From the Cornell Nanofabrication Facility, Cornell
University, Ithaca, New York 14853, the ¶ Neurosciences Program,
Stanford University School of Medicine, Stanford, California 94305, the ** Department of Biomedical Sciences, Institute of Medical
Sciences, University of Aberdeen, Foresterhill,
Aberdeen AB25 2ZD, United Kingdom, and the
Department of Molecular Biology, Flanders
Interuniversity Institute for Biotechnology, University of Gent,
Ledeganckstraat 35, B-9000 Ghent, Belgium
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ABSTRACT |
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The TF-1 human erythroleukemic cell line exhibits
opposing physiological responses toward tumor necrosis factor- Cells have the capability of responding to a multitude of signals
that it encounters in its extracellular environment. One such signal
with widespread pleiotropic actions is the cytokine tumor necrosis
factor- One action of TNF, the induction of apoptosis, is characterized by a
discrete set of cellular events regulated by gene expression (7, 8).
The physiological events accompanying apoptosis include
condensation of the chromatin, degradation of DNA through the
activation of endogenous nucleases, and dissolution of the cell into
small membrane-bound apoptotic vesicles (9, 10). In vivo,
these vesicles are phagocytosed by macrophages or other phagocytic
cells. Cell death by apoptosis is essential in many physiological
processes, including embryonic development of the nervous system (11),
oncogenic pathology (12), and clonal selection of hematopoietic cells
(13).
Conversely, TNF has also been shown to stimulate cellular proliferation
in a variety of systems (14, 15). Although apoptosis as a physiological
phenomenon has only recently received widespread attention, it is
apparent that proper development, organismal homeostasis, and oncogenic
transformation are all thought to be contingent on a delicate balance
between these opposing processes. The importance of both can be seen in
a number of pathological diseases that are a consequence of either
uncontrolled proliferation or apoptosis. Whereas proliferation is
clearly linked to cell cycle progression, the role of the cell cycle
and cell cycle-related proteins, such as cyclins, in apoptosis is less
clear. Whether TNF-induced cell proliferation or apoptosis demonstrated
in different model systems is also connected to the cell cycle
apparatus has yet to be determined.
We have used the human erythroleukemic cell line TF-1 as a model system
to study TNF actions in early hematopoietic progenitor cells. TF-1
cells are a CD34+ myeloid progenitor stem cell line
originally isolated from a human erythroleukemia patient (16). These
cells are factor-dependent and thus require a cytokine such
as granulocyte-macrophage colony stimulating factor (GM-CSF) or
interleukin-3 for survival and proliferation. In this study, we
demonstrate that the TF-1 cell line displays differential sensitivity
toward TNF depending on the growth state of the cell. We show that
mitotic activity is required for TNF-induced apoptosis, whereas
conversely, mitotically quiescent cells respond to TNF by cellular
proliferation. In addition, we demonstrate an important role for the
p75TNFR in addition to the p55TNFR in mediating apoptosis.
Reagents and Cell Culture--
Unless otherwise noted, chemicals
were purchased from Sigma. Recombinant human TNF was obtained from R & D Systems (Minneapolis, MN). Recombinant human GM-CSF was procured from
Boehringer Mannheim. Sea Kem GTG agarose was purchased from FMC Corp.
(Rockland, ME). TF-1 cells were cultured as described previously in the
presence of 1.0 ng/ml GM-CSF unless otherwise indicated (17). Log
growth or stationary cultures were grown from a starting cell density of 2.0 × 104 cells/ml. Cell counts were performed
daily over a period of 8 days to generate the TF-1 cell growth curves
seen in Fig. 1, A and B (insets).
Measurement of Metabolic Activity by
Microphysiometry--
Description and operation of the Cytosensor
microphysiometer (Molecular Devices Corp., Sunnyvale, CA) have been
detailed elsewhere (18, 19). Briefly, TF-1 cells were collected from
cultures of either 5.0 × 103 to 1.0 × 104 cells/ml (log growth) or 1.0-1.2 × 106 cells/ml (stationary). Approximately 2.5 × 105 cells were confined between two microporous
polycarbonate membranes separated by a 50-µm-thick annular spacer
immobilized within a fibrin clot described previously (20). The
microporous membranes and annular spacer form a disk-shaped chamber of
6 mm diameter and 50 µm height (volume, ~2 µl) that, together
with the fibrin immobilizing reagent, traps cells during the fluid
perfusion of microphysiometer experiments. This cell capsule was placed
in a flow- and temperature (37 °C)-regulated sensing chamber of the microphysiometer so that the lower membrane was in diffusive contact with the surface of the light-addressable potentiometric sensor chip,
and pH changes were monitored at the sensor surface in an area of 2.5 mm (2) in the center of the chamber. Cells were perfused with low
phosphate-buffered RPMI medium (Irvine Scientific, Irvine, CA) in a
cyclic manner automatically controlled by a peristaltic pump. The pump
cycle was 150 s long, comprising a flow-on period (100 µl/min
for 110 s) followed by a flow-off period (40 s). The 110 s
duration of the flow-on period was necessary to allow for complete
renewal of the medium in the sensing chamber. Extracellular acidification rate was determined from the slope of a linear
least-squares fit to the relation of sensor output voltage
versus time during the central 30 s of each flow-off
period. The light-addressable potentiometric sensor used was Nernstian
(61 mV ~ 1 pH unit at 37 °C), so a rate of change of sensor
output voltage of 1 µV/sec corresponds closely to an extracellular
acidification rate of 1 × 10 Analysis of [3H]Thymidine
Incorporation--
[3H]Thymidine incorporation was
performed as described (21). Briefly, equal numbers of TF-1 cells were
pulsed with 1 µCi/ml [methyl-3H]thymidine (5 Ci/mmol, Amersham Pharmacia Biotech) for 1 h at 37 °C. Cells
were collected onto glass fiber filters under vacuum and washed three
times with 5 ml of ice-cold PBS. Filters were then washed with ice-cold
5% trichloroacetic acid followed by a wash with 10 ml of ice-cold
absolute ethanol. Dry filters were placed in scintillation vials, and
radioactivity was determined by counting in a Beckman LS1801 liquid
scintillation counter (Beckman Instruments, Fullerton, CA).
