[CANCER RESEARCH 32, 323-325,
February 1972]
Lethal Effect of Adriamycin
Cells1
on the Division Cycle of HeLa
S. H. Kim and J. H. Kim
Division of Biophysics, Sloan-Kettering Institute [S. H. K.] and Department of Radiation Therapy, Memorial Hospital for Cancer and Allied
Diseases [J. H. K.J New York, New York 10021
SUMMARY
elsewhere (3). Tests for contamination of the HeLa cultures
with mycoplasma were negative.
Synchronous cultures were obtained by selective collection
and plating of mitotic cells (5). Labeling procedure,
autoradiography,
and determination
of 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
Adriamycin, 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 of 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 of the generation time. With the use of a
synchronous population of 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 of 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.
violet, and counted after projection with a photographic
enlarger. A colony containing more than 50 cells was
considered to be reproductively intact.
INTRODUCTION
RESULTS
Adriamycin is an antibiotic of the anthracycline group
isolated from Streptomyces var. caesius (1). The antibiotic has
a chemical structure similar to that of daunomycin, differing
from daunomycin only in the replacement of a hydrogen atom
in the acetyl radical of the aglycone moiety by a hydroxyl
group (9). Adriamycin has recently been reported to be an
effective growth inhibitor of several human tumors as well as
of leukemic cells. Preliminary clinical studies seem to indicate
that the adriamycin might have a higher therapeutic index (the
ratio of the normal tissue tolerance dose to the tumor lethal
dose) than does daunomycin (2, 7). The effect of daunomycin
on the nucleic acid metabolism and viability of 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 of the
biological activities of adriamycin and daunomycin.
Effect of Adriamycin on Nucleic Acid and Protein
Synthesis. In view of the close structural similarity of
adriamycin to daunomycin, which has been shown to be an
effective inhibitor of nucleic acid synthesis in HeLa cells (4),
an experiment was carried out in which asynchronously
growing cells were exposed for various times to concentrations
of adriamycin in the range of 0.01 to 1.0 Mg/ml. Chart 1 shows
that the rate of DNA and RNA synthesis was promptly
reduced to about 20 and 50% of the control, respectively,
within 1 hr after exposure to the drug (1.0 Mg/ml). The rate of
protein synthesis as measured by tritiated valine incorporation
into the acid-insoluble fraction was not significantly reduced.
Measurements of the total DNA, RNA, and protein content in
replicate cultures showed a reduction in the amount of DNA
and RNA following exposure of cells to the drug for a period
of 12 hr, while that of protein is not appreciably reduced.
Effects of Adriamycin on Cell Division. For observation of
the effect of adriamycin on cell multiplication, randomly
growing cells were exposed to various concentrations of the
drug for 32 hr. The control cells were growing exponentially,
with a doubling time of 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).
Effects of Adriamycin on Cell Viability. The lethal effect of
adriamycin in terms of colony formation of single cells was
studied both in asynchronous and synchronous cultures of
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 of adriamycin
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 of 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.
FEBRUARY
1972
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1972 American Association for Cancer Research.
323
S. H. Kim and J.H.Kim
Thymidine~3H
Val i ne ~3H
Chart 1. Effects of adriamycin on the incorporation
of
tritium-labeled thymidine, uridine, and valine into DNA, RNA, and
protein, respectively. Twenty-two hr after the plating of 5 X 10s
trypsinized cells, adriamycin was added and, at indicated times
following the drug addition, 15-min pulses of thymidine-3 H (1.9
Ci/mmole, 1 /jCi/ml), 10-min pulses of uridine-3 H (20 Ci/mmole, 1
fiCi/ml) or 30-min pulses of 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.
2
Hours
Chart 3. Survival of asynchronously growing HeLa cells exposed to
varying concentrations of adriamycin as a function of time of exposure.
The drug was added to cells 20 hr after plating. Each point represents
an average of 6 replicate plates. The plating efficiency of the control
cells was 60%.
Gì
32
30
Chart 2. Changes in the number of cells per plate following exposure
of HeLa cells to adriamycin. o, control (no drug); »,adriamycin 0.01
Mg/ml; A, adriamycin, 0.1 Mg/ml; »,adriamycin, 1.0 Mg/ml.
100
80
during the division cycle revealed that the drug was most toxic
during S phase (Chart 4). A relatively high degree of
synchrony and a normal rate of cell progression through the
cell cycle were obtained, as demonstrated by the graph of the
percentage of cells labeled with tritiated thymidine (Chart 4).
