Download Mechanism of Growth Arrest of Chemically

[CANCERRESEARCH38, 2807-2812,September1978]
0008-5472/78/0038-0000$02.00
Mechanism of Growth Arrest of Chemically Transformed Cells in Culture1
HaroldL. Moses,2JacquelineA. Proper,Mary E. Volkenant,DavidJ. Wells, and MichaelJ. Getz
Departments of Pathology and Anatomy (H. L. M., J. A. P., M. E. V., D. J. W., M. J. G.J and Molecular Medicine (H. L. M.J, Mayo Clinic/Foundation,
Rochester, Minnesota 55901
or they growth arrest at subconfluence in serum-deficient
medium (17,20).The quiescentcells
can thenbe stimulated
to undergo a round of DNA synthesis by adding serum (26,
ABSTRACT
The mechanism of growth arrest was studied in two
mouse embryo-derivedcell lines and two transformed 29) or purified mitogens including EGF3(3, 11, 23), FGF
clones obtained through 3-methyicholanthrenetransfor (10), or the tumor promoter, TPA (21, 25). DNA virus
mation of both of the parent nontransformedlines. The transformed cells, on the other hand, do not growth arrest
two transformed lines were AKR-MCA derived from the
in G, under usual monolayer culture conditions. They either
nontransformedAKR-2B cell line and the C3H/MCA-58 become arrested throughout the cell cycle or continue
derived from the C3H/10T1/2cell line. Both transformed proliferating until they die even when placed in serum
clones reach a saturation density approximately 2-fold deficient medium (18, 19). However, Holley et a!. (12) have
that of their respectivenontransformedparent lines. The reported that a chemically transformed line of 3T3 cells
chemically transformed cells were found to reach low does become quiescent when grown in medium with 0.2%
base-line levels of [3Hjthymldineincorporationbefter if serum and that these cells growth arrest in the G, phase of
allowedto remainin highserum(10%)thanin lowserum the cell cycle. O'Brien and Diamond (16) have shown that
(0.5%) while the nontransformed
cells growth arrested
chemically transformed hamster cells can be growth ar
befter in low serum. Changingto fresh mediumwith 10% rested in medium with 10% serum and that the cells can be
serum caused the quiescent nontransformedand trans stimulated to undergo a waveof DNAsynthesis beginning 6
formed cells to undergo a wave of DNA synthesis, as hr following stimulation in a manner similar to that of their
determined by [3H]thymidine incorporation, suggesting nontransformed cells, suggesting that the transformed cells
that the transformedcells, like the nontransformedcells, were arrested in G,.
were growth arrested in the G@,/G,phase of the cell cycle.
The present study was carried out in order to confirm that
The time interval between the stimulationto proliferate chemically transformed cells, unlike DNA virus-transformed
and the onsetof DNA synthesiswas considerablyshorter cells, can become quiescent in G, and to examine the
in the transformedcells than in the nontransformedcells. mechanism for growth arrest in the chemically transformed
The quiescent transformedcells were not stimulatedto cells. For these studies we have used the nontransformed
undergoDNA synthesisby the additionof serum, epider AKR-2B cell line and a 3-methylcholanthrene-transformed
mal growth factor, fibroblastgrowthfactor, or the tumor clone (AKR-MCA)derived from the AKR-2B cells (9). In
promoter, 12-O-tetradecanoylphorbol-13-acetate. They addition, we have examined the C3H/1OT'/2 cell line and a
were stimulated by the addition of fresh medium contain
3-methylcholanthrene-transformed
derivative, cabled C3H/
ingno serum.Thenontransformed
cellsgrowtharrested MCA-58 (22). Both chemically transformed cells became
inthepresenceof10%serumwerestimulated
to undergo quiescent in G1 at a point closer to the onset of DNA
a round of DNA synthesis by serum, epidermal growth synthesis than that for their nontransformed counterparts.
factor, fibroblast growth factor, 12-O-tetradecanoylphor The data indicate that the chemically transformed cells
bol-13-acetate.
