[CANCER RESEARCH 53. 2349-2357.
May 15. 1993)
Etoposide-induced Apoptosis in Human HL-60 Cells Is Associated with Intracellular
Acidification1
Michael A. Barry, Jason E. Reynolds, and Alan Eastman2
Department of Pharmacology.
Dartmouth Medical School. Hanover. M-vr Hampshire 03755-3835
ABSTRACT
Apoptosis was originally defined by morphological criteria (16).
One of the earliest morphological events in apoptotic cells is chro-
Apoptosis is a pathway of cell death characterized by internucleosomal
digestion of genomic DNA. Such DNA digestion can be induced by both
physiological stimuli and cytotoxic treatment with many anticancer
agents. This digestion has generally been considered to be mediated by a
Ca2VMg2*-dependent endonuclease that is activated by increases in intracellular ( 'a; '. However, we suggest that an alternate endonuclease, DNase
matin condensation to the nuclear membrane. This morphological
change has been shown to be caused by internucleosomal DNA di
gestion by an endogenous endonuclease (1. 17). Activation of this
endonuclease has been proposed to play a central role in apoptosis,
since it is one of the earliest events in the death process. More
importantly, the DNA double-strand breaks produced by this endonu
II, may be a more likely candidate. In these studies, apoptosis was induced
in human HL-60 cells by a 30-min incubation with the topoisomerase II
inhibitor etoposide. DNA digestion characteristic of apoptosis began
within 3 h of removal of etoposide. Morphological indication of apoptosis
was observed concurrently. Only about 20% of the cells underwent apo
ptosis at this time; these appeared to be cells in S phase at the time of
etoposide treatment. The remainder of the cells progressed to the G2 phase
and arrested there for at least 48 h. Intracellular Ca2* and pH were
measured in individual cells by flow cytometry. No changes in intracellular
(.'a2' were observed, but an acidification of up to l pH unit occurred in
about 15% of the cells and correlated with the time course of appearance
of DNA digestion. Cells were sorted on the basis of intracellular pH and
only the acidic cells showed the morphology and DNA digestion charac
teristic of apoptosis. These results demonstrate the involvement of DNase
II in apoptotic DNA digestion and suggest mechanisms of pH homeostasis
as regulators of apoptosis.
INTRODUCTION
Apoptosis is the mechanism of cell death activated in mammalian
cells following exposure to a wide variety of stimuli. Apoptosis is
activated in physiological processes such as glucocorticoid killing of
thymocytes (1), selection of immature thymocytes (2), growth factor
withdrawal (3), antibody binding to cell surface proteins (4), and
cytotoxic T-cell killing (5). In addition, a wide variety of cytotoxic
agents kill cells by apoptosis despite the fact that each damages
different cellular targets (6, 7). These observations suggest that apo
ptosis may represent an underlying mechanism for cell death regard
less of the initial stimuli. If this is true, then each of these divergent
stimuli must converge on a fundamental signal transduction pathway
of cell death.
The significance of apoptosis has been emphasized recently by
reports that certain genes involved in oncogenic transformation appear
to do so by suppressing cell death. These genes include the hcl-2
oncogene (8, 9), the adenovirus El B gene (10), and the Epstein-Barr
virus latent genes (11). Furthermore, genetic changes such as overexpression of c-myc (12), or reintroduction of the wild-type p53 tumor
suppressor gene (13) have been shown to induce apoptosis, but this
can be overcome by appropriate growth factors or by other compen
satory oncogenic changes (13-15). The signal transduction pathways
involved in oncogenic transformation are therefore closely associated
with the pathway of apoptosis.
Received 12/7/92; accepted 3/11/93.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
1This study was supported by N1H grant CA50224 and Cancer Center Support Grant
clease represent a point of irreversible commitment towards death.
Therefore, an understanding of the events which trigger activation of
this endonuclease is critical, for it is these events which determine the
fate of the cell.
Mammalian cells contain a variety of endonucleases which could be
involved in apoptosis (reviewed in Ref. 18). A Ca2+/Mg2 +-dependent
endonuclease was first implicated in apoptosis because of its presence
in the nuclei of cells that undergo apoptosis even though other endo
nucleases were also known to be present (19). It was thought that this
endonuclease could be activated by a sustained rise of intracellular
Ca2+ seen during apoptosis (20-22). However, the resulting intracell
ular free Ca2 + concentrations were 1000-fold lower than required to
activate the endonuclease in vitro (23-25). In addition, DNA digestion
occurs in many models of apoptosis with no apparent Ca2+ require
ment and in cells with no apparent Ca2+/Mg2* -dependent endonu
clease (26-28).
Studies from our laboratory demonstrated that CHO' cells undergo
endonuclease activation in response to a variety of cytotoxic anticancer agents (6), but they do not contain detectable Ca2+/Mg2^dependent endonuclease (29). In contrast, the nuclei of these cells had
high levels of the ubiquitous endonuclease DNase II which has no
Ca2+ requirement. DNase II mediates internucleosomal DNA diges
tion when cells or nuclei are incubated at acidic pH values (29). For
DNase II to be involved in apoptosis. dying cells would have to
undergo intracellular acidification.
