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[CANCER RESEARCH 35, 2340-2349, September 19751
@
Preferential Inhibition by Homopolyribonucleotides of the
Methylation of Ribosomal Ribonucleic Acid and Disruption
of the Production of Ribosomes in a Rat Tumor'
Ming C. Liau, Don W. Smith, and Robert B. Huribert
Department of Biochemistry,
The University of Texas System Cancer Center, M. D. A nderson Hospital and Tumor Institute, Houston, Texas 77025
SUMMARY
growth of experimental tumors (1 , 4, 11, 20—22).Further
more, it has been reported that poly(I).poly(C)
can also
The literature indicates that some mechanism other than inhibit the replication of normal cells characterized by rapid
growth such as embryonic tissues ( 1), regenerating liver
the interferon
or host-mediated
immune enhancement
proliferation of salivary epithe
might also be responsible for an antitumor
effect of (15), or hormone-induced
polyinosinate-polycytidylate
[poly(I) . poly(C)]. We have ex hum (37). Attempts to elucidate the mechanism of antitu
mor effect by this compound are complicated by the wide
amined the effect of this drug on the synthesis of ribosomes
variety of biological effects exerted by this compound in
and other macromolecules
in a rat tumor, the Novikoff
animals. Some of these effects, notably the induction of
ascites hepatoma. The nucleolus was one of the primary
targets affected by the administration
of poly(I). poly(C) in interferon (12, 17), the activation of macrophages (2), and
the enhancement
of immune response (10), have been
vivo. A progressive decline of the activity of nucleolar
ribosomal RNA methylases began within 2 hr, followed by considered to play an important role in the destruction of
a decline of the nucleolar RNA content. The activity of tumor cells in vivo. However, the correlation between the
nucleolar RNA polymerase was inhibited only at later time induction of interferon and the antitumor effect of inter
feron inducers has not been consistent (4, 9, 35, 43). The
intervals. Labeling of tumor macromolecules
in vivo re
that poly(I). poly(C) was active against cer
vealed that the methylation of ribosomal RNA and the demonstration
tam cells in culture (29) and against solid tumors (20, 21),
production of ribosomes, particularly the small subunits,
were immediately and progressively affected, followed by and was as effective against tumors of immunosuppressed
inhibition of the synthesis of DNA, RNA, and protein at hosts (4, 11), indicated that some mechanism other than
host-mediated immune enchancement is responsible for the
later times. In addition, poly(I). poly(C) also induced disag
Thus, the antitumor
gregation of polyribosomes and restricted the movements of antitumor effect of poly(I).poly(C).
nuclear RNA to cytoplasm and of cytoplasmic protein to effect of poly(I) . poly(C) cannot be satisfactorily attributed
solely to the interferon-induced
or host-mediated
mech
nucleus. These in vivo effects of poly(I) poly(C) on tumor
cells were observed neither on the host livers nor on livers of anisms. It is our intention to explore still unknown mech
anisms ofthe antitumor effect mediated by poly(I). poly(C).
normal rats.
This report provides evidence that the nucleolus of rat
Studies on isolated nucleoli showed that the in vitro
Novikoff ascites hepatoma was one of the primary targets
addition of polyinosinate
and several other compounds
of poly(I) . poly(C) in vivo.
actively inhibited tumor ribosomal RNA methylases but affected by the administration
The inhibition of the methylases was the earliest detectable
were devoid of inhibitory effect against liver ribosomal
RNA methylases; these results augment other studies in the event, followed by inhibition of the synthesis of DNA,
RNA, and protein at later time intervals. It appears that
literature in suggesting a selective effect ofthe polyinosinate
initial impairment of the methylation of ribosomal precur
moiety on tumor cells.
We conclude from this study that initial impairment of sor RNA, following exposure of tumor cells to poly(I).
poly(C), is responsible for the destruction of ribosomes,
the methylation of ribosomal precursor RNA, following
preferentially
the small subunits at early time intervals,
exposure of tumor cells to poly(I).poly(C),
is responsible
for the destruction of ribosomes, preferentially the small during maturation processes. Failure to provide new ribo
somes thus triggers the events limiting the growth of tumor
subunits, during the maturation processes. Failure to pro
vide new ribosomes thus triggers the events limiting the cells. Evidence is also provided to indicate that rRNA
methylases are probably regulated differently during nor
growth of tumor cells.
mal and malignant growth. The sensitivity of tumor en
INTRODUCTION
zymes and insensitivity of liver enzymes toward several
Poly(I).poly(C)2
has been shown to block the induction
inhibitory compounds
suggests that the use of rRNA
of tumor by various agents (16, 18, 19), and to inhibit the
2 The
1 This
work
was
supported
by
American
Cancer
Society
and National Cancer Institute Grant CA-l5086-Ol.
Received September 23, 1974; accepted May 7, 1975.
2340
Grant
NP-97B
abbreviations
used
are:
poly(l)
. poly(C),
polyinosinate-polycyti
dylate; [‘H]S-Ado-Met, [methyl-3H]-S-adenosyl-i-methionine;
poly(I),
polyinosinate; PCA, perchloric acid; TCA, trichloroacetic
acid; poly(C),
polycytidylate; cAMP, cyclic 3,5-AMP.
CANCER
RESEARCH
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VOL. 35
Antitumor
methylase
inhibitors as antitumor
agents
favorable differential effects on tumor cells.
MATERIALS
may
provide
AND METHODS
Materials. [3HJS-Ado-Met (4.5 Ci/mmole) and [methyl
3H]-L-methionine (5 Ci/mmole) were obtained from Amer
sham/Searle Corp., Arlington Heights, Ill. NaH232PO4 (0.5
Ci/mmole)
was obtained from New England Nuclear,
Boston, Mass. [5-3H]CTP (12.6 Ci/mmole)
and [5-3H]orotic acid (5 Ci/mmole)
were obtained from Schwarz/
Mann, Orangeburg,
N. Y. Poly(I).poly(C),
sodium salt
(52o.u.
>
12),
> 100,000)
and
were
poly(I)
obtained
and
from
polyguanylate
P-L
(both
Biochemicals,
M.W.