Theywerenotstimulated
bymediumwith growth arrest due to depletion of multiple amino acids and
out serum. The data indicate that the chemically trans
glucose from the chemically defined medium. In contrast to
the nontransformed cells, they are unresponsive to serum,
than do the nontransformedcells. The nontransformed EGF, FGF,and TPA.
cells growth arrest due to depletion of essential serum
growth factors, whereas the chemically transformed cells
MATERIALSAND METHODS
formed cells growth arrest in G1by a different mechanism
appeartogrowtharrestduetodepletionofmultipleamino
acids and glucosepresentin the chemicallydefined Cell Culture. AKR-2Band AKR-MCAcells are grown in
McCoy's Medium 5A supplemented with 10% fetal bovine
medium.
serum (Reheis Chemical Co. , Phoenix, Ariz.) without anti
biotics (9). C3H/10T1/2and C3H/MCA-58cells are grown in
INTRODUCTION
Eagle's basal medium also supplemented with 10% fetal
Nontransformed animal cells in culture growth arrest in
the G1 phase of the cell cycle when they reach a certain
density due to depletion of essential serum growth factors
bovine serum and without antibiotics (8). Experimentswere
carried out both in 490-sqcm plastic roller bottles (Corning
Glass Works, Corning, N. Y.) or Falcon No. 3002 Petri
dishes with a 21-sq cm growth surface. All experiments are
carried out within 10 passages of the frozen stock from
I This
investigation
was
supported
by
Grant
CA
16816
from
the
National
Cancer Institute, Department of Health, Education, and Welfare, and the
which cells are recovered periodically. These stock cells
MayoFoundation.
2 To
whom
requests
for
reprints
should
be
addressed.
Received April 3, 1978; accepted May 30, 1978.
SEPTEMBER
1978
3 The
abbreviations
used
are:
EGF,
epidermab
growth
factor;
FGF,
fibro
blastgrowthfactor; TPA,12-O-tetradecanoylphorbol-13-acetate.
2807
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H. L. Moses et a!.
have been shown to be free of Mycop!asma contamination
by culture methods, by scanning and transmission electron
microscopy (9), and by staining with the Hoechst No. 33258
stain (4). Cells are examined after Hoechst No. 33258
Staining monthly to ensure that they have remained free of
Mycoplasma.
The AKR-MCA and C3H/MCA-58 cells are transformed as
indicated
by growth morphology,growth insoftagar,and
tumorigenicity in nude mice (8, 9, 22). The AKR-2B and
C3H/10T1/2 cells do not grow in soft agar and do not form
tumors innude mice (8,9,22).
Growth Arrest of Transformed and Nontransformed
of the AKR-2B and AKR-MCA cells showed that the 2 cell
lines had virtually identical generation times (Chart 1; Table
1). However, the AKR-MCA cells reached a saturation den
sity that was approximately 2.5-fold greater than that of the
AKR-2B cells (Table 1). The same saturation density was
achieved in each cell line regardless of whether the initial
inoculation was 10@or 5 x 10@cells/dish (Chart 1). The
generation times for the 2 C3H cell lines is shown in Table
1. The C3H/1OT'/2 cell line had a slightly longer generation
time than did the chemically transformed counterpart; how
ever, the difference was probably not significant. Again the
saturation density of the transformed cells was over 2-fold
Cells. The methodfor growtharrestof the AKR-2Bcellsin
low serum has been described previously (1, 7). For growth
arrest in 10%serum, both transformed and nontransformed
greater than that of the nontransformed cells, and the
saturation density of both cell lines was similar to that
cells are subcultured with a dilution of 1:30 for the AKR
cells or 1:10 for the C3H cells, medium is changed on Day 4
adding 35 ml forroller
bottlesand 2 ml fordishes,and
[3Hjthymidine incorporation is determined as described
The saturation density of the AKR-2B cells was slightly
greater than that of the transformed C3H/MCA-58cell line
(Table 1). This does not, however, mean that the AKR-2B
cells pile up; they form monolayers of closely packed
previously reported for these cells (22).
