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(Received for publication, September 6, 1995; and in revised form, October 30, 1995) From the
Treatment of the mouse fibrosarcoma cell line L929 with tumor
necrosis factor (TNF) induces necrotic cell death. A crucial step in
the cytotoxic action mechanism of TNF involves perturbation of
mitochondrial functions leading to the formation of reactive oxygen
intermediates (ROI). L929 cells have energy requirements adapted to a
high proliferation rate. Glutamine (Gln) is utilized as a major energy
source and drives mitochondrial ATP formation, while glucose is mainly
converted to lactate through glycolysis. We investigated the role of
the bioenergetic pathways involved in substrate utilization on the
cytotoxic action of TNF and established a link between Gln oxidation
and TNF-induced mitochondrial distress. Omission of Gln from the medium
desensitizes the cells to TNF cytotoxicity, while the lack of glucose
in the medium does not alter the TNF response. Sudden depletion of Gln
from the culture medium results in a sharp decline in mitochondrial
respiration in the cells, which might explain the decreased TNF
responsiveness. However, when L929 cells are adapted to long term
growth under conditions without Gln, these so-called
L929/Gln
Tumor necrosis factor (TNF) ( In the mouse L929 fibrosarcoma cell line, TNF
signaling leads to necrotic cell death(6, 7) . A major
step in the cytotoxic mechanism is the formation of ROI in the
mitochondria. Their crucial role was demonstrated by the interference
of specific inhibitors of the electron transport chain with necrotic
cell response (8) and by the correlation between sensitivity to
TNF cytotoxicity and mitochondrial activity in the cell(9) .
More recently, we followed directly the TNF-induced mitochondrial ROI
and correlated it with cytotoxicity(10) . Hence the change from
a sensitive to a resistant phenotype observed under hypoxic growth
conditions (11, 12) can be explained by the inability
to generate ROI in the mitochondria. Besides oxygen, another
important parameter that affects mitochondrial functionality is the
availability of energy substrates. Normal and malignant cells often
exhibit different metabolic requirements (13, 14, 15, 16, 17) ; the
exuberant proliferation rate of transformed cells demands an adapted
energy metabolism. Therefore, the amino acid Gln, instead of the
Glc-derived pyruvate, is used preferentially as a substrate for ATP
production by oxidative phosphorylation. The underlying adaptations of
the intermediary metabolism include (i) an increased activity of the
mitochondrial matrix enzyme glutaminase, converting Gln to glutamate,
(ii) formation in the mitochondria of the citric acid cycle
intermediate Here we describe
the result of the above changes in mitochondrial enzyme composition and
substrate utilization to TNF cytotoxicity. We show that L929 cells use
Gln and not Glc as the major energy substrate and that this particular
energy metabolism promotes the cytotoxic response of the cells to TNF.
Our results also demonstrate that the dependence of TNF cytotoxicity on
Gln is not due to the overall rate of mitochondrial respiration per
se. Enzymatic pathways specifically utilized in mitochondrial
oxidation of Gln appear to sensitize the mitochondria to TNF-induced
perturbation of their activity and thereby amplify the resulting
production of cytocidal ROI.
As shown in Fig. 1, the presence of both Glc and Gln, i.e. normal
growth conditions, supports a metabolism that combines a high rate of
respiration with a high production of lactate. A high lactate release
indicates that a considerable part of the pyruvate generated from Glc
by glycolysis does not enter the citric acid cycle but is reduced to
lactate. Nevertheless, the cells show a high rate of respiration.
