[CANCER RESEARCH 34, 588-593, March 1974]
Effect of Aminonucleoside on Serum Stimulation of Nonhistone
Nuclear Protein and DNA Synthesis in Normal and
SV40-transformed Human Fibroblasts1
Jolanta J. Cholon and George P. Studzinski2
Department of Pathology, Jefferson Medical College of Thomas Jefferson University. Philadelphia, Pennsylvania 19107
SUMMARY
Addition of medium containing fresh serum to crowded,
starved cultures of normal fibroblasts is known to induce
DNA synthesis in a large proportion of the cells, preceded
by stimulation of the synthesis of nonhistone chromosomal
proteins. We now show that after prolonged culture, human
lung fibroblasts transformed by the oncogenic virus SV40
also show a wave of DNA synthesis when exposed to fresh
serum and that the synthesis of nonhistone nuclear proteins
is stimulated by this treatment with the same temporal
relationship to feeding and subsequent DNA synthesis as
that seen in normal cells.
The SV40-transformed cells differ from their normal
counterparts by their resistance to the inhibitory effects of
aminonucleoside. The observed rapidly occurring serum
stimulation of nonhistone protein synthesis takes place in
the presence of this antimetabolite, and the cells go on to
synthesize DNA and divide. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of nonhistone nuclear pro
teins shows that the synthesis of some rapidly migrating
classes of these proteins correlates with subsequent initia
tion of DNA synthesis.
entry of these cells into the S phase (and to a lesser extent
into mitotic phase), i.e., the inhibition takes place princi
pally in the Gì
phase of the cell cycle (25). Since it has been
shown that the most likely trigger for DNA synthesis is the
activation of the genome by newly synthesized nonhistone
chromosomal proteins (16 19, 28), we inquired whether the
inability of AMS to restrain rapidly the proliferation of
neoplastic cells is due to its lack of effect on the synthesis of
these proteins. The experiments presented here suggest that
this, indeed, is the case.
MATERIALS
AND METHODS
Cell Culture. WI38 diploid human lung fibroblasts (7)
were purchased from the Cell Culture Fund, Stanford
University, Stanford, Calif., and used for experiments at
passages 15 to 28. Their SV40-transformed counterparts,
WI38-VA13 (5), were kindly given to us by F. Jensen and
were cultivated in this laboratory for 2 years through
Passages 196 to 258. The cells were routinely grown in 32-oz
glass bottles in Eagle's minimal essential medium supple
mented with 10% fetal calf serum and 1% glutamine but
without antibiotics. All tissue culture supplies were obtained
from Flow Laboratories, Rockville, Md.
Tests for Mycoplasma contamination were performed at
INTRODUCTION
intervals by the method of Hayflick (6) and every week by
Treatment of cultured mammalian cells with AMS,3 a the recently described autoradiographic screening test (26).
Cells were enumerated by dispersal of the monolayer in
structural analog of adenosine, produces inhibition of
0.25% trypsin in Versene (balanced salt solution containing
cellular RNA synthesis (3, 22, 27) and of cell proliferation
(23). This effect is, however, clearly different in normal and 0.02% EDTA). A portion of the suspension was diluted with
neoplastic cultured cells. In a variety of normal fibroblasts, 10 ml of Versene and counted in a Coulter counter Model B
AMS arrests cell proliferation within 1 generation time, (Coulter Electronics, Inc., Hialeah, Fla.).
Autoradiographic Determination of DNA Synthesis.
whereas cells cultured from malignant tissues or trans
W138
and WI38-VA13 cells were grown on glass coverslips
formed in vitro, spontaneously or by oncogenic viruses,
in
Petri
dishes as described previously (25). The monolayers
continue to proliferate for several cell generations in the
were maintained without a change of medium for 7 days
presence of this inhibitor (4, 22-25).
The inhibition of growth of normal fibroblasts by AMS is (16) and then exposed to fresh medium containing 10% fetal
not due to a direct effect of this antimetabolite on DNA calf serum. In some cases the fresh medium contained 100
synthesis or mitosis, but is the result of inhibition of the ¿igAMS per ml, and other experimental groups were
pretreated for various times with this concentration of AMS
1Supported by USPHS Research Grant CA12334-2 from the National
as well. One series of cultures was continuously exposed to
Cancer Institute and American Cancer Society Grant T554. These results 0.5 /iCi of thymidine-3H per ml (specific activity, 6.7
were reported previously in a preliminary form (I).