below on Day7 after subculture. If the level of [3H]thymidine epithelioid-type cells (9).
incorporation
is not at base-line level, the cells are incu
Growth Arrest of Chemically Transformed Cells. The
bated for an additional 24 hr and the level of [3H]thymidine plateau of cell density reached by the nontransformed cells
incorporation is again determined. In most cases the level
of [3H]thymidmne incorporation reached base-line levels by
7 days after subculture, and in virtually all cases by 8 days
after subculture.
Stimulationof DNA Synthesisin QuiescentCells. Rest
ing cells are stimulated
to grow by changing
to fresh
medium with 10% serum. Since the act of changing
medium
(i.e., removing and readding the depleted medium) causes
some small but significant stimulation of DNA synthesis,
is due to growth arrest of the cells (17, 20). The similar, but
higher plateau of cell density achieved by the transformed
cells could be due to either growth arrest of the transformed
cells or the attainment of a balance between cell replication
and cell death. For examination of these possibilities, the
level of [3H]thymidineincorporation was determined in the
AKR-MCAcells as they reached their saturation density. As
shown in Chart 2, the level of [3Hjthymidine incorporation
10@
experiments concerning the differential effect of serum,
purified mitogens, or chemically defined medium on stimu
lation of DNA synthesis are carried out by adding the factors
to the depleted medium. Stock solutions of EGF and FGF
(Collaborative Research, Inc., Waltham, Mass.) are either 1
or 10
@g/ml in Hanks'
balanced
salt solution
containing
0.5% bovine serum albumin. TPA (obtained from the lIT
Research Institute, Chicago, Ill. , through the NCI Carcino
genesis Research Program) is dissolved in acetone at a
@
i06_
concentration
of10 pg/mI.
Studies on stimulation of AKR-MCA cells with different
components of the chemically defined medium are carried
out with the GIBCO RPMI-1640 Select-Amine Kit (Grand
Island Biological Co., Grand Island, N. Y.). Media are
formulated
deleting
single
amino
acids,
glucose,
.—-*
AKR-281,10'
.--o AKR@2B
5 10@
AKR-MCA1. iO@
AKR-MCA5,jO'
or vita
mins, and 2 ml are added to the 2 ml of depleted medium
with serum in Petri dishes. [3H]Thymidineincorporation is
determined 18 hr after addition.
Days
Chart 1. Growth curves for AKR-2B and AKR-MCAcells. Calls were
seeded in 21-sq cm dishes at an initial concentration of 10' or 5 x 10@celbs/
Callswereharvestedbytrypsinizationandcountedin a hemacytometer
Assay for DNA Synthesis. Cells are pulsed for 60 mm dish.
at the indicated times.
with
1 .0
@Ci[methyl-3H]thymidine
(6.7
@Ci/mmol; New
England Nuclear, Boston, Mass.) per ml of medium, and
the incorporation into acid-precipitable material is deter
mined as previously described (1, 7). DNA is determined by
the diphenylamine reaction of Burton (2), and the results
are expressed as cpm [3H]TMPincorporated per @g
DNA
per hr or @gDNA per roller bottle. Light microscope
autoradiography is performed as described previously (1).
Comparison
of generation
Table 1
time and saturation
density of
nontransformedand chemically transformedcells
density
time (hr)
(cells/sq cm)
AKR-2BGeneration 16.8 ±1.6°Saturation1 .05 x
10'AKR-MCA16.4
10'C3H-10T1/2305b3.44 ±0.9w'2.86
x
[email protected]
x
x 10@
RESULTS
a Mean ±S.D. , 3 separate experiments.
SaturationDensityand GenerationTime. Growthcurves
2808
b Mean
of 2 separate
experiments.