Inc.,
Efftcts
ofPolv(!).Poly
(C)
were injected per rat, again i.p. The rats were sacrificed 3 hr
later, and tumor cells and livers were removed for the
analyses of incorporation
of radioactive precursors into
macromolecules.
Tumor cells were washed 3 times with 0.14 M NaCl and
0.01 M Tris-chloride (pH 7.8) by low-speed centrifugation
(300 x g, 5 mm). Fractionation of tumor cells into nuclear
and cytoplasmic fractions was accomplished by homogeni
zation of tumor cells in 6 volumes of 0.01 M Tris-chloride
(pH 7.8)-0.Ol M NaCl-l.5
mM MgCI2 containing 0.1%
Triton X-lOO in a Potter-Elvehjem
homogenizer with a
loose-fitting Teflon pestle until most of the cells were
broken. Nuclei were sedimented at 600 x g for 5 mm and
resuspended in one-half the amount of 0.01 M Tris-chloride
(pH 7.8)-0.Ol M NaCI-l.5
mrvi MgCl2 used. One-tenth
volume of 3.3% deoxycholate-6.7% Triton X- 100 was added
to the nuclear suspension, stirred by Vortex mixer, and
centrifuged at 600 x g for 5 mm to yield pure nuclei. The
nuclei were free of cytoplasmic tags and contamination
by
whole cells. The RNA/DNA
ratios of these nuclear prepa
rations were 0.38 to 0.55, whereas those of the tumor cells
were 2.9 to 3.3. The combined supernatant (50.6) was further
centrifuged at 20,000 x g for 15 mm to yield the superna
tant postmitochondrial
extract (S20).
For the fractionation of liver, the rats were sacrificed by
decapitation, and the excised livers were rinsed with isotonic
sucrose solution. The livers were pressed through a Harvard
tissue press, then were homogenized in 5 volumes ofO.25 M
sucrose, 5 mM MgCl2, and 0.01 M Tris acetate (pH 7), and
were centrifuged
at 600 x g for 10 mm to separate
cytoplasm from nuclei. Nuclei were further purified by
suspension of the 600 x g sediment in 5 volumes (with
respect to starting tissue volume) of 2.2 M sucrose, 5 mM
MgCI2, and 0.01 M Tris acetate (pH 7) and were centrifuged
at 88,700 x g for 15 mm. The RNA/DNA
ratios of the
purified liver nuclear preparations were 0.2 to 0.24, whereas
those of whole livers were 2.8 to 3.3.
For the determination
of DNA synthesis through incor
poration of 32P into DNA, an aliquot of purified nuclear
preparations was precipitated and washed with 0.4 N PCA.
The precipitate was digested with 0.3 M KOH at 37°for 18
hr. The alkaline solution was acidified to 0.4 N PCA. The
precipitate containing DNA and protein was washed once
with cold 0.4 N PCA. The DNA was hydrolyzed in 0.4
Milwaukee,
Wis. Novikoff ascites hepatoma cells were
grown in 150- to l75-g Sprague-Dawley
rats. The experi
ments were conducted on the 4th or 5th day after transplan
tation of the tumor cells, when about 4 to 6 ml of packed
cells per rat were obtained.
Preparation of Nucleoli. Nucleoli from Novikoff ascites
hepatoma were prepared as described previously (24, 25).
Nucleoli from host or normal rat liver were prepared by a
similar procedure. Rats were sacrificed by decapitation, and
livers were perfused in situ with cold 0.25 M sucrose, 5 mM
MgCl2, and 0.01 M Tris acetate (pH 7). The operations
thereafter were conducted in ice-bath temperature.
The
livers were pressed through a stainless steel Harvard tissue
press (Harvard Apparatus Co. Dover, Mass.) to remove
connective tissues. The minced livers were homogenized
with 3 volumes (assuming I g tissue to be I ml) of 0.01 M
Tris acetate (pH 7), 10 mM MgCl2, and 0.2 mM CaCl2. The
homogenate was filtered through 8 layers of cheesecloth and
pressed through a chilled French pressure cell (American
Instrument Co., Silver Spring, Md.) at 4000 to 6000 psi.
The pressed homogenate was made 0.25 M sucrose and was
centrifuged at 900 x g for 10 mm. The sediment was
suspended by homogenization
with a glass-Teflon Potter
Elvehjem homogenizer in 4 volumes (with respect to starting
tissue volume) of 2.1 M sucrose, 5 mM MgC12, and 0.01 M
Tris acetate (pH 7), and was centrifuged at 88,700 x g for
15 mm to sediment nucleoli.
In most cases, very clean nucleolar preparations
from
tumor cells could be obtained easily. The preparations were
N PCA at 100° for 15 mm. The hot PCA-soluble
superna
free of contamination
by nuclei or large subcellular frag
tant was analyzed for DNA content and 32P.
ments, except that slight contamination by amorphous and
For the determination
of RNA synthesis through incor
fibrous particles was occasionally observed. The nucleolar
preparations from liver were also free of contamination
by poration of 32P into RNA, an aliquot of tumor cells or liver
homogenate was deproteinized with phenol, according to
nuclei or large subcellular fragments, although the contami
nation by fibrous materials was more noticeable than in the
procedures
described
previously
(25). The ethanol
preparations from tumor cells. The RNA/DNA
ratios of precipitated RNA was washed with cold 0.4 N PCA and
the tumor nucleolar preparations
were I .46 ±0.05, and
then hydrolyzed in 0.3 N KOH at 37°for 18 hr. The alkaline
those of the liver nucleolar preparations were 0.65 ±0.05.
hydrolysate was acidified to 0.4 N PCA. The PCA-soluble
Analyses of the Effect of Poly(1). Poly(C) on the Synthe
supernatant
was taken for the determination
of RNA
sis of Macromolecules
in Tumor Cells and Host Liver.
content and “P.
