J. Cell Sci. 7S, 347-355 (1985)
347
Printed in Great Britain © The Company of Biologists Limited 1985
THE ALTERATION OF MITOTIC EVENTS BY
IONOPHORE A23187 AND CARBONYL CYANIDE
n-CHLOROPHENYLHYDRAZONE
MICHAEL L.ZIEGLER, JESSE E. SISKEN* AND SHARANJIT
VEDBRATf
Department of Medical Microbiology and Immunology, College of Medicine, University of
Kentucky, Lexington, Kentucky 40536, U.SA.
SUMMARY
A large quantity of published work indicates that calcium ions may be involved in the regulation of
mitotic events and recent reports suggest that the onset of chromosome movement is dependent
upon a transient increase in free cytosolic calcium ions. In this paper we examine the effects of two
agents known to perturb intracellular calcium pools on mitosis in HeLa cells. These were the
calcium-selective ionophore A23187 and carbonyl cyanide n-chlorophenylhydrazone (CCCP),
which is a protonophoric inhibitor of oxidative phosphorylation. Owing to a stimulation of
glycolysis, the latter agent does not decrease intracellular ATP in HeLa but does cause mitochondria
to release calcium ions. Our data show that, at low concentrations, both agents prolong metaphase
but differ in their effects on anaphase and cytokinesis. Studies with chlorotetracycline, a commonly
used probe for membrane-associated calcium, verify that these agents do affect calcium pools under
the conditions of our experiments. The data presented are consistent with the idea that increased
cytosolic calcium levels can directly or indirectly affect mitotic events but, contrary to other
suggestions, cause a prolongation of metaphase, i.e. they delay the onset of chromosome movement.
INTRODUCTION
The role of calcium in cell division has been a subject of considerable interest for
many years. A substantial quantity of published work suggests that calcium ions may
play a regulatory role in mitotic events and that the availability of these ions is itself
regulated, both temporally and spatially, during the division process (e.g. see Hepler
& Palevitz, 1974; Harris, 1975; Rebhun, 1977; Silver, Cole & Cande, 1980; Sisken,
1980; Salmon & Segal, 1980; Wolniak, Hepler & Jackson, 1980, 1983; Kiehart, 1981;
Izant, 1983). The exact role of these ions, their source(s) and the mechanisms by
which they are regulated remain open questions.
In previous experiments we showed that treatment of HeLa cells with low levels of
nicotine can delay the onset of chromosome movement (i.e. prolong metaphase) and
accelerate the rate of furrowing once it begins (VedBrat, Sisken & Anderson, 1979).
Since nicotine stimulates muscle contraction and calcium-dependent stimulus—
• Author for correspondence.
f Present address: Department of Microbiology, College of Physicians and Surgeons, Columbia
University, New York, N.Y., U.S.A.
Key words: calcium ions, mitosis, ionophores, metaphase, HeLa cells.
348
M. L. Ziegler, J. E. Sisken and S. VedBrat
secretion coupling by releasing calcium from intracellular pools (e.g. see Weiss, 1968;
Tjalve & Papov, 1973), we suggested that nicotine might have similar effects on HeLa
cells and that this release could be responsible for the changes in duration of mitotic
events (VedBrat et al. 1979).
In order to gain a better understanding of the nicotine effect and of the role of
calcium in cell division, we have studied the effects of two other agents that release
calcium ions (Ca2+) from intracellular sources: carbonyl cyanide n-chlorophenylhydrazone (CCCP), which causes release of Ca2+ from mitochondria and the calciumselective ionophore A23187. Both cause changes in mitotic parameters in HeLa cells
when used at non-toxic levels and both drastically reduce fluorescence in cells stained
with the fluorescent chelate probe, chlorotetracycline (CTC), a commonly used probe
for membrane-associated Ca2+ (e.g. see Caswell, 1979).
MATERIALS AND METHODS
HeLa (GEY) cells (Microbiological Associates) were grown in plastic T-flasks in Eagle's minimal
essential medium (MEM) with Hanks' salts supplemented with 10% calf serum and 2mMglutamine. In some cases penicillin (100 units/ml) and neomycin (0 - l /ig/ml) were also added to the
medium.
