(CANCER RESEARCH 33, 2965 2971, November 1973]
Differences in RNA Formation and Polyribosome Metabolism in
Serum-starved Normal and Transformed Cells
Michihiko Kuwano, Hideya Endo, and Yukio Ikehera
Cancer Research Institute, Faculty of Medicine [M. K., H. £.],and Department of Physiological Chemistry, Faculty of Pharmacuetical Sciences ( Y. /.],
Kyushu University, Fukuoka 812, Japan
SUMMARY
Comparative studies of RNA synthesis and polyribosome
metabolism in normal (RFL) and transformed (RFL-T)
cells under serum starvation show that their growth is more
or less dependent upon serum factor. By limiting serum in
the medium, 80 to 90% of RNA or protein synthesis is
inhibited in RFL cells, while in RFL-T cells macromolecular synthesis proceeds at one-third to one-fourth the rate of
control. Ribosomal RNA synthesis is more preferentially
blocked in untransformed cells under decreased protein
synthesis caused by serum deprivation than in transformed
cells. However, when both RFL and RLF-T cell lines are
treated with cycloheximide, 2 /xg/ml, which inhibits over
90% protein synthesis, little if any ribosomal RNA is
produced. Also, analysis of cytoplasmic extracts with
isokinetic sucrose density gradient centrifugation shows
the greater transition of polyribosomes into monosomes in
RFL-T cells compared to that of RFL cells with time of
serum deprivation. The prominent redistribution of polyri
bosomes, which occurred in serum-starved RFL-T cells but
not in RFL cells, is restored by addition of 0.2 /ig/ml to
2.0 /ig/ml cycloheximide. Since nascent peptides are be
ing made in polyribosomes of RFL-T starved for serum
but little if any peptide chains are made in RFL, the ribo
somal transition requires peptide formation at a reduced
level.
INTRODUCTION
In bacterial cells, especially in Escherichia coli, RNA
regulation has been shown to be controlled differently
—¿"relaxedand "stringent" strains since the proposal by
Stent and Brenner (28). Recent detailed studies provide
additional data that rRNA synthesis was specifically de
pressed in the stringent strain (15, 20), process that could be
mediated by the presence of a tetraphosphate derivative of
guanine (2).
On the other hand, in mammalian cells, RNA regulation
must be complex since this problem must be considered with
respect to the correlative reaction of nucleus and cytoplasm
(4, 25). For growing animal cells, serum is an indispensable
growth factor (10, 29) and serum requirement has been
considered to be one of important parameters differentiat
Received April 2, 1973; accepted August 7, 1973.
ing untransformed cells from transformed cells (3, 5). In
various cultured cells, serum or amino acid deprivation
diminished bulk RNA formation or induction of enzyme
possibly at the translation level (6, 7, 17). Growth limitation
by lack of serum caused a greater coordinate reduction in
various macromolecular metabolisms such as DNA,
RNA, and protein synthesis in untransformed cells than it
did in transformed cells, which were interpreted in relation
to pleiotypic response (9).
Comparative studies on macromolecular metabolisms
proceeding in transformed and untransformed cells during
serum starvation might help us to understand the growth
control as well as RNA-regulatory mechanisms. This study
reports physiological studies of normal fibroblast and its
counterpart transformed with oncogenic virus under the
serum starvation, in which RNA species that were formed
and polyribosomal metabolism were analyzed.
MATERIALS
AND METHODS
Cells. Normal RFL cells derived from rat fibroblast and
its transformed derivative, RFL-T (12, 13), inducing sar
coma in Wistar-Furth rats (donated to us by Dr. M. Kohga)
were cultured in a glass Petri dish (6 cm in diameter) at 37°
in 3 ml Eagle's essential medium (Nissui Seiyaku Co.,
Tokyo, Japan), containing 10% calf serum unless otherwise
indicated. RFL shows contact-inhibited growth, and RFL-T
forms characteristic multilayered cultures.
