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<strong>Lethal</strong> <strong>Effect</strong> <strong>of</strong> <strong>Adriamycin</strong> on the Division Cycle <strong>of</strong> HeLa Cells
S. H. Kim and J. H. Kim
Cancer Res 1972;32:323-325.
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[CANCER RESEARCH 32, 323-325, February 1972]
<strong>Lethal</strong> <strong>Effect</strong> <strong>of</strong> <strong>Adriamycin</strong> on the Division Cycle <strong>of</strong> HeLa
Cells1
S. H. Kim and J. H. Kim
Division <strong>of</strong> Biophysics, Sloan-Kettering Institute [S. H. K.] and Department <strong>of</strong> Radiation Therapy, Memorial Hospital for Cancer and Allied
Diseases [J. H. K.J New York, New York 10021
SUMMARY
<strong>Adriamycin</strong>, a new antitumor antibiotic in the anthracycline
group, promptly inhibits DNA and RNA synthesis and arrests
cell division. The cell viability (defined as the capacity <strong>of</strong> a
single cell to grow out into a macroscopic clone) is reduced
sharply following exposure to adriamycin, 0.1 fig/ml, for a
fractional period <strong>of</strong> the generation time. With the use <strong>of</strong> a
synchronous population <strong>of</strong> HeLa cells, it is shown that the
maximum loss in cell viability takes place when exposure to
adriamycin occurs during the DNA-synthetic phase (S). The
relative dose-response curves <strong>of</strong> HeLa cells exposed to either
adriamycin or daunomycin show that daunomycin is
significantly more effective in reducing the cell viability than is
adriamycin on a molar basis.
elsewhere (3). Tests for contamination <strong>of</strong> the HeLa cultures
with mycoplasma were negative.
Synchronous cultures were obtained by selective collection
and plating <strong>of</strong> mitotic cells (5). Labeling procedure,
autoradiography, and determination <strong>of</strong> nucleic acids and
protein have been described in detail elsewhere (6).
Cell counts were performed with a Model B Coulter
counter. Plating for colony counts was carried out with 60-mm
plastic Petri dishes. Control and adriamycin-treated plates
prepared from trypsinized single cell suspensions or harvested
mitotic cells (500 cells/plate) were incubated for 12 days at
37°.Colonies were fixed with methanol, stained with crystal
violet, and counted after projection with a photographic
enlarger. A colony containing more than 50 cells was
considered to be reproductively intact.
INTRODUCTION
<strong>Adriamycin</strong> is an antibiotic <strong>of</strong> the anthracycline group
isolated from Streptomyces var. caesius (1). The antibiotic has
a chemical structure similar to that <strong>of</strong> daunomycin, differing
from daunomycin only in the replacement <strong>of</strong> a hydrogen atom
in the acetyl radical <strong>of</strong> the aglycone moiety by a hydroxyl
group (9). <strong>Adriamycin</strong> has recently been reported to be an
effective growth inhibitor <strong>of</strong> several human tumors as well as
<strong>of</strong> leukemic cells. Preliminary clinical studies seem to indicate
that the adriamycin might have a higher therapeutic index (the
ratio <strong>of</strong> the normal tissue tolerance dose to the tumor lethal
dose) than does daunomycin (2, 7). The effect <strong>of</strong> daunomycin
on the nucleic acid metabolism and viability <strong>of</strong> HeLa cells has
recently been reported from this laboratory (4). The studies
reported in this paper, in which a mitotically synchronized
culture was used, provide some information on cell viability
and nucleic acid synthesis in HeLa cells following treatment
with adriamycin and make possible a comparison <strong>of</strong> the
biological activities <strong>of</strong> adriamycin and daunomycin.
MATERIALS AND METHODS
Experiments
were carried out with HeLa S-3 cells in Eagle's
minimum essential medium supplemented with 15% fetal calf
serum. Details <strong>of</strong> the cell culture procedure were described
'This work was supported in part by Grant AT(30-1)910 from the
United States Atomic Energy Commission and by National Cancer
Institute Grant CA 08748.
Received May 13, 1971;accepted October 26, 1971.
