[CANCER RESEARCH 48,496-502,
February I, 1988]
Effects of Acute pH 6.6 and 42.0°CHeating on the Intracellular pH of Chinese
Hamster Ovary Cells1
John A. Cook2 and Michael H. Fox3
Department of Radiology and Radiation Biology, Colorado State University, Fort Collins, Colorado SOS23
ABSTRACT
Incubation of Chinese hamster ovary cells in pH 6.6 medium for 4 h
prior to and during 42.11'( heating enhanced thermal cell killing compared
to cells heated under normal pH 73 conditions. We examined the
relationship between the extracellular pH and intracellular pH (pH¡)of
Chinese hamster ovary cells using a flow cytometer with the pH-sensitive
fluorescent molecule 2,3-dicyanohydroquinone. Using either normal (7.3)
or low (6.6) pH conditions, the mean pH¡and population pHi heteroge
neity was studied as a function of time at 42.0°C.Cells incubated at pH
6.6 for 4 h had a resting pi I, 0.14 to 0.19 pH units lower than cells at
normal pH 7.3, indicating the presence of an active pH¡
regulatory system.
Heating l h at 42.0V at normal pH caused an increase in the pii-, of
0.14 pH units. With further heating the cells gradually returned to the
unheated (7.3) control levels. Similar pi 1, changes were observed with
the cells incubated and heated at pH 6.6. However, the mean pH¡was
always more acidic than cells heated at normal pH. Active pi I, regulation
was still possible for a substantial (>30%) number of cells even after 10
h of heating under low pH conditions. These results suggest that a
breakdown in pH¡regulation is not the mechanism of low pH-induced
heat sensitizaron.
INTRODUCTION
It has become increasingly apparent over the last few years
that the success of hyperthermia in cancer treatment is highly
dependent on tumor environmental factors (1, 2). A reduction
in tumor blood flow can create regions of localized hypoxia,
nutrient deprivation, and low pHe" (3-5). Because the above
conditions are necessarily interdependent it could be difficult
with i/i vivo systems to identify the relative effect each one has
on hyperthermic sensitivity. However, utilizing in vitro cell
cultures it has been possible to analyze each condition sepa
rately and examine in detail its specific interaction with heat
(6-9).
Several investigators (8-10) have reported that incubation
and heating of CHO cells at 42.0°Cin low pH medium greatly
The cellular mechanism by which low pHc can sensitize cells
to hyperthermia is unknown. The influence of pH on cellular
metabolism, protein assembly, and enzyme kinetics, however,
suggests the necessity of preventing intracellular acidification
from occurring (12-16). In recent years the concept of pH¡
regulation in biological systems has received increased attention
(13-15). It is known that the pH¡can influence a variety of
biochemical pathways (17-19) and may be important in growth
factor stimulation of quiescent cells (20). The possibility, there
fore, that the combination of low pi I, and hyperthermia could
induce intracellular acidification and thus have a profound
effect on survival seemed a reasonable hypothesis.
In order to examine the intracellular pH as a function of
heating, we refined a technique utilizing a pH-sensitive fluores
cent molecule DCH in conjunction with a flow cytometer (21,
22). The advantages of using DCH with flow systems are the
following, (a) The fluorescence spectrum of DCH shifts in a
pH-dependent manner. Thus, a fluorescence ratio technique
can be utilized to provide pi I, distributions independent of dye
concentration. Because asynchronous cell populations have
large volume variations, and heating induces further volume
changes (23), the ratio technique is crucial in eliminating this
variable from the pH¡profiles, (b) Under proper conditions the
pH¡reproducibility and resolution are excellent, (c) DCH comes
in an esterified form which rapidly crosses cellular membranes.
Hence, a rapid, nondestructive means of delivering the probe
molecule into cells is possible. (</) Single cell statistics are
available which would allow examination of any potential pi I,
heterogeneity induced by heating.
In this report we examined how the intracellular pH of CHO
cells was altered by 42.0°C heating. We measured the pH¡
distributions of cells heated in medium at a normal pHe of 7.3,
or incubated at a low pi 1, of 6.6 for 4 h prior to and during
heating. It was determined that pH¡changes occurred under
both pHc conditions. However, the changes noted at either pH
were similar in both the magnitude and direction of change.