Ci/mmole, New England Nuclear, Boston, Mass.) dissolved
2To whom reprint requests should be addressed.
in the complete medium, and triplicate coverslips were fixed
3The abbreviations used are: AMS, aminonucleoside of puromycin;
at intervals thereafter. In another series the cultures were
SDS, sodium dodecyl sulfate.
pulse labeled by 30-min exposure to thymidine-3H at 5
Received September 28, 1973; accepted December 3, 1973.
588
CANCER RESEARCH VOL. 34
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A MS Effect on Nonhistone
l. The coverslips were prepared for autoradiography
as described previously (25). The proportion of labeled cells
was determined by counting 1000 cells in each coverslip.
Incorporation of Radioactive Leucine into Residual Nu
clear Proteins. Confluent monolayers of both WI38 and
WI38-VA13 cells were maintained for 7 days in 32-oz
bottles without a change of medium. One experimental
group served as a control, another received 100/¿g
AMS per
ml into the depleted medium, while Groups 3 and 4 received
fresh medium or fresh medium with 100 pg AMS per ml.
After 1 hr each group was exposed to leucine-L-3H (specific
activity, 5 Ci/mmole) or leucine-L-14C (specific activity,
0.311 Ci/mmole) (New England Nuclear). Concentrated
leucine-3H was added to the complete medium bathing the
cell sheet to the final concentration of 2 fiC'i of medium per
Proteins
tetramethylethylenediamine and 0.1% ammonium persulfate. Electrophoresis was performed at 22°for 11 hr at
constant current of 5 ma/gel at 90 V in 0.1 M sodium
phosphate buffer, pH 6.8-0.1% SDS.
After electrophoresis each gel was frozen at -20°,
fractionated by mechanical means, and placed into scintilla
tion vials. The slices were dissolved in 0.5 ml of 30% H2O2,
1 ml of NCS was added, and the radioactivity was counted
in 10 ml of toluene-based scintillation fluid in a liquid
scintillation counter.
RESULTS
Effect of AMS on Serum Stimulation of DN A Synthesis in
Starved
Cultures.Confluent cultures of W138 fibroblasts are
ml for total incorporation experiments and at 20¿Ã-Ci/ml
for
gel electrophoretic separation. Leucine-l4C was added to 2 known to respond to refeeding with medium containing
/¿Ci/ml.At the end of 30 min the monolayers were washed 3 fresh serum by showing a wave of DNA synthesis, which is
times with ice-cold 0.2 M phosphate-buffered solution, pH maximal approximately 18 hr after addition of the serum
7.0, and harvested by trypsinization. The trypsinized cells (14, 16). The lower part of Chart 1 shows that when AMS is
were collected by centrifugation at 1500 x g for 10 min at added with the fresh serum this stimulation of DNA
4°.
synthesis does not take place, which is consistent with the
Isolation of Nuclei and Fractionation of Nuclear Proteins. previous finding that normal exponentially growing fibro
Nuclei were isolated as described by Tsuboi and Baserga blasts cannot pass through the Gìphase when AMS is
(29), except that 0.5% Triton X-100 was used in place of 1% present (25). Unlike the normal cells, SV40-transformed
Triton N-101. Generally 7 x IO6cells were used for total WI38 fibroblasts are not quiescent when they reach conflu
incorporation experiments, and 2.4 x IO7cells were used for ence; the upper part of Chart 1 shows that between 30 and
gel electrophoresis of the extracted proteins. The absence of
too
gross cytoplasmic contamination of the isolated nuclei was
monitored by phase-contrast microscopy, and when neces
sary the 0.5% Triton X-100 wash was repeated. The nuclei
were extracted exactly as described by Rovera and Baserga
(16), and the tightly bound residual protein pellet was
dissolved in l N NaOH or in 0.01 M sodium phosphate
buffer, pH 6.8, containing 1%SDS for gel electrophoresis.
The residual nuclear protein fraction was dissolved in 1
ml of l N NaOH, 1 ml of NCS and 10 ml of toluene-based
scintillation fluid were added, and the radioactivity was
determined in a Nuclear Chicago Mark I scintillation
counter (Searle Analytics, Cherry Hill, N. J.). The effi
ciency of counting was 26% for tritium and 60% for 14C.
Electrophoresis of Nuclear Protein Fraction. The proteins
were separated according to their molecular weights (or the
molecular weights of their subunits) by SDS-polyacrylamide gel electrophoresis (11, 12), by resuspending the tightly
bound residual protein pellet in 1% SDS and dialyzing
overnight at room temperature against 0.01 M sodium
phosphate, pH 6.8, containing 0.1% SDS, with 2 changes of
12
18
24
8
32
the buffer. After dialysis, the amount of protein in the
HOURS ON SERUM
suspension was determined by the method of Lowry et al.
