(CANCER RESEARCH 53. 1456-1460. March 15. 199.1]
Expression of BCL-2 Protein Enhances the Survival of Mouse Fibrosarcoid Cells in
Tumor Necrosis Factor-mediated Cytotoxicity1
Thierry Hennet, Giuseppe Bertoni, Christoph Richter, and Ernst Peterhans2
Institute of Veterinary- Virology, University of Berne. Länggass-Strasse 122. CH 3012 Berne ¡T.H., G. B., E. P.\: and Laboratory of Biochemistry I. Swiss Federal Institute of
Technology, Zurich ¡C.R.¡,Switzerland
ABSTRACT
Tumor necrosis factor (TNF) kills some types of tumor cells in vitro and
participates in tumor elimination in vivo. TNF has been shown to kill cells
by altering their mitochondria structurally and functionally. The oncogene
l<( I -2 codes for a protein located in the inner membrane of mitochondria
which is able to inhibit the commitment to cell death in various cell types.
We have therefore investigated whether TNF-mediated killing of the cell
line L929 could be modulated by expression of the protein BCL-2. We
report here that L929 cells transfected with a BCL-2 expression vector
have an increased survival compared to wild type cells after TNF chal
lenge. The protective effect is greatest at moderate TNF concentrations
and is still significant at concentrations that killed 100% of wild type cells.
The action of BCL-2 is selective inasmuch as cells are not protected against
other cytotoxic agents blocking various mitochondria! functions. We show
that cells expressing BCL-2 have a higher mitochondria! membrane po
tential iAM'i than wild type cells. The increase in AM'could be linked with
the enhanced survival of cells after TNF challenge. Indeed, we found that
treatment of wild type L929 cells with the ionophore nigericin, which
increases AM7,protects them even at high TNF concentrations.
INTRODUCTION
In human follicular B-cell lymphoma the BCL-2 gene is translo
cated from chromosome 18 to chromosome 14 and its expression
becomes regulated by the potent immunoglobulin heavy chain pro
moters resulting in overexpression of the oncogene (1,2). The BCL-2
gene codes for a M, 26,000 membrane protein with no similarity to
any other known protein (3). Its localization in the inner membrane of
mitochondria suggests that it could influence some functions of this
organelle (4). Beside its mitochondrial location, BCL-2 has the unique
properly for an oncogene to enhance cell survival rather than to
promote cell proliferation (5) as demonstrated by its protective effect
against apoptotic cell death (6). However, the precise function of
BCL-2 within mitochondria has not been elucidated. BCL-2 is of
physiological importance in the establishment of B-cell memory (7),
and its pattern of expression in different tissues suggests that it could
be involved in the life span control of various cell types (8, 9).
We were interested whether the expression of certain rescue pro
teins can protect cells against immune effector mechanisms. This
could be a way used by some tumor cells to escape immunosurveillance. In a first attempt, we focused our attention on the role of BCL-2
expression in tumor cells challenged by the cytotoxic cytokine TNF3
in vitro.
The mechanism of TNF-mediated cell death varies according to the
target cells, some dying by necrosis and others by apoptosis (10).
Oxidati ve stress (11, 12), induction of endogenous nucleases (13), and
depletion of ATP/NADH stores (14) have been proposed to contribute
Received 9/14/92; accepted 1/5/93.
The costs of publication of this article were defrayed in pan by the paymenl of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicale this fact.
1This work was supported by the Swiss National Fund, grants No 31-28810.90 to E.P.,
and No 31-26254.89 to C.R.
2 To whom requests for reprints should be addressed.
' The abbreviations used are: TNF, tumor necrosis factor; cDNA, complementary
DNA; AM', mitochondrial membrane potential; O2 . Superoxide anión; TNF-R55, TNF
receptor I (M, 55.000); TNF-R75. TNF receptor II (M, 75,000); NAO. IO-/V-nonylacridine
orange; WT. wild type.
to cell killing. Further, some groups have observed that cytotoxicity is
related to structural and functional alterations of mitochondria in cells
treated with TNF (15-17). We have shown that mitochondria of TNFtreated L929 cells generate increased amounts of O2 (18). Mitochon
dria are the first cellular target to show damage, and the loss of
mitochondrial integrity leads to cell death apparently because of a
depletion of energy stores.
