[CANCER RESEARCH 35, 3014 3020, November 1975]
The Inhibition of Ribosomal R N A Synthesis and Maturation in
Novikoff Hepatoma Cells by 5-Fluorouridine I
David S. Wilkinson, 2 Thea D. Tlsty, and R. J a n e Hanas
Department of Biochemistry, College of Medicine, University of South Florida, Tampa, Florida 33620
SUMMARY
The inhibition of ribosomal R N A (rRNA) maturation by
5-fluorouridine (FUrd) in Novikoff hepatoma cells appears
to depend upon the incorporation of the analog into the 45 S
r R N A precursor. Precursor synthesized in the presence of
FUrd is not processed into mature rRNA, but precursor
synthesized in the absence of the analog is processed
normally after the addition of the drug. The effect of FUrd
on r R N A maturation is concentration dependent. At a
concentration of 1 • 10 -4 M, the analog completely inhibits
the formation of mature 18 S and 28 S r R N A ; while at a
concentration of 1 • 10 -v M, the analog has no significant
effect on r R N A maturation. These results suggest that some
minimum degree of analog substitution is necessary to
inhibit the maturation process. In addition to its inhibition
of maturation, FUrd also inhibits the transcription of 45 S
r R N A precursor. However, this effect of the drug is less
complete and more time dependent than the effect on
maturation. The inhibition of r R N A maturation by FUrd
persists after removal of the analog from the culture
medium. Cells that had been exposed to FU rd for 2 hr were
unable to process 45 S r R N A precursor 20 hr after removal
of the drug from the medium.
INTRODUCTION
5-Fluorodeoxyuridine and FUra 3 have been used extensively in the treatment of certain types of cancer (9-11) since
they were first synthesized in 1957 (5). It is generally
thought that the cytotoxicity of these and several related
compounds, such as 5-fluoroorotic acid and FUrd, is related
to the fact that they are all metabolically converted to
5-fluoro-2'-deoxyuridine -5'-monophosphate, which is a
potent inhibitor of thymidylate synthetase and, hence, of
DNA synthesis (8). However, a number of recent reports
have demonstrated that certain of these compounds are also
potent inhibitors of r R N A metabolism. Since the synthesis
of ribosomes during the G1 phase of the cell cycle is a
~'Research supported by Grants DRG-1220 from the Damon RunyonWalter Winchell Cancer Fund and F73FS-2 from the American Cancer
Society, Florida Division, Inc.
2To whom correspondence should be addressed.
3The abbreviations used are: FUra, 5-fluorouracii; FUrd, 5-fluorouridine; Urd, uridine; PCA, perchloric acid.
Received May 12, 1975; accepted July 17, 1975.
3014
prerequisite for D N A synthesis during the S phase (1), the
inhibition of r R N A maturation by these fluorinated pyrimidines may be of equal or even greater importance than the
inhibition of DNA synthesis.
In mammalian cells, mature 28 S and 18 S rRNA are
produced in the nucleolus from a common precursor by a
complex maturation process (2). The initial r R N A transcript product is a 45 S molecule, which contains the
sequences of both 28 S and 18 S rRNA, plus additional
sequences the functions of which are currently undefined.
The 45 S molecule is methylated, in both base and sugar
moieties, either during or soon after its synthesis. After
methylation, the 45 S molecule is converted to a 41 S
molecule, which also contains the sequences for both 28 S
and 18 S rRNA. The 41 S molecule is cleaved to a 32 S
molecule and a 20 S molecule, which are the immediate
precursors of mature 28 S and 18 S rRNA, respectively.
Throughout the maturation process the 45 S r R N A precursor and the various intermediates exist as ribonucleoprotein
particles. FUra inhibits r R N A synthesis in bacteria (7),
yeast (4), and rat liver (26). 5-Fluoroorotic acid inhibits the
production of mature 28 S and 18 S r R N A in the livers of
rats (24) and mice (6). Recent studies demonstrated that
both FUrd and FUra inhibit the maturation of r R N A in
Novikoff hepatoma cells (25). In all of these studies the
inhibition appeared to occur at a posttranscriptional site.