Rats bearing Novikoff ascites hepatoma were given single
For the determination
of RNA methylation
through
i.p. injections of poly(I). poly(C) (5 to 6 mg/kg) dissolved in incorporation of methyl-3H into RNA, an aliquot of tumor
0.9% NaC1 solution. After various periods of exposure, 50 cells, subcellular fractions, or liver homogenate was depro
@zCiof [32P]phosphate or 0.3 mCi of [methyl-3H]methio
teinized with phenol as mentioned above. The RNA was
nine, together with 1 @tmoleof adenosine and guanosine,
incubated in 0.5 M Tris-chioride (pH 8.8) at 37°for 1 hr,
SEPTEMBER
1975
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2341
M. C. Liau et al.
precipitated,
and washed in 0.4 N PCA, and then was
hydrolyzed in 0.3 N KOH at 37°for 18 hr. The alkaline
hydrolysate was acidified to 0.4 N PCA. The PCA-soluble
supernatant
was taken for the determination
of RNA
content and 3H.
For the determination of protein synthesis through incor
poration of [methyl-3H]methionine
into protein, an aliquot
of tumor cells or liver homogenate was precipitated with 0.4
N PCA
and
extracted
with
0.4
N PCA
at
100° for
15 mm.
The hot PCA-insoluble pellet was dissolved in 1 N NaOH at
100°for 10 mm. An aliquot of NaOH-soluble
protein was
precipitated with 5% TCA and collected on a Millipore filter
for the determination of 3H; another aliquot was taken for
the determination
of protein according to the method of
Lowry et al. (27).
Since the amount of ascites fluid and tumor cells and
probably the amount of labeled precursor taken up varies
from rat to rat, it is difficult to compare precisely the
relative activities of the labeled macromolecules.
Wherever
possible, data were referred to an amount of cells containing
1 mg of DNA. In order to further normalize the data, the
cold 0.4 N PCA-soluble radioactivity (cpm/mg of DNA) of
the washed tumor cells or washed liver was measured in
every case, and the activities of macromolecular
synthesis
(cpm incorporated per mg of RNA, DNA, or protein) were
then adjusted proportionally to represent the same amount
of acid-soluble radioactivity as the control. Thus, the effect
of poly(I). poly(C) on the uptake of precursor or the pool
size of acid-soluble intermediates has been cancelled out in
the data reported.
For the study of polysomal profiles, 3 ml of S20 fraction
were layered onto 24 ml of 15 to 30% (w/w) sucrose gradient
containing 0.02 M Tris-chloride (pH 7.8), 2 mr@iMgC12, and
0.05 M KC1 on top of a 2-mi cushion of 50% sucrose, and the
gradient was centrifuged at 15,000 rpm for 16 hr in an SW
25. 1 rotor. The gradient was then fractionated into l-ml
fractions by an automatic fractionator
(Instrumentation
Specialties Co., Lincoln, Neb.). The ribonucleoprotein
was
filtered onto Millipore filters (0.45 sm), washed with cold 5
determined by Burton's diphenylamine
reaction (8), and
protein by the procedure of Lowry et a!. (27). The determi
nation of radioactivity on Millipore filters or in aqueous
solution was as previously described (26).
Assay of Nucleolar RNA Polymerase, rRNA Methylases,
and Cell-soluble tRNA Methylases. To study the effect of
poly(I).poly(C)
on the nucleolar enzymes involved in the
synthesis of ribosomes and cell-soluble tRNA methylases,
rats bearing Novikoff ascites hepatoma were given 5 to 6
mg poly(I).poly(C)
per kg i.p. Poly(I).poly(C)
was dis
solved (1 mg/mI) in 0.9% NaCI solution by incubation at
370
for
1
hr.
with
occasional
vigorous
shaking.
Nucleoli
and
high-speed cellular extract (5226) from control rats, as well
as from poly(I) . poly(C)-treated
rats, were prepared as
described previously (24, 26). Isolated nucleoli were incu
bated in the complete RNA-synthesizing
medium (24),
either with [3HJCTP for the assay of RNA polymerase or
with [3H]S-Ado-Met
for the assay of rRNA methylase
activities. [3H]S-Ado-Met,
stored in dilute acid solution to
prevent decomposition,
was neutralized with NH4OH be
fore use. The assay of tRNA methylases in 5226 was
conducted as described previously (26).
RESULTS
The Nucleolus as the Primary Target following the
Administration
of Poly(I). Poly(C) in vivo. It has been
reported by Margolis and Levy (29) that exposure of
cultured cells to poly(I). poly(C) under certain conditions
led to the inhibition of the synthesis of ribosomal precursor
RNA. The nucleolus of Novikoff ascites hepatoma
is
apparently also one of the primary targets affected by the
administration
of poly(I).poly(C)
into the rat in vivo. The
effect of poly(I) . poly(C) on the tumor nucleolus was studied
by exposing Novikoff ascites hepatoma to poly(I) . poly(C)
in vivo, followed by the isolation of nucleoli and assay in
vitro of the activity of enzymes involved in the synthesis of
ribosomes. The results summarized in Table I indicate that
the rRNA methylases showed a definite decline in activity
mM MgCl2,
and dried for the measurement
of radioactivity
within 2 hr and progressively declined in activity as exposure
(34).
For the study of RNA profiles, 3 ml of 520 fraction were time increased. The RNA content of isolated nucleoli
made 0.3% with respect to Sarcosyl NL 97 (K & K diminished within 4 hr and also progressively decreased in
amount. In contrast, the activity of RNA polymerase was
Laboratories,
Inc., Plainview, N. Y.) and were deprotein
ized by shaking with an equal volume of phenol at room altered relatively little for the Ist 4 hr of exposure to
temperature as described (25). The RNA was layered onto poly(I). poly(C). Although a considerable decrease of RNA
28 ml of 10 to 40% (w/w) sucrose gradient containing 0.01 M polymerase activity was observed between 4 and 8 hr and
NaCI, 1 mM EDTA, and 0.01 M sodium acetate (pH 5.1) thereafter, the degree of inhibition was never as great as that
and was centrifuged at 22,500 rpm for 16 hr in an SW 25.1 of rRNA methylases. The same dosage of poly(I). poly(C)
rotor. The gradient was then fractionated into l-ml frac
given to a normal rat did not cause an appreciable change of
tions. The RNA was precipitated with 5% TCA, filtered the activity of liver nucleolar enzymes.
onto Millipore filters (0.45 am), washed with 5% TCA,
The tumor nucleolar RNA formed in the presence of
and dried for the measurement of radioactivity.
poly(I).poly(C)
showed various degrees of degradation on
Analysis of Methylated
Nucleotides. The analysis of sucrose gradients. An example is shown in Chart 1. Upon
base-methylated
mononucleotides
of tRNA liberated by incubation of isolated nucleoli in vitro, both nonlabeled and
labeled RNA from in vivo poly(I).poly(C)-treated
tumor
alkaline hydrolysis was carried out as previously described
nucleoli underwent extensive degradation,
indicating that
(24).
