Selective Effects by Valinomycin on Cytotoxicity ... - Cancer Research

<strong>Selective</strong> <strong>Effects</strong> <strong>by</strong> <strong>Valinomycin</strong> on Cytotoxicity and Cell Cycle
Arrest of Transformed versus Nontransformed Rodent
Fibroblasts in Vitro
Beate Kleuser, Hubert Rieter and Gerold Adam
Cancer Res 1985;45:3022-3028.
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[CANCER RESEARCH 45, 3022-3028, July 1985]
<strong>Selective</strong> <strong>Effects</strong> <strong>by</strong> <strong>Valinomycin</strong> on Cytotoxicity and Cell Cycle Arrest of
Transformed versus Nontransformed Rodent Fibroblasts in Vitro1
Beate Kleuser, Hubert Rieter, and Gerold Adam2
Fakultättur Biologie, UniversitätKonstanz, D-7750 Konstanz, Federal Republic of Germany
ABSTRACT
The effect of submicromolar concentrations of the K+ iono-
phore valinomycin on proliferation, viability, distribution of cell
population over phases of the cell cycle, and cellular adenosine
triphosphate content of different permanent rodent cell lines in
vitro was investigated. <strong>Valinomycin</strong> inhibits proliferation of all cell
lines tested with a saturating effect at about 20 to 100 nw. The
effect of valinomycin on nontransformed 3T3 mouse and Rat-1
cells is nontoxic, whereas it acts with increasing toxicity on the
transformed cells in the order 3T6 mouse, polyoma-3T3 mouse,
temperature-sensitively Rous sarcoma virus-transformed Rat-1
at permissive temperature, and SV40-3T3 cells. According to
these and some other criteria, the essential action of valinomycin
appears to be to impose on the cells at low growth densities a
state of limiting growth condition which normally is encountered
only at high cell densities and/or low serum concentration.
Nontransformed cells are proliferation arrested <strong>by</strong> valinomycin
essentially in the G, phase of the cell cycle, whereas all trans
formed cells under this condition are not arrested selectively in
G,. In all cell lines tested (3T3, 3T6, and SV40-3T3), cellular
adenosine triphosphate content is decreased <strong>by</strong> about 33% upon
treatment with 20 nw valinomycin. Evidence is presented for a
mitochondrial site of action of valinomycin.
INTRODUCTION
It is well known that untransformed cells, upon encountering
limiting growth conditions, shift reversibly into a quiescent state
of proliferation with a Gìequivalent of DNA ("restriction point
control"), whereas cells transformed <strong>by</strong> DNA viruses generally
are arrested in all phases of the cell cycle and eventually die (1-
4). Exploitation <strong>by</strong> reinforcement of such differences of prolifer
ation behavior between transformed and normal cells appeared
promising in attempts to devise procedures selectively toxic for
tumor cells (4-6). However, subsequent work demonstrated that
various transformed cell lines differ widely with respect to the
effect of serum deficiency or drug treatment on cell growth and/
or phase of cell cycle in which proliferation arrest occurs (5, 6).
In particular, spontaneously, chemically, or RNA virus-trans
formed cell lines appear to be arrested predominantly in G, phase
upon serum reduction (5). In our search for an agent innocuous
for normal cells but affecting selectively the proliferative state of
transformed cells in general, interference with the energized state
of the mitochondria appeared most promising because it deter
mines both ATP level (7) and calcium homeostasis of the cell (8).
This direction of proceeding is strongly supported <strong>by</strong> recent
findings on the selective cytotoxicity of the cationic dye rhoda-
' Supported <strong>by</strong> grants from Stiftung Volkswagenwerk (Schwerpunkt Synergetik)
and Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 156).
2 To whom requests for reprints should be addressed.
Received 12/26/84; revised 3/18/85; accepted 4/4/85.
mine 123 on carcinoma cells (9,10). This compound is retained
tightly <strong>by</strong> the mitochondria of transformed but not of nontrans
formed epithelial cells (11). However, it is not retained significantly
<strong>by</strong> transformed or nontransformed fibroblasts (11). Correspond
ingly, we have not detected significant inhibitory effects on the
proliferation of SV40-3T3 cells.3
The macrocyclic ionophore VAL4 at submicromolar concentra
tion is known to dissipate the mitochondrial transmembrane
potential (12-14) and there<strong>by</strong> to affect functions depending on
this, such as ADP-ATP translocation (15), calcium fluxes (16),
and, under certain conditions, oxidative phosphorylation (12-14,
17). In line with our present approach, VAL at submicromolar
concentrations was found to inhibit DNA synthesis in chicken
embryo fibroblasts (18) and, furthermore, was reported in a brief
summary to exhibit antitumor activity in tests in vivo (19, 20).
