Download Amino Acid Analogs, and Nucleases on the Synthesis of DNA in

Autoradiographic
Studies of the Effects of Antibiotics,
Amino Acid Analogs, and Nucleases on the Synthesis
of DNA in Cultured Mammalian Cells*
VIN0D C. SHAHt
(Zoolo@ryDepartment,
Columbia University,
New York, New York)
SUMMARY
Autoradiographic
studies of the effects of some antibiotics, amino acid analogs, and
nucleases on the synthesis of DNA have been carried out to gain information on the
regulatory
mechanism
of DNA replication
within the cell. Cultured cells of the Chinese
hamster
and human cancer (HeLa) were used as experimental
material.
Puromycin,
chloramphenicol,
p-fluoro-phenylalanine,
and methylated
tryptophan
analogs inhibited in varying degree the synthesis of protein. They also affected the
synthesis of deoxyribonucleic
acid (DNA) by changing the number of cells able to in
corporate thymidine-H3 as well as by reducing the rate of thymidine incorporation in
the replicating cells. The relationship of histone and DNA synthesis was studied cyto
chemically, and data obtained indicate that histones do not seem to play any direct
role in DNA replication. Actinomycin D inhibited intracellular ribonucleic acid (RNA)
synthesis
but did not affect
DNA
synthesis.
This
suggests
that
there
is no necessary
coupling of RNA and DNA syntheses. The nucleases (DNase I, DNase II, and RNase),
within a short time after contact with the cells in culture medium, caused an increase
in the number of cells able to incorporate thymidine. After 3 hours of treatment both
DNase II and RNase resulted in decreases in DNA synthesis.
The results are discussed with regard to the hypothesis that RNA may act as re
pressor for DNA synthesis and that the repressor activity is regulated by amino acids or
proteins.
The requirements
free systems
have
for DNA replication
been defined
in cell
by Kornberg
and
others (17) . However, the mechanisms which regu
late DNA synthesis
within the cell are still
obscure (920).These control mechanisms could con
ceivably operate at three levels : (a) in the forma
tion of precursors—i.e. , the conversions of ribo
nucleotides to deoxyribonucleotides
(392), the for
mation of thymidylate
(192), or the phosphoryla
tion of deoxyribonucleotides
to the corresponding
* A portion
of a dissertation
submitted
in partial
fulfillment
triphosphates
(921); (b) by the synthesis or activa
tion of DNA polymerase; and (c) by the formation
of an active primer or template for synthesis. In
1954
Cohen
and
Barner
(8) suggested
that
protein
and/or ribonucleic acid synthesis might be direct
ly involved in the replication of DNA on the basis
of the behavior of a thymine-requiring
strain of
Escherichia
coli. Protein
and RNA
synthesis
with
out concomitant DNA synthesis was found lethal,
but the cell death was prevented if protein syn
thesis was also blocked. Various studies since then
of the requirements for the degree of Doctor of Philosophyin
(7,
the Faculty of Pure Science, Columbia University.
This investigation
was supported in part by a grant from
shown
13, 924) have
extended
a requirement
these
for protein
observations
synthesis
and
to initi
ate but not for the continuation of DNA synthesis
in E. coli and its phages.
In addition, studies of bacterial mutants re
t Present
address:
Medical
Research
Center,
Brookhavenquiring various amino acids have indicated a re
National Laboratory, Upton, L.I., New York.
quirement
for amino acids in the synthesis of
RNA, apparently
in some process that does not
Received for publication March 25, 1963.
the Atomic Energy Commission AT (30-1)-1304 to J. Herbert
Taylor and by a Faculty Fellowship from Columbia Univer
sity.
1137
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Cancer Research
1138
involve protein synthesis (35). A recent report by
Kellenberger et al. (16) indicates that synthesis of
both RNA and DNA in E. coli operates by a
mechanism that requires amino acids. However,
“relaxedstrain― mutants of E. coli were found in
which DNA synthesis would proceed in the ab
sence of a required amino acid. Chloramphenicol
could be substituted
for various amino acids
except serine. Bendich and Rosenkranz
(3) have
found phosphoserine linked to DNA by a covalent
bond which suggests that serine may be directly
involved in the synthesis of DNA.
In the somatic cells of higher forms, DNA ap
pears to be conjugated
with the basic proteins
(histones). Bloch and Godman (5) showed that
DNA and histones are synthesized simultaneously
during the interphase. It has been shown recently
that protein synthesis is necessary for DNA syn
thesis in mammalian cells, as was found earlier for
bacteria (923,927).On the basis of the above obser
vations, one might suspect that one type of protein
necessary for the DNA synthesis in higher forms
may be the histones.
The present investigations
were undertaken to
examine, in mammalian cells in culture, the effects
of various agents which alter one or more of the
processes suspected to be involved in the control
of DNA replication.
1963
hydrated.
The cover glasses with cells exposed
were then attached
to microscopic slides with
“Euparol―
according to the method described by
Taylor (36). After 92days, the surface to which the
cells were attached was covered with Kodak AR
10 stripping
70
C.
for
film
1—3
and
exposed
weeks.