Since the BCL-2 protein is located within mitochondria, we inves
tigated whether it can protect TNF-treated cells by preventing mito
chondrial damage. To this end, we concentrated on the mouse fibrosarcoid L929 cell, one of the most thoroughly studied targets of TNF
cytotoxicity. We report that expression of BCL-2 in L929 cells en
hances the resistance of these cells to TNF-mediated cytotoxicity.
Protection is specific inasmuch as cells are still sensitive to an array
of other cytotoxic agents. In addition, we show that the effect of
BCL-2 is not due to a change in the expression of TNF receptors or
to a decreased ability to produce O2 after TNF addition. Since
BCL-2 is located in mitochondria, we also investigated the mitochon
drial electron transport system in BCL-2-transfected cells. While the
activity of the mitochondrial respiratory chain remains unchanged, we
observed a higher Aty in cells expressing BCL-2. This parameter
could be linked to the TNF resistance inasmuch as we found that the
ionophore nigericin, known to increase A^, protects the cells against
killing by TNF.
MATERIALS
AND METHODS
Materials. Recombinant mouse TNFa was purchased from Genzyme
(Boston, MA). The activity was 4 X IO7 units/mg protein. The mouse BCL-2
cDNA was a generous gift of S. J. Korsmeyer (Howard Hughes Medical
Institute, St. Louis, MO). W. Lesslauer (Hoffmann LaRoche Ltd., Basel,
Switzerland) kindly provided the cDNA of mouse TNF receptors. The expres
sion vector BCMGSNeo was obtained from F. Melchers (Basel Institute for
Immunology, Basel, Switzerland) (19). Unless indicated the chemicals were
from Sigma Chemical Co. (St. Louis, MO).
Cell Culture. The mouse fibrosarcoid L929 cell line was obtained from the
American Type Culture Collection (Rockville, MD). The cells were subcloned
by means of a cloning ring and the derived subline (WT) was used for all
further experiments. The cells were cultured in Dulbecco's modified Eagle's
medium (Giòco, Paisley, United Kingdom), supplemented with 2 mw
glutamine,
10 mm 4-(2-hydroxyethyl)-l-piperazineethanesulfonic
acid
(pH 7.4), penicillin (100 units/ml), streptomycin (100 ug/ml), and 7% heatinactivated fetal calf serum.
Plasmid Construct. All enzymes and buffers were from Boehringer Mann
heim (Mannheim, Germany). The BCL-2 gene was excised from the pN2-MBcl-2 vector with tfmdIII and EcoRl (5) and ligated into the polylinker of the
Bluescript plasmid at the WmdIII and EcoRI sites (Stratagene, La Jolla, CA).
The resulting construct was then digested with Xhol and Noti and the 926-base
pair BCL-2 fragment was inserted into the corresponding sites of the BCMG
SNeo vector.
Electroporation and Selection of G-418-resistant Cells. Cells were har
vested after trypsinization, washed twice with ice cold phosphate-buffered
saline and resuspended at 1 x IO7 cells/ml. Plasmid DNA (25 ug) was added
to the cells which were electroporated after 10 min incubation on ice using a
Gene Pulser apparatus (Bio-Rad, Richmond, CA) set at 1000 V, 25 uF. Cells
were then cultured in Dulbecco's modified Eagle's medium-7% fetal calf
serum. The antibiotic G-418 (Gibco, Madison, WI) was added at 0.5 mg/ml to
the culture medium after 48 h. Batches of 10,000 cells were transferred to
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BCL-2 PROTEIN PROTECTION
IN TNF CYTOTOXICITY
Not!
culture dishes (Falcon; Becton Dickinson, Oxnard, CA) and cell clones were
isolated with cloning rings after 15 days of culture.