This paper describes in detail the nature of the inhibition of
r R N A maturation by FUrd in Novikoff hepatoma cells.
MATERIALS AND METHODS
Cells and Media. Novikoff hepatoma cells (strain N I-S 1)
were grown at 37 ~ in suspension culture under an atmosphere of 5% CO2:95% air in a gyratory incubator rotating at
150 rpm (12). The cells were grown in Swim's Medium 77,
supplemented with calf serum (100 ml/liter) and Pluronic
F-68 (1 g/liter) and modified to contain 4 mM t~-glutamine.
This medium is hereafter designated as Medium $69.
Extraction and Electrophoretic Analysis of RNA. Upon
completion of the experimental incubation period, the cell
suspensions were transferred to centrifuge tubes and chilled
to 0 ~ The cells were harvested by centrifugation at 3000 • g
for 5 min at 4 ~. R N A extraction (25) and analysis by
polyacrylamide:agarose gel electrophoresis (24) were performed as described previously. After electrophoresis, the
gels were scanned at 260 nm with a recording spectrophotometer equipped with a linear transport module and then
CANCER RESEARCH VOL. 35
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Research.
Inhibition o f r R N A Metabolism by FUrd
cut with a Mickel gel slicer ( B r i n k m a n n I n s t r u m e n t s , Inc.,
W e s t b u r y , N . Y.) into 2 - m m slices. The gel slices were
incubated overnight at 37 ~ in 0.5 ml of 0.5 N N a O H and
then assayed for radioactivity after the addition of 0.5 ml of
0.5 N HC1 and either 5 ml of P C S solubilizer or 10 ml of
Scintiverse.
Incorporation of [aH]Guanosine into the Acid-soluble
Fraction. Cells were exposed to [8-aH]guanosine under
various conditions. U p o n completion of the incubation
period, the cells were harvested by centrifugation at 270 • g
for 3 rain. The cell pellets were washed by mixing with a
vortex mixer in fresh M e d i u m $69, and the cells were
collected again by centrifugation at 27,000 • g for 5 min.
The washed cell pellets (containing a p p r o x i m a t e l y 25 • 106
cells) were suspended in 2 ml of cold 0.2 y P C A and allowed
to sit at 0 ~ for 10 rain. The resulting precipitate was
collected by centrifugation (27,000 x g, 5 min) and washed
twice with 1 ml of cold 0.2 N P C A . The combined
s u p e r n a t a n t fluids are the acid-soluble fraction.
Chromatographic Analysis of Urd Nucleotides in the
Acid-soluble Fraction. Cells were exposed to [5-aH]uridine
under various conditions. The acid-soluble fraction was
prepared as described above, except that trichloroacetic acid
(6 g / 1 0 0 ml) was used in place of 0.2 N P C A . The combined
s u p e r n a t a n t fluids for each sample were extracted with ether
until the p H was raised to a p p r o x i m a t e l y 5. S a m p l e s were
then lyophilized, and the residue was dissolved in water (5
# l / m l of acid-soluble fraction). Five #1 of each sample were
spotted on W h a t m a n No. 3 M M p a p e r (23 x 57 cm). The
uridine nucleotides were separated by descending c h r o m a tography, using a solvent containing 1 M a m m o n i u m
acetate:95% ethanol (30:70, v/v) for 14 hr. A f t e r air drying,
the c h r o m a t o g r a m s were cut into 1- x 3-cm segments,
which were placed in scintillation vials and rocked with 1 ml
of 0.01 Y HC1 for 1 hr at 37 ~ . Five ml of a dioxane-based
counting cocktail were added for the d e t e r m i n a t i o n of
radioactivity in the eluate.
Radioactivity Measurements. All radioactivity measurements were p e r f o r m e d by a P a c k a r d Model 3375 liquid
scintillation spectrometer. A u t o m a t i c external standardization was used to correct for quenching. The dioxane-based
counting solution contained 8 g of O m n i f l u o r ( N e w England
Nuclear, Boston, Mass.) and 60 g of n a p h t h a l e n e (J. T.