RNA and Protein Synthesis. RFL or RLF-T, 4 to 5 x 10"
cells/ml, were exposed to leucine-3H, 2 ^Ci/ml and uridine-14C, 0.05 /iCi/ml, for 120 min as indicated in Chart
3; and RNA and protein synthesis were measured by countthe TC A'-insoluble fraction as described previously (12,
13). To see RNA formation as shown in Charts 1 and 4,
cells were labeled for 120 min with uridine-14C, 0.5 ¿iCi/ml,
under various conditions.
Chemicals and Isotopes. Actinomycin D (Merck Sharp
and Dohme, Rahway, N. J.), cycloheximide (Ishizu Phar
maceutical Co., Osaka, Japan), and Triton X-100 (Wako
Pure Chemical Industries, Osaka, Japan) were used as
chemical agents. Uridine-3H, 5 Ci/mmole, and leucine-3H,
32 Ci/mmole, from Daiichi Pure Chemical Co., Tokyo,
Japan, and undine-14C 50 /iCi/230 ^g, from New Eng
land Nuclear, Boston, Mass., are used as isotopes.
1The abbreviation used is: TCA, trichloroacetic acid.
NOVEMBER 1973
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2965
Michihiko Kuwano, Hideya Endo, and Yukio Ikehara
Preparation of Cell Lysate and Polyribosomes. After
labeling, cells were released from the glass of the Petri
dishes with a rubber scraper and collected by centrifugation
at 1500 x g for 5 min. After being washed once with
phosphate-buffered saline solution, the cell pellets were
suspended in 0.4 ml of 50 mM Tris/100 mM KC1/5 mM
MgCl2, pH 7.4, containing 0.5% Triton X-100 and incu
bated at 2—4°
for 10 min with agitation. With this method
almost 100% of cells were lysed, but cell nuclei remained
intact under microscopic observation. Cell lysates were
centrifuged at 2000 x g for IOmin, and the supernatant was
layered onto an isokinetic density gradient of 11 ml of 10 to
40% sucrose (w/v) in 50 mMTris/25 mM KC1/5 mM MgCl2,
pH 7.4 (8, 24). The gradients were spun at 39,000 rpm in a
Spinco SW41 rotor for 70 min with the brake on. The
bottoms of the tubes were puncutured and 0.4-ml fractions
were collected by a Beckman fraction recovery system.
Each fraction was precipitated with 10% TCA, plated on
glass filter paper (Whatman, England), and washed twice
with 5% TCA and 0.1 N HC1, respectively. The filters were
dried and counted in 5 ml of toluene-based scintillation
liquid with a Beckman scintillation spectrometer.
Nascent Peptide Formation. In order to show the forma
tion of nascent peptide chains on polyribosomes, cells were
incubated with leucine-3H, 20 ^Ci/ml, for 10 min at 37°.
Polyribosomes of cell lysates were prepared as described
above and 0.5-ml fractions of the gradients were analyzed
for TCA-precipitable radioactivity. To release the labeled
nascent polypeptides from polyribosomes, the lysate super
natant was treated with puromycin at 10 mM final concen
tration in the presence of 0.5 M KC1 and incubated at 2-4°
for 15 min (l). Under our conditions, about 70% nascent
peptides could be released from polyribosomal regions by
puromycin treatment. The polyribosomal patterns were
obtained as performed above, and then radioactivities
responsible for nascent peptides in each fraction were
calculated by subtracting the radioactivity of puromycintreated polyribosomes from that of the untreated.
while RFL-T cells still grew at a reduced rate.
Results of RNA and protein synthesis comparable with
that of growth curve were obtained, when both cell lines
were examined by incorporation of luecine-3H and uridineMCinto acid-insoluble fraction in the absence or presence of
serum in the medium. As shown in Chart 1, formation of
both RNA and protein was sharply curtailed by starvation
for serum of the control cell, while the transformed cell
continued to produce RNA and protein at a reduced rate.