The
in the dark
preparations
at about
were
de
veloped in D-19 (half strength) for 5 minutes at
18°C., rinsed in water, and fixed in Kodak acid
fixer (half strength) for 10 minutes. They were
again rinsed in water briefly and transferred to a
diluted solution (9 : 1) of Kodak hypo clearing
agent for 92minutes. After two rinses (3 minutes
each), they were dried and stained by azure B
bromide at pH 4, for 10 minutes. After a short
rinse in distilled water, the slides were dried again.
The thymidine-H3
(specific activity, 1.88 c/
mmole) and cytidine-H3 (specific activity, 0.36 c/
mmole) were obtained from Schwarz Laboratory,
Mount Vernon, New York. The lysine-H3 (spe
cific activity,
1.5 c/mmole)
was a gift to Dr.
Taylor from Dr. Klaus Hempel, Institute
of
Chemical and Medical Isotope Research, Univer
sity
of Köln, Germany.
The
leucine-H3
(specific
initiated
from embryonic
tissues of Chinese
hamster (Cricetulus griseus), was kindly supplied
by Dr. George Yerganian, Children's Cancer Re
search Foundation,
Boston. A strain of human
cancer cells (HeLa) was obtained from Dr. Berge
Hampar, Department
of Microbiology, College of
Physicians and Surgeons, Columbia University,
New York. The stock cultures of both the strains
were maintained in milk dilution bottles in Puck's
activity, 3.57 c/mmole) was purchased from New
England Nuclear Corporation,
Boston, Massa
chusetts.
A reticle with concentric circles was placed in
one of the eyepieces of the microscope. At a magni
fication of 19250X , its smallest circle with a
diameter of 5.1 delimited an area of 920.492sq. j@,
which was taken as the unit area for grain count
ing.
Antibiotics, amino acid analogs, and nucleases.—
Puromycin, chloramphenicol,
and actinomycin D
were generous gifts from the laboratories
of
American Cyanamid Co., Chas. Pfizer & Co., and
Merck, Sharp and Dohme, respectively. $-Methyl
tryptophan, isomers A and B, were kindly supplied
by Dr. H. R. Snyder, Department
of Biochemis
(929) medium
try,
MATERIALS
@
Vol. 923, September
AND
METHODS
Cell culture.—A strain of hamster cells (Al),
containing
8 per cent
calf serum,
8
per cent fetal calf serum, 500 units/nd penicillin,
and 100 @g/ml streptomycin.
For most experi
ments the cells were transferred
to screw-cap
Leighton
tubes (Bonus Laboratory
Products,
P.O. Box 66, North Andover, Massachusetts)
and
were grown attached to 10 X 50-mm. No. 1 cover
glasses. Each tube contained 1 ml. of medium
with 4—5X l0@cells (counted by hemocytometer).
Experiments were performed after 50—70hours of
growth in the culture tubes.
Autoradiography.—The
cultures on the cover
glasses were routinely fixed in Duboscq-Brassil
fixative (11) for 92hours; however, other fixatives
were also used, depending on the nature of an ex
periment. They were then rinsed in water and de
University
of Illinois,
Urbana,
Illinois.
The
other amino acid analogs, 5-methyl tryptophan
and p-fluorophenylalanine,
were purchased from
Nutritional
Biochemical Corporation,
Cleveland,
Ohio. The nucleases, RNase, DNase I, and DNase
II, were bought from Worthington
Biochemical
Corporation, Freehold, New Jersey.
The compounds were dissolved in glass-distilled
water and used to dilute the double-strength
medium to give the final appropriate
concentra
tion.
RESULTS
Generation
method,
studying
time
of the celLi.—A pulse-labeling
described by Taylor (36), was used for
the generation cycle of the hamster cells.
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1963 American Association for Cancer Research.
SHAH—DNA Synthesis in Cultured Mammalian
A group of cultures was incubated for 10 minutes
in a medium containing thymidine-H3 (1 j@c/ml),
washed briefly with Tris buffer, and placed in a
conditioned medium containing unlabeled thymi
dine at a concentration
50 X the radioactive thy
midine. The cells were fixed at intervals in for
maldehyde, 40 per cent solution, 5 parts; 70 per
cent ethanol, 90 parts; and glacial acetic acid, 5
parts, for 1 hour and stained by the Feulgen
method after a hydrolysis of 10 minutes. After an
exposure of 92 weeks to the stripping film, the
autoradiograms
were developed and examined for
labeled interphase and division stages by random
sampling. The ratio of labeled interphases was de
termined by examination of @50—300
cells at each
time interval. To obtain the ratio of labeled divi
sion stages 150—9200
division figures on each slide
were scored.
Cells
1189
cells dropped during the first 1—92
hours and then
increased to reach a peak of approximately
64 per
cent at 8 hours after labeling. It again dropped,
became equal to the fraction initially labeled by
192 hours—which
was one generation
sults are similar to the measurements
cycle.
The
re
by Hsu et at.
(14)in a similar strain of hamstercells,sincethey
have found the mean generation time of 11—192
hours with S-period of 5.8 hours, with the use of
McCoy's 5a medium supplemented
with 920 per
cent fetal calf serum.