To analyze the potential involvement of intracellular acidification
or increased intracellular Ca2+ in endonuclease activation, apoptosis
was induced in HL-60 human promyelocytic leukemia cells by the
Ca2+ ionophore ionomycin (30). Although this type of apoptosis was
expected to be Ca2+ dependent, endonuclease activation occurred
with no correlation to changes in intracellular Ca2 * and even occurred
in the absence of free intracellular Ca2 +. In contrast, intracellular
acidification occurred in these cells with excellent correlation to the
amount of endonuclease activation. This correlation suggested that
DNase II could be involved in apoptosis, but it suffered from the fact
that only marginal (0.2-0.3 pH units) decreases of intracellular pH
were observed. The measured pH values in the dying cells were still
higher than pH 6.5 which is about the maximum pH where DNase II
will mediate significant internucleosomal DNA digestion in vitro (29).
The intracellular pH measurements in those studies represented the
average intracellular pH of the entire population of cells. Therefore, it
is possible that the marginal acidification observed in the cell popu
lation could be an underestimate of a subpopulation of more acidic
cells.
CA23108. M. A. B. and J. E. R. were supported by National Cancer Institute Cancer
Biology Training Grant CA09658.
2 To whom requests for reprints should be addressed.
1The abbreviation used is: CHO. Chinese hamster ovary.
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The present study of apoptosis activated by the topoisomerase II
inhibitor etoposide addresses this problem by using flow cytometry to
measure intracellular pH. By this method we observed the production
of a subpopulation of cells the intracellular pH of which was as low
as pH 6.4. The appearance of these highly acidic cells coincided with
both endonudease activation and morphological apoptosis. More sig
nificantly, when the etoposide-damaged cell population was sorted by
intracellular pH. only the acidic cells were morphologically apoptotic
and undergoing endonuclease activation. In contrast, no detectable
changes in intracellular Ca2 ' occurred during etoposide-activated
apoptosis.
MATERIALS
APOPTOSIS
Intracellular pH Measurement. Intracellular pH measurements were per
formed as detailed (30) with modifications to allow carboxy-SNARF-1 loading
and measurement by flow cytometry in complete medium containing bicar
bonate at 37°C.For the final hour of each experimental protocol. I x 10" cells
were loaded with l UM of the acetoxymethylester
derivative of carboxySNARF- 1 in 2 ml of complete medium at 37°Cin a CO2 incubator. The cells
were then pelleted and resuspended in fresh, complete medium and assayed
from a vial maintained at 37°Con a Becton Dickinson Facstar Plus flow
cytometer. Intracellular carboxy-SNARF-l
was excited at 488 nm and the
emission measured at both 585 and 640 nm with 5-nm band-pass filters using
linear amplifiers. The 585/640 ratio was calculated electronically as an instru
ment parameter. Data were analyzed using Lysys II software. Cells with
carboxy-SNARF- 1 fluorescence of less than 50 units were excluded to prevent
the derivation of artifactual ratio values. Intracellular pH was estimated by
comparison of the mean ratio values of a sample to a calibration curve of
intracellular pH generated by incubation of carboxy-SNARF- 1-loaded HL-60
AND METHODS
Materials. Etoposide was generously supplied by Bristol-Myers Squibb
(Wallingford, CT). The aceloxymethyl esters of INDO-I and carboxySNARF-I were purchased from Molecular Probes (Eugene, OR). Cell culture
materials were obtained from Gibco BRL (Grand Island. NY). LeukoStat was
purchased from Fisher Scientific (Pittsburgh. PA). All other drugs, chemicals,
and enzymes were purchased from Sigma Chemical Co. (St. Louis. MO).
Cell Culture and Drug Treatment. HL-60 human promyelocytic leuke
mia cells were obtained from American Type Culture Collection (Rockville,
MD). The cells were maintained below 5 x IO5 cells/ml in suspension at
36.5°Cin a humidified atmosphere of 5% CO2-95% air in RPMI 1640. sup
plemented with 10% fetal bovine serum, penicillin and streptomycin.
bicarbonate. IO ITIMA'-(2-hydroxyethyl)piperazine-A''-(2-ethanesulfonic
DURING
18 m.M
acid).
Cell numbers were measured with a Coulter Counter (Hialeah. Fl).
Etoposide was prepared immediately prior to each use as a 40 msi stock in
dimethyl sulfoxide. The cells were pelleted by centrifugation for 5 min at 1000
rpm. the supernatant was removed, and the cells were resuspended at 1 x IO6
cells/ml in complete medium containing the indicated concentrations of eto
poside. After 30 min. the cells were repelleled. washed, and resuspended in
fresh medium. All samples, including controls, contained 0.2% dimethyl sul
foxide which had no observed effect on any of the assays performed.
Cytotoxicity was assessed by a limited dilution colony formation assay. For
each concentration of etoposide. the cells were incubated as above for 30 min
and washed, and 4000 cells were added to each of 8 wells in a 96-well plate.
