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[CANCERIRESEARCH
27, 124-129,January 1967]
Inhibition
of DNA Synthesis in HeLa Cells by Hydroxyurea1
S. E. PFEIFFER*AND L J. TOLMACH
Committee on Molecular Biology, and the Edward Mallinckrodt Institute of Radiology, School of Medicine, Washington University,
St. Louis, Missouri
Summary
Materials
Hydroxyurea is a highly specific, rapidly acting inhibitor of
DNA synthesis in HeLa S3 cells. It is without detectable effect
on cells that are not synthesizing DNA. Incubation in inhibitory
concentrations for as long as 19 hr does not kill cells in any
phase of the division cycle. Rapid and complete reversal is
accomplished simply by removing the drug from the culture
medium.
HeLa S3 cells were grown in Medium N16HHF by conven
tional methods (3). Synchronization was carried out by the
mitotic cell selection procedure (20), using a modified medium
(N16FCF) containing 10% calf and 5% fetal calf sera in place
of the human and horse sera, in order to achieve stronger ad
herence of interphase cells to the growth vessel (12, 15); in
addition, calcium was eliminated until the time of collection to
improve the yield (16). All experiments were carried out in
Medium N16FCF unless otherwise noted and were timed from
collection of mitotic cells.
The rates of DNA, RNA, and protein synthesis were deter
mined from measurements of the incor]x>ration of radioactive
precursors. Small volumes (10-20 /j,\) of "C-labeled thymidine
(0.05 pc; 33 mc/mmole), uridine (0.1 /ic; 230 mc/mmole), or
leucine (0.2 ¡j,c;198 mc/mmole) were added to 1 ml of culture
medium for pulse (10-30 min) labeling of DNA, RNA, or pro
tein, respectively. Additional unlabeled thymidine (10~5n)
Introduction
The introduction of hydroxyurea as a potentially useful agent
in leukemia chemotherapy (e.g., Ref. 21) has led to several
studies of its effects at the cellular level. Mohler (8) found that
this drug both inhibits division and causes death of Chinese
hamster cells and that it inhibits division of HeLa S3 cells as
well. Young and Hodas (24) reported that synthesis of DNA in
HeLa cells is inhibited by hydroxyurea, that neither RNA nor
protein synthesis is inhibited, and that the inhibition of DNA
synthesis is readily reversible 30 min after the initiation of
treatment. They suggested, as did Frenkel et al. (2), that inhibi
tion of DNA synthesis arises from interference with ribonucleotide diphosphate reduction. Finally, Sinclair (18) has recently
reported that the toxicity of hydroxyurea for Chinese hamster
cells is restricted to cells in the DNA-synthesizing (S) phase of
the division cycle. He also found that the drug is without effect
on the progression of these cells through the non-DNA-synthesizing portions of the cycle, so that cells incubated in its presence
accumulate at the end of the pre-DNA-synthetic (Gì)
phase. This
accumulation, together with the killing of the S-phase cells,
suggested that hydroxyurea might be a useful agent for pro
ducing synchronous populations of cells.
The generality of the foregoing effects has not, however, been
widely tested; indeed, Mohler (8) reported that while thymidine
prevents the inhibitory action of hydroxyurea on Chinese
hamster cells, it is without effect on hydroxyurea-inhibited HeLa
S3 cells. It would appear, therefore, that different mammalian
cell types may respond in qualitatively different ways to the
drug. We have carried out the experiments reported here to
define further the behavior of HeLa S3 cells treated with hy
droxyurea, so as to assess its utility as a selective inhibitor of
DNA synthesis.
1This investigation was supported by TJSPHS Research Grant
No. CA-04483 from the National Cancer Institute.
* Trainee supported by USPHS Training Grant 5T1 GM-714.
Received May 27, 1966; accepted August 18, 1966.
124
and Methods
was added for continuous labeling of DNA. At the end of the
labeling period the growth medium was withdrawn and the
cultures were quickly rinsed twice with buffered saline and
fixed with acetic acid-ethanol (1:3) for at least 30 min. Cultures
labeled with uridine-14C were fixed as above and washed twice
with 0.2 N perchloric acid for at least 1 hr/wash, all at 4°C.