We have therefore investigated the effect of VAL on cellular
proliferation for a spectrum of transformed and nontransformed
rodent fibroblasts. We will show in the following that VAL arrests
nontransformed rodent fibroblasts in Gìbut various transformed
fibroblasts nonselectively with respect to d. Furthermore, it is
shown that VAL renders virus-transformed cells at low cell
densities to enter a state leading to cell death after 1 to 2 days,
whereas it is reversibly cytostatic for nontransformed fibroblasts
and for a spontaneously transformed line (3T6 cells). Preliminary
accounts of part of this work appeared previously (21, 22).
MATERIALS AND METHODS
CANCER RESEARCH VOL. 45 JULY 1985
3022
Cell Cultures. Swiss 3T3 and 3T6 cells were obtained from Flow
Laboratories (Bonn, Federal Republic of Germany). SV40 virus-trans
formed Swiss 3T3 cells (SV40-3T3, line 101) and polyoma virus-trans
formed Swiss 3T3 cells (PY-3T3) were kindly provided <strong>by</strong> Dr. M. M.
Burger, Basel, Switzerland. Fisher F2408 (Rat-1) rat cells and Rat-1 cells
transformed <strong>by</strong> mutant ts LA334m of strain B77 of Rous sarcoma virus
(ts RSV-Rat-1) were kindly given <strong>by</strong> Dr. J. Wyke, London, England. Stock
cultures of these cells were maintained in plastic dishes (Greiner, Nürtingen,
Federal Republic of Germany) with antibiotic-free Dulbecco's mod
ification of Eagle's medium (Flow Laboratories) containing 5% heat-
inactivated NBCS (Gibco Europe, Glasgow, Scotland) at 37°C and a
humidified atmosphere of 10% CO2 in air. Nontransformed cells were
passaged at subconfluency, and transformed cells were passaged after
confluency, 3 times weekly <strong>by</strong> splitting about 5:1. After about 40 pas
sages, cells were renewed from frozen stocks.
Experimental cultures and controls were grown as described above,
except that penicillin and streptomycin (each 30 M9/ml) was added and
the medium was renewed daily. The ts RSV-Rat-1 cells were grown at
35°C in medium containing fetal calf serum, buffered with 20 mw W-2-
hydroxyethylpiperazine-AT-2-ethane-sulfonic acid, and sodium bicarbon
ate (0.6 mg/ml), and maintaining the atmosphere at 2% CO2 in air. The
3 Unpublished observations.
4 The abbreviations used are: VAL, valinomycin; NBCS, newborn calf serum; ts
RSV-Rat-1, Rat-1 cells temperature-sensitively transformed <strong>by</strong> mutant LA334m of
Rous sarcoma virus strain B77.
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procedures for seeding and Coulter counting of the experimental cultures
were as described previously (23), yielding cell densities of parallel plates
reproducible within 5 to 10%. All the data given are averages from
duplicate plates. VAL was obtained from Serva, Heidelberg, Federal
Republic of Germany, and was applied from a 20 ,UMstock solution in
ethanol, keeping the ethanol concentration in the medium below 0.1%.
Controls were treated equally, but without VAL.
Cell Cycle Analysis. Fractions of cells in different phases of the cell
cycle were determined flow cytometrically <strong>by</strong> the fluorescence of the
DNA-specific bisbenzimide stain Hoechst 33258, using the impulse cy-
tophotometer ICP22 (Biophysics Systems, Bensheim, Federal Republic
of Germany) as described previously (23). Cells were trypsinized, fixed
for 15 min in 50% ethanol, and stained for 10 min in Hoechst 33258 (8
¿ig/ml).The DNA histogram was evaluated graphically in terms of the
relative fraction GiM of cells with a Gìequivalent of DNA, defined as the
ratio of the area of the Gìpeak to the total area of the fluorescence
histogram. For separation of the area of the d peak in the region of
overlap with the S-phase area of the histogram, the criteria given in (24)
were applied, allowing, if necessary, for skewed S-phase distributions
<strong>by</strong> nonhorizontal extrapolation of the S-phase area up to the abscissa of
the G! maximum.