No attempt was made to find the exact genera
tion time of the HeLa cells in culture. According
to the estimation of Painter and Drew (926), the
generation time of HeLa cells was approximately
928 hours, with 8@ hours for the DNA synthetic
period 8, 14 hours for Gi, and 3—10hours for
G92.The cycle for the HeLa cells in these cultures
U
0
z
U
U
U
0@
CHART 1.—Frequency
treatment
I
2 3 4
of labeled
interphases
with thymidine-H3
in hamster
5 6 7 8 9 10 II 2 3 14 15 16 17 18 19 20
TIME IN HOURS
(circles)
and labeled
division
stages
(bars) at various
After a lag of 1 hour (Chart 1) a small percent
may be slightly
shorter,
age of labeled dividing cells began to appear, and here is richer in nutrients
@
@
intervals
after
10 minutes,
cells.
by the 6th hour ca. 97 per cent of the division
figures were labeled. The frequency of the labeled
divisions then declined until the 11th hour, when
it reached 19 per cent, after which there was again
a rise and a second peak at 18 hours, with approxi
mately 78 per cent of mitotic cells labeled. The
average generation time, therefore, was about 192
hours, the interval between the two peaks of
labeled division stages. The DNA synthetic
period S obtained by multiplying the fraction of
labeled interphase cells, after a short contact with
thymidine,
by the generation time (0.48 X l@,
Chart 1) was 5.76 hours. Taking 1—92
hours as the
initial lag time for the appearance of labeled divi
sion figures as post-DNA synthetic period G92and
approximately
1 hour for mitosis M, the pre
DNA synthetic time Gi can be estimated to be
about 4 hours. The frequency of labeled interphase
Painter and Drew.
Effects of puromycin,
since the medium used
than the one used by
chioramphenicol,
and two
amino acid analog8 on the incorporation of amino
acids into protein.—The amino acids leucine-H3
and lysine-H3 were used in these experiments. Pre
liminary studies showed that leucine-H3, even
with a high specific activity (3.57 c/mmole), failed
to label HeLa and hamster cells, if used in concen
trations below 10 sc/ml for
hour with an ex
posure to film for
weeks. However, lysine-H5
(10
@sc/ml), after
a treatment
of 10 minutes,
pro
duced satisfactory autoradiographs. Thus, the
leucine pool in these cells seemed to be consider
ably greater than that of the lysine.
The hamster cells were incubated with differ
ent concentrations of puromycin, chioramphenicol,
5-methyltryptophan,
and p-fluorophenylalanine
in the culture medium and at certain intervals
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1963 American Association for Cancer Research.
Cancer Research
1140
@
were labeled with lysine-H3 (10 /Lc/nil) for hour.
They were then fixed and autoradiograms
were
prepared as described previously with an exposure
of 92weeks to the film.
Charts 92and 3 show the effects of puromycin
and chloramphenicol
on the incorporation
of
PUROMYCIN
-J
-J
Vol.923,
September 1963
The amino acid analogs p-fluorophenylalanine
and 5-methyl tryptophan also inhibit protein syn
thesis (Charts 4, 5). The 5-methyl tryptophan
in
the concentration of 1 mg/ml (about 500 times the
tryptophan
concentration
in Puck's
medium)
re
duced protein synthesis more than 90 per cent
after 924hours of incubation. However, p-fluoro
phenylalanine
even in higher concentration
(1.5
mg/mi) (about 9200 times the phenylalanine
con
centration in Puck's medium) inhibited no more
than 40 per cent after similar incubation. In both
U
U
p -FLUOROPHENYLALAN NE
(1)
z
4
-J
-J
Ui
U
CD
-j
4I-.
0
Vi)
z
4
CD
-J
4
6
9
HOURS OF INCUBATION
0
I-
Ca&RT2.—Effect of various concentrations of puromycin
on the incorporation of lysine-H3 (10 @sc/ml—30
minutes) in
hamster cells. Average of SOcells for each time interval.
CHLORAMPHENICOL
3
6
9
2
5 18 21
HOURS OF INCUBATION
CHART 4.—Effect of various
phenylalanine
on incorporation
minutes)
in hamster
24
concentrations
of p-fiuoro
of lysine-H1
(10
@@c/ml—30
cells. Average
of 30 cells for each time
interval.
@
-J
-J
U
C.)
5- METHYL TRYPTOPHAN
I
I
I
I
35
C/)
z
C/)
-J
.J 30
Ui
U
4
0
%,.
S@@@O@7RoL
25
U)
z
@
@
0
1-
CD
@
4
@
6
9
I
HOURS OF INCUBATION
of various
concentrations
of chioram
phenicol on the incorporation of Iysine-H1 (10 pc/ml—S0mm
utes) in hamster
•%___. 2Oug
N@@'%@%lr..@_
5
.
10
ug
.
@S%%%
‘@‘
5
@
CHART 5.—Effect
@20 -
cells. Average of SO cells for each time interval.
lysine-H3. Puromycin in concentrations
of 925,50,
and 100 @.ig/m1rapidly inhibited protein synthesis,
although the effects increased in intensity with the
increasing concentrations.