Chart 1. The proportion of cells synthesizing DNA following addition of
(10). Equal amounts (100 to 150 fig) of residual proteins
from 2 experimental groups, one labeled with leucine-3H fresh medium to coverslip cultures of WI38 or WI38-VA13 cells which
and the other labeled with leucine-14C, were mixed in the have been starved for 7 days. Medium with fresh serum was added to all
, WI38 cells: ®,control, pulse label; x, control,
total volume of 0.3 ml, and 60% sucrose was added to this cultures at Time 0.
continuous label; O, 100 jig AMS per ml applied together with fresh serum,
mixture to a final concentration of 10% sucrose. The continuous label; A, 100 fig AMS per ml applied 12 hr before the fresh
mixture was subjected to electrophoresis on 7.5% acrylamserum, then together with the fresh serum, continuous
label.
,
ide-1% bisacrylamide gels, 20 cm long, 6 mm in diameter, WI38-VA13 cells: ®,control, pulse label; x, control, continuous label; O,
made up in 0.1 M sodium phosphate, pH 6.8 0.1% SDS 100 ¡igAMS per ml applied together with fresh serum, continuous label; A,
electrophoresis buffer. The polymerization of the gel mix 100 Mg AMS per ml applied 12 hr before the fresh serum, then together
ture was catalyzed by the addition of 0.025% N, N,N' ,N'- with the serum, continuous label. Vertical bars, S.D. of 3 determinations.
MARCH 1974
589
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Jolanta J. Chalónand George P. Studzinski
40% of transformed cells under these conditions are in S
phase. Many mitotic figures can be seen when these cultures
are examined by phase-contrast or direct light microscopy.
However, addition of serum produces a stimulation of DN A
synthesis which resembles the wave of DNA synthesis
induced by serum in WI38 cells both in regard to the
magnitude of the effect—approximately 50% of the cells in
the cultures are stimulated—and in regard to the time
parameters—the maximal effect is seen at 18hr. It may also
be noted that the curves for pulse-labeled cultures of
transformed cells and for transformed cultures exposed to
thymidine-3H continuously are almost identical during the
1st 18 hr after serum addition, indicating that very few cells
leave the S phase during that time. AMS does not inhibit
cell stimulation of DNA synthesis in transformed cells
(Chart 1).
Effect of AMS on Serum Stimulation of the Synthesis of
Nonhistone Nuclear Proteins in Starved Cultures. Serum
stimulation of DNA synthesis in confluent cultures of WI38
fibroblasts is preceded by several hr by an increased
synthesis of nuclear nonhistone proteins, as was shown by
Rovera and Baserga (16). Aminonucleoside prevents this
increase (Table 1). The synthesis of these proteins is also
stimulated when starved, confluent cultures of transformed
fibroblasts are exposed to fresh serum, but in this case AMS
appears to be ineffective (Table 1). However, these conclu
sions are based on incorporation of exogenously supplied
leucine-3H into the nuclear proteins and would be valid only
if the specific activities of intracellular pools of leucine were
unaltered during different experimental conditions. To test
this point, incorporation of leucine-3H into nonhistone
proteins was related to its rate of incorporation into all
cellular proteins as has been done by others (e.g., Ref. 17).
The results presented in Table 2 confirm that serum does
appear to stimulate the synthesis of nuclear nonhistone
proteins in both normal and transformed fibroblasts and
even in the presence of AMS in the transformed cells.
Effect of AMS on Serum-inducedChanges in the Patterns
of Synthesis of Residual Nonhistone Nuclear Proteins. SDS
gel electropherograms of the residual protein fractions from
nuclei of normal and transformed fibroblasts are shown in
Charts 2 and 3. Qualitative differences can be seen between
these cell types (and this was confirmed by coelectrophoresis
of these proteins), but serum did not produce a qualitative
Effect of AMS
Table 1
on serum stimulation of leucine-3 H incorporation
residual nonhistone
into
nuclear proteins
The values represent the means ±S.E. of 4 experiments, normalized
to 100 for starved cultures.
Relative incorporation into residual
proteins in dpm//¿gof total
nuclear proteins
Experiment
W138
Starved
cultures1
1390
±
serumStarved,
hr on fresh
AMS°1 1 hr on
571±
hr on fresh serum + AMS°100136 ±12100193
1Dose at 100 jig/ml.
590
WI38-VA13
2762
±
45217
±
±77
Effect of AMS
on leucine-3H
Table 2
incorporation
into residual nuclear proteins
relative to incorporation into total cellular proteins
The values represent the means of 2 experiments, each performed in
triplicate, with the range of values in parentheses. Incorporation of leucine3H into residual protein in dpm/jig protein is expressed as the ratio of
incorporation of leucine-3H into all cellular proteins in dpm//jg protein.