Baker Chemical Co., Phillipsburg, N. J.) per liter of
dioxane ( I C N Isotope and N u c l e a r Division, Cleveland,
Ohio).
Materials. S w i m ' s M e d i u m 77 and calf serum were
purchased f r o m G r a n d Island Biological Co., G r a n d Island,
N. Y., and I n t e r n a t i o n a l Scientific Industries, Inc., C a r y ,
Ill., respectively. Pluronic F-68 was a gift from the W y a n dotte C h e m i c a l Co., W y a n d o t t e , Mich. P C S solubilizer,
[8-aH]guanosine (15.8 C i / m m o l e ) , and [5-aH]Urd (5 C i /
ramBle) were obtained from A m e r s h a m / S e a r l e Corp., Arlington Heights, Ill. [2-~4C]Urd (45 m C i / m m o l e ) was purchased from C a l a t o m i c , Los Angeles, Calif. Scintiverse was
obtained from Fisher Scientific, Pittsburgh, Pa. Actinomycin D was supplied by C a l b i o c h e m , La Jolla, Calif.
U r d was purchased from S i g m a C h e m i c a l Co., St. Louis,
Mo. F U r d was a generous gift from H o f f m a n n - L a R o c h e ,
Inc., Nutley, N.J.
NOVEMBER
RESULTS
Concentration Dependence of the Inhibition of rRNA
Maturation by FUrd. Identical cultures of N o v i k o f f hcpat o m a cells were exposed to [3H]Urd for 90 rain in the
presence of various concentrations of F U r d . C h a r t 1 shows
the results of the electrophoretic analysis of the R N A
extracted from these cultures. At a c o n c e n t r a t i o n of I •
l0 -7 M ( C h a r t 1A), F U r d had little or no effect on the
incorporation of the labeled nucleoside into m a t u r e 18 S or
28 S r R N A , since essentially the same radioactivity profile
was obtained in control cultures treated with i • 10 7 xl
U r d (results not shown). Although some inhibition of
m a t u r e r R N A f o r m a t i o n was observed at 1 • 10 -6 M F U r d
( C h a r t I B), significant a m o u n t s of 32 S, 28 S, and 18 S
r R N A were still produced. There was essentially no f o r m a tion of m a t u r e 18 S or 28 S r R N A at l • 10 s M F U r d
( C h a r t I C), although significant production of the 45 S and
32 S r R N A precursors was observed. At 1 • 10 4 M F U r d
10000[
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Chart 1. Effect of different concentrations of FUrd on the incorporation of [aH]Urd into rRNA. Novikoffhepatoma cells at a density of 1.42 •
106/ml were harvested and resuspended in one-fifth the original volume of
fresh Medium $69. Identical 5-ml cultures were treated with [aH]Urd (1
~,Ci/ml) and different concentrations of FUrd (A, 10 7 M; B, 10 6 ~I; C,
10 5 M; D, 10 4 M). After incubation for 90 rain, total cellular RNA was
extracted and 1 A26o unit of each sample was applied to a composite gel
containing 2.4% acrylamide and 0.6% agarose. Gels were subjected to
electrophoresis for 3 hr at 6 ma/gel, scanned at 260 nm, and cut into 2-ram
slices: each slice was assayed for radioactivity. The location of the various
rRNA peaks was verified by comparing the radioactivity profile to the A~6o
scan.
1975
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1975 American Association for Cancer
Research.
3015
D. S. W i l k i n s o n et al.
( C h a r t l D), the initial 45 S r R N A precursor was the only
species into which a significant a m o u n t of labeled nucleoside was i n c o r p o r a t e d . F o r each of the F U r d - t r e a t e d
cultures discussed here, c o n t r o l cultures were run that
contained the c o r r e s p o n d i n g c o n c e n t r a t i o n s of Urd. In each
case the radioactivity profile was qualitatively identical to
that seen in C h a r t 1A.
Effect of FUrd on the Rate of 45 S r R N A Synthesis.