By 192hours 100 @g/ml
of puromycin reduced the incorporation
into the
total protein by about 90 per cent. Higher concen
trations of chioramphenicol
were required to in
hibit protein synthesis significantly.
It may be
noticed that as much as 92mg/ml chioramphenicol
for 924hours was required to reduce protein syn
thesis by 95 per cent.
0
I
I
3
6
CHART 5.—Effect
I
I
I
I
I
9 12 5
8 21 24
HOURS OF INCUBATION
of various
concentrations
of 5-methy
tryptophan
on incorporation
of lysine-H' (10 j@c/ml—S0 mm
utes) in hamster cells. Average of 30 cells for each time interval.
cases concentrations
lower than 1 mg/mi pro
duced little effect.
In all the above experiments, although the pro
tein synthesis may be reduced as much as 95 per
cent, the effects on cell morphology
were not
severe. However, treatments for longer time than
mentioned above with maximum concentration of
inhibitors resulted in loss of cells by detachment
from the glass.
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1963 American Association for Cancer Research.
SHAH—DNA Synthesis in Cultuned Mammalian
Ei7e@t of the inhibitors
of protein
sijnthesis
on
DNA replication.—Cultures of hamster cells were
incubated after changing to fresh medium with
appropriate
amounts of inhibitors. Control cul
tures also received fresh medium without the in
hibitors at the same time. At several intervals cul
tures were rinsed and incubated in conditioned
medium containing thymidine-H3, 1 @ic/mlfor 30
minutes. They were then washed briefly with Tris
buffer, fixed, and autoradiograms
were made as
described before.
Puromycin at a concentration
of 100 zg/ml in
hibited the incorporation of thymidine-H3 within
a short time of incubation (Chart 6, Fig. 1). The
effect was gradual and cumulative, since the de
crease in incorporation (which may be interpreted
as rate of synthesis) occurred slowly until at 192
hours, with 100 @g/ml,the incorporation of thymi
dine was negligible. Chloramphenicol
(92mg/nil)
also reduced the rate of incorporation
of thymi
dine-H3 (Chart 6). After a lag of 3 hours there was
a rapid decrease in the incorporation of thymidine
up to 192hours. Afterward there was slower de
crease, and by 924hours DNA synthesis was hard
CelLi
1141
incorporation of thymidine-H3 was observed with
5-methyl tryptophan.
Both isomers A and B of
$-methyl tryptophan
were less effective than 5methyl tryptophan,
since no significant decrease
of grains was found with them even up to 924
hours of incubation.
Effects of the protein inhibitors on DNA syn
thesis were also studied by examining the per
centage of labeled cells in the above experiments.
The results
areshown inChart7.
The percentage of labeled cells after treatment
with puromycin (50 @ug/ml)increased by about 18
per cent during the first 4 hours and then rapidly
dropped to 77 per cent at 924hours of incubation.
Cliloramphenicol
(92 mg/mi) also had a similar
4
Ui
2
4
6
8 20
HOURS OF INCUBATION
4
CHART 7.—Frequency
z
of labeled
hamster
22
24
cells after treat
D
ment with various protein inhibitors for different lengths of
U)
time
z
and
then
incubated
in medium
containing
thymidine-H3
4
(1 j@c/ml—3O
minutes). Counted about 200 labeled cells for
CD
each time interval of each compound.
5 grains were counted.
Cells having
more than
effect, except that no initial rise in the percentage
3
6
9
12
15
18
HOURS OF INCUBATION
CHART 6.—Shows
incorporation
21
of thymidine-H3
24
in ham
ster cells after treatment with the protein inhibitors for various
lengths of time. Average of 80 cells for each time interval.
ly detectable.
The amino acid analog p-fluoro
phenylalanine
(Chart 6) also produced a reduction
in the incorporation of thymidine-H3. The inhibi
tion began from the very onset of the treatment
with this analog, and, after an incubation of 924
hours, p-fluorophenylalanine
(1 mg/mi) reduced
the DNA synthesis by about 80 per cent.
No inhibition in the incorporation
of thymi
dine-H3 was noticed until 18 hours of incubation
in tryptophan
analogs (Chart 6). After 18 hours
for hamster cells an abrupt change in the rate of
of
labeled
cells
was
found
in
this
case.
No
sig
nificant decrease was observed with 5-methyl
tryptophan
(1 mg/mi) up to 18 hours, after which
a slight decrease was observed. There was a
gradual drop from the beginning of the treatment
in the percentage of labeled cells treated with
p-fluorophenylalanine.
Isomers of $-methyl tryp
tophan seemed to be slightly more effective than
5-methyl tryptophan. In this case, after 6 hours of
treatment, a slow decrease was observed. Between
6 and
18 hours
of incubation,
@9-methyl tryptophan
B appeared to have more effect, but after 18 hours
the A isomer was more effective than the B.