From these 8 wells, serial 2-fold dilutions of the cells were performed in the
remaining 11 rows of 8 wells and the plates were returned to the CO2 incubator.
Fourteen days later, the wells were stained with 100 ul of 0.4% trypan blue and
wells with living cells were counted. The relative survival was expressed as the
minimum number of inoculated cells required to produce surviving colonies in
all 8 rows for that dilution of cells. The concentration of etoposide which
required twice the number of inoculated cells to produce a row of surviving
colonies was designated as the 50% lethal concentration of drug.
Measurement of DNA Digestion. DNA digestion in cells was measured as
previously described (29). In this electrophoretic assay, whole cells are lysed
in the wells of the gel and high molecular weight genomic DNA (>20 kilobase
pairs) remains trapped in or near the well. Smaller fragments down to 180 base
pairs in length are resolved in the gel. DNA is visualized with ethidium
bromide.
Morphology. Apoptotic morphology of HL-60 cells was assessed as de
scribed by Cotter et al. (31). Briefly, 100 ul of HL-60 cells at 1 x IO6 cells/ml
were loaded in a cytospin centrifuge and centrifuged on to a glass microscope
slide at 350 rpm for 5 min. The cells were fixed with melhanol and stained
using LeukoStat. a modified Wright stain.
Cell Cycle Analysis. Cell cycle analysis was performed on cells stained
with propidium iodide as previously detailed (32). The cells were analyzed on
a Becton Dickinson Facscan How cytometer (Braintree. MA) using Cell Fit
software. The relative distributions of cells in normal phases of the cell cycle
were calculated using RF1T cell cycle analysis software when the S phase
distribution was normal and nearly rectangular. When the S phase population
was not rectangular. SFIT was used to avoid overestimation of the S phase
cells. Only cells in G,. S, and G2-M can be analyzed by either SFIT or RFIT.
Therefore to estimate the percentage of cells with DNA content less than G,.
cells in this region were gated as was the sum of cells in G,. S, and G2-M. From
these gates the numbers of cells in each region were used to correct the percent
of total cells in each of the normal cell cycle phases.
cells in varied pH buffer in the presence of the proton ionophore nigericin (Ret.
33: Fig. \A). It should be noted that some cells (e.g.. CHO cells and rat
thymocytes) inefficiently load carboxy-SNARF- 1 and other fluorescent probes
making these types of assays cell line dependent.
To assess the intracellular pH of individual cells in the population, the
fluorescence of each cell was displayed on a 2-dimensional dot plot with 585
nm fluorescence on the .»-axisand 640 nm fluorescence on the v-axis (Fig. Iß).
Since the distance of each cell from the origin is directly proportional to the
amount of carboxy-SNARF- 1 loaded in the cell, cells on the same line out from
the axis possess the same intracellular pH. A shift to the right represents cells
with lower intracellular pH while a shift to the left represents cells with higher
intracellular pH. To quantify the number of acidic cells produced following
etoposide, two regions were placed on the dot plot. Region 1 contained 99% of
the control cells. Region 2 was placed directly to the right of region 1 such that
all of the acidic subpopulation was enclosed. The percentage of acidic cells was
calculated by dividing the number of cells in region 2 by the sum of the number
of cells in both region I and region 2.
Cells were sorted by their 585/640 ratio on the Facstar Plus. To minimize
any overlap between the cells, the 25% most acidic and the 25% most alkaline
cells were collected. The sorted cells were collected into complete medium in
tubes on ice and then tested for DNA digestion and morphology within 2 h. No
changes in DNA content or morphology occur during this storage.
Intracellular Ca2+ Measurement. Intracellular Ca2+ was measured using
l UM of the fluorescent Ca2^ probe INDO-1 (30) but modified for flow
cytometry as above for carboxy-SNARF-1. Intracellular Ca2* was measured
by excitation at 355 nm with ratioing of the emission at 405 and 485 nm.
Intracellular Ca2* was calculated using the Ca2* dissociation equation:
.(R - Rmm)
where [Ça2"*"],,,
is intracellular [Ca2*], K¿is the Ca2* dissociation constant for
INDO-1 (Kj = 250 n.w), Rmax is the ratio of emission fluorescence of cells
treated with 20 U.Mof the Ca2 * ionophore ionomycin in 440 U.Mextracellular
Ca2 * . R is the fluorescence ratio of the sample of interest, and R,,,m is the ratio
of fluorescence of cells treated with ionomycin and addition of ethyleneglycol
bis(/3-aminoethyl ether)-/V.AW./v"-tetraacetic acid from a 0.5 Mstock until the
fluorescence ratio reaches its minimum value (34). Intracellular Ca2* calcu
lated from this equation is displayed in Fig. 1C. The dot plot distributions of
cells treated with ionomycin or ionomycin with ethyleneglycol bis(ß-aminoethyl ether)-iV,A',A''JV'-tetraacetic acid are shown in Fig. ID. Cells with in
creasing intracellular Ca2* shift to the right in the INDO-1 dot plot, while cells
with less intracellular Ca2* shift to the left.