Identical cultures were exposed to [~H]guanosine for 15 min
in the presence of either 1 x 10 -4 M Urd or l x 10 -4 M
F U r d . In o n e - h a l f of the cultures, the nucleosides were
added s i m u l t a n e o u s l y with the labeled precursor; in the
other half, the nucleosides were added l hr before the
addition of labeled precursor. The e l e c t r o p h o r e t i c analysis
of R N A extracted from these cultures is shown in C h a r t 2.
W h e n F U r d was added s i m u l t a n e o u s l y with the [3H]guanosine, the i n c o r p o r a t i o n of radioactivity into 45 S r R N A
precursor was not inhibited. However, when F U r d was
added 1 hr before the addition of [3H]guanosine, the
i n c o r p o r a t i o n of radioactivity into 45 S r R N A precursor
was significantly inhibited. The d a t a in Table 1, which
shows the i n c o r p o r a t i o n of radioactivity into the acid-solu-
Table 1
Effect of FUrd on the incorporation 0]" [8-3H]guanosine into the
acid-soluble fraction
Eight 25-ml aliquots from a stock culture containing 0.93 • 10e cells/ml
were centrifuged, and the cell pellet derived from each aliquot was
resuspended in 5 ml of fresh medium. Each experimental 5-ml culture was
exposed to [3H]guanosine(i #Ci/ml) for 15 min. In addition to the labeled
precursor, either 1 x 10-' MUrd or I • 10-4 M FUrd was added to each
culture according to the protocol indicated below. At the end of the 15-min
pulse, the amount of radioactivity in the acid-soluble fraction was
determined.
Treatment
Preincubation
(min)
cpm/l0 e cells
U rd
Urd
FUrd
FUrd
0
60
0
60
17,099 + 114a (100)~
17,761 • 112 (104)
18,619•
(109)
24,809 • 927 (145)
a Mean • the average deviation for values obtained from duplicate
cultures.
o Numbers in parentheses, each value expressed as the percentage of the
corresponding value obtained in the cultures that received Urd and
[3H]guanosine simultaneously.
ble pool, indicate that decreased uptake of [3H]guanosine
was not responsible for the decreased i n c o r p o r a t i o n of
radioactivity into 45 S r R N A precursor.
45S
500
A
-- i
B
Effect of FUrd on the Urd Nucleotide Pool. Identical
il4tle
cultures were incubated with either 1 x 10 -4 M Urd or I •
400
10 -4 M F U r d . Both cultures contained [3H]Urd, 1.25
45S
# C i / m l . After various periods of time, the acid-soluble pool
was analyzed by paper c h r o m a t o g r a p h y . The results ob~S
_
300
tained are shown in C h a r t 3 and ]'able 2. The relative
distribution of radioactivity a m o n g the various uridine
200
nucleotides was not affected by the presence of F U r d .
A p p r o x i m a t e l y 75% of the radioactivity was in the form of
U T P in each culture at all time points examined.
I00
Effect of FUrd on the Maturation of 45 S r R N A Precursor
Labeled in the Absence of Analog. Identical cultures were
exposed to [~H]Urd for 15 rain. This pulse length allows
45 S
label to be i n c o r p o r a t e d only into the 45 S r R N A precursor
==600
D
( C h a r t 2). At this point, one culture was made I • 10 -4 ~1
848
with respect to Urd and a n o t h e r was made 1 x 10 -4 M with
~,
500
respect to F U r d . Both cultures received a c t i n o m y c i n D at
the time of addition of the nucleoside to inhibit further
R N A synthesis. T w e n t y min following the addition of
400
a c t i n o m y c i n and unlabeled nucleoside, R N A was extracted
28S
28S
i
and analyzed by gel electrophoresis. In both control ( C h a r t
300
4A) and F U r d - t r e a t e d ( C h a r t 4 B ) c u l t u r e s , essentially all of
''
i'
-the 45 S molecules that had been labeled during the initial
45 S
15-min pulse were processed to 32 S, 28 S, or 18 S r R N A .