RESULTS
<strong>Effect</strong> <strong>of</strong> <strong>Adriamycin</strong> on Nucleic Acid and Protein
Synthesis. In view <strong>of</strong> the close structural similarity <strong>of</strong>
adriamycin to daunomycin, which has been shown to be an
effective inhibitor <strong>of</strong> nucleic acid synthesis in HeLa cells (4),
an experiment was carried out in which asynchronously
growing cells were exposed for various times to concentrations
<strong>of</strong> adriamycin in the range <strong>of</strong> 0.01 to 1.0 Mg/ml. Chart 1 shows
that the rate <strong>of</strong> DNA and RNA synthesis was promptly
reduced to about 20 and 50% <strong>of</strong> the control, respectively,
within 1 hr after exposure to the drug (1.0 Mg/ml). The rate <strong>of</strong>
protein synthesis as measured by tritiated valine incorporation
into the acid-insoluble fraction was not significantly reduced.
Measurements <strong>of</strong> the total DNA, RNA, and protein content in
replicate cultures showed a reduction in the amount <strong>of</strong> DNA
and RNA following exposure <strong>of</strong> cells to the drug for a period
<strong>of</strong> 12 hr, while that <strong>of</strong> protein is not appreciably reduced.
<strong>Effect</strong>s <strong>of</strong> <strong>Adriamycin</strong> on Cell Division. For observation <strong>of</strong>
the effect <strong>of</strong> adriamycin on cell multiplication, randomly
growing cells were exposed to various concentrations <strong>of</strong> the
drug for 32 hr. The control cells were growing exponentially,
with a doubling time <strong>of</strong> about 18 hr, while the cell population
exposed to the drug (0.01 /ig/ml) increased slightly for 12 hr
and then remained stationary (Chart 2).
<strong>Effect</strong>s <strong>of</strong> <strong>Adriamycin</strong> on Cell Viability. The lethal effect <strong>of</strong>
adriamycin in terms <strong>of</strong> colony formation <strong>of</strong> single cells was
studied both in asynchronous and synchronous cultures <strong>of</strong>
HeLa cells. The survival curves obtained with asynchronous
cells showed less effect at short exposure times to the drug
than at longer times (Chart 3). The lethal action <strong>of</strong> adriamycin
FEBRUARY 1972 323

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Research.
S. H. Kim and J.H.Kim
Thymidine~3H Val i ne ~3H
Chart 1. <strong>Effect</strong>s <strong>of</strong> adriamycin on the incorporation <strong>of</strong>
tritium-labeled thymidine, uridine, and valine into DNA, RNA, and
protein, respectively. Twenty-two hr after the plating <strong>of</strong> 5 X 10s
trypsinized cells, adriamycin was added and, at indicated times
following the drug addition, 15-min pulses <strong>of</strong> thymidine-3 H (1.9
Ci/mmole, 1 /jCi/ml), 10-min pulses <strong>of</strong> uridine-3 H (20 Ci/mmole, 1
fiCi/ml) or 30-min pulses <strong>of</strong> valine-3H (0.6 Ci/mmole, 3 juCi/ml) were
given to cells, which were then processed, and radioactivity was
measured in a liquid scintillation counter, o, control (no drugs); •¿,
adriamycin, 0.01 Mg/ml; A, adriamycin, 0.1 Mg/ml; »,adriamycin, 1.0
Mg/ml.
Hours
Chart 3. Survival <strong>of</strong> asynchronously growing HeLa cells exposed to
varying concentrations <strong>of</strong> adriamycin as a function <strong>of</strong> time <strong>of</strong> exposure.
The drug was added to cells 20 hr after plating. Each point represents
an average <strong>of</strong> 6 replicate plates. The plating efficiency <strong>of</strong> the control
cells was 60%.
2
Gì
32
30
Chart 2. Changes in the number <strong>of</strong> cells per plate following exposure
<strong>of</strong> HeLa cells to adriamycin. o, control (no drug); »,adriamycin 0.01
Mg/ml; A, adriamycin, 0.1 Mg/ml;»,adriamycin, 1.0 Mg/ml.
during the division cycle revealed that the drug was most toxic
during S phase (Chart 4). A relatively high degree <strong>of</strong>
synchrony and a normal rate <strong>of</strong> cell progression through the
cell cycle were obtained, as demonstrated by the graph <strong>of</strong> the
percentage <strong>of</strong> cells labeled with tritiated thymidine (Chart 4).
Comparative Study <strong>of</strong> <strong>Adriamycin</strong> and Daunomycin on Cell
Viability. Chart 5 shows the relative dose response curves <strong>of</strong>
asynchronously growing HeLa cells exposed for 1 hr to
adriamycin or daunomycin. It is evident that daunoymcin is
significantly more effective in reducing the cell viability than
adriamycin on a molar basis.