For time-lapse cinemicrography, cells were scraped or trypsinized from the inner surfaces of the
flasks, injected into Rose chambers and allowed to grow for 16—48 h. The medium was then removed
from the chambers and replaced with a warm sample of treatment or control medium. Control
cultures were run in parallel with or in tandem to treated cultures. Time-lapse photography began
immediately after the initiation of treatment at the rate of two frames per min for up to 33 h using
methods previously described (Sisken, 1964). Frame-by-frame analyses of the films were done on an
analytical projector (Photo Instrumentation Corp. Burbank, Calif.).
Criteria for determining durations of mitotic stages were as follows. Metaphase was judged to
begin when chromosomes were first observed to be aligned on the equator of the mitotic spindle as
seen in lateral view, and to end when chromatids began anaphase movement. The time from
beginning of chromatid movement to the initiation of cytokinesis was taken as an approximation of
anaphase since it was previously shown that in cultured human amnion cells, cytokinesis began,
under several different experimental conditions, when the chromatids had moved approximately
85 % of their final distance apart (Sisken, 1973). The beginning of cytokinesis was identified as that
point at which dimpling of the cell surface could first be clearly seen and its completion was taken as
that point when the separation of the two daughter cells, with the exception of the intercellular
bridge, was completed. The data were analysed using a statistical model that takes into account both
intra- and interexperiment variances (VedBrat et al. 1979).
A23187 (obtained as a gift from Eli Lilly Co. or purchased from Calbiochem) was dissolved in
dimethylsulphoxide (DMSO) and diluted to 10~4M in 8 5 % DMSO, 15% distilled water. This
stock was added to media to give final concentrations of 1-0 or 2 ' 0 / / M A 2 3 1 8 7 and 0-85 or 1-7%
DMSO, respectively. Separate control experiments indicated that these concentrations of DMSO
had no effects on the mitotic parameters we measured. CCCP (Sigma) was dissolved in ethanol to
give a 5 X 10~3 M stock solution and diluted appropriately in media. CTC (Sigma) was prepared fresh
for each experiment and dissolved in distilled water to give a stock solution of 1 -0 mM. For staining
with CTC, cells were incubated in Rose chambers for at least 24 h in complete culture medium.
They were then rinsed twice and incubated for 1 h in MEM containing 10 /iM-CTC, 1 % calf serum,
20mM-HEPES buffer (pH7-l) and, for experimentals, either A23187 or CCCP.
Fluorescence microscopy was performed with a Leitz microscope fitted with an epi-illumination
system. A Leitz D filter cube was used along with a 400 nm narrow band pass filter in the excitation
pathway to enhance the selectivity for the calcium chelate (see Fabiato & Fabiato, 1979).
For doubling time measurements, cells were seeded in replicate plastic flasks with 5xlO 4
cells/25 cm2 flask. Cells were grown for 4 days in the MEM described above or in MEM containing
349
Calcium affectors and mitotic events
CCCP. At the end of this time, cells were trypsinized and counted with a haemocytometer.
Doubling times were calculated from average cell counts from two flasks.
RESULTS
A23187 is a divalent cation ionophore whose biological effects are related to its
capacity to bind to cell membranes and allow calcium to diffuse through them along
concentration gradients. The specific membranes most affected by this agent and the
direction of flow of calcium is dependent upon cell type, A23187 and exogenous Ca 2+
concentrations and the duration of treatment. For example, Babcock, Chen, Yip &
Lardy (1979) found that at low concentrations, the agent released Ca2+ from internal
stores and caused Ca2+ efflux from liver cells and bull sperm, while at higher
concentrations it stimulated uptake of exogenous Ca + . Jensen & Rasmussen (1977),
on the other hand, concluded that in human peripheral lymphocytes the initial effect
was to stimulate uptake of Ca2+ through the plasma membrane followed by a timedependent redistribution of the ionophore leading to an efflux of calcium from the
cell.
