Studies of DNA,
RNA,
Human
MYRON
KARON,
and Protein Synthesis in Cultured
Cells Exposed
SHERMAN
WEISSMAN,
to 8-Azaguanine
CAROL
(Medicine Branch and Metabolism Service, Department
MEYER,
AND
PATRICK
HENRY
of Health, EducaLion, and Welfare, U.S.P.H.S.
Na@ionai Cancer Institute, National Institutes of Health, Bethesda, Maryland)
SUMMARY
Studies of the kinetics of DNA, RNA, and protein synthesis in KB cell spinner
cultures exposed to 8-azaguanine were performed at doses of the analog which pro
duced little or no inhibition of cell multiplication.
The results indicated that, of
the three factors studied, protein synthesis is most sensitive to inhibition by aza
guanine.
As in the case of Bacillus
cerens, the analog was preferentially
incorpo
rated into soluble RNA (sRNA).
8-Azaguanine inhibits growth in a variety of living
systems. The analog is incorporated into bacterial, viral,
and mammalian RNA, replacing up to 5 % of the guanine
residues (9). In B. cereus Smith and Matthews (21)
found that
40 % of total RNA guanine
can be replaced.
Using the same organism, Mandel and Markham (14)
showed that the analog appeared primarily in short-chain
RNA, and, more recently, Levin (10) demonstrated that
most of this 8-azaguanine is incorporated into sRNA.
This paper reports studies of some of the molecular
events preceding azaguanine-induced
growth inhibition
washed 3 times with ice-cold normal saline to remove
adherent
serum,
and frozen
until
time
for analysis.
The
DNA, RNA, and protein were separated by a modifica
tion of the Schmidt-Thannhauser procedure (20). The
cell pellet was resuspended in 0.5 M perchloric acid (PCA)
at 4°C.for 15—20mm. and then centrifuged.
The result
ing precipitate was washed twice with 0.5 M PCA; once
with 95 % ethanol:water, 4 : 1; and then digested for 18
hr. with 0.5 M KOH at 37°C. The digest was carefully
neutralized at 4°C.to pH 7—7.5and the KC1O4 removed
by centrifugation. An equal volume of cold 1 M PCA
in a human cell line maintained in suspension culture.
Such a system is ideal for the evaluation of cellular DNA,
RNA, and protein synthesis, as well as for the study of
was added to precipitate the DNA and protein and the
supernatant
was removed, neutralized,
and assayed for
azaguanine
counter
distribution
in the various
species of RNA.
The concentration of inhibitor can easily be adjusted to
produce any desired growth effect.
MATERIAlS
AND
Bethesda,
Md.
They
were
in a Packard-Tri-Carb
that had an absolute
liquid scintillation
efficiency of 7 % for tritium
when a dioxane-containing phosphor was used (6). ISV
absorption was determined at 260 m@on a Beckman DU
spectrophotometer.
METHODS
The
precipitate
was washed
twice
in cold 0.5 M PCA and the DNA hydrolyzed by treatment
with 0.5 M PCA at 90°C. for 30 min.
KB cells growing in suspension culture with a genera
tion time of 30 hr. were obtained from Microbiological
Associates,
radioactivity
maintained
The hydrolysate
was assayed for radioactivity and optical density as
above. The protein precipitate was redissolved in 0.5
at
cell counts of 80,000-400,000/mi
in spinner flasks with
N NH4OH
Eagle's medium
with 5 % horse serum,
less steel planchets in a gas flow low-background
(2 c.p.m.)
j3-counter that had an absolute efficiency of 20—27%, de
(4), supplemented
glutamine 0.3 gm/100 ml, and penicillin and streptomy
clii. All experiments were performed during log phase
growth. Cell counts were made on a hemacytometer.