Determination of Cellular ATP. Cellular levels of ATP were deter
mined <strong>by</strong> the luciferin-luciferase assay. Cells were scraped off the plate
with a rubber policeman and suspended in 5 ml calcium-magnesium-free
phosphate-buffered saline containing 1 mw EDTA. A volume of 0.1 to
0.2 ml of this cell suspension was added to 0.6 ml of distilled boiling
water and heated for 10 min in a boiling water bath. Cellular ATP was
measured in a luminometer (Luminometer 1250; LKB, Gräfelfing,Federal
Republic of Germany) after adding a 10- to 1OÛ-M!aliquot of this solution
to a volume of 200 n\ reagent solution of the luciferin-luciferase system
(ATP Bioluminescence CLS; Boehringer, Mannheim, Federal Republic of
Germany). Measurements were calibrated <strong>by</strong> adding 5 ^l of a standard
with known ATP concentration.
RESULTS
Effect of VAL on Proliferation and Viability. The effect of
VAL on the proliferation and viability of different normal and
transformed cell lines is shown in Charts 1 to 9. In all these
experiments, cells were seeded at a density of 1000 to 3000/sq
Chart 1. Effect of VAL on proliferation and viability of 3T3 cells maintainedwith
daily medium renewals containing NBCS. O, untreated control (5% NBCS);•, cells
during daily application of 20 nw VAL (5% NBCS); A, cells after VAL treatment,
maintainedwith 5% NBCS; A, same with 20% NBCS; •cells after VAL treatment
reseeded at 5% NBCS. Top, density of cells adhering to the plates; bottom,
percentage of cells released into the supernatant medium since the last renewal
relative to the number of adhering cells; abscissa, time of growth in days (t/d).
GROWTH INHIBITION AND CYTOTOXICITY BY VAL
CANCER RESEARCH VOL 45 JULY 1985
3023
Chart 2. Effect of VAL on proliferationand viabilityof SV40-3T3cells maintained
with daily medium renewals containing NBCS. O, untreated controls (5% NBCS);
».cells during daily application of 20 ni«VAL (5% NBCS); A, cells after VAL
treatment maintainedwith 5% NBCS; A, same at 20% NBCS; •cells after VAL
treatment reseeded at 5% NBCS. Top, density of cells adhering to the plates;
bottom, percentage of cells released into the supernatant medium since the last
renewal relative to the number of adhering cells; abscissa, time of growth in days
(t/d).
cm in Dulbecco's modification of Eagle's medium containing 5%
NBCS and maintained with daily medium renewals. From the
time indicated <strong>by</strong> the first arrow until the time indicated <strong>by</strong> a
second arrow (whenever this occurs), VAL was applied daily at
20 nw (except in the experiments shown on Charts 3, 7, and 8,
where variant concentrations of VAL were used). In all cases,
proliferation of the treated populations is completely arrested
after about 2 days of severely slowed-down growth. Even at a
concentration as low as 2 nw, there is substantial inhibition of
proliferation as shown in Chart 7 for 3T6 cells (similar data were
obtained for 3T3 and SV40-3T3 cells, not shown).
Toxic effects of VAL on the cell lines investigated are indicated
<strong>by</strong> decreases of cell densities with time. In addition, we have
determined <strong>by</strong> Coulter counter the number of cells per nuclei
released into the medium during the 1-day interval since the last
medium exchange. These data are plotted in the lower parts of
Charts 1 to 4 as a percentage of cells in the supernatant medium
relative to adhering cells, and they agree well with corresponding
decreases of numbers of (adhering) cells given in the upper parts
of these charts. At 20 to 100 nv VAL, there is little effect on the
viability of 3T3, 3T6, and Rat-1 cells (Charts 1, 3, 5, 7, and 8).