Finally, the culture dishes were rinsed with 70% ethanol and
air dried. The bottoms were punched out for counting in a
low-background Geiger counter.
Cell division was monitored by repeated microscopic observa
tions of delineated fields (7). The mean doubling time of the
cells in Medium N16FCF was about 24 hr. Cell survival was
determined by scoring colonies containing 50 or more cells
after 10-14 days of incubation.
Hydroxyurea, obtained originally from Miss Barbara Stearns
of The Squibb Institute for Medical Research, was dissolved in
double distilled water and stored at -20°C until just before
use. A calculated volume of a 100-fold concentrated stock solu
tion was added to a culture to obtain the concentration desired.
To reverse inhibition, the drug-containing medium was with
drawn, the culture was rinsed once with medium, and fresh,
pre-gassed medium was replaced.
Results
CONCENTRATION DEPENDENCE
OF INHIBITION.
The
inhibition
of DNA synthesis as a function of hydroxyurea concentration
was determined by measuring thymidine-14C incorporation in
a series of randomly dividing cultures over a 3-hr period in the
CANCER
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Inhibition of DNA Synthesis
"O
0.2
0.4
0.6
CONCENTRATION
CHART 1. Rate of thymidine-14C incorporation (% of untreated
hydroxyurea concentration (see text for details of the method).
0.8
1.0
5,0
OF HYDROXYUREA (mM)
control) into randomly dividing HeLa cell cultures as a function of
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12
16
20
AFTER COLLECTION
.0
CHART2. Effect of increasing concentrations of hydroxyurea on cellular progression of synchronous cultures of HeLa cells (2 experi
ments combined) as examined by the continuous incorporation of thymidine-14C into DNA (open symbols) and cell division (closed
symbols; 100-160 cells present at 7 hr). Hydroxyurea concentrations: 0 mm (O), 0.025 mm (A), 0.25 mM (O), 1 HIM (V), and 5 mM (D).
presence of the drug. Semilogarithmic plots of the cpm incorpo
rated as a function of the duration of exposure to hydroxyurea
and thymidine-14C were drawn, the exponential rate constant for
each concentration, relative to that for the untreated control,
being taken as a measure of the degree of inhibition of DNA syn
thesis (Chart 1). The 50% inhibition concentration of 5 X 10~5 M
is close to that found by Young and Hodas (24) for HeLa cells
(1 X 10~4M), but is less than Mohler (8) found for Chinese ham
ster cells (6.5 X 10~4 M) or Yarbro et al. (23) found for ascites
tumor cells (about 3 X 10~4 M). At about 1 mM the rate of in-
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125
12
126
16
20
24
28
HOURS AFTER COLLECTION
32
36
1.0
40
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Inhibition of DNA Synthesis
corporation is reduced to 2% of that in untreated cultures; it
remains constant at that level to at least 5 mu. In subsequent
experiments in which maximal inhibition was desired, 2.5 n»i
hydroxyurea was used.
Chart 2 shows that when increasing concentrations of hy
droxyurea are added to synchronous cultures immediately after
the harvest of mitotic cells, the subsequent rate of DNA syn
thesis is correspondingly reduced, as would be predicted from
the data of Chart 1. As expected, the nearly complete inhibition
of DNA synthesis at concentrations above 1 mM prevents cell
division; no division can be detected by 25 hr, while 80% of
the cells in untreated cultures have divided by this time.
RESTRICTION
OF INHIBITORY
ACTION TO THE S PHASE. Because
of experimental uncertainty arising from the low levels of
thymidine-14C incorporated at early times, it is difficult to de
termine from the data of Chart 2 whether hydroxyurea has any
effect on the progression of cells through G\. To test this point,
synchronous cells were treated continuously with 2.5 mM hy
droxyurea from 0 to 5 hr, i.e., before the normal start of the S
phase (Chart 3A). There was no lag in the onset of DNA syn
thesis, nor any delay in progression through the S phase or
cell division; treatment confined to Gìis without detectable
effect on cell progression.