Kinetic 8tudles.—Kmetic studies of protein, RNA, and
DNA synthesis were performed using replicate 300 ml
cultures in 500 ml spinner flasks containing
three different
doses of 8-azaguanine-C'@. Thirty milliliter aliquots were
removed at various intervals following the addition of
10 @cof uridine-H5 (specific activity 1.3 c/mmoles) and
1 Mcof frleucine-C'4 (200 @zc/pmo1e)
and immediately cooled
to 4°C.by being poured over crushed ice made from 0.85%
NaC1. The cells were centrifuged at 4°C.for 10 mm.,
Received
for publication
August
19, 1964.
and
assayed
for
radioactivity
on
2.5
cm
stain
pending on the thickness of the window used, and for
protein content by the Lowry method (12). The ratio
of H5/C14 in the incubation media was such that the con
tamination of tritium with C'4 was less than 1 % of the
total counts.
The gas flow $-counter used in these ex
periments does not detect tritium.
Similar short-term
kinetic experiments of RNA, DNA, and protein synthesis
employing a single isotope were performed in separate
replicate cultures using uridine-H,
thymidine-H3,
and
leucine-C'4 for periods of 6—Shr.
Long-term kinetic experiments were performed in
replicate 3-liter cultures maintained in 4-liter spinner
185
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1965 American Association for Cancer Research.
186
Vol. 25, February
Cancer Research
1965
re-extracted with hot phenol. The solution containing
the RNA was then adjusted to 2 % in KAc and 0.001 M
NaEDTA, and the RNA precipitated by the addition of
2 volumes of 95 % ethanol. After it had stood at —30°C.
for at least 1 hr., the precipitate was collected and washed
once with 70 % ethanol-2 % KAc at 4°C.and redissolved
in a pH 6.7 buffer:
M MgCl2,
and
0.01 M NaAc,
polyvinal
sulfate
0.05 M NaCl, 0.0001
(PVS)
2 pg/mi
(1).
This
RNA was stable for at least 4 weeks when stored at —30°C.
A concentration
of 3 X 108 cells yielded from 140 to 160
optical density units at 260 rn@; less than 5 % was DNA.
Macromolecular RNA separations.—RNA
separations
on sucrose gradients were performed by carefully layering
0.5-1.5 mg of the sample in a volume of 0.6 ml on top of a
5—20
% linear gradient and centrifuging at 22,500 r.p.m.
in the SW 25.1 swinging bucket rotor of a Spinco model L
0
‘C
ultracentrifuge
E
for 13—17hr.
Fractions
of 0.5 ml were
removed a drop at a time, after the bottom of the tube
..-%
U)
-J
was punctured.
Separations
.J
UI
0
on
methylated
bovine
(MAK
column)
albumin were performed by a modification of the method
of Mandel and Hershey (15). A 3-layered, 39.1 X 0.9
cm methylated albumin column was prepared as follows:
the first 23.5 cm contained 23 mg of methylated albumin
with 6 gm of Celite (Johns-Manvffle); the next 13 cm
contained 2 mg methylated albumin and 3 gm Celite;
the last 2.6 cm contained Celite alone. The column was
packed under a pressure of 3 lb/sq in. The RNA was
eluted by a linear gradient of 0.3—1.4M NaCl in 0.05 M
phosphate
buffer at pH 6.8; and 3 ml fractions
for analysis.
Assay of &ucrose gradients and MAK
RNA
0
12
24
48
36
CHART 1.—Growth
of KB
cells
in spinner
represent
± 2 standard
bottles.
Aliquots
culture
with
and
with
The limits for each point
deviations.
of 80 ml were removed
8—12hr. over a 65-hr. period.
at intervals
of
Chemical measurements
of RNA were made by the orcinol procedure
(2) and of
DNA by the diphenylamine reaction (3).
RNA extraction.—RNAwas extracted by a modifica
tion of the method
of Scherrer
and Darnell
(19).
Cells
were collected by centrifugation at 4°C.at 1500 r.p.m.
for 10 mlxi. and washed once with 200 volumes of cold
0.85 % NaCl.