RNA formed under the influence of poly(I).poly(C)
was
Assay of RNA, DNA, Protein, and Radioactivity. RNA
was determined by the orcinol reaction (14), DNA was more unstable than control RNA. The preferential inhibi
2342
CANCER RESEARCH
VOL.35
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@
T
A ntitumor
Efftcts
of Poly(J) . Poly (C)
Table 1
The efftct ofpoly(f) .poly(C) in vivo on the nucleolar RNA content, on activities ofnucleolar enzymes involved
in the synthesis of rRNA , and on activities of cell-soluble tRNA methylases
Rats (150 to 160 g/rat) bearing Novikoff ascites hepatoma on the 4th day (8 and 16 hr of exposure) or 5th
day (2 and 4 hr ofexposure)
were given I i.p. dose of poly(I).poly(C)
(5 to 6 mg/kg,
dissolved in 0.9% NaCI
solution) and were killed at the end of the indicated time periods. Poly(I). poly(C) was also given i.p. to a nor
mal rat (liver, 16 hr) at the same dosage. Nucleoli and ultracentrifugal cell extract (S,.@)were prepared from
control tumors cells and liver, and from poly(I) . poly(C)-treated tumor cells and liver. Control livers were from
normal rats. Nucleolar RNA content: Aliquots of nucleoli were taken for the determination
of RNA and
DNA content. Nucleolar enzyme activity: Aliquots of nucleoli were incubated in the complete RNA-synthesiz
ing medium (25) either with 0.2 mM [‘H]CTP or 2.2
@M
[‘HIS-Ado-Met for the assay of RNA polymerase
or rRNA methylase activities, respectively. The incubation was conducted at 30°for 15 mm. The nucleoli
were precipitated with cold 0.4 N PCA, neutralized, extracted with 2 M NaCI at 100°,and the nucleic acids
were chilled and reprecipitated with cold 0.4 N PCA. RNA was hydrolyzed in 0.3 N KOH at 37°for 18 hr,
and DNA was precipitated by acidifying to 0.4 N PCA for the separate determination of RNA, DNA, and
radioactivity.
The RNA polymerase
[‘H]CMP incorporated
into RNA
activity of control tumor nucleoli was in the range of 11.3 ±I .8 nmoles of
per mg DNA,
and the rRNA
methylase
activity
was in the range of0.27
0.006 nmole of methyl-'H incorporated into RNA per mg DNA. The corresponding
nucleoli were 2.26 ±0.045 and 0.043 ±0.005 nmoles, respectively.
±
values for liver control
Cell-soluble tRNA methylase activity: The assay of tRNA methylases was conducted as described
previously (26). The standard assay mixture in a volume of 0.25 ml contained 0.05 M Tris-chloride (pH 7.8),
0.04 M NH4F, 0.5 mM MgCl,, 1 msi dithiothreitol,
I mM ATP, 50 @gE. coli tRNA (sodium salt), 0.9 @tM
[‘HIS-Ado-Met,and an amount of S@,econtaining 85 to 100 @g
of protein. The tRNA methylase activity of
tumor control 522. was in the range of 39 to 45 pmoles of methyl-3H
incorporated
into tRNA per mg protein.
The data are the average of 2 to 3 determinations, where expressed as mean ±SE., and represent a single
determination
elsewhere.
Nucleolar
RNA contentNucleolar
tRNAmethylaseRNARNATime
(%to ofexposurepolymerasemethylaseactivity
poly(I).poly(C)RNA/DNA%
control)Tumor0
ofcontrol(%
enzyme activityCell-soluble
ofcontrol)(%
ofcontrol)of
0.05a1001001001002hr1.38±0.079496±3.585±3.4944hr1.24±0.088593±3.877±
hr. control1.46
±
1.78hr1.09±0.037559±1.535±0.5113l6hr0.60±0.054130±0.213±0.8123Liver0
0.05100100100l6hr0.68104104112a
hr. control0.65
±
Mean
±
SE.
so,
@
@
200
I
Cut .
@
:
::
.-. 100
@
i$s?ssass
100
_
I
tion
P.11I—C
115 205
;:
,.
455
0—@
200
PsIyI-C
of
nucleolar
rRNA
methylases
and
loss
of
nucleolar
RNA following the administration
of poly(I).poly(C)
in
vivo are consistent with the observation of Vaughan el al.
(41) that submethylated ribosomal precursor RNA is more
susceptible to degradation.
We have previously shown that poly(I) was capable of
inhibiting both nucleolar rRNA methylases and tRNA
methylases in vitro (24). In contrast to the progressive
decline of the activity of nucleolar rRNA methylases, the
activity of cell-soluble tRNA methylases, when assayed on
100
mCi of [3H]methionine for 3 hr. Nucleoli were prepared and divided into 2
equal parts. One part was suspended in 0.5 ml ofO.05 si Tris (pH 7.8), 0.25
M sucrose,
510520
IRA
Chart
RNA.
CII
0 0 S
I. The effect of poly(I).poly(C)
One rat bearing
Novikoff
ascites
on the stability
hepatoma
was given
of nucleolar
I i.p. dose of
poly(I).poly(C)
(5 mg/kg, dissolved in 0.9% NaCI solution) for 16 hr. The
poly(I).poly(C)-treated
rat and I control rat were then labeled with 0.3
SEPTEMBER
5 mM MgCl2,
0.04
@iNH4F,
and 0.9 mg ofbentonite
per ml, and
incubated at 30°for 15 mm. RNA was prepared from the control nucleoli
(a, nonincubated; b, incubated) and from nucleoli from the poly(I). poly(C)
treated rat (c, non-incubated;
d, incubated) by phenol deproteinization
(25) and resolved in 10 to 40% sucrose gradients containing 0.01 MTris (pH
7.4). 0. 1MLiCI. 0.01 MEDTA, and 0.2% Sarcosyl NL 97 by centrifugation
at 45,000 rpm for 4 hr in an SW 50 rotor.