In a similar experiment, hydroxyurea was added to synchro
nous cells 18 hr after collection, when the majority were in the
post-DNA-synthetic
(Gì)phase. Subsequent monitoring of
cell division (Chart 3B) showed that cells in the treated culture
began to divide at the same time as in the control culture and
that division proceeded at the control rate for about 4 hr, the
duration of Gz for these cells (20). The division rate then de
clined in the treated cultures, finally reaching zero when 54%
of the cells had divided; in contrast, more than 90% of the control
cells divided. An estimate of the number of cells still in the S
phase at 18 hr was made from the ratio of the rate of DNA
synthesis at this time relative to the rate 14 hr after collection,
when DNA was being synthesized most rapidly.3 The ratio
indicates that approximately 40% of the cells were still syn
thesizing DNA at the time of addition of hydroxyurea. This
value is in agreement with the fraction of cells, relative to control,
that did not divide (0.4). Thus, it appears that the progression
of cells in GÎ,as in Gì,
is not affected by 2.5 niM hydroxyurea.
Since the cells proceeding to mitosis divided normally, the
same conclusion can be drawn for progression through mitosis.
SPECIFICITYOF ACTION.If the rate of uridine incorporation
into RNA is measured in hydroxyurea-treated synchronous G¡
cells, little or no inhibition of RNA synthesis can be detected.
Similarly, no inhibition of leucine incorporation into protein
' Autoradiographic determination of the fraction of thymidinelabeled cells at 18 hr would give a direct measure of the fraction
of cells in S. Since the rate of incorporation of thymidine-14C by
the culture is the product of the fraction of labeled cells and rate
of synthesis per cell, the use of this parameter tends to underesti
mate the fraction of cells still in S (see Ref. 20, Fig. 6).
can be detected under these conditions. Table 1 presents data
illustrating this for cells to which hydroxyurea was added at
3.75 hr and which were tested at later times. Analogous results
were obtained with randomly dividing cultures in which DNA
synthesis was inhibited 98% by addition of the drug and tested
immediately thereafter.
It would appear from these results, from the report of Young
and Hodas (24), and from the restriction of the inhibitory action
of hydroxyurea to the S phase, that the drug affects only DNA
synthesis. However, as will be described elsewhere, treatment
with hydroxyurea severely inhibits the increase in the rate of
RNA synthesis that normally occurs early in the S phase of this
system. Nevertheless, as the rate of RNA synthesis is not de
creased, it is likely that the effect is an indirect consequence of
the inhibition of DNA synthesis; a similar effect on acceleration
of RNA synthesis is brought about by other DNA inhibitors as
well (Pfeiffer, unpublished data).
SPEED OF ACTION, REVERSIBILITY,
AND TOXICITY. It
ÃŒS
often
desirable that an inhibitor act rapidly and be highly (and,
preferably, rapidly) reversible. In addition, if the studies in
which it is to be used involve cell survival (colony-forming
ability) measurements, the temporary inhibition must cause
little or no cell killing.
The speed with which hydroxyurea inhibits DNA synthesis is
shown hi Chart 3(7. When added to synchronous cells synthe
sizing DNA at the peak rate, maximal inhibition occurs within
the time required for experimental manipulation of the cultures.
Reversibility was examined by exposing mitotic cells to
hydroxyurea for 16.6 hr and then removing the inhibitor. Chart
3D shows the effect on both DNA synthesis and cell division.
The following observations are pertinent: (a) DNA synthesis
(closed circles) resumes immediately after the drug is removed,
even after a period of inhibition much longer than that studied
by Young and Hodas (24); i.e., reversal is rapid. (6) DNA syn
thesis proceeds more rapidly in the treated culture than in the
control (open circles), but the areas under the curves are the
same within the limits of accuracy of the measurements; this
TABLE 1
INCORPORATION
OF RADIOACTIVELY
LABELEDPRECURSORSINTO
PROTEINANDRNA IN NORMALANDHYDROXYUREATREATEDCULTURES
AFTERCOLLECTION0
PRECURSORSLeucine-I4CUridine-14CTIME
(hr)61068RATE
OF INCORPORATION'
(cpm)Control10134655Treated12145350
0 2.5 mM hydroxyurea was added at 3.75 hr.
