[CANCER RESEARCH 37, 514-523, February 1977]
Effects of Serum Concentration on the Expression of
Carcinogen-induced Transformation in the
C3H/IOT'/2 CL8 Cell Line'
John S. Bertram
Department of Experimental Therapeutics, Grace Cancer Drug Center, Roswell Park Memorial Institute, Buffalo, New York 14263
SUMMARY
The G3H/10T1/2 CL8 cell line (10T1/2) is being widely
used as a quantitative assay system for chemical and physi
cal carcinogens. 10T1/2 cells, but not their transformed
counterparts, exhibit a decreased final saturation density
with decreasing serum concentration. Exposure of carcino
gen-treated cultures to 5% serum 8 days posttreatment led
to a 2- to 6-fold enhancement in transformation frequency in
comparison with cultures maintained in 10% serum
throughout. Exposure 14, 21, or 28 days posttreatment also
caused enhancement of transformation frequency, pro
vided a sufficient time fan expression of the malignant phe
notype was allowed. Exposure to 5% serum 1 or 2 days
posttreatment did not lead to significant enhancement of
transformation frequency. In contrast, exposure to 15 or
20% serum after 8 days virtually abolished the expression of
malignancy; however, this inhibition could be reversed by
5% serum. Momphobogically transformed foci isolated from
cultures exposed to 5% serum produced clones in agarose
with the same frequency as did foci isolated from cultures
exposed to 10% serum.
Reconstruction experiments, utilizing confluent mono
layers of 1OT'/2 cells overlaid with transformed cells, dem
onstrated that the growth of transformed cells decreased
proportionally with the log of serum concentration. This
effect was not caused by depletion of medium and was
dependent upon the presence of 1OT'/2cells. It is concluded
that the expression of malignancy in this system is governed
by the serum-modulated cell density of the mass of non
transformed cells in the culture.
malignant transformation induced by a variety of agents,
including polycyclic hydrocarbons (27), chemothenapeutic
agents (17), X-rays (33), and methylating agents (4). It is also
being used to study procedures or agents which modify the
carcinogenic response, such as phase of the cell cycle (4),
tumor promoters (24), actinomycin D (3), modifiers of car
cinogen metabolism (25), and interactions between chemi
caband physical carcinogens (32).
A disadvantage of the lOT'!2 system and other established
transformable cell lines (9, 19) is that 3 to 5 weeks are
required from the time of carcinogen treatment to the devel
opment of transformed foci. Although the reasons for this
long latent period are not known, it seemed possible that
the use of a selective medium for the growth of transformed
cells might improve this assay system by increasing its
speed and sensitivity. A limited study had previously shown
that the saturation density achieved by 1OT'/2 cells was
sensitive to serum concentration (unpublished results), that
transformed cells exhibited a much higher saturation den
sity than did 10T1/2cells (4, 5, 27), and that the TF was in
versely proportional to cell density at the time of treatment
(27). It therefore appeared feasible to use a serum concen
tration suboptimal fan nontransformed 1OT'/2 cells as a se
lective medium to aid the development of transformed cells.
This paper describes the development of such a selective
medium and demonstrates that in the 10T1/2system non
transformed cells have the capacity to inhibit replication of
transformed cells, this capacity being modulated by serum
concentration. A preliminary report of the results has been
presented (2).
INTRODUCTION
MATERIALS AND METHODS
In 1973, a new mouse fibmoblast cell line designated
10T1/22was developed, which was highly sensitive to post
confluence inhibition of cell division and in which malig
nant transformation could be induced by chemical carcino
gens in a dose?dependent manner (27, 28). This cell line is
currently being used in many laboratories as a tool to study
Cells. The 1OT'/2 cell line derived from mouse embryo
fibroblasts (27) was used exclusively for the experiments
involving assay of chemically induced malignant transfon
mation. Stock cultures were maintained as previously de
scnibed (28), and cells were used between the 5th and 15th
passage. In the experiments measuring the growth of malig
nantly transformed cells under different conditions, mom
phobogically transformed colonies were isolated by ring
cloning from cultures previously treated with MGA amNacetoxyacetylaminafluomene and were subcultured in a
manner similar to that of the 1OTV2cells. These transformed
lines were shown to be tumomigenic by injection into immu
, Supported
in part by Grant CA 13038 from the National Cancer
Institute,
USPHS.
2 The
abbreviations
used
are:
lOT'!2,
C3H/1OT'/2
CL8;
TF,
transformation
frequency; MCA, 3-methylcholanthrene; BME, Eagle's basal medium; HIFCS,
heat-inactivated fetal calf serum ; DMBA, 7,12-dimethylbenz(a)anthracene.
Received June 16, 1976; accepted November 12, 1976.
514
CANCERRESEARCHVOL. 37
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1977 American Association for Cancer Research.
Serum Effects on Transformation
nosuppressed G3H mice as described previously (27). All
cell lines were negative for mycoplasma contamination as
judged by routine screening for growth on mycoplasma
agar (Grand Island Biological Co., Grand Island, N. Y.) and
by absence of cytoplasmic labeling as shown by automa
diography after a 1-hr pulse with tnitiated thymidine, 1 pCi/
ml, as previously described (4).
Culture Medium. BME and HIFCS were obtained from
Grand Island Biological Co. Stock cultures were maintained
in BME supplemented with 10% HIFGS without antibiotics
as previously described (27). Experimental cultures were
maintained in BME containing penicillin (100 units/mI) and
streptomycin (50 @g/ml)and supplemented with concentra
tions of HIFCS varying from 0.1 to 20% by volume as stated
in individual experiments.
Growth Studies. Cells were harvested from late logamith
mic-phase cultures by 4 mm exposure to 0.1% trypsin
(Grand Island Biological Co.) in phosphate-buffered saline
(0.8% NaGI, 0.115% Na2HPO4, 0.02% KH2PO4, 2H@O,and
0.02% KGb,pH 7.4). Cells were seeded at either 5 x 10@,10k,
or 5 x 10@cells/60-mm Petni dish, as indicated, in 5 ml of
BME supplemented with the appropriate serum concentra
tion. All counts were performed at the stated intervals after
seeding, by trypsinizing cells to form a monodispemse sus
pension and diluting to 10 ml with phosphate-buffered sa
line containing 10% HIFGS to prevent agglutination. Sus
pensions were electronically counted using a Model Z
Coultem Counter. Results represent the mean of 2 counts
each on 4 dishes.
Transformation Assay. Cells taken from late logarithmic
phase cultures by trypsinization were plated at a concentra
tion of 10@celbs/60-mm Petni dish in 5 ml BME supple
mented with 10% HIFCS and antibiotics. A 2nd series of
replicate cultures containing 200 cells/dish was set up at
this time from the same cell suspension. After 24 hr, when
the cells had attached but had not yet begun to divide,
dishes were treated with the appropriate carcinogen. Rou
tinely, fan any given concentration of carcinogen, 12 mepli
cate cultures seeded at 10@cells/dish were treated for the
transformation assay, while 4 cultures seeded at 200 cells/
dish were used to assess carcinogen-induced toxicity.
