(Received for publication, December 19, 1994)
From the
Apoptosis is a form of cell death associated with DNA
fragmentation and chromatin condensation. We recently established that
intracellular acidification occurred during apoptosis following
cytotoxic insult. The current studies were designed to determine
whether intracellular acidification was more generally associated with
apoptosis, specifically in a model of growth factor withdrawal. Upon
withdrawal of interleukin-2, CTLL-2 cells accumulated in the G phase of the cell cycle and started to fragment their DNA around
12 h concurrent with both decreased pH and increased
Ca
. Chelation of Ca
did not inhibit
DNA digestion, whereas incubation with a calcium ionophore prevented
both acidification and DNA digestion. Hence, acidification rather than
increased Ca
was associated with apoptosis. The
acidified cells represented a discrete population up to 0.7 pH units
below normal. The extent of acidification depended upon the
extracellular pH; above pH 6.3, intracellular pH was significantly
below extracellular pH, whereas below pH 6.3, the cells still regulated
their pH. Inhibition of the
Na
/H
-antiport prevented the apoptotic
cells from regulating their intracellular pH under these acidic
conditions. These results demonstrate that apoptotic cells retain a
functional antiport but that its set-point has changed. Many survival
factors are known to phosphorylate and activate the antiport, hence
apoptosis is likely to be associated with dephosphorylation. Although
acidification always occurred during apoptosis, maintaining
intracellular pH above 7.2 did not prevent apoptosis, suggesting that
an acid pH is not essential for apoptosis. We hypothesize that other
critical regulators of apoptosis must be subject to dephosphorylation.
Apoptosis is a form of cell death defined by morphological criteria(1, 2) . The earliest event observed in a cell undergoing apoptosis is chromatin condensation at the nuclear membrane, whereas other organelles remain intact. This is generally followed by nuclear fragmentation, cell surface blebbing and cell shrinkage. Loss of membrane integrity is a late event. These changes occur as a consequence of many initiating stimuli such as glucocorticoid-mediated killing of thymocytes(3) , growth factor withdrawal(4) , and cytotoxic T-cell killing(5) . In addition, a wide variety of cytotoxic agents induce apoptosis(6, 7) .
Much interest in apoptosis has been generated by the observation that genes such as bcl-2 can prevent cells from dying(8) . There are now many genes and drugs that can either cause or prevent apoptosis, but there is little understanding of the pathways that induce this form of cell death. Accordingly, there is no framework for understanding how any gene or drug modulates the eventual cell death. The current experiments were designed to address this important gap in our understanding.
The origin of the chromatin condensation and therefore an initiating event in apoptosis is thought to be DNA fragmentation. Usually, this fragmentation arises from internucleosomal digestion of genomic DNA (1) . Several reports that apoptosis occurs without such DNA digestion can be explained by the finding that DNA breaks at >50-kilobase pair frequency are sufficient to induce the morphological changes of apoptosis(9, 10) . To understand the signal transduction pathways leading to apoptosis, we have attempted to identify an endonuclease that produces the DNA digestion, so that we can subsequently understand the upstream events that regulate its activation.
Mammalian cells contain a variety of
endonucleases any of which could be involved in apoptosis(11) .
A Ca/Mg
-dependent endonuclease has
frequently been implicated in apoptosis, but many cells appear to lack
this endonuclease or lack the increase in Ca
required
to activate it. Even former proponents of a
Ca
/Mg
-dependent endonuclease have
recently suggested that Ca
may be involved in a
signal transduction pathway rather than as a direct activator of the
endonuclease(12) . Our laboratory identified an alternate
endonuclease, deoxyribonuclease II, that was activated by decreased
intracellular pH with no requirement for
Ca
(13) . In human HL-60 cells, apoptosis can
be induced rapidly by many drugs. Using the topoisomerase II inhibitor
etoposide, we established that apoptotic cells underwent selective
inhibition of pH regulation leading to a 0.8 pH unit acidification with
no change in intracellular Ca
(14) . Cells
were sorted on a flow cytometer, and only the acidic cells had the
morphology and DNA digestion of apoptosis. These results were
consistent with apoptosis resulting from intracellular acidification.