.,
I~ ~
200
Since nucleosides are t r a n s p o r t e d into the cell and phosphorylated in the presence of a c t i n o m y c i n D (14), these d a t a
I00
indicate that the presence of F U r d or its low-molecularweight metabolites did not prevent the m a t u r a t i o n of 45 S
I
- __:_~- --molecules synthesized prior to its addition.
I0
20
30
I0
20
30
These results are significantly different from those shown
FRACTION
in
C h a r t 4D, which were obtained from a culture in which 1
Chart 2. Inhibition ofrRNA synthesis byFUrd. Cells were exposed to
x
10 -4 M F U r d was present together with the [3H]Urd
[SH]guanosine for 15 min. Urd (A and C) or FUrd (B and D) was added
either simultaneously with (A and B) or I hr before (C and D) the addition during the initial 15-rain pulse. As before, a c t i n o m y c i n D
of the radioactive compound. RNA was extracted and analyzed by was added 15 rain after the addition of label and R N A was
extracted 20 min after the addition of a c t i n o m y c i n D. The
electrophoresis as described in the legend of Chart I.
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CANCER RESEARCH VOL. 35
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1975 American Association for Cancer
Research.
Inhibition
I
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1
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l
'1
, UTP
[
I
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I
Control
&3
15 min
FUrd
15 min
x
UDP
I0
UDP
UDPG
20
30
Migration
I0
20
Distance (cm)
Urd
1600
30
Chart 3. Effect of F U r d o n / h e relative d i s t r i b u t i ~ 1 7 6 1 7 6
in the acid-soluble pool. Cells that had grown to a density of 1.38 ?< 10~/rnl
werc harvested and resuspendcd in one-fifth the original volume of fresh
Medium $69. Identical cultures were then exposed to [3H]Urd (1.25
#Ci/ml) in t h c p r e s e n c e o f l x 10 4st Urd(control) or I x I0 4y~ FUrd.
After incubating for 15 rain, the acid-soluble fraction was prepared and
analyzed by paper chromatography.
UMP
b v FUrd
I
I
[
I
1
I
1
328
~
52S
~ 800
lOS
-- 12001
--
B
~ _ @
0
5 10 IS 20 25 30 35
200
40
1
0
S
200
1 1 1 1 1 1 1
--
800
400
400
I
1
1
I0 tS 20 25 30 35 40
1
1
J
1
1
1
45s
150
-
C
-
IS0
32S
=. I00
Distribution of radioactivity
UTP ~ UDP UDPglucose
1600
l l l l l l l
1200 I
Table 2
The e['fect 0[ FUrd on the distribution q[ Urd nucleotides in the
acid-soluble pool
Two 40-ml aliquots from a stock culture containing 1.38 • IOs cells/ml
were centrifuged, and the cell pellet derived from each aliquot was
resuspended in 8 ml of fresh medium. One culturc was treated with 1 •
10 4xl Urd and the other with 1 • 10-'M FUrd. Both cultures contained
[3H]Urd (1.25 #Ci/ml). One-ml aliquots were removed alter 15, 30, and 60
min of incubation. The trichloroacetic acid-soluble fraction was extracted
with ether, lyophilized, and chromatographed. The amount of radioactivity
associated with each nucleotide fraction is reported as the percentage of the
total counts recovered from the chromatogram.
Treatment
Metabol&m
w a s e x t r a c t e d a n d a n a l y z e d by e l e c t r o p h o r e s i s ( C h a r t 5, A
a n d D ) . T h e cells in t h e r e m a i n i n g p o r t i o n o f e a c h c u l t u r e
were harvested by centrifugation,
washed twice, resusp e n d e d in f r e s h m e d i u m d e v o i d o f [ 3 H ] g u a n o s i n e o r u n l a b e l e d n u c l e o s i d e , a n d a l l o w e d t o i n c u b a t e f o r an a d d i t i o n a l
20 hr. E a c h c u l t u r e w a s t h e n i n c u b a t e d w i t h [ 1 4 C ] U r d in t h e
a b s e n c e o r p r e s e n c e o f a c t i n o m y c i n D, a f t e r w h i c h i n c u b a t i o n R N A w a s e x t r a c t e d a n d a n a l y z e d by e l e c t r o p h o r e s i s .