The effects of 1-0 and 2-0^iM-A23187 on the durations of metaphase, anaphase and
cytokinesis in HeLa cells are presented in Table 1 and Fig. 1, which show that each
phase of mitosis responded differently. Treatment with this ionophore caused an
average increase of 24-33 % in the duration of metaphase and, in the same cells, a
21-33% decrease in the duration of cytokinesis. These effects were essentially
Table 1. The effects ofA23187 on the duration of mitotic stages
Drug (M)
109
81
27
73
79
99
Avg. 486
25-4± 1-91
19-2 ±2-02
18-9 ±2-75
20-6 ±2-07
21-5 ±2-03
22-5 ±1-94
21-7 ±0-85
109
84
38
81
95
100
507
5-15±0-31
4-73 ±0-31
4-83 ±0-33
5-20 ±0-31
5-20 ±0-31
4-99 ±0-31
A23187
79
(1X1O"6M) 121
Avg. 210
26-2 ±2-03
27-5 ±1-87
26-9 ±1-38
79
121
200
None
(2X10~ 6 M)
Avg.
53
45
40
138
28-4 ±2-24
28-0 ±2-34
30-2 ±2-42
28-8 ±1-35
P = 0-001
502 ±0-13
109
84
40
81
100
100
514
3-12 ±0-20
2-96 ±0-20
2-91 ±0-21
3-01 ±0-21
3-04 ±0-20
2-97 ± 0-20
300 ±008
5-35 ±0-31
5-43 ±0-31
5-39 ±0-22
79
121
200
215 ±0-21
1-87 ±0-20
2-00 ±0-15
P = 0-001
P = 0-18
P = 0-01
A23187
Cytokinesis
mean duration
±S.E.(min)
Anaphase
mean duration
±S.E.(min)
Metaphase
mean duration
±S.E.(min)
67
50
50
167
5-89 ±0-32
5-33 ±0-32
5-05 ±0-32
5-43 ±018
P=0-l
67
50
50
167
2-29 ±0-21
2-35 ±0-21
2-45 ±0-21
2-36 ±0-12
P = 0-001
M. L. Ziegler, J. E. Sisken and S. VedBrat
350
constant throughout the 33 h of continuous treatment (Fig. 1A,B) and nearly identical
to those produced by nicotine (VedBrat et al. 1979). Alterations in chromosome
alignment or movement were not apparent in these studies or in those involving
CCCP, which follow.
Although the data in Table 1 indicate that A23187 might cause a small overall
prolongation of anaphase, the differences, according to the conservative statistical
analysis used in this work, were not significant. However, a time-course analysis of the
data (Fig. lc) indicates that the effects of this ionophore on anaphase are somewhat
complex. A concentration of 1-0/XM A23187 produced no time-dependent effects,
while at 2-0 /XM A23187 caused a 24 % increase in anaphase duration during the first 8 h
of treatment followed by a decrease to, or possibly below, control levels.
Specific mitochondrial inhibitors such as the phenylhydrazone uncouplers of
oxidative phosphorylation have been shown to cause mitochondria to lose calcium
ions in other systems (Luthra & Olsen, 1976; BabcockeJ al. 1979) as well as in HeLa
cells (Ikehara et al. 1984). We were, therefore, interested in the effects of CCCP on
40
Metaphase
30
—I
20
4
Cytokinesis
-6
o
I 2
ra
3
Q
Anaphase
1
_L
10
20
Duration of treatment (h)
30
Fig. 1. The effects of A23187 on metaphase, cytokinesis and anaphase as a function of the
O)
duration of treatment. (O
O) Controls; ( •
• ) 1-0/<M-A23187; (O
A23187. Cells were photographed at the rate of two frames per min for 33 h.
Calcium affectors and mitotic events
351
Table 2. Effect ofCCCP on mitotic parameters
Metaphase
mean duration
±S.E.(min)
n
Anaphase
mean duration
±S.E.(min)
n
Cytokinesis
mean duration
±S.E.(min)
4-30 ±0-08
59
3-30 ±0-07
5-85 ±0-14
5-46±0-13
5-70 ±0-07
21
33
54
4-12±0-15
3-89±0-ll
Drug (M)
n
None
60
22-8 ±1-19
60
CCCP
(5xlO" 7 M)
Avg.