Apparently, the substrate that fuels this high respiration is Gln,
since cells maintained exclusively with Gln showed an equal or even
slightly increased respiration compared with the control condition. As
expected, lactate production in these cells dropped to background
levels due to the absence of substrate suitable for glycolysis. When
Gln was omitted, Glc being the only metabolic substrate available to
the cells, both the rates of lactate release and of oxygen consumption
dropped below 50% of the control value. Apparently, under this culture
condition, part of the pyruvate that otherwise is reduced to lactate
now enters the citric acid cycle to drive respiration. Finally, when
both Gln and Glc were omitted from the medium, all cells died within 48
h (results not shown), indicating that no other energy-providing
substrate was available to the cells under this condition. These
results clearly indicate that in L929 cells Gln is metabolized in the
mitochondria through the citric acid cycle and hereby fuels oxidative
phosphorylation, whereas the metabolism of Glc is limited to
glycolysis, resulting in a high lactate production. These
characteristics are as expected for a tumor cell-type energy metabolism
as described before for other cell lines(14, 15) .
Figure 1:
Analysis of the energetic pathways used
by L929 cells. For lactate production (open bars), 2
Figure 2:
Comparison of TNF-induced cell death in
parental L929 and L929/Gln
Figure 3:
Analysis of TNF-induced ROI response and
cell death. Analysis was performed in parental L929 cells (
Binding of TNF to its p55 receptor in malignant, nonlymphoid
cells triggers a complex signal transduction mechanism leading to
cytostasis, necrosis, or apoptosis, depending on the cell type and the
physiological condition. In this report we analyzed the necrotic death
induced by TNF in the mouse fibrosarcoma cell line L929 and show that
one of the factors that modulates the responsiveness of the cells to
the cytotoxic activity of TNF is the availability of energy substrate
and, more specifically, the species of respiratory substrate used by
the cell at the time of TNF treatment. Neoplastic cells have been
reported to have a changed metabolic behavior characterized by the
preferential use of Gln, instead of Glc, as the major energy source.
Gln is oxidized at a high rate in tumor mitochondria, while Glc is
primarily converted to lactate. We established that L929 cells grown in
culture indeed use Gln preferentially as a substrate for oxidative
metabolism and as a corollary produce high amounts of lactate from Glc.
Furthermore, we demonstrated that modulating the cellular energy
metabolism by altering substrate availability in the culture medium
resulted in significant changes in TNF response. More specifically,
omission of Gln from the medium shortly before TNF treatment
desensitized the cells to TNF cytotoxicity. This treatment led to a
sharp decrease in electron flow through the mitochondrial electron
transport chain. Since several lines of evidence previously established
that TNF cytotoxicity in L929 cells requires functional mitochondria (8, 9) , apparently as a source of cytocidal
ROI(10) , this loss of mitochondrial activity might explain the
reduced TNF responsiveness of Gln-deprived L929 cells. Therefore, we
adapted L929 cells to grow without Gln, thus enforcing the use of Glc
as major respiratory substrate. The resulting stable
L929/Gln Previous reports from this laboratory have
shown that TNF induces excess ROI production in the mitochondria of
L929 cells and that these ROI are directly cytocidal and/or necessary
for downstream events leading to cell death. In the absence of Gln,
this ROI response was markedly attenuated, whereby both the absolute
levels of ROI generated as well as the rate of appearance of cells
exhibiting increased ROI levels were abated. Apparently, the oxidation
of Gln creates a metabolic condition in the mitochondria that
facilitates mitochondrial production of excess ROI upon TNF stimulation
and that enhances the cytotoxic response of the cells to TNF. This
facilitation is not based on the overall rate of electron flow in the
mitochondria, since L929 cells using Glc or Gln as a respiratory
substrate showed similar rates of oxygen consumption. Possibly, the
kind of substrate used affects the formation of multienzyme complexes
of a higher order (14) that are differentially sensitive to
regulatory mechanisms (in)activated by TNF. Alternatively, by-products
of Gln oxidation through the citric acid cycle, such as
citrate(14) , a precursor of fatty acid and cholesterol
biosynthesis, may affect upstream signaling events and amplify the
activation signal delivered to the mitochondria. Further experiments
will be needed to clarify this mechanism. Since both the use of Gln
as an oxidative substrate and sensitivity to the cytotoxic action of
TNF are tumor cell-specific features (the first facilitating the
latter), the validity of a causal link between tumor cell bioenergetics
and tumor cell responsiveness to TNF cytotoxicity has to be considered.