The results suggested that a breakdown in pi I, regulation by
hyperthermia was not the mechanism of low pHe-induced heat
sensitization.
increased hyperthermic cell killing compared to heating at
normal pH. In the absence of heat, incubation under low pH
(6.6-6.8) conditions was not cytotoxic but did perturb cellular
growth and division (10). Since it was known that certain
tumors are perfused with an extracellular fluid 0.2 to 0.8 pH
units lower than the interstitial fluid (11), the concept of pH as
a crucial factor in tumor response to hyperthermia was devel
oped (8-10).
MATERIALS
Received 6/15/87; revised 9/30/87; accepted 10/30/87.
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 investigation was supported by USPHS Grant CA2S636, awarded by
the National Cancer Institute, Department of Health and Human Services; the
United States Department of Agriculture Animal Health and Disease Program;
and NIH General Medical Sciences Shared Instrumentation Program for the
purchase of an SI .M 4800 spectrofluorometer.
1This work is submitted by this author in partial fulfillment for the degree
Doctor of Philosophy. Present address: National Cancer Institute, Bldg. 10, Rm.
B3B69, Bethesda, M D 20892.
3 To whom requests for reprints should be addressed.
4 The abbreviations used are: pi I,, extracellular pH; CHO, Chinese hamster
ovary; pH¡,intracellular pH; DCH, 2,3-dicyanohydroquinone; HEPES, 4-(2hydroxyethyl)-l-piperazineethanesulfonic
acid; PIPES, piperazine-JV,yV'-bis(2ethanesulfonic acid; ADB, l,4-diacetoxy-2,3-dicyanobenzol; MDADS, multiple
data acquisition and display system.
AND METHODS
Cell Line and Culture Conditions. CHO 10B2 cells were routinely
grown in Ham's F,2 (Gibco, Grand Island, NY) medium supplemented
with 10% fetal bovine serum. Stock cultures were maintained at pH 7.3
with 14 MINIbicarbonate plus 5% <'<)...All stock cultures were grown
in a humidified 37.0°Cincubator.
Heating Procedures. Stock cultures were trypsinized with 0.25%
trypsin 14 to 16 h prior to heating, and appropriate numbers of cells
were plated out into tissue culture flasks (Falcon 125) for both survival
and pi I, measurements. Low pH treatments commenced 4 h prior to
heating at 42.0°C.Plates to receive low pH medium had their normal
pH (7.3) medium removed and low pH (6.6) medium substituted. Low
pH was accomplished in all cases by reducing the bicarbonate from 14
HIM to 2.4 mM and incubating at 37.0°Cwith 5% CO2. All pH
measurements were made with either an Orion pH meter (Cambridge,
MA) or a Beckman Model 71 pH meter (Irvine, CA), both of which
497
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INTRACELLULAR pH AFTER 42'C HEATING AT pH 6.6
were accurate to within 0.001 pH units. For each heating experiment
the pH was monitored prior to heating and also periodically during
heating by using replicate plates. Once caps to the T-2S flasks were
sealed for water immersion the pH remained within 0.05 to 0.10 pH
units of the initial preheating pH values over the entire 10 h of heating.
The temperature of the water bath was 42.0 ±0.1 °Cin all cases.
NORMAL pH
Survival Procedures. Survival plates were placed in a 37.0V incuba
tor for S to 15 min immediately after heating. Medium from both the
normal and low pH flasks was then replaced with normal medium (pH
7.3), and the plates were returned to the 37.0°Cincubator for colony
formation. Three survival plates for each heat point were used for
statistical purposes. Colony growth was allowed for 8 to 11 days.
Colonies were fixed with a 3:1 mixture of methanol:acetic acid and
stained with crystal violet for colony counting. Colonies containing
more than SOcells were counted and scored. The number of cells per
colony was determined prior to heating, and a multiplicity correction
was made according to standard procedures (24).