The current experiments were designed to investigate whether
intracellular acidification was more generally associated with the
induction of apoptosis. We have therefore extended our studies to a
common physiological model of apoptosis; specifically, a system in
which apoptosis is induced by removal of a growth factor. We report on
an IL-2()-dependent cytotoxic T lymphocyte cell line CTLL-2
and establish that intracellular acidification, but not increased
Ca
, also correlates with apoptosis in this model. We
further demonstrate that the acidification can be explained by
alteration in the set-point of the
Na
/H
-antiport. However, we also show
that an acidic pH is not required for apoptosis. This has led to a
hypothesis that may explain the regulation of apoptosis.
As an additional assay for apoptosis, cells were
incubated with 1 µg/ml Hoechst 33342 for 5 min and analyzed on a
flow cytometer with excitation at 355 nm and emission measured at 440
nm. This assay detects an increased rate of accumulation of Hoechst
33342 into apoptotic cells which is thought to be due to very early
changes in membrane permeability(19) . pH and
Hoechst 33342 fluorescence were measured simultaneously by first
incubating cells with carboxyl-SNARF-1 AM for 1 h in complete medium,
then washing once and resuspending the cells in medium at defined
pH
containing Hoechst 33342. As needed, the medium also
contained 20 µM EIPA to inhibit the
Na
/H
-antiport. Analysis was performed
after 5 min, a time shown in initial experiments to equilibrate the
pH
, and results were displayed as a two-dimensional contour
plot of 585/640 ratio for pH versus Hoechst 33342
fluorescence.
Figure 1:
DNA digestion induced in CTLL-2 cells
following withdrawal of IL-2. 10 cells were electrophoresed
in each lane except where stated. A, time course of appearance
of DNA fragments following withdrawal of IL-2. B, following 16
h without IL-2, cells were analyzed by flow cytometry for intracellular
pH and Ca
and sorted into the 40% most alkaline and
40% most acidic cells (see Fig. 3) or the 40% lowest
Ca
and 40% highest Ca
(see Fig. 4). In the case of the sorted cells, 2
10
cells were analyzed in each lane. C, cells were
incubated with either 5 mM EGTA or 1 µM ionomycin
for 16 h, with or without IL-2.
Figure 3: Flow cytometric analysis of intracellular pH changes in individual CTLL-2 cells following withdrawal of IL-2. At the indicated times following withdrawal of IL-2, cells were loaded with carboxyl-SNARF-1, then analyzed by flow cytometry, and displayed as a two-dimensional scatter plot according to the fluorescence intensities at emission wavelengths of 585 and 640 nm. Quantitation was performed by drawing regions around each population excluding the cells with fluorescence <50 and presenting the results as histograms: A, cells with normal pH; B, acidic cells.
Figure 4:
Flow
cytometric analysis of intracellular Ca changes in
individual CTLL-2 cells following withdrawal of IL-2. At the indicated
times following withdrawal of IL-2, cells were loaded with indo-1, then
analyzed by flow cytometry, and displayed as a two-dimensional scatter
plot according to the fluorescence intensities at emission wavelengths
of 405 and 485 nm. Quantitation was performed by drawing regions around
each population, excluding the few cells with fluorescence <50, and
presenting the results as histograms: A, cells with normal
Ca
; B, cells with increased
Ca
.
The cell cycle phase dependence of apoptosis
following removal of IL-2 was assessed. Cells were analyzed for DNA
content by flow cytometry (Fig. 2). By 4 h following removal of
IL-2, cells began to accumulate in the G phase, and by 8 h,
most of the cells had exited the S phase. At 16 h, the time at which
DNA digestion appeared maximum, most of the cells remained arrested in
the G
phase. Not until 20 h did a significant number of
cells begin to appear in the sub-G
population as is
frequently observed in apoptotic cells. However, these sub-G
cells rapidly disintegrate into many fragments, so quantitation
of this population is not possible. Up to the time that the cells
disintegrate, they also exclude trypan blue. These results show that
DNA digestion occurs several hours before loss of membrane integrity
and cell disintegration.
Figure 2: Cell cycle analysis of CTLL-2 cells following withdrawal of IL-2. Relative DNA content was assessed by fixing cells, staining with propidium iodide, and analyzing by flow cytometry.