DMBA and MCA (Sigma Chemical Go. , St. Louis, Mo.) were
freshly dissolved in dry acetone before use and were di
rectly applied to the cultures in 25 @b
of solution delivered
with a micropipet. Control cultures received 25 @l
acetone
(0.5% final concentration), which was nontoxic. DMBA and
MCA were removed after 16 hm,and fresh medium contain
ing 10% HIFCS was added. X-inmadiation was applied at a
dose mateof 170 mads/min from a G.E. Maxitmon 300 Unit
(300 kV, 20 ma, 0.25-mm Cu filter). Cultures were irradiated
in aim under about 5 mm medium. Controls were sham
irradiated.
For studies an the temporal effects of different serum
concentrations on the final expression of transformation,
cell plating and treatment were performed under identical
conditions of 10% HIFCS; dishes were only randomized
after a minimum period of 24 hr after removal of cancino
gen. In this way exposure to carcinogen was uniform for all
experiment groups, the only variable being the subsequent
manipulation of serum concentration. For studies designed
to validate these results for other batches of HIFCS, cells
were plated, treated, and maintained in the indicated Se
rum. All cultures used fan the transformation assay were
mefedweekly for 5 weeks with BME, supplemented with the
appropriate serum concentration. Cultures used to assess
carcinogen-induced
toxicity were treated identically to
matched cultures used for the transformation assay but
were maintained fan 8 days only. At the end of these time
periods cultures were washed with 0.9% NaGI solution,
fixed with methanol, and stained with 10% giemsa in phos
phate-buffemed saline diluted 10-fold with water.
Scoring for Morphological Transformation and for Toxic
ity. All dishes were examined under a Bausch and Lomb
Stereo Zoom microscope, and transformed foci were
scored as Type II or Type Ill as previously described (4, 27).
Total surviving colonies were counted in the cultures
seeded at 200 cells/dish and fixed after 8 days. The
plating efficiency and TF were calculated as previously
described (4).
Both Type II (moderately oncogenic) and Type Ill (highly
oncogenic) foci have been included in the total for the
following reasons: (a) Type II foci always comprised 10 to
20%
of
the
total
transformed
foci
(the
remainder
being
Type
Ill) regardlessofcarcinogen
orserum concentration; (b) low
serum may alter the morphology of transformed foci and
bias scoring; and (C)the previously observed conversion of
Type II to Type Ill may be accelerated or delayed by low
serum again biasing scoring.
Colony area determinations were made using a standard
stemeological technique (cf. Ref. 35). Stained plastic Petni
dishes were placed in a photographic enlarger (Besselem
Model 45M) and projected on a white surface containing a
uniform grid of points (1 point = 8.25 sq mm at x4). Total
colonies and points coincident with them were counted for
each dish. The average colony area was then calculated.
Cloning in Soft Agarose. An adaptation of the method of
MacPhemson and Montagnier (23) was used. Cells were
suspended in 4 ml of 0.4% agarose (Type II; Sigma) in BME
and 10% HIFGS and rapidly poured oven 4 ml of a bottom
layer of 0.8% agarose in BME and 10% HIFGS in 60-mm
Petni dishes. Dishes were incubated at 37°in 5% CO2for up
to 4 weeks. Colonies were examined under dark-field illumi
nation, and those containing at least 32 cells were scored as
positive. In some experiments cells were stained using 2-(piadophenyl)-3-p-nitrophenyl-5-phenyltetrazolium
chloride
(Aldrich Chemical Go., Milwaukee, Wis.) essentially as de
scnibed by Schaeffem and Friend (29), except that a 48-hr
incubation with dye was required.
Reconstruction Experiments. Confluent cultures of 10T1/2
cells were prepared by seeding 10@cells in the stated serum
concentrations. When confluent, as judged micmoscopi
cabby,5 ml of fresh medium supplemented with the appra
pniate serum concentrations were added to the confluent
manolayers and to an equal number of empty dishes. All
dishes were seeded 24 hr later with the stated number of
transformed cells contained in 0.1 ml BME and 10% HIFCS.
Dishes were incubated for about 8 days without further
medium change. At this time dishes were fixed and stained
far analysis of colony size and number, while in the case of
the mixed cultures, 2 additional dishes were trypsinized,
and the cell pellet, obtained by centnifugation, was plated
into agarose as described above.
FEBRUARY1977
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1977 American Association for Cancer Research.
515
J.S. Bertram
RESULTS
Effects of Serum Concentration on Growth Rate and Satu
ration Density
In Charts
10T1/2 and
1 and 2 are presented
growth curves far parental
2 malignantly
transformed
cell lines grown
in
SemaA and B, respectively. Several conclusions can be
drawn from these growth curves. First, increasing the se
rum
concentration
from
2.5 to 20%
had
no effect
an the
growth rate of 10T1/2cells or T1OT1/2cells; doubling times
over
the
logarithmic
portion
of the
growth
curves
were
about 16 hr for Sera A and B. Second, transformed cells
have the same doubling time as 1OT'/2cells. Third, the final
saturation density achieved by 10T1/2cells is dependent upon
serum concentration. In Chart 2 it can be seen that reducing
the serum concentration from 20 to 10%, to 5%, and to 2.5%
caused a reduction in final saturation density of 38, 50, and
68%, respectively. Fourth, T1OT1/2cells show no change in
saturation density with decreasing serum concentration
from 20 to 2.5%. This is mast evident for MGA T1OT1/2cells.
Fifth, while different
commercial
lots of serum may be iden
tical in their abilities to promote growth and allow high
saturation densities when tested in 10T1/2cells at a cancen
tration of 10%, differences became apparent at lower serum
concentration. Thus 5 and 1% Serum A achieved a satuma
tion density equivalent to 10 and 2.5% Serum B, mespec
tively.
With this as a background,
we next investigated
whether
serum levels producing low saturation levels in lOT1!2cells
could be used as a selective agent far recently transformed
cells. In a preliminary experiment utilizing Serum A, cells
I
U)
0
E
E
0
@0
U)
-J
-J
Id
C)
DAYS AFTER SEEDING
Chart 2. Growth curves for cells cultured in Serum B. On Day 0, 5 x 10@
1OT'/2cells (leftpanel) or MCA-transformed cells (rightpanel) were seeded in
60-mm Petri dishes in BME supplemented with the following concentrations
of HIFCS, Lot A750318: 20%, 0; 10%, L@;5%, 0; 2.5%, x; 1%, 0; and 0.1%,
..
Cultures
were
refed
with
the
appropriate
serum
every
3 to 4 days.
Total
cells per dish were determined starting 24 hr after seeding. Results repre
sent the means of 4 dishes.
were plated and treated with a transforming dose of DMBA
in 10% HIFCS, maintained for 7 days at this serum level, and
then maintained for a further 4 weeks on 2.5% HIFCS. The
result of this manipulation was that the TF increased by a
factor of 14. Unfortunately, a limited supply of Serum A
precluded repeating this observation. All subsequent exper
iments, except those reported in Table 2, were performed
with Serum B.