Spectrofluorometry. Emission spectra were obtained with 2 ^g/nil
(12.5 UM) DCH (Molecular Probes, Inc., Junction City, OR) in high
K* buffers. High K+ buffer contained 120 mM KC1, 30 HIMNaCl, 0.5
HIMMgSO4, 1 HIMCaCl2, 1 mM NaHPO4, 5 mM glucose, 10 mM
HEPES (Research Organics, Cleveland, OH), and 10 mM PIPES
(Calbiochem-Behring, La Jolla, CA). Spectra were collected with an
SLM 4800 spectrofluorometer (Urbana, IL). Excitation of dye was at
351 nm to match the laser line on the cell sorter.
pHi Measurements. Cells for pH, measurements (3 to 5 x 10s cells/
T-25 flask) were trypsinized, washed once with normal saline buffer,
and then resuspended in 1 ml of normal saline buffer at the appropriate
pH (7.3 or 6.6). Five n\ of the acetoxy form of DCH (ADB; Molecular
Probes, Inc.) were added from a stock of 2 mg/ml of ADB dissolved in
dimethyl formamide. The final concentration of ADB was 42 /¿M.
Cells
were incubated at room temperature for 19 to 20 min before analyzing
with the flow cytometer. Normal saline buffer contained 145 mMNaCl,
5 mM KC1, 0.5 mM MgSO4, 1 mM CaCl2, 1 mM NaHPO4, 5 mM
glucose, 10 HIMHEPES, and 10 HIMPIPES.
The technique of calibrating and measuring intraccilular pH with the
flow cytometer has been described in more detail elsewhere.5 However,
a brief explanation is given below. A Coulter EPICS V cell sorter
(Hialeah, FL) was used for all measurements. Dye excitation was
accomplished using 200 mW of the 351 and 364 nm UV doublet laser
line. Fluorescence from each cell was collected and integrated over two
spectral band widths: 418 to 440 nm (Fl) and 469 to 485 nm (F2). The
analog ratio of the two signals (F1/F2) was obtained by the analog
function board which in turn was part of the system's MDADS. The
resultant ratio signal was stored and processed by the MDADS unit.
Calibration for the pH¡measurements was accomplished by incuba
tion of CHO cells in buffers of high potassium (120 mM KCl) with the
proton ionophore nigericin (Calbiochem-Behring). This produces the
condition that pH, = pH¡(25). Hence, by determining the pH. of the
buffers, we could assign appropriate pH values to each of the 256
channels over which the ratio signal was recorded. All pi I, measure
ments were carried out at room temperature. In general, 10,000 cells
were analyzed for each pH, measurement.
RESULTS
Effect of Low pH on Survival at 42.0°C.Incubation of CHO
cells at pH 6.6 for 4 h (acute low pH) at 37.0°Chad no effect
on survival as determined by plating efficiencies of 70 to 80%.
In fact, CHO cells were able to grow under low pH conditions
(6.6 to 6.7), albeit with a doubling time increased from 12 h at
pH 7.3 to 25 h at pH 6.6. Heating at 42.0°Cin low pH (6.6)
medium induced a rapid increase in cell killing as compared to
cells heated in normal pH (7.3) medium (Fig. 1). Heating for 5
h under low pH conditions reduced survival to <10~5. In con
trast, for cells heated at a normal pH of 7.3 for 5 h, survival
9J. A. Cook and M. H. Fox. Intracellular pH measurements using flow
cytometry with l,4-diacetoxy-2,3-dicyanobenzene, submitted for publication.
,0
-3
10
ACUTE LOW pH
-5
10
6
TIME AT 42'C
8
10
12
14
(HR)
Fig. 1. Survival of CHO cells as a function of time at 42*C under either pH
7.3 (•)
or pH 6.6 (A) conditions. Points, mean; bars, SE (not plotted when smaller
than the symbol).
was reduced to only 23%. Continued heating to 10 h at pH 7.3
had only minimal additional effects on survival, demonstrating
the phenomenon of thermotolerance. These findings are in good
agreement with other studies on low pH and 42.0°Cheating
with CHO cells (8, 9).