Inhibition of proliferation <strong>by</strong> VAL in these cells is reversible after
a certain time lag, as shown <strong>by</strong> discontinuing the VAL treatment
after 3 days and applying fresh medium containing 5% or 20%
NBCS with daily medium renewal (Charts 1 and 3). The lag time
of recovery of proliferation is somewhat shorter, when cells are
trypsinized and reseeded at lower cell density into fresh medium
containing 5% NBCS (Charts 1 and 3). Essentially all of the cells
nontoxically exposed to VAL will resume proliferation after 2-5
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GROWTH INHIBITION AND CYTOTOXICITY BY VAL
Chart 3. Effect of VAL on proliferation and viability of Rat-1 cells maintained
with daily medium renewals containing NBCS. O, untreated control (5% NBCS); •,
cells during daily application of 100 nM VAL (5% NBCS); A, cells after VAL treatment
(5% NBCS); A, same at 20% NBCS; D, cells after VAL treatment reseeded at 5%
NBCS. Top. density of cells adhering to the plates; bottom, percentage of cells
released into the supernatant medium since the last renewal relative to the number
of adhering cells; abscissa, time of growth in days (t/d).
days under these conditions, as was ascertained for 3T3 and
3T6 cells <strong>by</strong> the 5-bromodeoxyuridine-Hoechst 33528 method
(25); data are not shown here. In contrast, the effect of VAL on
the DNA and RNA virus-transformed cell lines (SV40-3T3 and ts
RSV-Rat-1 at permissive temperature) is strongly cytotoxic
(Charts 2, 4, and 9, broken curves). This toxic effect continues
for >7 days, even if the VAL treatment is replaced <strong>by</strong> daily
application of fresh medium containing 5% or 20% NBCS (Charts
2 and 4, solid curves after second arrow). Recovery in the
medium with a higher concentration of NBCS (20%) turns out to
be more efficient (less cell death) and faster. Interestingly, recov
ery after reseeding into fresh medium at lower cell density
appreciably alleviates cell loss but still requires 4 to 5 days
(Charts 2 and 4).
Phases of Cell Cycle of VAL-induced Inhibition of Prolifer
ation. Normal cells are arrested in G,. when proliferationis limited
<strong>by</strong> serum factors and/or cell density (cf. controls in Charts 5 and
8). However, there are spontaneously, chemically, or RNA virus-
transformed cell lines which are also arrested in d phase, if
growth conditions are limiting. An example of this behavior is
3T6 cells (see control in Chart 7). It has therefore been of interest
whether the extent of cytotoxicity of VAL correlates with the
phase of cell cycle of VAL-induced growth arrest. We have
determined histograms of cellular DNA contents <strong>by</strong> flow cytometry,
as described in "Materials and Methods," and evaluated the
relative fraction G1wof cells with a d equivalent of cellular DNA.
This quantity is plotted in parallel to cellular population dynamics
10
106
20
Chart 4. Effect of VAL on proliferation and viability of Rat-1 cells transformed
<strong>by</strong> ts RSV-Rat-1 at permissive temperature (35°C)maintained with daily medium
renewals containing fetal calf serum. O, untreated controls (5% fetal calf serum);
•,cells during daily application of 20 nM VAL (5% fetal calf serum); A, cells after
VAL treatment maintained at 5% fetal calf serum; A, same at 20% fetal calf serum;
•,cells after VAL treatment reseeded at 5% fetal calf serum. Top, density of cells
adhering to the plates; bottom, percentage of cells released into the supernatant
medium since the last renewal relative to the number of adhering cells; abscissa,
time of growth in days (f/d).
0.65
CANCER RESEARCH VOL. 45 JULY 1985
Chart 5. Effect of daily application of 20 nM VAL in fresh medium containing 5%
NBCS on density ((op) of 3T3 cells and their relative fraction G,ra with G, equivalent
of DNA (bottom). Abscissa, time of growth in days (f/o). •,untreated control; A,
VAL-treated cells.