These results indicate that RFL and RFL-T cell lines
correspond to normal and transformed cells based on the
reported criterion of serum requirement (3, 5).
Comparison of Rate of RNA Synthesis and Inhibition of
rRNA Formation. Recently, contact-inhibited cells were
found to produce little rRNA that was recovered by
addition of fresh serum (6). Valine deprivation from HeLa
cell culture also suppresses 45 S rRNA formation (17). To
examine RNA formation during serum deprivation in RFL
and RFL-T cell lines under serum deprivation, cells prelabeled with undine-14C for 18 hr were exposed to uridine-3H
for 60 min with actinomycin D, 0.03 ¿¿g/ml,
or 120 min after
serum starvation for various periods. With a low level of
actinomycin D, 0.02 to 0.04 jig/ml, which specifically
suppresses rRNA formation (23), 50 to 60% RNA labeled
for 120 min in growing cells was inhibited. Calculated ratio
(3H/14C) paralleled the time for serum deprivation espe
cially when normal cells were labeled with uridine-3H for
120 min (Chart 2). A constant rate of actinomycin D-insen(a)
(b)
15.000
7.500
1QOOO
5000
CL
RESULTS
Serum Requirements for Cellular Growth, RNA, and
Protein Synthesis. In our layer-cultured fibroblastic cell
lines, untransformed (RFL) and transformed (RFL-T),
total cellular RNA synthesis was markedly affected by
suppressed protein synthesis in normal cells compared to
transformed cells (12). In mammalian cells, serum has been
shown to be an important factor for cellular growth as well
as in discriminating between transformed and untrans
formed cells (3, 5). We investigated the physiological
responses of normal RFL and transformed RFL-T to serum
deprivation. Growth of cultured cells was usually inhibited
in both cell lines by serum starvation, although the cells
grew exponentially in the medium containing 10% calf
serum. RFL cells ceased to grow by contact inhibition after
3 days, while transformed RFL-T cells continued exponen
tial growth for 4 days. However, serum-starved RFL cells
stopped growing almost completely after the starvation
2966
2500
5.000
1
1
Time ( day )
Chart 1. RNA and protein synthesis of RFL and RFL-T. From 2 to 3 x
IO5 cells/ml were inoculated into a Petri dish containing 3 ml minimal
essential medium plus 10% serum, and the next day 1 part of the plates in
serum-free medium continued incubation with uridine-14C, 0.01 ¿iCi/ml,
and leucine-3H, 0.2 /jCi/ml. At the indicated time after serum starvation (0
time in chart), 1 Petri dish carrying layer-cultured cells was removed and
their 10%TCA-insoluble fractions were counted as described (12). a, RFL;
b RFL-T. Incorporation of leucine-'H (O, •¿)
and uridine-"C (A, A) into
acid-insoluble fraction in absence (•,A) or presence (O, A) of serum.
CANCER RESEARCH VOL. 33
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RNA Synthesis and Polyribosomes
sitive RNA (mRNA) formation proceeds in both cell lines
during serum starvation.
Then RNA synthesized in untransformed RFL and
transformed RFL-T cells with or without serum starvation
for 6 hr was analyzed against various doses of this
antibiotic. As shown in Chart 3, 50 to 70% of RNA labeled
for 120 min with uridine-14C in exponentially growing RFL
and RFL-T cells under the subconfluent growth condition
was inhibited for their synthesis by the presence of ac
tinomycin D, 0.02 to 0.04 /ig/ml. Within a drug concentra
tion ranging from 0.02 to 0.04 /ig/ml inhibited RNA is
considered to be rRNA. Therefore, Chart 3 indicates
greater suppression of rRNA synthesis in untransformed
cells than that in transformed RFL-T cells.