CANCER RESEARCH VOL. 38
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Growth Arrestof TransformedCe!!s
AXR-2B
.@
0
‘;@
AKR-MCA
0
I
c
@)
.@-
E@-
0
t.)
0
20
24
48
Hoursafter stimulation
Hours after medium change
Chart 2. Growtharrestof AKR-MCAcells in 0.5 or 10%serum.Cellswere
seeded in 490-sq cm plastic roller bottles (1.5 to 2.0 x 10' cells/bottle), and
the medium was changed on Day 4. The incorporation of [‘H]thymidineinto
DNAfollowing 1-hrpulseandthe DNAcontentper bottleweredeterminedat
24, 48, and 72 hr after mediumchange.Virtually identical resultswere ob
tamed with cells grown in dishes.
Chart 3. Stimulationof AKR-2BandAKR-MCAcells. Cellswereseededin
21-sqcm dishes(7 x 10' cells/dish), and mediumwaschanged4 dayslater
at which time the mediumvolumewas reducedfrom 5 to 2 mI/dish. Cells
were stimulatedto proliferate at zero time by changing to fresh medium
containing 10% serum. Incorporation of [3H]thymidine and quantity of DNA
were determined as described under ‘Materials
and Methods.―[3H]Thymidine
was added 1 hr prior to harvestat the time indicated. Very similar results
wereobtainedwith the useof robberbottles.
decreased markedly in the AKR-MCA cells grown both in
0.5 and 10%serum. The cells in 0.5%serum showed a high
rate of cell death with sboughing of dead cells into the
medium which is reflected by the declining DNA content
per bottle as shown in Chart 2. Lower levels of
[3H]thymidine incorporation into the AKR-MCA cells was
.——*C3H/SOT'/Z
— C3H/MCAS8
obtained consistently with medium containing 10% serum
as opposed to medium containing 0.5% serum (Chart 2).
All 4 cell lines used in this study generally attained
minimal
levels of [3H]thymidine
incorporation
in medium
with10% serum at7 daysfollowing
subculture
as illustrated
at the zero time point in Charts 3 and 4. Incubation for
Hours after stimulation
longer times usually gave no further decrease in incorpora
Chart4. Stimulation of C3H/1OT'12
and C3H/MCA-58cells. Cells were
seeded in 490-sq cm roller bottles (2 to 3 x 10' cells/bottle), and the medium
tion. Autoradiographic studies of the 2 AKR cell lines
waschanged4 dayslater.Cellswerestimulatedto proliferateat zerotime by
following a 1-hr pulse with [3H]thymidine after 7 days of
changing to fresh medium containing 10% serum. [3H]Thymidine incorpora
culture in medium with 10% (3 days following medium
tion and DNA content were determined at the indicated times as described in
legendto Chart3 and in ‘Materials
and Methods.'
change) revealed that 0.8% of the nuclei of the AKR-2B
cells and 1.1% of the nuclei of the AKR-MCA cells were
labeled. Standard deviations were 0.2 and 0.4% for the the AKR-MCA cells labeled for 1 hr, at 18 hr following
AKR-2B and the AKR-MCAcells, respectively, with 6 sepa stimulation 58 ±3% of the cell nuclei were labeled.
Effect of Medium, Serum, GrowthFactors, and TPA on
rate determinations for each cell line.
Stimulationof DNA Synthesisin Growth-arrestedCells.