However, as expected for a very pleiotropic cytokine like TNF, there is
no evidence for a strict correlation between the use of Gln as a
respiratory substrate and the sensitivity to TNF cytotoxicity. For
example, the TNF sensitivity of cells that die in an apoptotic mode,
was not diminished after Gln omission (not shown). This may be related
to the early inactivation of mitochondrial activity observed during
programmed cell death(26) , rendering an active contribution of
mitochondria impossible. In contrast, when mitochondria actively
contribute to the cytotoxic process, such as in L929 or WEHI 164 cl 13
cells (not shown), Gln enhanced the TNF-cytotoxic response. Hence we
may conclude that the tumor cell-characteristic use of Gln as an
oxidative substrate contributes to the TNF responsiveness of those
tumor cells in which mitochondria play an active role in the cytotoxic
process. Since Gln is abundantly present in body fluids and since its
concentration may alter during the progression of neoplastic disease,
maintaining high concentrations in circulation may, for certain tumors,
positively affect the therapeutic value of TNF.
Volume 271,
Number 1,
Issue of January 5, 1996 pp. 192-196
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
A MODULATOR OF REACTIVE OXYGEN INTERMEDIATE-MEDIATED CYTOTOXICITY
OF TUMOR NECROSIS FACTOR IN L929 FIBROSARCOMA CELLS (*)
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
cells have restored respiration, but they
still display a decreased sensitivity to TNF cytotoxicity. Thus the TNF
responsiveness of L929 cells depends on bioenergetic reactions that are
specifically involved in the oxidation of Gln. This is further
confirmed by the desensitizing effect of specific inhibitors of these
Gln-linked enzyme reactions on TNF cytotoxicity in the parental cells,
but not in the L929/Gln
cells. Analysis of the
induction of mitochondrial ROI formation by TNF in parental and
L929/Gln
cells suggests that the effect of Gln on the
sensitivity to TNF cytotoxicity involves a mechanism that renders the
mitochondria more susceptible to TNF-induced mediators, resulting in
enhanced ROI production and accelerated cytotoxicity.
)is a pleiotropic
cytokine mainly produced by activated macrophages. Besides its role in
the host defense against microorganisms and bacterial pathogens, TNF is
involved in the pathology of various diseases, such as the systemic
inflammatory response syndrome(1, 2, 3) .
Furthermore, TNF is specifically cytotoxic for many types of
transformed cells, especially in the presence of interferon. Most
TNF-mediated activities, including cytotoxicity, are initiated by
ligand-induced cross-linking of the p55 TNF receptor; only in
T-lymphocytes has a role of the p75 TNF receptor in cell proliferation
and in cytotoxicity been unambiguously demonstrated(4) .
Cross-linking of the receptors initiates signal transduction, possibly
through association of the intracellular death domain of the p55
receptor (5) .
-ketoglutarate via transamination of glutamate, and
(iii) the presence of malic enzyme, generating intramitochondrial
pyruvate(14, 18, 19) .
Cell Culture
L929, a murine fibrosarcoma cell
line, was grown in Dulbecco's modified Eagle's medium
supplemented with 10% heat-inactivated fetal calf serum, 2 mM Gln, 100 units/ml penicillin, and 0.1 mg/ml streptomycin.
Adaptation of the cells to Gln-free culture conditions was performed by
a stepwise decrease in Gln concentration in the medium to zero over a
2-month period. The resulting L929/Gln population was
routinely maintained in Dulbecco's modified Eagle's medium
free of Gln (and glutamate) (Gibco Bio-Cult, Paisley, UK), supplemented
with 10% dialyzed heat-inactivated fetal calf serum and antibiotics.
Dulbecco's modified Eagle's medium without Gln and Glc was
supplemented with Gln (2 mM), Ino (100 µM), dThd
(40 µM) and Urd (100 µM), or Glc for use in
short term culture experiments. Suspension cultures of the normally
adherent-growing L929 and L929/Gln
cells were set up
when required for flow-cytometric experiments. To that end, cells were
harvested from adherent cultures grown in tissue culture flasks by
trypsinization at 37 °C, washed, and resuspended in culture medium.