RNA Analysis. Total cell RNA was purified according to the method of
Chomczynski and Sacchi (20) using a kit (Promega, Madison, WI). RNA (5 ug)
was dissolved in 50% formamide/6.6% formaldehyde, separated by electro-
Xhol
Sail
Xbal
phoresis in a 1.5% agarose/6.6% formaldehyde gel, and then transferred to
Hybond-C extra membranes (Amersham, Amersham, United Kingdom) fol
lowing standard procedures (21). Blots were hybridized using the following
probes: HindlWEcoRl BCL-2 fragment (5); EcoRUEcoRl TNF-R55 fragment;
BamHl/EcoR\ TNF-R75 fragment (22). After removal of the primary probes by
boiling, the membranes were rehybridized with a chicken ß-actinprobe.
Hindlll
TNF Cytotoxicity Assay. Cells were incubated with TNF and actinomycin
D (1 ng/ml) at 37°Cin Iscove's modified Dulbecco's medium (Gibco) sup
HindHI
plemented with 0.2% Bacto-peptone (Difco, Detroit, MI), glutamine (2 ITIM).
pyruvate (1 ITIM),penicillin (100 units/ml), streptomycin (100 ug/ml), nonessential amino acids (Gibco), minimal essential medium-vitamin solution
(Gibco), and 10 HIM4-(2-hydroxyethyl)-l-piperazineethanesulfonic
acid (pH
7.4). Cell viability was measured by the 3-|4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide cleavage assay as described by Mosmann (23).
The percentage of cylotoxicity was defined as the ratio
A
A ,
Fig. 1. BCL-2 expression vector. The BCL-2 gene was cloned inlo the XhoUNotl sites
of the BCMGSNeo vector (19) after subcloning in the //indlll/EroRI siles of the Bluescript vector from the pN2-M-BCL-2 vector (5). The cytomegalovirus panactive (CMVP)
promoter controls the transcription of the BCL-2 gene. The neo gene confers resistance
towards the antibiotic G-418. Genetic elements of bovine papilloma virus (BPV} allow the
episomal replication of the vector at high copy number (30-100 copies/cell).
X 100
where At. is the absorbance of 100% viable cell control minus background and
As is the absorbance of the tested sample minus background (100% cytotoxicity). The clone WT expressed the same TNF sensitivity as the uncloned L929
cell population.
Membrane Potential. A^ was analyzed by flow cytometry using a FAC-
WT
Scan (Becton Dickinson) equipped with the software Lysis II. Fluorescence
was recorded after 30 min staining of cells in suspension with rhodamine 123
at 0.2 ug/ml (24, 25). The amount of mitochondria within cells was evaluated
by staining cells with 0.2 UMNAO (Molecular Probes, Eugene, OR) for 30 min
as described (26).
Mitochondrial Enzyme Assays. Oxygen consumption was measured using
a Clark-type electrode (YSI model 5331). Cells were set to a density of 6 x
106/ml in 25 mv Tris-HCl (pH 7.4)-0.25 M sucrose-2 HIM EDTA-5 mm
MgCl2-10 HIMK2HPO4 and incubated under constant stirring at 37°C.Oxygen
B28
B38
B
TNF-R75
phenylenediamine/ascorbate
was used as a measure of cytochrome c oxidase
activity (complex IV) (27). Respiratory activity was expressed in ng atoms
oxygen/min/IO6 cells.
ß-actin
was detected in WT cells and in cells transfected with the BCMGSNeo
vector alone (Fig. 2A). We selected for further investigations three
clones, Bl 1, B22, and B28, with high BCL-2 mRNA levels, and one
clone, B38, with low BCL-2 mRNA level. These clones were com
pared to the original WT clone and to a clone (V3) transfected with the
BCMGSNeo vector alone. As an essential control for TNF cytotoxicity assays, we compared the expression of TNF receptors in BCL-2and mock-transfected cells. Fig. 2, fi and C, shows that transfection of
L929 cells had no effect on the expression of TNF-R55 and TNF-R75
as assayed in Northern blotting. We rehybridized the filters with a
probe for ß-actinto standardize our results (Fig. ID).