Twelve hours following removal of IL-2, a second
population of cells began to appear to the right of the control
population. As previously seen with HL-60 and ML-1
cells(14, 20) , this population was not a continuum of
cells with decreasing pH, but rather a distinct population
with a more acidic pH
. The mean pH
of this
acidic population was 6.7. The fluorescence intensity of the acidic
cells was lower than the normal cells, suggesting that the acidic cells
have a reduced uptake or increased efflux of carboxyl SNARF-1. However,
the measurement of pH
is independent of the intracellular
dye concentration as it is the ratio of fluorescence emissions that is
used to calculate pH. Quantitation of the number of cells in each
population was obtained by drawing regions around these cells; these
values are presented as histograms in Fig. 3.
The number of acidic cells increased throughout the experiment with concomitant decrease in the control population. At 12 h, approximately 7% of the cells were in the acidic population. The number of acidic cells increased to 20, 30, and 48% at 16, 20, and 24 h, respectively. The time of acidification correlated closely with the time of the DNA digestion and occurred prior to fragmentation of the cells or loss of membrane integrity as assayed by trypan blue.
Figure 5:
Modulation of intracellular Ca by EGTA and ionomycin in CTLL-2 cells following withdrawal of
IL-2. Cells were incubated with 5 mM EGTA or 1 µM ionomycin for 16 h in the presence or absence of IL-2 and analyzed
by flow cytometry for intracellular Ca
. Histograms
are as described in legend to Fig. 4.
Figure 6: Modulation of intracellular pH by EGTA and ionomycin in CTLL-2 cells following withdrawal of IL-2. Cells were incubated with 5 mM EGTA or 1 µM ionomycin for 16 h in the presence or absence of IL-2 and analyzed by flow cytometry for intracellular pH. Histograms are as described in legend to Fig. 3.
Calcium ionophores often induce
apoptosis(21, 22) , but in some systems they have also
been shown to prevent apoptosis(23, 24) . Similarly,
calcium ionophores can cause either intracellular acidification or
alkalinization(22, 25) . Accordingly, we investigated
the effect of ionomycin on intracellular pH and Ca,
as well as on DNA digestion. Ionomycin (1 µM) in the
presence of IL-2 increased intracellular Ca
in the
majority of cells to 400 nM, and after removal of IL-2, a
subpopulation was observed with even higher Ca
(Fig. 5). In contrast, ionomycin inhibited intracellular
acidification following removal of IL-2 (Fig. 6). Ionomycin
appeared to decrease the carboxyl-SNARF-1 fluorescence in many cells,
but there was clearly no cells in the acidic population. Ionomycin also
markedly inhibited DNA digestion (Fig. 1C). These
results confirm that acidification rather than increase in
intracellular Ca
concentration correlates with
apoptotic DNA digestion.
Activation of protein kinase C by phorbol
esters TPA can also protect cells from
apoptosis(26, 27) . The addition of 10 nM TPA
to CTLL-2 cells at the time of removal of IL-2 inhibited both the
acidification and DNA digestion up to at least 24 h (data not shown).
These cells still accumulated in the G phase but produced
no sub-G
population of apoptotic cells. Intracellular pH
could also be modulated by the readdition of IL-2 at any time point,
which immediately prevented more cells from undergoing intracellular
acidification as well as preventing further DNA digestion, although
readdition of IL-2 could not reverse acidification (data not shown).
Figure 7:
Identification of proton transport
mechanisms operating in CTLL-2 cells. Cells were loaded with
carboxyl-SNARF-1 for 1 h, then analyzed continuously by flow cytometry
in the absence of bicarbonate; the lines represent the mean pH of the
cell population. Ammonium chloride (10 mM) was added to cells
at the indicated time, then removed rapidly by centrifugation after 10
min followed by one wash. The first part of the curve is representative
of the pH changes that occur prior to the
acid-loading step. Following acid loading, cells were allowed to
recover either in control medium (Cont) or medium containing 1
µM bafilomycin (Baf) or 20 µM EIPA.
A more detailed analysis of the pH
changes that occur in CTLL-2 cells was performed. Following the
induction of apoptosis, cells were equilibrated for 5 min at different
pH and the pH
was measured (Fig. 8).