6 cpm/culture after 20 (leucine)- or 10 (uridine)-min
labeled precursor.
pulses with
CHART 3. Rate of DNA synthesis (circles; thymidine-14C incorporation, 30-min pulses) and relative cell number (squares; 100-200
cells present at the 1st reading) versus time after collection of mitotic cells in control (open symbols) and hydroxyurea-treated
(closed
symbols; 2.5 HIM) cultures of synchronous HeLa S3 cells. Hydroxyurea was present: A, 0-5 hr; B, continuously from 18 hr; C, continu
ously from 12 hr; and D, 0-16.6 hr.
JANUARY
1967
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127
S. E. Pfeiffer and L. J. Tolmach
T "'
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'l
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'
100—¿—o—c*0-H-»n
axa' —
80
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er
\
40
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20
0
4
DURATION
8
12
16
20
24
28
OF TREATMENT
WITH HYDROXYUREA IHR)
CHART4. Survival (% of untreated control) of HeLa cells after exposure to 2.5 mia hydroxyurea for various durations of treatment.
Control plating efficiencies were routinely 90-100%. The different symbols indicate survival of both randomly dividing cultures (•)
and synchronous S (O) or Gì(D) cultures growing in medium N1CFCF and randomly dividing cultures growing in Medium N16HHF
(A).
ndicates that delaying the onset of DNA synthesis does not
affect the total amount of DNA synthesized, (c) The fraction of
cells ultimately dividing (squares) is nearly the same in both
cultures (control, 93%; treated, 90%). Observations (6) and (c)
indicate that reversal of inhibition is complete on removal of
the drug.
The increased rate of DNA synthesis observed in Chart 3D
is due to 2 factors. First, an increase in the rate of DXA synthesis
in the culture as a whole may result from an increase in the de
gree of synchrony arising from the accumulation of cells at the
end of Gìin the presence of the inhibitor. Sinclair has demon
strated such behavior conclusively by autoradiography in
Chinese hamster cells (18) treated with hydroxyurea, and, in
fact, synchronization has been effected or enhanced with a
number of DNA inhibitors in a variety of systems. Second, there
is actual shortening of S for at least some cells. Treated cells
began to divide as early as 8 hr after reversal of inhibition,
whereas the minimum duration of S plus G 2 in the untreated
cells was 10 hr. (Since the duration of S plus Gj is quite constant
among the cells of a given population (19, 20), it is presumed
that the first cells to divide were the first to commence DNA
synthesis.) Thus, at least the most rapidly progressing cells which
have undergone temporary inhibition at the end of Gì
appear to
traverse the S and GÕphases more quickly than do untreated
cells. This is not unexpected, inasmuch as the rate of DNA syn
thesis is faster after reversal of inhibition, while the amount of
DNA synthesized is the same. Similar behavior has been noted
by others in experiments in which DNA synthesis was delayed
by treatment with different inhibitors (17, 22). Such distortion
of the normal rate of cell progression must be taken into con
128
sideration when methods of synchronization based on inhibition
of DNA synthesis are employed.
The toxicity of hydroxyurea was examined under a wide
variety of conditions: randomly dividing cells, or synchronous
cells in either S or Gì,
were exposed to hydroxyurea for jjeriods
ranging from 0.5 to 31.5 hr (Chart 4). Only after inhibition for
more than 19 hr is the plating efficiency definitely reduced, in
marked contrast to the reported behavior of Chinese hamster
cells (8, 18). This delayed effect may be attributed to the longterm inhibition of DNA synthesis rather than to a specific
effect of hydroxyurea; the effect of long-term inhibition with
fluorodeoxyuridine has been examined in detail by others (17).
The foregoing plating efficiency measurements were carried
out in Medium N16FCF, which is known to contain thymidine
(presumably as a component of the fetal calf serum). Since
Mohler had rejiorted (8) that thymidine protects Chinese
hamster cells from the inhibitory action of hydroxyurea, we
examined the possibility that it might protect HeLa cells from
loss of reproductive capacity, even though the ex¡>eriments
reported here show that it is unable to prevent inhibition of
DNA synthesis by hydroxyurea in this cell type.4 When plating
efficiencies were measured in synchronous populations growing
either in Medium N16HHF (which lacks significant amounts of
thymidine) or in N16HHF with 10~5M thymidine added no
cell killing was found after treatment for 4.5 hr during the S
4 In Mohler's experiments (8), thymidine appeared to protect
Chinese hamster cells more against loss of viability than against
inhibition of division.