The resulting
cell pellet was thoroughly
resuspended in 10-20 volumes of a pH 5.2 buffer: 0.01
M NaAc,
and 0.01 M NaEDTA
containing
of sucrose
gradient
columns.—The
and chromatographic
fractions was assayed in alternate tubes by measurement
of ISV absorption at 260 m@after dilution to 1.5 ml with
water. For assay of radioactivity, 2 mg of yeast RNA
60
HOURS
out the addition of 8-azaguanine.
content
were taken
0.05 % bentonite
(17). Ten per cent sodium dodecyl sulfate (SDS) was
added to a final concentration of 0.5 % and the cells ad
lowed to lyse for approximately 30 sec. at 4°C. An equal
volume of redistilled 90 % phenol, containing 0.1 % 8hydroxyquinoline (7), was added to the cell suspension
and the material shaken vigorously in a water bath at
60°C. for 3 mm. The resulting suspension was then
cooled rapidly to 4°C. in a dry ice-alcohol mixture and
centrifuged for 5 mm. at 20,000 g. An additional 0.5 %
SDS by volume was added to the aqueous phase and
(Nutritional
Biochemical Corp.) were added to the re
maining alternate fractions and the material precipitated
with an equal volume of cold 10 % trichioroacetic
acid
(TCA). The precipitate was washed once with cold 5 %
TCA and then extracted for 30 min. at 90°C.in 3 ml 5 %
TCA. This technic removed 99 % of the acid soluble
counts. Thrice-ether-extracted 1 ml aliquots were assayed
for radioactivity on planchets for C14and 0.5 ml aliquots
were assayed in the liquid scintillation counter for H3.
Correction was made for the C'4 appearing in the tritium
samples by use of a C'4 standard
counted
ments.
Methods based on the acid precipitabiity
on both instru
of RNA were
used throughout these studies only after preliminary ex
periments had indicated that neither guanine-C14 nor
azaguanine-C'4 was released from RNA by 5 % TCA treat
ment for periods as long as 3 hr. The later precaution
was particularly pertinent because of the reported acid
lability
of azaguanine
containing
RNA
(10).
From
45
to 55 % of the azaguanine counts found in the sRNA
region of the sucrose gradient, as compared with less than
5 % found in the ribosomal fraction, were acid soluble.
The acid solubiity of uridine H3 and guanine C'4 was
always less than 5 % in both fractions.
Stability
studies with actinomycin
D.—Uridine-H3, 3.5
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KARON
200
et al.—DNA,
RNA,
and
DNA
Protein
1.5 -
187
Synthesis
DNA
‘.@@70.5-RNA
0
@0
e%J
0
0
RNA
aC-,
1.5 —
1.0
0 None
. /xIO7M
0
A 3x1O7M
A
0.5 —
H
. Ix/ci'M
A 3x/0'M
/xIO@6M
U)
0.
C,)
1000
Pro/em
A /x1@T6M
1.5 -
I.00.5-I
50:@
t
@
I
2
a
b
HOURS
CHART
2.—The
kinetics
of DNA,
RNA,
and
protein
synthesis
following
I
4
3
HOURS
the
addition
5
of azaguanine
to three of four replicate cultures containing uridine-H' and leucine-C―. (a) Specific activity V8.time;
(b) ratio of specific activity with azaguanine, to that without azaguanine vs. time.
@c,
was added to four replicate 300-nil spinner cultures at
zero time. In addition, 0.1 @c(3 X 10@ M) azaguanine
C'@ was added to two of the four spinners. Aliquots
of 30 ml were removed at various intervals before and
after the addition
cultures,
of actinomycin
one containing
D 0.1
@gJmito two of the
both uridine-H3
and azaguanine
C―,and the other containing uridine alone. The dose of
actinomycin D selected had been shown in preliminary
experiments
to inhibit
20-30 mm.
