisphosphate and P-ribose-PP. Both metabolites inhibit the pp60v-src kinase activity and the epidermal growth factor receptor kinase (83, 84). Since levels of both metabolites decrease under glucose starvation, phosphotyrosine should increase but not decrease under those conditions. Glycolytic phosphometabolites and their synthetic products, e.g. glucose 6-phosphate, phosphoserine, and ribose-5-P, can be used as alternative substrates by tyrosine phosphatases instead of phosphotyrosine (85). A decrease in those metabolites may make the phosphate in tyrosine more accessible to phosphatases. Another candidate for the inhibition of tyrosine phosphorylation is Ap4A, which is a potent inhibitor of pp60v-src kinase (84). However, the reduction of the glycolytic carbon flow to synthetic processes and the decrease in the NADH levels correlate best with the decrease in phosphotyrosine and the inhibition of cell proliferation (2, 47, 86). Therefore, it is likely that a synthetic product of the glycolytic carbons such as NAD and/or NADH or AMP itself either regulates tyrosine kinases or tyrosine phosphatases.
General ConclusionsThe MCF-7 and MDA-MB-453 cell system represents a valuable model for identification of metabolites that regulate the protein kinase cascade and cell proliferation. In addition, the data presented in this paper provide useful suggestions regarding the therapeutic consequence linked to the individual metabolic characteristics of those cell lines.
MCF-7 cells have a high glycolytic capacity that allows survival under hypoxic conditions (26). Under glucose starvation cell proliferation is reduced (Fig. 1A). Cell proliferation is a process that consumes a great deal of energy with a 2-4-fold increase over nonproliferating cells (87-89). Therefore, the inhibition of cell proliferation under glucose starvation saves energy, and all of the ATP produced by glycolysis is used for the survival of the cells. MCF-7 cells have an optimal metabolism to survive in solid tumors with variable glucose supply (2). This advantage of MCF-7 cells is linked to the disadvantage that glycolytic energy production is highly sensitive to reduction in NAD levels (49).
In MDA-MB-453 cells glycolytic energy production is not impaired by lowering of the NAD levels. Several tumor therapeutic drugs and peroxides produced by natural killer cells lower NAD levels and thereby impair energy regeneration and induce apoptosis (2, 90-92). Therefore, it would be interesting to investigate whether MDA-MB-453 cells are generally more resistant to such attacks than MCF-7 cells.