Modulation of TF by Serum Concentration
I
U)
0
E
E
0
tO
U)
-J
-J
Id
U
DAYS AFTER SEEDING
chart 1. Growth curves for cells cultured in Serum A. On Day 0, 10@1OT'/a
cells (left panel) or N-acetoxy-acetylaminofluorene-transformed
cells (right
panel) were seeded in 60-mm Petri dishes in BME supplemented with the
following concentrations of HIFCS, Lot C944126: 10%, i@;5%, 0; 1%, 0; 0%,
A. Cultures were refed with tha appropriate serum every 3 to 4 days. Total
cells per dish were determined starting 24 hr after seeding. Results represent
the means of 4 dishes.
516
Temporal Effects. To investigate the temporal relation
ship between the time of treatment, the time of exposure to
a bow concentration of serum, and the final TF, a large
number of replicate cultures were treated with DMBA at 1.0
p.g/mI in 10% serum, and at intervals after treatment ran
dam groups of 12 dishes were exposed to medium contain
ing 5% serum. Cultures exposed to 5% serum up to 8 days
after treatment were all fixed on Day 36. The remaining
dishes were further randomized; half of each group were
fixed on Day 36, and the remainder were fixed after 28 days
in 5% serum to allow time fan expression of malignancy.
This concentration of Serum B was chosen to give approxi
mately the same final saturation density as 2.5% Serum A,
which in preliminary experiments markedly enhanced the
TF. A biphasic response in the expression of morphological
transformation was observed. When cells were placed in
low serum shortly after treatment (Day 1 or 2), the observed
transformation was not significantly higher than that seen in
controls maintained throughout in 10% serum. However,
when cells were placed in low serum on Day 4 on later,
significant enhancement of transformation was observed
(Table 1). In cultures placed in 5% serum on Day 21 or later
and fixed after the standard 36-day period, transformation
CANCERRESEARCHVOL. 37
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1977 American Association for Cancer Research.
Serum Effects on Transformation
Table 1
Enhancement of transformation as a function of time of addition of 5% HIFCS
Replicatecultures in BME + 10%HIFCSwere treated 24 hr after plating with DMBA(1
pg/mI) or acetonealone. After 16hr (Day1) all cultures were supplied with fresh medium
containing 10% or 5% HIFCS. At increasing times posttreatment, the group of 12 cultures
were exposedto 5% HIFGSas indicated. All cultures were fixed, stained, and scored for
malignantly transformed foci (types lb + Ill) 36 days after treatment or after 28 days in 5%
HIFCS.
Dayspost
treat
in
of
10% se
in
5% se
TreatmentNo.
dishesment rumDays rumFoci/dish―Area/focus―
mm)0.5%Acetone11360000.5%Acetone1282800DMBA123602.9
@
(sq
2.41DMBA121353.9
±0.7323.6
±
3.52DMBA122343.7
±0.4530.4
±
0.98DMBA124316.1
±
0.83DMBA128287.5
±0.6328.2
±0.65―27.9
3.06―DMBA6
±078b34@7
±
±2.2
44.7 ±
±2.42
5.0DMBA3
614
1421
284.7
5.0 ±0.4922.3
13.4DMBA6
421
2114
283.3
±0.40
6.0 ±1.94―24.8 66.8 ±
628
287
21―3.0 6.2 ±0.96―25.9 65.3 ±12.70
±6.75
±0.69
a Mean
± SE.
b Significantly
C Fixed
after
different;
only
21
p
days
=
to
0.05
prevent
from
cultures
maintained
overlapping
declined to control levels. However, if an extended period
was allowed for the expression of the malignant phenotype,
the degree of transformation was again about twice that
seen in the treated controls maintained in 10% serum
throughout. Thus the repression of expression of the trans
formed phenotype by 10% serum appears reversible. In
these experiments results have been expressed as trans
formed foci/dish, since all cultures received identical treat
ment with DMBA. The plating efficiency was 29.8 ±0.7 far
controls and 14.8 ±1.1 for dishes treated with DMBA (1 @g/
ml). These values were not significantly altered by exposure
to 5% serum on Days 1, 2, on 6 after treatment.
The reversibility of the serum-induced repression was
further examined in a separate experiment in which cultures
were treated with a 0.5 j.@g/mIdosage of DMBA under
standard conditions and after 8 days were exposed to 5, 10,
on 15% HIFGS, to allow maximum, moderate, or zero
expression of the transformed phenotype, respectively. It
will be seen in Table 2 that even after 22 days in 15% serum,
which was nonpermissive for the transformed phenotype,
exposure to the permissive concentration of 5% serum al
bowed full expression of all the initiated but latently trans
formed cells. The apparent loss of some of the transformed
cells in cultures maintained for 28 days in 15% HIFCS may
be an artifact, since these cultures were fixed after 3 weeks
instead of the usual 4 weeks in 5% serum, because of
problems in maintaining cultures for these long periods.
When scoring fixed and stained cultures for momphabogi
cabtransformation, it became apparent that, not only were
there more foci in cultures placed in 5% serum after 8 days,
but the foci tended to be larger. This was confirmed by
estimating the area of each focus (Table 1). While a tend
ency for larger foci was seen in all cultures exposed to 5%
of
throughout
in
10%
serum.
foci.
2Reversibility
Table
transformedphenotypeReplicate
of the serum-induced inhibition of the
DMBA(0.5
cultures in BME + 10% serum were treated with
12cultures
j@g/ml) or acetone as a control. After 8 days groups
culturesexposed
were exposed to 5, 10, or 15% serum. Those
of
afurther to 5 and 10%serum were maintained at that level for
culturesexposed
28 days, then fixed,
to5%
stained,
and scored.
Those
to 15%serumwereeither fixed after 28daysor exposed
to5%serum at increasing time intervals after treatment. Exposure
5%serum
serum was for 28 days, except for those cultures exposed to
andstained
after 36 days which for technical reasons were fixed
after only 21 days.Serum
Final se
concentra- rum con
tion
centrationposttreat8 days
foci/Treatment
S.E.Acetone
ment
0Acetone control
10
(days post-
Transformed
treatment)
dish ±
10
0DMBA control
5
5
(NS)@DMBA
5
5
0.47DMBA
10
10
0DMBA
15
15
(NS)DMBA
(NS)DMBA
15
15
5 (15)―
5 (22)
1.7 + 0.35
1.9 + 0.41
(NS)DMBA
15
15
5 (29)
5 (36)
1.6 + 0.39
1.2 + 0.36
a NS,
not
b Numbers
increasing
significantly
in
parentheses,
time intervals
2.0 + 0.31
1.2 +
different.
cultures
after treatment
exposed
to
5%
serum
at
with 15% serum.
serum, only cultures that received 5% serum 8 days after
treatment had statistically significantly larger foci than
those developing in cultures maintained in 10% serum
throughout. An analysis of the distribution of the area/focus
in treated cultures maintained in 10% serum throughout
and in those exposed to 5% serum after 8 days (Table 1,
FEBRUARY1977
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1977 American Association for Cancer Research.