After the initial 2-hr incubation period, [3H]guanosine was
UTP
4
Of rRNA
I00
o
Urd
50
50
15 min
u rd
FU rd
77
76
8
7
6
8
9
9
o
0
1 I l Ik,,#',,x/[ L l l , J
5 10 tS 20 2s 30 3S 40
FRACTION
30 rain
U rd
FUrd
73
71
12
14
9
8
6
7
13
20
5
3
60 min
U rd
FU rd
78
72
4
5
presence of FUrd during the initial labeling period resulted
in t h e s y n t h e s i s o f 45 S r R N A p r e c u r s o r w h i c h w a s n o t
c o n v e r t e d to 32 S , 28 S, o r 18 S r R N A
during the
s u b s e q u e n t a c t i n o m y c i n D t r e a t m e n t . In t h e c o n t r o l c u l t u r e ,
w h i c h c o n t a i n e d I • 10 -~ M U r d d u r i n g t h e i n i t i a l p u l s e , all
o f t h e l a b e l e d 45 S r R N A p r e c u r s o r w a s c o n v e r t e d t o t h e s e
more mature rRNA species during the 20-min actinomycin
D treatment (Chart 4C).
Persistent Effects of FUrd on rRNA Maturation. Identic a l c u l t u r e s w e r e e x p o s e d t o [ 3 H ] g u a n o s i n e in t h e p r e s e n c e
o f e i t h e r I • 10 -~ M U r d o r I • 1 0 - ' M F U r d f o r 2 hr. F r o m
e a c h c u l t u r e , a n a l i q u o t o f cells w a s t a k e n f r o m w h i c h R N A
NOVEMBER
0 - I
0 5
I 1 IX,4,/ ' k ~ l -10 t5 20 25 30 3S 40
NUMBER
Chart 4. Effect of FUrd on the maturation of 45 S rRNA precursor
during a 20-rain treatment with actinomycin D. Novikoff cells were grown
to a density of1.14 • 106/ml, harvested, and resuspendedin one-fitththc
original volume of fresh Medium $69. Five ml of the resulting cell
suspension were placed in each of 4 experimental flasks. At 0 time, 1
culture was treated with I x 10 4 ~,1Urd (C) and another was treated with I
• 10-' .sl FUrd (D). All 4 cultures received [3H]Urd (0.5 #Ci/ml) at 0 time.
Fifteen rain following the addition of [3H]Urd, actinomycin D (5 #g/ml)
was added to each culture. At this time, in addition to the actinomycin D, I
culture also received I • 10 4 ~,I Urd (A) and another received I • 10 -4 .',1
FUrd (B). Twenty min after the addition of actinomycin D, RNA was
extracted and analyzed by electrophoresis as described in the legend of
Chart 1. A diagram of the experimental protocol is shown below.
Culture
flask
A
B
C
D
[~H]Urd
1
1
1+ Urd
.[+ FUrd
I'15rain
Actinomycin D
~+ Urd
1+ FUrd
],
~
-~l'
20rain
1975
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1975 American Association for Cancer
Research.
Extract
RNA
1
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3017
D. S. Wilkinson et al.
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30
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-
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20
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,~
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13
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IO
FRACTION
20
1
. . . . .
1
i
--~
8 ~
x
i,
"
F
,::,
"0
.
,,,s
---'
30
6~
-
-,,.
,
IO
30
30
NUMBER
Chart 5. Persistent effects of FUrd on r R N A maturation. Two experimental cultures were prepared by harvesting cells from a stock culture
containing 0.67 • 10 ~ cells/ml and resuspending the cell pellets in one-fifth the original volume of fresh medium. One culture received 1 • 10-' x.1 Urd (A
to C) and the other culture received I x 10 4 M FUrd (D to F). Both cultures received [aH]guanosine (1 # C i / m l ) . After incubation for 2 hr, aliquots were
taken from each culture for the extraction of R N A (A and D). Cells were then harvested by centrifugation from the remaining portions of each culture,
washed twice in cold medium, and resuspended at a density of 0.67 x 106 cells/ml in medium devoid of [aH]guanosine or unlabeled nucleosides. After
incubating overnight, the cells in each culture were harvested by centrifugation and resuspended in one-fifth the original volume of fresh medium. Both
cultures were divided into 2 equal portions. Exactly 20 hr following the removal of [aH]guanosine from the medium, each culture received [14C]Urd (0.1
# C i / m l ) either in the presence (C and F) or absence (B and E) of actinomycin D (0.04 # g / m l ) . Two hr after the addition of [14C]Urd (22 hr after the
removal of [aH]guanosine) R N A was extracted and analyzed by electrophoresis as described in the legend of Chart 1.