The cells were seeded in 90- or 30-mm diameter bacterial-grade Petri
dishes at 3-5
10
cells/ml and incubated
overnight at 37 °C in a humidified, 5% CO
incubator.
Under these conditions, the cells no longer adhered to the plastic
surface and remained in suspension. TNF sensitivity of the cells was
not extensively altered in these suspension cultures(6) . All
cells were mycoplasma-free as judged by an enzymatic immunoassay
(Boehringer, Mannheim, Germany).TNF and Reagents
Recombinant murine TNF was
produced in Escherichia coli and purified to at least 99%
homogeneity (20) . The preparation had a specific activity of
1.2 10
IU/mg protein and contained <4 ng of
endotoxin/mg of protein. TNF activity was determined as described
previously(21) , using an international standard TNF
preparation (code 88/532, obtained from the Institute for Biological
Standards and Control, Potters Bar, UK) as a reference. Unless
otherwise mentioned, TNF was added at time zero to cell cultures at a
final concentration of 1000 IU/ml. Cycloheximide was dissolved in
culture medium and added to a final concentration of 50 µg/ml. PI
(Becton-Dickinson, Sunnyvale, CA) was prepared as a 100 stock
solution (3 mM) in NaCl/P
and stored at 4 °C.
DHR123 (Molecular Probes, Eugene, OR) was prepared as a 5000
stock solution (5 mM) in dimethyl sulfoxide and stored at
-20 °C. Amino-oxyacetate (0.2 mM), L-glutamate--hydrazide (5 mM),
6-diazo-5-norleucine (1 mM), and acivicine (1 mM)
were purchased from Sigma and dissolved in medium. Butylated
hydroxyanisole (Sigma) was prepared as a 1000
stock solution
(100 mM) in ethanol.Lactate Production
The medium of exponentially
growing adherent cultures was replaced with fresh medium, and the cells
were cultured for a further period of 10 h at 37 °C in a humidified
CO incubator. The amount of lactate released by the cells
into the supernatant was assayed with a commercial diagnostic test
(Sigma) on the basis of lactate dehydrogenase activity. The lactate
content was expressed in mM normalized to a standard of
10
cells/well.Oxygen Consumption
Oxygen consumption of intact
cells was measured with a Clark-type microelectrode in a 1-ml cell
(Strathkelvin Instruments, Glasgow, UK) equilibrated at 37 °C.
Cells, grown in suspension culture overnight, were harvested, counted,
and resuspended in fresh medium saturated with O. O consumption was followed for 5-15 min, depending on the
rate of consumption. Background O consumption in cell-free
medium was 0.25% per min. S.D. on independent preparations was <5%.PI Exclusion Assay and Quantitative Flow
Cytometry
Cell samples were taken from suspension cultures, and
PI was added 3-10 min before analysis on an EPICS 753 flow
cytometer (Coulter Electronics, Luton, UK). Data acquisition was
triggered on a forward angle light scatter signal. Cell debris and
multicell aggregates were electronically gated out. The PI dye was
excited with a water-cooled argon-ion laser (250 mW) at 488 nm. PI
fluorescence was measured above 610-nm wavelength using a long pass
filter. Routinely, 5000 cells were analyzed. S.D. on duplicate samples
was consistently <5%.Measurement of ROI Formation by Flow Cytometry
The
ROI probe DHR123 was added to suspension cultures at the onset of the
experiment. Cell samples were taken from the suspension cultures at
regular time intervals and analyzed on an EPICS 753 flow cytometer as
described before(10) . The DHR123-derived R123 fluorescence was
excited with a water-cooled argon-ion laser (250 mW) at 488 nm and
detected between 515- and 550-nm wavelength. Cell debris and multicell
aggregates were electronically gated out. R123 fluorescence was gated
on the viable cell population and was measured on 3000 viable
cells/sample.