Analysis of Cell Number--
In order to assess proliferation or
cell death from within the same assay, measurement of cell activity
(and thus proportionately cell number) was determined by using the
3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) or
3'-(1-phenyl-amino-carbonyl)-3,4-tetrazolium)-bis (4-methoxy-6-nitro)
benzene sulfonic acid hydrate (XTT) (Boehringer Mannheim) colorimetric
assays. Basically, TF-1 cells (100 µl of growth medium/well) were
seeded into a round-bottomed 96-well plate at the indicated density.
After 18-24 h of recovery, cells were treated with an additional 100 µl/well of growth medium containing drug and/or TNF and incubated for
24 h more. Each well then received a further 20 µl of growth
medium with 5 mg/ml of MTT or XTT. The cells were then allowed to
incubate for 3-4 h to allow the metabolism of substrate to form the
colored metabolic product. Cells in the 96-well plates were pelleted by
centrifuging for 5 min at 1500 rpm (800 RCF). The supernatant medium
was carefully aspirated off with a bent 24 gauge needle under low
vacuum. To each well was added 100 µl of DMSO, and after 15-30 min,
the absorbance for the MTT-formazan product was read at 540 nm (450 nm
for XTT) with background subtraction at 690 nm using a Thermomax
microplate reader (Molecular Devices Corp.). The results are expressed
in arbitrary absorbance units or as a percentage of the absorbance in
control-treated cells.
Epifluorescence Apoptosis Analysis--
TF-1 cells had various
agents added directly to the culture medium for the indicated time.
Cells were then washed once by resuspension-centrifugation in culture
medium (4 °C), and DNA was stained by inclusion of 5 µg/ml Hoechst
33342 stain as described (22). A 5-µl aliquot of the stained cell
sample was examined by epifluorescence using a Leitz Aristoplan
microscope equipped with an A cube, and photographed at a magnification
of × 320. Fields with large numbers of cells (>150) were used to
ascertain the percentage of cells that were intensely fluorescing under ultraviolet excitation (characteristic of nuclear condensation due to
the chromosomal compaction and nuclear karyorrhexis seen in apoptosis).
The existence of nonfluorescent cells was confirmed by visible light
microscopy. Poorly fluorescing cells that had passed through the
greatest nuclear condensation were not included in calculations.
DNA Fragmentation Assay--
TF-1 cultures at either 8.0 × 104 to 1.0 × 105 cells/ml (log growth) or
1.0-1.2 × 106 cells/ml (stationary) were collected
and treated with or without 30 ng/ml TNF for 3 and 18 h. Cells
were pelleted at 500 × g for 5 min and washed twice in
ice-cold PBS. Total DNA was extracted using the G NOME DNA kit from Bio
101 (La Jolla, CA) as per the manufacturer's instructions. DNA
concentrations were calculated using 260 nm absorbance with a
Hewlett-Packard 8451 spectrophotometer (Hewlett-Packard, Palo Alto,
CA). Equal amounts of DNA (10 µg) were loaded per well on a 1.2%
agarose gel and run at 7 V/cm for 1 h. DNA bands were visualized
and photographed under UV illumination using a PhotoPrep system from
Fotodyne (Hartland, WI). Extent of DNA fragmentation was quantitated
using the cellular DNA fragmentation enzyme linked-immunoassay kit by
Boehringer Mannheim. Briefly, TF-1 cells were labeled with
5-bromo-2'-deoxyuridine for 24 h prior to treatment with TNF at
the indicated concentrations for 3 h. Cells were lysed, and
fragmented DNA was separated from genomic DNA by centrifugation at
250 × g for 10 min. The lysate was removed and tested
by the enzyme-linked immunoassay as described previously (23).
TNF Radiolabeling and Binding Measurements--
Mutational
analysis of human TNF revealed that certain mutations of the wild-type
sequence could enable the mutated protein to selectively bind to either
of the TNF receptor subtypes. The specific double mutation of R32W/S86T
(termed R1-TNF) allows selective activation by this mutant protein
("mutein") of the p55TNFR only, whereas the D143N/A145R (termed
R2-TNF) double mutation allows selective activation of the p75TNFR
subtype only (24, 25). The TNF receptor-subtype-selective muteins were
125I-radiolabeled essentially as described (26) for use in
radioligand binding studies to determine the TNF receptor subtype
composition of TF-1 cells under varying conditions. Essentially, 5 µg
of mutein was dissolved in 10 µl of 20 mM phosphate
buffer in a siliconized tube. To the protein solution was added 1 µl
(100 µCi) of 125I (Amersham Pharmacia Biotech) plus 10 µl of chloramine T (freshly dissolved in 10 mM phosphate
buffer), which was then mixed and incubated for 30 s at room
temperature before the addition of 10 µl of sodium metabisulfite
(freshly dissolved in 10 mM phosphate buffer) and 100 µl
of 1% albumin in PBS. The mixture was then loaded onto a G25 Sephadex
column (equilibrated in 1% albumin in PBS). Radiolabeled protein was
eluted with 1% albumin in PBS, collecting 1-ml fractions, which were
tested for incorporation. Specific radioactivity of the muteins was
1-5 µCi/µg. TNF binding occurred in 200 µl of culture medium
(4 °C) for 4 h before separation of unbound label in a Brandel
filtration system (Semat, St. Albyns, United Kingdom) by washing in 50 mM Tris (pH 7.4) plus 1 mg/ml bovine serum albumin
(4 °C). Nonspecific binding accounting for up to 80% of the total
binding as determined in the presence of 200 nM (4 µg/ml)
excess unlabeled wild-type human TNF. Saturation (0.01-30
nM label) binding measurements were performed to allow Scatchard analysis of the TNF receptor subtypes on whole TF-1 cells
(3 × 106 cells/tube).