Calibration and Measurement of Intracellular pH. Because
intracellular acidification has been identified with the loss of
functional and reproductive integrity in cells (12, 15), we ex
amined the relationship between the extra- and intracellular pH
in both unheated and heated cells. The pH¡was measured by
utilizing the fluorescence properties of the molecule DCH (21,
22). DCH has two proton-dissociable groups (pKi = 5.5 and
pK2 = 8.0) which influence its absorption and fluorescence
properties in solution.5 pH induces changes in emission wave
length as well as intensity, as shown in Fig. 2. The ratio of
intensities at 418-440 nm (Fl) to 469-485 nm (F2) is propor
tional to the buffer pH value.
The pH-dependent fluorescence spectral shifts of DCH make
it a suitable pH, indicator when used with flow cytometric
instrumentation. Calibration of the flow system is demonstrated
in Fig. 3. Using the proton ionophore nigericin in the presence
of high K1 buffers (>120 mM), it is possible to equilibrate the
pH¡of cells with the pHc (25). Fig. 3/4 shows that nigericin
acted uniformly on the entire population, since the pi I¡histo
grams had coefficients of variation in the range from 2 to 4%
and were shifted without distortion. As the pHe was increased,
the ratio channel number decreased. The mean channel number
of each histogram was determined and plotted as a function of
the buffer pH value (Fig. 3Ä). It can be seen that, over the
range of pH values of 6.9 to 7.7, the calibration curve is linear
with a slope of 58 channels/pH increment. We have determined
that relative changes in pH¡can be measured within 0.05 pH
units.5
Absolute pH, measurements with this technique require pre
cise knowledge of the intracellular potassium levels. However,
because the slope of the pH calibration curve was found to be
independent of intracellular potassium levels,5 relative pH¡
changes can be accurately determined. Therefore, unheated
cultures at either pH 7.3 or 6.6 were processed simultaneously
with the heated cultures. This procedure facilitated direct com
parisons between samples and allowed for accurate measure
ments of any pH, changes which occurred.
498
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INTRACELLULAR pH AFTER 42'C HEATING AT pH 6.6
Ill
O
Fig. 2. Emission spectra of DCH in high
K" buffers at various pH values. Fl and F2
represent the approximate wavelength band
widths which are measured with the flow cytometer.
B
O
III
>
¿
WAVELENGTH
(run)
Effects of 42.0°CHeating on the pH¡.Fig. 4 shows the pH¡ 6). It was apparent that, while cells under acute low pH condi
distributions for CHO cells heated at 42.0°Cfor various times
tions were always more acidic than cells at normal pH, the
under normal pH (7.3) conditions. All samples were processed
relative changes which occurred during heating were remarka
immediately after heating at room temperature. After l h of bly similar. Examination of the mean pH¡changes which oc
heating at 42.0°C,an increase in the pH¡of approximately 0.10 curred at low pH between S and 10 h of heating also demon
to 0.15 pH units was observed. This increase appeared to strated a distinct advantage of utilizing the ADB technique for
pH¡measurements. After 10 h of heating at 42.0°C,the mean
involve the entire population as the pH¡histogram increased
uniformly in channel number when compared to the unheated
pH¡for the low pH cells was 0.29 to 0.31 pH units lower than
control population at pH 7.3. Further heating of up to 10 h at the unheated pH 7.3 control cells. However, as Fig. 5D clearly
42.0°Cproduced a gradual reacidification back to the resting
shows, this reduction in the pH, was primarily due to a skewing
pH¡of the unheated control cultures.
of the pHj distributions towards more acidic pH values, and
Fig. 5A represents the pH¡distributions for CHO cells cul
not to any uniform pH, shifts of the population. In fact, even
after 10 h of heating (which reduced survival to < 10"'), the
tured at normal pH 7.3 or incubated for 4 h at pH 6.6. Each
culture was prepared and run in normal saline buffer with the pH¡values of approximately 30 to 40% of the heated low pH
pH of the buffer matched to the pH of the growth medium (7.3 cells still overlapped the unheated control cells at pH 7.3.
or 6.6). The mean resting pH, determined for CHO cells grown
at pH 7.3 varied between 7.10 and 7.20, with a population
DISCUSSION
heterogeneity of approximately 0.3 pH units.