Studies on the rate of incorporation
of thymi
dine-H3, after treatment with the various protein
inhibitors, have also been made on HeLa cells
(Chart8).The results
werelargely
inagreement
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1963 American Association for Cancer Research.
Cancer Research
11492
with the observations
(50
@@g/mi)and
on hamster
cells. Puromycin
cbloramphenicol
effective inhibitors
(92 mg/mi)
were
of both protein and DNA syn
85
Effect of puromycin and 5-methyl tryptophan on
75
70
65
60
55
50
@:45
z
@
1963
thesis in HeLa cells. The analog p-fluorophenylal
anine (1 mg/mi) had some effect, whereas 5methyl tryptophan
(1 mg/mi) had no effect on
DNA synthesis until 9292
hours of incubation. After
9292
hours the effect was marked.
80
@
Vol. 923, September
40
@
I
2
3
4
5
6
7
8
HOURS OF INCUBATION
CHART 8.—HeLa
cells showing
H1 after the treatment
9
incorporation
of protein inhibitors
0
II
12
of thymidine
for various lengths
of time. Average of SOcells having more than 5 grains. Puro
mycin, 100 pg/ml;
chloramphemcol,
2 mg/mI;
hi@tone synthesis.—Since the histones in mam
malian cells do not contain tryptophan
(9), and
since the tryptophan
analogs will inhibit the syn
thesis of tryptophan-containing
proteins, it was
thought desirable to study the effects of 5-methyl
tryptophan
on histone synthesis.
Rasch and Woodard
(30) showed that his
tones can be extracted by 1/100 N HC1 at 925°
C.
for 4 hours, after removal of nucleic acids in
methanol-fixed
tissues. De (10) has modified this
method by using methanol
freeze-substitution,
which provides better preservation and extraction
of histones.
A few experiments were conducted to study the
effects of 5-methyl tryptophan
and puromycin on
histone synthesis in hamster cells. A group of cul
tures were incubated in the medium containing
5-methyl tryptophan
(1 mg/mi) or puromycin (50
hg/mi) for 192,18, and 924hours. At the end of each
time they were incubated in a conditioned medium
containing lysine-H3 (10 Mc/ml) for
hour and
fixed as described by De (10) with methanol
freeze-substitution.
Controls were similarly pre
pared, except that they were not treated with the
inhibitors. All the cultures were then hydrolyzed
p-fluorophenyl
with
5 per cent
920 minutes
alanine, 1 mg/mi; 5-methyl tryptophan, 1 mg/ml.
TABLE
trichloroacetic
to remove
nucleic
acid at 900 C. for
acids.
The
prepara
1
EFFECT OF 5-METHYLTRYPTOPHAN(1 MG/MI) ANDPUROMYCIN(50 pG/ML@
ON HISTONE
SYNTHESIS
IN HAMSTER
tryptophan
± S. E.Puromycin
Hours of
E.12A
incubation5-Mthyl
B
13.6±1.2
CELLS
±S. E.Control
7.2±0.8
±S.
17.1±1.8
4.5±2.618AHistone18.3±1.4
2.4±1.321.6±1.19
4.7±1.89.6±1.1
B
10.1±0.9
4.3±0.6
15.8±1.2
4.1±1.924AHistone13.9±1.2
1.5±1.019.9±1.5
3.8±1.55.8±0.8
3.6±0.5
B
Histone8.5±0.8
15.3±1.1
5.8±0.4
3.2±0.3
2.7±0.9
0.4±0.5819.6±1.3 4.3±1.7
Mean grains='average
of 50 cells of each group. Lysine-H' (10 pc/mI, SO minutes).
Grains represent nuclear incorporation. Exposure to the film, 10 days.
A=treated
with 5 per cent trichioroacetic acid at 9O@C. for 20 minutes.
B=treated
with 5 per cent trichioroacetic acid at 900 C. for 20 minutes and 1/100
N HC1 for4 hoursat 25°C.
Histoneindicated
by difference
between A and B.
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1963 American Association for Cancer Research.
SHAH—DNA
@
Syntheth
in Cultured
tions were then rinsed with 70 per cent alcohol and
later with water. The cover glasses with the at
tached cells were now transferred to small tubes
which contained 1/100 N HC1. They were marked
previously by cutting one corner and placed in
such a manner that only the lower half was dipped
in the acid. They were left there for 4 hours at
9250
C.
Later
they
were
rinsed
in
water,
incorporation
of lysine-H3
dehy
Puromycin
inhibited
synthesis
in the histone
histone
synthesis
remained
essentially
unaffected
by
3).
No
significant
number
of grains
was
observed
over the nucleolus after 1 hour of incubation in the
drug. The cytoplasmic grains were not recorded in
these experiments,
since cytidine-H3 at concen
mar
kedly; after 18 hours of incubation there was a!
most complete cessation of the histone synthesis.
Effect of actinomycin D on the synthesis of DNA
and RNA.—Actinomycin D was dissolved in
@
1143
actinomycin D at the concentration
used here.