The determination of RNA-speciflc activity
was made on alkaline
scribed above.
RNA synthesis
hydrolysates
completely
within
by the methods
de
presence of carrier albumin, and the amount of radio
activity solubiized measured.
Total precipitable radio
activity was determined by hot acid hydrolysis of a thrice
0.5 M PCA-washed precipitate.
sis of this sucrose gradient
RNA digestion experiment.s.—Separate
1-liter cultures
to a 2 % concentration
Paper chromatographyof an alkaline digest.—A1,750-
of KAc,
M azaguanine-C'4,
and
the
entire
Following
Aliquots
of PCA-precipitable,
and RNase-releasable
radioactivity.
@jk@linedigestion was performed in 0.3 M KOH for 18
hr. at 37°C.,and the amount of radioactivity made acid
after
precipitation
in the presence of carrier albumin.
was carried
out with 10 @igRNase
with 0.5 M PCA
RNaee digestion
(Worthington)
in a
cell
pellet
the removal
of KC1O4 by precipitation
in the
cold, three separate 0.5 ml aliquots were incubated for 4
hr. at 37°C.after the following additions: (a) 5 mg dried
(c) adjustment to pH 7.0 only.
in 0.1 M Tris buffer pH 8.1.
saline-washed
processed by the modified Schmidt-Thannhauser
pro
cedure to separate the alkaline hydrolysable material.
KAc and redissolved
were taken for the determination
determined
fraction
manner.
whole-snake
soluble
the sRNA
of the azaguanine labeled RNA, separated by chromatog
raphy on methylated albumin, was studied in a similar
the RNA was precipitated with 2 volumes of 95 % ethanol
at —30°C.
in the presence of 2 mg of yeast RNA. The
precipitates were washed once with 70 % ethanol-2 %
KOH-hydrolysable,
In addition to the analy
material,
ml spinner culture was incubated for 10 hr. with 3 X 10@
were exposed to 3 @icof azaguanine-C'4 (1.5 X 106 M)
and to 2 @cguanine-C14. The RNA was isolated by the
hot phenol method. The ribosomal RNA (rRNA) was
separated from sRNA on a sucrose gradient. After each
fraction had been adjusted
volume of 1 ml for 30 mm. at 37°C. The reaction was
stopped by the addition of 1 ml of cold 1 M PCA in the
venom after adjustment
to pH 8.8 with con
centrated NILOH; (b) 50 mg of E8cherit@hiacoli alkaline
phosphatase (Worthington) after adjustment to pH 7.0;
Aliquots of 10 ml from each of the three different frac
tions were then spotted over both guanosine and 2' , 3'guanylic acid standards and chromatographed in a de
scending fashion in isobutyric acid :water: conc. NHIOH:
0.1 M EDTA at 100:55.8:4.2: 1.6, on Whatman no. 1
paper
(22).
The IN
absorbing
spots were cut out and
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1965 American Association for Cancer Research.
Cancer Research
188
@
Pro/em
Vol. 25, February
by the Cancer Chemotherapy
Bethesda,
Md. Uridine-H8,
None
. IX/07M
a 3X107M
1965
National Service Center,
1.3 c/mmole;
leucine-C'4,
200 @ic/j@mole;
guanine-C'4, 5 @ic/Mmole;and thymidine
H3, 360 mc/mmole were products of New England Nu
clear Corp.
RESULTS
a.
The effect on cell growth of different concentrations
of
azaguanine over a 60-hr. period is shown in Chart 1. A
E
concentration of 1 X 10@ M azaguanine did not inhibit
growth,
whereas
1 X 10_6 M was not only inhibitory
but
proved progressively lethal. By 60 hr., 3 X 10@ M
showed a 30-40 % (two separate experiments) inhibition.
This difference
DNA
in growth
rate first became
manifest
at
16-20 hr.