3024
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10

Chart 6. Effect of daily application of 20 DMVAL in fresh medium containing 5%
NBCS on density (fop) of SV40-3T3 cells and their relative fraction G,„,with Gt
equivalent of DMA (bottom). Abscissa, time of growth in days (r/d). •,untreated
control; A, VAL-treated cells.
in Charts 5 to 9. Interestingly, all nontransformed lines tested
approach a Giw near 1 under the action of VAL, whereas all
transformed cells tested are not arrested selectively in d if VAL
is applied. The same result was found for PY-3T3 cells (data not
shown). The kinetics of transition of VAL-treated cells through
the cell cycle is different for different cells. Chart 5 shows that
3T3 cells during 2 to 3 days after application of VAL gradually
run through the cell cycle and slowly accumulate in G,, whereas
the transit of Rat-1 cells through the cycle under these condi
tions, albeit only for a fraction of the population, occurs faster
(Chart 8). According to Charts 6, 7, and 9, all transformed cells
upon VAL treatment do not accumulate selectively in Gì.How
ever, the details of the resulting changes of the DMA histograms
differ somewhat for different transformed cell lines (data not
shown); whereas SV40-3T3 and ts RSV-Rat-1 (35°C)after VAL
treatment accumulate mostly in early S phase, 3T6 and PY-3T3
are arrested in all phases of the cycle but within about 5 days
partly move out of S phase and accumulate preponderantly in
Gìand G:>.
Effect of VAL on Cellular ATP Content. Because a long-term
effect of VAL on cellular potassium content cannot be observed
at the doses applied here (21), mitochondria! uncoupling might
be the basis of its proliferation-inhibiting effect. We have there
fore determined the effect of VAL on cellular ATP contents of
normal and transformed mouse cells. Results for 3T3 (3 inde
pendent experiments), 3T6, and SV40-3T3 cells (2 independent
experiments each) are shown in Table 1. Every determination
was made in duplicate. Untreated controls within experimental
error were indistinguishable at the time of VAL application and
24 h thereafter; these data were therefore lumped together and
given as control. According to the data given in Table 1, the
cellular ATP content of normal and transformed cells is lowered
GROWTH INHIBITION AND CYTOTOXICITY BY VAL
0.75
0.25
CANCER RESEARCH VOL. 45 JULY 1985
3025
Chart 7. Effect of daily application of VAL in fresh medium containing 5% NBCS
on density (top) of 3T6 cells and their relative fraction G,,„with G, equivalent of
DMA (bottom). Abscissa, time of growth in days (f/d) O, untreated control; •,cells
treated with 2 â„¢ VAL; A, cells treated with 20 nu VAL.
Charts. Effect of daily application of 100 HM VAL in fresh medium containing
5% NBCS on density ((op) of Rat-1 cells and their relative fraction G,M with Gì
equivalent of DNA (bottom). Abscissa, time of growth in days (tjd). O, untreated
control; •.VAL-treated cells.
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10
15

GROWTH INHIBITION AND CYTOTOXICITY BY VAL
10 15
Chart 9. Effect of daily application of 20 MMVAL in fresh medium containing 5%
fetal calf serum on density (top) of ts RSV-Rat-1 cells at 35°Cand their relative
fraction G,w with G, equivalent of DNA (bottom). Abscissa, time of growth in days
(f/d). O, untreated control; A, VAL-treated cells.
Table 1
Effect of 24-h treatment with VAL on cellular ATP contents
Cells3T3SV40-3T33T6TreatmentControl20
VALControl20 nw
VALControl20 nw
content(fmol/cell)5.66
(12f3.84 + 0.82"
(7)3.05 ±0.52
(8)2.23 ±0.27
(4)3.72 ±0.30
nmiVALATP
(7)2.11 ±0.34
±0.20 (4)%
* Average ±SD.
6 Numbers in parentheses, number of determinations.
of de
creaserelative
tocontrol322743
<strong>by</strong> about one-third relative to control if 20 nMVAL is applied for
24 h. Cellular ATP contents 48 h after application of 20 nM VAL
(data not shown) within experimental error do not differ from
those at 24 h of exposure.
DISCUSSION
Our present work has shown that daily application of VAL at
>2 nM leads to inhibition of proliferation of all cell lines tested.
This inhibitory effect saturates at about 20 to 100 nw VAL. The
action of VAL is not cytotoxic to nontransformed cells, such as
3T3 and Rat-1, but is cytotoxic to transformed cells with increas
ing impact in the order 3T6, PY-3T3, ts RSV-Rat-1 (35°C),and
SV40-3T3 cells. The low cytotoxic effect on 3T6 and PY-3T3 in
contrast to SV40-3T3 cells correlates with their fairly short time
lag of 5 to 20 days for induction of tumors upon inoculation into
nude mice as compared to >80 days for SV40-3T3 cells (26).5
Possibly, the long latency for tumor development of SV40-3T3
inocula in nude mice results from the death of a majority of the
inoculated SV40-3T3 cell population before tumor development
starts from a surviving minority of cells.