Thus, the observed preferential control of rRNA forma
tion might be limited by a difference in the growth rate of
the cells or by a difference in the level of cellular protein
synthesis or stability of responsible protein between 2 cell
lines. To examine these possibilities, we analyzed RNA
synthesized under conditions of depressed protein synthesis
by using cycloheximide, an inhibitor of protein synthesis. At
a low dose of cycloheximide, 20 to 30% RNA synthesis was
blocked. However, with cycloheximide, 1 ng/m\, to inhibit
over 90% protein synthesis, RNA synthesis fell to 50% of
control in both cell lines. Therefore, RNA's synthesized
under inhibited protein synthesis by cycloheximide, 0.2 or
2.0 ¿¿g/ml,
were again analyzed for their sensitivity to a low
(a)
(b)
8
0.08 0
Time ( hr )
Chart 2. Effect of serum starvation on ratio of RNA synthesis in RFL
and RFL-T. From 4 to 5 x 10s cells/ml in 3 ml culture medium in Petri
dishes of RFL (O, •¿)
and RFL-T (A, A) prelabeled with uridine-"C, 0.01
¿iCi/mlfor 18 hr were deprived of serum for various times (0, 2, 4, 6, and
8 hr) and then exposed to uridine-3H, 0.2 /iCi/ml, in RFL-T and to 0.4
/iCi/ml in RFL. 3H labeling continued for 60 min in the presence of
actinomycin D, 0.03 jig/ml (•,A), and also for 120 min (O, A, respec
tively. 14Ccpm is usually 4000 to 6000 and 3H/14C was calculated after
counting acid-insoluble fraction.
NOVEMBER
Actinomycin
0.04
0.08
D ( pg/ml )
Chart. 3. RNA species formed during serum starvation in RFL and
RFL-T cells. Exponentially growing RLF (a) and RFL-T (b), 4 to 5 x 10*
cells/ml each, cultured in 3 ml minimal essential medium •¿
10%calf serum
in a Retri dish were serum starved for 6 hr, followed by labeling with
uridine-"C, 0.5 /iCi/ml (50 /iCi/230 //g), for 120min in presence of various
concentrations of actinomycin D. TCA-insoluble fractions (10%) of cells
treated without (O) or with (•)serum starvation were counted on glass
filter paper.
1973
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2967
Michihiko Kuwano, Hideya Endo, and Yukio Ikehara
dose of actinomycin D. Cells from RFL and RFL-T
pretreated for 5 hr with cycloheximide were examined for
their RNA species labeled for 120 min (Chart 4). Ac
tinomycin D-insensitive RNA species seemed to increase
relatively in both cell lines, RFL (Chart 4a) and RFL-T
(Chart 46), which suggested a decrease of net rRNA
synthesis in cycloheximide treatment. Unlike the effect of
serum starvation, cycloheximide seemed to abolish rRNA
formation equally in both RFL and RFL-T cell lines (Chart
3). Chart 4 indicates rates of mRNA synthesis in cycloheximide-treated cells. Under serum deprivation in normal cell
culture, formation of rRNA but not of mRNA fell, which
suggests a differential control of RNA synthesis.
Alterations of Polyribosomal Pattern by Serum Depriva
tion and the Restoration by Cycloheximide. We examined
how RNA formed in the nucleus is utilized in cytoplasm
under serum deprivation, as well as serum effects upon
polyribosomal cycles. Recently diminishing polyribosomal
size caused by lack of serum was reported (7, 11), and we
obtained polyribosomes from 0.5% Triton X-100-treated
cytoplasmic extracts by sucrose density gradient centrifugation when cells prelabeled for 18 hr with uridine-MC were
starved for serum for 4 hr, followed by labeling for another
2 hr with uridine-3H. From a comparison of the polyriboso
mal pattern of RFL with that of RFL-T, a characteristic
change of profiles was found in RFL-T different from those
of RFL (Chart 5). Usually in exponentially growing cells,
monosomes were not prominent in polyribosomal profiles.