The influence of these same concentrations of analog
on the early time-course of DNA, RNA, and protein syn
a.
thesis, following simultaneous
exposure to uridine-H3
and
leucine-C'4, is shown in Chart 2a. At 1 X 1O@M,inhibition
of protein synthesis began within 2 hr., and of DNA syn
thesis within 4 hr., while that of RNA was unaffected.
In addition (Chart 2b), there was an early increase in the
synthetic rate of all three components, except for protein
at 1 X 10-s M, as compared with the azaguanine-free
a.
culture.
Similar
employing
separately.
0
10
20
30
50
40
60
70
HOURS
CHART
3.—Chemical
measurement
of DNA,
RNA,
and
protein
results
were
obtained
in experiments
thymidine-H3, uridine-H3, and leucine-C'4,
Long-term experiments also demonstrated
that the synthesis of protein was inhibited earlier than
that of RNA even at 1 X 10@ M azaguanine—a dose which
produces no inhibition in cell multiplication
(Chart 3).
The nature of the RNA formed at various times follow
ing the addition of azaguanine to the media is shown in
Chart 4. The distribution of azaguanine-C'4 and un
eluted with 0.1 N HC1 and radioactivity determined in dine-H3 in the total cellular RNA was analyzed after
sedimentation on a sucrose gradient. The curve of
the scintillation counter.
represents the
Effect of guanine on uptake of azaguanine-C'4.—Eightoptical density measurements at 260 m@&
200-mi replicate spinner cultures were treated with 3 X separation of stable RNA into the three usual components,
10@, 3 X 10—6
and 3 X 10@ Mguanine-C'2 in the presence 28s, 18s and 4—6s. The distribution of acid-precipitable
of 0.6 @c
azaguanine-C'4 (3 x 10@ M). After 4 hr., 80-mi radioactivity represents the new RNA formed in the time
aliquots were poured immediately into saline ice and the periods indicated. At 30 mm., the unidine3-containing
RNA sedimented well in front of the 28s peak, but by
acid-precipitable,
alkaline-hydrolysable
RNA, and the
acid-precipitable, acid-hydrolysable DNA extracted and 1.5 hr. 28s, 18s, and 4—6scomponents were already present
in addition to this heavier material. This transition
counted in the Tri-Carb. These studies were undertaken
after preliminary experiments using uridine-H3 and 3 X was completed by 3 hr., at which time there was no longer
any rapidly sedimenting RNA detectable. In contrast,
10—b
M guanine-C'2 revealed no inhibition of uridine up
take by this high dose of guanine.
there was an early and rapid incorporation of azaguanine
there
Effect of azaguanine-C'2on distributionof guanine-C'4 C'4 into RNA of the 4—6sregion. Subsequently,
in RNA .—Approximately 3 @cguanine-C'4 (6 X 10@ M) was some incorporation into 28s and 18s material, but
were added to two replicate 1-liter cultures, one of which this RNA is always of much lower specific activity than
had also received, 1.5 hr. previously, a dose of 3 X 10@ that of sRNA. Chromatography on bovine methylated
M azaguanine-C'2.
After
3 hr.,
the
RNA
was
extracted
albumin of an aliquot of RNA from the 3-hr. experiment
shown in Chart 4 demonstrated close coincidence of the
with hot phenol and analyzed on a sucrose gradient.
uridine-H3 and azaguanine-C'4 in the ribosomal material,
Aliquots for radioactivity were counted directly without
prior acid precipitation.
but a more broadened distribution of uridine than aza
per 100 ml of culture fluid over 65-hr. period.
measurement
Samples for DNA
at zero time were misplaced.
Materials.—8-Azaguanine-2-C'4,
2
@&c/@mole,was ob
tained from Isotope Specialties Co., Burbank, California.
Of this material, 99 % moved as 8-azaguanme in electro
phoresis
at pH 3.4 and 7.0.
Measurements
of concen
guanine
in the sRNA
region
(Chart
5).