517
J. S. Bertram
Lines 3 and 7, respectively) is shown in Chart 3. The in
creased number of transformed foci is clearly evident, and it
can be seen that the majority of this increase is associated
with foci of an area equal to or greater than those foci
developing in 10% serum. The increased frequency of small
and large foci developing in 5% serum may be a conse
quence of late development of foci in the former case and,
in the latter case, to an increased tendency fan large foci to
merge and be scored as 1 focus. The gross appearance of
stained control and treated cultures is shown in Fig. 1.
Cloning Efficiency in Soft Agarose. To determine
whether foci developing in 5% serum had the same poten
tial for tumonigenicity as foci developing in 10% serum, the
capacity for transformed cells to produce foci in soft aga
rose was measured. Momphobagically transformed foci in
I
Areo/foajs(mm)
Chart3. Sizedistributionof transformedfoci developingin 5%serumand
in 10% serum. The area of each transformed focus developing in DMBA (1
@g/ml)-treatedcultures was determined for cultures exposed to 5% serum 8
days after treatment (open bars) and for cuftures exposed to 10% serum
(dottedbars) throughoutthe 36-dayduration of the experiment.For further
details see Table 1. Error bars represent means ±SE.
Fig. 1. Gross appearance of fixed and
stained 60-mm Petri dishes. a, Control cul
tures exposed to 5% HIFCS 8 days after
acetone treatment; b , DMBA-treated cul
tures exposed to 5% serum 2 days after
treatment; c, DMBA-treated cultures ex
posed to 5% serum 8 days after treatment;
d, DMBA-treated cultures
maintained
throughout in 10% serum. All dishes were
duced by DMBA were isolated at random from Experiments
A and B in which treated cultures had been exposed to
either 10% serum throughout or to 5% serum 8 days after
treatment as described above. The results shown in Table 3
demonstrate that about 70% of the transformed lines were
cbonogenic in soft agarose 4 weeks after isolation. The
morphology of the lines that were cbonogenic in aganose
differed from those that were not. Clonogenic lines showed
a high degree of piling up and crossing oven of cell proc
esses, whereas nonclonogenic lines exhibited high cell
densities but little tendency to pile up. Parental 10T1/2cells
obtained by passaging of the original line used to seed
Experiments A and B failed to replicate in soft agarose and
remained as single cells. However, cells were still viable at
this time as judged by staining with a tetnazolium salt as
described.
Dose-Response Relationships. The dose-response char
actenistics of this effect were next examined using a range
of doses of DMBA from 0.01 to 5.0 pg/mI and a mangeof
serum concentrations from 2.5 to 20%. All cultures were
treated and maintained in 10% HIFCS until 8 days after
treatment when the appropriate concentration of serum in
BME was added. The results plotted in Chart 4 clearly show
that 2.5 and 5.0% serum caused an equivalent enhancement
of TF over most of the dose mangetested in comparison with
cultures maintained in 10% serum. At the DMBA level of
0.01 and 0.05 pi/ml, the 3 concentrations of serum gave
almost identical results; this may be simply due to sampling
errors of the small number of transformed foci produced by
these low doses of carcinogen. This experiment also dem
onstrated that if cultures were placed in double the serum
concentration (20%) 8 days after treatment, the expression
of the malignant phenotype was virtually abolished. Only 1
transformed focus was observed in the entire series of 84
dishes exposed to the wide range of DMBA concentrations.
Groups of 12 (solvent only) control dishes maintained in the
b
takenfrom theexperimentreportedin Table
1. Note the greater number of foci, their
greater size, and the decreased staining
density of the background in c as compared
to d. All dishes were fixed and stained with
giemsa 36 days after treatment. For further
details, see the text.
I
518
CANCERRESEARCHVOL. 37
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1977 American Association for Cancer Research.
Serum Effects on Transformation
3Enhancement
Table
carcinogensCells of TFfor different serum batches and different
groupsof
wereplated in BMEsupplementedwith 10%of the appropriate HIFCS,and
16hr
12 cultures were exposed to the stated carcinogen 24 hr after plating. After a further
aftertreatment
all cultures received fresh medium + 10% of the appropriate HIFCS. Eight days
all cultures wereexposedto BMEsupplementedwith the statedfinal concentra
tion
treatment.Plating
of the appropriate HIFCS.All cultures were fixed and stained36 daysafter
celldensity
efficiencies were determined in cultures seeded in 10% HIFCSat a bower
observedin
and were fixed and stained 8 days posttreatment.
No transformation
was
control cultures treated with solvent alone or sham irradiated
shown).Plating
(data not
En
effi-
mentHIFCSFinal HIFCSconcenfactorBlot
tration (%)
2.5A750318 Experiment1 10
3.5B
5
Carcinogen
DMBA(1 pg/mI)
hance
ciency %
control
44
TF
1.4
Experiment 2 10
1.75
DMBA (1 @g/ml)
47
1.8
Experiment3 10
2.45
DMBA (1 pg/mb)
53
1.6
Experiment4 10
1.85
DMBA(1 @g/ml)
40
1.1
10
2.05
MCA(5 @g/ml)
95
0.5
10
4.25
X-ray(400 rads)
33
0.2
10
5
10
2.15
DMBA(1 @g/ml)
51
0.5
MGA(5 @.&g/mI)
96
0.9
10
DMBA(2.5 @g/ml) 36
0.2
3.0B
3.8B
2.0B
1.0B
1.4C
1.9A156619
0.9C
2.0D
5.8A560126
5
1.4
Validation with Different Sera and Different Carcinogens
>.
w
a
Id
z
0
0
Ii.
U)
z
4
I-
DMBA pg/mb
Chart 4. Effect of serum concentration on expression of the transformed
phenotype in cells treated with a range of DMBA concentrations. Replicate
cultures in BME supplemented with 10% Serum B were treated for a period of
16 hr with the stated concentrations of DMBA. Eight days after treatment
groups of 12 cuftures for each DMBA concentration were exposed to the
followIng concentration of HIFCS, Lot A750318 for the duratIon of the experi
ment: 20%, 0; 10%, @;
5%, 0; and 2.5%, X. The TF was calculated as de
scribed in the text.
appropriate serum concentrations contained no tnans
formed colonies. Saturation densities obtained by trypsiniz
ing and counting 4 control cultures from each group were
8.1, 6.9, 4.7, and 2.7 x 10@cells/60-mm dish for cultures
maintained for 4 weeks in 20, 10, 5, and 2.5% HIFCS, me
spectively.