5 Unpublished data.
If SV40-3T3 cells are grown in vitro to saturating cell densities,
massive cell death is observed after a delay of 1 or 2 days (1, 2,
4). The growth behavior of 3T6 cells is quite different, because
their saturation is reached more gradually, is regulated <strong>by</strong> the
serum concentration, and does not exhibit substantial cell death.
Similarly, proliferation of 3T6 is inhibited <strong>by</strong> different concentra
tions of VAL at graduated cell densities and without substantial
cell death (Chart 7). Thus, comparing the growth behavior of
different cell lines with and without VAL, the essential action of
VAL appears to be to impose on the cells at low growth densities
a state of limiting growth condition which normally is encountered
only at high cell densities and/or low serum concentration. This
interpretation can be formulated quantitatively <strong>by</strong> using a simple
population-dynamic model allowing for only 2 cellular subpopu
lations, dividing and long-term growth-inhibited (quiescent) cells
with a cell density-dependent probability of transition from the
dividing to the quiescent subpopulation (27, 28). We have shown
earlier that proliferation of 3T3 and SV40-3T3 cells under the
usual in vitro conditions may thus be described adequately and
that the specific features of SV40-3T3 cells may be accounted
for <strong>by</strong> a somewhat lower transition probability (about one-fifth
as compared to normal 3T3) to the growth-inhibited state and,
above all, <strong>by</strong> a larger rate constant (0.5 to 3.0/day) of death of
the growth-inhibited cell population (27, 28). If the major effect
of saturating concentrations of VAL is a massive increase of the
probability of transition to the growth-inhibited state, cells will
accumulate in this state. Then, the rate constant of overall cell
death is given <strong>by</strong> that of the inhibited cell subpopulation which,
according to Chart 2, is at least 0.6/day in reasonable agreement
with the figures for untreated SV40-3T3 cells given above. Even
more detailed features, such as the interplay of cell density and
action of VAL at different concentrations (cf. Chart 7), may be
specified quantitatively according to this model.6
Our data on the effect of VAL on the cellular distribution over
the phases of the cell cycle generally agree with this interpreta
tion but allow for a more detailed characterization of the mode
of growth arrest. Most significantly, all the nontransformed cells
are growth inhibited <strong>by</strong> VAL in Gì, whereas all transformed cells
are inhibited predominantly in S and G2,although some of them
(3T6 and, to a lesser extent, PY-3T3) without VAL are growth
arrested in Gì. This apparently general difference between nor
mal and transformed cells of the cell cycle phase of growth arrest
<strong>by</strong> VAL appears to be relevant for a possible exploitation in
phase-specific selective killingof tumor cells in antitumor therapy.
The cycle phase of growth arrest <strong>by</strong> VAL generally does not
correlate with its cytotoxic effect; e.g., 3T6 cells are arrested in
S and G2 <strong>by</strong> VAL but experience only relatively little cytotoxic
effect, if compared with SV40-3T3 or ts RSV-Rat-1 (35°C)cells.
According to our still incomplete data, a better correlation is
observed between viability in the presence of VAL and the ability
of cells to down-regulate their rRNA metabolism. We have
observed5 that 3T3, Rat-1, and 3T6 cells under the action of
VAL exhibit a decrease of cellular rRNA similar to that of the
untreated controls (23). For 3T6 cells, this result is quite unex
pected, inasmuch as they down-regulate their rRNA metabolism
but are growth arrested mostly outside the Gìphase of the cell
cycle (Chart 7). In contrast, both SV40-3T3 (23) and ts RSV-Rat-
1 (35°C)cells5 are not able to regulate their rRNA content
• To be published elsewhere.
CANCER RESEARCH VOL. 45 JULY 1985
3026
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dependent on cell density (without application of VAL) and thus
exhibit features of "unbalanced growth" (29, 30). We have not
yet obtained data on the rRNA content of these cells under the
action of VAL but would expect that they also exhibit unbalanced
growth under these conditions.