RESULTS
DNA Digestion. HL-60 cells undergo rapid DNA digestion follow
ing exposure to a variety of cytotoxic agents (30, 35). To assess the
extent of DNA digestion following a short exposure to etoposide,
HL-60 cells were incubated for 30 min with a range of concentrations
of etoposide and DNA digestion was measured 3.5 h after removal of
the drug (Fig. 2A). Untreated control cells contained only high mo
lecular weight DNA. In contrast, etoposide at 20 ug/ml (the 50% lethal
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B.
A.
800
1.Û1
pH7.8
pH6.2
:¿$P
.o
600
CO
0.8
E
c
o
to
C
IO
400
0.6
35
m
S
0.4
6.0
6.5
7.0
7.5
100
150
200
8.0
Intracellular pH
50
200
D.
200
Intracellular Calcium (nM)
250
400
600
800
585 nm Fluorescence
200
400
600
800
405 nm Fluorescence
Fig. I. Calibration of intracellular pH and Ca: ' measurements by flow cytometry. In A and B. carboxy-SNARF-1-loaded HL-60 cells were incubated in varied pH buffer in the
presenceof the proton ionophore nigericin. The average ratio of the emission wavelengths is shown in A and the distribution on a two-dimensional dot plot according to the fluorescence
intensities at the two emission wavelengths is shown in B. In C and D, INDO- l-loaded HL-60 cells were incubated with the calcium ¡onophoreionomycin in the presence of 440 usi
extracellular dì2' or in the presenceof elhyleneglycol bistß-aminoethyletnerÃ--W.A'.WJV'-tetraaceticacid to chelate all the Ca-~. The standard curve in C was obtained from the Ca:*
dissociation equation, and the two-dimensional dot-plots are shown in D.
demonstrates that short exposure to etoposide causes internucleoso
concentration of drug) and higher concentrations caused the produc
tion of lower molecular weight DNA fragments consisting of multimal DNA digestion characteristic of apoptosis.
Morphology. The morphology of etoposide-treated cells was an
mers of 180 base pairs. These multimene fragments are the product of
alyzed in cytospin preparations. The nuclei of control cells were
internucleosomal DNA digestion by an endogenous endonuclease
nearly round and consisted of mottled red-purple staining with one or
considered characteristic of apoptotic cell death (22, 36). At this time,
membrane integrity as assayed by trypan blue exclusion was 100% for
two nucleoli present (Fig. 3A). Within 4 h of exposure to etoposide,
apoptotic cells appeared in the population with chromatin condensa
all of the cells.
The kinetics of DNA digestion was tested in cells incubated with 80
tion either to the periphery of the nuclear membrane or in clumps
|ag/ml of etoposide (99.9% lethal concentration of drug) (Fig. 2B). At
within the cell (Fig. 3, B and C). The amount of condensed chromatin
the end of exposure to etoposide (0.5 h), there was an increased
varied markedly with some cells showing little if any chromatin
amount of high molecular weight DNA fragments which migrated
staining, probably indicating total degradation and loss of cellular
about 5 mm into the gel. This DNA consists of fragments of 6-20
DNA. However, by 24 h these latter cells had disappeared from the
kilobase pairs which are too large to be resolved in the 2% agarose gel.
population, suggesting that they had disintegrated.
Cell Cycle Analysis. Early apoptotic HL-60 cells appear in the cell
The amount of DNA in this band decreased after etoposide removal
until the 3-h time point, when it reappeared along with faint nucleocycle distribution as cells with DNA content less than G, (37). This
some fragments. Continued incubation appeared to conven the large
gross alteration in DNA content results from degradation of cellular
DNA fragments to the smaller nucleosome fragments with maximal
DNA by activation of the endogenous endonuclease during apoptosis.
Similar cell cycle analysis was performed on HL-60 cells following 30
digestion within 5 h. The observation of high molecular weight DNA
fragments immediately after exposure to etoposide suggests that these
min exposure to 80 ug/ml etoposide (Fig. 4). In control cells, approx
fragments represent the etoposide-topoisomerase
II cleavable com
imately 50% were in G, phase, 40% were in S phase, and 10% were
in the G2-M phase of the cell cycle (Fig. 4fi). Four h after exposure
plex. The large fragments observed at later time points probably
represent early products of apoptotic endonuclease digestion which
to etoposide, cells began to appear with DNA content of less than G,
as described for apoptotic HL-60 cells (37) and consistent with the
are converted to nucleosome fragments with continued digestion. This
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B.
etoposlde (iig/ml)
0 10 20 40 80 160
time (h)
0
0.5
12345678
Fig. 2. DNA digestion induced in HL-60 cells following incubation with etoposide. In A, cells were incubated with 0-160 ug/ml etoposide for 30 min. the drug was removed, and
(he cells were incubated for an additional 3.5 h. Cells were then harvested. The DNA was separated by gel electrophoresis and visualized with ethidium bromide. In B, cells were
incubated with 80 ug/ml etoposide for 30 min (0.5 h time point), the drug was removed, and cells were incubated for additional times up to 7.5 h (8 h time point). The 0 point reflects
undamaged cells. DNA fragmentation was then analyzed by electrophoresis.