To determine whether enhancement of expression of
morphological transformation by low serum levels would
occur with other commercial lots of HIFCS, several serum
lots were tested for growth promotion properties. Those
supporting saturation densities of about 5 and 3 x 10@celbs/
60-mm dish when tested at concentrations of 10 and 5%,
respectively, were purchased. Two of 4 lots tested satisfied
these criteria; growth curves obtained using these sema,
designated SemaC and D, demonstrated saturation densi
ties in 10% serum of 4.8 and 5.1 x 10@,and 3.0 and 2.8 x 10@
in 5% serum, respectively. 1OT'/2 cells were grown in these
semafor 1 week; then they were plated, treated, with carcin
ogen, and maintained for a further 8 days in 10% serum as
before. After this period cells were maintained in 5% of the
respective semauntil fixed and stained. In Table 4 is pre
sented data on the 3 different semaused in this investigation,
together with data on the use of 3 different carcinogens.
Enhancement of TF on exposing cells to 5% serum 8 days
after treatment was observed for all tested combinations of
carcinogens and serum. Enhancement factors ranged be
tween 1.9 for DMBA and Serum C to 5.8 for DMBA and
Serum D.
The data far 4 experiments utilizing Serum B and DMBA
at 1 @g/mlare also presented in Table 4. Although expeni
ments were conducted many months apart it can be seen
that the variation in TF and in the enhancement factor is
small. When these data are combined with the data pre
sented in Table 1 and Chart 4, the mean TF in 10% serum is
1.5 ±0.2, in 5% serum it is 3.1 ±0.4, while the mean
enhancement factor is 2.2 ±0.2.
FEBRUARY1977
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1977 American Association for Cancer Research.
519
J. S. Bertram
Table 4
fociMCA-induced,
Cloning efficiency in soft agarose of morphologically transformed
ExperimentsA
morphologicalby transformed foci were isolated by ring cloning from
cells/dish
and B. Cloneswere cultured for 4 weeksin BME + 10%HIFCSand werethen plated at 10@
fromcultures
into soft agarose, as described in the text. Clones Al to A4 and B1 to B7 were isolated
toB14
exposed to 5% HIFCS 8 days after carcinogen treatment, while clones A5 to A7 and B8
inagarose
were isolated from cultures maintained in 10% HIFCS throughout.
Cloning efficiency
wererecorded.Serum
was scored 4 weeks after plating;
32 cells or more)
only barge colonies
(approximately
effiClone
centratian (%)
Serumcon-
con
centraClone
ciencyAltion (%)
l0@A2
5
Soft agarose cloning
ciency
Soft agarose cloning
1 .5 x 10@
A5
10
1 .2 x
10@A3 5
1.4 x 10@
A6
10
4.2 x
10A4
5
1.6 x l0@
A7
10@B2
5
4.2 x lO@
B8
10
1.5 x
l0@B3
5
2.3 x l0@
B9
10
6.3 x
4.3 x 10-i
BlO
10
2.1 x
10
10
2.1 x
2.5 x
effi
5Bl
l0@B4
5
10B5
5
10B6
10@B7
5
5
5
<10
Bll
1.7 x l0@
1.5 x 10@
l0@Overall
<10@
Bl2
B13
B14
8/11 positive
Overall 7/10 positive
Reconstruction Experiments
@
Representative experiments in which the effects of in
creasing serum concentration an the growth of 2 trans
formed cell lines are measured in the presence and in the
absence of parental 1OT'/2cells are shown in Chart 5. Trans
formed Line A was a long-established tumomigenic line mi
tially isolated from a MCA-treated culture and was in its
72nd passage when tested. Its growth curves in different
serum concentrations are shown in Chart 2. Transformed
Line B was in its 2nd passage when tested , having been
isolated 4 weeks previously from a MGA-tmeatedculture.
On increasing the serum concentration from 2.5 to 20% in
the presence of 1OT'/2 cells (Chart 5, lower panels), both
lines showed an approximately straight-line decrease in
colony size and in the number of cbonogenic cells develop
ing in soft agarose. In both cases the number of colonies
remained constant within the limits of error on increasing
the serum concentration from 2.5 to 10%. In 20% serum,
Clone A produced no colonies, although viable transformed
cells were present as shown by the production of clones in
soft agarose. The production of clones in soft agarose is
limited to transformed cells and has been shown in separate
experiments to be approximately quantitative with incmeas
ing seeding density. In the absence of 10T1/2cells (Chart 5,
upperpanels), the response to increasing serum concentra
tion was biphasic and quite different from that observed in
the presence of 1OT'/2cells. Both lines showed an increase
in colony size and number when grown in 5% as opposed to
2.5% serum, while growth in 20% serum produced colonies
similar in size and number to those developing in 2.5%
serum.
The inhibitory effect on the growth of transformed cells
exhibited by high serum concentration is thus clearly me
diated via 10T1/2cells. Since at confluency in 20% serum the
saturation density of 10T1/2cells is 10 times that attained in
520
.@
I
3
20
t
I000
I
I
4000
20
,0
B2
I
1000
2
@0
I
Sirum
Concsnlrotio,, S
Ssrum Concintration S
Chart 5. Effect of serum concentration on the growth of transformed cells
in the presence and in the absence of 1OT'/2 cells. Confluent cultures of
10T1/2cells were prepared by seeding 10-' cells in the stated serum concen
trations. When confluent, as judged microscopically, 5 ml of fresh medium
supplemented with the appropriate serum concentration were added to the
confluent monolayers and to an equal number of empty dishes. All dishes
were seeded 24 hr later with transformed cells. Dishes were incubated for
about 8 days withoutfurther medium change. At thistime dishes were scored
for colony number (ti) and colony size (0), and in the case of the mixed
cultures, the contents of 2 dishes were seeded into agarose for determination
of the number of cbonogenic cells (0). Clone A (left panels) was a malignantly
transformed MCA line at Passage 72 when used; Clone B (right panels) was a
recently isolated MCA-transformed line. Upperpanels (Al, 81), cells seeded
in the absence of 1OT'/2 cells; lower panels (A2, 82) cells seeded onto
confluent monolayers of l0TV@cells. Results represent the mean ±SE. for
colony number and area and the mean of 2 cultures each for the cbonogenic
ity in agarose.
2.5% serum, (see Chart 2), there appeared a possibility that
this large cell population may be causing rapid depletion of
the growth medium, thereby inhibiting the growth of the
transformed cells. To exclude this possibility, the growth
promoting properties of new medium containing 5 on 20%
serum were compared with those of used medium contain
CANCERRESEARCHVOL. 37
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1977 American Association for Cancer Research.
Serum Effects on Transformation
ing 5 or 20% serum. The used medium had been removed
from cultures permissive amnonpermissive, respectively, for
the growth of transformed cells. The results shown in Table
5 demonstrated cleanly that the used medium was superior
to new medium in supporting the attachment and growth of
transformed cells. Thus nutritional depletion of the growth
medium cannot explain the results presented here.