Incubation at pH 6.6 reduced the resting pH, by 0.19 pH
In this paper, we attempted to demonstrate whether intracelunits. Generally, we have found that incubation at pH 6.6 will
lular acidification was the mechanism of acute low pH-induced
reduce the resting pH¡by 0.14 to 0.19 pH units. The pH¡
heat sensitization of CHO cells. From the data in Figs. 5 and
heterogeneity of the low pH cells was similar to that determined
6, it appeared that a small acidification was produced by low
for cells at normal pH. The mean resting pH¡values determined
pH and heating, but only after heating times greater than 5 h.
for cells at pH 7.3 and pH 6.6 are consistent with results
published by Gonzalez-Mendez et al. (26) for a CHO cell line In addition, a skewing of the pH¡distributions towards more
acidic pH values was responsible for the small reduction in the
using a different pH¡measuring technique.
Fig. 5, B to D, shows the pH¡distributions for CHO cells mean pH¡of the population (Fig. 5D).
It can be argued that the initial acidification of 0.14 to 0.19
heated at 42.0°Cfor various times under acute low pH (6.6)
pH
units of the unheated low pH cells was a crucial event in
conditions. Similarly to the cells at normal pH, there was an
heat sensitization. However, several kinds of evidence were
increase in the pH¡by approximately 0.1 pH units (relative to
found to contradict this hypothesis, (a) It was evident that, even
the unheated controls at pH 6.6) after l h of heating. The pH¡
after 10 h of heating under low pH conditions, 30 to 40% of
then slowly decreased, approaching the unheated control resting
the cells had the same pH¡levels as unheated normal pH cells.
pH, value (at pH 6.6) after 5 h of heating. Continued heating
(b) While we reported relative pH¡changes in order to improve
to 10 h caused substantial increases in the pH¡heterogeneity,
measurement accuracy, calculation of the absolute pH¡levels is
such that the pH, distribution was broadened and skewed to
reasonably accurate. We calculated that the unheated low pH
wards more acidic pH¡values.
cells would have steady-state pHr levels between 6.90 and 7.05.
In order to quantitate the pH¡changes which occurred during
We have tentatively verified this measurement with another
heating, the differences between the mean pH, values for both
fluorescence pH technique.6 (c) This value falls within the
heated and unheated cells were calculated (using the calibration
' Unpublished observations.
curve) and plotted as a function of time at 42.OT heating (Fig.
499
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INTRACELLULAR pH AFTER 42'C HEATING AT pH 6.6
A/HEATED-III.-UNHEATEDmVL
••T
ID O K
r» » •-co
1^ K K <D
"mmS3Z
_lUloUI>
PB.HEATED-JML-UNHEATED_V
c/•HEATED-y-UNHEATED-
300
150
RATIO CHANNEL NUMBER
175
150
RATIO CHANNEL NUMBER
300
Fig. 4. Ratio (pH,) histograms of CHO cells heated at 42V under normal pH
(7.3) conditions. Heated cells were analyzed along with unheated normal pH
cells. Heating times were:.(. 1 h; B, 5 h; C, 10 h. Increasing pH is indicated by
decreasing ratio channel numbers.
oc
m
III
125
U
O
pH<7.3)- -pH(6.6>
NO H (7.3HH-H
(6.6)
OC
75
6.50
7.25
8.00
pH
NO H (7.3X
NO H (7.3>
Fig. 3. A. ratio (pH,) histograms of CHO cells incubated in high K buffers
with S Mg/m' of nigérianat various pH values. In B, the mean ratio channel
number of each histogram in A was calculated and plotted as a function of the
buffer pH. The regression equation (
) was calculated to be y = 155 —58.4(.x),
where x = (pH, - 6.87) and r2 was 0.99.
(6.6)
(6.6)
300 0
bounds of other published results for CHO cells under similar
conditions (26). However, our results show a larger differential
between extracellular and intracellular pH than that reported
by Chu and Dewey (27) using a dimethyloxazolidinedione tech
nique.