Changing to fresh medium with 10% serum resulted in
stimulation of a wave of increased thymidine incorporation
in all 4 cell lines (Charts 3 and 4). The prereplication interval
Stimulationof DNA Synthesis in Growth-arrestedNon
transformed and Chemically Transformed Cells. The data
cells was shortened considerably to approximately 8 hr
(Chart 3). A similar shortening of the prereplicative interval
presented in Charts 3 and 4 indicate that the chemically
transformed cells are growth arrested at a point closer to
the onset of DNA synthesis than that for the corresponding
nontransformed cells. In order to examine the possible
mechanism of growth arrest in the transformed and non
transformed cells, we added various factors to quiescent
cells, and evaluated their effect on DNA synthesis at 18 or
for the chemically
C3H cells (4 hr) relative to
20 hr following addition by determining [3H]thymidmnein
the nontransformed C3H/1OT'/2 cells (10 hr) was also ob
served (Chart 4). Autoradiography following a 1-hr pulse
with [3H]thymidineat 20 hr following stimulation in the AKR
2B cells revealedthat 61% of the nuclei were labeled with a
corporation. As shown in Chart 5, the AKR-2B cells were
stimulated by fetal bovine serum alone; the growth factors,
EGF and FGF; and the tumor promoter, TPA. The AKR-2B
cells were not stimulated by the chemically defined medium
standard deviation of 13% for 3 separate determinations.
without serum. In contrast, the chemically transformed
between stimulation and the onset of DNAsynthesiswas 12
hr for the AKR-2B cells (Chart 3) as previously
7). However,
the
SEPTEMBER1978
prerepbication
transformed
interval
for
reported
the
(1,
AKR-MCA
In
2809
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H. L. Moses et a!.
AKR-MCAcells were not stimulated by serum, EGF, FGF,or
6
TPA (Chart 5). These cells were stimulated very effectively
by the chemically defined medium without serum (Chart 5).
In order to determine whether the presence of serum was
necessary for stimulation of DNA synthesis in the AKR-MCA
cells, the experiment shown in Chart 6 was performed.
These data show that, if the depleted medium containing
10% serum is removed from the cells by rinsing with
medium without serum at room temperature, DNA synthesis
is not stimulated by the medium. If medium containing 10%
serum is added after rinsing,
the cells are stimulated
C-)
(Chart
6). However, the addition of EGF instead of serum to the
medium does not cause stimulation. These data indicate
that some factor present in serum is necessary for stimula
tion of DNA synthesis in the AKR-MCA cells but that this
factor is not depleted by the cells as they become growth
E
a
arrested.
Chart 7 shows that the C3H/1OT'/2 cells behave essentially
the same as do the AKR-2B cells being responsive to serum,
EGF, and TPA. Similar to the AKR-MCA cells, the chemically
transformed C3H/MCA-58 cells are stimulated only by me
dium without serum. The C3H/1OT'/2 cells are also stimu
bated by FGF (data not shown); the effect of FGF on C3H/
MCA-58cells was not determined.
Determinationof Factors Depleted in Medium by AKR
MCA Cells. The observation that growth-arrested AKR-MCA
Control Add ChangeRinse—
Rinse—
Rinse
mediummediummedium mediummedium
+
+
serum EGF
Chart 6. Effect of serum and EGF on stimulation of DNA synthesis In AKR
MCA cells. Same as Chart 5, except as follows. Change of serum-free
McCoy's Medium 5A. Rinse-medium, spent medium removed, cells rinsed 3
times with serum-free medium, and 2 ml of serum-free medium added;
Rinse-medium+ serum, sameas precedingexcept2 ml mediumcontaining
10% serum added; Rinse-medium + EGF, same as preceding except 2 ml
medium containing EGF (10 ng/mb) added.
20
4
10
0 C3H/1OTV,
0 C3H/MCA—58
AKR-2B
AKR-MCA
7
fiflE:..
6
0
5
3
0
2
E
I
0
Control Change Medium Serum
EGF
TPA
medium alone alone lOng/mblOOng/mI
LL@Lftft
ControlMediumMediumSsrumEGF EGF FGF FGF TPA
+
serum
lOng
lOOng lOng lOOnglOOng
/mb /ml
/mI
/mb /ml
Chart 5. Effect of medium, serum, growth factors, and TPA on stimulation
of DNA synthesis in growth-arrested AKR-2B cells and AKR-MCA cells. Cells
in dishes were brought to a quiescent state as described in “Materials
and
Methods.―Control bars, base-line incorporation of [‘Hjthymidine.For the
otherbars, the indicatedmaterialwasaddedto the 2 ml of depletedmedium
containing 10% serum, and the level of @H]thymidineincorporated was
determined 20 hr later for the AKR-2B cells and 18 hr later for the AKR-MCA
cells. Medium + serum, 2 ml of fresh medium with 10% serum added;
Medium, 2 ml of McCoy's Medium 5A without serum; Serum, 200 M1serum.