On the other hand, centrifugation through a sucrose
gradient for 41 hr. revealed no difference in the distribu
tion of acid-precipitable uridine-H3 and azaguanine-C14
tration were based upon a molar extinction coefficient
of 7,500 at 262 m,@in 0.1 N HC1. 8-Azaguanine-C'@,
in light-weight RNA (Chart 6). Similarly, there was no
difference in the stability of newly synthesized azagua
NSC 749, and actinomycin
nine-C'4- and uridine-H3-containing whole RNA following
D, NSC 3053, were supplied
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KARON
et al.—DNA,
RNA,
and
Protein
Synthesis
189
(—)
.
140
S 120
100
8O@
...-
30 6O@
2040
10 20
0
0
>I—
U)
@
3 hrs.
w
II
b—.Op//cal
Densily
0
.
. Azoguonme
C'4
-J
—
Urid/ne
H3
0
I—
a-
2400
0
502000
40 1600
C)
@0
30
@
e,A,)
@,v -..@
20 800
10 400
f
..S..4
@
S.
0
20
0
40
20
00
40
TUBE NUMBER
CHART
4.—Analysis
of double-labeled
RNA
on sucrose
gradients
at 0.5,
1.5,
3.0,
and
11.0
hr.
after
the addition of radioactivity. Sedimentation was at 22,500 r.p.m. in a SW 25.1 swinging bucket rotor
for 15, 15, 13, and 17hr., respectively. Added to the 3- and 11-hr. incubations were 0.18@@c
of azaguanine
C'4 (3.6 X 1O@M) and 7.5 @c
uridine-H1; 1 @ic(1 X 10@ M) azaguanine
the 0.5 and 1.5 hr. incubations.
and 20 @curidine were added for
The first two peaks represent rRNA; the third, sRNA.
Radioactivity
is expressed per milliliter of hot acid hydrolysate.
.—.
-----.Azoguonine C/I
°—°U,idine/13
C-)
@0
S
‘Ii
0.050
0
CHART 5.—Separation
20
TUBE NUMBER
10
of RNA
firstpeak representsmaterial
and the third, rRNA.
on a column
0
30
of methylated
bovine
albumin.
The
which did not adhere to the column; the second, sRNA:
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1965 American Association for Cancer Research.
Cancer Research
190
Vol. 25, February 1965
(0)
160
0@
0
120
C)
-V
8O@
—4
I
rn
aCO
C@J
I-
40
U)
c@j
0
‘C
-J
a-
0
0
I—
0
30
TUBE NUMBER
Cii4.RT6.—A41-hr. sucrose gradient, analyzed like those in
Chart 4.
0
the inhibition of RNA synthesis by actinomycin D (Chart
7). In addition,
the percentages
of acid-precipitable
CHART
I
2
7.—Stability
3
of
4
iOURS
5
azaguanine-C14
6
and
ministered
inhibited
minimal inhibition of cell multiplication.
thesis was the most sensitive to inhibition
16-20 % by the addition
of guanine
3.75 hr. after
at concentrations
corporation
long-term cultures.
the amount incorporated into RNA, making an evalua
tion of the effect of exogenous
guanine
technically
im
possible. Azaguanine-C'2 at 3 X 10@ M did not change
the distribution of guanine-C'4 (1 X 10@ M) in the RNA
synthesized after 3 hr., when compared with a replicate
culture not exposed to azaguanine (Chart 8).
Following treatment of an aliquot of an alkaline digest
of azaguanine labeled RNA with whole snake venom,
99 % of the recovered counts moved on paper chromatog
raphy with 2' , 3'-guanylic acid.
associated with guanosine.
After
phosphatase, however, no activity
GMP region and 100 % of the
No counts were found
treatment with E. coil
was found in the 2' , 3'recovered counts were
eluted with the guanosine standard.