The absolute pH¡measurements are useful because of the
remarkably similar pH¡response of CHO cells to the first 5 h
of heating under either pH protocol (Fig. 6). Of particular
interest was an increase of 0.10 to 0.15 pH units after l h of
heating. This response was independent of the incubation pH
and appeared to involve the entire population. Hence, we cal
culated that the low pH cells went from a pH¡of 6.90-7.05 to
7.00-7.15. It is true that the normal pH cells also became
alkaline and, hence, still maintained a higher pH¡level than the
low pH cells. However, if this small acidification was the
important factor in heat sensitization, then to our thinking, this
low pH effect must be very subtle indeed.
Finally, we have obtained data with low pH cells and 45°C
heating which indicate that these cells undergo large alkalinizations as a function of time at this temperature.7 In fact, the
7J. A. Cook and M. H. Fox. Intracellular pH of Chinese hamster ovary cells
heated at 45.0V at pH 6.6 measured by flow cytometry, submitted for publication.
RATIO CHANNEL NUMBER
Fig. 5. Ratio (pH.) histograms of CHO cells heated at 42'C under low pH
(6.6) conditions. In B to D, heated low pH cells (//) were analyzed along with
unheated control cells ( V»//). 1, unheated low pH cells versus unheated normal
pH cells. Heating times were: B, 1 h; C, 5 h; D, 10 h.
pHj of the low pH cells can actually increase to levels greater
than the heated normal pH cells. The importance of these
observations is that the low pH cells were still more sensitive
to 45°Cheating than are the normal pH cells. Thus, we would
argue from the above evidence that intracellular acidification is
not the primary mechanism of heat sensitization under our
experimental conditions.
It is pertinent at this point to discuss potential artifacts caused
by the ADB technique. While the fluorescence ratio technique
is less susceptible to alteration with environmental factors than
overall fluorescence intensity, several factors can distort the
ratio signal. The initial hydrolysis of ADB revealed the presence
of an intermediate which had a blue-shifted fluorescence spec
trum as compared to the fluorescence spectrum of DCH.5 The
temporary presence of this compound can alter the ratio signal
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INTRACELLULAR
pH AFTER 42'C HEATING AT pH 6.6
at 43°C,although they did not ascribe any importance to the
observation, which might not be statistically significant with
their technique. A recent paper (28) suggests that the increases
in pH¡may be related to the induction of thermotolerance. The
authors determined that amiloride, a diuretic drug, enhanced
thermal cell killing at 42.0°Cwith V-79 cells at either pH 7.3
or 6.6 (28). Amiloride is a known inhibitor of the Na+/H+
exchange activity at the plasma membrane surface (29). While
amiloride can have pleiotropic effects at the concentrations
used in this study, it was concluded that an inhibition of the
Na+/H+ pump was at least partially responsible for the inhibi
TIME AT 42.0°C (HR)
Fig. 6. Relative pH¡changes as a function of 42'C heating for CHO cells
incubated under either pH 6.6 or pH 7.3 conditions. The ordinate is the difference
in the mean pi I, of heated (//) cells venus unheated control (C) cells. Heated
cells were processed and analyzed along with unheated normal pH cells in order
to obtain relative pH, changes. Cells were heated at pH 7.3 (D) or pH 6.6 (A).
such that an acidic or decreased pH¡value would be recorded.
Unstable or rapidly changing ratio signals could also indicate
possible interference by this species. Because complete hydrol
ysis of this intermediate to DCH will be enzymatically depend
ent, agents which can perturb enzyme function (such as heat)
could lead to erroneous pH, measurements. Some of our initial
experiments with short hydrolysis times (<10 min) with either
42.0°Cor 45.0°Cheating indicated that this was a serious
source of error. However, we have determined that with longer
hydrolysis times (20 min), stable pH measurements are ob
tained.7 Furthermore, this would operate in the opposite direc
tion of the alkaline shifts seen consistently within the tirsi hour
of heating.
Other problems, such as partitioning into nonpolar environ
ments, would shift the equilibrium to favor the diprotonated
form of DCH. However, no localization of DCH was detectable
with fluorescence microscopy of either heated or unheated cells.
While it is difficult to completely eliminate these problems
when increased ratio channel numbers (decreased pH¡values)
are recorded, decreased ratio channel numbers (increased pHr
values) cannot be explained by this mechanism.