However, during serum starvation, monosomes started to
increase, with a reciprocal decrease of polyribosomes
(larger than monosomes) as observed in Chart 5, c and d.
Chart 6 presents percentage of polyribosomes, monosomes,
and free ribosomal subunits against total ribosomes summa
rized from cpm of 14C of 3 independent trials in each cell
line. About one-half of the polyribosomes (larger than
monosomes) transited into monosomes in transformed cells
with time for serum starvation, while only slight altered
redistribution of polyribosomes occurred in normal RFL
(Chart 6, a and b). In both cell lines, little or no change of
the subunit pool was observed, as shown in Chart 6c. We
also tried longer starvation of serum (18 hr) in transformed
RFL-T cells, and similar ribosome distribution as in the
case of 7-hr starvation was observed. However, compared to
shorter starvation, smaller amounts of mRNA entered
polyribosomes after prolonged deprivation of serum (Y.
Ikehara and M. Kuwano, unpublished data).
Accumulation of monosomes seemed to be characteristic
in serum-deprived transformed cells, while protein synthesis
still continued at a measurable rate (Chart 1). To test
whether protein synthesis is a requisite for changes in
ribosome distribution, an inhibitor of protein synthesis,
(b)
(a)
10
0
0.04
0.080
Actinomycin
0.04
0.08
D (pg/ml)
Chart 4. RNA species formed during presence of cycloheximide in RFL
and RFL-T cells. Growing RFL (a) and RFL-T (b) cells, 4 to 5 x 10!
cells/ml, pretreated without (A) or with cycloheximide, 0.2 jig/ml (O), and
2.0 ¿ig/ml(•),for 5 hr were labeled with uridine-"C, 0.5 ¿tCi/ml,in the
presence of various concentrations of actinomycin D.
2968
Fraction
20
30
no.
Chart 5. Ribosome distribution in serum-starved RFL and RFL-T.
Growing RFL and RFL-T cells, 4 to 5 x IO5cells/ml, prelabeled with
uridine-"C in a Petri dish containing 3 ml media were treated as follows: a,
RFL without starvation; b, 4-hr serum-starved RFL; c, RFL-T without
starvation; d, 4-hr serum-starved RFL-T. Cells with or without serum
starvation (5 plates for each experiment) were exposed further to uridine3H, 5 fiCi/ml, for 120 min. Cytoplasmic extracts were analyzed by
isokinetic sucrose density gradient analysis as described in "Materials and
Methods." Arrow, monosomal region; O, cpm 3H; •¿.
cpm "C.
CANCER RESEARCH
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VOL. 33
RNA Synthesis and Polyribosomes
fraction (Chart 8). Little polypeptide formation was ob
served in serum-starved RFL cells, whereas the transfor
mant could clearly produce nascent peptidic chains. How
ever, the nascent peptides formed in the starved RFL-T cells
were smaller than those of the control (Chart 86). This
evidence is reasonably explained by the shift of mRNA peak
indicated in Chart 7, since the distribution of specific
(b)
8
Time
( hr )
Chart 6. Summary of changes in ribosome distribution caused by serum
starvation. As a function of hr of serum deprivation, percentage of total
ribosomes in polyribosomes (a), in monosomes (b), and in free ribosomal
subunits (c) were calculated from 3 repeated experiments on each cell line,
RFL (A) and RLF-T (O), as shown in Charts 5 and 6. These curves were
obtained by counting long labeling with uridine-"C. Under our conditions,
polyribosomes larger than monosomes were ranged from 60 to 80% in
exponentially growing cultures.
cycloheximide, was added to culture medium during serum
deprivation. A remarkable accumulation of monosomes
observed at 7 hr after serum starvation was almost com
pletely blocked by the presence of cycloheximide, 0.2 to 2.0
Mg/ml, a dose that inhibits 50 to 90% of protein synthesis
(data not shown). This might suggest that some peptide
formation is involved in the transition of polyribosome that
appeared during starvation for serum.