B22
TNF-R55
ascorbate (0.8 mM/4 ITIM).The activity of the mitochondrial respiratory chain
downstream of NADH dehydrogenase (complex I) was defined as the rate of
oxygen uptake after addition of the substrate succinate. The rate of oxygen
uptake in the presence of the electron donors yV.AW./V'-tetramethyl-/?-
We cloned the mouse BCL-2 gene into the Xhol/Notl site of the
BCMGSNeo vector (Fig. 1). This plasmid replicates in transfected
cells as an episome at elevated copy numbers (19). The panactive
promoter cytomegalovirus that regulated BCL-2 expression ensured a
high degree of transcription (28). We found elevated BCL-2 mRNA
levels in most G-418-resistant L929 clones screened while no mRNA
B11
BCL-2
disappearance was monitored after consecutive addition of (final concentra
tions): digitonin (0.2 mg/107 cells); rotenone (0.2 UM); succinate (5 IHM);
antimycin A (0.2 UM) and /V./V.Ar.JV'-tetramethyl-p-phenylenediamine/
RESULTS
V3
fe.
Fig. 2. mRNA levels of BCL-2. TNF receptor p55, TNF receptor p75, and ß-actin.
RNA from clones WT, V3, B11, B22, B28. and B38 was hybridized with a fragment of
mouse BCL-2 (A ), mouse TNF-R55 cDNA (B), mouse TNF-R75 cDNA (C), and chicken
ß-aclin(D). The exposure time was 12 h for the BCL-2 hybridization. 24 h for the ß-actin,
and 4 days for the TNF receptor hybridizations. Only one band was visible on each
autoradiogram, except for TNF-R75 which produced no signal.
TNF kills L929 cells in a dose-dependent manner. The cytotoxic
effect can be further enhanced by inhibiting protein synthesis in target
cells (29). We observed that BCL-2 expression protected L929 cells
from TNF cytotoxicity (Fig. 3) in both the presence and absence of the
transcriptional inhibitor actinomycin D. The protective effect was
most pronounced at concentrations that killed about 50% of WT cells
and was still significant at the highest concentrations tested (1000
pg/ml and 20 ng/ml). In the presence of actinomycin D, the survival
of cell clones treated with TNF correlated with the extent of BCL-2
expression (Fig. 3A). In contrast, all clones transfected with BCL-2
had a similarly increased resistance when challenged with TNF in the
absence of actinomycin D (Fig. 3fi). Additional experiments showed
that the enhanced survival of BCL-2 expressing cells treated with TNF
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BCL-2 PROTKIN PROTECTION
IN TNI- CYTOTOXICITY
sumption, which reflects the activity of complexes II, III, and IV. In all
clones we found rates of oxygen uptake ranging from 2.5 to 3.3 ng
atoms O/min/IO6 cells (results not shown). When measuring oxygen
consumption through complex IV alone, we also failed to detect
significant differences between clones expressing BCL-2 and WT
cells. In all clones the rate of oxygen uptake through complex IV was
between 4.6 and 5.0 ng atoms O/min/106 cells (results not shown).
TNF (pg/ml)
TNF (ng/ml)
Fig. 3. Survival of L929 cells transacted with BCL-2 after TNF challenge. M) Cells
were incubated in serum-free medium with various TNF concentrations in the presence of
actinomycin D. Cell viability was measured 20 h after addition of TNF using the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide cleavage assay. (B) Higher TNF
concentrations were necessary to kill cells in the absence of actinomycin D. Cell viability
was measured 72 h afler TNF addition. Each symbol represents a typical experiment of
five experiments. O. Clone WT; A, clone V.I; •¿
clone B11 ; A, clone B22: »,clone B28;
•¿
clone B38.
was not related to a decreased O2 production as measured by lucigenin-dependent chemiluminescence (results not shown).