above morphological observations of cells with little if any chromatin
staining. The accumulation of these cells appeared to plateau at 8 h
with little additional increase up to 48 h. Approximately 18% of the
cells had DNA content less than G,. Although there was not an
obvious change in the total number of S phase cells over 8 h (Fig. 4ß),
there was a decrease in the number of late S phase cells at the same
time that cells appeared with DNA content less than G, (Fig. 4A). This
suggests that the late S phase cells may be the subpopulation of cells
undergoing apoptosis rapidly following exposure to etoposide. These
cells were presumably in early S phase at the time they were incubated
with etoposide. There was also an increase in the number of cells
entering S phase 6-8 h after etoposide which suggests the occurrence
of a transient G, arrest. The majority of the cells which did not
undergo apoptosis became arrested in the G2 phase within 24 h. With
extended time, the number of G2 arrested cells gradually decreased
until eventually all of the cells had died (data not shown). Similar G2
arrest prior to cell death has been observed in other cells following
exposure to etoposide (38). This delayed cell death may also be due to
apoptosis since internucleosomal DNA digestion has also been seen at
later times (6). In summary, cell death after a 30-min exposure to 80
ug/ml etoposide occurred in two phases. Rapid apoptosis occurred
within Shin only a subpopulation of the cells, whereas the remaining
cells underwent G2 arrest and died asynchronously from G2-M with
out reentering normal cell cycle.
Intracellular pH. We have previously observed intracellular acid
ification concomitant with endonuclease activation in HL-60 cells
damaged by the Ca2+ ionophore ionomycin (30). These measure
ments yielded acidification of approximately 0.3 pH units relative to
untreated cells. In the current studies, intracellular pH measurement
was performed by flow cytometry. This facilitated intracellular pH
measurements without removing the cells from their normal medium,
serum, or bicarbonate; such conditions give unacceptable background
fluorescence when samples are analyzed in a spectrofluorimeter.
Intracellular pH was first assayed in the total population of HL-60
cells following incubation with a range of concentrations of etoposide
(Fig. 5A). Control cells maintained an intracellular pH of 7.24 when
extracellular pH was 7.25. Within 3.5 h after exposure to etoposide,
the cells underwent concentration-dependent
intracellular acidifica
tion. The intracellular pH reached maximal acidification of 0.15 pH
units after exposure to 80 and 160 (jg/ml etoposide. When the intra
cellular pH was measured with time following exposure to 80 ug/ml
of etoposide, the pH decreased within 4 h by approximately 0.2 pH
units (Fig. 5ß).
The observed intracellular pH values in Fig. 5, A and B, reflect the
average intracellular pH of the entire population of cells similar to the
type of results which are obtained using spectrofluorimetry (30).
However, one advantage of measuring intracellular pH by flow cy
tometry is that the relative intracellular pH values of each cell can be
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Fig. 3. Morphology of HL-60 cells undergoing
apoptosis. A, control cells. In B and C, cells were
incubated with 80 ug/ml etoposide for 30 min. the
drug was removed, and the cells were incubated for
an additional 3.5 h before fixing and staining. In
1) and £.cells that been damaged with etoposide
3.5 h earlier were analyzed and sorted by flow
cytometry. The 25^- most alkaline cells (D) and the
25% most acidic cells (E) were collected, fixed,
and stained.
displayed in a two-dimensional dot plot according to their fluores
cence intensities at two different wavelengths (Fig. 1). When dis
played in this format, cells with the identical intracellular pH lie on the
same line extending outward from the origin. More acidic cells lie to
the right whereas more alkaline cells lie to the left. When the intra
cellular pH of the individual cells were represented in this fashion for
etoposide-treated cells, an increasing number of cells appeared to the
right of the control population (Fig. 6). At the lower concentrations of
etoposide, these cells formed a continuum of cells to the right of the
control population. At 80 and 160 ug/ml of etoposide. these cells
resolved into a distinct subpopulation. The cells in the control popu
lation maintained intracellular pH of approximately 7.24. In contrast,
the mean intracellular pH of the subpopulation to the right of the
control population was approximately 6.4. When the percentage of
cells in the acidic population was calculated, accumulation of acidic
cells occurred in a concentration-dependent manner (Fig. 5C). At the
3.5-h time point, the maximum number of cells in the acidic popula
tion was 12% for the 160 (jg/ml etoposide sample. When this analysis
was performed on cells at varied times after exposure to 80 ug/ml
etoposide (Fig. 5D), a similar accumulation of acidic cells occurred
with a maximum of 15% of acidic cells at 5 h. In summary, following
exposure to etoposide, a subpopulation of cells with intracellular pH
below pH 6.5 accumulated with similar kinetics and concentration
dependence as was observed for endonuclease activation. In addition.
the observed number of acidic cells was very similar to the number of
apoptotic cells which had DNA content less than G,.