Association of mRNA with Monosomes under Serum
Starvation and Formation of Nascent Peptides. Newly
synthesized mRNA was detected in polyribosomes (14, 21);
this mRNA was known to enter polyribosomes after 20 min
of labeling (22). Under serum starvation, both RFL and
RFL-T cells still continued mRNA formation at a constant
rate, as indicated in Chart 3. Cells starved for serum were
exposed to uridine-3H for 60 min in the presence of
actinomycin D, 0.03 ng/m\, and were analyzed by sucrose
density gradient centrifugation. As compared with control
(Chart la), peaks of 3H-labeled RNA were shifted from the
heavy polyribosome to the light polyribosome region, and
major radioactivity was found to be associated with mono
somes that accumulated by serum starvation (Chart Ib).
This might be attributed to preferable blockage of ribosome
cycle by the starvation of protein synthesis at an early stage,
possibly initiation.
Next, peptide formation coupled to polyribosome metab
olism was studied on cells cultured in serum-free medium
with or without puromycin treatment. Radioactivity due to
nascent polypeptides on polyribosomes was obtained by
subtracting the radioactivity remaining in the ribosomes
after puromycin treatment from total radioactivities in each
NOVEMBER
rti
I
10
10
30
20
Fraction
20
30
no.
Chart 7. mRNA distribution in polyribosomes after serum starvation.
Three plates of 4 to 5 x 10s cells/ml prelabeled with undine- 14Cfor 18 hr
were starved for serum as follows: a, control; b, serum starved for 6 hr, then
labeled with uridine-3H, 10 ¿iCi/ml,for 60 min in presence of actinomycin
D, 0.03 /ig/ml. Polyribosomal patterns were obtained as described in Chart
4. Arrow, monosomal region; O, cpm 3H, •¿,
cpm "C.
(b)
(a)
'o
X
e
o.
o
I
m
10
10
20
Fraction
20
no.
Chart 8. Nascent peptide formation in RFL and RFL-T. Eight plates
containing 3 ml cultured medium of RFL (a) and RLF-T (b), 4 to 5 x 10"
cells/ml, with or without serum starvation for 7 hr were exposed to
leucine-3H, 20 ¿»Ci/ml,
for 10 min and divided into 2 parts. Polyribosomal
patterns of 1 part of cells (4 plates) are shown, respectively, after
subtraction from polyribosomal patterns of other cells (4 plates) treated
with puromycin in vitro as described in "Materials and Methods." O,
control; •¿.
serum starved for 7 hr; arrow, monosome.
1973
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2969
Michihiko Kuwano, Hideya Endo, and Yukio Ikehara
nascent peptide-like albumin was found in accordance with
polyribosomal size (Y. Ikehara and H. C. Pilot, submitted
for publication). Thus, this analysis of peptide being formed
in addition to kinetic studies of protein synthesis described
above would support the theory that there is ribosomal
movement to make peptides in transformed RFL-T starved
for serum even though the rate of synthesis or polyriboso
mal size diminishes. Much less protein synthesis is proceed
ing in normal RFL starved for serum. The polyribosomes
observed after serum starvation of normal RFL cells might
be "frozen," resulting in no detectable peptide formation.
DISCUSSION
The effect of serum deprivation will be discussed in 2
major parts: transcription and translation levels.