In the experiments in which cells were treated
with cytidine-H3 after 1, 92,and 4 hours of incuba
tion with actinomycin D (5 X l08 M) some reduc
tion in the grains per cell was observed. Although
both chromatin and nucleolar RNA were affected,
there was a greater effect on nucleolar RNA (Table
fraction was low but significant;
92. There was no detectable effect of 5-methyl
tryptophan on the synthesis of histone, since even
after 18 hours or 924hours the rate of incorporation
of lysine-H3 into histone remained unaffected;
3.
CelL!@
Later they were dehydrated, dried, and autoradio
grams were prepared. The film was exposed for 92
weeks.
Table 92shows the results from cells incubated
in actinomycin D for 1, 92,and 4 hours and thymi
dine-H3 for
hour. There was no significant de
crease in grains per cell in the cultures treated with
the drug. Thus, for 4 hours the rate of DNA
drated in alcohol, dried, and autoradiograms
were
prepared. The results of these experiments
are
shown in Table 1. The data show that:
1. The
Mammalian
TABLE
water (l0@ M with pH adjusted to 7) and then
added to the medium to give a final concentration
of 5 X 108 M. The HeLa cells were incubated in a
medium containing actinomycin D for 1, 92,and 4
hours and then transferred
to a conditioned
medium containing thymidine-H3
(0.5 @@c/ml)
or
cytidine-H3 (1 j@c/mi) for hour. They were then
fixed in ethanol :acetic acid (3 : 1) for 1 hour. The
cover glasses with cells attached,
which were
treated with cytidine-H3, were subjected to hy
drolysis in 1 N HC1 at 60°C. for 7 minutes to re
move RNA on the lower half of the cover glass.
Cells on the other half retained the labeled RNA.
2
EFFECTS OF AcTIN0MYCIN
INCORPORATION
D (5X108
M) ON
OF THYMIDINE-H3
IN HELA CELLS
0.5 @sc/mlfor SO minutes; number of grains per
unit
area
(20 sq. p) is an average
of
50 cells having more than 5 grains.
Hours of
incubationControl
S.D.1
±
±
S.D.Experimental
55.46±4.1
2
29.21±3.5
33.16±4.3
434.11±5.6
38.23±5.651.45±3.2
TABLE S
EFFECT
OF ACTIN0MYcIN
D (5X108
M) ON INCORPORATION
OF CYTIDINE-H3
(A) Total grains of cytidine-H3 (1 pc/mI for SO minutes);
(B) Grains after hydrolysis in 1 N HC1 for 7 minutes at 60°C.; figures represent
each time interval.
grains before
RNANucleolus
extractionMean
IN HELA
CELtS
mean from about
grains after
RNA extractionLabel
50 cells at
in
HoussABA—BMean
±
2
4
±
S. D.Chromatin
D.1
±
S. D.Nucleolus
±
S. D.Chromatin
±
S. D.Nucleolus
±
S. D.Chromatin
S.
Control
Experimental
1.15±0.15 16.06±2.8
0.90±0.17
5.84±1.0
0.25±0.22
10.22±2.9
Control
4.78±0.6
22.56±5.6
Experimental
0.95±0.4
16.29±2.7
0.83±0.15
0.80±0.15
8.25±1.6
5.16±1.4
3.95±0.25
0.13±0.19
14.31±5.2
11.13±5.0
Control
4.36±0.7
22.53±3.1
0.72±0.11
7.08±1.6
3.64±0.22
15.45±4.5
Experimental4.83±0.6
0.82±0.0918.91±3.2
16.68±2.50.89±0.12
0.78±0.096.52±1.2
11.27±2.5
5.41±1.153.94±0.22
0.04±0.01212.59±4.4
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1963 American Association for Cancer Research.
Cancer Research
1144
@
tration 1 @zc/mlfor hour did not show much in
corporation into the cytoplasm.
Vol.923,
September 1963
and 48 per cent with RNase, compared with the
controls incubated under similar conditions but
without the enzymes in the medium. Later there
was a gradual drop in the percentage of cells able
to incorporate thymidine-H3 except those growing
in DNase I. Between the period of 192hours and
Effects of nucleases on DNA synthesis.—The ef
fects of the nucleases (DNase I, DNase II, and
RNase) on the incorporation
of thymidine-H3
were studied with HeLa cells. The cultures were
incubated in Puck's medium containing one of
these enzymes at a concentration
of 1 mg/mi.
After various intervals the enzymes were removed
792 hours
a reduction
of about
924 per
cent
was
ob
served with DNase II, whereas within the same
period a reduction of 60 per cent was found with
RNase. During this period no significant varia
by rinsing
thecultures
inTrisbuffer,
and a pre
(1)
-J
-j
U
U
0
U
-J
U.
0
U
CD
z
U
U
U
aI
CHART 9.—Frequency
2 3 4
of thymidine-H@-labeled
5 6
7 8 9 10 II
HOURS OF INCUBATION
HeLa
200 labeled cells having more than 5 grains were counted
cells after
incubation
in enzymes
for each time interval
TABLE
for various
lengths
of time.
About
of each enzyme.
4
EFFECT OF NUcLEASES ON INCORPORATION OF THYMIDINE-H'
IN HELA CELLS
Grains per unit area (20 sq. ii); average of SO cells having more than five grains.