The Metabolic Effects of TNF on TF-1 Cells Differ Depending upon
Growth State--
To investigate the functional responses of TF-1
cells to TNF, we monitored changes in metabolic activity during
continuous TNF exposure with a Cytosensor microphysiometer. Based on a
pH-sensitive, light-addressable potentiometric sensor (18), the
Cytosensor renders a general representation of ligand induced receptor
activation by monitoring the rate of extracellular acidification (27). Basal extracellular acidification rates depend on the energy-producing metabolic pathways, glycolysis, and aerobic respiration. Both metabolic
pathways produce protons through formation of the acidic byproducts
lactic acid and CO2, respectively. Thus, to a first approximation, extracellular acidification rate represents the sum of
cellular glycolytic and respiratory activity and is therefore a measure
of cellular metabolic activity. Stimulation by TNF or any growth
factor, if sufficiently coupled to energy dependent cellular processes,
perturbs extracellular acidification rates and is thus is detected by
the Cytosensor (27).
Preliminary experiments indicated a differential sensitivity of TF-1
cells to TNF depending on culture condition. Therefore, we investigated
TNF effects on cells at the extremes of both log growth and stationary
phase. As seen in Fig. 1A,
TF-1 cells obtained from a culture undergoing log growth (8 × 104 to 1.0 × 105 cells/ml, see Fig.
1A, inset) responded to TNF with a
concentration-dependent decrease in metabolic activity over a
period of 3 h. At 30 ng/ml TNF, an initial transient increase in
metabolic activity (~ 5%) was followed by a decrease of greater than
30% when compared with untreated control cells (Fig. 1A,
Table I). Continuous exposure to 30 ng/ml
TNF for 18 h decreased metabolic activity by approximately 90% of
the initial basal activity (data not shown).
In contrast, TF-1 cells obtained from the same culture, but in the
stationary phase of their growth curve (1.0-1.2 × 106 cells/ml; see Fig. 1B, inset) responded to
TNF with a stimulation of metabolic activity (Fig. 1B, Table
I). At high concentrations (>3 ng/ml), the response was biphasic: a
dramatic initial burst followed by a secondary sustained elevation of
metabolic activity (Fig. 1B, Table I). This response was
reminiscent of the metabolic response seen with GM-CSF (28), a cytokine
required for TF-1 cell proliferation, suggesting that TNF may induce
cellular proliferation under these conditions. Maximal effects of TNF
were seen at 30 ng/ml TNF (data not shown) consistent with other known
responses (22, 29).
Differential Effects of TNF upon Cell Viability and Proliferation
Depend on Cell Growth State--
We hypothesized three possible
mechanisms of decreased metabolic activity resulting from TNF exposure.
These included (i) a general suppression of cellular metabolism; (ii) a
conversion from glycolytic to aerobic metabolism, resulting in reduced
proton production per ATP turned over (19); and (iii) a cytotoxic mode of action. We observed that TNF exhibited dramatically different effects on cell viability as a function of the growth state. Log growth
cells responded to a 48-h treatment with TNF by a
concentration-dependent decrease in viable cell number as
compared with untreated cells (Fig.
2A). Alternatively, cells
initially in a stationary growth state showed a
concentration-dependent increase in viable cell number
following a 48-h TNF treatment when compared with untreated controls
(Fig. 2B).
Increased cell number in response to TNF could either be due to rescue
from cell death as a result of growth factor depletion or actual
induction of cellular proliferation. To distinguish between these
possibilities, we measured cell growth over a period of 3 days in the
presence and absence of TNF. The data indicate that the number of
viable TF-1 cells derived from a stationary culture decreased slightly
over time in the absence of TNF (Fig. 3),
but with 1 ng/ml TNF treatment, the number of viable cells increased
over the course of the experiment (Fig. 3). This appeared to represent
a TNF stimulation of cellular proliferation with TF-1 cells collected
from a stationary culture. In addition, the ability of TNF to induce
proliferation in quiescent TF-1 cells is due to its actions directly on
the cells and not through its induction of other secondary mitogenic
factors. This was confirmed in conditioned medium experiments in which
the medium from 24 h, 50 ng/ml TNF-treated cells in the extra
presence of 0.1 mg/ml of a neutralizing anti-TNF polyclonal antibody
was unable to elicit proliferation of quiescent TF-1 cells as measured
by MTT assay (104 ± 5% of control cell number). Medium with
neutralizing antibody alone resulted in 97 ± 8% of control,
whereas medium conditioned for 24 h with TNF alone (no
neutralizing antibody included) still partly retained its ability to
induce proliferation (122 ± 6% of control cell levels (mean ± S.D. from representative experiments repeated three other times with
similar findings)). Thus, the proliferative effect of TNF is directly
on quiescent TF-1 cells and not via induction of some other TNF-induced
mitogenic factor.