Another problem with the ADB technique was the time
needed to prepare and analyze the heated samples. Unfortu
nately, with 20-min sample preparation times and 20-min hy
drolysis times, over 40 min elapsed before pH¡measurements
could be taken. Hence, we cannot rule out the possibility that
pH¡changes may have taken place during heating which were
not measurable after 40 min. For this reason we decided to
both prepare and run all samples at room temperature with the
hope of preventing any reequilibration from occuring. The
efflux of DCH from CHO cells was so rapid that it was
impossible to accumulate intracellular DCH prior to heating.
There have been several reports which indicate that the initial
increases in pH¡measured in this study are in fact real events.
Calderwood and Dickson (2) measured an increase in pi I, of
approximately 0.25 pH units with a Yoshida sarcoma tumor
heated in vivo at 42.0°Cfor 1 h. Although they did not attach
tion of thermotolerance measured.
The gradual return to the unheated resting pH, with contin
ued heating may reflect alterations in the rate of protein syn
thesis. Hahn and Shiu (30) have demonstrated that there is an
initial inhibition of the rate of protein synthesis in CHO cells
by 40 to 50% after l h of heating at 42.0°C.Thereafter the rate
returns to normal with 6 h of continuous heating. Since in
creases in the pH¡have been correlated with the activation of
the rate of protein synthesis in quiescent cells (19), it is possible
that 42.0T heating can also stimulate this general (growth
factor-activated) pathway. With growth factor stimulation of
quiescent cells, one of the first events which occurs is the
activation of Na+/H+ antiport, leading to increases in the rest
ing pH¡by 0.1 to 0.2 pH units (31). The reacidification may
simply reflect the increased proton-producing metabolic activity
brought about by the reinitiation of biosynthetic pathways.
Our results and interpretations disagree with the finding of
Hofer and Mivechi (32). They reported that a reduction in the
pHe of the growth medium of BP-8 murine sarcoma cells was
not a major factor in heat sensitivity. However, when they
artificially decreased the pi I, of these cells a substantial amount
of heat sensitivity was recorded. We agree that artificially
inducing intracellular acidification could potentiate heat dam
age, but whether this actually occurs in well-oxygenated and
metabolically competent cells (as in our study) is another matter
entirely. For tumors which have impaired blood flow and as a
consequence are under nutrient stress, intracellular acidification
may play an active role in hyperthermic cell killing. This is the
rationale for combined hyperglycemia and hyperthermia treat
ments. From the excellent review by Calderwood and Dickson
(2), at least one tumor (Yoshida sarcoma) with hyperglycemia
demonstrated large decreases in both the pi I«and pi I, and
indeed was curable by 42.0°Chyperthermia. However, other
observations by these authors tend to deemphasize this finding,
(a) The Yoshida sarcoma tumor was curable with 42.0°Cheat
ing even without
carcinoma tumor
in the pi I, while
still curable with
hyperglycemia treatments, and (b) a D23 rat
with hyperglycemia showed large decreases
still maintaining a high pi I,, but was in fact
42.0°Cheating. Our results also support this
second finding that the extracellular pH can play an active role
in heat sensitization.
In summary, we have examined the relationship between the
extra- and intracellular pH of CHO cells incubated at either
pH 7.3 or 6.6. We have determined that CHO cells actively
resist pH¡changes such that reducing the pHe from 7.3 to 6.6
produces a corresponding change in the resting pH¡of only
0.14 to 0.19 pH units. Heating at 42.0"C under acute low pH
conditions did not induce a large intracellular acidification. In
fact, heating produced transient increases in the pi I, (at either
pHe) which could be important for thermotolerance induction.
any importance to the changes, the pi I, was still elevated 3 h Finally, our results indicate that enhanced thermal cell killing
after the heat treatments. Chu and Dewey (27) also showed a with low pH treatments may not depend upon large reductions
small increase in pH¡for CHO cells heated at pH 6.72 for l h in the internal pH of cells.
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INTRACELLULAR pH AFTER 42'C HEATING AT pH 6.6
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Effects of Acute pH 6.6 and 42.0°C Heating on the Intracellular
pH of Chinese Hamster Ovary Cells
John A. Cook and Michael H. Fox
Cancer Res 1988;48:497-502.
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