incorporated predominantly into mature 28 S and 18 S incubation resulted in a 14C profile that was strikingly
r R N A in the Urd-treated cultures (Chart 5A). Little or no similar to the tritium profile seen after the initial 2-hr
[aH]guanosine was incorporated into these mature r R N A exposure to [aH]guanosine. This result demonstrates dispecies in the FU rd-treated cultures (Chart 5D). The tritium rectly the continued ability of the Urd-treated cells to both
present in the R N A extracted from the Urd-treated cultures transcribe and process r R N A (Chart 5B) and the persistent
inhibition of r R N A processing in the FUrd-treated cells
2 2 hr after removal of [aH]guanosine from the medium was
found to reside almost exclusively in 28 S and 18 S r R N A (Chart 5E). However, the FUrd-treated cells retained the
(Chart 5B). No tritium was associated with the 45 S region. ability to synthesize significant amounts of 45 S r R N A
In contrast, significant amounts of tritium were still associ- precursor. The lack of [14C]Urd incorporation into 45 S
ated with the 45 S r R N A precursor in the FUrd-treated r R N A precursor during the actinomycin D treatment in
cells (Chart 5E). Furthermore, there was no distinct associa- either the Urd-treated cultures (Chart 5C) or the FUrdtion of tritium with mature 18 S or 28 S r R N A in the treated cultures (Chart 5F) verifies that the concentration
analog-treated cultures. The significant decrease in the of actinomycin D used was sufficient to inhibit new r R N A
amount of tritium associated with the 45 S region observed transcription.
after exposure of the FUrd-treated cells to actinomycin D
(Chart 5F) indicates that the FUrd-substituted precursor D I S C U S S I O N
undergoes metabolic turnover even in the absence of a
FUrd is but I of a number of analogs which can inhibit
normal maturation pathway. The actinomycin D treatment
did not affect the distribution of tritium in R N A extracted the formation of mature r R N A (3, 13, 15, 16, 18, 20, 21). It
from the Urd-treated cells (Chart 5C). This finding may be has been postulated (22, 25) that the ability of analogs to
attributed to the relatively slow turnover of the mature 18 S inhibit r R N A maturation may be related to their ability to
be incorporated into the 45 S r R N A precursor and the
and 28 S r R N A species.
Exposure of the cells to [~4C]Urd during the last 2 hr of extent to which that incorporation alters the physiocochemi3018
CANCER RESEARCH VOL. 35
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Inhibition
cal characteristics of that molecule. Recently, Weiss and
Pitot (22) d e m o n s t r a t e d that the incorporation of 5-azacytidine into the 45 S and 32 S r R N A precursors alters their
secondary structure in such a way that the analog-substituted molecules possess a decreased electrophoretic mobility. If incorporation is the basis of their inhibitory effect,
there should be an obligatory requirement for R N A synthesis in the presence of the analogs. The d a t a shown in C h a r t 4
suggest that the inhibition of r R N A m a t u r a t i o n by F U r d
does depend upon the incorporation of the analog into 45 S
r R N A precursor. P r e c u r s o r molecules synthesized before
the addition of F U r d were processed normally. In contrast,
certain intercalating agents inhibit the m a t u r a t i o n of normal 45 S r R N A precursor (17). However, these c o m p o u n d s
can bind directly to double-helical regions of R N A , thereby
disrupting its secondary structure.