CANCER
RESEARCH
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VOL. 27
Inhibition of DNA Synthesis
phase (Chart 4, triangles). It may be concluded, therefore, that
HeLa cells respond differently from Chinese hamster cells to
treatment
with hydroxyurea,
in regard not only to toxicity
during S but also to the effect of thymidine (8, 18).
Discussion
The present experiments confirm the report of Young and
Hodas (24) concerning the specific inhibition by hydroxyurea of
DNA synthesis in HeLa cells. In addition, information is pre
sented regarding the absence of inhibitory effects on cells in
phases of the division cycle other than S, the speed with which
inhibition and reversal of this inhibition occurs, and the length
of time that inhibition may be sustained before cell killing is
observed. In this last respect these results with HeLa cells are
in marked contrast to the killing effect of the drug observed in
Chinese hamster cells (8) inhibited in the S phase of the division
cycle (18). Whereas Sinclair (18) found that exposure of S-phase
cells to hydroxyurea for 1 hr was sufficient to kill 80-90% of
the population, Chart 4 indicates that HeLa cells can withstand
up to 19 hr of exposure to the drug without significant toxic
effects. Such divergent behavior between these 2 cell strains is
unexplained. However, considerations of such differences are of
possible significance in the application of hydroxyurea to tumor
therapy.
In conclusion, hydroxyurea
ap¡>ears to closely resemble
fluorodeoxyuridine
(17, 22), deoxyadenosine (5, 6, 10, 13), and
high concentrations
of thymidine (5, 14) in being a rapidly
acting inhibitor of DNA synthesis in animal cells; however, it
possesses a particularly
useful combination
of properties for
experimental
studies. Thus, unlike inhibition by fluorodeoxyuridine, the action of hydroxyurea is readily reversed simply by
removing the drug from the culture medium, and hydroxyurea
has the added advantage of exerting its inhibitory effect in the
presence of concentrations of thymidine that reverse the action
of fluorodeoxyuridine.
Further, the inhibitory action of hydroxy
urea is specific for DNA synthesis, whereas thymidine at high
concentrations
also inhibits incorporation of undine (4, 9, 11)
or cytidine (1) into RNA. Thus, hydroxyurea
would appear
to be an important addition to the list of agents available for
the study of growth dynamics in animal cells.6
References
1. Feinendegen, L. E., Bond, V. P., and Hughes, W. L. RNA
Mediation in DNA Synthesis in HeLa Cells Studied with
Tritium Labeled Cytidine and Thymidine. Exptl. Cell Res.,
25: 627-47, 1961.
2. Frenkel, E. P., Skinner, W. N., and Smiley, J. D. Studies on a
Metabolic Defect Induced by Hydroxyurea. Cancer Chemo
therapy Rept., 40: 19-22, 1964.
3. Ham, R. G., and Puck, T. T. Quantitative Colonial Growth of
Isolated Mammalian Cells. In: S. P. Colowick and N. O.
Kaplan (eds.), Methods in Enzymology, Vol. 5, pp. 90-119.
New York: Academic Press, 1962.
4. Kasten, F. H., Strasser, F. F., and Turner, M. Nucleolar and
Cytoplasmic Ribonucleic Acid Inhibition by Excess Thymi
dine. Nature, 207: 161-64, 1965.
6 Some of the data presented in Charts 2 and 4 are from experi
ments performed by Dr. Robert A. Phillips and Dr. Barbara G.
Weiss.
JANUARY
5. Klenow, H. On the Effect of Some Adenine Derivatives on the
Incorporation
in Vitro of Isotopically Labeled Compounds
into the Nucleic Acids of Ehrlich Ascites Tumor Cells.
Biochim. Biophys. Acta, 35: 412-21, 1959.
6. Langer, L., and Klenow, H. The effect of Some Purine Deoxyribosides on the Incorporation
of [HC]-Formate and
l32?]-Orthophosphate into DNA of Ascites Tumor Cells in
Vitro. Ibid., 37: 33-37, 1960.