L929 Fibrosarcoma Cells Use Gln as a Respiratory
Substrate
L929 cells are routinely maintained in synthetic
medium containing 25 mM Glc and 2 mM Gln. In order to
evaluate the balance between the utilization of Gln and Glc as
substrates for oxidative phosphorylation, the rate of oxygen
consumption and lactate production was measured in cells maintained for
18-24 h before analysis in the presence of either substrate alone
or combined. Thus, the measurement of these parameters on cells
maintained on either Gln or Glc or both allows to evaluate the
contribution of each substrate to the oxidative metabolism and to
glycolysis, respectively. Since Glc is an essential precursor of ribose
moieties for nucleic acid biosynthesis, Ino, dThd, and Urd were added
to the medium, where Glc was omitted in order to replace the anabolic
function of Glc(15) . Gln, on the contrary, is a nonessential
amino acid and can be omitted from the culture medium.
10 cells were seeded in 6-well dishes in the respective
media for 18 h. Then the medium was refreshed in all wells, and the
lactate released in the medium was measured after 10 h of incubation.
The 100% value of lactate equals a concentration of 5.2
mM/10 cells. For oxygen consumption (filled
bars), cells were cultured in suspension cultures for 18 h in the
respective media, collected by centrifugation, and resuspended in a
small volume of O-saturated medium to measure oxygen
consumption with a Clark-type oxygen electrode at 37 °C. The 100%
value of oxygen consumption equals 115 ng oxygen/min/10 cells. ITU refers to Ino, dThd, and
Urd.
Sensitivity to TNF-induced Cytotoxicity Is Modulated by
the Oxidative Substrate
Cells kept overnight in the presence of
Glc, Gln, or both were assayed for their sensitivity to the cytotoxic
activity of TNF. Cytotoxicity was measured on individual cells by flow
cytometry on the basis of loss of plasma membrane integrity, detected
by means of uptake of the fluorogenic exclusion dye PI. Loss of plasma
membrane integrity has previously been shown to represent an early and
irreversible step in the necrotic process induced by TNF in L929 cells (6) . As shown in Table 1, replacement of Glc by its
anabolic, but energetically neutral, counterparts Ino, dThd, and Urd
(thus leaving Gln as the sole energetic substrate) did not affect the
sensitivity of the cells to TNF-induced cytotoxicity. However, when Gln
was omitted from the culture medium, a markedly decreased cytotoxic
response was observed. This desensitization was equally pronounced when
cells were treated with TNF in the presence of cycloheximide. This
protein synthesis inhibitor is known to enhance the response of L929
cells to the cytotoxic action of TNF, presumably by preventing the
synthesis of rescue factors that counteract TNF activity(22) .
Furthermore, the sensitizing effect of cycloheximide was observed
equally in the presence or absence of Gln, suggesting that TNF
signaling leads to comparable rescue activity in either culture
condition.
Adaptation of L929 Cells to Growth in the Absence of Gln
Reversibly Alters Their TNF-induced Cytotoxic Response
As shown
above, Gln deprivation results in a drop in the rate of oxygen
consumption exceeding 50% (Fig. 1) and hence in mitochondrial
activity. Since earlier reports established that mitochondrial electron
transport activity is a prerequisite for the TNF cytotoxic mechanism in
L929 cells(8) , the reduced mitochondrial respiration observed
in the absence of Gln might explain the diminished responsiveness of
these cells to TNF. In order to verify this explanation, L929 cells
were adapted to growth on Glc as sole energy-providing substrate. They
were grown with gradually lowered concentrations of Gln, the
concentration of Glc remaining unaltered, so that after 2 months a
population was obtained that stably grew on Gln-free medium. These
adapted L929/Gln cells grew slower and showed a more
elongated, fibroblast-like morphology as compared with the scattered,
dedifferentiated morphology of the parental cell population. These
changes in morphology are in agreement with previous reports that
utilization of Gln may affect the phenotype of cells(23) . Also
metabolically, L929/Gln
cells differ from the
parental cells in that Glc fully supported the respiration, reaching an
equal or even slightly elevated rate of oxygen consumption as compared
with parental L929 cells; the only difference was that marginal amounts
of lactate were produced by the adapted cells. Thus adaptation restored
the metabolization pathway of the cells driven by oxidation of Glc
instead of Gln. However, despite the restored mitochondrial activity,
L929/Gln
cells still were desensitized with respect
to TNF cytotoxicity (Fig. 2), similar to the desensitization
after a brief Gln deprivation. This impeded TNF responsiveness is a
stable feature and cannot be ascribed to an altered genotype of the
adapted cells, as demonstrated by the nearly complete restoration of
TNF response after 2 weeks of culture in the presence of Gln (Fig. 2). Thus, switching between a sensitive and an insensitive
phenotype appears to depend on enzymatic pathways involved in the
oxidation of Gln as opposed to Glc and not on the overall electron flow
generated by either substrate in the mitochondria.