Differential Control of RNA Formation in Normal and
Transformed Cells. Decreased rRNA formation that ap
peared in contact-inhibited normal cells was thought to be
related to growth rate or the rate of transcription, and the
suppressed formation of rRNA is restored to a higher level
by the addition of fresh serum (6). However, our data
indicate that serum deprivation during subconfluent cellular
growth reduced peptide formation more in untransformed
RFL than in its transformed counterpart, RFL-T. Another
possible explanation might be considered, that nucleolar
RNA polymerase is degraded rapidly when protein synthe
sis stops. This possibility is supported by evidence that the
turnover of the nucleolar protein responsible for rRNA
formation is rapid in rat liver treated with cycloheximide
(30), where specific inhibition of rRNA formation was
observed (19). In tissue culture cells of RFL and RFL-T
under almost complete blockage of protein synthesis, a
similar extent of depressed rRNA formation occurred in
both cell lines (Chart 4). Thus, the blockage of rRNA
synthesis in serum-starved untransformed fibroblast might
be due to rapid degradation of nuclear protein initiated by
the stop of formation of the synthetic apparatus. Also, since
protein synthesis still proceeds in serum-starved trans
formed cells even at a reduced rate, formation and degrada
tion of rRNA could be balanced under such a condition. Of
course, at present other possibilities cannot be excluded,
including difference of transcriptional rate, difference of
transport speed, and RNA maturation difference between 2
cell lines used under serum deprivation.
Differences in the Ribosome Distribution in Normal and
Transformed Cells. Comparative studies of polyribosomes
in normal (RFL) and transformed (RFL-T) cells under
serum starvation show that most polyribosomes are inert in
RFL, while polyribosomes in RFL-T cells are functioning,
for the following reasons: (a) newly formed mRNA con
tinues to enter polyribosomes of starved RFL-T cells even
though polyribosomal sizes diminish; (b) nascent peptide
and total protein are formed after starvation for serum in
RFL-T cells. Little, if any, peptide synthesis is observed
in serum-starved RFL cells in which polyribosomes might
be frozen.
In the presence of peptide bond formation proceeding at
an appreciable rate in serum-starved transformed RFL-T
2970
cells, ribosomal redistribution, increase of smaller polyribo
somes and monosomes, and reciprocal decrease of larger
polyribosomes appeared. Here, serum factors seemed to be
involved in initiational steps of peptide formation, since
newly formed mRNA could be found in a complex with
monosomes that accumulated on serum starvation (Chart
7). However, this possibility might be only suggestive until a
specific factor involved in this step is purified from serum.
The polyribosome disaggregation and appearance of mono
somes were also shown in serum-starved Ehrlich ascites cells
(11), in puromycin-treated hamster cells (27), or in NaFtreated reticulocytes (18). The redistribution that occurred
in serum-starved RFL-T might require a certain rate of
protein synthesis, because this transition did not seem to
occur in starved RFL cells where transpeptidation was
greatly depressed. Also, an inhibitor of protein synthesis,
cycloheximide, almost completely blocked the transition of
polyribosomes into monosomes and stabilized polyribo
somes. If cycloheximide could diminish elongation of
peptide synthesis as discussed (26), an overall balance of
slow initiation (caused by serum starvation) and lowered
rate of elongation could result in reforming of polyribo
somes. Regulatory mechanisms of macromolecular synthe
sis underlying mammalian cells were recently presented in
relation with that of bacterial systems (9), in which cyclo
heximide was thought to be similar to the action of chloramphenicol in bacterial systems. Effects of serum deprivation
observed here led us to compare the blockage in the ribosome cycle to that caused by streptomycin (16). Mono
somes and mRNA accumulated in streptomycin-sensitive
Escherichia coli treated with the drug, which eventually
stopped protein synthesis. However, the complex of mono
somes and mRNA accumulated after serum starvation
seemed to be more dynamic, since it can easily be changed
into polyribosomes by suppression of protein synthesis
with cycloheximide (see text) or by addition of fresh
serum (11).
ACKNOWLEDGMENTS
We thank Katsuko Matsui and Mayumi Ono for skillful and devoted
help. We thank Dr. Henry C. Pilot and Dr. David Schlessinger for
critically reading this manuscript.
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RNA Synthesis and Polyribosomes
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2971
Differences in RNA Formation and Polyribosome Metabolism in
Serum-starved Normal and Transformed Cells
Michihiko Kuwano, Hideya Endo and Yukio Ikehera
Cancer Res 1973;33:2965-2971.
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