DISCUSSION
The data clearly show that fetal calf serum depresses, in a
dose-dependent manner, the expression of the transformed
phenotype in cells transformed by a variety of carcinogens
and that this depression can be reversed by exposing cells
to low concentrations of serum for a time period sufficient to
allow for the expression of transformation in latent cells and
for the subsequent division of these latent cells to farm
morphalagically transformed foci. Although the time course
of expression of the transformed phenotype after exposure
to 5% serum has not been closely studied, Table 1 indicates
that the process requires 3 to 4 weeks. Routine microscopic
examination of cultures indicated that the macroscopic foci
present after 3 to 4 weeks arose from microscopic foci as
previously observed in the standard assay system. Thus it
can be concluded that the transformed foci, revealed by
exposure to law serum, arise from individual cells or small
clusters of cells and not from preformed macroscopic foci.
This implies that the event leading to loss of contact inhibi
tion and, thus, the development of transformed foci occurs
later than 8 days after treatment and that high serum levels
either inhibit this event on inhibit some subsequent event
beading to entry of cells into the division cycle.
Table 5
Comparativeeffects
thegrowth of new medium versusused medium on
cellsSeven-day-old
and plating efficiency of transformed
ofl0T@
culture
medium
was aspirated
from
cultures
andsubsequently
cells grown to confluency in 20% and in 5% serum
thetime
overlaid with the transformed cell line A or B. At
of aspiration,
the transformed
cells had produced
large cob
seeChart
nies in 5% serum but not in 20%serum (for experimentdetails
emptyPetni
5). The used medium was centrifuged and poured in
mediumcontaining
dishes; identical dishes were prepared using fresh
placedin 5% or 20%serum. Transformedcells were then
ofnew
respectivedishes of used medium and corresponding dishes
withoutmedium
medium, and cell growth was measuredafter 6 days
change.Aga
Cell
roseline
cbonesbAMedium
1000A 5% new
2100A 5% used
1200A 20% new
1900B 20% used
Plating efficiency (%)“Size (SEmm)―
46 ± 2.5
0.8 ±0.2
58 ±12.0
2.0 ±0.2'
53 ± 4.2
1.0 ±0.1
50 ± 6.3
1.7 ±0.1C
380B 5% new
44@B 5% used
90B
20% new
20% used
51 ± 2.8
78 ± 3.5'
32 ± 2.1
60 ± 4.2'
2.2 ±0.2
3.0 ±0.3e
2.2 ±0.2
2.1 ±0.2
1 focus/dish.
420
a Mean ±S.E. of 4 dishes.
‘I
Mean
of
total
colonies
in
2
dishes.
r Statistically different from corresponding value in new me
dium.
Untreated cultures become confluent and cease replica
tion about 8 to 10 days after seeding (Charts 1 and 2; Ref. 4),
and while the situation in treated cultures has not been
closely studied, the lack of dense foci and mitotic figures
until 3 to 4 weeks after treatment implies that the initiated
but latent cells are also subject to postconfluence inhibition
of cell division. Thus it must be provisionally assumed that
the serum-sensitive event leading to loss of contact inhibi
tion occurs in the absence of cell division. Presumably, this
event is responsible for the long latent period between
treatment and development of transformed foci previously
reported for mouse prostate cells (9) and BALB/3T3 cells
(19).
A direct action of serum on latently transformed cells in
carcinogen-treated cultures appears most unlikely for the
following reasons. First, malignant cells are not responsive
to changes in serum beveloven the entire range of concen
trations causing expression or repression of the malignant
phenotype (2.5 to 20%) (Charts 1 and 2). Although growth
curves constructed using established transformed cells may
not be valid models for the study of recently transformed
cells, the growth of neithenthe parent 10T1/2cells nor T1OT'/2
cells is stimulated by law serum. The finding that fetal calf
serum retards the development of spontaneous transforma
tion is probably not applicable to the present investigation,
since those studies were conducted with primary cultures
(8, 13, 26). Second, in reconstruction experiments utilizing
confluent monobayers of 10T1/2 cells overlaid with estab
lished lines of transformed cells with or without a confluent
monolayer of 1OT'/2 cells, the transformed cells were in
hibited by increasing serum concentration only in the pres
ence of 10T1/2cells (Chart 5), and this effect was not due to
nutritional depletion of the medium (Table 5).
Low serum could potentially increase the apparent TF by
causing increased seeding from preexisting foci and, thus,
enhance the formation of satellite foci. This is also consid
ered most unlikely, since a size analysis of transformed foci
developing in 10% as opposed to 5% serum demonstrated
that the majority of the increased number of foci developing
in 5% serum occurred in a size bracket equal to or larger
than the majority of foci developing in 10% serum (Chart 3).
Seeded foci should be smaller than the parent focus. If
seeding is responsible for the increased number of foci in
low serum, then the use of higher serum concentrations
should be expected to decrease the TF to a plateau nepre
senting the true TF. This did not occur; serum concentra
tions of 15 and 20% decreased the TF to essentially zero
(Table 2; Chart 4). Seeding requires the presence of at least
Finally, reconstruction
experiments
have dem
anstrated unequivocally that established transformed cells
are also inhibited by serum in the presence of 1OT'/2 cells.
Furthermore, these studies show that the number of foci
remains constant until high serum concentrations are
reached (Chart 5). Thus seeding is not observed in recon
struction experiments.
This study was originally initiated to determine whether
recently transformed cells could be placed at a selective
advantage over 10T1/2cells by the use of low serum . While a
selective advantage between the 2 cell types will certainly
be accentuated by placing cells in law serum, the virtually
FEBRUARY1977
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1977 American Association for Cancer Research.
521
J. S. Bertram
complete inhibition of growth of transformed cells caused
sponse. Kakunaga (20) concluded from his studies on
BALB/3T3 cells that cell division is the only requirement for
ments (Chart 4; Table 2) and in reconstruction experiments
fixation and expression of the transformed phenotype, the
(Chart 5) cannot be explained in this way, especially since present results point to a 2nd process which may not be
the medium was adequate for the growth of these cells dependent on cell division, in which initiated cells break
(Table 5). It can thus be concluded from these studies that away with varying degrees of success from endogenous am
exogenous control mechanisms. These results using cell
the inhibitory effect of serum on the growth of transformed
cells is mediated via the mass of nontransformed cells in the culture systems may be compatible with the idea of a mini
mum clone size fan tumor autonomy recently put forward by
culture. Therefore it appears that transformed 10T1/2cells
Bell (1). Clinically and experimentally (11), metastases can
are still responsive to the growth control mechanisms open
ative in 10T1/2cells, but external control is required. In lOT1!2 remain dormant for prolonged periods; it is hoped that the
cell system described here will prove a useful model for
cells these controls cause the phenomena of postconflu
ence inhibition of cell division, in which cells are arrested in studying interactions between normal and transformed
a reversible state of G1 arrest (4). It seems likely that the cells, in particular as related to latency of metastases.