Intracellular Ca2f. To test whether increased intracellular Ca2 *
occurs during etoposide-induced endonuclease activation, we mea
sured intracellular Ca2 + by flow cytometry in a similar manner to
intracellular pH measurement described above. Control cells main
tained intracellular Ca2+ at approximately 15 nin (Fig. 5£).Three and
one-half h after a 30-min incubation with a range of concentrations of
etoposide, the cells had intracellular Ca2 ' levels essentially identical
to that of the control cells. The levels of intracellular Ca2 4 were also
measured following exposure to 80 ug/ml etoposide and showed no
significant alteration of intracellular Ca2 * with time (Fig. 5F). When
intracellular Ca2 * was expressed in a two-dimensional dot plot format
as for intracellular pH, there was no evidence of any subpopulation of
cells with higher intracellular Ca2< (Fig. 7). By this method, intra
cellular Ca2 + increases of 50 nM and larger are detectable. Therefore,
there was no detectable change of intracellular Ca2 ' at the time that
endonuclease activation occurred in HL-60 cells dying by apoptosis.
Cells Sorted by Intracellular pH. HL-60 cells were incubated for
30 min with 80 ug/ml etoposide and postincubated for 3.5 h. At this
time, the cells were sorted on the flow cytometer based on their
intracellular pH. Of the total population, the 25% most acidic cells and
the 25% most alkaline cells were collected and DNA digestion and
morphology were assessed in both groups. Nucleosome fragments
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from 18-65 kiludaltons. None of these has yet been fully character
ized, nor has it been adequately established that any of them is
involved in apoptosis.
CHO cells undergo apoptotic DNA digestion following incubation
with a variety of cytotoxic agents (6). When this laboratory attempted
to purify a Ca2 ' /Mg2 ' -dependent endonuclease from these cells, little
it any was found. However, we found high levels of DNase II (29).
DNase II has maximum activity around pH 5 but also exhibits suffi
cient activity up to pH 6.5 to produce internucleosomal DNA diges
tion when added back to nuclei. This endonuclease was previously
overlooked because incubation conditions were always kept above pH
7.4. Perhaps the only information to date that might suggest DNase II
could not be involved in apoptosis derives from an analysis of the
products of DNA digestion. It has been reported that the DNA di
gested during apoptosis has 3'-hydroxy and 5'-phosphate termini
characteristic of digestion by DNase I-type enzymes (26). However,
the presence of chromatin-associated
polynucleotide kinase/3'phosphatase (39) would yield these termini even if the DNA were first
A.
a.
t:
7.3-1
7.3
7.2-
7.2-
7.1-
7.1-
7.0 J i
0
c.
36
48
60
0
TJ
72
Time (h)
i-
40
—¿.1 . .
80
120 160
7.0 J r0
D.
15
i
24
.
10
151
10
I
Fig. 4. Cell cycle analysis of HL-60 cells following damage with etoposide. Cells were
incubated with SOug/ml etoposide for 30 min (0.5 h time point), the drug was removed,
and cells were incubated for additional times up to 63.5 h (64-h time point). The cells were
fixed and stained with propidiurn iodide and the DNA content was analyzed by flow
cylomeiry. The number of cells in each phase of the cell cycle seen in the histograms (A )
was calculated (B). The cell cycle phases were G¡(•).S {•),G:-M (A), cells with less
than G, DNA content (O).
1
5H
0J
40
E.
were present only in the acidic cells (Fig. 8). The amount of DNA
digestion in IO5 acidic cells was similar to that observed in 4 X IO5
O
of the total cells, thereby confirming that all of the DNA degradation
derived from the acidic cells. When the morphology of the acidic and
alkaline cells were compared, the alkaline cells were morphologically
normal (Fig. 3D), whereas the acidic cells contained almost exclu
sively apoptotic cells (Fig. 3£).This demonstrates a perfect correla
tion between intracellular acidification, DNA digestion, and apoptosis
that occurs during rapid death of HL-60 cells.
80
120
160
F.
30-
30-
20
20
10-
10
a
3
O
g
40
80
120
etoposide (ug/ml)
160
2
4
Time (h)
Fig. 5. Changes in intracellular Ca2* and pH induced by etoposide in HL-60 cells. In
A. C. and £.cells were incubated with 0-160
DISCUSSION
Mammalian cells contain a variety of endonucleases any of which
could be involved in apoptosis (reviewed in Ref. 18). ACa2'/Mg2tdependent endonuclease has been implicated frequently in apoptosis
but, as discussed in the "Introduction," it is clear that this endonu
ng/ml etoposide for 30 min. the drug was
removed, and the cells were incubated for an additional 3.5 h prior to analysis of
intracellular pH or Ca2*. In B. D. and f-'. cells were incubated with 80 ug/ml etoposide for
30 min. the drug was removed, and cells were incubated for additional times up to 5.5 h
(6-h time point) prior to analysis. The 0 h point reflects undamaged cells. In A and B. cells
were loaded w ith carboxy-SN ARF-1 and analyzed by flow cytometry lor intracellular pH.
