International Journal of Hyperthermia

ISSN: 0265-6736 (Print) 1464-5157 (Online) Journal homepage: https://www.tandfonline.com/loi/ihyt20

Correlation between cell killing effect and cell

membrane potential after heat treatment:
analysis using fluorescent dye and flow cytometry

T. Nishida, K. Akagi & Y. Tanaka

To cite this article: T. Nishida, K. Akagi & Y. Tanaka (1997) Correlation between cell
killing effect and cell membrane potential after heat treatment: analysis using fluorescent
dye and flow cytometry, International Journal of Hyperthermia, 13:2, 227-234, DOI:
10.3109/02656739709012385

To link to this article: https://doi.org/10.3109/02656739709012385

Published online: 09 Jul 2009.

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INT. J. HYPERTHERMIA, 1997, VOL. 13, NO. 2, 227-234

Correlation between cell killing effect and cell membrane potential after
heat treatment: analysis using fluorescent dye and flow cytometry
T. NISHIDAt, K. AKAGI and Y. TANAKA
Department of Radiology, Kansai Medical University 10-1 5 , Fumizonocho,
Moriguchi City, Osaka, Japan

(Received 7 December 1995; revised 27 September 1996; accepted 3 October 1996)

Exposure of mammalian cells to a temperature between 42 and 44°C produces

multiple effects on cellular metabolism. However, none of these effects have been
demonstrated to be casually related to cell death. The plasma membrane has also
been suggested to be one of the targets for hyperthermia. Hyperthermia influences
the membrane fluidity and membrane fluidity affects both passive diffusion and
active transport process. Here, we examined the effect of hyperthermia on the
transmembrane potential V-79 cells using a lopophilic probe and flow cytometry.
The underlying principle on which this method is based is that a freely diffusible
cation distributes itself across the membrane according with the transmembrane
potential and with the concentration gradient. By these methods, we tried to find
the relation between the change of surviving fraction and membrane potential.
We revealed that the membrane potential becomes to be depolarized by heat
treatment between 41 and 44°C immediately after heat treatment. In the
relationship between the change in membrane potential and cell surviving
fraction, the difference between 43 and 44°C was not statistically significant but
between 42 and 43°C was statistically significant. These results imply that the cell
membrane (potassium ion channel) is one of the targets of heat treatment and
that duration of depolarized condition leads to cell death.
Key words: Tumour cells, membrane potential, hyperthermia, fluorescent dye,
flow cytometry.

1. Introduction
Hyperthermia has been generally accepted as an effective treatment modality for
cancers. Exposure of tumour cells to a temperature between 43 and 46°C produces
multiple effects on cellular metabolism. It inhibits the synthesis of some proteins
(Henle and Leeper 1979), and the metabolism of high-energy phosphates (Sinesky et
al. 1979), and induces heat-shock proteins such as hsp70 and 72 (Kelley and
Schlesinger 1978). Although there have been many reports regarding the possible
mechanisms of the cell killing effect of hyperthermia, the actual mechanism has not
been clarified yet even a t the cellular level.
The cell membrane may be the critical target for hyperthermia. Many reports
have noted that hyperthermia causes structural and functional changes in the cell
membrane (Papahadjopoulos et al. 1973, Wallach et al. 1979, Quirt and Mackillop
1991, Dynlacht and Fox 1992). Microelectrodes have been employed to measure the
cell membrane potential. However, it is difficult to examine the changes of mem-

t To whom correspondence should be addressed.

02654736/97 $12.00 0 1997 Taylor & Francis Ltd
228 T. Nishida et al.

brane potential by hyperthermia because of technical difficulties and limitations in

the number of cells for measurement. Recently, some simple methods for measuring
the membrane potentials have been developed using fluorescent dyes, and the
changes of membrane potential can now be easily assessed from the changes of
fluorescence intensity measured using flow cytometry (FCM). To investigate a role
of cell membrane damage in the cell killing by hyperthermia, we measured the
changes in membrane potential using a lipophilic cationic fluorescent dye.