Multiple studies have shown that fairly high concentrations
(>1 ÿM) of VAL are required to affect 1C transport through the
plasma membrane (31, 32). Mitochondrial function is much more
sensitive to VAL; here, concentrations of about 7 to 70 nw have
been found to affect the mitochondrial membrane potential (13,
33). In order to arrive at meaningful comparisons between effec
tive VAL concentrations of different studies, the strongly lipophilic
nature of this compound must be taken into account. The
partition coefficient of VAL between phospholipid and water has
been determined in bilayer studies and was found to be in the
range of 10" to 10s (34). Using the data given in Ref. 13, we
have a nearly saturating effect of VAL at 10 ¿¿g VAL/g mitochon
drial protein suspended at 7 mg protein/ml, yielding an overall
concentration of VAL of about 70 nw. Taking into account 200
tig lipid/mg protein for these mitochondria (35) and a lipid density
of about 0.5 g/ml, we arrive at an effective concentration of
about 25 UM VAL in mitochondrial
nearly saturating effect if a partition
lipid under conditions of a
coefficient of 104 to 105 is
used. If VAL is applied to cell cultures, it is partitioned between
water and all lipid phases present, such as serum lipids and
cellular lipids. Using cellular lipid contents determined earlier (36),
a serum concentration of 5% with total lipid at 2.5 mg/ml serum,5
a lipid density of 0.5 g/ml, and a partition coefficient of 104 to
105 for VAL in all lipids, we arrive at concentrations of VAL in
(mitochondrial) lipid of 8 to 80 MMat overall VAL concentrations
of 2 to 20 nw, respectively. Thus, the effective VAL concentra
tions in (mitochondrial) lipid under the conditions of our present
measurements are identical <strong>by</strong> order of magnitude with those
found to be effective in reducing mitochondrial membrane poten
tial (13), whereas much higher concentrations were needed to
affect K+ transport through the plasma membrane (31, 32).
Further arguments for a mitochondrial site of action of VAL stem
from experiments6 on analogous proliferation-inhibiting effects of
mitochondrial uncouplers, such as m-chlorocarbonyl-cyanide
phenylhydrazone, at about 10 fiM and on the fast and substantial
inhibition of mitochondrial uptake of the cationic fluorescent dye
rhodamine 123 upon cellular treatment with 20 nw VAL. Our
data given in Table 1 are also consistent with this interpretation,
because the ATP content of all cells tested decreases <strong>by</strong> about
30% upon treatment with 20 nw VAL.
In order to account for the selective toxic effect of VAL on
virus-transformed cells (in the presence of nonselective effects
on ATP content), the following possibilities may be discussed. If
the action of VAL is mediated via a decrease of cellular ATP
content, different cell lines may react differentially to an about
equal decrease of cellular ATP. In this context, it is of interest
that in both reticulocytes and ascites tumor cells a decline of
cellular ATP level <strong>by</strong> 30% leads to inhibition of protein synthesis
(37). Alternatively, VAL may act differently on the mitochondrial
contribution to intracellular Ca2+ homeostasis (8, 16). Cellular
ATP contents may be adjusted <strong>by</strong> concomitant stimulation of
glycolysis (38, 39). We have started work to test this second
possibility <strong>by</strong> characterizing the effect of VAL on intracellular
calcium compartments. By analysis of 45Ca efflux, 3 kinetic
calcium compartments may be separated (40), whereas the
GROWTH INHIBITION AND CYTOTOXICITY BY VAL
intracellular free Ca2+ concentration may be determined using
the fluorescent indicator Quin 2 (41). For 3T3 cells, all of these
calcium pools were shown to decrease significantly <strong>by</strong> treatment
with 20 nw VAL;7 the transformed cell lines are investigated
presently. In addition, the effect of VAL on the following cellular
reactions appears to represent fruitful issues for further study:
interplay between glycolysis and oxidative phosphorylation (37,
38); protein synthesis (37, 42); polyamine metabolism (43); and
rRNA metabolism (Ref. 14; own unpublished results briefly dis
cussed above). Studies on the exploitation of the cycle phasespecific
arrest of tumor cell proliferation also appear to be useful
and are in progress in our laboratory using in vitro and in vivo
systems.
ACKNOWLEDGMENTS
We wish to thank Dr. F. Gruber for collaboration and advice in animal experi
ments, Dr. G. Stark for discussion, and F. Braun and A. Kesper for their excellent
technical assistance.
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