20
33
S3
37-6 ±2-9
34-5 ±3-3
36-7 ±1-47
20
33
53
CCCP
(5xlO~ 6 M)
Avg.
22
15
37
26-9 ±1-98
29-4 ±2-84
27-9 ±1-17
P = 0-002
P = 0-001
P = 0-001
P = 0-001
22
15
37
4-66 ±0-13
4-53 ±0-16
4-59 ± 0 0 7
P= 0-002
400 ± 0 0 6
22
15
37
4-14±0-12
3-57 ±0-13
3-82 ± 0 0 6
P= 0-002
mitotic parameters of HeLa cells because it affects intracellular Ca2+ pools by a
different mechanism from that of A23187. Table 2 shows that in the presence of
0-5 /XM-CCCP, metaphase duration was increased by 61 % (from 22-8 min for controls
to 36-7 min). In addition, and in contrast to the effects of A23187, both cytokinesis
and anaphase were somewhat prolonged. Higher concentrations of CCCP (5-0/ZM)
consistently produced less prolongation of mitotic parameters though all phases were
still significantly longer than in control cultures. Neither concentration was overtly
toxic over a 4-day period, as doubling times in controls and cultures treated with 0-5
and 5-0//M-CCCP were 27-1, 27-2 and 27-7 h, respectively.
To establish that these treatments actually alter intracellular Ca2+ pools under the
conditions of our experiments, we examined their effects on the fluorescence of CTC.
Both agents were found to reduce CTC fluorescence to very low levels. Control cells
demonstrate fibrillar fluorescence due to mitochondria and diffuse fluorescence
presumably resulting from the association of CTC with elements of the endoplasmic
reticulum and perhaps other intracellular membranous components (Figs 2, 6). As
seen in Figs 3 and 7, in the presence of A23187 very little of thefluorescenceremains in
either metaphase or interphase cells after 1 h of treatment. Similarly, both interphase
and mitotic cells show very low levels offluorescencewhen treated with 0-5 /iM-CCCP
and even less when treated with 5-0/XM-CCCP (not shown). That this loss of
fluorescence is not simply due to a deficiency in ATP is indicated by the finding that
treatment of cells with toxic levels of antimycin A (5-5 fXM), an inhibitor of electron
transport, does not result in decreased CTC fluorescence (Figs 5, 9).
DISCUSSION
Both A23187 and CCCP were observed to increase significantly the duration of
metaphase (i.e. delay the onset of anaphase chromosome movement). Since they
release Ca2+ from intracellular stores (e.g. see Luthra & Olsen, 1976; Babcock, Furst
352
M. L. Ziegler, J. E. Sisken and S. VedBrat
Figs 2-9
Calcium affectors and mitotic events
353
& Lardy, 1976; Babcock et al. 1979) and reduce CTC fluorescence (Figs 3, 4, 7, 8),
the data are consistent with the idea that the prolongation of metaphase induced by
these agents is mediated by altered cytosolic concentrations of Ca + .
It is reasonable to suggest that increased Ca2+ levels could increase metaphase
durations since microtubules that are major components of the mitotic spindle are
known to be Ca2+-sensitive (Weisenberg, 1972). It has also been observed that
A23187 can cause the breakdown of cytoplasmic (though not mitotic spindle)
microtubules in other mammalian cells (e.g. see Fuller & Brinkley, 1976). However,
to explain all of the effects of A23187 and CCCP on the basis of the direct action of
increased cytosolic Ca2+ is probably too simplistic. The fact that these agents affect
cytokinesis differently, A23187 shortening it while CCCP prolongs it somewhat,
indicates that other intracellular changes induced by these agents are also affecting the
dividing cell. Thus, while the effects on metaphase could result directly from
increased Ca 2+ levels, the possibility remains that other effects of one or both of these
agents, perhaps secondary to calcium release, might account for some of the
alterations in mitotic parameters. For example, an increase in cytosolic Ca + levels
could cause an increase in intracellular pH due to loss of protons, as occurs in other
systems (Steinhardt, Shen&Zucker, 1978; Tilney, Kiehart, Sardet & Tilney, 1978),
and it could be this alteration that affects mitotic parameters. It is known that
microtubules can be disassembled at alkaline pH (Regula et al. 1981) and that
polymerization of microfilaments is enhanced by increases in pH (Spudich, Spudich
&Amos, 1979).