To determine if the protective action of BCL-2 was broad or rather
selective for TNF we also investigated the effect of various cytotoxic
agents. Because of the localization of BCL-2 in mitochondria, we
focused on agents acting on this organelle. We challenged L929 cells
expressing BCL-2 with rotenone (1-100 UM),antimycin A (5-100 JIM),
and sodium azide (1-70 ITIM),which inhibit, respectively, mitochon
dria! complexes I, III, and IV. We also investigated the effect of the
uncoupler carbonyl cyanide /j-trifluoromethoxyphenylhydrazone
(1-50 UM), the ATPase inhibitor oligomycin (2-50 ug/ml), and the
oxidant H2O2 (1-250 UM)on the viability of cell clones. In addition,
we applied l-methyl-4-phenylpyridinium
(0.1-10 ITIM),which inhibits
complex I activity and increases Superoxide anióngeneration at this
site (30), and dequalinium (10-500 UM),a cytotoxic drug accumulat
ing in mitochondria (31). Except for antimycin A, we failed to detect
any change in the sensitivity of the clones towards these agents
(results not shown). Others have shown that T-cells overexpressing
BCL-2 were protected not only from apoptosis but also from necrotic
death induced for example by azide (32). Because this agent blocks
mitochondria! electron transport and consequently ATP production,
this suggested that BCL-2 could prevent the loss of mitochondrial ATP
and thereby rescue cells from dying. We failed to detect any difference
in the sensitivity for azide of cells expressing BCL-2 compared to WT
cells. Noteworthy is the observation that L929 cells needed a higher
concentration of azide (3 ITIM)than T-cells to be killed. Surprisingly,
the clones B11, B22, B28, and B38 were more sensitive to the action
of the inhibitors of mitochondrial complex III than WT cells. Fig. 4A
shows that all clones expressing BCL-2 were killed after 24 h incu
bation in the presence of 25 UMantimycin A while 75% of WT cells
still survived at this concentration of drug. Interestingly, clone B38,
which had the lowest BCL-2 mRNA level, was also the least sensitive
to antimycin A. To establish whether the action of antimycin in fact
reflected its inhibition of complex III activity, we investigated the
action of myxothiazol, another inhibitor of the same mitochondrial
complex. In agreement with the results obtained with antimycin A, we
found that clones B11, B22, B28, and B38 were killed at lower
concentrations of myxothiazol than the cells not expressing BCL-2
(Fig. 4ß).
To determine if the enhanced sensitivity toward antimycin A was
due to a decreased complex III activity in clones expressing BCL-2,
we measured O2 consumption by means of a Clark-type electrode. No
change was observed in the rate of succinate-dependent oxygen con
In addition to determining the activity of mitochondrial electron
transport, we compared the membrane potential A^ in the BCL-2
clones with that in WT cells. The fluorescent dye rhodamine 123
permits the determination of Aty without permeabilizing cells or
isolating mitochondria. This lipophilic cation is taken up by mito
chondria in proportion to the membrane potential (negative inside),
allowing thus to determine the latter parameter by measuring the
fluorescence intensity issued from rhodamine 123. Compared to WT
cells and clones transfected with the BCMGSNeo vector alone we
detected an increased A'*!' in clones Bll, B22, B28, and, to a lesser
extent, clone B38 (Fig. 5A). Because the difference in fluorescence
intensity between the clones tested could originate from a difference
in the amount of mitochondria within cells, we also measured this
parameter in our cell clones. The fluorescent dye NAO specifically
binds to mitochondria by interacting with lipids of the inner mito
chondrial membrane (33) and binding was shown to be independent of
A^. NAO staining of cells revealed no difference in the amount of
mitochondria between the different cell clones (Fig. 5B), indicating
that the higher rhodamine 123 fluorescence measured in clones Bll,
B22, B28, and B38 was indeed due to an increase in ASK
To determine the role of A^ in TNF-mediated cytotoxicity, we
treated WT L929 cells with the ionophore nigericin which increases
A'*!'by dissipation of the pH gradient across the mitochondrial mem
brane. Fig. 6 shows that nigericin protected cells from cytotoxicity in
a dose-dependent fashion even at high concentrations of TNF, which
otherwise resulted in killing of more than 95% of the cells. Nigericin
alone was not cytotoxic at the concentrations used to modulate TNFmediated cytotoxicity (results not shown). Treatment of cells with
uncouplers like carbonyl cyanide p-trifluoromethoxyphenylhydrazone
and the potassium-ionophore valinomycin failed to affect TNF-medi
ated cytotoxicity (results not shown), indicating that breakdown of
A^ did not further enhance cell death induced by TNF. In addition, we
found that treatment of cells with monensin, which is structurally
related to nigericin but lacks its capacity to enhance Aty, did not
influence cytotoxicity after TNF challenge (results not shown). This
latter finding further supports the interpretation that the effect of
nigericin is due to its action on A^.