1975
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2343
M. C. Liau et a!.
heterologous
Escherichia coli tRNA, was found to be
diminished only slightly at the early time point (2 hr). When
exposure time was extended to 8 hr or longer, the specific
activity of tRNA methylases was slightly enhanced. The
same results were also obtained when an assay of tRNA
methylases was conducted in the presence of a stimulatory
concentration of NH4C1 (0.2 M). An analogous observation
has been reported that the methylation of rRNA and tRNA
was noncoordinately inhibited when cells were infected with
foot-and-mouth disease virus (3, 40).
The Effect of Poly(I). PoIy(C) on the Synthesis of Macro
molecules in Tumor Cells and Host Liver. There was a
technical difficulty in using rat-carried tumor cells for the
measurement of the rate of synthesis of macromolecules.
The synthetic rate was affected by the age and concentration
of the tumor cells and the physiological condition of the rat,
such as the amount of ascites hemorrhage.
In general,
younger tumor cells, more concentrated tumor cells, and
less hemorrhagic conditions tended to permit a higher rate
for the synthesis of macromolecules.
To minimize nonuni
formity, rats carrying approximately
the same amount of
ascites fluid were selected among many transplanted rats for
the experiments, and labeling with radioactive precursors
was conducted on the 5th day after transplantation.
Data
were further adjusted to the control level of acid-soluble
radioactive intermediates.
The possible differences in the
uptake of radioactive precursors and their conversion to
acid-soluble radioactive intermediates were thereby greatly
reduced. There was, however, an apparently significant
(although difficult to quantitate) increase of the acid-soluble
radioactivity (both 32P and [methyl-3Hjmethionmne deriva
tives) stimulated by poly(I) . poly(C). The increase Was 20 to
25% for the 2-hr exposure and 6 to 18% for the 8 to 16-hr
exposure.
As shown in Table 2, the 3-hr incorporation of [32P]phos
phate into DNA in tumor cells was initially stimulated, and
then was inhibited after more than 8 hr of exposure to
poly(I).poly(C).
The stimulation of DNA synthesis is in
accord with the observation of Brown and Coffey (6) that
poly(I) was capable of stimulating
DNA synthesis in
isolated nuclei and soluble chromatin. The incorporation of
phosphate into RNA and of methionine into protein was
immediately and gradually inhibited to almost the same
extent. The effect of poly(I).poly(C)
on the methylation of
RNA was similar to that on RNA synthesis; however, the
inhibition of RNA methylation was greater than that of
RNA synthesis.
In contrast to the inhibitory effects of poly(I). poly(C) on
macromolecular
synthesis in tumor cells, Table 2 shows that
the synthesis of macromolecules in host liver in vivo was not
inhibited. In a separate experiment, when poly(I).poly(C)
was given i.p. to a normal rat, followed 16 hr later by
labeling of RNA with 0.2 mCi of [3H]orotic acid for 3 hr.
there was only 11% inhibition of liver RNA synthesis in the
treated rat. These results, considered together with the
differential effect of poly(I) . poly(C) on the activities of
tumor and liver nucleolar enzymes (Table 1), suggest that
the effect of poly(I) . poly(C) is relatively specific for these
tumor cells in vivo. In this in vivo system, it was not possible
to make direct comparisons of effects on liver and ascites
tumor, because the same accessibility ofthe drug to the liver
is not assured. However, it has previously been documented
in other systems that poly(I) . poly(C) exerts a greater effect
on tumor cells and rapidly proliferating cells than on normal
resting cells (1, 15, 18, 20), and in a later section we present
results ofexperimentation
in vitro in which these physiologi
cal factors were avoided. Evidently, the relatively specific
antitumor effect of poly( I) . poly(C) could not be attributed
to selective uptake of this compound into the tumor (20).
The inhibition of protein synthesis by poly(l).poly(C)
correlates with the effect of poly(I). poly(C) on the polyso
mal profiles, as shown in Chart 2A. In these experiments,
Novikoff ascites cells were vigorously homogenized in order
to obtain clean nuclei. As a result, polysomes were not well
preserved. Nevertheless, the amount of polysomes as mea
sured by UV absorption was noticeably diminished, and the
incorporation
of 32P into the polysomal
fraction was
markedly decreased in response to the treatment of the rat
with poly(I).poly(C).
The effect was more prominent in
those cells exposed to poly(I).poly(C)
for longer periods of
Table 2
The effect ofpoly(I).poly(C)
on the synthesis ofmacromolecules in tumor cells and host liver
Rats bearing Novikoff ascites hepatoma were given poly(I).poly(C)
as described in Table I. After the indicated periods of exposure, 50 zCi of
[‘2P]phosphate or 0.3 mCi of [methyl-'H]methionine,
together with I @zmoleof adenosine and guanosine, were injected i.p. The rats were sacrificed 3 hr
later, and the tumor cells and liver were removed for the analyses of macromolecular
synthesis detailed in “Materials and Methods.― The data represent
the average of 2 or 3 separate determinations.
All of the data have been calculated to represent the same amount of cold 0.4 N PCA-soluble radioactivity.
In the case of RNA methylation, no correction was made for the possible incorporation of radioactivity into normal purine bases. Previously, we have
shown that, under the present labeling conditions, only 5% of the labeled cytoplasmic rRNA was due to the labeling of normal purine bases.
withDNA
activity (cpm/mg
([methvl-3H]methionine)Tumor
synthesis (32P)RNA
Time ofexposure
to poly(I) . poly(C)
LiverTumor
before LiverControl
labeling (hr)Specific
synthesis (32P)RNA
16'13,350
I2l±5.8b
33±2.4
22±2.2
103±8.4
110±8.9
96±4.545,000
methylation
(methyl-3H)Protein
LiverTumor
590
2@
8'
DNA, RNA, or protein)
synthesis
LiverTumor
93±4.3
45±3.3
12,000
118±5.9
97±6.8
86±1.7
37±2.1
97±6.3
127±8.8
790
97±3.1
45±2.9
100±5.9
103±5.5
32±2.0
108±4.13,900
24± 1.9
104±4.29,900
34±2.2
118 ±7.4
1,380
a The data are expressed as % of control activity.
b Mean
2344
± SE.