Mitotically Active TF-1 Cells Undergo TNF-dependent
Apoptosis--
TF-1 cells in log growth responded to TNF by both a
decrease in metabolic activity (Fig. 1A) and a decrease in
viable cell number (Fig. 2A). These findings suggested a
cytotoxic mode of action for TNF, which we further examined by using
inhibitors of protein synthesis and gene transcription. Treatment with
2 µg/ml cycloheximide or actinomycin D completely eliminated the decrease in metabolic activity seen with TNF (data not shown), implying
that the cytotoxic action of TNF required active macromolecular synthesis consistent with an apoptotic mechanism. Also intriguing was
the transient increase in metabolic activity during the first 30 min of
treatment with high concentrations of TNF (Fig. 1A). This
was indicative of an energy-requiring process consistent with other
work showing that the progression of apoptosis requires cellular energy
(30). Taken together with previous reports of TNF action, this suggests
that in mitotically active cells, TNF may be inducing apoptosis.
To more directly evaluate the possibility of TNF-induced apoptosis, we
employed two independent assays. Chromatin condensation and DNA
fragmentation, two physiological stages in the progression of apoptosis
(8), were assessed by analysis of Hoechst 33342 staining and gel
electrophoresis, respectively. As seen in Fig. 4A, a
concentration-dependent increase in the percentage of
highly fluorescent ("epifluorescent") Hoechst-stained apoptotic
cells occurred following TNF stimulation. The time course of
TNF-mediated apoptosis is shown in Fig. 4B. These results
indicated that 40% of the cell population was apoptotic after only
3 h of treatment with 50 ng/ml TNF as measured by Hoechst stain.
Decreases in the percentage of apoptotic cells seen at later time
points were due to advancement beyond the state of chromatin
condensation, and cells beyond this stage of apoptosis are not scored
by the Hoechst stain assay (see under "Experimental Procedures").
As an independent assay of apoptosis, we also determined the extent of
DNA fragmentation by agarose gel electrophoresis after TNF treatment.
As seen in Fig. 4C, cells treated with 30 ng/ml TNF
exhibited significant DNA fragmentation after 3 and 18 h of
treatment when compared with untreated controls. Both the time course
and concentration dependence of DNA fragmentation and Hoechst
stain were consistent with measurements of cell viability (Fig.
2A) and changes in metabolic activity (Fig.
1A).
DNA Synthesis and the Effects of TNF--
The previous data
demonstrate that TF-1 cells in a state of log growth respond to TNF by
induction of apoptosis, whereas induction of proliferation occurs with
the same cell population when allowed to grow to a stationary phase.
This suggested that only a mitotically active cell, i.e. one
undergoing active DNA synthesis, was sensitive to TNF-induced
apoptosis. Similarly, a quiescent cell not actively undergoing DNA
synthesis should not be sensitive to TNF-induced apoptosis. To test
these predictions we induced mitogenesis in a stationary culture by
GM-CSF supplementation and then compared the extent of TNF-induced
apoptosis in the proliferating culture relative to a non-GM-CSF
stimulated stationary culture.
Mitotic activity was determined by measuring
[3H]thymidine incorporation into DNA. TF-1 cells
initially obtained from a stationary culture, when supplemented with 1 ng/ml GM-CSF for 18 h, showed similar levels of
[3H]thymidine incorporation into DNA when compared with
cells from a log growth culture (Fig.
5A). Both of these conditions
were significantly greater than that seen with control cells from a stationary culture (Fig. 5A). This indicates that the
addition of GM-CSF to mitotically quiescent cells induces mitogenesis
to levels comparable to log growth cultures. The extent of TNF-induced apoptosis was determined under these culture conditions by quantitating the amount of DNA fragmentation as described under "Experimental Procedures." Results for log growth cultures showed approximately a
6-7-fold increase in DNA fragmentation with TNF concentrations greater
than 3 ng/ml (Fig. 5B). We did not detect a significant amount of TNF-induced DNA fragmentation in the stationary cultures (Fig. 5B). In contrast, GM-CSF supplementation of these
cells caused a dramatic enhancement of TNF-induced DNA fragmentation to
levels comparable to log growth cultures (Fig. 5B).
Furthermore, experiments utilizing drugs that block mitosis in HeLa
cells: nocodazole (31) and aphidicolin (32) preincubated for 1 h before 1 ng/ml GM-CSF treatment for 24 h, prevented the ability of
GM-CSF to render TF-1 cells susceptible to TNF-induced apoptosis. The
added inclusion of 20 µM nocodazole or 3 µg/ml
aphidicolin reduced control TNF-induced apoptosis levels after 15 h from 59 ± 2% to 18 ± 5 and 17 ± 3%, respectively
(apoptosis levels measured by epifluorescence in cells without 50 ng/ml
TNF for 15 h were 9 ± 2% (data are mean ± S.E.,
n = 5)). These results provide strong evidence
supporting the claim that active mitogenesis is required for TNF to
induce apoptosis.
p75TNFR Is Involved in TNF Signaling for Apoptosis--
To begin
assessing which receptor subtype(s) mediates the TNF apoptotic effect,
we performed immunoblot analysis on whole cell homogenates and
determined that TF-1 cells express both p55TNFR and p75TNFR (Fig.
6). Importantly, we found that expression
levels for p75TNFR were consistently higher in cells collected from log growth cultures than from stationary cultures, whereas p55TNFR expression levels did not vary significantly between these two culture
conditions. To confirm the rise in functional levels of p75TNFR
receptors in log growth cells in addition to increased p75TNFR protein
levels (Fig. 6), we utilized mutations of human TNF that are useful in
selectively recognizing the two TNF receptor subtypes (24-26).