The values have been normalized to 100 for starved cultures.
Ratio of leucine-3H incorporation
Experiment
Starved cultures
1 hr on fresh serum
Starved, 1 hr on AMS"
1 hr on fresh serum + AMS"100
WI38-VA13
WI38
196 (183-209)
70 (63-77)
53 (57-49)100
139 (132-146)
35 (27-48)
142(127-157)
" Dose at 100 ^g/ml.
change in the electrophoretic pattern of nuclear residual
proteins from either normal or transformed fibroblasts. A
distinct quantitative change was induced in these profiles by
fresh serum, as first noted by Tsuboi and Baserga (29),
namely, there is a relative increase in the synthesis of some
classes of rapidly migrating, therefore of low molecular
weight in SDS, residual nuclear proteins. This may be seen
more clearly in Charts 4A and 5A where data from these
experiments are shown after recalculation which stresses
relative rates of incorporation. Charts 4B and 5B show that
AMS prevents the serum-induced relative stimulation of the
synthesis of rapidly migrating residual nuclear proteins in
normal but not in transformed fibroblasts. Similar results
were obtained in 2 further identically performed experi
ments. Thus there appears to be a direct relationship
between the synthesis of the low-molecular-weight residual
nonhistone nuclear proteins that occurs shortly after serum
addition and the subsequent initiation of DNA synthesis in a
large number of cells in the population, normal or trans
formed.
DISCUSSION
The results presented here obtained with WI38 cells
confirm and extend the experiments reported by Rovera and
Baserga (16, 17) and Tsuboi and Baserga (29). We found
that the early increase in the synthesis of nonhistone nuclear
proteins which follows addition of serum to starved conflu
ent monolayers of these fibroblasts is inhibited by the mild
inhibitor of RNA synthesis, AMS, an action which is
similar to the effects of 5-azacytidine and low doses of
actinomycin D (16, 29). We also showed that AMS
inhibition of serum stimulation is directed at the rapidly
migrating classes of nonhistone nuclear proteins thus
strengthening the view that the synthesis of these protein
species is causally related to subsequent initiation of
cellular DNA synthesis.
WI38 fibroblasts transformed by the oncogenic SV40
virus were not inhibited by AMS either with regard to the
early serum stimulation of nonhistone nuclear proteins
synthesis or with regard to the subsequent wave of DNA
synthesis in the culture. Although these cells did not show
CANCER
RESEARCH
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VOL. 34
A MS Effect on Nonhistone
Proteins
labeled precursors is the possibility that the specific activity
of intracellular precursor pool may be subject to change
when experimental conditions are altered. However, pool
effects do not influence the results of gel electrophoretic
experiments as performed here, which clearly show that
2O
3O
40
SO
IÃŽO-I
SLICE NUMBER
Chart 2. SDS gel electrophoretic profiles of residual nonhistone proteins
from nuclei of WI38 cells. Residual proteins from starved cells (
)
labeled with leucine-"C have been coelectrophoresed with residual proteins
from cells exposed
to fresh medium
for 1 hr (
) and labeled with
leucine-"H. The values charted should be multiplied by 100 to give the
correct value.
0
10
20
SLICE
10
70
3O
4O
SLICE NUMBER
40
50
Chart 4. Effect of serum on the relative rates of incorporation of
leucine-3H and leucine-"C into residual nonhistone proteins of WI38 cells.
The radioactivity in each slice in the serum-stimulated group shown in
Chart 2 was calculated as percentage of average radioactivity of all the
slices in that gel, then expressed and plotted here as the ratio of the
similarly obtained value for the incorporation into nonhistone nuclear
proteins from starved cultures in the same slice. A, residual nuclear
proteins labeled in the absence of AMS. B, residual nuclear proteins
exposed to fresh medium and labeled in the presence of 100 ng AMS per
ml. x 100.
iVvA;
0
30
NUMBER
JO
6O
Chart 3. SDS gel electrophoretic patterns of residual nonhistone
proteins from nuclei of WI38-VA13 cells. Residual proteins from starved
cells (
) labeled with leucine-"C have been coelectrophoresed with
residual proteins from cells exposed to fresh medium for 1 hr (
) and
labeled with leucine-3H. The values charted should be multiplied by 100 to
give the correct value.