CANCER RESEARCH
VOL.35
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@
@
I
U
A ntitumor
Effects of Po/v(J) . Polv (C)
small subunits and free messenger ribonucleoprotein
sedi
ment (30, 34), was also diminished.
RNA's prepared from this 520 fraction also revealed
considerably different profiles among control and poly(I).
poly(C)-treated
specimens, as shown in Chart 2B. The
labeling of rRNA was more greatly inhibited relative to the
a
I
labeling of 4 to 6 5 RNA, and the labeling of 18 5 rRNA
was more greatly inhibited relative to the labeling of 28 S
rRNA.
In addition, the radioactive
peak sedimenting
U
U
between
was
FRACT
185
8 hr
Control
B
300
The
U 28$
150
1OSj
500
in
‘.
p
:
I
f
a
‘
,
@
U
,
.
.
16 hr
2 hr
.4
@
0
-.
5
10
“
100
\ i\
a:
.4
:
...,.
.
15
200
5
10
.‘
.
15
20
F R A C T I ON S
Chart 2. The effect of poly(l).poly(C)
on the profiles of cytoplasmic
ribonucleoprotein
and RNA. The rats were treated with poly(I).poly(C)
and labeled with [32P]phosphate, according to the protocol described in
Table
2. Postmitochondrial
extracts
(S,@) were prepared.
A, For the study
of ribonucleoprotein
profiles, 3-mi aliquots were layered onto a 24-ml I5 to
30% (w/w) sucrose gradient containing 0.02 M Tris-chloride (pH 7.8)-2 mM
MgCI3-0.05 M KCI, on top of 3 ml of 50% sucrose solution as cushion, and
centrifuged at 15,000 rpm for 16 hr in an SW 25.1 rotor. The gradients
were fractionated
on an ISCO automatic
fractionator,
collecting
I
ml/fraction.
Radioactivity in each fraction was determined by filtration
onto a Millipore filter (0.45 sm). The filter was well washed with cold 5 mM
MgCl,,
dried, and counted
in a toluene
phosphor
medium.
B, For the study
of RNA profiles, RNA was prepared from the S,0 fraction, layered onto a
28-mI 10 to 40% (w/w) sucrose gradient in 0.1 M NaCl-l msi EDTA-O.Ol M
sodium acetate (pH 5.1) and centrifuged at 22,500 rpm for 16 hr in an SW
25.1 rotor. RNA in the 1st 20 fractions of each gradient was precipitated
with 5% TCA in the presence ofO. I mg albumin per tube and collected onto
a Millipore filter for the determination
of radioactivity.
time. Responding in parallel to the effect of poly(I).poly(C)
to disaggregate polysomes, the appearance of radioactive
ribonucleoprotein
in the 45 S peak of the cytoplasm, where
SEPTEMBER
characteristic
ofcytoplasmic
extracted
from
mRNA
cells
exposed
to
Effect
of
Poly(I).
Poly(C)
on
the
Intracellular
Move
-
tumor
cells
but
also
abberrations
in
movement
of
radioactive RNA in the cytoplasm and the relative amount
of
radioactive
ifl
response
protein
to
in
increasing
the
nucleus
exposure
progressively
decreased
time.
The Effect of Poly(I) . PoIy(C) Administered in Vivo on
the Base Methylation of tRNA. One of the remarkable
!@:i@
‘
200
/‘
?@
@
@
S
RNA
@“I@oppt'
)
1000
500
18
in
macromolecules
from the site of synthesis to their destina
tion. As shown in Tables 3 and 4, the relative amount of
450••_'
\@
@
and
ment of Macromolecules in Tumor Cells. Poly(I). poly(C)
caused not only the inhibition of macromolecular
synthesis
r@
@
5
poly(I). poly(C) for 8 hr or longer. It appears that poly(I).
poly(C) has preferential inhibitory effect toward the pro
duction of ribosomes, and more selectively on the produc
tion of small subunits. Labeling of RNA with [methyl
3H]methionine produced a similar trend (Table 3), namely,
that a diminished formation of methylated cytoplasmic
rRNA is observed after 2 hr of treatment by poly(I).
poly(C). Selective inhibition of the methylation of small
subunits was apparent only during early intervals (2 to 4 hr@
of exposure.
I ONS
1001
4
diminished
features
of
poly(I)
as
tRNA
methylase
inhibitor
is
its
selective inhibition of adenine methylase when directly
included in the in vitro enzyme assay (24). The analysis of
tRNA methylated
with [methyl-3H]methionine
in vivo
(Table 5) indicates that poly(I).poly(C)
caused a small but
noticeable preferential
inhibition of the methylation
of
tRNA adenine during early time intervals of exposure to
poly(I) . poly(C), although this inhibition of the methylation
of tRNA adenine was no longer detectable when cells were
exposed to poly(I)-poly(C)
for 8 hr or longer. An assay of
cell-soluble tRNA methylases, prepared from tumor cells
treated in vivo with poly(I). poly(C), on heterologous E. coli
tRNA (Table 1) also revealed similar results. Preferential
inhibition of the adenine methylase was detectable only in
enzyme extracts prepared from tumor cells treated for a
short time (2 hr), but not for a longer time (10 hr).
Different Responses of Tumor and Liver Nucleolar rRNA
Methylases toward Compounds Affecting Their Activities.
The results shown in Tables 1 and 2 indicate that poly(I).
poly(C) in vivo exerted its inhibitory effect selectively on
tumor cells. We regard this selective effect to be a very
important feature of this type of antitumor agent. The
problem is to determine what causes this kind of selective
action. Since tumor rRNA methylases are apparently one
of the primary targets affected by the administration
of
poly(I). poly(C) in vivo, we therefore carried out a study to
determine whether there was any difference in the behavior
of enzymes in nucleoli from tumor or liver. The results
1975
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1975 American Association for Cancer Research.