Radioligand binding studies using the TNFs that selectively bind
p55TNFR (R1-TNF) or p75TNFR (R2-TNF) showed an increase in the p75TNFR
(but not p55TNFR) receptor level in log growth TF-1 cells (Fig.
7). The Bmax
values for R1-TNF were 2438 receptors/cell for log growth TF-1 cells
and 2356 receptors/cell for stationary phase cells; in contrast, the
Bmax values for R2-TNF were 3516 receptors/cell
for log growth TF-1 cells and 2873 receptors/cell for stationary phase
cells. These data, taken together, indicate that p75TNFR subtype levels
are modulated in mitotically active TF-1 cells; however, the p55TNFR
subtype remains relatively constant.
Because p75TNFR receptor levels seemed to parallel changes in
TNF-mediated cell death, we next sought to examine its individual role
in mediating apoptosis. We utilized a neutralizing anti-p75TNFR monoclonal antibody (33) and quantitated TNF-mediated apoptosis in the
presence and absence of anti-p75TNFR by Hoechst staining. Immunoblot
analysis indicated no cross-reactivity with p55TNFR by this antibody
(Fig. 6), so inhibition of TNF responses by this antibody should be
specific for p75TNFR. Fig. 8 shows that
pretreatment with anti-p75TNFR antibody inhibits TNF-induced apoptosis
of TF-1 cells in log growth by approximately 80%. Nonspecific rat IgG had no significant effect on TNF-induced apoptosis of TF-1 cells, and
neither antibody activated apoptosis in the absence of TNF (Fig. 8). In
addition, the cytotoxic action of TNF as seen by the Cytosensor assay
was significantly inhibited by the anti-p75TNFR neutralizing antibody
(data not shown). These results again implicated a role for p75TNFR in
TNF-induced apoptotic death in log growth TF-1 cells.
Further detailed analysis using the p55TNFR-selective mutein R1-TNF and
the p75TNFR-selective mutein R2-TNF (as well as unmutated wild-type TNF
and TNF procured from a commercially available source as control
measurements) indicated a role for both the TNF receptor subtypes in
TNF-induced TF-1 cell proliferation or death (Fig. 9). Activation of each TNF receptor
subtype individually could lead to a partial proliferative response or
cytotoxic response, which was, once again, dependent on metabolic
status of the cells (Fig. 9). The ability of each individual receptor
to partially perform the TNF-mediated proliferative or death actions in
TF-1 cells does not extend to all lymphoid cells that express both TNF
receptor subtypes and die in response to TNF. For example, the U937
cell line, which expresses approximately equal proportions of TNF
receptor subtypes (Fig. 6), seems to die in response to TNF treatment
through only a p55TNFR-mediated mechanism (Table II). These investigations again
implicated a partial role for both TNF receptor subtypes in TNF-induced
apoptotic death in log growth TF-1 cells. Taken together, these data
strongly implicate a role for p75TNFR in mediating the TNF-induced
apoptosis observed in TF-1 cells.
Although initially recognized and named for its ability to cause
necrotization of tumor masses, TNF has since been shown to also have
dramatic systemic effects. Chronic TNF production occurs in cachexia, a
wasting disease of cancer (1). TNF also plays a primary role in
inflammation and at extreme physiological concentrations is responsible
for septic shock (1). Understanding how TNF can have such extreme
physiological consequences requires an understanding of the cellular
basis of its action. At the cellular level, TNF has been shown to
modulate the fundamental processes associated with development,
including cell proliferation, differentiation, and apoptosis. However,
the molecular mechanisms by which TNF induces these events are only now
beginning to be understood, involving a complex array of
TNFR-associating factors. Indeed, although both TNF receptors are
believed to be involved in TNF-mediated signaling, it has been widely
thought that the majority of the responses are mediated by the p55TNF
receptor subtype (34).
In assessing the affects of TNF, we initially used the Cytosensor
microphysiometer, an instrument that measures cellular metabolic activity by detecting changes in rate of extracellular acidification. We found that metabolic activity decreased after TNF treatment of log
growth phase cells. From this finding, we predicted a cytotoxic mode of
action for TNF on cells in log growth. TNF treatment of stationary
phase cells resulted in a dramatic increase in metabolic activity;
thus, we were able to predict a proliferative response to TNF that was
subsequently confirmed. Although changes in metabolic activity above or
below initial basal levels are not definitive for apoptosis or
proliferation, in this study, we have shown that they can be
predictive. The microphysiometer results correlated well with more
traditional indexes of apoptosis, which confirmed the validity of this
method for monitoring progression of apoptosis in this system. Hoechst
staining and analysis of DNA fragmentation by gel electrophoresis,
although definitive, only measure the end stages of apoptosis; early
signaling events of apoptosis are not observed. Other measurements such
as intracellular calcium have also been used (35). Some forms of
apoptosis may be independent of changes in intracellular calcium and
thus may not be a clear indicator of apoptosis (29).
Extracellular acidification rate or metabolic activity reflects
homeostasis in ATP generating pathways. Activation of either apoptosis
or proliferation should also concomitantly change cellular ATP
homeostasis because these processes require energy. Indeed, using
microcalorimetry, Wallen-Ohman et al. (36) have obtained similar results in examining apoptosis in the KM-3 pre-B acute lymphocytic leukemia cell line. Microcalorimetric measurements revealed
an increase in metabolic activity, which preceded detectable DNA
fragmentation by several hours. Clearly, as cells undergo death, their
contribution to the metabolic activity of a population of cells
disappears and metabolic activity decreases. Rapid, continuous monitoring of metabolic activity may offer a way of screening agents
that affect early mechanisms in the apoptotic pathway.
