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

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
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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).
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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.
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