DISCUSSION
It is evident from the present experiments that adriamycin
promptly inhibits the synthesis <strong>of</strong> DNA and RNA in HeLa
cells (Chart 1). The rate <strong>of</strong> protein synthesis is not
10
--o-'
6 12 18
Hours after mitosis
24
100
80 9
Chart 4. Survival <strong>of</strong> synchronously growing HeLa cells exposed to
either adriamycin (0.3 Mg/ml) or daunomycin (0.5 Mg/ml) for 1 hr
during the different phases <strong>of</strong> the division cycle, o o, percentage <strong>of</strong>
cells labeled with tritiated thymidine (10-min pulse) during the division
cycle in the controls. AM, adriamycin; DM, daunomycin. The plating
efficiency <strong>of</strong> the control cells was 55%. The data represent the average
<strong>of</strong> 2 separate experiments.
60
20
1
324 CANCER RESEARCH VOL. 32

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Research.
<strong>Lethal</strong> <strong>Effect</strong> <strong>of</strong><strong>Adriamycin</strong>
on HeLa Cells
•¿AM
systems (9). The lethal action <strong>of</strong> adriamycin during the
division cycle shows that the drug is most effective in reducing
the reproductive capacity <strong>of</strong> cells engaged in DNA synthesis
(Chart 4). Again, the result with adriamycin is similar to that
with daunomycin in our previous studies (4). The apparent
mechanism for the differential lethal activity occurring during
the division cycle is not known. Whether the accessibility to
the drug <strong>of</strong> DNA in chromosomes may vary or whether the
efficiency <strong>of</strong> the repair may change during the cycle cannot be
determined without further experimentation.
<strong>Adriamycin</strong> has been shown, in several experimental animal
tumor systems, to have a higher therapeutic index than
daunomycin (2, 7). <strong>Adriamycin</strong> is, in fact, less effective in
killing HeLa cells than daunomycin on a molar basis. A recent
in vivo comparative study <strong>of</strong> daunomycin and adriamycin
shows that adriamycin is less cytotoxic than daunomycin to
normal hematopoietic colony-forming cells in mice, although
the lethal effect on the leukemic cells was more pronounced
with adriamycin than with daunomycin (8).
ACKNOWLEDGMENTS
0.2 0.3
Drug concentration lyg/ml)
Chart 5. Comparative survival in random HeLa cells exposed to
various concentrations <strong>of</strong> either daunomycin or adriamycin for 1 hr.
Drug was added to cells 20 hr after plating. The plating efficiency <strong>of</strong>
the control cells varied from 60 to 65%. Each point represents an
average <strong>of</strong> 3 separate experiments. AM, adriamycin; DM, daunomycin.
significantly reduced, at least for the 1st 12 hr after exposure
<strong>of</strong> cells to the drug. These results demonstrate that
replacement <strong>of</strong> a hydrogen atom at the acetyl radical <strong>of</strong> the
aglycone by a hydroxyl group does not appreciably alter the
pattern <strong>of</strong> the inhibition <strong>of</strong> nucleic acid and protein synthesis.
Although no in vitro studies on the interaction <strong>of</strong> the drug
with DNA were carried out, as was the case with daunomycin
(4), it may be conjectured that the basic inhibitory
mechanisms <strong>of</strong> action <strong>of</strong> adriamycin and daunomycin are
similar, on the basis <strong>of</strong> the rather close structural similarity
between the 2 antibiotics.
The cell viability, defined here as the capacity <strong>of</strong> a single
cell to grow out into a macroscopic clone, is reduced sharply
following exposure <strong>of</strong> the asynchronous cell population to
adriamycin (0.1 /Jg/ml) for a fractional period <strong>of</strong> the
generation time (Chart 3). However, a comparison <strong>of</strong> the
relative lethality <strong>of</strong> adriamycin with daunomycin shows that
adriamycin is less toxic than daunomycin (Chart 5). The less
toxic effect <strong>of</strong> adriamycin was also observed in in vivo animal
0.4
We extend our gratitude to Dr. J. S. Laughlin and Dr. G. J. D'Angio
for encouragement and support throughout this study. We also thank
Dr. J. Fried for assistance in the preparation <strong>of</strong> the manuscript.
REFERENCES
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(14-Hydroxydaunorubycin), a Novel Antitumor Antibiotic.
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