Our results demonstrate that TNF stimulates proliferation of TF-1 cells
when cells are in a growth-arrested state. Increased cellular number
was due to true cellular proliferation and not rescue or prevention of
cell death as compared with control cultures. The stimulation of
proliferation by TNF was not as potent as GM-CSF, a growth factor known
to promote growth and survival of TF-1 cells (37). This suggests that
the mitogenic signal of TNF may be insufficient for long term survival
of TF-1 cells, although this was not established in this study. In
contrast, our data also indicate that the TF-1 cell line is only
sensitive to TNF-mediated apoptosis when mitotically active or actively
progressing through the cell cycle, although we have yet to explore the
effects of TNF sensitivity on cells arrested in specific phases of the
cell cycle, such as G1/S phase-arrested cells. These data
suggest a common link between proliferation and apoptosis. Both may
intersect at the level of mitosis and the cell cycle, as our results
indicate that the biological effect of TNF is strongly influenced by
cell cycle progression. This argues that cell cycle-related proteins may be involved in governing the physiological consequences of TNF.
Although a direct relationship between TNF and cell cycle-related molecules has not been reported, some studies have shown either an
acceleration or inhibition of the cell cycle in the presence of TNF
(38, 39).
The intracellular second messenger ceramide is a possible mechanism by
which TNF may affect cell cycle progression and apoptosis (40-42).
Signaling by the p55TNFR stimulates a neutral sphingomyelinase, which
in turn generates ceramide and sphingosine. A role for ceramide in
inducing apoptosis was originally shown in HL-60 myeloid leukemia cells. Subsequent work has shown that ceramide also induces cell cycle
arrest, which may activate apoptosis in some cell types. Ceramide then
may be an effector molecule in the cell cycle and apoptotic effects
seen with TNF.
More generally, a number of recent studies have reported a connection
between the cell cycle and induction of apoptosis (43-45). Meikrantz
et al. (44) showed that agents that promote premature mitosis in HeLa cells induce apoptosis and that sensitivity for induction of apoptosis paralleled the activation of cyclin
A-dependent kinases. In another study, premature activation
of p34cdc2 kinase was sufficient for induction
of apoptosis of cytotoxic T lymphocytes (45). Clearly, an
association exists between the cell cycle and apoptotic sensitivity.
The c-myc gene, in addition to its well established role in
mitogenesis, is a potent inducer of apoptosis and that expression of
Bcl-2 inhibits c-myc-induced apoptosis (46). Bcl-2 and other
proteins known to participate in apoptosis may be important in defining
a physiological role for c-myc (47-51). Although the
involvement of c-myc in TNF-mediated cytotoxicity has been
suggested (52), TNF-induced mitogenesis via a c-myc
mechanism has yet to be established.
Our results have also demonstrated that p75TNFR is involved in inducing
apoptosis in the TF-1 cell line. A role for p75TNFR in mediating
cytotoxicity is controversial (26, 29, 53-56). Our data neither rule
out a role for p55TNFR nor contradict the ligand-passing model proposed
by Tartaglia et al. (57). Indeed, the role for p75TNFR in
TF-1 cell apoptosis or proliferation may be only as a
"ligand-passing" role, because we have not attempted to demonstrate
a signaling function of p75TNFR here. Although a "death domain" has
been defined for p55TNFR (54) and not for p75TNFR, this does not rule
out the involvement of associated signaling proteins. In fact, a
signaling role for p75TNFR has been demonstrated by the discovery of
TNFR-associating factor proteins that associate with the receptor (58,
59). Whether these proteins are involved in apoptotic signaling remains
to be fully determined. The finding that p75TNFR is involved in
apoptosis and our results demonstrating that levels of p75TNFR
expression correlate with sensitivity to TNF-induced apoptosis may
explain why TNF induction of cell proliferation in mitotically
quiescent TF-1 cells does not lead to sensitivity to apoptosis.
However, the continued presence of p75TNFR subtype in conditions where TNF does not induce cell death (i.e. stationary phase cells)
is obvious from this study, suggesting that its mere presence does not
dictate sensitivity of TF-1 cell to TNF-induced cell death. Therefore,
just as the role of the p55TNFR solely to induce apoptosis in this
study cannot be ruled out, neither can the role of p75TNFR in inducing
cell death be proven as exclusive. Indeed, it seems that TNF-induced
cell proliferation or cell death in TF-1 cells does not occur
exclusively through one of the TNF receptor subtypes, unlike
TNF-mediated cell death in U937 cells (see Table II). It is now
becoming clear that, in some cell systems at least, the relative
proportion of p75TNFR subtype (acting in co-operation with the p55TNFR)
may be decisive in predetermining the apoptotic cell death that occurs
in response to TNF (26, 60, 61). Clearly, there are other cellular
factors that are regulated and contribute to the distinct TNF effects
that we see here, and it will be important to distinguish the subtle
cellular changes that occur in order to fully understand the principle
or cell life or death.