Wellauer and Dawid (23) have shown with the electron
microscope that H e L a cell 45 S r R N A precursor contains a
series of hairpin loops. F o r m a l d e h y d e d e n a t u r a t i o n studies
mdicate that base pairing is responsible for loop formation.
it is possible that the a r r a n g e m e n t of loops is one of the
signals that the m a t u r a t i o n enzymes recognize. A n y t h i n g
that interferes with base pairing, such as analog substitution, could alter the loop m o r p h o l o g y , thereby altering the
m a t u r a t i o n process. O u r results are consistent with the
hypothesis that F U r d incorporation disrupts the physicochemical characteristics of the precursor molecule, rendermg it unfit for p r o p e r processing. The concentration dependence of the inhibition by F U r d indicates that a certain
degree of substitution m a y be necessary to elicit the effect.
In addition to its ability to inhibit m a t u r a t i o n , F U r d also
inhibits the transcription of r R N A . However, this effect of
the drug is much less complete and does not manifest itself
as rapidly as the effect on m a t u r a t i o n . Therefore, it m a y be
an indirect consequence of impaired m a t u r a t i o n , whereby
inhibited processing of 45 S precursor decreases the synthesis of new 45 S molecules. Studies on the acid-soluble
fraction indicate that impaired nucleoside t r a n s p o r t
or uridine nucleotide metabolism is not responsible for the
inhibition of 45 S r R N A synthesis by F U r d .
There is no significant a c c u m u l a t i o n of 45 S r R N A
precursor in the presence of F U r d , despite the continuous
synthesis and impaired processing. This implies that nonspecific nucleases are constantly degrading the fraudulent
R N A or that n o r m a l cleavage enzymes are breaking down
the analog-substituted R N A in a nonspecific fashion. The
nonproductive d e g r a d a t i o n of F U r d - s u b s t i t u t e d 45 S r R N A
precursor in the presence of actinomycin D supports this
hypothesis.
The inhibition of r R N A m a t u r a t i o n by F U r d persisted
even after prolonged incubations following the removal of
the drug from the medium. This finding is probably related
to the fact that the d i s a p p e a r a n c e of F U r d nucleotides from
the acid-soluble fraction is extremely slow (unpublished
data). This persistent effect on the f o r m a t i o n of ribosomes
must be t a k e n into account when considering the overall
cytotoxic activity of F U r d . However, the inhibition of
r R N A m a t u r a t i o n is p r o b a b l y not the p r i m a r y site of action
of all of the fluorinated pyrimidines. We have found, for
example, that even after preincubation with the drug for 2 hr
NOVEMBER
of rRNA
Metabolism
by FUrd
1 • 10 -4 M fluorodeoxyuridine had no effect on r R N A
m a t u r a t i o n . Essentially all of the cell-killing activity of this
c o m p o u n d is probably related to the inhibition of D N A
synthesis. One must also be cautious about e x t r a p o l a t i n g
from our results with F U r d in one strain of N o v i k o f f c e l l s to
other cell lines. As suggested previously by U m e d a and
Heidelberger (19), the exact m e c h a n i s m of toxicity m a y well
vary according to the cell line and culturing conditions. For
example, 1 • 10 -7 M F U r d , which had no effect on r R N A
m a t u r a t i o n in our study, was sufficient to prevent the
growth of a fluorodeoxyuridine-sensitive N o v i k o f f cell line
(19); but 1 • 10 4 M F U r d , which completely inhibited
r R N A m a t u r a t i o n in our system, was required to suppress
completely the growth of a flurodeoxyuridine-resistant
N o v i k o f f cell line. In the sensitive cells, the inhibition of
D N A synthesis m a y have been the critical site of inhibition,
while in the relatively resistant cells, the inhibition of r R N A
m a t u r a t i o n m a y have been the critical site,
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CANCER
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Research.
V O L . 35
The Inhibition of Ribosomal RNA Synthesis and Maturation in
Novikoff Hepatoma Cells by 5-Fluorouridine
David S. Wilkinson, Thea D. Tlsty and R. Jane Hanas
Cancer Res 1975;35:3014-3020.
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