The values derive from the average fluorescence ratio of the whole population of cells. In
C and /). the fluorescence of individual cells loaded with carboxy-SNARF-1
was recorded
clease cannot be involved in all forms of apoptosis. and it may be
questionable whether it is involved in any. The involvement of a
specific Ca2 */Mg2 ' -dependent endonuclease is confounded by nu
in a dot plot format as described in Fig. I. The percentage of cells that shifted into the
acidic region was calculated. The results in C are derived from the dot plots shown in Fig.
6. In £ and F, cells were loaded with INDO-1 and analyzed by flow cytometry for
intracellular Ca-* concentration. The values derive from the average fluorescence ratio for
merous reports of such endonucleases with molecular masses ranging
the whole population of cells.
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ENDONUCLEASE
O ug/ml etoposlde
ACTIVATION
1000-fold discrepancy between this and the concentration required.
To understand the origin of this intracellular acidification, it is
important to emphasize that the cells are still metabolically viable.
First, the acidic cells are able to take up fluorescent dye, deesterify it,
and then retain it, thereby demonstrating membrane integrity. The
cells also exclude trypan blue. Second, to maintain a low intracellular
Ca2+ against a Ca2 ' gradient requires metabolic activity. A metabol
ically inactive or necrotic cell would have intracellular pH and Ca2+
S
400
600
600
200
10 ng/ml etoposlde
O
eoo
eoo
60 uà /mietoposlde
200
20 tig/ml etoposlde
400
400
600
APOPTOSIS
ditions of apoptotic cells satisfy enzymatic requirements of DNase II.
In contrast, the intracellular Ca2 ' in these cells stayed around 30 riM
which is approximately 10.000-fold lower than the Ca2 ' required for
Ca2+/Mg2t-dependent
endonuclease activity in vitro. Even in cases
where increased intracellular Ca2 ' reportedly occurs, there is still a
40 ugymletoposlde
E
c
200
DURING
equal to the extracellular environment. These cells therefore maintain
an electrochemical gradient across the cytoplasmic membrane. Under
conditions of a normal electrochemical gradient, selective inhibition
of pH regulation would cause pH to drop l pH unit (41). This is
consistent with the level of intracellular acidification observed in these
studies. Therefore, the large acidification in the apoptotic cells could
be due to selective inhibition of pH regulation without interference of
the homeostasis of other ions. Such selective inhibition of pH regu
lation has been reported following hyperthermia damage (42). In this
case, one-half of the observed decrease of intracellular pH was re
portedly due to inhibition of the Na^/H*-exchanger.
BOO
160 iig/ml etoposlde
The subpopulation of cells that die rapidly after exposure to eto
poside represents those cells damaged at a particularly vulnerable
phase of the cell cycle. These cells appear to be in early S phase at the
time they are damaged, which is consistent with toxicity arising from
400
200
400
600
585 nm Fluorescence
585 nm Fluorescence
0 ug/ml etoposlde
40 iig/ml eloposlde
to ng/ml etoposlde
60 Mg/mletoposide
20 iig/ml etoposlde
160 ucj/mletoposlde
Fig. 6. Row cylometry analysis of inlracellular pH changes in individual HL-60 cells
damaged with etoposide. Cells were incubated with (>-160 ug/ml etoposide for 30 min. the
drug was removed, and the cells were incubated for an additional 3.5 h prior to analysis.
The cells were loaded with SNARF-l
during the final hour, analyzed by flow cytometry.
and displayed as a two-dimensional dot plot according to the fluorescence intensities al
emission wavelengths of 585 and 640 nm.
digested to a 3'-phosphate and 5'-hydroxy termini by a DNase II-type
endonuclease. Accordingly, there is no evidence that obviates DNase
II as an endonuclease associated with apoptosis. On the contrary, our
current analysis of intracellular pH changes strongly implicates DNase
II in apoptotic DNA digestion.
To test the hypothesis that acidification might be a signal for DNA
digestion, we used HL-60 cells because of the rapidity with which
they undergo apoptosis following a variety of toxic insults. Evidence
first came from the observation that intracellular acidification occurs
during Ca2+ ionophore-induced apoptosis (30). Cells respond to in
creased Ca2 + by pumping it into intracellular organelles or out
through the cytoplasmic membrane. Ca2+ export is mediated by Na+/
Ca2+ exchange or by Ça24/ATPase, the latter has an obligate require
ment for a proton as a counterion (40) which would lead to intracell
ular acidification if the protons are not subsequently exported. This
could explain activation of DNase II under conditions reported to
increase intracellular Ca2 +.
The current studies provide much more demonstrative evidence for
the involvement of intracellular acidification in apoptotic DNA diges
tion because we measured the intracellular pH in individual dying
cells. Following damage by etoposide, the intracellular pH decreased
to pH 6.4 only in a subpopulation of HL-60 cells. When sorted, this
acidic subpopulation was shown morphologically to have the chromatin condensation characteristic of apoptosis, as well as apoptotic
DNA degradation. Cells with normal pH did not show these changes.