2345
M. C. Liau et al.
Table 3
The efftct ofpoIv(J).po1@v(C) on the methylation ofRNA in tumor cells
Rats bearing Novikoff ascites hepatoma were given poly(I)- poly(C) and were subsequently labeled with [methyl-'Hjmethionine,
as described in Tables
1 and 2. Tumor cells were fractionated into nuclear and cytoplasmic fractions as described in “Materialsand Methods.― RNA's were purified by
deproteinization
with phenol and precipitation
with ethanol; tRNA was separated from rRNA, in the case of cytoplasmic RNA preparation,
by
precipitation of rRNA in 2 M NaC1 (25); tRNA was recovered from 2 M NaCI supernatant by ethanol precipitation and was further incubated in 0.5 so
Tris-chloride (pH 8.8) at 37°for I hr. The determination
of specific activity of methylated RNA and the correction based on the same amount of
acid-soluble radioactivity were as described in Table 2. The data represent the average of 2 or 3 separate determinations
expressed as mean ±SE.
cytoplasmicrRNA%
tRNAMethylated
Time
ofexposureTotal
totalto
poly(I).(C)methylation
controlbefore
SControll0@Y'58
labeling (hr)control
RNAMethylated cytoplasmicMethylated
as % ofRNA
as % oftotal%
ofcontrolmethylated%
activitymethylated
RNAactivitycytoplasmic
0.01286±
±1.8lO0@47
±0.08445±
1.753
±2.578
±0.05837±2.142±2.237±1.363±1.620±3.21.05±0.02
1.950±0.944±3.054±
a 3,9(J@
b I 1,000
C 1,520
cpm/mg
cpm/mg
cpm/mg
The Effect ofpoly(f)
±4.852
cytoplasmic
of
of
RNAactivity18
±[email protected]
±2.764
±3.30.73
1.632±4.60.89
5/28
±
RNA.
RNA.
RNA.
Table 4
.p@1y(@)on the transport ofmacromolecules
cells
in tumor
The experiments were the same as described in Table 2. However, the
tumor cells were fractionated into cytoplasmic and nuclear fractions, as
described in “Materials and Methods.― RNA's were prepared from both
fractions by the phenol procedure and assayed as described in “Materials
and Methods.― The assay of [methyl-3H]methionine-Iabeled
protein
among cytoplasmic and nuclear fractions was as described in “Materials
and Methods.―
Time ofexposure
to poly(I).poly(C)
[32P]RNA
labeled protein as % of
beforeprotein0,control54732517784380163783
labeling (hr)Cytoplasmic
as % of total [32P]RNA[methyl-'Hjmethionine
total ‘H
(Table 6) show that many compounds that are inhibitory
against tumor nucleolar rRNA methylases were without
inhibitory effects against liver nucleolar rRNA methylases.
interferon has been demonstrated
in cultured cells (5). In
this case, a prolonged contact for at least 18 hr with
interferon was required to produce a significant inhibition of
the synthesis of protein and RNA, while the formation of
ribosomal subunits was hardly affected. Thus, the action of
poly(I).poly(C)
on Novikoff ascites hepatoma is distinctly
different from the action of interferon on cultured cells.
Inconsistent correlation between the levels of the interferon
induced and the antitumor activities of the inducers (4, 35,
43) also suggests that different mechanisms are perhaps
involved in the actions of the interferon and the interferon
inducers possessing antitumor activity.
The immediate effects detectable within a few hr of
exposure of tumor cells to poly(I).poly(C)
correspond
remarkably well to the biological effects produced in vitro
by poly(I). These are the inhibition of RNA methylation,
the selective inhibition of adenine methylase of tRNA, and
the stimulation of DNA synthesis (6, 25). The idea that
poly(I) is probably the active strand of poly(I) . poly(C) has
been suggested on the basis of uptake of poly(I) and the
degradation
of poly(C) by the tumor cells (36) and the
On the contrary, there were various degrees of stimulation, critical requirement for size of poly(I) but not poly(C) for
possibly resulting from inhibition of RNase activity. l'he the demonstration
of antitumor and antiviral activities as
sensitivity of tumor enzymes and the insensitivity of liver well as interferon induction (9, 32, 39). The requirement for
enzymes toward inhibitory compounds may be the main complex formation with poly(C) for the demonstration of its
reason behind the potentially selective inhibitory effect of biological activities in vivo may indicate that poly(C)
rRNA methylase inhibitors on tumor cells. The desirability
facilitates the uptake of active poly(I) or its interaction with
of using rRNA methylase inhibitors as antitumor agents is a receptor site on or within the cell, or that the complex
indicated.
confers resistance to degradation by nucleases (31).
In procaryotic and eucaryotic microorganisms,
the rate
DISCUSSION
of synthesis of rRNA and the number of ribosomes/genome
are proportional to the rate of exponential growth (28, 33),
Several factors may be involved in the antitumor effects
and these characteristics
are maintained by several regula
of poly(I). poly(C). Considering the inhibitory effect of
tory processes, including methylation of RNA. In mamma
poly(I). poly(C) against solid tumors (20, 21), against
tumors in immunosuppressed
animals (4, 11), and against
rapidly proliferating cells in tissues ( I, 15, 37), a direct effect
of poly(I). poly(C) or an effect mediated through interferon
is likely. A direct inhibition of cell multiplication
by
han cells, the ability of cells to replicate is specifically
correlated to the ability of cells to provide needed ribosomes
(33, 38). The formation of ribosomes is greatly affected by
the methylation processes. It has been demonstrated
that
the 45 S pre-rRNA synthesized without methylation under
2346
CANCER RESEARCH
VOL.35
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A ntitumor
Efftcts
of Poly(I) . Poly (C)
Table 5
The effect ofpoly(I).polv(C)
on the pattern of base methylation of tRNA
Rats bearing Novikoff ascites hepatoma were treated with a single dose of 5 to 6 mg of
poly(I). p@@ly(C)per kg for the indicated time intervals. Rats either were labeled with [methyl.
