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

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
Downloaded from
cancerres.aacrjournals.org on December 2, 2012
Copyright © 1985 American Association for Cancer Research