0
S
10
25
SO
Antimycin A (¡M)
75
0
S
10
25
50
100
Myxothiazol (uM)
Fig. 4. Differential sensitivity of BCL-2 clones toward inhibitors of mitochondrial
complex III. Antimycin A (A) or myxothiazol (ß)were added to cells which were
subsequently incubated for 24 h at 37°C.After this time, cell viability was measured with
the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide cleavage assay. Shown
are means of triplicates; SD was less that 5% of the signal and is not shown. Each s\tnhol
represents a typical experiment of four experiments. O, clone WT; A, clone V3; •¿,
clone
Bll; A. clone B22; *, clone B28; •¿
clone B38.
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BCL.2 PROTEIN PROTECTION IN TNF CYTOTOX1CITY
Fig. 5. Increased mitochondrial membrane poten
tial in clones expressing BCL-2. Cells were stained
after trypsinization with rhodamine 123 for 30 min
(A) or with NAO for 30 min (Ö)and analyzed with
flow cytometry. Excitation wave length was at 488
nm and fluorescence emission was recorded at 530
nm. Three representative clones (WT, V3, and B22)
are shown. The results of one experiment of five are
shown.
200
400
600
800
Relative fluorescence Intensity
SO
(0
110
330
1000
TNF (pg/ml)
Fig. 6. Nigericin protection of WT L929 cells against TNF-mediated cytotoxicily.
Nigericin was added to WT cells in the presence of actinomycin D and various TNF
concentrations. Cell viability was measured after 20 h incubation at 37°C.Columns, mean
±SD of six measurements. Nigericin was not toxic alone at the concentrations
no nigericin; O. O.I UMnigericin; ffl, 0.25 UMnigericin.
DISCUSSION
used. EH,
1000
0
600
800
1000
Relative fluorescence intensity
BCL-2 (38), we are unable to detect endogenous BCL-2 transcripts in
L929 cells. As expected, the transfection of L929 cells with the BCL-2
gene had no influence on the transcription of TNF receptor genes and
on the proliferation of transfected cells (38). The increased survival of
TNF-treated cells was observed in the presence and absence of protein
synthesis inhibitors. However, when actinomycin D was added, clone
B38, which had the lowest BCL-2 mRNA level, was more susceptible
to TNF-mediated cytotoxicity. This is possibly linked to a decrease of
the BCL-2 protein level under the threshold needed to protect cells
against TNF once transcription is blocked by actinomycin D. We have
not measured the half-life of the BCL-2 protein in transfected L929
cells, but data from others indicate that the level of this protein
decreases within 12 h after arrest of synthesis in hemopoietic cells (5).
Because we detected the same amount of O2 produced by cells
expressing BCL-2 and WT cells, we conclude that BCL-2 confers
protection rather than modulating the effector mechanisms of TNFmediated cytotoxicity.
The enhanced susceptibility to the cytotoxic agents antimycin A and
myxothiazol of BCL-2-transfected cells argues for a role of BCL-2 as
a regulator of mitochondrial metabolism. These two drugs inhibit the
mitochondria! complex III, which transfers electrons from CoQ to
cytochrome c and participates in the build-up of the proton gradient
across the mitochondrial membrane (39). Since antimycin A binds
stoichiometrically to the cytochrome be, of complex III, the higher
sensitivity to antimycin A and myxothiazol of cells expressing BCL-2
suggests that these cells had a decreased amount of complex III in
mitochondria. However, proof for this is lacking because it was tech
nically not possible to obtain sufficient mitochondria of L929 cells to
study the composition of the respiratory chain enzymes. Along this
line, we did not find any change in the activity of complexes II, III.
and IV in L929 cells transfected with BCL-2 compared to WT cells.