3H]methionine
for 3 hr. as described in Table 2, or were sacrificed for the preparation
of
ultracentrifuged
cell-soluble extracts (S,,@) as sources of tRNA methylases. In viva-labeled tRNA
was prepared from cytoplasmic fraction, as described in Table 3. The purified tRNA was hydro
lyzed in 0.3 M KOH at 37°for 18 hr. and the mononucleotides
liberated were separated by paper
electrophoresis
(26). For the correction of radioactivity incorporated
into normal purine bases,
radioactivity
associated with adenine and guanine nucleotides was recovered from paper by
extraction with 0. 1 N HCI. The extracts were dried in a vacuum desiccator. Purine bases were
liberated from nucleotides by hydrolysis in I N HCI at 100°for 1 hr. Adenine and methylated
adenine were separated by descending chromatography
in butanol/concentrated
NH4OH (86/14)
for 18 hr. and guanine and methylated guanines were separated by descending chromatography
in
2-propanol/12
N HCI/H30 (68/17.6/14.4)
for 90 hr. Under the present labeling conditions, 6 to
I 1% of the radioactivity in the adenine nucleotide and 4 to 8% of the radioactivity in the guanine
nucleotide were found to be due to purine ring synthesis, which values were corrected for in the data
presented. The incubation of S,21 extracts with E. coli tRNA and analysis of methylated bases were
conducted as previously described (24, 26).
Distribution
extractMethylated
tRNAmethylated
of label as % of total
methylatedin vitro
coli tR
bNA
y 5266
in vivoE.
poly(C)ControlPoly(I).poly(C)2
basesControlPoly(l).
hrC2224
15.2A1612
20.6G5659
63.5U66
hr
8 hr2
10
2117.217.5
1619.114.7
5964.267.2
40.50.6
goes total degradation (41). We believe that the disruption
of the formation of ribosomes through initial inhibition of
the methylation of ribosomal precursor RNA plays one of
the key roles in the antitumor effect of poly(I).poly(C)
in
the case of Novikoff ascites hepatoma, but we certainly do
not seek to discount antitumor effects mediated by other
factors such as interferon, activated macrophages,
and
host-mediated immune mechanism.
The inhibitory effect of poly(I).poly(C)
on tumor cells
and rapidly proliferating cells, in comparison with lesser
effects on resting cells, may be a very important feature of
the action of poly(I) . poly(C). There is reason to believe that
the interactions between poly(I).poly(C)
and surface mem
brane of various cells are different. The induction of
interferon was shown to be specifically antagonized
by
concanavalin
A (13), indicating that both compete for
common receptor sites. It has been amply demonstrated
that concanavalin A has a tendency to agglutinate tumor
and transformed
cells better than their normal parental
cells, and that the agglutinability of normal cells is greatly
enhanced during mitosis (7). Thus, the sensitivity of tumor
and mitotic cells toward poly(I).poly(C)
may be partly
related to the characteristics
of their cell surfaces. Our
demonstration
that liver rRNA methylases responded dif
ferently toward many compounds
inhibitory to tumor
enzymes seems to provide a rationale whereby rRNA
methylase inhibitors may exert a selective inhibitory effect
on tumor cells. The basis for the different response of tumor
and liver enzymes toward these inhibitory compounds is
currently under investigation. Since we have found that
SEPTEMBER
hr
0.7
Table 6
Different responses oftumor and liver nucleolar rRNA methylases toward
compounds affecting their activities in vitro
Nucleoli were prepared from Novikoff ascites hepatoma cells or host
livers as described in “
Materials and Methods.― The standard assay
mixture in a volume of 0.25 ml contained: 0.05 M Tris-chloride (pH 7.8),
0.25 M sucrose, I mM MgCl2, 0.2 mM EDTA, 0.04 M NH4F, nucleoli
containing approximately
50 zg of DNA. and 1.24 MM [‘H]S-Ado-Met
without (control) or with compounds, as listed. The inc@ibation and assay
were conducted as described in Table I. The tumor nucleolar rRNA
methylase activity assayed under these conditions was in the range of 85 to
I 13 pmoles of methyl-3H incorporated into RNA per mg DNA, and the
liver nucleolar rRNA methylase activity was in the range of 22 to 30
pmoles of methyl-3H incorporated into RNA per mg DNA. The data are
the average of 2 to 4 determinations.
activityTumorLiverPoly(l),2@M'
ofcontrol
Compounds%
Polyguanylate, 2 @M
ApA,'2mM
2'-O-methylated
dinucleotides,
cAMP,2mM
2 mM
AMP,4mM57±3.0'@
a Concentration
S Mean
(
ApA,
±
is based on minimum
67 ±3.9
51 ±1.6
55 ±3.8
62±3.2
65±2.5139±4.5
molecular
144 ±4.0
109± 1.6
105 ±4.9
118±5.1
113 ±4.7
weight.
SE.
adenosyl(3',S'-phospho)adenosine.
most mono-, di-, and oligonucleotides
show inhibitory
activity against tumor rRNA methylases in isolated nucleoli
(23), we postulate that excessive RNA degradation resulting
from the initial inhibiton of rRNA methylases is probably
1975
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1975 American Association for Cancer Research.
2347
M. C. Liau et al.
most responsible for the observed inhibition of macromolec
ular synthesis occurring at later time intervals. The inhibi
tion of macromolecular
synthesis is more prominent at a
time when the continued existence of poly(I) in its original
inhibitory form becomes doubtful.
In addition to the
likelihood that poly(I) is degraded
intracellularly,
the
specific biological effects manifested by poly(I), such as the
inhibition of tRNA adenine methylation and stimulation of
DNA synthesis, could no longer be demonstrated
at the
longer time intervals. The argument follows then that an
initial inhibition of rRNA methylases is sufficient to trigger
the regulatory mechanism of ribosome production operat
ing under normal growth.
The inhibition of tumor rRNA methylases by cAMP
(Table 6) is of particular interest, since it has been proposed
by Webb et al. (42) that the antitumor effect of synthetic
polynucleotides may be mediated by their stimulation of the
formation of cAMP.
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2349
Preferential Inhibition by Homopolyribonucleotides of the
Methylation of Ribosomal Ribonucleic Acid and Disruption of
the Production of Ribosomes in a Rat Tumor
Ming C. Liau, Don W. Smith and Robert B. Hurlbert
Cancer Res 1975;35:2340-2349.
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