(TNF)
treatment, dependent upon the mitotic state of the cells. Mitotically
active cells in log growth respond to TNF by rapidly undergoing
apoptosis whereas TNF exposure stimulates cellular proliferation in
mitotically quiescent cells. The concentration-dependent
TNF-induced apoptosis was monitored by cellular metabolic activity and
confirmed by both DNA epifluorescence and DNA fragmentation. Moreover,
these responses could be detected by measuring extracellular
acidification activity, enabling rapid prediction (within ~ 1.5 h of TNF treatment) of the fate of the cell in response to
TNF. Growth factor resupplementation of quiescent cells, resulting in
reactivation of cell cycling, altered TNF action from a proliferative
stimulus to an apoptotic signal. Expression levels of the type II TNF
receptor subtype (p75TNFR) were found to correlate with sensitivity to
TNF-induced apoptosis. Pretreatment of log growth TF-1 cells with a
neutralizing anti-p75TNFR monoclonal antibody inhibited TNF-induced
apoptosis by greater than 80%. Studies utilizing TNF receptor
subtype-specific TNF mutants and neutralizing antisera implicated
p75TNFR in TNF-dependent apoptotic signaling. These
data show a bifunctional physiological role for TNF in TF-1 cells that
is dependent on mitotic activity and controlled by the p75TNFR.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(TNF)1 (1). TNF
has been shown to modulate proliferation, differentiation, and
apoptotic or necrotic cell death in a number of different cell types
(2-4). These disparate responses to TNF are mediated by TNF binding to
specific cell surface receptors. Two distinct TNF receptors, type I
(p55TNFR) and type II (p75TNFR) (Mr
55,000-60,000 and 70,000-80,000 in human cells, respectively), have
been identified (5, 6), although it remains unclear which of the many
responses reported for TNF can be attributed to a specific receptor
subtype (4). Moreover, the precise signal transduction pathways for each of these receptor subtypes have yet to be fully delineated.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3 pH unit/min.
Acidification rate data were mathematically normalized to 100% at the
data point just prior to TNF treatment (basal acidification rate).
Normalization of acidification rates allows for direct comparison of
rate data collected from separate chambers with different initial rates
and takes into account the variation in the number of cells deposited
above the active sensing region of the light-addressable potentiometric
sensor chip.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Effect of TNF on the metabolic activity of
TF-1 cells. The metabolic activity of TF-1 cells derived either
from a culture in log growth (A, see inset) or
from a stationary culture (B, see inset) was
monitored using a Cytosensor microphysiometer as described under
"Experimental Procedures." Metabolic activity is expressed as
acidification rate and normalized as described under "Experimental
Procedures." At time 0, cells were treated with TNF at the indicated
concentration for the duration of the experiment. Inset,
TF-1 cells were cultured as described under "Experimental
Procedures" at an initial cell density of 2 × 104
cells/ml. Cell counts were performed daily over a period of 8 days. The
data represent typical experiments that were performed at least five
separate times with similar findings.
Metabolic effects of TNF on TF-1 cells
percent acidification rate of control cells). Data
represent the mean ± S.E. from at least five independent
experiments.
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Fig. 2.
The effect of TNF on TF-1 cell
viability. TF-1 cells either in a state of log growth
(A) or stationary (B) were cultured in a 96-well
microtiter plate at a starting cell density of either 1 × 104 (A) or 1 × 106
(B) cells/well in the presence of TNF at the indicated
concentrations, and viable cell number was determined after 48 h
using the XTT colorimetric assay described under "Experimental
Procedures." Data represent the means ± S.E. of at least three
independent experiments performed with triplicate determinations.
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Fig. 3.
TNF induces cellular proliferation of
quiescent TF-1 cells. TF-1 cells were seeded at 1 × 106 cells/ml in the presence (shaded columns) or
absence (open columns) of 1 ng/ml TNF in 96-well microtiter
plates, and viable cell number was determined at the indicated time
points using the XTT colorimetric assay described under "Experimental
Procedures." Data represent the means ± S.E. of at least three
independent experiments performed with triplicate determinations.
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Fig. 4.
TF-1 cells in log growth respond to TNF by
induction of apoptosis. A, apoptosis was determined
after treating log growth TF-1 cells with TNF at the indicated
concentrations for 15 h, staining with Hoechst 33342, and scoring
for condensed chromatin as described under "Experimental
Procedures." Percentage of apoptosis was calculated by dividing
apoptotic cells by the total number of cells scored. Data represent the
mean ± S.D. of triplicate determinations from a representative experiment that was repeated at least twice with similar results.
A, inset, a representative field of highly fluorescent
Hoechst-stained cells scored in the Hoeschst stain apoptosis assay as
described under "Experimental Procedures" is shown. The
arrows point to TF-1 cells undergoing TNF-induced apoptosis
and displaying characteristic nuclear condensation, which results in
the epifluorescence measured. B, log growth TF-1 cells were
treated with 50 ng/ml TNF, and apoptosis was determined using Hoechst
stain at the indicated time points as described. Percentage of
apoptosis was calculated by dividing apoptotic cells by the total
number of cells scored. Data represent the mean ± S.D. of
triplicate determinations from a representative experiment that was
repeated at least twice with similar results. C, log growth
TF-1 cells were treated with or without 30 ng/ml TNF for the indicated
time periods, and total DNA was purified as described under
"Experimental Procedures." DNA size standards are shown. 10 µg of
DNA was loaded per well and separated on a 1.2% agarose gel. DNA was
visualized by UV illumination following staining with ethidium bromide.
This figure shows representative data from three independent
experiments.
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Fig. 5.