Nevertheless, it remains possible that BCL-2 is interacting with
mitochondrial complex III without affecting its activity but making
complex III more susceptible to inhibition by antimycin and myxothi
azol.
A role for BCL-2 in mitochondrial function was further supported
by the finding that L929 cells expressing BCL-2 have a higher Aty
than WT cells. This increase is related to BCL-2 expression since all
clones investigated are derived from a clone of L929 cells (WT) which
displayed constant values for A^ over passages. According to the
chemiosmotic theory of Mitchell (40) A'*!'is the driving force for ATP
Cell death has emerged to be as important as cell proliferation in
controlling biological functions. Cell death is crucial in processes like
tissue modeling during development (34), immune tolerance (35), and
immune killing mechanisms (36). Effectors responsible for the latter
mechanisms, comprising cytotoxic T-cells and soluble factors like
TNF, act by inducing cell death of target cells in vitro. It was therefore
of interest, as suggested by Williams (37), to investigate whether
genes that inhibit the commitment to cell death could protect tumor
cells from killing by immune mediators such as TNF. To answer this
question, we have transfected a TNF-sensitive clone from L929 cells
with a BCL-2 expression vector and investigated the sensitivity of the
transfected cells to the cytotoxic cytokine TNF. WT L929 cells were
killed by pg amounts of TNF when their protein synthesis was
blocked. When challenged with TNF, L929 cells expressing BCL-2 synthesis and is essential for viability of cells as its breakdown leads
showed enhanced resistance compared to WT cells. In agreement with to cell death. In addition, A^ can vary according to the physiological
observations made by others in NIH3T3 fibroblasts transfected with status of cells. For example, it is increased in activated cells compared
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BCL-2
PROTEIN
PROTECTION
to resting cells (41). Aty is also increased in a variety of tumor cell
types (42, 43). Although this increase is not sufficient to cause neo
plasia, it indicates that these cells have a higher rate of ATP synthesis,
which could be related to their elevated proliferative activity. Further,
it is tempting to link a higher Aty with an increased resistance to stress
caused by a decrease of ATP within cells. This could be especially true
for TNF, which has been shown to act in that way (14, 18). To verify
this hypothesis we used nigericin, a drug known to increase A^ by
eliminating the pH gradient across the mitochondria! membrane. As
expected, nigericin-treated cells were protected against a TNF chal
lenge, indicating that an enhancement of A1? is an important factor in
determining the sensitivity of cells to TNF.
The expression of BCL-2 may give a double advantage to tumors in
vivo. It can enhance cell survival in low growth factor conditions (38)
or even growth factor deprivation as observed for some myeloid cells
(5). Similar to our results in vitro, BCL-2 expression could render
tumor cells more resistant to killing mechanisms mobilized by the
host, like TNF. Along this line, it could be of interest to investigate
whether BCL-2 expression can protect cells from lytic viral infections.
This possibility seems attractive in view of the pattern of BCL-2
expression in cells like neurons (8, 9), which cannot be replaced when
killed. In order to verify this hypothesis we are currently assaying the
resistance of cells transfected with BCL-2 against various cytolytic
IN TNF CYTOTOXICITY
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
viruses.
ACKNOWLEDGMENTS
26.
We thank S. Korsmeyer, S. Hahn, and P. Erb for providing us with the mouse
BCL-2 cDNA; F. Melchers for the BCMGSNeo vector; and W. Lesslauer for
27.
giving us the mouse TNF receptor cDNA. Our thanks also go to I. M. Nauta
and P. Kuhnert for their help.
28.
29.
REFERENCES
30.
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Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1993 American Association for Cancer Research.
Expression of BCL-2 Protein Enhances the Survival of Mouse
Fibrosarcoid Cells in Tumor Necrosis Factor-mediated
Cytotoxicity
Thierry Hennet, Giuseppe Bertoni, Christoph Richter, et al.
Cancer Res 1993;53:1456-1460.
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