2. Materials and methods

2,1. Cells and culture conditions
An established cell-line from Chinese hamster (V-79-379-A) was routinely grown
and subcultured in eagle-MEM medium (Nikken Biomedical Laboratory, Kyoto,
Japan) supplemented with 10% fetal bovine serum (Gibco Canada, Burlington,
Ont., Canada). Stock cultures were maintained in 10-cm diameter plastic tissue
culture dishes (Becton Dickinson & Co., USA) at 37f0.5"C under 5% COz in air.
Cells in the logarithmic growth phase were used in this study.

2.2. Heat treatment

Cells in stock dishes were trypsinized with 0.05% trypsin. To prevent the effect of
trypsin on the cell membrane, cells were washed 2x with complete culture medium
by centrifugation at 1500 rpm for 3 min. Cells were resuspended in test tubes with 2
ml of phosphate buffered saline (PBS) at a cell concentration of 1 x lo6 cells/ml.
Then, incubation for 10 min at 37°C was followed by heat treatment at a tempera-
ture between 41 and 44°C for 5-120 min in a water bath, Advantec, (Tokyo
Seisakusho, Osaka, Japan) regulated precisely with f 0.5"C of the fixed tempera-
ture.

2.3. Fluorescent dye as a probe for membrane potential

Lipophilic cationic probe (3,3'-dipentyloxacarbocyanine iodide (DiOC5(3))
(MOLECULAR PROBE, INC., OR., USA) was selected as an indicator of the
membrane potential. DiOC5(3) was dissolved in dimetyl sulphoxide (DMSO) to
yield a 1 0 0 stock
~ ~ solution. In each experiment, 5 0 0 n ~DiOC5(3) was freshly
prepared. No cytotoxic effect was observed in the colony formation at this concen-
tration.

2.4. Staining method

Immediately after heat treatment, the fluorescent dye (DiOC5(3)) was added to
the cell suspension (lo6 cells/ml PBS) from the 1 0 0 stock~ ~ solution to achieve a
final concentration of 500 nM. Staining was carried out by incubation at 37°C for 20
min. Immediately after staining, the fluorescence intensity was measured by FCM.
In the experiment to investigate the changes in fluorescence intensity for 6 h after
heat treatment, DiOC5(3) was added at 2,4 and 6 h after heat treatment.

2.5. Equipment for flow cytometry and measurement conditions

The fluorescence intensity was measured with the FACScan (Becton Dickinson
Co., USA) under the following conditions. The excitation wavelength was 488 nm
and the fluorescence wavelength was 530130 nm using a band-pass filter. The fluor-
escence intensity was measured in 10 000 cells. The sensitivity of the photomultiplier
 Hyperthermia and membrane potential 229

was adjusted so that the mean value of fluorescence intensity before heat treatment
should be 300 channels.

2.6. Evaluation of heat sensitivity of tumour cells

Heat sensitivity was evaluated by the colony formation assay.
The following subjects were examined:
(1) the changes of fluorescence intensity, i.e., the changes in membrane potential,
by heat treatment at 41, 42, 43 and 44°C for 5 to 120 min;
(2) the changes of survival rate assayed by the colony formation assay after heat
treatment at 41, 42, 43, and 44°C for 5 to 120 min;
(3) correlation between the changes in membrane potential and cell survival rate;
(4) the changes in membrane potential for 6 h after heat treatment: changes in
fluorescence intensity were investigated for 6 hours after heat treatment at
42°C for 30 rnin and at 44°C for 10 min.