For the remaining columns, sufficient EGF, FGF, or TPA was added to give
the final concentrationsindicated.
2810
serum
Chart 7. Effect of medium,serum,EGF,andTPAon stimulation of C3H/
1OT'/2and C3H/MCA-58cells. Same as Chart 5, except 13H]thymidlne
incorporation determined at 18 and 16 hr after stimulation for the C3H/
1OT'/aand C3H/MCA-58 cells, respectively.
and C3H/MCA-58cells are stimulated by addition of chemi
cabbydefined medium and not serum indicate that growth
arrest in these cells results from depletion of some essential
factors from the medium. In order to determine those
components responsible for growth arrest, we determined
the degree of stimulation of quiescent AKR-MCA cells
obtained by adding medium deficient in specific single
amino acids or other components. The addition of media
CANCERRESEARCH
VOL. 38
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Growth Arrestof TransformedCells
deficient in cystine, glutammne, isoleucine, leucine, lysine,
resting AKR-MCA cells to proliferate; however, this factor is
methionine, tryptophan, or vabineto growth-arrested AKR
MCA cells cause virtually no stimulation of the cells, while
not depleted when transformed cells are growth arrested
(seeChart 6).
It is well known that artificial deprivation of bow-molecu
lar-weight nutrients in the cell culture medium can cause
growth arrest of cells (15, 17, 20). Leucine and methionine
media deficient in the other amino acids stimulate to a level
43 to 100% of that obtained with a complete medium (Table
2). The degree of stimulation of AKR-MCAcells with media
deficient in glucose, phosphate, or the vitamin mixture was
also determined (Table 2). The deletion of glucose resulted
deprivation has been reported to cause cessation of growth
stimulation almost equal to that obtained with complete
medium. The data indicate that at least 8 amino acids and
at random points in the cell cycle (14, 24). On the other
hand, phosphate or a combination of histidine and gluta
mine deficiency leads to growth arrest in G, (13, 14, 24).
The net effect of the multiple amino acid plus glucose
deficiency observed in the chemically transformed cells in
glucose are depleted by the AKR-MCAcells to the point that
this study appears to be G, arrest.
their replacement is essential for stimulation of growth. The
addition of these 8 amino acids (cystine, glutamine, isobeu
to a level 34% of that obtained with complete medium.
Cells arrested in G, due to amino acid deficiency are
different from cells arrested in G0due to depletion of serum
growth factors. Enger and Tobey (6) have reported that
Chinese hamster ovary cells growth arrested by isoleucine
deficiency maintain high levels of rRNA and mRNAsynthe
DISCUSSION
the same cells growth arrested due to growth factor deple
We have presented data indicating that the nontrans
formed and chemically transformed cells growth arrest in
tion show a marked reduction of RNA and protein accumu
lation. We have shown that the AKR-MCA cells maintain a
in no stimulation of DNA synthesis in the AKR-MCA cells
while the deletion of vitamins or phosphate resulted in
cine, leucine, lysine, methionine, tryptophan, and valine) in
balanced salt solution with glucose did stimulate the cells
sis as well as protein synthesis in the resting state, whereas
the G1 phase of the cell cycle due to different mechanisms.