DISCUSSION
These studies were undertaken to investigate the rela
tionship between DNA, RNA, and protein synthesis, and
to obtain information on the nature of the RNA formed
the
start
of the
experiment.
of azaguanine
after
into DNA was only 2 % of
RNA
The
legend
refers to the concentration of azaguanine-C'4 in the cultures.
C'4 to the media. This inhibition, however, occurred
only at a concentration of 3 X 10@M,which was 100 times
greater than that of the azaguanine (Table 2). The in
of azaguanine-C'4
uridine-H3
8
following inhibition of RNA synthesis with actinomycin D ad
guanine-C'4 and azaguanine-C'4 rendered acid-soluble
by KOH or RNase treatment, from both the ribosomal
and RNA fractions, were quite similar (Table 1.)
The uptake of azaguanine-C'4 into total RNA was
by about
7
analog.
This was demonstrated
short
intervals
and
by
which produce only
Protein syn
by this punine
both by isotope studies
chemical
measurements
in
Inhibition of protein synthesis has
been observed by others (13) in a variety of animal and
viral systems, as well as more recently in HeLa cells (8).
In addition to inhibiting protein synthesis, azaguanine
is incorporated
into RNA.
(10), this incorporation
This phenomenon
As in the case of bacteria
occurs primarily
occurs rapidly
into sRNA.
and at concentrations
of analog which produce no inhibition in either DNA,
RNA, or protein synthesis for as long as 3 hr. The de
crease
in azaguanine-specific
activity
after
11 hr.,
how
ever, does reflect the fact that 3 X 10@ M is mhibitory by
that time. Azaguanine does not interfere with the orderly
progression of uridine-H3 from its early association with
rapidly sedimenting RNA (greater than 28s) into ribo
somal and sRNA as the incubation is continued—a proc
ess which is still imperfectly understood
(5); nor does
azaguanine
alter
the
distribution
of newly
incorporated
guanine-C'4 in RNA.
Several lines of indirect evidence indicate that the
radioactivity
sedimenting
with RNA, on the sucrose
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1965 American Association for Cancer Research.
KARON et al.—DNA,
RNA,
and Protein
TABLE
PER CENT OF RNA
Synthesis
191
1
RENDERED ACID-SOLUBLE BY KOH
AND RNase
TREATMENT
P1ACTIONSolubleUntreatedKOHRNaseUntreatedLOBRNaseAzaguanine
or SEPARATIONRNA
C'4-isoropzMETHOD
110
Gradient
,760
(66%)
(9(J%)b
MAK Column
—
—
Gradient5,680
(87%)
313
(97%)
321
—
15,8005, 12,600
(80%)3
GuanineSucrose Sucrose
044
9,330
(59%)1
cent
of
untreated
(untreated
=
total
acid
precipitable)
266
(83%)
2,800910 2,420
(86%)900
a The period of radioactive assay was such that ±2standard deviations was
countrate.
b Per
(86%)
counts.
2,180
(78%)
3% of the indicated
•
TABLE 2
EFFECT
OF GUANINE
ON THE UPTAXE
OF AZAGUANINE
C'@(3X 107M) INTO RNA
of azaguaninc
(cpmfE26O)None435
Concentration
of guanineUptake
53X107M383±5
5a460±
±
43X10'M460±5
450 ±
53X10'M376±4
450 ±
E
378 ±4
0
CO
C'J
a±2
Standard
deviations.
C)
>-
-V
U,
-4
UI
gradient following phenol extraction of KB cells exposed
to 8-azaguanine-2-C'4, most likely represents the incor
poration of analog into RNA chains. The distribution
of radioactivity on these gradients is peculiar to azagua
nine. This unique distribution is not observed in either
unidine-H3 or guanine-C'4 in RNA synthesized in the
presence
of azaguanine,
making
transfer
of the
a
w
I,,
-J
C-)
aa
2-C'4
atom from the analog to a normal RNA precursor unlikely
as an alternative.