3. Results
Figure 1 shows histograms of the distribution of fluorescence intensity obtained
by flow cytometry. Histograms shifted to the right with heating time. Although, we
only show histograms obtained by heat treatment at 43°C here, the increase in
fluorescence intensity was also revealed at all heating temperature from 41 to 44°C.
The relative change of fluorescence intensity was calculated from the mean fluor-
escence intensity of 10 000 cells after heat treatment and that before heat treatment,
and expressed in percentage.
Figure 2 shows changes in fluorescence intensity immediately after heat treat-
ment at 4144°C. The fluorescence intensity at each heating temperature increased
with heating time. The changes in fluorescence intensity at 30 min were 122% at
41"C, 128.5% at 42"C, 135% at 43"C, and 152% at 44°C and changes at 60 rnin
were 132% at 41°C 140.4% at 42"C, 156.2% at 43"C, and 162.7% at 44°C.
Figure 3 shows the survival rate curves after heat treatment at 42,43 and 44°C as
assayed by the colony formation method. The survival rate at 30 min was 90% at
42"C, 50% at 43°C and 5% at 44°C. And at 60 min, it was 70% at 42"C, 4% at
43"C, and 0.09% at 44°C.
Figure 4 shows the relationship between the fluorescence intensity immediately
after heat treatment and the survival rates as assayed by the colony formation
method after heat treatment, i.e., the relationship between the change in membrane
potential and the degree of cell killing effect. When the survival rate was 50 and 6O%,
the fluorescence intensity was 122.8 and 126.7% (heated at 43°C for 20 min, at 44°C
for 5 min), when the survival rate was 25 and do%, the fluorescence intensity was
131.3 and 135% (heated at 43°C for 30 min, at 44°C for 10 min), and when the
survival rate was 4 and 5 % , the fluorescence intensity was 152 and 156.2% (heated at
43°C for 60 min, at 44°C for 30 rnin). The relationship between the increase in
fluorescence intensity and the decrease in survival rate at 43°C also compared
with that at 42°C. When the fluorescence intensity increased to even 130% at
42"C, the survival rate fell to only 50%. In the relationship between the change of
survival rate and fluorescence intensity, the statistical significance was examined
between heat temperatures using student t test. There were no statistically significant
differences between heat treatment at 43°C and that at 44°C. However, there was a
230 T. Nishida et al.

Fluorescence Intensity

Figure 1. The histograms obtained by flow cytometry immediately after heating. The hor-
izontal axis indicates fluorescence intensity and the vertical axis indicates number of cells.
The heating temperature is 43°C: (A) preheating; (B) heated for 10 min; (C) heated for 20
min; (D) heated for 30 min; (E) heated for 60 min. These histograms shifted to the right
with heating time. This phenomenon indicates the depolarization of membrane potential.

lSO)
*s
h
Y
160
a2
Y
E
U
8 140

x2 120
0
1
G; 100
0 30 60 90 120
Heating Time (min.)
Figure 2. The changes of fluorescence intensity after heat treatment. The fluorescence inten-
sity are expressed as percentages of the control (preheating). The fluorescence intensity
increased with heating time at all heating temperature. 41°C 0, 42°C 0, 43°C A and
44°C *.
 Hyperthermia and membrane potential 23 1

1
0 10 20 30 40 50 60 70 80 90 1 0

Heating Time (min.)

43°C A
Figure 3. The survival rate curves after heat treatment at 42,43 and 44°C. 42°C 0,
and 44°C +

n 10-
&
v
!

.
c3
)
u
m 1-
k

0.1-

. _
U.Ul :
100 125 150 175

Fluorecsence Intensity (%)

Figure 4. Correlation between the changes of fluorescence intensity and changes of surviving
fraction. The changes in fluorescence intensity is plotted on the horizontal axis and the
surviving fraction is plotted on the vertical axis. The data of figure 3 and 4 are applied. In
the relation between the survival rate and rate of fluorescence change, there was no
statistically significant differences between heat treatment at 43 and at 44°C. However,
there was statistically significant differences between heat treatment at 42 and at 43 or
44°C. 42°C 0, 43"A and 44°C +.
232 T. Nishida et al.

0 2 4 6 8
Time After Heat treatment (hrs.)
Figure 5. The changes of fluorescence intensity measured at 2,4 and 6 h after heat treatment
at 42 and 44°C. At 6 h after heat treatment at 44°C for 10 min, the fluorescence intensity
+
remained at 122 3%, while, it recovered rapidly to about 114 f 3% at 2 h after heat
treatment and returned to the pre-treatment level at 6 h after heat treatment at 42°C for
30 min. 42°C 0, 44°C +.

statistically significant difference between heat treatment at 42°C and that at 43 or

44°C.
Figure 5 shows the changes in fluorescence intensity for 6 h after heat treatment
once obtained an increase in fluorescence intensity to 131 k 1% at 42°C and
*
129 1% at 44°C. The fluorescence intensity after heat treatment at 44°C slightly
recovered to 121 & 3% at 4 h after heat treatment, but remained 122 f 3% even at 6
h after heat treatment. On the other hand, after heat treatment at 42"C, the fluor-
escence intensity recovered rapidly to 114 & 3% at 2 h and to the pre-treatment level
at 6 h after heat treatment.