As with other nontransformed cell lines, the AKR-2B and
C3H/1OT'/2 cell lines growth arrest due to depletion of
higher level of polyadenylic acid-containing polysomal RNA
in the resting state than do AKR-2B cells (9). The shorter
lag phase between the stimulation to proliferate and the
onset
of DNA synthesis observed in the chemically trans
essential serum growth factors; the quiescent cells are
formed
cells could possibly result from these cells having
stimulated by the addition of growth factors (EGFor FGF),
to
accomplish
less macromolecular synthesisthan the non
serum, or the tumor promoter, TPA. The transformed cells
transformed cells in order to replicate their DNA.
(AKR-MCAand C3H/MCA-58) either do not require or do
It has also been shown that 3T3cells growth arrested due
not deplete the serum growth factors and are insensitive to
stimulation by these factors when in the quiescent state.
The AKR-MCAcells appear to growth arrest due to deple
tion of multiple amino acids plus glucose from the medium
and are stimulated to proliferate by the addition of the
chemically defined medium without serum. Some factor
present in serum appears to be necessaryfor stimulation of
withMedium
mediumL-Arginifle
component deleted
of quiescent
cells%
AKR-MCA
maximumstimulation
complete
68L-Asparagine
87L-Asparatic
100L-CyStine
acid
<1L-Glutamic
100L-Glutamine
acid
4L-GlyCine
100L-Histidine
100L-Hydroxyproline
59L-lsoleucine
EGFto cells. However,this effect appearsto be specific for
sarcoma virus transformation since DNAvirus-transformed
cells, spontaneously transformed cells, and chemically
transformed cells show EGF receptor levels comparable to
those of their normal counterparts (5, 27, 28).
The data suggest that the major alteration present in the
transformed cells observed in this study is in their response
to growth factors. The transformed cells could either be
hypersensitive to serum growth factor effect so that much
lower concentrations are required for growth stimulation,
or the cells could havean altered capability for degradation
of the growth factors so that they do not become depleted
<1L-Leucine
under normal culture conditions. It can be hypothesized
that the growth factor response remains sustained in the
transformed cells, causing the cells to continue probiferat
ing beyond the point that nontransformed cells would
growth arrest until amino acids are depleted from the
medium. This results in growth arrest of the transformed
cells at a much higher saturation density than the nontrans
formed cells.
<1L-Lysine
5L-Methionine
4L-Phenylalanine
100L-PrOline
100L-Senine
52L-Threonine
43L-TryptOphan
<1L-Tyrosine
100L-Vabine
<1Glucose
ACKNOWLEDGMENTS
2Vitamins
100Phosphate
89
SEPTEMBER
reported in this paper, the quiescent chemically trans
formed cells are not stimulated to proliferate by the addition
of growth factors to growth arrested cells. Whether this
lack of responsiveness is due to absence or alteration of
the EGFreceptor is not known at present. Todaro et a!. (27,
28) have reported that murine or feline sarcoma virus
transformation of mouse cells specifically blocks binding of
Table2
Effectfrommedium
of single amino acid, glucose, or vitamin deletion
on stimulation
to amino acid deficiency are not responsive to FGF (14). As
The authors wish to thank Dr. Wallace Rowe and Dr. Natalie Teich for
seed stocks of the AKR-2B cell line, Dr. Charles Heidelberger for seed stocks
1978
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1978 American Association for Cancer Research.
2811
H. L. Moses et a!.
of the C3H/1OT'/2 and C3H/MCA-58 cell lines, and Patricia H. Hart for
assistance In preparation of the manuscript.
Synthesis in 3T3 Cells: Low-Molecular Weight Nutrients. Proc. NatI.
Aced.Sci. U. 5., 71: 2942-2945,1974.
14. Kamely,D., and Rudland, P. Nutrient-dependentArrest of Fibroblast
Growth is Partially Reversed by Insulin but Not Fibroblast Growth Factor.
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CANCER RESEARCH VOL. 38
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1978 American Association for Cancer Research.
Mechanism of Growth Arrest of Chemically Transformed Cells
in Culture
Harold L. Moses, Jacqueline A. Proper, Mary E. Volkenant, et al.
Cancer Res 1978;38:2807-2812.
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