In addition, exogeneous guanine
interfered only minimally with the uptake of azaguanine.
The fact that approximately the same percentage of
guanine-C'4 and azaguanine-C'4
is released with either
alkaline digestion or RNase treatment
from ribosomal
and sRNA is good evidence that the analog is incorporated
into polyribonucleotide
material.
In addition, the find
TUBE NUMBER
CHART
8.—Comparison
of the
distribution
of
guanine-C14
in
lag that, following the treatment of an alkaline hydrolysate
of azaguanine-containing RNA with snake venom, which
RNA after 3 hr., with and without azaguanine in the media.
primarily
r.p.m.
hydrolyzes
5'-monophosphate
nucleotides,
all
Analysis was made on a sucrose gradient
for
17
sedimented
at 22,500
hr.
the recoverable activity moved as 2' , 3'-guanylic acid on
paper chromatography
and that, following treatment
with
E. coli phosphatase, which hydrolyzes only 2' , 3'-phos
pb.ate linkages, the activity
was associated with guanosine,
is still further evidence that the analog is held in classical
RNA
linkage.
This paper
chromatographic
system
did
not distinguish between azaguanylic and guanylic acids.
Assuming, as is the case in B. cereus (10), that the
azaguanine incorporated
into the sRNA of KB cells re
places guanine mole for mole, one can estimate the extent
of the replacement using the following information: a
30-hr. doubling time; 1 mg RNA/m1 = 25 optical density
units at 260 in,@;guanine = 30 % of total sRNA nucleo
tides (16); 1 Mmole azaguanine-C'4
= 1.12 X [email protected].
on the low-background j@-counter. In the 3-hr. experi
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1965 American Association for Cancer Research.
Cancer Research
192
ment illustrated in Chart 4, tube 51 contained 47 X 2
c.p.m. (total activity/E260) in about 0.3 optical density
units, but if all the guanine residues ordinarily incorporated
into sRNA during this period were replaced with aza
guanine there should have been 1100 c.p.m. in tube 51
(3/30 x 0.3 X 1/25 x 300/363 x 1.12 X 10@). Therefore,
only 8—9
% (94/1100 x 100) of the guanine residues were
replaced. This is about 1/5 the guanine replacement
observed in bacteria by Levin, but is of the same order
as that observed in mammalian systems (21).
A most attractive hypothesis would be that the incor
poration of azaguanine into sRNA is the cause of the
inhibition of protein synthesis. Such an effect could be
produced either by interfering with the enzyme systems
concerned with attaching amino acids to sRNA or by
preventing the proper reading of the genetic code (11).
An important question is whether the analog is replacing
8 % of all the guanine residues in@ll the species of sRNA,
100 % of the guanine in 8 % of the species, or, perhaps,
some intermediate
distribution.
In fact, an unusual 5s
RNA component has recently been isolated from nibo
somes (18). All the evidence developed here, however,
indicates
that the azaguanine
is incorporated
into species
of RNA which are indistinguishable by density gradients,
RNase and KOH digestion, and stability after treatment
with actinomycin D, from that of ordinary RNA.
There
is a suggestion, however, following chromatography
on
columns of methylated bovine albumin that the distribu
tion of the analog
in sRNA
may not be uniform.
are now under way to investigate
Studies
some of these questions.
ACKNOWLEDGMENTS
We wish to thank Dr. Jack Davidson for his help in testing the
radiopurity
of
the
azaguanine.
Mrs.
Clara
Horton
gave
indis
pensable advice on the technics for growing KB cells in suspension
culture, and Dr. Norman Salzman graciously provided warm room
space at rather critical times.
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Studies of DNA, RNA, and Protein Synthesis in Cultured Human
Cells Exposed to 8-Azaguanine
Myron Karon, Sherman Weissman, Carol Meyer, et al.
Cancer Res 1965;25:185-192.
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