Comparative Study of Adriamycin and Daunomycin on Cell
Viability. Chart 5 shows the relative dose response curves of
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 of DNA and RNA in HeLa
cells (Chart 1). The rate of protein synthesis is not
324
9
60
1
10
20
--o-'
6
12
18
24
Hours after mitosis
Chart 4. Survival of 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 of the division cycle, o
o, percentage of
cells labeled with tritiated thymidine (10-min pulse) during the division
cycle in the controls. AM, adriamycin; DM, daunomycin. The plating
efficiency of the control cells was 55%. The data represent the average
of 2 separate experiments.
CANCER RESEARCH VOL. 32
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1972 American Association for Cancer Research.
Lethal Effect ofAdriamycin on HeLa Cells
•¿AM
systems (9). The lethal action of adriamycin during the
division cycle shows that the drug is most effective in reducing
the reproductive capacity of 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 of DNA in chromosomes may vary or whether the
efficiency of the repair may change during the cycle cannot be
determined without further experimentation.
Adriamycin has been shown, in several experimental animal
tumor systems, to have a higher therapeutic index than
daunomycin (2, 7). Adriamycin is, in fact, less effective in
killing HeLa cells than daunomycin on a molar basis. A recent
in vivo comparative study of 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
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 of the manuscript.
0.2
0.3
Drug concentration lyg/ml)
0.4
Chart 5. Comparative survival in random HeLa cells exposed to
various concentrations of either daunomycin or adriamycin for 1 hr.
Drug was added to cells 20 hr after plating. The plating efficiency of
the control cells varied from 60 to 65%. Each point represents an
average of 3 separate experiments. AM, adriamycin; DM, daunomycin.
significantly reduced, at least for the 1st 12 hr after exposure
of cells to the drug. These results demonstrate that
replacement of a hydrogen atom at the acetyl radical of the
aglycone by a hydroxyl group does not appreciably alter the
pattern of the inhibition of nucleic acid and protein synthesis.
Although no in vitro studies on the interaction of the drug
with DNA were carried out, as was the case with daunomycin
(4), it may be conjectured that the basic inhibitory
mechanisms of action of adriamycin and daunomycin are
similar, on the basis of the rather close structural similarity
between the 2 antibiotics.
The cell viability, defined here as the capacity of a single
cell to grow out into a macroscopic clone, is reduced sharply
following exposure of the asynchronous cell population to
adriamycin (0.1 /Jg/ml) for a fractional period of the
generation time (Chart 3). However, a comparison of the
relative lethality of adriamycin with daunomycin shows that
adriamycin is less toxic than daunomycin (Chart 5). The less
toxic effect of adriamycin was also observed in in vivo animal
FEBRUARY
REFERENCES
1. Arcamone, F., Franceschi, G., Tenco, S., and Selva, A. Adriamycin
(14-Hydroxydaunorubycin),
a Novel Antitumor
Antibiotic.
Tetrahedron Letters, 13: 1007-1010, 1969.
2. DiMarco, A., Gaetani, M., and Scarpinato, B. Adriamycin (NSC-123,
127): A New Antibiotic with Antitumor Activity. Cancer
Chemotherapy Kept., 53: 33-37, 1969.
3. Kim, J. H., and Eidinoff, M. L. Action of 1-0-DArabinofuranosylcytosine on the Nucleic Acid Metabolism and
Viability of HeLa Cells. Cancer Res., 25: 698-702, 1965.
4. Kim, J. H., Gelbard, A. S., Djordjevic, B., Kim, S. H., and Perez, A.
G. Action of Daunomycin on the Nucleic Acid Metabolism and
Viability of HeLa Cells. Cancer Res., 28: 2437-2442, 1968.
5. Kim, J. H., and Perez, A. G. Ribonucleic Acid Synthesis in
Synchronously Dividing Populations of HeLa Cells. Nature, 207:
974-975, 1965.
6. Kim, S. H., Kim, J. H., and Djordjevic, B. Effects of X-irradiation on
RNA and Protein Synthesis in HeLa Cells. Radiation Res., 42:
577-589, 1970.
7. Monfardini, S., Bonadona, G., DiPietro, S., Guindani, A.,
Fossati-Balani, F., and De Lena, M. Sperimentazione clÃ-nica
preliminare con Adriamicina in Pazienti Affetti da Leucemia e
Tumori solidi mi fase Avansata. Tumori, 55: 197-216, 1969.
8. Razek, A. A. A Comparative Study of Daunomycin (D) and
Adriamycin (A). Proc. Am. Assoc. Cancer Res., 12: 20, 1971.
9. Sandberg, J. S., Howsden, F. L., DiMarco, A., and Goldin, A.,
Cancer Chemotherapy Rept., 54: 1-7, 1970.
1972
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1972 American Association for Cancer Research.
325
Lethal Effect of Adriamycin on the Division Cycle of HeLa Cells
S. H. Kim and J. H. Kim
Cancer Res 1972;32:323-325.
Updated version
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/32/2/323
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1972 American Association for Cancer Research.