In Table 1 it can be seen that exposure of cultures to 5%
serum-induced high saturation density of 1OTV2 cells is
responsible fan the growth inhibition of transformed cells.
serum shortly after treatment (1 on 2 days) fails to cause the
The increased cell crowding could then cause increased
enhancement in TF observed in cultures exposed at later
membrane interactions and allow nantmansformed cells to times. Since these cultures received identical carcinogen
modify the growth of an adjacent transformed cell (21, 30). treatment and were subsequently exposed to 5% serum for
a time sufficient to allow expression of the transformed
In support of this concept, it has been shown that increas
ing the ratio of normal to transformed 3T3 cells causes phenotype, it can be concluded that fixation of the original
increasing growth inhibition of the transformed cells and chemical lesion as a stable biological lesion is inhibited by
that this inhibition can be reversed by tumor promoters
exposure to 5% serum. In our original publication on the
oncogenic transformation of 1OT'/2 cells, we showed that
thought to be membrane-active agents (31). In addition,
while this paper was in preparation it was found that Haben exposure to 5% serum (postconfluency) reduced the TF in
et al.,3 working independently with the 1OTV2system and comparison with cultures maintained in 10% serum (27).
using different methodologies, had reached similar canclu
Furthermore, in the paper by Reznikoffetal. (28) describing
sions regarding the maleof cell density in the expression of the properties of the lOT'!2 cell line, these cells were stated
not to respond to serum concentration by reaching a higher
morphological transformation. The role of the membrane
saturation density. I have no explanation for these incon
could be direct, by preventing a change in conformation
(12), or indirect, by competing for a protease required for a sistencies except that the early studies were conducted with
modification of membrane structure (7, 10). An alternative
only 1 batch of serum and at only 2 concentrations. Fixation
mechanism whereby a high cell density of nontransformed
of DMBA damage in lOT'!2 cells appears essentially cam
cells can cause growth inhibition of transformed cells plete 4 days after exposure (Table 1). Kakunaga has shown
in a line of 3T3 cells that 1 cell division was required to fix
would be if nontransfommed cells secrete a growth-inhibi
tory substance to which the transformed cells respond but the oncogenic lesion induced by MCA and that another 3 or
cannot themselves secrete or accumulate. Such an inhibi
4 generations are required before transformation can be
tory substance could be a normal cell metabobite, in which expressed at a later date (20). This time period in 3T3 cells is
case the inhibition would be analogous to that of metabolic
similar to the 4-day period required for fixation in lOT'!2
cells reported here. The mechanism by which 5% serum can
cooperation observed in assay systems for mammalian mu
tagenesis (14, 34), or could be a specific mitatic inhibitor
inhibit this fixation is not clean, since rates of growth of
having characteristics of a chabone (6). From the data nontreated, nontransfommed cells do not differ in 5 am10%
shown in Table 5, any inhibitor must have a short biological
serum (Charts 1 and 2).
Transformed foci developing in 5% serum are cbonogenic
half-life. However, such inhibitors have been detected in
conditioned medium from other cell cultures (16, 22).
in soft agamosewith approximately the same frequency as
Decreases in TF with increasing initial cell plating density are foci developing in 10% serum. The ability to grow with
have been reported for the A31-714 line of BALB/3T3 cells out attachment to a solid substrate shows a very strong
(19), for mouse prostate cells (9), and for 10T1/2cells (27, 33). correlation with tumomigenicity (15), and this relationship
In our original publication an the transformation of lOT'!2 appears to apply to lOT'!2 cells and their transformed coun
cells we also demonstrated that, as tile initial plating density temparts (18). In the present studies about 70% of trans
increased, an increasing number of foci were found on the formed foci later grew in agamose; this value agrees well
edge of the Petni dish (i.e. , in the region of beastcell den
with a theoretical value of 78% which can be predicted from
sity). This was interpreted as being due to a requirement for the following considerations: (a) in these experiments about
20% of the transformed foci were of type II morphology and
cell division for expression of malignant transformation
(27). The results presented here would suggest that cell 80% were type Ill, and (b) about 50% of type II and 85% of
density per se was the important determinant of this me type III are tumamigenic in vivo (27). Thus these data imply
strongly that exposure to low serum is allowing expression
of the transformed phenotype in initiated cells which would
3 D. A. Haber,
D. A. Fox,
W.
F. Dynan,
and
W. G. Thilly.
Cell
Density
otherwise
remain latent and that serum enhancement of
dependent Expression of Neoplastic Transformation in the C3H/1OT'/2 Line,
transformation can be validly used to measure TF.
submitted to publication to CANCER RESEARCH.
by high
522
serum
concentrations
in transformation
experi
CANCERRESEARCHVOL. 37
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1977 American Association for Cancer Research.
Serum Effects on Transformation
Over the last 6 months this new protocol fan enhancing
the sensitivity of the 1OTV2assay system has been routinely
used in this laboratory with excellent results. The increased
yield of transformed foci leads to better statistics; the
greater contrast between transformed foci and the back
ground of nontransfarmed cells lead to more rapid scoring
of these foci (Fig. 1); the smaller consumption of serum
leads to obvious budgetary savings. The only problem that
may be encountered is in the strict enforcement of this new
protocol. In Table 1 is seen that the TF is depressed if low
serum is supplied too soon after treatment. Presumably,
this delay is related to a requirement for a certain number of
cell divisions. If carcinogen treatment induces a prolonged
delay in cell cycle transit time, cultures should be main
tamed for extended periods in 10% serum. This delay
should be apparent as a delay in the development of cola
nies in the assay for toxicity. Thus if routine fixing and
staining of surviving colonies in the toxicity assay must be
delayed, exposure to 5% serum in the transformation assay
should also be delayed fan the same period of time.
ACKNOWLEDGMENTS
I wish to thank Aurelie Mulhern and Roberta Roberts for diligent technical
assistance.
REFERENCES
1. Bell, G. I. Modelsof Carcinogenesisas an Escapefrom Mitotic Inhibi
tore.Science,192:569—574,
1976.
2. Bertram, J. S. Effect of Serum Concentration on Expression of Chemi
cally Induced Neoplastic Transformation in Culture. Proc. Am. Assoc.
Cancer Res., 17: 51, 1976.
3. Bertram, J. S. Enhancement by Actinomycin D of Dimethylbenzanthra
cene Induced Cell Killing and Malignant Transformation in Cultured
C3H/1OT'/2CL8Cells. FederationProc.,35: 410,1976.
4. Bertram, J. S., and Heidelberger, C. Cell Cycle Dependency of Onco
genic Transformation Induced by N-Methyl N'-nitro-N-nitrosoguanidine.
Cancer Aes., 34: 526-537, 1974.
5. Bertram, J. S., and LeStourgeon, W. M. Failure of Malignantly Trans
formed C3H/1OT'/2 CL8 Cells to Accumulate Nuclear Actin in Response
to High Cell Density. Proc. Am. Assoc. Cancer Aes., 16: 71, 1975.
6. Bullough, W. S. Chabone Control Systems. ln: J. LoBue and A. S.
Gordon (eds.), Humoral Control of Growth and Differentiation, pp. 1-8.
New York: Academic Press, Inc. , 1973.
Growth. Science, 192: 218-226, 1976.