While the possibility that one or more effects of these agents might result from a
reduction in intracellular ATP, this does not appear to be the case for CCCP, at least,
since cells incubated in CCCP continue dividing for at least three doublings at control
rates. Further, due to a stimulation of glycolysis, CCCP concentrations as high as 5 /iM
do not reduce ATP levels in HeLa cells in the presence of glucose or glutamine
(Ikehara et al. 1984), both of which were present in our medium. This is consistent
with the observations that a related agent, £-trifluoromethoxyphenylhydrazone
(FCCP), causes collapse of the mitochondrial membrane potential, release of calcium
and disruption of microtubules in HeLa cells (Maro & Bormans, 1982). This was also
not a simple result of ATP depletion, since neither azide nor oligomycin produced the
same effect. We have no explanation for the reverse dose effect of CCCP seen in our
studies.
With respect to the role of Ca2+ in mitosis, the data in this and our previous paper
(VedBrat et al. 1979) indicate that increased intracellular Ca2+ levels cause a
prolongation of metaphase. However, the degree of prolongation, up to 33% for
A23187 and 61 % for CCCP, is perhaps less than might have been expected given the
Figs 2-9. Fluorescence of cells exposed to 10^M-CTC as described in Materials and
Methods. Exposures for photography and printing were the same for all photographs. Figs
2-5 are of metaphase cells as follows: Fig. 2, CTC alone; Fig. 3, CTC plus 1-O^MA23187; Fig. 4, CTC plus 0-5 ^M-CCCP; Fig. 5, CTC plus 5-5 fiM-antimycin A. Figs 6-9
are of interphase cells as follows: Fig. 6, CTC alone; Fig. 7, CTC plus l-0/iM-A23187;
Fig. 8, CTC plus 0-5/iM-CCCP; Fig. 9, CTC plus 5-5^M-antimycin A. X560.
354
M. L. Ziegler, J. E. Sisken and S. VedBrat
profound reduction in CTC fluorescence caused by both agents. One possible
explanation for this is that increased cytosolic Ca2+ levels caused by any release are
buffered by other Ca regulatory systems such as a plasma membrane Ca + dependent ATPase, which may pump much of the excess Ca2+ to the exterior of the
cell. On the other hand, there are published data that suggest that we might have
expected to see a decrease in metaphase durations. Izant (1983) has reported that
injection of Ca2+ into Ptkl cells speeds up the metaphase/anaphase transition, i.e.
shortens metaphase, and Wolniakei al. (1983) have reported a transient decrease in
chlorotetracycline fluorescence (indicating a release of membrane-associated Ca ) in
late metaphase, just before the onset of anaphase. The suggestion offered in both cases
was that an increase in free Ca2+ might be involved in the trigger for anaphase
movement. While one might reconcile all of the data by suggesting, for example, that a
transient increase in Ca2+ levels might be stimulatory while a prolonged increase is
inhibitory, more data are required to establish that increases in Ca2+ are general
occurrences at metaphase/anaphase transitions and, if they are, that they are involved
in the triggering of chromosome movement.
The final point to be made concerns the fact that doubling times of cells were
essentially unaffected by levels of CCCP that eliminate most of the CTC fluorescence
in HeLa cells. The data suggest: (a) that most of the CTC fluorescence in these cells is
mitochondrial; and (b) that this pool of Ca2+ plays no role in the progression of cells
through the cell cycle.
The authors acknowledge the expert technical assistance of Mrs Munira Nasser and Mrs Sally D.
Grasch. The work was supported in part by grant CA27399 from the National Cancer Institute,
National Institutes of Health, by a grant from the University of Kentucky Tobacco and Health
Research Institute and by a Biomedical Research Support grant to the College of Medicine,
University of Kentucky.
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{Received 4 September 1984 -Accepted 5 December 1984)