During the final 5 min, the cells were also incubated with Hoechst
33342; this helps to discriminate the normal from apoptotic cells as
the latter show enhanced Hoechst fluorescence. When pH
was
between 7 and 7.9, the undamaged cells were able to maintain their
pH
close to 7.4; at lower pH
, these cells still
maintained their pH
above pH
. In contrast, the
pH
in apoptotic cells was lower than pH
except
under the most acidic conditions. If these apoptotic cells were
completely unable to regulate pH
, then the line for
pH
would be parallel to, but more acidic than the pH
by an amount dependent upon the electrochemical gradient across
the cytoplasmic membrane. However, the two lines were not parallel,
rather they converged around pH 6.3. To determine whether the
convergence of these two lines was due to the
Na
/H
-antiport, cells were
equilibrated in the presence of EIPA; under these conditions, the
pH
dropped dramatically at acidic pH
. In
contrast, incubation of control cells with EIPA for 5 min had no effect
on the pH
because EIPA only prevents recovery from an acid
load as occurs in the apoptotic cells, but does not itself cause
acidification. Parallel experiments in the presence of bicarbonate had
negligible effect on these profiles (data not shown). These results
demonstrate that the apoptotic cells still possess a functional
Na
/H
-antiport, but that its set-point
has changed.
Figure 8:
The dependence of pH
on pH
in normal and apoptotic CTLL-2 cells.
Fifteen hours after withdrawal of IL-2, cells were loaded with
carboxyl-SNARF-1 for 1 h, then equilibrated in medium of various
pH
containing 1 µg/ml Hoechst 33342 and as
required, 20 µM EIPA. After 5 min, pH
was measured by flow cytometry. A-F represent
examples of the experimental results. Cells at pH
= 7.40 (A, C, E) or 6.45 (B, D, F)
were equilibrated either in the absence (A, B) or presence (C, D) of EIPA. pH
is recorded on the abscissa and Hoechst fluorescence on the ordinate.
The pH
profiles are overlapped in E and F, to emphasize that EIPA only influences pH
in apoptotic cells at low pH
. The
summary of the complete experiment is shown in G; the diagonal line represents pH
=
pH
.
Figure 9:
The influence of pH
on DNA digestion in CTLL-2 cells following withdrawal of IL-2. Cells
were incubated at the indicated pH
in the presence
or absence of IL-2 for 16 h. DNA digestion was then analyzed by gel
electrophoresis.
Growth factors are usually required for passage of cells
through the G phase of the cell cycle. Upon removal of
IL-2, CTLL-2 cells initially accumulated in the G
phase.
Between 8 and 12 h after IL-2 withdrawal, these cells began to digest
their DNA in a manner characteristic of apoptosis. No other indicator
of toxicity was evident until around 20 h at which time a population of
cells was observed with sub-G
DNA content. Until this time,
the cells maintained membrane integrity.
The flow cytometer was used
to measure intracellular H and Ca
as
these two ions have been variously implicated in apoptosis. Although
the apoptotic cells exhibited increases in both of these ions, only
increased H
consistently correlated with the
appearance of DNA digestion. In particular, when EGTA was used to
chelate extracellular Ca
, no increase in
intracellular Ca
was observed even though the cells
still underwent intracellular acidification and apoptotic DNA
digestion. Furthermore, prevention of intracellular acidification with
ionomycin prevented DNA digestion.
Perhaps the most significant
observation is that the intracellular acidification produced a discrete
population of cells with a pH about 0.7 below that for control cells.
This same observation was made when HL-60 and ML-1 cells underwent
apoptosis following incubation with the anti-cancer drug
etoposide(14, 20) . These acidic cells remain viable
as judged by their retention of the fluorescent dyes, exclusion of
trypan blue, and by their ability to maintain low intracellular
Ca against a Ca
gradient which
requires metabolic activity. It is assumed that an electrochemical
gradient is maintained across the cytoplasmic membrane as this provides
the only explanation for the observations. Under conditions of a normal
electrochemical gradient (-60 mV), the Nernst equation calculates
that complete inhibition of pH regulation will cause the pH
to drop 1 pH unit(28) . This is consistent with the
intracellular acidification observed in these studies. Any other origin
of intracellular acidification such as through metabolic production of
protons, would lead to cells exhibiting a continuum of decreasing pH.