cells. TNF-induced cell
death in parental L929 cells (
), L929/Gln
cells (
), and L929/Gln cells that subsequently
had been cultured in the presence of Gln for over 2 weeks (
) is
shown. Cells were grown in suspension cultures 18 h before TNF
treatment (1000 IU/ml); cell death was detected by PI uptake and flow
cytometry.
Specific Inhibitors of the Gln Metabolism Mimic the
Effect of Gln Removal
To further substantiate the involvement of
Gln-specific, enzymatic pathways in the sensitization of L929 cells to
the cytotoxic activity of TNF, we analyzed the interference with
TNF-induced cytotoxicity by specific inhibitors of Gln hydrolysis and
catabolism. The inhibitors were chosen on the basis of their
interference with sequential steps of Gln oxidation; they were assayed
on L929 cells, cultured in the presence of both Gln and Glc, as well as
on L929/Gln cells, the latter in the absence of an
exogenous source of Gln (Table 2). Acivicine, a Gln analogue that
cannot be metabolized and thus acts as a Gln antagonist, drastically
decreased the TNF sensitivity of L929 cells. Similarly, inhibition of
glutaminase activity by L-glutamate-
-hydrazide (24) or by 6-diazo-5-norleucine decreased the cytotoxic
response of L929 cells to TNF. Glutaminase catalyzes the conversion of
Gln to glutamate and is mainly present in the mitochondria.
Mitochondrial overexpression of this enzyme is a common feature in
tumor cells(16) . In a final step, the glutamate formed is
converted to the citric acid cycle intermediate
-ketoglutarate by
transamination, whereby aspartate or pyruvate act as ammonium
acceptors. Inhibition of the transaminase enzymes by amino-oxyacetate
again drastically reduced the cytotoxic response of L929 cells to TNF.
Contrary to acivicine, L-glutamyl-hydrazide, or
6-diazo-norleucine, amino-oxyacetate also significantly reduced the
cytotoxic response of L929/Gln cells, suggesting that
transamination of other amino acid intermediates contributes to the
cytotoxic response of L929/Gln
cells. Apparently,
transamination of intermediates derived from Gln, being the most
abundant amino acid, or from other amino acids in the absence of Gln,
represents a key step in the sensitization of L929 cells to the
cytotoxic action of TNF.