@
.H0@@@.5?f
incubationDNase
D.0
II
I
±
D.DNase
±
±
S. D.RNase
S. 1).Control
±
@.
35.57±3.4
S
55.08±5.2
41.26±4.9
58.45±5.6
56.52±5.8
6
37.85±5.5
39.12±5.8
34.48±3.6
41.18±5.6
12
53.51±5.4
52.27±5.3
58.95±4.7
57.02±5.2
24
4836.88±4.559.65±4.857.62±3.952.88±3.138.76±4.734.16±4.132.98±4.440.23±5.9
@
conditioned medium containing thymidine-H3
(1
ac/mi) was added for hour. They were fixed in
the Duboscq-Brassil
mixture for 92 hours, and
autoradiograms
were made as described previous
ly. The exposure to the film was for 10 days. After
the film was processed, the cells were stained with
azure B bromide at pH 4.
The initial effect of the nucleases was to induce
cells to incorporate
thymidine-H3
into nuclei
(Chart 9). By 3 hours the number of cells capable
of incorporating
thymidine increased by 17 per
cent with DNase I, 929per cent with DNase II,
tion was noticed with DNase I. The average
amount of thymidine incorporated per labeled cell
was equivalent to the controls at all times during
the 48 hours of incubation
with the enzymes
(Table 4). Similar amounts of other proteins, such
as bovine hemoglobin, ovalbumin, and horse heart
myoglobin, failed to produce any significant dif
ference in the ability of cells to incorporate
thymidine.
DISCUSSION
It is evident from the above studies that all
the protein inhibitors which were tried on hamster
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1963 American Association for Cancer Research.
SHAH—DNA Synthesis in Cultured Mammalian
and HeLa cells, and which inhibit protein syn
thesis, also affect to various degrees the synthesis
of DNA. The inhibitors differ widely in their mode
of action—for example, puromycin (37) and chlor
amphenicol
(925) act to block the formation of
polypeptides at ribosomes; p-fluorophenylalanine
CelLi
1145
One might suspect that the inhibition
synthesis
would
exert
a control
of protein
through
the
ab
sence of some enzymes necessary in the following
three steps of DNA synthesis : (a) the regulation of
the supply of precursors, (b) polymerization
of the
nucleoside triphosphates,
or (c) the activation of
(92)isincorporated
to formfalseproteins,
whereasthe DNA template.
the methyl tryptophan
analogs (33) prevent the
Kornberg and his associates (18) found that,
incorporation
of tryptophan
into proteins by immediately after an infection of E. coli with T2
competition with it for enzyme activation. Thus
phage, three new enzymes are formed and can be
it is interesting that, though the inhibitors differ detected within 4 minutes after infection. They
in their
actions
and operate
at different
levels
of
the protein synthetic pathway,
they all affect
DNA synthesis. These observations suggest that
it is a polypeptide
or a finished protein rather
than individual amino acids which is necessary for
the DNA synthesis in this system. If it were
otherwise the antibiotics,
puromycin and chior
amphenicol, which are known to prevent polypep
tide formation, would not be effective. The in
volvement of amino acids as shown for E. coli by
Kellenberger et al. (16) is not, of course, ruled out.
The inhibition of protein synthesis reveals its
effects on DNA synthesis in two ways in auto
radiographs of hamster cells. There is a progres
sive reduction in the number of cells which in
corporate
thymidine-H3
(Chart 7) and also a
gradual decrease in the amount of thymidine-H3
incorporated per cell per unit time (Chart 6, Fig.
1). In an asynchronously
dividing
system,
like
the
hamster cells in tissue culture, at any one time ap
proximately 48 per cent of the cells are synthesiz
ing DNA (Chart 1). The reduction in the number
of cells synthesizing DNA indicates that the pro
tein inhibition prevents cells from entering the
S-phase—i.e., prevents the initiation of DNA syn
thesis. Apparently
cells already in the S-phase
continue to incorporate
thymidine-113, and this
accounts
for the cells which continue
to synthesize
DNA. The reduction in rate of thymidine incor
poration per cell suggests that protein synthesis is
necessary for the continuation
of the duplication
of the chromosome complement even in cells which
have entered the S-phase. These experiments do
not reveal whether those cells which enter syn
thesis
before
or during
the early
period
of treat
ment continue to complete replication
of the
chromosome complement. However, if synthesis is
completed, the reduced rate of thymidine incor
poration indicates that the S-period would be ex
tended. Mueller et al. (923) have obtained data on
HeLa cells with puromycin inhibition following
synchronization
by aminopterin,
which indicates
that continued protein synthesis is necessary for
the acceleration of synthesis which occurs upon
release of the aminopterin block.