Furthermore, complete loss of ion regulation would lead to equalization
of the intracellular and extracellular pH.
Cells regulate their pH
through a variety of ion transport mechanisms, including
Na/H
-antiports, ATP-driven
H
-pumps, and several bicarbonate
exchangers(28) . The Na
/H
antiport is a major H
-extruding mechanism which
is driven by the inward-directed Na
gradient. This
antiport has a high affinity for H
at pH 6, but is
inoperative at neutral pH. However, a wide variety of external signals,
including growth factors enhance the affinity of the antiport for
H
at neutral pH leading to alkalinization of the
cells. This modulation of intracellular pH is brought about by
phosphorylation of the antiport (29, 30, 31) . Activation of protein kinase C
by phorbol esters has also been shown to phosphorylate and activate the
Na
/H
-antiport(30, 32) .
CTLL-2 cells appear to regulate their pH primarily with
the Na
/H
-antiport as demonstrated by
the ability of EIPA to inhibit recovery from an acid load. In contrast,
an inhibitor of the H
-ATPase did not prevent recovery
of pH
. A detailed analysis of the pH
over a
range of pH
also suggested that a modification of the
Na
/H
-antiport was responsible for the
acidification observed during apoptosis. A similar experiment has been
performed in mutant Chinese hamster ovary cells selected for lack of
the Na
/H
-antiport; the pH
in these cells was well below the pH
even under
acidic conditions(33) . These results can only be obtained if
the cells retain an electrochemical gradient. This pattern is exactly
what was observed here when apoptotic cells were equilibrated with
EIPA. However, in the absence of EIPA, the apoptotic cells were still
able to regulate their pH
under acidic conditions,
demonstrating that they still possess a functional antiport but that
its set-point has been altered. These observations are consistent with
the hypothesis that apoptosis is associated with dephosphorylation of
the antiport.
It has previously been suggested that the
Na/H
-antiport is important in
apoptosis. Inhibitors of the antiport have been shown to overcome the
protective effect of growth factors or phorbol esters in an
IL-3-dependent leukemia cell line as well as inhibiting the
alkalinization caused by IL-3 addition(34) . The previous
results only observed pH
changes of 0.2 units upon addition
or removal of growth factor, whereas the current results show a much
greater acidification as cells undergo apoptosis. This suggests that
previous work observed the fine tuning of the antiport, whereas we
suggest that the acidification arises from a complete loss of pH
regulation in the cells, possibly as a result of complete
dephosphorylation of the antiport. It should be noted that the
acidification occurs both in the presence and absence of bicarbonate,
so presumably bicarbonate exchangers contribute little to the
regulation of intracellular acidification during apoptosis.
Finally,
we questioned whether the observed acidification was required for DNA
digestion and apoptosis. Cells incubated in the absence of IL-2 at
pH 7.9 still underwent intracellular acidification, but
only to pH
7.2. Under these conditions, deoxyribonuclease
II would not be expected to be active, yet DNA digestion was still
observed. These results suggest that an acidic pH
per
se is not required for activation of any component of an apoptotic
pathway, whether it be an endonuclease, a protease, or some other
component. Rather, the results suggest that the signal for
acidification may be the same as the signal for apoptosis. The signal
implicated in these experiments is a phosphatase that might
dephosphorylate both the antiport and some other critical regulator of
apoptosis.
In summary, these studies have demonstrated that
intracellular acidification occurs during apoptosis following
withdrawal of an essential survival factor. Together with our previous
observations that acidification also occurs during apoptosis following
incubation of HL-60 and ML-1 cells with cytotoxins, these results
suggest that intracellular acidification is frequently associated with
apoptosis. The origin of the acidification appears to be alteration in
the set-point of the Na/H
-antiport,
possibly as a result of its dephosphorylation. This is consistent with
the known function of many survival factors that phosphorylate and
activate the antiport. However, the acidic pH
does not
appear essential for apoptosis, so we hypothesize that other critical
regulators of apoptosis must be subject to dephosphorylation. In this
regard, there are reports that phosphatase inhibitors can protect cells
from apoptosis(35, 36) .