13. Evans, V. J., and Andersen, W. F. The Effect of Serum on Neoplastic
Transformation In Vitro. J. NatI. Cancer Inst., 37: 247-249, 1966.
14. Fox, M. Factors Affecting the Quantitation of Dose Response Curves for
Mutation Induction in V79 Chinese Hamster Cells after Exposure to
Chemical and Physical Mutagens. Mutation Aes., 29: 449-466, 1975.
15. Freedman, V. H., and Shin, S. Cellular Tumorigenicity in Nude Mice:
Correlation with Cell Growth in Semi-solid Medium. Cell, 3: 355-359,
1974.
16. Horowitz, I. A., Nowint, H., and Hall, E. J. conditioned Medium from
Plateau-phase Cells. Radiology, 114: 723-726, 1975.
17. Jones, P. A., Benedict, W. F., Baker, M. S., Mondal, S., Rapp, U., and
Heidelberger, C. Oncogenic Transformation of C3H/IOTVa Clone 8
Mouse Embryo Cells by Halogenated Pyrimidine Nucleosides. Cancer
Res., 36: 101-107, 1976.
18. Jones, P. A., Laug, W. E., Gardner, A., Nye, C. A., Fink, L. M., and
Benedict, W. F. In Vitro Correlates of Transformation in C3H/1OT'/2
Clone 8 Mouse Cells. Cancer Res., 36: 2863-2867, 1976.
19. Kakunaga, T. Quantitative Assay System of Malignant Transformation by
ChemicalCarcinogensUsinga CloneDerivedfrom BALB/3T3.Intern.J.
Cancer, 12: 463-473, 1973.
20. Kakunaga, T. The Role of Cell Division in the Malignant Transformation
of Mouse Cells Treated with 3-Methylcholanthrene. Cancer Res., 35:
1637-1642, 1975.
21. Levine, E. M., Jeng, D., and Chang, V. Contact Inhibition, Polyribosomes
and Cell Surface Membranes in Cultured Mammalian Cells. J. Cellular
Physiol., 84: 349-364, 1974.
22. Little, J. B. Repair of Potentially
Lethal Radiation
Damage in Mammalian
Cells: Enhancement by Conditioned Medium from Stationary Cultures.
Intern. J. Radiation Biol., 20: 87-92, 1971.
23. MacPherson,
I., and Montagnier,L. AgarSuspensionCulturefor the
Selective Assay of Cells Transformed by Polyoma Virus. Virology, 23:
291-294, 1964.
24. Mondab, S., and Heidelberger, C. Transformation of C3H/1OT'/a CL8
Mouse Embryo Fibroblastsby Ultraviolet Irradiation and a Phorbol Ester.
Nature,260: 710-711, 1976.
25. Nesnow, S., and Heidelberger, C. The Effect of Modifiers of Microsomal
Enzymes on Chemical Oncogenesis in Cultures of C3H Mouse Cell Lines.
Cancer Res., 36: 1801-1808, 1976.
26. Price, F. M., Gantt, A. A., and Evans, V. J. Effect of Fractions
of Horse
and Fetal Bovine Serum on Neoplastic Conversion of C3H Mouse Cells in
Tissue Culture. In Vitro, 6: 437—440,
1971.
27. Reznikoff,
C. A.,Bertram,J.S.,Brankow,D. W., and Heidelberger,
C.
Quantitative and Qualitative Studies on Chemical Transformation of
Cloned C3H Mouse Embryo Cells Sensitiveto Postconfluence Inhibition
of Cell Division. Cancer Res., 33: 3239-3249, 1973.
28. Reznikoff,C. A.,Brankow,D. W., andHeidelberger,
C. Establishment
and Characterization of a Cloned Line of C3H Mouse Embryo Cells
Sensitive to Postconfluence Inhibition of Cell Division. Cancer Aes. , 33:
3231-3238, 1973.
29. Schaeffer,
W. I. , and Friend, K. Efficient
Detection
of Soft Agar Grown
Colonies using a Tetrazobium Salt. Cancer Letters, 1: 259-262, 1976.
30. Sivak, A. Induction
of Cell Division:
Role of Cell Membranes
Sites. J.
Cellular Physiol., 80: 167—174,
1972.
31. Sivak, A., and VanDuuren, B. L. A Cell Culture System for the Assess
ment of Tumor-promoting Activity. J. NatI. Cancer Inst., 44: 1091-1097,
1970.
7. Burger,M. M. A Differencein the Architectureof the SurfaceMembrane
32. Terzaghi, M. , and Little, J. B. Interactions between Radiation and
of Normal and Virally Transformed Cells. Proc. Natl. Acad. Sci. U. S., 62:
Benzo(a)pyrene in an In Vitro Model for Malignant Transformation. In: E.
994—1001,1969.
Karbe and J. F. Park (eds.), Experimental Lung Cancer Carcinogenesis
8. Carbone, G., Piazza, A., and Parmiani, G. Effect of Different Sera on
and Bioassays,pp. 497-506. Berlin: Springer-verlag, 1974.
Growth and Spontaneous Neoplastic Transformation on Mouse Fibro
33. Terzaghi, M., and Little, J. B. X-radiation-induced
Transformation
in a
blasts In vitro. J. NatI. Cancer Inst., 52: 387-392, 1974.
C3H Mouse Embryo-derived Cell Line. Cancer Res., 36: 1367-1374, 1976.
9. Chen, T. T. , and Heidelberger, C. Quantitative Studies on the Malignant
Transformation of Mouse Prostate Cells by Carcinogenic Hydrocarbons
34, Van Zeeband, A. A., van Diggelen, M. C. E., and Simons, J. W. I. M. The
In Vitro. Intern.J. Cancer,4: 166-178,1969.
Robe of Metabolic Cooperation in Selection of Hypoxanthine-guanine
10. Collard,
J.G.,and Smets,L.A.Effect
ofProteolytic
Inhibitors
on Growth
Phosphoribosyl Transferase (HGPRT) Deficient Mutants from Dipboid
and SurfaceArchitectureof Normaland TransformedCells. Exptl. Cell
Mammalian Cell Strains. Mutation Ass., 14: 335—363,
1972.
Aes., 86: 75-80, 1974.
35. Weibel,E. A., and Bolender,A. P. Stereobogicab
Techniquesfor Electron
11. Eccles, S. A. , and Alexander, P. Immunologically-mediated Aestraint of
Microscopic Morphometry. In: M. A. Hayat (ed), Principles and Tech
Latent Tumor Metastases. Nature, 257: 52-53, 1975.
niques of Electron Microscopy, pp. 237-296. New York: Van Nostrand
12. Edelman, G. M. Surface Modulation in Cell Recognition and Cell
Reinhold Co., 1973.
FEBRUARY
1977
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1977 American Association for Cancer Research.
523
Effects of Serum Concentration on the Expression of
Carcinogen-induced Transformation in the C3H/10T½ CL8 Cell
Line
John S. Bertram
Cancer Res 1977;37:514-523.
Updated version
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/37/2/514
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1977 American Association for Cancer Research.