also found
an increase
of about
twe!vefo!d
in the
DNA polymerase. The nature of the bases incor
porated in phage DNA during its synthesis is
governed by a specific kinase system (34), since
each of the four nucleotides has its own kinase
which regulates the rate of production of triphos
phates for the polymerization
system (15). A con
trol mechanism might then operate by inhibiting
some of these enzymes along the path. However,
the measurements of DNA polymerase and deoxy
nucleotide kinases in bacteria have shown their
presence in adequate amounts in cells in which
protein or DNA synthesis has been blocked for
considerable length of time (4). Powell (928) found
in mouse L-fibroblasts that, despite the marked
depression in intracellular
DNA synthesis ob
served 5—8hours after the treatment with puromy
cm, virtually no inhibition of the phosphorylation
of thymidine to thymidine triphosphate
or the
activity of DNA polymerase could be detected at
the same time. These observations seem to suggest
that the control mechanism is not likely to operate
at the first two steps—namely, the regulation of
precursors or the inhibition of the nucleotide
polymerizing enzymes. In view of these results,
Billen (4) suggested that DNA replication requires
protein synthesis to make primer DNA available
to the Kornberg enzyme system.
Because the histones in higher forms are closely
conjugated with DNA and are synthesized simul
taneously with it during the interphase,
many
have speculated that the histones act as gene
modifiers
or as inhibitors
of gene functions.
Since
there exists some genetic control of the sequence of
the replication
of chromosomes
in mammalian
cells (36), one might suspect that at least in higher
forms the histones may play an important role in
the control of DNA synthesis in these cells. The
cytochemical studies (Table 1) suggest that there
is no detectable
effect
of 5-methyl
tryptophan
on
histone synthesis. The observations on DNA syn
thesis after the treatment
with 5-methyl trypto
phan show that the DNA synthesis is affected
after 18 hours (1@ generation cycle) in hamster
(Chart 6) and 9292hours (about one generation
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1963 American Association for Cancer Research.
Cancer Research
1146
cycle) in HeLa cells. These results suggest that the
histones are not the proteins necessary for the
synthesis of DNA, since there is considerable
histone synthesis when a marked reduction (60—70
per cent) in DNA synthesis occurs.
The experiments with actinomycin D on HeLa
cells (Tables 92,3) show that, although there is a
progressive decrease in the rate of RNA synthesis,
DNA synthesis remains essentially unaffected for
4 hours. Reich et al. (31) have also reported in
L-9929mouse fibroblasts that the actinomycin D
which selectively inhibits the synthesis of cellular
RNA has no effect on DNA synthesis. These re
sults show that there is no necessary coupling of
RNA and DNA synthesis. However, a possible
role of RNA as inhibitor or modulator
of the
DNA synthetic process is not excluded.
The initial increase and subsequent decrease in
the number of cells able to incorporate thymidine
with nucleases, particularly RNase (since DNase I
has little effect and DNase II is known to contain
some RNase), can be explained in at least two
ways. The first possibility is that the RNase may
reduce the rate of DNA replication without pre
venting cells from entering
the S-phase and
thereby result in the accumulation
of thymidine
H3—incorporating cells. However, this seems un
likely, since the rate of increase in number of cells
in synthesis during the first 3 hours exceeds the
rate at which cells normally enter the S-phase. A
second possibility is a modification
of the hy
pothesis of Kurland and Maa1Øe (19), based on
regulatory mechanisms proposed by Monod and
Jacob (9292)(see also [16]), in which they proposed
that transfer RNA might act as a repressor only
when it is not coupled with activated amino acid.
The amino acids thus act as inducers which corn
bine with RNA to inactivate its repressor func
tion. Alfert et at. (1) have shown that, in the living
onion root tips exposed to RNase, the enzyme
forms
a complex
with
RNA
but
does not remove
it and that this complex formation
results in
masking of RNA-phosphate
groups and suppres
sion of basophilia.
It could be assumed that
RNase in this system could also combine with
RNA and inactivate its repressor function. The
later depression in the number of cells incorporat
FIG. 1.—Effect of puromycin on incorporation of lysine -H'
(25 pc/mI) and thymidine-H3
(1 pc/mi) in HeLa cells treated
forlhour.ExposuretothefilmforS weeks.Preparedwithhigh
grain density
for photographic
purposes.
Approximately
2,SOOX.
Vol.923,
September 1963
ing thymidine-H'
is probably due to its subse
quent effect on protein synthesis (6).
In conclusion it can be said that the observa
tions in these studies are consistent with the fol
lowing hypothesis for the control mechanism of the
DNA synthesis in the intact cell: RNA (type un
specified) is involved in the control mechanism of
DNA synthesis in the form of repressor at the
template level, and the repressor activity is regu
lated by amino acids (E. coli) or perhaps by some
proteins by combining with the RNA to inactivate
its repressor function.
ACKNOWLEDGMENTS
The author is deeply grateful to Professor J. H. Taylor for
his advice and guidance throughout the course of this investi
gation. My sincere appreciation
to Professor A. W. Pollister
for his valuable
suggestions
and encouragement.
To Dr. W. L.
Hughes,
Brookhaven
National
Laboratory,
I am thankful
for
help in the preparation of the manuscript.
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Autoradiographic Studies of the Effects of Antibiotics, Amino
Acid Analogs, and Nucleases on the Synthesis of DNA in
Cultured Mammalian Cells
Vinod C. Shah
Cancer Res 1963;23:1137-1147.
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