contact or starvation
inhibition of DNA synthesis or
mitosis, as loss of such restraints is a well-known conse
quence of neoplastic transformation,
they provided a useful
population for comparison with WI38. The system was
chosen because it is of great importance
to perform
20
30
lo
experiments on homogeneous cell populations when com
SLICE
NUMBER
plex patterns are to be compared, for supporting tissues,
Chart 5. Effect of serum on the relative rates of incorporation of
necrotic cells, and so forth, would introduce uncontrollable
variables and additional complexity. In this specific in leucine-3H and leucine-'"C into residual nonhistone proteins of WI38VAI3 cells. The radioactivity in each slice in the serum-stimulated group
stance, the 2 cell strains chosen for study differ principally
in that the SV40 virus has been incorporated
into the shown in Chart 3 was calculated as percentage of average radioactivity of
all the slices in that gel, then expressed and plotted here as the ratio of the
genome of one of them. When these cell strains were treated
similarly obtained value for the incorporation into nonhistone nuclear
identically, in each case serum produced a wave of DNA proteins from starved cultures in the same slice. A, residual nuclear
synthesis preceded by stimulation of low-molecular-weight
proteins labeled in the absence of AMS. B, residual nuclear proteins
nonhistone nuclear proteins. A perennial problem in the exposed to fresh medium and labeled in the presence of 100 in%AMS per
interpretation
of results obtained after incorporation
of ml. x 100.
MARCH 1974
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591
Jolanta J. Cholon and George P. Studzinski
AMS does not prevent the preferential synthesis of lowmolecular-weight nonhistone proteins which follows addi
tion of fresh serum to starved cultures.
Comparison of the effects of AMS on normal and
transformed human fibroblasts is only valid if this com
pound enters each type of cell with equal facility. This
indeed has been shown recently to be the case by measuring
the uptake of tritiated AMS by WI38 and WI38-VA13 cells
(4).
Nonhistone chromosomal proteins can be considered to
comprise 3 functional groups of proteins (8): the structural
components of the chromosomes; enzymes associated with
the chromosomes (e.g., Ref. 2); and proteins with regulatory
effects on the genome, perhaps analogous in some respects
to the activator and repressor proteins of microorganisms
which bind to unique DNA sites in a highly specific manner
(e.g., Refs. 13 and 15). Residual nucleolar proteins may
have similar functions. The nuclear proteins studied here are
probably of both chromosomal and nucleolar origin, but
irrespective of their source the results suggest that the
rapidly migrating classes of nonhistone proteins serve as
gene activators, and findings in some other systems are
consistent with this thesis. Stein and Matthews (21) showed
that the decreased synthesis of nonhistone chromosomal
proteins which occurs during Gìin HeLa cells treated with
actinomycin D during mitosis affects principally the pro
teins which migrate most rapidly on polyacrylamide gel
electrophoresis. Wakabayshi et al. (30) found that the DNA
binding fraction of nonhistone proteins from rat liver which
confers immunospecificity to nonhistone proteins consists of
low-molecular-weight proteins. In lymphoid cells stimulated
to proliferate by phytohemagglutinin several specific classes
of protein were preferentially stimulated during the activa
tion, and some of these proteins were of low molecular
weight (9). On the other hand, when leukemic cells were
stimulated in this way there was a progressive disappear
ance of low-molecular-weight proteins (31). This discrep
ancy could be due to the fact that the measurements on the
leukemic cells were performed 22 hr after addition of
phytohemagglutinin, a considerable interval of time, yet
further studies appear to be indicated to establish this point.
It would appear therefore that AMS inhibits the prolifer
ation of WI38 cells by interfering with a process which
precedes the increase in the synthesis of low-molecularweight nonhistone nuclear proteins which are required to
activate the genome. The inhibition of the synthesis of these
nuclear proteins may explain the arrest of exponentially
growing WI38 cells in d and G2 phases of the generation
cycle (25), since, at least in some cells, nonhistone protein
synthesis is maximal at those times (20). The previous
findings of the lack of direct effect of AMS on DNA
synthesis in normal cells and the lag in the inhibitory action
of AMS also become understandable. On the other hand,
transformed cells continue to proliferate in the presence of
AMS because the synthesis of the small nonhistone nuclear
proteins is not inhibited by AMS. This raises the intriguing
question by what mechanism the synthesis of regulatory
nonhistone proteins is protected in transformed cells. Per
haps the answer to this question will bring us closer to the
solution of the nature of neoplastic transformation.
592
ACKNOWLEDGMENTS
We thank Janet Shoemaker for enthusiastic and skillful technical
assistance.
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Effect of Aminonucleoside on Serum Stimulation of Nonhistone
Nuclear Protein and DNA Synthesis in Normal and
SV40-transformed Human Fibroblasts
Jolanta J. Cholon and George P. Studzinski
Cancer Res 1974;34:588-593.
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