Since DNase II mediates internucleosomal DNA digestion up through
at least pH 6.5 in vitro, this demonstrates that the intracellular con
405 nm Fluorescence
405 nm Fluorescence
Fig. 7. Flow cytometry analysis of intracellular Ca2* changes in individual HL-60 cells
damaged with etoposide. Cells were incubated with 0-160
ug/ml etoposide for 30 min, the
drug was removed, and the cells were incubated for an additional 3.5 h prior to analysis.
The cells were loaded with INDO-1 during the final hour, analyzed by flow cytometry, and
displayed as a two-dimensional dot plot according to the fluorescence intensities at
emission wavelengths of 405 and 485 nm.
2355
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ENDONUCLEASE
ACTIVATION
DURING
APOPTOSIS
(1 x 106)
(4x105)
I.
Total Cells
I
Total Cells
Alkaline Cells (1 x 10s)
Acidic Cells
(1 x 10s)
Fig. 8. DNA fragmentation in HL-60 cells sorted on the basis of intracellular pH. Cells were incubated with 80 ug/ml etoposide for 30 min, the drug was removed, and cells were
incubated for an additional 3.5 h. The cells were loaded with SNARF-1 during the final hour and then analyzed and sorted by flow cytometry. The 25% most alkaline cells and the
25% most acidic cells were collected and analyzed by electrophoresis for DNA fragmentation. Lanes contain IO6 unsorted cells. 4 X IO5 unsorted cells. 10s alkaline cells, and 10s acidic
cells, respectively.
interaction between topoisomerases and the replication fork (43).
However, all the cells incubated with etoposide at 80 ug/ml for 30 min
eventually died. After removal of etoposide, the majority of cells
progressed through the cell cycle and arrested in the G2 phase for at
least two days after which time they also died by apoptosis as DNA
digestion has been seen at these extended times (6). The intracellular
pH can not be readily measured during this delayed cell death as too
few cells die at any specified time to exhibit an acidic population. The
acidification is transient; the cells rapidly lose membrane integrity and
their pH can no longer be measured as the fluorescent dye leaks, and
the pH rises to equal the extracellular pH. The results suggest two
pathways for inducing apoptosis, one in which there is rapid, presum
ably more direct disruption of pH homeostasis and a later indirect
pathway which is associated with transition through the G2-M phase
of the cell cycle and which is presumably also associated with intra
cellular acidification. The importance of this latter transition for cytotoxicity has previously been discussed (44).
These results do not address whether DNA digestion is required for
cell death. When internucleosomal DNA digestion is observed, it
seems obvious that the cell would be unable to survive. However,
there are several reports that DNA digestion does not always occur
when cells undergo the morphological changes characteristic of apo
ptosis (45-47). This might be explained by limited DNA digestion that
did not resolve into detectable DNA fragments. It is not necessary to
destroy a large portion of the genome to kill a cell; indeed, one
unrepaired DNA double-strand break is thought to be sufficient to kill
a cell (48). Therefore, lethal endonuclease-mediated
DNA doublestrand breaks can be present in cells without being detected by the
assays for internucleosomal DNA digestion. Other techniques have
been used to detect infrequent breaks in cells undergoing apoptosis
without overt DNA digestion to nucleosome-length fragments (47).
DNase II is probably a ubiquitous endonuclease. Early studies in
mouse, chicken, and horse found DNase II in the brain, pancreas,
liver, intestinal mucosa, thymus, and kidney, with highest levels in the
spleen (49). More recent studies, have identified DNase II in CHO
cells (29), human lymphoblasts (50), human gastric mucosa, and
cervix (51), as well as in human urine, saliva, semen, and milk (52).
DNase II has generally been considered a lysosomal enzyme, yet it
was originally reported to be present in nuclei (53), and we have
detected it in both CHO and HL-60 nuclei (29).4 Accordingly, it is
possible that DNase II may be involved in apoptotic DNA digestion
following many initiating stimuli. It may not be the only endonuclease
associated with apoptosis. Given the large number of endonucleases
that exist in cells (18), it is possible that activation of any of them
would yield apoptotic DNA digestion. The work here does not obviate
Ca2 */Mg2 '-dependent endonucleases in apoptosis, but it makes a
strong case for the involvement of DNase II. It remains to be deter
mined in which apoptosis systems DNase II or other endonucleases
are involved. However, the results do suggest steps in which growth
4 Unpublished observations.
factors or oncogenes may be able to protect cells. Incubation with
growth factors, as well as activation of protein kinase C both lead to
phosphorylation of ion exchangers such as the Na*/H '-antiport
which leads to alkalinization of cells (54, 55). pH regulation also
appears important in oncogenic transformation as evidenced by the E5
oncoprotein of bovine papillomavirus which binds to the vacuolar
HVATPase (56). Also, transfection of cells with a yeast HVATPase
can lead to alkalinization and transformation (57). Increased intracell
ular pH is commonly associated with proliferative stimuli and onco
genic transformation, and our results suggest that decreased intracell
ular pH may be associated with apoptotic cell death.
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2357
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Etoposide-induced Apoptosis in Human HL-60 Cells Is
Associated with Intracellular Acidification
Michael A. Barry, Jason E. Reynolds and Alan Eastman
Cancer Res 1993;53:2349-2357.
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