Gln Metabolism Enhances TNF-induced Mitochondrial ROI
Production
We described previously that TNF treatment of L929
cells results in the production of intracellular ROI. These ROI
represent an essential step in the TNF-cytotoxic pathway and are of
mitochondrial origin(10) . Hence, bioenergetic pathways
involved in mitochondrial oxidation of Gln might affect ROI formation
and thus explain the sensitizing effect exerted by this amino acid on
TNF-induced necrotic cell death. In order to verify this hypothesis, we
analyzed the levels of ROI generated upon TNF treatment in L929 and
L929/Gln cells. ROI production was probed with the
cell-permeable, fluorogenic dye DHR123. Oxidation of the nonfluorescent
DHR123 by the reactive oxygen species H
O or
superoxide anion yields the cationic, fluorescent R123, which is
subsequently sequestered by active mitochondria(25) . Analysis
by flow cytometry of the DHR123-derived R123 fluorescence in
TNF-treated cultures showed that the number of cells exhibiting
increased ROI levels (Fig. 3A) as well as the amplitude of
ROI increment (Fig. 3B) relative to untreated controls were
dramatically decreased in the L929/Gln culture
deprived of exogenous Gln. The number of cells exhibiting increased ROI
levels evolved in parallel with the cytotoxic response for both cell
types (Fig. 3C). The lower ROI levels generated by TNF
in the absence of Gln provide an explanation why these cells require
more time before sufficient ROI have accumulated to inflict cell death.
The link between ROI production and cell death, as reported previously
in L929 cells, was also confirmed in L929/Gln
cells
by the complete arrest of both responses following addition of the free
oxygen radical scavenger butylated hydroxyanisole (Fig. 3, A-C). These results indicate that sensitization of L929
cells to TNF cytotoxicity by oxidative metabolism of Gln involves a
mechanism that renders the mitochondria more susceptible to a cytosolic
TNF signal, resulting in an enhanced activation of the ROI-producing
pathway, which in turn leads to accelerated cytotoxicity.
) and
L929/Gln cells (
) in the absence (full
line) or presence (dashed line) of the radical scavenger
butylated hydroxyanisole. A, percentage of TNF-responsive
viable cells (DHR123 profiles of TNF-treated cells minus DHR123
profiles of untreated cells at the corresponding time points), showing
an increased DHR123-derived R123 fluorescence intensity after TNF
treatment. B, TNF-induced increment in ROI formation: mean
R123 fluorescence intensity of TNF-responsive viable cells compared
with untreated control cells. C, TNF-induced cell death:
percentage of PI-positive cells in TNF-treated cultures as compared
with untreated cultures. The arrows indicate the time of
addition of butylated hydroxyanisole to the cell
cultures.
population had the appearance of
differentiated fibroblasts, as opposed to the dedifferentiated,
scattered morphology of parental L929 cells; moreover, it showed a
reduced proliferation rate and in fact nearly behaved like
untransformed fibroblasts. Also the metabolic parameters, namely low
lactate release and high oxygen consumption, corresponded to those of
normal cells. These parameters indicate that Glc supports in
L929/Gln
cells a restored electron flow through the
mitochondrial electron transport chain. Nevertheless, these adapted
cells still displayed the TNF-resistant phenotype observed after short
term depletion of Gln. We therefore conclude that a factor responsible
for sensitivity of the TNF-cytotoxic response in the presence of Gln
resides in the activity of enzyme systems linked to the oxidative Gln
metabolism and not in the overall mitochondrial electron flow. This
conclusion was confirmed by the mimicking effect observed with
inhibitors that block, at various steps, the enzymatic oxidation of Gln
through the citric acid cycle in the mitochondria. Thus the use of Gln
as an energy substrate facilitates TNF signal transduction leading to
necrotic cell death.
)
We thank Drs M. Vincx and J. Vanfleteren for technical
assistance.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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J. G. Pastorino, G. Simbula, K. Yamamoto, P. A. Glascott Jr., R. J. Rothman, and J. L. Farber The Cytotoxicity of Tumor Necrosis Factor Depends on Induction of the Mitochondrial Permeability Transition J. Biol. Chem., November 22, 1996; 271(47): 29792 - 29798. [Abstract] [Full Text] [PDF]
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R. J. Rogers, J. M. Monnier, and H. S. Nick Tumor Necrosis Factor-alpha Selectively Induces MnSOD Expression via Mitochondria-to-Nucleus Signaling, whereas Interleukin-1beta Utilizes an Alternative Pathway J. Biol. Chem., June 1, 2001; 276(23): 20419 - 20427. [Abstract] [Full Text] [PDF]
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