4. Discussion
For direct measurement of membrane potentials, the method with microelec-
trodes has been employed. However, the changes in membrane potential by heat
treatment was difficult to examine because of technical difficulties, difficulties in
measuring the potential immediately after heat treatment, and limitations in the
number of cells for measurement by this direct method. However, it is possible to
indirectly measure the changes in membrane potential by observing changes in
fluorescence intensity using cyanine dyes which move across the membrane accord-
ing to the K+ gradient, since the membrane potential is proportional to the intra-
cellular Kf concentration. When the cells are depolarized, the fluorescence intensity
increases and when they are hyperpolarized, it decreases. These phenomena have
been demonstrated in electrophysical experiments carried out using K+ ionophores
in culture medium at different K+ concentrations (Shapiro 1988).
 Hyperthermia and membrane potential 233

It has been reported by many investigators that the fluorescence intensity of

cyanine dyes change in parallel with the membrane potential (Hoffman and Laris
1974, Sims et al. 1974, Waggoner 1979). Among the many fluorescent probes avail-
able for studying the membrane potential, we selected DiOC5(3) because it is not
toxic at a concentration of lOmM or less and also it is now commercially available
because of its lack of hypofluorescent complex formation.
In this experiment, the changes in membrane potential could be measured easily
and rapidly using FCM analysis and the fluorescent probe for each suspended cells
as one cell group. Immediately after heat treatment, fluorescence intensity increased,
i.e., intracellular K+ increased at every heating temperature from 41 to 44°C.
Intracellular K+ is mainly regulated by active influx of K+ through the Na+/K+
pump and passive influx and efflux of K+ depending on the concentration gradient
through K+ leak channels in the cell membrane. Discussing the fluorescence change
immediately after heat treatment, there is a possibility that heat treatment acceler-
ates the function of Na+/K+ pump. Bates et al. (1985) investigated the effects of heat
treatment on the Na+/K+ pump by measuring the influx and efflux of radioactive
isotope (56RbC1)in Chinese hamster ovary cells during heat treatment (at 3 1-50°C).
They reported that the influx of K+ increased with temperature from 31 to 45°C and
concluded that the Na'/K+ pump is not damaged at a temperature between 3 1 and
45°C. Stevenson el a!. (1983) also reported a similar finding; measurement of the
uptake of 42KCl in CHO cells with heat treatment at 42°C for 30 min revealed that
the passive influx of K+ via the Na/K+ pump increased. Our results are consistent
with these papers in that the function of the Na+/K+ pump is accelerated and the
membrane is depolarized immediately after heat treatment.
If functional effect by heat treatment on the cell membrane leads to cell death, the
changes in membrane potential after heat treatment and the survival rate should
correlate. However, this subject has not been discussed previously. An identical
correlation between the change in fluorescence intensity which indicates the changes
in membrane potential immediately after heat treatment and change in survival rate
were observed in 43 and 44°C. At these heating temperatures, functional damage of
the cell membrane results in membrane potential change correlating with survival
rate. On the other hand, statistically significant differences were revealed between 42
and 43 or 44°C. This is because even at 41 and 42°C of the temperature which has
little cell killing effect, fluorescence intensity increased immediately after heat treat-
ment.
Considering the possibility of reversibility of the fluorescence intensity, the fluor-
escence change was measured for 6 h at 42 and 44°C. The increased fluorescence
intensity immediately after heat treatment was reversible a t 42"C, while it was irre-
versible at 44°C.
Although we did not directly demonstrate the impairment of K+ leak channels, it
was suggested that the degree of impairments of Kf leakage channels differ between
42 and 44°C.
These results imply that the cell membrane (potassium ion channel) is one of the
targets of heat treatment and that irreversible depolarized condition (increase in
intracellular cation concentration, mainly K+) due to impairments of K+ leakage
channels leads to cell death.
234 Hyperthermia and membrane potential

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