TNF induced apoptosis in mitotically
stimulated TF-1 cells. A, TF-1 cells were collected
from cultures either in log growth (open column) or
stationary (shaded column) growth phase (see Fig. 1, A
or B, inset). Cells in stationary phase were
supplemented with 1 ng/ml GM-CSF (striped column) for
18 h prior assessment of mitotic nuclear activity. Cells under
each of these conditions were subjected to [3H]thymidine
incorporation measurements as described under "Experimental
Procedures." B, TF-1 cells were collected from cultures
either in log growth ( ) or in stationary growth phase either with
(
) or without (
) addition of 1 ng/ml GM-CSF for 18 h prior
to treatment with TNF at the indicated concentrations for 3 h
before DNA fragmentation was quantitated by the enzyme-linked
immunosorbent assay described under "Experimental Procedures." The
data (means ± S.D. of triplicate determinations) are from an
determination that is representative of at least four independent
experiments.
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Fig. 6.
Immunoblot analysis of p55TNFR and
p75TNFR. Cells were solubilized in SDS-polyacrylamide gel
electrophoresis sample buffer, and immunoblot analysis was performed as
described under "Experimental Procedures" using a mouse monoclonal
antibody to the human p55 TNF receptor subtype (left panel)
or a rat monoclonal antibody to the human p75 TNF receptor subtype
(both antisera from Genzyme Corp., Cambridge, MA) (right
panel). Left lanes, U937 cells known to express both
p55TNFR and p75TNFR (62); middle lanes, TF-1 cells obtained
from a culture in log growth (see Fig. 1A,
inset); right lanes, TF-1 cells obtained from a
stationary culture. Immunoblotting was repeated three times with
similar results, and a representative experiment is shown. Shown at the
left are the locations of molecular mass standards.
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Fig. 7.
TNF receptor subtype-specific radioligand
binding. Scatchard analyses of the radioligand saturation binding
to TF-1 cells of mutant TNF ligands that specifically recognize either
the p55TNFR (R1-TNF) (A) or the p75TNFR (R2-TNF)
(B). Whole TF-1 cells in either log growth (open
symbols) or stationary (closed symbols) phase had the
TNF receptor subtype composition measured as described under
"Experimental Procedures." The data represent the binding from
experiments, which is representative of at least three independent
determinations (all experiments gave similar results). In the
experiment shown, the Kd values for R1-TNF and
R2-TNF binding were 50 and 117 nM, respectively, for log
growth TF-1 cells and 49 and 127 nM in stationary phase
TF-1 cells. The Bmax values for R1-TNF and
R2-TNF binding were 2438 and 3516 receptors/cell, respectively, for log
growth TF-1 cells and 2356 and 2873 receptors/cell in stationary phase
TF-1 cells.
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Fig. 8.
p75TNFR is involved in mediating apoptosis in
TF-1 cells. Cells derived from a culture in log growth (see Fig.
1A, inset) were treated with or without 30 ng/ml TNF in the
presence of either 10 µg/ml rat monoclonal anti-p75TNFR (Genzyme) or
10 µg/ml nonspecific rat IgG (Calbiochem, San Diego, CA) as
indicated. Hoechst stain analysis was performed after 15 h of
treatment as described under "Experimental Procedures." Percentage
of apoptosis was calculated by dividing apoptotic cells by the total
cell number. Data represent the means ± S.E. of four independent
experiments.
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Fig. 9.
TNF receptor subtype action in TNF-mediated
TF-1 cell proliferation or cell death. TF-1 cells were seeded into
a 96-well culture plate at the indicated density to allow cells to be
in a quiescent or log growth state (Fig. 1, A or
B, inset) 18 h after seeding. The cells were
then treated with a maximal (50 ng/ml) concentration of TNF (from a
commercial source, R & D Systems), wild-type TNF, or the R1-TNF and
R2-TNF mutants alone or in combination. After 24 h, the cell
number was determined with the MTT cell assay as described under
"Experimental Procedures." The data represent the mean ± S.D.
of eight determinations from an experiment that is typical of findings
in at least three other individual experiments.
TNF receptor subtype-specific actions on TF-1 and U937 cell apoptosis
DISCUSSION
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ABSTRACT
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RESULTS
DISCUSSION
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ACKNOWLEDGEMENTS |
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We thank Alexis Davis for technical assistance and Dr. Michael Anderson for many helpful suggestions. We are indebted to Dr. Meenu Wadwha for cells and reagents, to Dr. Kath Shennan for assisting in radiolabeling TNF, and to Dr. John Ngai for the generous use of his Cytosensor microphysiometer. We are grateful to Drs. Stuart Feinstein and Kathleen Foltz for critical review and comments regarding the manuscript.
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FOOTNOTES |
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* This work was supported by the Defense Advanced Research Projects Agency (MDA 972-92-C-0005) (to G. T. B.), Fonds voor Wetenschappelijk Onderzoek-Vlaanderen (9005097N) (to P. V.), the Royal Society of London, the Nuffield Foundation, and the Wellcome Trust (to D. J. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ These authors contributed equally.
A Howard Hughes Medical Institute Predoctoral Fellow
previously supported by an Arnold T. Beckman Fellowship.
§§ To whom correspondence should be addressed. Tel.: 44-1224-273154; Fax: 44-1224-273019; E-mail: david.macewan{at}abdn.ac.uk.
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ABBREVIATIONS |
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The abbreviations used are:
TNF, tumor necrosis
factor-;
TNFR, TNF receptor;
GM-CSF, granulocyte-macrophage
colony-stimulating factor;
MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide;
PBS, phosphate-buffered saline;
p55TNFR, type I 55-kDa TNF receptor subtype;
p75TNFR, type II 75-kDa TNF receptor subtype;
R1-TNF, R32W/S86T human
TNF mutant protein;
R2-TNF, D143N/A145R human TNF mutant protein;
XTT, sodium 3'-(1-phenyl-amino-carbonyl)-3,4-tetrazolium)-bis
(4-methoxy-6-nitro) benzene sulfonic acid hydrate.
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