7. Marcus, P. I., and Puck, T. T. Host-Cell Interaction of Animal
Viruses. I. Titration of Cell-Killing by Viruses. Virology, 6:
405-23, 1958.
8. Mohler, W. C. Cytotoxicity of Hydroxyurea Reversible by
Pyrimidine Deoxyribosides in a Mammalian Cell Line Grown
in Vitro. Cancer Chemotherapy Rept., 34: 1-6, 1964.
9. Morris, N. R., Reichard, P., and Fisher, G. A. Studies Con
cerning the Inhibition of Cellular Reproduction by Deoxyribonucleosides. I. Inhibition of Synthesis of Deoxycytidine
by Phosphorylated
Derivatives
of Thymidine.
Biochim.
Biophys. Acta, 68: 84-92, 1963.
10. Overgaard-Hansen,
K., and Klenow, H. On the Mechanism of
Inhibition of Deoxyribonucleic
Acid Synthesis in Erhlich
Ascites Tumor Cells by Deoxyadenosine in Vitro. Proc. Nati.
Acad. Sei. U. S., 47: 680-86, 1961.
11. Painter, R. B., Drew, R. M., and Rasmussen, R. E. Limita
tions in the Use of Carbon-Labeled
and Tritium-Labeled
Thymidine in Cell Culture Studies. Radiation Res., el: 355-66,
1964.
12. Phillips, R. A., and Tolmach, L. J. Repair of Potentially
Lethal Damage in X-Irradiated
HeLa Cells. Ibid., 89: 41332, 1966.
13. Prusoff, W. H. Further Studies on the Inhibition of Nucleic
Acid Biosynthesis by Azathymidine and by Deoxyadenosine.
Biochem. Pharmacol., 8: 221-25, 1959.
14. Puck, T. T. Studies on the Life Cycle of Mammalian Cells.
Cold Spring Harbor Symp. Quant. Biol., 24: 167-76, 1964.
15. Puck, T. T., Marcus, P. I., and Cieciura, S. J. Clonal Growth
of Mammalian Cells in Vitro. Growth Characteristics
of
Colonies from Single HeLa Cells with and without a "Feeder"
Layer. J. Exptl. Med., 103: 273-83, 1956.
16. Robbins, E., and Marcus, P. I. Mitotically Synchronized
Mammalian Cells: A Simple Method for Obtaining Large
Populations. Science, 144: 1152-53, 1964.
17. Rueckert, R. R., and Mueller, G. Studies on Unbalanced
Growth iu Tissue Culture. I. Induction and Consequences of
Thymidine Deficiency. Cancer Res., 20: 1584-90, 1960.
18. Sinclair, W. K. Hydroxyurea: Differential Lethal Effects on
Cultured Mammalian Cells During the Cell Cycle. Science,
160: 1729-31, 1965.
19. Sisken, J. E., and Morasca, L. Intrapopulation
Kinetics of
the Mitotic Cycle. J. Cell Biol., 25 (Part 2).-179-89,1965.
20. Terasima, T., and Tolmach, L. J. Growth and Nucleic Acid
Synthesis in Synchronously Dividing Populations of HeLa
Cells. Exptl. Cell Res., SO: 344-62, 1963.
21. Thurman, W. G. (ed.). Symposium on Hydroxyurea. Cancer
Chemotherapy Rept., 40: 1-78, 1964.
22. Till, J. E., Whitmore, G. F., and Gulyas, S. Deoxyribonucleic
Acid Synthesis in Individual L-Strain Mouse Cells. II. Effects
of Thymidine Starvation. Biochim. Biophys. Acta, 72: 277-89,
1963.
23. Yarbro, J. W., Kennedy, B. J., and Barnum, C. P. Hydroxy
urea Inhibition of DNA Synthesis in Ascites Tumor. Proc.
Nati. Acad. Sei. U. S., 53: 1033-35, 1965.
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1967
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129
Inhibition of DNA Synthesis in HeLa Cells by Hydroxyurea
S. E. Pfeiffer and L. J. Tolmach
Cancer Res 1967;27:124-129.
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